Figure 1 ( A Total PD - L1 , PARP from colon ( Colo - 205 ) , lung ( A549 ) , pancreatic ( PANK1 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 GAPDH is loading controlTotal PD - L1 , PARP from triple negative breast cancer cell ( MDA - MB - 436 ) lysates treated with indicated DR5 agonist antibodies named Lexa ( lexatumumab ) , KMTR2 , BaCa , GAPDH is loading controlC ) Total PD - L1 , PARP , cleaved caspase - 3 from ovarian cell ( Cavo - 3 ) lysates treated with indicated DR5 agonist antibodies named tigatuzumab . GAPDH is loading control ( D ) Total PD - L1 , CD47 , Calreticulin , PARP from MDA - MB - 436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z - VAD . GAPDH is loading control . ( E ) Total PD - L1 western blotting signal from the lysates of OVCAR - 3 ( n = 3 ) and MDA - MB - 436 ( n = 3 ) cells after 6 hrs treatment of 100nM lexa ( DR5 agonist antibody ) was normalized to GAPDH . ( F ) Surface biotinylation of PD - L1 from indicated tumor cells after indicated DR5 agonist treatments . Cleaved caspase - 3 and PARP indicates activation of DR5 signaling . In lane 3 and 4 , KMTR2 was pre - neutralized either with recombinant DR5 ( rDR5 ) or recombinant FOLR1 ( rFOLR1 ) . ( G ) Representative flow cytometry plots of PD - L1 from two different tumor cell lines treated with indicated DR5 antibodies . Secondary alone and IgG1 control are included . ( H ) Relative surface PD - L1 % cells , after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean ( n = 3 - 4 ) . ( J ) Relative surface PD - L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD - L1 in IgG1 treated tumors and horizontal line in samples indicated mean ( n = 2 - 6 ) ( K ) ER ( - ) , PR ( - ) and HER2 ( - ) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD . Cg - Prkdcscid Il2rgtm1Wjl / SzJ mice , following by treatment with IgG1 or KMTR2 ( 50μg , 4 doses ) . Harvested tumors were analyzed for PD - L1 , CD47 and PARP in lysates . ( L ) KMTR2 treated ( 50μg , 4 doses ) UCD52 TNBC PDX tumors were stained for PD - L1 using immunohistochemistry ( IHC ) . ( M ) Total PD - L1 and CD47 blotting analysis from DR5 resistant MDA - MB - 436 cell lines after indicated DR5 agonist treatment ( 50nM , 6hr ) .

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 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 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 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 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 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 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 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 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 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 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

Figure 1(A Total PD-L1, PARP from colon (Colo-205), lung (A549), pancreatic (PANK1) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2 GAPDH is loading controlTotal PD-L1, PARP from triple negative breast cancer cell (MDA-MB-436) lysates treated with indicated DR5 agonist antibodies named Lexa (lexatumumab), KMTR2, BaCa, GAPDH is loading controlC) Total PD-L1, PARP, cleaved caspase-3 from ovarian cell (Cavo-3) lysates treated with indicated DR5 agonist antibodies named tigatuzumab. GAPDH is loading control(D) Total PD-L1, CD47, Calreticulin, PARP from MDA-MB-436 cell lysates treated with indicated DR5 agonist antibodies ± caspase inhibitor Z-VAD. GAPDH is loading control.(E) Total PD-L1 western blotting signal from the lysates of OVCAR-3 (n=3) and MDA-MB-436 (n=3) cells after 6 hrs treatment of 100nM lexa (DR5 agonist antibody) was normalized to GAPDH.(F) Surface biotinylation of PD-L1 from indicated tumor cells after indicated DR5 agonist treatments. Cleaved caspase-3 and PARP indicates activation of DR5 signaling. In lane 3 and 4, KMTR2 was pre-neutralized either with recombinant DR5 (rDR5) or recombinant FOLR1 (rFOLR1).(G) Representative flow cytometry plots of PD-L1 from two different tumor cell lines treated with indicated DR5 antibodies. Secondary alone and IgG1 control are included.(H) Relative surface PD-L1 % cells, after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated cells in corresponding tumor cells and horizontal line in samples indicated mean (n=3-4).(J) Relative surface PD-L1 % cells in indicated tumors after DR5 agonist treatments Relative signal is normalized to surface PD-L1 in IgG1 treated tumors and horizontal line in samples indicated mean (n=2-6)(K) ER (-), PR (-) and HER2 (-) UCD52 patient derived tumor tissue was xenografted in breast fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, following by treatment with IgG1 or KMTR2 (50μg, 4 doses). Harvested tumors were analyzed for PD-L1, CD47 and PARP in lysates.(L) KMTR2 treated (50μg, 4 doses) UCD52 TNBC PDX tumors were stained for PD-L1 using immunohistochemistry (IHC).(M) Total PD-L1 and CD47 blotting analysis from DR5 resistant MDA-MB-436 cell lines after indicated DR5 agonist treatment (50nM, 6hr).


