Figure 3Left graph shows the multiplication rate of P493 - 6 ( - Tet ) cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA determined by viable cell counting 3 days after seeding of equal number of viable cells ( determined by Trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) . Right graph shows percentage of apoptotic P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or a TSC1 - specific sh - RNA determined by FACS analysis of AnnexinV / 7AAD stained cells ( mean ± st . dev . , n = 3 biological replicates ) . Rapamycin treatment recovers survival of TSC1 knockdown in P493 - 6 cells . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 14 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 30pM rapamycin where indicated ( mean ± st . dev . , n = 3 biological replicates ) . TSC1 knockdown is synthetic lethal with MYC deregulation . U2OS - MYC - ER cells expressing either scrambled control sh - RNA or TSC1 - specific sh - RNA were treated with hydroxytamoxifen to induce MYC and rapamycin ( 100 nM ) where indicated . Percentage of apoptotic cells was determined with Annexin / 7AAD staining 4 days after MYC induction ( mean ± st . dev . , n = 3 biological replicates ) . Survival rate of different BL cell lines upon TSC1 knockdown . Graphs show numbers of viable cells expressing either a scrambled control sh - RNA or a TSC1 - specific sh - RNA 3 days after seeding of equal number of viable cells ( determined by trypan blue exclusion ; mean ± st . dev . , n = 3 biological replicates ) . Immunoblots of control - or TSC1 - shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity ( 10nM ) or titrate the activity to control levels ( 30 pM ) , and survival rate of these cells over 7 days ( mean ± st . dev . , n = 3 biological replicates ) ; ( Bl2 cells were selected for stable knockdown with Puromycin , DG75 cells without selection ) . Ramos cells expressing either a TSC1 - specific or a control shRNA were inoculated into NOD / SCID mice and tumor volume was measured regularly . The immunoblot shows the level of knockdown of TSC1 ( sh - TSC1b ) compared to control ( sh - contr ) , and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation . Tumor growth curves show mean ± SEM ( n = 8 / group ) . Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation . The top panels show tumors derived from Ramos cells stably expressing control sh - RNA ( sh - contr ) ; the lower panels show tumors derived from Ramos cells stably expressing TSC1 - specific sh - RNA ( sh - TSC1b ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates).Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates).Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA 3 days after seeding of equal number of viable cells (determined by trypan blue exclusion; mean ± st.dev., n=3 biological replicates).Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection).Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n=8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control sh-RNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific sh-RNA (sh-TSC1b).


Figure 4TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or TSC - specific sh - RNA , treated with 20 nM rapamycin for 12 h , or tetracycline to repress MYC for 48 h where indicatedTSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner . Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated ( B ) ( mean ± st . dev . , n = 6 biological replicates ) . Ratio of mitochondrial to genomic DNA determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) . expression of cytochrome C ( CYCS ) or ATP5G1 mRNAs ( D ) determined by qRT - PCR in P493 - 6 ( - Tet ) cells expressing either scrambled control sh - RNA or a TSC1 - specific sh - RNA , and treated with 20 nM rapamycin for 12 h where indicated ( mean ± st . dev . , n = 3 technical replicates ) . TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner . FACS analysis of DCF - DA stained P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated ( mean ± st . dev . , n = 3 biological replicates ) . TSC1 knockdown results in increased phosphorylation of SAPK / JNK . Immunoblot of SAPK / JNK Thr183 / Tyr185 - phosphorylation in P493 - 6 ( - Tet ) cells expressing scrambled control sh - RNA or a TSC1 - specific sh - RNA . α - tubulin expression serves as loading control . Antioxidant treatment rescues cells from death caused by TSC1 knockdown . Relative viable cell number counts of P493 - 6 ( - Tet ) cells expressing scrambled control shRNA or TSC1 - specific sh - RNA 3 days after seeding equal number of viable cells ( Trypan blue exclusion ) , in the presence of 10 μM of the antioxidant BHA where indicated ( mean ± st . dev . , n = 3 biological replicates ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicatedTSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates).Ratio of mitochondrial to genomic DNA determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates).TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control.Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).


Figure 5Accumulation of EU - labeled ( Click - It , Invitrogen ) TSC1 - mRNA over 3 h in high MYC ( - Tet ) versus low MYC ( + Tet ) P493 - 6 cells ( mean ± st . dev . , n = 3 technical replicates ) . Drawing of the TSC1 promoter and luciferase - reporter constructs with the predicted E - box ( CACGTG , pos . - 317 / - 322 ) indicated . Relative luciferase units ( RLU ) of co - transfection experiments using the indicated TSC1 - promoter - reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells ( mean ± st . dev . , n = 3 ) . Immunoblot shows MYC overexpression and α - tubulin as loading control . TSC1 mRNA turnover determined by pulse and chase ( Click - It , Invitrogen ) over 8 h in P493 - 6 cells , either treated with tetracycline to repress MYC or left untreated ( mean ± st . dev . , n = 3 technical replicates ) . miR - 15a expression determined by qRT - PCR in P493 - 6 cells treated with tetracycline for 3 days ( + Tet ) to suppress MYC expression or in untreated ( - Tet ) cells ( mean ± st . dev . , n = 3 technical replicates ) . Immunoblots showing expression levels of TSC1 , S6K / P - S6K and α - tubulin in P493 - 6 and HEK293T cells overexpressing miR - 15a or a control miRNA . At the right miR - 15a overexpression was determined by qRT - PCR in P493 - 6 and HEK293T cells ( mean ± st . dev . , n = 3 technical replicates ) . Immunoblot showing TSC1 expression , S6K / P - S6K and α ‑ tubulin in MCF - 7 cells transfected with LNA modified anti - sense miR - 15a oligos or control LNAs . At the right , successful miR - 15a knockdown determined by qRT - PCR in MCF - 7 cells is shown ( mean ± st . dev . , n = 3 technical replicates ) . Relative luciferase activities ( RLU ) derived from reporter constructs containing TSC1 - 3 ´ UTR wild type sequences or with mutated miR - 15a seed sequence binding site on co - transfection with miR - 15a in MCF - 7 cells ( mean ± st . dev . , n = 3 biological replicates ) . The drawing at the left shows the TSC1 - 3 ' UTR with the position of the miR - 15a / 16 - 1 seed sequence and used mutation indicated . Rate of oxygen consumption in P493 - 6 ( - Tet ) cells ectopically expressing miR - 15a or control - miRNA vector , under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated ( mean ± st . dev . , n = 8 biological replicates ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates).Drawing of the TSC1 promoter and luciferase-reporter constructs with the predicted E-box (CACGTG, pos. -317/-322) indicated. Relative luciferase units (RLU) of co-transfection experiments using the indicated TSC1-promoter-reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells (mean ± st. dev., n=3). Immunoblot shows MYC overexpression and α-tubulin as loading control.TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates).miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates).Immunoblots showing expression levels of TSC1, S6K/P-S6K and α-tubulin in P493-6 and HEK293T cells overexpressing miR-15a or a control miRNA. At the right miR-15a overexpression was determined by qRT-PCR in P493-6 and HEK293T cells (mean ± st. dev., n=3 technical replicates).Immunoblot showing TSC1 expression, S6K/P-S6K and α‑tubulin in MCF-7 cells transfected with LNA modified anti-sense miR-15a oligos or control LNAs. At the right, successful miR-15a knockdown determined by qRT-PCR in MCF-7 cells is shown (mean ± st. dev., n=3 technical replicates).Relative luciferase activities (RLU) derived from reporter constructs containing TSC1-3´UTR wild type sequences or with mutated miR-15a seed sequence binding site on co-transfection with miR-15a in MCF-7 cells (mean ± st. dev., n=3 biological replicates). The drawing at the left shows the TSC1-3'UTR with the position of the miR-15a/16-1 seed sequence and used mutation indicated.Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).