Figure 2 ( C ) Luciferase activity of reporter lines from tumor cell - Jurkat cell co - culture assay using MDA - MB - 436 and OVCAR3 cells after treatment ( 50nM ) with indicated DR5 agonists ( tiga : tigatuzumab , AMG : AMG655 , KMTR1 , Lexa : Lexatumumab ) . The background luciferase signal from untreated cells ( due to basal surface PD - L1 ) was subtracted . Various controls treatments ( IgG1 , anti - PD - L1 , anti - EGFR ) are also shown ( n = 3 ) . ( D ) Same as ( C ) except tumor cells were pre - treated with indicated inhibitors for AKT , ERK , mTOR , MEK , STAT3 , PARP - i and NF - Kβ along with DR5 agonist KMTR2 antibody prior to co - culture . MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 GAPDH is loading control in EMDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 phosphorylated p65 , total p65 , cleaved caspase - 3 and PARP . Tubulin is loading control in F . MDA - MB - 436 cells were treated with TNFα and indicated DR5 agonists for indicated times . Lysates were analyzed for PD - L1 , CSN5 , phosphorylated p65 and PARP . GAPDH is loading control in G . ( H ) Flow cytometry analysis of MDA - MB - 436 cells treated with TNFα ± MG132 ( top ) and indicated DR5 agonist ± MG132 ( bottom ) . ( I ) Total PD - L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132 . ( J ) Flow cytometry surface PD - L1 analysis of OVCAR - 3 cells treated with indicated DR5 agonist ± MG132 . ( K ) MDA - MB - 436 cells were treated with MG132 for indicated times and total PD - L1 from lysates was analyzed . GAPDH is loading control . ( L ) Immunoblotting of DR5 confirming generation of knock out ( DR5 - KO ) cell lines . ( M ) Cell viability analysis of DR5 - KO and DR5 - WT cells ( n = 3 ) . ( N ) DR5 - KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD - L1 specific antibodies . ( O ) TNBC WT and DR5 - KO ( MDA - MB - 436 ) cells were treated with either DR5 agonist or TNFα as indicated . Lysates were analyzed for PD - L1 , S5a and DR5 . GAPDH is loading control . ( P ) OVCAR - 3 and MDA - MB - 436 ( WT and DR5 - KO ) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates . GAPDH is loading control .

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 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 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 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 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 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 B-PANEL_START 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 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 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 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

Figure 2(C) Luciferase activity of reporter lines from tumor cell-Jurkat cell co-culture assay using MDA-MB-436 and OVCAR3 cells after treatment (50nM) with indicated DR5 agonists (tiga: tigatuzumab, AMG: AMG655, KMTR1, Lexa: Lexatumumab). The background luciferase signal from untreated cells (due to basal surface PD-L1) was subtracted. Various controls treatments (IgG1, anti-PD-L1, anti-EGFR) are also shown (n=3). (D) Same as (C) except tumor cells were pre-treated with indicated inhibitors for AKT, ERK, mTOR, MEK, STAT3, PARP-i and NF-Kβ along with DR5 agonist KMTR2 antibody prior to co-culture. MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5 GAPDH is loading control in EMDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1 phosphorylated p65, total p65, cleaved caspase-3 and PARP. Tubulin is loading control in F.MDA-MB-436 cells were treated with TNFα and indicated DR5 agonists for indicated times. Lysates were analyzed for PD-L1, CSN5, phosphorylated p65 and PARP. GAPDH is loading control in G.(H) Flow cytometry analysis of MDA-MB-436 cells treated with TNFα ± MG132 (top) and indicated DR5 agonist ± MG132 (bottom).(I) Total PD-L1 from OVCAR3 cell lysates was analyzed after treatment of indicated DR5 agonist ± MG132.(J) Flow cytometry surface PD-L1 analysis of OVCAR-3 cells treated with indicated DR5 agonist ± MG132.(K) MDA-MB-436 cells were treated with MG132 for indicated times and total PD-L1 from lysates was analyzed. GAPDH is loading control.(L) Immunoblotting of DR5 confirming generation of knock out (DR5-KO) cell lines.(M) Cell viability analysis of DR5-KO and DR5-WT cells (n=3).(N) DR5-KO and WT cells were treated with TNFα and indicated DR5 agonist followed by flow cytometry using PD-L1 specific antibodies.(O) TNBC WT and DR5-KO (MDA-MB-436) cells were treated with either DR5 agonist or TNFα as indicated. Lysates were analyzed for PD-L1, S5a and DR5. GAPDH is loading control.(P) OVCAR-3 and MDA-MB-436 (WT and DR5-KO) cells were treated with KMTR2 followed by ubiquitin and S5a immunoblotting from total lysates. GAPDH is loading control.