figf1a , Polychromasia ( Wright - Giemsa stain , indicated by arrows ) and increased reticulocytes ( brilliant cresyl blue stain ) revealed by staining blood smears from 6 - week - old wild - type ( WT ) or Nix - / - mice ( scale bar , 20 µm ) . b , c , Ter119 ( b ) or Mitotracker deep red ( c ) versus CD71 staining of RBCs from 3 - or 6 - week - old mice . Cell counts were also calculated ( mean ± s . e . m . ; n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . b , c , Ter119 ( b ) or Mitotracker deep red ( c ) versus CD71 staining of RBCs from 3 - or 6 - week - old mice . Cell counts were also calculated ( mean ± s . e . m . ; n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . d , Analyses of RBCs from 3 - or 6 - week - oldmice by immunocytochemistryAnalyses of RBCs from 3 - or 6 - week - oldmice by transmission electron

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O

figf1a, Polychromasia (Wright-Giemsa stain, indicated by arrows) and increased reticulocytes (brilliant cresyl blue stain) revealed by staining blood smears from 6-week-old wild-type (WT) or Nix-/- mice (scale bar, 20 µm).b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.d, Analyses of RBCs from 3- or 6-week-oldmice by immunocytochemistryAnalyses of RBCs from 3- or 6-week-oldmice by transmission electron microscopy


figf2a , Quantification of NHS - biotin - labelled RBCs or transferred CMFDA - labelled RBCs ( n = 3 ) . b , Western blot analyses of caspases in RBCs after in vitro culture . Asterisks denote processed caspases . FL , full length . c , The relative luminescence units ( RLU ) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix - / - RBCs after 12 - h culture in the presence of qVD - oph or solvent control ( DMSO ) ( n = 3 ) . d , Mitotracker versus annexin V staining of RBCs after in vitro culture . e , Mean fluorescent intensity ( MFI ) of ROS staining in RBCs after in vitro culture , and caspase activities in Nix - / - RBCs after 24 h culture with solvent control , ROS scavengers or qVD - oph ( n = 3 ) . BHT , butylated hydroxytoluene ; CAT , catalase ; TEM , tempol . f , Haematoxylin and eosin ( HE ) staining of spleen sections of 9 - week - old wild - type and Nix - / - mice . Arrows denote iron deposits within macrophagecytoplasm . Iron deposits were also stained with Prussian blue , followed by counterstain with nuclear - fast red . Scale bar , 20 µm . g , Real - time RT - PCR of erythropoietin ( Epo ) ( n = 12 ) . For all relevant panels , statistical significance of the data ( mean ± s . e . m . ) is : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3).b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length.c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3).d, Mitotracker versus annexin V staining of RBCs after in vitro culture.e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol.f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophagecytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm.g, Real-time RT-PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P < 0.05; **P < 0.01; ***P < 0.001.


figf3a , Ter119 + CD71 + reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 ( top row ) , 2 ( middle row ) or 4 ( bottom row ) days , followed by Mitotracker deep red and Ter119 staining . b , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue . Scale bar , 20 µm . c , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy . Scale bar , 5 µm . The Pearson coefficiency for LC3 and COX IV co - localization at day 0 ( mean ± s . e . m . ) is : wild type , 0 . 81 ± 0 . 001 ; Nix - / - , 0 . 44 ± 0 . 036 ( n = 35 , P = 0 . 0006 ) . d , CD71 + Ter119 + reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy . Scale bar , 0 . 5 µm .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3a, Ter119+CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining.b, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 µm.c, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 µm. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; Nix-/-, 0.44 ± 0.036 (n = 35, P = 0.0006).d, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy. Scale bar, 0.5 µm.


figf4a , Ter119 + CD71 + reticulocytes were sorted from the peripheral blood of wild - type and Nix - / - mice at day 6 after PHZ treatment . The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT - 737 , followed by staining with Mitotracker deep red and phycoerythrin - conjugated anti - Ter119 . b , c , Reticulocytes were cultured with FCCP ( b ) or ABT - 737 ( c ) for 24 h and analysed by transmission electron microscopy . Scale bar , 0 . 5 µm .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4a, Ter119+CD71+ reticulocytes were sorted from the peripheral blood of wild-type and Nix-/- mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119.b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 µm.


Figure 2 ( B ) BW25113 ( ycbB , relA ' ) and its derivatives obtained by individual deletion of endopeptidase genes . BW25113 ( ycbB , relA ' ) is an abbreviated name for BW25113 ΔrelA pKT2 ( ycbB ) pKT8 ( relA ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2  (B) BW25113(ycbB, relA') and its derivatives obtained by individual deletion of endopeptidase genes. BW25113(ycbB, relA') is an abbreviated name for BW25113 ΔrelA pKT2(ycbB) pKT8(relA').


Figure 3 Growth of BW25113 ( ycbB , relA ' ) , BW25113 ( ycbB , relA ' ) ΔmepM , and its derivatives harboring plasmids encoding MepM , MepM H393A , and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg / ml ( + ceftriaxone ) or in the absence of the drug ( - ceftriaxone ) in BHI agar plates supplemented with 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively . The genes encoding MepM , MepM H393A , and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter , which is inducible by 2 , 4 - diacetylphloroglucinol ( DAPG ) . Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 Growth of BW25113(ycbB, relA'), BW25113(ycbB, relA') ΔmepM, and its derivatives harboring plasmids encoding MepM, MepM H393A, and MepM ΔLytM were tested in the presence of ceftriaxone at 8 µg/ml (+ ceftriaxone) or in the absence of the drug (- ceftriaxone) in BHI agar plates supplemented with 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively. The genes encoding MepM, MepM H393A, and MepM ΔLytM were inserted into the vector pHV9 under the control of the PphlF promoter, which is inducible by 2,4-diacetylphloroglucinol (DAPG). Basal level of expression of mepM under the control of PphlF was sufficient to restore ceftriaxone resistance in the absence of the inducer.