Figure 3ApoEVs isolated after IgG1 and KMTR2 treatment ( OVCAR - 3 ) were blotted against CD63 and PD - L1 in dot blots . ApoEVs isolated after IgG1 and tigatuzumab treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 . ApoEVs isolated after IgG1 and lexatumumab ( D ) treatment ( MDA - MB - 436 ) were run on western and blotted against CD63 and PD - L1 . ( F ) ApoEVs isolated from DR5 sensitive tumor cells grown in Met - HPG + media were added on to DR5 - KO cells ( growing in regular media ) . After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye . HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met - HPG + ( DR5 - WT ) to DR5 - KO cells . ( G ) ApoEVs isolated from DR5 - WT OVCAR - 3 cells ( KMTR2 treated ) were added on to DR5 - KO ( MDA - MB - 231 ) cells for indicated times ( 24 - 96 hrs ) to analyze PD - L1 transfer kinetics from ApoEVsafter addition of MDA - MB - 436 derived ApoEVs ( treated with combination of KMTR2 + lexatumumab DR5 agonists or IgG1 ) on to DR5 - KO MDA - MB - 231 cells , total lysates were immunoblotted for PD - L1 after 24 hrs . KMTR2 + lexatumumab ( n = 3 ) , IgG1 ( n = 2 ) . ( I ) Left : PD - L1 surface histogram shows direct antibody treatment on DR5 - KO cells . Right : PD - L1 surface histogram from DR5 - KO cells after treatment ( 24 hrs ) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment . ApoEVs treated tumor cells were analyzed in PD - 1 reporter co - culture assays for luciferase signal ( K ) MDA - MB - 436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times . - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor . Lysates were immunoblotted for cleaved caspase - 3 , 8 , PARP , PD - L1 , S5a , ROCK1 and GAPDH ( n = 3 ) . u . c : uncleaved , c : cleaved , n . s : non - specific band ( L ) Relative caspase - 8 and caspase - 3 activity assays caspase - 8 maintained steady activity , while caspase - 3 activity required at least 40 ( + ) mins of DR5 agonist treatment without ZDEVD ( n = 3 ) . ( M ) After indicated treatments with KMTR2 ( DR5 agonist ) and ZDEVD ( caspase - 3 preferred inhibitor ) , cells were allowed to grow 24 hrs , followed by cell viability analysis .

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 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 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 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 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 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 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

Figure 3ApoEVs isolated after IgG1 and KMTR2 treatment (OVCAR-3) were blotted against CD63 and PD-L1 in dot blots.ApoEVs isolated after IgG1 and tigatuzumab treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.ApoEVs isolated after IgG1 and lexatumumab (D) treatment (MDA-MB-436) were run on western and blotted against CD63 and PD-L1.(F) ApoEVs isolated from DR5 sensitive tumor cells grown in Met-HPG+ media were added on to DR5-KO cells (growing in regular media). After 48 hrs flow cytometry analysis was carried out with HPG catalyzing dye. HPG incorporation from flow cytometry data confirms ApoEVs transfer from cells growing in Met-HPG+ (DR5-WT) to DR5-KO cells.(G) ApoEVs isolated from DR5-WT OVCAR-3 cells (KMTR2 treated) were added on to DR5-KO (MDA-MB-231) cells for indicated times (24-96 hrs) to analyze PD-L1 transfer kinetics from ApoEVsafter addition of MDA-MB-436 derived ApoEVs (treated with combination of KMTR2+lexatumumab DR5 agonists or IgG1) on to DR5-KO MDA-MB-231 cells, total lysates were immunoblotted for PD-L1 after 24 hrs. KMTR2+lexatumumab (n=3), IgG1 (n=2).(I) Left: PD-L1 surface histogram shows direct antibody treatment on DR5-KO cells. Right: PD-L1 surface histogram from DR5-KO cells after treatment (24 hrs) with ApoEVs isolated from DR5 sensitive cells after either Lexa or KMTR2 treatment.ApoEVs treated tumor cells were analyzed in PD-1 reporter co-culture assays for luciferase signal(K) MDA-MB-436 cells were treated with KMTR2 for 6 hrs and along with ZDEVD for indicated times. - ZDEVD shows final time of KMTR2 exposure to cells in absence inhibitor. Lysates were immunoblotted for cleaved caspase-3, 8, PARP, PD-L1, S5a, ROCK1 and GAPDH (n=3). u.c: uncleaved, c: cleaved, n.s: non-specific band(L) Relative caspase-8 and caspase-3 activity assays caspase-8 maintained steady activity, while caspase-3 activity required at least 40 (+) mins of DR5 agonist treatment without ZDEVD (n=3).(M) After indicated treatments with KMTR2 (DR5 agonist) and ZDEVD (caspase-3 preferred inhibitor) , cells were allowed to grow 24 hrs, followed by cell viability analysis.