Figure 4 The mepM gene was expressed under the control of TIS1 or TIS2 ( translation initiation ) and of PrhaBAD ( the L - rhamnose - inducible promoter of vector pHV30 ) . Growth of BW25113 ( ycbB , relA ' ) ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg / ml , IPTG 40 µM ( induction of ycbB ) and L - arabinose 1 % ( induction of relA ' ) . ( A ) For TIS2 , growth around paper disks containing 1 or 2 mg of L - rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance . In contrast , a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer . ( B ) The experiment was repeated with bacteria pre - exposed to L - rhamnose that were harvested at the vicinity of the disk containing 2 mg of L - rhamnose . Expression of ceftriaxone resistance remained dependent on the presence of L - rhamnose indicating that the requirement for MepM is not transient . The diameter of the growth zones is larger in panel ( B ) than in panel ( A ) as expected from the fact that bacteria in the inoculum used in ( B ) had been grown in the presence of the inducer and already contained MepM . In ( A ) growth is only possible after diffusion of L - rhamnose in the medium prior to the action of ceftriaxone . At a distance from the disk , diffusion was not sufficiently rapid to observe resistance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.


Figure 5 ( A ) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme ( upper panel ) or by lysozyme and 5 µM MepM ( lower panel ) . Absorbance was monitored at 205 nm ( mAU , milli - absorbance unit ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 (A) rpHPLC chromatograms of sacculi isolated from BW25113 grown in minimal medium to stationary phase and digested by lysozyme (upper panel) or by lysozyme and 5 µM MepM (lower panel). Absorbance was monitored at 205 nm (mAU, milli-absorbance unit).


Figure 6 Functional complementation of the mepM deletion in BW25113 ( ycbB , relA ' ) ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal . Induction of endopeptidase ( ED ) genes was performed with 0 . 2 % L - rhamnose in the presence or absence of 8 µg / ml ceftriaxone . BHI agar plates contained 40 µM IPTG and 1 % L - arabinose for induction of ycbB and relA ' , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 Functional complementation of the mepM deletion in BW25113(ycbB, relA') ΔmepM was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter and of the TIS1 translation initiation signal. Induction of endopeptidase (ED) genes was performed with 0.2% L-rhamnose in the presence or absence of 8 µg/ml ceftriaxone. BHI agar plates contained 40 µM IPTG and 1% L-arabinose for induction of ycbB and relA', respectively.


Figure 8 ( B ) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53 ( mepM ) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter . In this assay , functional replacement of MepM is detected based on growth in media containing 0 . 2 % or 1 % L - arabinose for expression of the endopeptidase gene carried by vector pHV7 , while by - passing the requirement for induction of the mepM copy of pHV53 by L - rhamnose . Complementation was observed with both concentrations of inducer for mepH , mepS , and pbpG . Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L - arabinose ( 1 % ) was lethal . The right panel presents the growth control performed in the presence of 0 . 2 % L - rhamnose for induction of the mepM copy carried by pHV53 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 (B) The plating efficiency assay was performed with derivatives of BW25113 Δ8EDs pHV53(mepM) harboring the vector pHV7 or recombinant plasmids carrying each of the eight endopeptidase genes under the control of the ParaBAD promoter. In this assay, functional replacement of MepM is detected based on growth in media containing 0.2% or 1% L-arabinose for expression of the endopeptidase gene carried by vector pHV7, while by-passing the requirement for induction of the mepM copy of pHV53 by L-rhamnose. Complementation was observed with both concentrations of inducer for mepH, mepS, and pbpG. Overproduction of mepM encoded by the pHV7 derivative in the presence of the high dose of L-arabinose (1%) was lethal. The right panel presents the growth control performed in the presence of 0.2% L-rhamnose for induction of the mepM copy carried by pHV53.


Figure 9 ( B ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9 (B) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding each of the eight endopeptidases under the control of the PrhaBAD promoter. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


Figure 10 ( A ) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63 ( ycbB ) was performed with the pHV30 vector or recombinant plasmids encoding mepA , mepK , mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter . ( B ) The complementation assay was repeated for BW25113 Δ7EDs pHV63 ( ycbB ) , which was obtained by deletion of all chromosomal endopeptidase genes except mepM . Induction of ycbB and of endopeptidase ( ED ) genes was performed with 0 . 2 % L - arabinose and 1 % L - rhamnose , respectively . BHI agar plates contained chloramphenicol ( 20 µg / ml ) to counter - select loss of pHV63 ( ycbB ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10 (A) Functional complementation of the mepK deletion of BW25113 ΔmepK pHV63(ycbB) was performed with the pHV30 vector or recombinant plasmids encoding mepA, mepK, mepS or derivatives encoding catalytically inactive endopeptidases under the control of the PrhaBAD promoter. (B) The complementation assay was repeated for BW25113 Δ7EDs pHV63(ycbB), which was obtained by deletion of all chromosomal endopeptidase genes except mepM. Induction of ycbB and of endopeptidase (ED) genes was performed with 0.2% L-arabinose and 1% L-rhamnose, respectively. BHI agar plates contained chloramphenicol (20 µg/ml) to counter-select loss of pHV63(ycbB).


Figure 11 ( A ) Growth curves of ( i ) BW25113 ( ycbB , relA ' ) and its ΔsltY derivative ; and ( ii ) strain M1 and its derivatives ( M1 . 1 to 1 . 4 ) harboring mutations in sltY . The growth medium contained ( i ) 40 µM IPTG , 1 % L - arabinose , and 8 µg / ml ceftriaxone for BW25113 ( ycbB , relA ' ) and its ΔsltY derivative or ( ii ) 50 µM IPTG and 16 µg / ml ampicillin for M1 and its derivatives .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 11 (A) Growth curves of (i) BW25113(ycbB, relA') and its ΔsltY derivative; and (ii) strain M1 and its derivatives (M1.1 to 1.4) harboring mutations in sltY. The growth medium contained (i) 40 µM IPTG, 1% L-arabinose, and 8 µg/ml ceftriaxone for BW25113(ycbB, relA') and its ΔsltY derivative or (ii) 50 µM IPTG and 16 µg/ml ampicillin for M1 and its derivatives.


f1a , Pancreatic sections stained with Atg7 , LC3 and p62 showing Atg7 − / − next to Atg7 + / + regions after Pdx - Cre - mediated recombination . Right - side images are cropped and magnified regions ( boxed ) from left - side images . The magnification is × 2 . 5 . b , Haematoxylin and eosin ( HE ) , p53 and caspase - 3 staining of exocrine tissue in Atg7 + / + and Atg7 − / − pancreases . c , Quantification ( median , s . e . m . ) of p53 and caspase - 3 activation in Atg7 - deficient acinar tissue . d , Kaplan - Meier analysis comparing overall survival of mice either wild type ( blue ) or Atg7 − / − ( red ) in the pancreas . Mantel - Cox test was used for statistics . ' n / a ' , 14 / 16 mice were killed > 500 days when completely healthy . e , Biochemical analysis of endocrine function in moribund Atg7 − / − or age - matched healthy control mice . Detailed information about the mice is tabulated . A Mann - Whitney U - test was used for statistics . Scale bars : 50 µm . * * P 0 . 01 and * * * P 0 . 001 .

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f1a, Pancreatic sections stained with Atg7, LC3 and p62 showing Atg7−/− next to Atg7+/+ regions after Pdx-Cre-mediated recombination. Right-side images are cropped and magnified regions (boxed) from left-side images. The magnification is × 2.5.b, Haematoxylin and eosin (HE), p53 and caspase-3 staining of exocrine tissue in Atg7+/+ and Atg7−/− pancreases.c, Quantification (median, s.e.m.) of p53 and caspase-3 activation in Atg7-deficient acinar tissue.d, Kaplan-Meier analysis comparing overall survival of mice either wild type (blue) or Atg7−/− (red) in the pancreas. Mantel-Cox test was used for statistics. 'n/a', 14/16 mice were killed >500 days when completely healthy.e, Biochemical analysis of endocrine function in moribund Atg7−/− or age-matched healthy control mice. Detailed information about the mice is tabulated. A Mann-Whitney U-test was used for statistics. Scale bars: 50 µm. **P 0.01 and ***P 0.001.


f2a , Alcian blue / PAS ( AB / PAS ) staining in pancreases from 150 - day - old Atg7 - proficient or Atg7 - deficient KrasG12D / + mice ( wild - type mice with expression of KrasG12D in their pancreases ) . b , Quantification of PanIN from mice of the indicated genotype and age . Fold increase and male / female ratios are shown . Box plots express the first ( Q1 ) and third ( Q3 ) quartiles set by the upper and lower limits of the box , whereas the median is shown by the horizontal line inside the box . The whiskers extend upward and downward to the highest or lowest observation within the upper ( Q3 + 1 . 5 × the interquartile range ) and lower ( Q1 − 1 . 5 × interquartile range ) limits . Values outside the upper and lower limits are ' outliers ' and are shown by individual symbols . c , d , Kaplan - Meier analysis comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + mice . c , d , Kaplan - Meier analysis comparing overall survival ( c ) or PDAC - free survival ( d ) of Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + micee , PanIN grading of pancreases from 150 - day - old Atg7 + / + or Atg7 − / − Pdx - Cre KrasG12D / + mice . Values are mean and error bars are s . d . b , e , A Mann - Whitney U - test was used for statistics . f , Representative p53 staining from 150 - day - old mice of both genotypes . g , PDAC - free survival of autophagy - proficient ( blue ) and Atg5 - deficient ( red ) KrasG12D / + mice . d , g , Median survival ± s . d . and male / female ( m / f ) ratio are provided . ' n / a ' , no animals succumbed to PDAC . A log - rank test ( Mantel - Cox ) was used for statistics . a , f , Scale bars : 500 µm ( a ) , 50 µm ( f ) . * P 0 . 05 , * * P 0 . 01 ; NS , not significant .

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f2a, Alcian blue/PAS (AB/PAS) staining in pancreases from 150-day-old Atg7-proficient or Atg7-deficient KrasG12D/+ mice (wild-type mice with expression of KrasG12D in their pancreases).b, Quantification of PanIN from mice of the indicated genotype and age. Fold increase and male/female ratios are shown. Box plots express the first (Q1) and third (Q3) quartiles set by the upper and lower limits of the box, whereas the median is shown by the horizontal line inside the box. The whiskers extend upward and downward to the highest or lowest observation within the upper (Q3 + 1.5 × the interquartile range) and lower (Q1 − 1.5 × interquartile range) limits. Values outside the upper and lower limits are 'outliers' and are shown by individual symbols.c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ mice.c, d, Kaplan-Meier analysis comparing overall survival (c) or PDAC-free survival (d) of Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ micee, PanIN grading of pancreases from 150-day-old Atg7+/+ or Atg7−/− Pdx-Cre KrasG12D/+ mice. Values are mean and error bars are s.d. b, e, A Mann-Whitney U-test was used for statistics.f, Representative p53 staining from 150-day-old mice of both genotypes.g, PDAC-free survival of autophagy-proficient (blue) and Atg5-deficient (red) KrasG12D/+ mice. d, g, Median survival ± s.d. and male/female (m/f) ratio are provided. 'n/a', no animals succumbed to PDAC. A log-rank test (Mantel-Cox) was used for statistics. a, f, Scale bars: 500 µm (a), 50 µm (f). *P  0.05, **P  0.01; NS, not significant.


f3a , Kaplan - Meier analysis comparing PDAC - free survival in Atg7 + / + ( blue ) or Atg7 − / − ( red ) KrasG12D / + p53 − / − mice . b , Pancreatic tumours of indicated genotypes stained for Atg7 , LC3 and p62 . Insets are magnified crops from the same images to show specifics of LC3 and p62 staining . The magnification is × 4 . c , Tumour incidence and tumour weight ( median , s . e . m . ) in mice from indicated genotypes , killed at 21 , 29 and 38 days of age . Numbers in the incidence diagram represent tumour - bearing mice versus all mice . Significance was assessed with Fisher ' s exact test ( incidence ) and Mann - Whitney U - test ( weight ) . d , Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes . e , Kaplan - Meier analysis comparing PDAC - free survival of Atg5 + / + ( blue ) or Atg5 − / − ( green ) KrasG12D / + p53 − / − mice . f , Kaplan - Meier analysis comparing PDAC - free survival of KrasG12D / + p53 − / − mice that were chloroquine - treated ( CQ , red ) , vehicle - treated ( PBS , black ) or untreated ( blue ) . a , e , f , The blue curve in all Kaplan - Meier plots represents the same colony of mice ( KrasG12D / + , p53 − / − , Atg7 + / + , Atg5 + / + ) . A Mantel - Cox test was used for statistics . b , d , Scale bars : 100 µm ( b ) , 200 µm ( d ) . * P 0 . 05 , * * P 0 . 01 .

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f3a, Kaplan-Meier analysis comparing PDAC-free survival in Atg7+/+ (blue) or Atg7−/− (red) KrasG12D/+ p53−/− mice.b, Pancreatic tumours of indicated genotypes stained for Atg7, LC3 and p62. Insets are magnified crops from the same images to show specifics of LC3 and p62 staining. The magnification is × 4.c, Tumour incidence and tumour weight (median, s.e.m.) in mice from indicated genotypes, killed at 21, 29 and 38 days of age. Numbers in the incidence diagram represent tumour-bearing mice versus all mice. Significance was assessed with Fisher's exact test (incidence) and Mann-Whitney U-test (weight).d, Representative haematoxylin and eosin stainings of pancreases from indicated time points and genotypes.e, Kaplan-Meier analysis comparing PDAC-free survival of Atg5+/+ (blue) or Atg5−/− (green) KrasG12D/+ p53−/− mice.f, Kaplan-Meier analysis comparing PDAC-free survival of KrasG12D/+ p53−/− mice that were chloroquine-treated (CQ, red), vehicle-treated (PBS, black) or untreated (blue). a, e, f, The blue curve in all Kaplan-Meier plots represents the same colony of mice (KrasG12D/+, p53−/−, Atg7+/+, Atg5+/+). A Mantel-Cox test was used for statistics. b, d, Scale bars: 100 µm (b), 200 µm (d). *P 0.05, **P 0.01.