Figure 4 ( A KMTR2 , KMTR2 + Z - VAD treated MDA - MB - 436 lysates for indicated early time points were analyzed for caspase - 8 , caspase - 3 , ROCK1 PARP as indicated . B ) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase - 3 , ROCK1 , pMLC , PARP , PD - L1 and CMTM6 etc . as indicated . ( C ) OVCAR3 cells were treated with pMLC activating ionophore A23187 ( positive control ) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1 . Lysates were later analyzed for ROCK1 , pMLC , caspase - 3 and PARP with GAPDH as loading control . Blue arrows indicate cleaved and activated ROCK1 and caspase - 3 . ( D ) MDA - MB - 436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment ( 20nM ) . After 6 hours flow cytometry was used to analyze surface PD - L1 . ( GSK269 : GSK269962A , GSK429 : GSK429286A ) ( E ) MDA - MB - 436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors , error bars indicate SD ( n = 2 ) . ( GSK269 : GSK269962A , GSK429 : GSK429286A ) Cultured MDA - MB - 436 cells were treated with KMTR2 ( IgG1 - Fc ) or IgG1 control ± ROCK1i ( GSK269962 ) for 6 hrs . After 6 hrs , 500nM anti - PD - L1 avelumab ( IgG4 - Fc ) was added to the media for additional 1 hr . Cellular lysates were pulled down with anti - hu - IgG4 specific beads . ( G ) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1 , PD - L1 and CMTM6 . Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples . Blue arrows indicate cleaved and activated ROCK1 . ( H ) Immunoprecipitation assays with anti - PD - L1 ( avelumab ) using OVCAR - 3 cell lines . Blue arrow indicates cleaved and activated ROCK1 . ( J ) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors ( n = 3 ) . ( K ) Tumor regression efficacy of MD5 - 1 ( 50μg ) , GSK269962A ( < 2mg / kg ) and MD5 - 1 + GSK269962A ( 50μg + 2mg / kg ) 4T1 tumors ( n = 4 - 7 ) . 6 - 8 weeks old BALB / c mice bearing 4T1 tumors ( ~ 100mm3 ) were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . GSK269962A ( ROCK1i ) was injected directly into the tumors . Tumor volumes were quantified at indicated days by caliper measurements .

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 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 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 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 O O O O O O O O O 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 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 4(A KMTR2, KMTR2+Z-VAD treated MDA-MB-436 lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1 PARP as indicated.B) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads. (G) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.(J) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors (n=3).(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.


Figure 5 ( B - D ) FACs plots confirming expression of human DR5 ( huDR5 ) and Chi - G4S - DR5 in mouse 4T1 cells . Chi - DR5 was not expressed on cell surface ( C ) . ( E ) Cell viability analysis of huDR5 and Chi - G4S - DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 ( n = 3 ) . ( F ) Comparison of tumor growth of grafted huDR5 and Chi - G4S - DR5 stable 4T1 cells in Balb / c mice ( n = 3 ) . ( G ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 150 - 200mm3 ) were treated with 4 doses ( 100μg ) of IgG1 , lexatumumab , lexatumumab + avelumab followed by tumor recovery and surface PD - L1 analysis using flow cytometry ( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . ( H ) Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . average tumor weight is shown ( n = 4 ) . ( J - K ) Confirmation of selective CD8 + T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti - PD - L1 avelumab alone or avelumab + anti - CD8a antibody . CD4 + T - cells remained unchanged . ( L ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , ROCK1i , lexatumumab + ROCK1i and anti - CD8 + lexatumumab + ROCK1i ( n = 4 - 6 , 50μg Lexatumumab , 2mg / kg ROCK1i , 6 doses ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections . Various treatments were started when tumors were ~ 100mm3 size . ( M ) Average of tumor weights ( harvested at same time ) from mice treated ( i . p . ) with IgG1 , lexatumumab , avelumab , lexatumumab + avelumab and anti - CD8 + lexatumumab + avelumab ( n = 4 - 6 ) . ( N ) 6 - 8 weeks old C57BL / 6 bearing Chi - G4S - DR5 expressing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Various treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements . Chi - G4S - DR5 stable 4T1 tumors ( after reaching ~ 100mm3 ) were treated with 6 doses of indicated treatment . Isolated tumors were imaged together ( n = 4 - 5 ) . ROCK1i indicates GSK269962A and was administered to animals via intra - tumor injections ( 2mg / kg ) . ( O ) KMTR2 ( 8 doses ) antibody used as the DR5 agonist for the experiment . ( P ) Average of tumor weights of data shown in ( O ) . ( Q ) Kaplan - Meier plot depicting the survival of syngeneic Chi - G4S - DR5 4T1 tumor bearing animals injected i . p . with 100μg of indicated antibodies such as IgG1 , KMTR2 and avelumab . Animals were injected with GSK269962A 2mg / kg ( in PBS ) directly into tumors wherever ROCK1i is indicated .

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 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 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 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 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 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 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 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 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

Figure 5(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C).(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).(F) Comparison of tumor growth of grafted huDR5 and Chi-G4S-DR5 stable 4T1 cells in Balb/c mice (n=3).(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). average tumor weight is shown (n=4).(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size.(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6).(N) 6-8 weeks old C57BL/6 bearing Chi-G4S-DR5 expressing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Various treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). (O) KMTR2 (8 doses) antibody used as the DR5 agonist for the experiment. (P) Average of tumor weights of data shown in (O). (Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.


Figure 6 ( A ) Chi - G4S - DR5 stable 4T1 tumors harboring mice were treated lexatumumab , lexatumumab + ROCK1i , and avelumab + lexatumumab and other controls as indicated . Antibodies were treated i . p at 100μg dose ( 6 total ) , ROCK1i ( in PBS ) was injected directly into tumors at 2mg / kg dose ( 6 total ) . Various treatments were started when tumors were ~ 400mm3 size . Harvested tumors were grouped together and sized matched ( 3 independent sets : n = 2 - 6 tumors in each set ) followed by TIL isolation CD8 / CD45 and CD4 / CD45 expressing cells were measured by flow cytometry . ( B ) Plots showing % of total double positive TILs ( CD8 + CD45 + + CD4 + CD45 + ) in right upper quadrant after combining 3 independent experiments . ( C ) Ratio of CD8 + CD45 + / CD4 + CD45 + isolated TILs from tumors in each indicated treatment ( n = 3 ) . Chi - G4S - DR5 stable MC38 tumors harboring mice were treated ( 6 total doses ) lexatumumab , avelumab , ROCK1i , lexatumumab + ROCKi , and avelumab + lexatumumab and IgG1 control as indicated . Harvested tumors homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , Foxp3 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments . ( H ) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5 - 1 and bispecific avelu - MD5 antibody . ( I ) Balb / c mice harboring luciferase stable 4T1 tumors were i . p . injected with indicated antibodies . 50μg dose and mice were imaged after 5 doses . ( J ) 6 - 8 weeks old C57BL / 6 mice bearing MC38 tumors were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day ( n = 4 - 6 ) . Indicated treatments were started when tumors were ~ 100mm3 size . Tumor volumes were quantified at indicated days by caliper measurements . ( K ) ~ 150 - 200mm3 size MC38 tumor bearing C57LB / 6 mice were treated with indicated MD5 - 1 , avelumab and bispecific antibodies along with control IgG1 , 6 total doses . Harvested tumors were homogenized followed by quantitation . Protein lysates were run on SDS - Page followed by immunoblotting using indicated CD8 , CD4 , caspase - 3 and granzyme - b antibody . GAPDH is loading control . Red arrows indicated cleaved caspase - 3 p19 and p17 fragments . ( L ) 6 - 8 weeks old C57BL / 6 mice were injected with MC38 cells . When tumors reached ~ 150 - 200mm3 , animals were intraperitoneally ( i . p . ) injected with 50μg of indicated antibody every third day . On day 18 , tumors were harvested , sized matched and pooled by treatment group , exposed to collagenase / DNase and were single cell suspensions enriched for CD8 + cells . Enriched CD8 + T - cells from various treatments were restimulated with anti - CD3 ( OKT3 ) antibody for 4 additional hours . CD8 gated cells were next analyzed for IFN - γ intracellular expression using flow cytometry . ( M ) Percentage of IFN - γ + CD8 + double positive cells from 3 independent experiments .