f4a , LC3 staining of representative pancreatic tumours from mice of indicated genotypes . Scale bars : 50 µm . b , Western blotting for LC3 , p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53 . c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR , c , d , Average oxygen consumption rate ( OCR , c ) and extracellular acidification rate ( ECAR , d ) of eight Atg7 + / + and seven Atg7 − / − cell lines revealed increased ECAR in autophagy - deficient cells . Error bars are s . e . m . e , Treatment with 2 - deocyglucose ( 2 - DG ) reduces ECAR and abrogates the difference between cell lines from Atg7 + / + and Atg7 − / − tumours . Values are the average of three biological replicates . Error bars are s . e . m . , g , LC - MS analysis revealed increased glucose consumption from medium ( f ) and extracellular lactate accumulation ( g ) in Atg7 - null cell lines . Values are the average of eight ( Atg7 + / + ) and seven ( Atg7 − / − ) biological replicates . h , LC - MS analysis shows increased accumulation of 2 - DG and its metabolite 2 - DG - 6 - phosphate in Atg7 - deficient cell lines . Values are averages of five biological replicates for each genotype . i , Background levels of 18F - FDG uptake in brain and heart can be seen in sagittal and coronal views ( middle and right panels , respectively ) of wild - type Atg7 mice ( top row ) . In contrast , intense 18F - FDG uptake is seen in pancreatic tumours of Atg7 deficient mice ( bottom row ) . j , LC - MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7 − / − tumour cell lines . Values are average of eight ( Atg7 + / + ) and six ( Atg7 − / − ) biological replicates . f - h , j , Error bars are s . e . m . a , f - h , j , A Mann - Whitney U - test was used to ascertain significance . * P 0 . 05 , * * P 0 . 01 .

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f4a, LC3 staining of representative pancreatic tumours from mice of indicated genotypes. Scale bars: 50 µm.b, Western blotting for LC3, p53 and Atg7 in cell lines derived from individual tumours which developed in the absence or presence of p53.c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR,c, d, Average oxygen consumption rate (OCR, c) and extracellular acidification rate (ECAR, d) of eight Atg7+/+ and seven Atg7−/− cell lines revealed increased ECAR in autophagy-deficient cells. Error bars are s.e.m.e, Treatment with 2-deocyglucose (2-DG) reduces ECAR and abrogates the difference between cell lines from Atg7+/+ and Atg7−/− tumours. Values are the average of three biological replicates. Error bars are s.e.m., g, LC-MS analysis revealed increased glucose consumption from medium (f) and extracellular lactate accumulation (g) in Atg7-null cell lines. Values are the average of eight (Atg7+/+) and seven (Atg7−/−) biological replicates.h, LC-MS analysis shows increased accumulation of 2-DG and its metabolite 2-DG-6-phosphate in Atg7-deficient cell lines. Values are averages of five biological replicates for each genotype.i, Background levels of 18F-FDG uptake in brain and heart can be seen in sagittal and coronal views (middle and right panels, respectively) of wild-type Atg7 mice (top row). In contrast, intense 18F-FDG uptake is seen in pancreatic tumours of Atg7 deficient mice (bottom row).j, LC-MS of intracellular glycolytic and pentose phosphate pathway intermediates shows enhanced accumulation in Atg7−/− tumour cell lines. Values are average of eight (Atg7+/+) and six (Atg7−/−) biological replicates. f-h, j, Error bars are s.e.m. a, f-h, j, A Mann-Whitney U-test was used to ascertain significance. *P  0.05, **P  0.01.


Figure 1C Subcellular fractionation of the WT and NLS - deficient FolSrpk1 - GFP proteins . N , nucleus ; C , cytosol . The amount of nuclear FolSrpk1 - GFP was set as 1 . Data information : For C , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining . D Quantitative measurement of H2O2 in tomato roots after infection by the WT and FolSrpk1p : : FolSrpk1 - GFP strains at 0 - 5 days post incubation ( dpi ) . Data are presented as mean ± SD . The presence of different letters above the mean values of three replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . E Subcellular fractionation of FolSrpk1 - GFP during the early infection stage . Total proteins were extracted from tomato roots at 0 ( conidia ) , 1 , 3 and 5 dpi . Equal amount of total , nuclear and cytoplasm proteins was separated by SDS - PAGE , and FolSrpk1 was detected using anti - GFP . The amount of total and nuclear FolSrpk1 - GFP at 0 dpi was each set as 1 . Data information : For E , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining . F Association of FolSrpk1 with FolAha1 under ROS stress . After treatment with 10 mM H2O2 for 0 , 1 and 2 h , proteins pulled down with GFP - Trap beads were probed with anti - GFP and anti - Flag ( top ) . Input proteins were shown by Western blotting with anti - GFP and anti - FLAG antibodies ( bottom ) . The amount of immunoprecipitated FolSrpk1 - Flag and FolAha1 - GFP at 0 h was each set as 1 . Data information : For , F each gel shown is a representative experiment carried out at least three times . G Fluorescence microscopy analysis of FolSrpk1 - GFP in mycelia of the FolSrpk1p : : FolSrpk1 - GFP strain treated with 10 mM H2O2 for 0 , 1 and 2 h . Arrows indicate the nucleus . Scale bars = 5 µm . I Subcellular fractionation analysis of FolSrpk1 - GFP in mycelia of the FolSrpk1p : : FolSrpk1 - GFP strain treated with 10 mM H2O2 for 0 , 1 and 2 h . The amount of nuclear FolSrpk1 - GFP at 0 h was set as 1 . Data information : For I , each gel shown is a representative experiment carried out at least three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and Coomassie brilliant blue ( CBB ) staining .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C Subcellular fractionation of the WT and NLS-deficient FolSrpk1-GFP proteins. N, nucleus; C, cytosol. The amount of nuclear FolSrpk1-GFP was set as 1. Data information: For C, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.D Quantitative measurement of H2O2 in tomato roots after infection by the WT and FolSrpk1p::FolSrpk1-GFP strains at 0-5 days post incubation (dpi). Data are presented as mean ± SD. The presence of different letters above the mean values of three replicates indicates a significant difference between different samples (P < 0.05, ANOVA).E Subcellular fractionation of FolSrpk1-GFP during the early infection stage. Total proteins were extracted from tomato roots at 0 (conidia), 1, 3 and 5 dpi. Equal amount of total, nuclear and cytoplasm proteins was separated by SDS-PAGE, and FolSrpk1 was detected using anti-GFP. The amount of total and nuclear FolSrpk1-GFP at 0 dpi was each set as 1. Data information: For E, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.F Association of FolSrpk1 with FolAha1 under ROS stress. After treatment with 10 mM H2O2 for 0, 1 and 2 h, proteins pulled down with GFP-Trap beads were probed with anti-GFP and anti-Flag (top). Input proteins were shown by Western blotting with anti-GFP and anti-FLAG antibodies (bottom). The amount of immunoprecipitated FolSrpk1-Flag and FolAha1-GFP at 0 h was each set as 1. Data information: For , F each gel shown is a representative experiment carried out at least three times.G Fluorescence microscopy analysis of FolSrpk1-GFP in mycelia of the FolSrpk1p::FolSrpk1-GFP strain treated with 10 mM H2O2 for 0, 1 and 2 h. Arrows indicate the nucleus. Scale bars = 5 µm.I Subcellular fractionation analysis of FolSrpk1-GFP in mycelia of the FolSrpk1p::FolSrpk1-GFP strain treated with 10 mM H2O2 for 0, 1 and 2 h. The amount of nuclear FolSrpk1-GFP at 0 h was set as 1. Data information: For I, each gel shown is a representative experiment carried out at least three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and Coomassie brilliant blue (CBB) staining.


Figure 2Co - IP ( C ) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1 . The amount of immunoprecipitated FolSrpk1 - Flag and FolAha1 - GFP in the first line was each set as 1 . Data information : For C , each gel shown is a representative experiment carried out at least three times . Y2H ( D ) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1 . F Fluorescence microscopy of FolSrpk1 - GFP and its mutant forms . Arrows indicate the nucleus . Bars = 5 µm . G Quantification of FolSrpk1 - GFP distribution in F . The calculation was from three independent replicates with three hyphae per replicate and three cells per hyphae . Different letters indicate statistically significant differences according to one - way ANOVA ( P < 0 . 05 ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean . H Subcellular fractionation of FolSrpk1 - GFP and its mutant forms . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining . The amount of nuclear FolSrpk1 - GFP was set as 1 . Data information : For H , , each gel shown is a representative experiment carried out at least three times . I K304 acetylation of WT and mutant FolSrpk1 proteins in the indicated strains after treatment with 10 mM H2O2 for 0 , 1 and 2 h . The amount of acetylated and total FolSrpk1 - GFP at 0 h was each set as 1 . Data information : For each gel shown is a representative experiment carried out at least three times .

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Figure 2Co-IP (C) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1. The amount of immunoprecipitated FolSrpk1-Flag and FolAha1-GFP in the first line was each set as 1. Data information: For C, each gel shown is a representative experiment carried out at least three times.Y2H (D) analyses showing the association of WT and mutant FolSrpk1 proteins with FolAha1.F Fluorescence microscopy of FolSrpk1-GFP and its mutant forms. Arrows indicate the nucleus. Bars = 5 µm. G Quantification of FolSrpk1-GFP distribution in F. The calculation was from three independent replicates with three hyphae per replicate and three cells per hyphae. Different letters indicate statistically significant differences according to one-way ANOVA (P < 0.05). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.H Subcellular fractionation of FolSrpk1-GFP and its mutant forms. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and CBB staining. The amount of nuclear FolSrpk1-GFP was set as 1. Data information: For H, , each gel shown is a representative experiment carried out at least three times.I K304 acetylation of WT and mutant FolSrpk1 proteins in the indicated strains after treatment with 10 mM H2O2 for 0, 1 and 2 h. The amount of acetylated and total FolSrpk1-GFP at 0 h was each set as 1. Data information: For each gel shown is a representative experiment carried out at least three times.


Figure 3A K304 acetylation ( top ) and amount ( middle ) of FolSrpk1 - GFP and FolSrpk1K304Q - GFP in WT and FolArd1 - OE strains treated with 10 mM H2O2 for 2 h . The over - expressed FolArd1 - Flag was detected by anti - Flag ( bottom ) . The amount of acetylated and total FolSrpk1 in the WT was each set as 1 . Data information : each gel shown is a representative experiment carried out three times . C FolArd1 directly acetylates FolSrpk1 in vitro . Purified GST - FolSrpk1 or its K304Q isoform ( 50 ng ) was incubated with or without 50 ng of purified FolArd1 and then analyzed by immunoblotting using anti - K304ac or anti - GST antibody . Anti - GST immunoblotting was used to show equal loading . The amount of acetylated and total GST - FolSrpk1 in the first lane was each set as 1 . Data information : each gel shown is a representative experiment carried out three times . E Subcellular fractionation analysis of FolSrpk1 - GFP in WT , ΔFolSir2 , and FolSir2 - OE strains . The amount of nuclear FolSrpk1 - GFP in the WT was set as 1 . Data information : each gel shown is a representative experiment carried out three times . The fractionation controls were Fol histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining . G , H Expression profile of FolArd1 ( G ) and FolSir2 ( H ) during the infection stage . Total RNA extracted from host roots at 0 ( conidia ) and 1 - 5 dpi was reverse transcribed . The expression levels were normalized to that of the Fol actin gene . Data are presented as mean ± SD . The presence of different letters above the mean values of three replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) .

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Figure 3A K304 acetylation (top) and amount (middle) of FolSrpk1-GFP and FolSrpk1K304Q-GFP in WT and FolArd1-OE strains treated with 10 mM H2O2 for 2 h. The over-expressed FolArd1-Flag was detected by anti-Flag (bottom). The amount of acetylated and total FolSrpk1 in the WT was each set as 1. Data information: each gel shown is a representative experiment carried out three times.C FolArd1 directly acetylates FolSrpk1 in vitro. Purified GST-FolSrpk1 or its K304Q isoform (50 ng) was incubated with or without 50 ng of purified FolArd1 and then analyzed by immunoblotting using anti-K304ac or anti-GST antibody. Anti-GST immunoblotting was used to show equal loading. The amount of acetylated and total GST-FolSrpk1 in the first lane was each set as 1. Data information: each gel shown is a representative experiment carried out three times.E Subcellular fractionation analysis of FolSrpk1-GFP in WT, ΔFolSir2, and FolSir2-OE strains. The amount of nuclear FolSrpk1-GFP in the WT was set as 1. Data information: each gel shown is a representative experiment carried out three times. The fractionation controls were Fol histone H1 (nucleus), Actin (cytosol) and CBB staining.G, H Expression profile of FolArd1 (G) and FolSir2 (H) during the infection stage. Total RNA extracted from host roots at 0 (conidia) and 1-5 dpi was reverse transcribed. The expression levels were normalized to that of the Fol actin gene. Data are presented as mean ± SD. The presence of different letters above the mean values of three replicates indicates a significant difference between different samples (P < 0.05, ANOVA).


Figure 4A Relative expression level of six antioxidant enzymes during the infection stage . The experiments were carried out as in Fig 3 . Data are presented as mean ± SD . The presence of different letters above the mean values of three biological replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . B Secretion of the FOXG _ 17180 - GFP , FOXG _ 15294 - GFP and FOXG _ 13788 - GFP proteins . The proteins extracted from mycelia ( M ) and culture supernatants ( S ) were analyzed by Western blotting with anti - GFP and anti - Actin . Each gel shown is a representative experiment carried out three times . The loading controls were Fol Actin and CBB staining . F qRT - PCR analysis of Fol EF - 1α transcript levels in tomato roots 14 days after infection by the indicated strains . The expression of tomato RCE1 was used as a control to ensure the use of equal amounts of RNA for RT - PCR . Data information : For F , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Relative expression level of six antioxidant enzymes during the infection stage. The experiments were carried out as in Fig 3. Data are presented as mean ± SD. The presence of different letters above the mean values of three biological replicates indicates a significant difference between different samples (P < 0.05, ANOVA).B Secretion of the FOXG_17180-GFP, FOXG_15294-GFP and FOXG_13788-GFP proteins. The proteins extracted from mycelia (M) and culture supernatants (S) were analyzed by Western blotting with anti-GFP and anti-Actin. Each gel shown is a representative experiment carried out three times. The loading controls were Fol Actin and CBB staining.F qRT-PCR analysis of Fol EF-1α transcript levels in tomato roots 14 days after infection by the indicated strains. The expression of tomato RCE1 was used as a control to ensure the use of equal amounts of RNA for RT-PCR. Data information: For F, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