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

Figure 6(A) Chi-G4S-DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab +ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2mg/kg dose (6 total). Various treatments were started when tumors were ~400mm3 size. Harvested tumors were grouped together and sized matched (3 independent sets: n=2-6 tumors in each set) followed by TIL isolation CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry.(B) Plots showing % of total double positive TILs (CD8+CD45+ + CD4+CD45+) in right upper quadrant after combining 3 independent experiments. (C) Ratio of CD8+CD45+/CD4+CD45+ isolated TILs from tumors in each indicated treatment (n=3).Chi-G4S-DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab +ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.(H) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5-1 and bispecific avelu-MD5 antibody.(I) Balb/c mice harboring luciferase stable 4T1 tumors were i.p. injected with indicated antibodies. 50μg dose and mice were imaged after 5 doses.(J) 6-8 weeks old C57BL/6 mice bearing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Indicated treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.(K) ~150-200mm3 size MC38 tumor bearing C57LB/6 mice were treated with indicated MD5-1, avelumab and bispecific antibodies along with control IgG1, 6 total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.(L) 6-8 weeks old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150-200mm3, animals were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day. On day 18, tumors were harvested, sized matched and pooled by treatment group, exposed to collagenase/DNase and were single cell suspensions enriched for CD8+ cells. Enriched CD8+ T-cells from various treatments were restimulated with anti-CD3 (OKT3) antibody for 4 additional hours. CD8 gated cells were next analyzed for IFN-γ intracellular expression using flow cytometry.(M) Percentage of IFN-γ+CD8+ double positive cells from 3 independent experiments.


Figure 1Median CSF protein abundance distribution as calculated from MS - intensities of quantified peptides of each protein . The top ten most abundant proteins and hemoglobin proteins are highlighted .

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Figure 1Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted.


Figure 2 A - C Protein AD / non - AD fold changes plotted vs statistical significance for Sweden ( A ) , Magdeburg / Kiel ( B ) , and Berlin ( C ) cohort . Proteins associated with the GO annotation neuron projection labelled in orange . Proteins above the dashed green line are statistically significant ( p < 0 . 05 ) and those above the black curves have a q - value below 0 . 05

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A-C Protein AD/non-AD fold changes plotted vs statistical significance for Sweden (A), Magdeburg/Kiel (B), and Berlin (C) cohort. Proteins associated with the GO annotation neuron projection labelled in orange. Proteins above the dashed green line are statistically significant (p < 0.05) and those above the black curves have a q-value below 0.05


Figure 3Correlation of protein AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Magdeburg / Kiel Proteins included differ significantly ( p < 0 . 05 ) and consistently in abundance by AD status in both cohorts each . Correlation of protein AD / non - AD fold changes in pairwise combinations of two cohorts each . Combinations are Sweden vs Berlin ( D ) , and Magdeburg / Kiel vs Berlin ( E ) . Proteins included differ significantly ( p < 0 . 05 ) and consistently in abundance by AD status in both cohorts each . Proteins that differ significantly ( p - value < 0 . 05 in E ; q - value < 0 . 05 in F ) in abundance by AD status across all three cohorts . Z - scored abundances of proteins in the AD and non - AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non - AD groups . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis . Black frames highlight proteins with consistent AD / non - AD fold changes across cohorts . Proteins that differ significantly ( p - value < 0 . 05 in E ; q - value < 0 . 05 in F ) in abundance by AD status across all three cohorts . Z - scored abundances of proteins in the AD and non - AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non - AD groups . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis . Black frames highlight proteins with consistent AD / non - AD fold changes across cohorts .

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 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 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

Figure 3Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Magdeburg/Kiel Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Berlin (D), and Magdeburg/Kiel vs Berlin (E). Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.


Figure 4 A - C ) Correlation of proteins with ELISA - measured t - tau concentration across samples within the Sweden ( A ) , Magdeburg ( B ) and Berlin ( C ) cohorts . Proteins with a q - value below 0 . 05 are labeled in yellow . Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non - AD CSF . D ) Three cohort summary of proteins significantly correlating with ELISA - measured t - tau . Protein names given for the 29 proteins out of the 40 protein signature with significant ( p < 0 . 05 ) correlation in each of the three cohorts . Pyruvat kinase PKM ( PKM ) was quantified in two isoforms and UniProt IDs are given in parenthesis . Protein abundance distribution of CSF showing the abundances of AD - modulated CSF proteins . Proteins of our 40 protein signature are highlighted in red ( elevated abundance in AD ) and blue ( elevated abundance in non - AD ) . Proteins linked to glucose metabolism are highlighted in purple and labelled . Protein abundance distribution of brain showing the abundances of AD - modulated CSF proteins . Proteins of our 40 protein signature are highlighted in red ( elevated abundance in AD ) and blue ( elevated abundance in non - AD ) . Proteins linked to glucose metabolism are highlighted in purple and labelled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A-C) Correlation of proteins with ELISA-measured t-tau concentration across samples within the Sweden (A), Magdeburg (B) and Berlin (C) cohorts. Proteins with a q-value below 0.05 are labeled in yellow. Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non-AD CSF.  D) Three cohort summary of proteins significantly correlating with ELISA-measured t-tau. Protein names given for the 29 proteins out of the 40 protein signature with significant (p < 0.05) correlation in each of the three cohorts. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis.  Protein abundance distribution of CSF showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.  Protein abundance distribution of brain showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.


Figure 2Circular representation of DNA methylation levels for mothers ( outer circle ) and children ( inner circle ) . The height of each bar indicates the methylation change between the smoking and non ‐ smoking group ( dark hue : hypermethylation , light hue : hypomethylation ) .

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Figure 2Circular representation of DNA methylation levels for mothers (outer circle) and children (inner circle). The height of each bar indicates the methylation change between the smoking and non‐smoking group (dark hue: hypermethylation, light hue: hypomethylation).


Figure 1 ( A ) Endogenous FAM134B forms oligomers . FAM134B knockout ( KO ) or wild type ( WT ) 293T cells were treated with 1μM of Thapsigargin ( Tg ) for different time and the cell lysates were prepared and analyzed by Western - blot . ( B ) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE . ( C ) In vitro liposome fragmentation assay . After the injection of recombinant proteins ( 100μg / 100μl ) into the chamber ( 500μl ) , the morphological changes of liposomes were monitored by live imaging for 20 min . The images at different time points as indicated are presented . Scale bars , 10 μm . " ns " means no significance , one - way ANOVA , error bars indicate SEM ( n = 3 ) . ( D ) Quantification of the time between protein addition and liposome fragmentation . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n = 3 ) . ( E , F ) Measurement of the intracellular ER - scission activity . U2OS cells transiently expressing EGFP - FAM134B ( WT ) , EGFP - FAM134B ( 84 - 233 ) or EGFP - FAM134B ( Δ84 - 233 ) at same levels , lysosomal degradation of EGFP - FAM134B was blocked by Bafilomycin A1 ( Baf A1 ) or DMSO . GFP - positive puncta were quantified for each cell in ( F ) . For Each group , at least 30 cells were counted . Scale bars , 10 μm . * * * P < 0 . 001 , " ns " means no significance , one - way ANOVA , error bars indicate SEM . ( G , H ) Measurement of the ER - phagy activity . U2OS cells transiently expressing mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( 84 - 233 ) or mCherry - EGFP - FAM134B ( Δ84 - 233 ) at same levels . ER - phagy is indicated by mCherry - positive but EGFP - negative puncta were quantified for each cell in ( H ) . For mCherry - EGFP - FAM134B ( WT ) , 50 cells were counted ( n = 50 ) ; for mCherry - EGFP - FAM134B ( 84 - 233 ) , n = 56 ; for mCherry - EGFP - FAM134B ( Δ84 - 233 ) , n = 66 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( I ) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co - expressing GFP - SEC61B and FAM134B WT - Flag , FAM134B ( 84 - 233 ) - Flag or FAM134B ( Δ84 - 233 ) - Flag .

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

Figure 1(A) Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild type (WT) 293T cells were treated with 1μM of Thapsigargin (Tg) for different time and the cell lysates were prepared and analyzed by Western-blot.(B) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE.(C) In vitro liposome fragmentation assay. After the injection of recombinant proteins (100μg/100μl) into the chamber (500μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. "ns" means no significance, one-way ANOVA, error bars indicate SEM (n=3).(D) Quantification of the time between protein addition and liposome fragmentation. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).(E, F) Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84-233) or EGFP-FAM134B (Δ84-233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For Each group, at least 30 cells were counted. Scale bars, 10 μm. ***P<0.001, "ns" means no significance, one-way ANOVA, error bars indicate SEM.(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (84-233) or mCherry-EGFP-FAM134B (Δ84-233) at same levels. ER-phagy is indicated by mCherry-positive but EGFP-negative puncta were quantified for each cell in (H). For mCherry-EGFP-FAM134B (WT), 50 cells were counted (n=50); for mCherry-EGFP-FAM134B (84-233), n=56; for mCherry-EGFP-FAM134B (Δ84-233), n=66. Scale bars, 10 μm. The scale bars in the magnification boxes are 2μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(I) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B WT-Flag, FAM134B (84-233)-Flag or FAM134B (Δ84-233)-Flag.