Figure 5B Relative growth reduction of the indicated strains representing the reduction in colony diameters between normal and stress conditions normalized to colony diameters under normal conditions ( four biological replicates ) . Data information : For B , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean . C Determination of H2O2 content in tomato roots infected by the indicted strains at 0 - 5 dpi . The experiments were carried out as in Fig 1 . Data are presented as mean ± SD of three biological replicates . F qRT - PCR analysis of Fol EF - 1α transcript levels in tomato roots 14 days after infection by the indicated strains . The experiments ( four biological replicates ) Data information : For F , the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Relative growth reduction of the indicated strains representing the reduction in colony diameters between normal and stress conditions normalized to colony diameters under normal conditions (four biological replicates). Data information: For B, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.C Determination of H2O2 content in tomato roots infected by the indicted strains at 0-5 dpi. The experiments were carried out as in Fig 1. Data are presented as mean ± SD of three biological replicates.F qRT-PCR analysis of Fol EF-1α transcript levels in tomato roots 14 days after infection by the indicated strains. The experiments (four biological replicates) Data information: For F, the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


Figure 6A Subcellular fractionation of BcSrpk1 - GFP in WT and BcSrpk1mutant strains . The amount of nuclear BcSrpk1 - GFP was set as 1 . Data information : , each gel shown is a representative experiment carried out three times . The fractionation controls were histone H1 ( nucleus ) , Actin ( cytosol ) and CBB staining . B Co - IP analysis revealing the interaction of BcAha1 with BcSrpk1 and its mutant forms . Data information : each gel shown is a representative experiment carried out three times . C K367 acetylation of BcSrpk1 during the early infection stage . Total proteins were extracted from tomato leaves at 0 ( spores ) , 12 , 24 and 36 hpi . Eluted proteins bound to the anti - GFP beads were probed by the anti - K304ac and anti - GFP antibodies . The amount of acetylated and total BcSrpk1 at 0 hpi was each set as 1 . Data information : each gel shown is a representative experiment carried out three times . F Relative density of DAB staining in E ( four biological replicates ) . Data information : For F the presence of different letters above the mean values of four replicates indicates a significant difference between different samples ( P < 0 . 05 , ANOVA ) . The whiskers of the boxplots indicate the upper and lower quartiles , the boxes indicate the interquartile range , and the plus sign indicates the mean .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Subcellular fractionation of BcSrpk1-GFP in WT and BcSrpk1mutant strains. The amount of nuclear BcSrpk1-GFP was set as 1. Data information: , each gel shown is a representative experiment carried out three times. The fractionation controls were histone H1 (nucleus), Actin (cytosol) and CBB staining.B Co-IP analysis revealing the interaction of BcAha1 with BcSrpk1 and its mutant forms. Data information: each gel shown is a representative experiment carried out three times.C K367 acetylation of BcSrpk1 during the early infection stage. Total proteins were extracted from tomato leaves at 0 (spores), 12, 24 and 36 hpi. Eluted proteins bound to the anti-GFP beads were probed by the anti-K304ac and anti-GFP antibodies. The amount of acetylated and total BcSrpk1 at 0 hpi was each set as 1. Data information: each gel shown is a representative experiment carried out three times.F Relative density of DAB staining in E (four biological replicates). Data information: For F the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.


Figure 2Two top - ranked SplashRNA - designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR - AB and miR - E lentiviral vectors with Venus as reporter . The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines . RNAi efficiency was determined by western blot ( WB ) analysis of target protein levels in the lysates of the transduced cells . - actin served as loading control . The RNAi efficiency of Pten . 1524 and Pten . 932 shRNAmir mediated by miR - AB vs . miR - E was tested in NIH - 3T3 cells in the context of < 30 % transduction efficiency ( GFP as reporter ) . Representative western blots of Pten protein expression of the FACS - sorted GFP + cells from two biological replicates were shown . β - actin served as loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Two top-ranked SplashRNA-designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR-AB and miR-E lentiviral vectors with Venus as reporter. The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines. RNAi efficiency was determined by western blot (WB) analysis of target protein levels in the lysates of the transduced cells. -actin served as loading control.The RNAi efficiency of Pten.1524 and Pten.932 shRNAmir mediated by miR-AB vs. miR-E was tested in NIH-3T3 cells in the context of <30% transduction efficiency (GFP as reporter). Representative western blots of Pten protein expression of the FACS-sorted GFP+ cells from two biological replicates were shown. β-actin served as loading control.


Figure 3Two top - ranked SplashRNA - designed shRNAmirs targeting human or mouse surface protein FAS were cloned into Venus - expressing miR - AB lentiviral vectors which harbor different promoters to drive the shRNAmir expression . The packaged lentiviruses were used to infect the indicated human or mouse cell lines . A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control . After > 96h of infection , FAS protein levels in the transduced Venus - positive cells were determined by surface staining and shown in histograms ( top panels ) and quantified by MFI of APC ( bottom panels ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Two top-ranked SplashRNA-designed shRNAmirs targeting human or mouse surface protein FAS were cloned into Venus-expressing miR-AB lentiviral vectors which harbor different promoters to drive the shRNAmir expression. The packaged lentiviruses were used to infect the indicated human or mouse cell lines. A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control. After >96h of infection, FAS protein levels in the transduced Venus-positive cells were determined by surface staining and shown in histograms (top panels) and quantified by MFI of APC (bottom panels).


Figure 4293T cells were co - transduced with four miR - AB lentiviruses at 1 : 1 ratio , which carries a neutral shRNAmir targeting CD4 or CD19 , with Azurite , GFP , Ametrine or mOrange as reporter respectively . Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated . Representative contour FACS plots cells on day 4 and day 22 shown ( top panels ) . Cell numbers of untransduced , single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4 , and plotted over time ( bottom panel ) . Data are shown as mean ± s . d . and n = 4 . NS indicates no significant difference ( P > 0 . 05 , two - tailed unpaired Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4293T cells were co-transduced with four miR-AB lentiviruses at 1:1 ratio, which carries a neutral shRNAmir targeting CD4 or CD19, with Azurite, GFP, Ametrine or mOrange as reporter respectively. Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated. Representative contour FACS plots cells on day 4 and day 22 shown (top panels). Cell numbers of untransduced, single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4, and plotted over time (bottom panel). Data are shown as mean ± s.d. and n=4. NS indicates no significant difference (P>0.05, two-tailed unpaired Student's t-test).


Figure 5CD8 + T cells were activated for 16h and then co - transduced with miR - AB retroviruses at 1 : 1 ratio , which carries a SplashRNA - designed shRNAmir , targeting surface protein CD127 , CD90 , CD44 or CD8 , with Azurite , GFP , Ametrine or mCherry2 as fluorescent reporters respectively for 4h . miR - AB retroviruses expressing CD19 - specific shRNAmirs with same fluorescent reporters were used as controls . Surface staining was performed 72h after transduction . FACS data were shown as overlaid histograms of control shRNAmirs ( shCD19s ) and target shRNAmirs ( shCD127 , shCD90 , shCD44 or shCD8 ) . Cells were categorized as sixteen populations expressing single , double , triple or quadruple fluorescent proteins or not ( none ) and fluorescent reporter - positive plots were highlighted with thick frames and white background . Each population ' s surface staining profile was shown on the right . The data was representative of two biologically independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5CD8+ T cells were activated for 16h and then co-transduced with miR-AB retroviruses at 1:1 ratio, which carries a SplashRNA-designed shRNAmir, targeting surface protein CD127, CD90, CD44 or CD8, with Azurite, GFP, Ametrine or mCherry2 as fluorescent reporters respectively for 4h. miR-AB retroviruses expressing CD19-specific shRNAmirs with same fluorescent reporters were used as controls. Surface staining was performed 72h after transduction. FACS data were shown as overlaid histograms of control shRNAmirs (shCD19s) and target shRNAmirs (shCD127, shCD90, shCD44 or shCD8). Cells were categorized as sixteen populations expressing single, double, triple or quadruple fluorescent proteins or not (none) and fluorescent reporter-positive plots were highlighted with thick frames and white background. Each population's surface staining profile was shown on the right. The data was representative of two biologically independent experiments.


Figure 1A ) ( left ) t - sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations . Red color corresponds to undifferentiated cells , light green corresponds to surface colonocytes ( enterocytes ) . t - sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8 , which also express the IFN - γ receptor IFNGR2 . B ) Immunofluorescence staining for ACE2 ( red ) and KRT20 ( green ) of human colon tissue section indicating ACE2 expression in surface colonocytes . Scale bar : 50 µm . C ) Human colon organoids cultured in full medium ( FM ) condition ( upper panel ) or FM treated with IFN - γ ( lower panel ) . Left : bright field images of 7 - day cultured human colon organoids , scale bar : 100 µm . Right : Immunofluorescence for KRT20 ( green ; note increased expression upon IFN - γ treatment ) . Scale bar : 25 µm . D ) Human colon organoids cultured in basic medium ( upper panel ) or additionally treated with IFN - γ ( lower panel ) . Left : bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN - γ ( lower panel ) scale bar : 100 µm . Middle : Immunofluorescence labelling for KRT20 ( green ) Right : Immunofluorescence labelling for E - cadherin ( green ) to visualize epithelial cell boundaries ; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN - γ . Scale bar : 25 µm . Comparison of E ) KRT20 , F ) MUC2 , G ) CHGA , H ) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons testACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN - γ ( n = 3 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons testJ ) Immunofluorescence staining for ACE2 ( red ) and KRT20 ( green ) of organoids grown in full medium ( upper ) or basic medium ( lower ) and additionally treated with IFN - γ ( right ) , indicating expression of ACE2 in differentiated enterocytes . White arrows point to ACE2 . Scale bar : 10

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) (left) t-sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations. Red color corresponds to undifferentiated cells, light green corresponds to surface colonocytes (enterocytes). t-sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8, which also express the IFN-γ receptor IFNGR2. B) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of human colon tissue section indicating ACE2 expression in surface colonocytes. Scale bar: 50 µm.C) Human colon organoids cultured in full medium (FM) condition (upper panel) or FM treated with IFN-γ (lower panel). Left: bright field images of 7-day cultured human colon organoids, scale bar: 100 µm. Right: Immunofluorescence for KRT20 (green; note increased expression upon IFN-γ treatment). Scale bar: 25 µm.D) Human colon organoids cultured in basic medium (upper panel) or additionally treated with IFN-γ (lower panel). Left: bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN-γ (lower panel) scale bar: 100 µm. Middle: Immunofluorescence labelling for KRT20 (green) Right: Immunofluorescence labelling for E-cadherin (green) to visualize epithelial cell boundaries; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN-γ. Scale bar: 25 µm.Comparison of E) KRT20, F) MUC2, G) CHGA, H) LGR5 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons testACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons testJ) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of organoids grown in full medium (upper) or basic medium (lower) and additionally treated with IFN-γ (right), indicating expression of ACE2 in differentiated enterocytes. White arrows point to ACE2. Scale bar: 10 µm


Figure 2A ) Immunofluorescence for N - protein ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Yellow arrows point to N - protein positive cells . Scale bar : 25 µm . B ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . basic medium organoids 48 h after infection and normalized to GAPDH ( n = 6 ) . C ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium vs . full medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 9 ) . D ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in basic medium vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . E ) qPCR data displaying the relative virus load of SARS - CoV - 2 measured by viral genome quantity in full medium + IFN - γ vs . basic medium + IFN - γ treated organoids 48 h after infection and normalized to GAPDH ( n = 4 ) . ( B - E ) Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by Student ' s t - test F ) qPCR data comparing the relative virus load of SARS - CoV - 2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium ( left ) and basic medium ( right ) untreated or treated with IFN - γ , indicating increase in virus load in differentiated and IFN - γ treated conditions ( n = 8 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons testG ) Electron microscopy imaging of organoids grown in basic medium , pretreated with IFN - γ for 3 days and infected for 48 h with SARS - CoV - 2 . Upper : Overview showing large virus - loaded vesicles in an epithelial cell . Scale bar : 500nm . Lower : Disintegrated organoid cell containing high virus load , scale bar : 2 . 5µm , higher magnification to visualize virus particles , scale bar : 100nm . H ) Immunofluorescence for cleaved - caspase3 ( red ) and E - cadherin ( green ) 48 h after SARS - CoV - 2 infection of human colon organoids cultured in full medium ( upper ) , full medium + IFN - γ ( upper right ) , basic medium ( lower left ) and basic medium + IFN - γ ( lower right ) . Scale bar : 50 µm . ( below ) Quantification of apoptotic cells , indicating increased cell death in the differentiated and IFN - γ treated condition after SARS - CoV - 2 infection ( n = 7 ) . Data are presented as mean + / - SD , * : p ≤ 0 . 05 , * * : p ≤ 0 . 001 , * * * : p ≤ 0 . 0001 , as determined by one - way ANOVA , followed by Tukey ' s multiple comparisons

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) Immunofluorescence for N-protein (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Yellow arrows point to N-protein positive cells. Scale bar: 25 µm.B) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. basic medium organoids 48 h after infection and normalized to GAPDH (n=6). C) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. full medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=9). D) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in basic medium vs. basic medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). E) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium + IFN-γ vs. basic medium+ IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4). (B-E) Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by Student's t-test F) qPCR data comparing the relative virus load of SARS-CoV-2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium (left) and basic medium (right) untreated or treated with IFN-γ, indicating increase in virus load in differentiated and IFN-γ treated conditions (n=8). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons testG) Electron microscopy imaging of organoids grown in basic medium, pretreated with IFN-γ for 3 days and infected for 48 h with SARS-CoV-2. Upper: Overview showing large virus-loaded vesicles in an epithelial cell. Scale bar: 500nm. Lower: Disintegrated organoid cell containing high virus load, scale bar: 2.5µm, higher magnification to visualize virus particles, scale bar: 100nm.H) Immunofluorescence for cleaved-caspase3 (red) and E-cadherin (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Scale bar: 50 µm. (below) Quantification of apoptotic cells, indicating increased cell death in the differentiated and IFN-γ treated condition after SARS-CoV-2 infection (n=7). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test