Figure 2 ( A ) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151 . ( B ) Comparison of the self - interaction of FAM134B mono - phosphorylation mutants using co - IP . ( C ) Comparison of the self - interaction of FAM134 tri - phosphorylation mutants using co - IP . ( D ) Comparison of membrane scission activity using in vitro liposome fragmentation assay . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n ≧ 3 ) . ( E ) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co - expressing GFP - SEC61B and FAM134B ( WT ) - Flag , FAM134B ( SA ) - Flag or FAM134B ( SD ) - Flag . ( F , G ) Measurement of the intracellular ER - scission activity . FAM134B knockout ( KO ) U2OS cells was engineered to express EGFP - FAM134B ( WT ) , EGFP - FAM134B ( SA ) or EGFP - FAM134B ( SD ) at endogenous levels , lysosomal degradation of EGFP - FAM134B was blocked by Bafilomycin A1 ( Baf A1 ) . GFP - positive puncta were quantified for each cell in ( G ) . For control , 25 cells were counted ( n = 25 ) ; For EGFP - FAM134B ( WT ) , n = 27 ; for EGFP - FAM134B ( SA ) , n = 29 ; for EGFP - FAM134B ( SD ) , n = 25 . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( H , I ) Measurement of the ER - phagy activity . FAM134B knockout ( KO ) U2OS cells was engineered to express mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( S151A ) or mCherry - EGFP - FAM134B ( S151D ) at endogenous levels . Lysosomal mCherry - positive but GFP - negative puncta were quantified for each cell in ( I ) . For mCherry - EGFP - FAM134B ( WT ) , 25 cells were counted ( n = 25 ) ; for mCherry - EGFP - FAM134B ( SA ) , n = 23 ; for mCherry - EGFP - FAM134B ( SD ) , n = 21 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM .

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

Figure 2(A) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151.(B) Comparison of the self-interaction of FAM134B mono-phosphorylation mutants using co-IP.(C) Comparison of the self-interaction of FAM134 tri-phosphorylation mutants using co-IP.(D) Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).(E) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B (WT)-Flag, FAM134B (SA)-Flag or FAM134B (SD)-Flag.(F, G) Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells was engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA) or EGFP-FAM134B (SD) at endogenous levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n=25); For EGFP-FAM134B (WT), n=27; for EGFP-FAM134B (SA), n=29; for EGFP-FAM134B (SD), n=25. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(H, I) Measurement of the ER-phagy activity. FAM134B knockout (KO) U2OS cells was engineered to express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (S151A) or mCherry-EGFP-FAM134B (S151D) at endogenous levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (I). For mCherry-EGFP-FAM134B (WT), 25 cells were counted (n=25); for mCherry-EGFP-FAM134B (SA), n=23; for mCherry-EGFP-FAM134B (SD), n=21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.


Figure 3 ( A , B ) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry - FAM134B and BAP31 upon Tg treatment . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . The colocalization was analyzed by Pearson ' s correlation coefficient ( PCC ) in ( B ) . For Ctrl , 16 cells were counted ( n = 16 ) ; for Tg treatment , 18 cells were counted ( n = 18 ) . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( C ) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot . Recombinant proteins for FAM134BWT and FAM134BS151A were purified from E . coil were incubated with purified CAMK2B by IP in kinase buffer . FAM134B phosphorylation was analyzed by a specific antibody recognizing phos - Serine151 of human FAM134B . ( D ) FAM134B S151 phosphorylation in cells treated with CAMK2 activator ( 100nM EB1089 for 1h ) or / and inhibitor ( 10μM KN93 for 2h ) . 293T or SKN - SH ( a cell line derived from neuroblastoma ) cells were treated with drugs as indicated , whole cell lysates were analyzed by phospho - FAM134B ( S151 ) antibody . ( E ) FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells . ( F ) In vitro reconstitution of CAMK2B - FAM134B - mediated membrane fragmentation using liposome assay . Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30 ° C for 10min , the resultant protein mixtures were transferred to chamber coated with liposomes . Liposome fragmentation was monitored by live imaging . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n = 3 ) . ( G , H ) Measurement of the ER - phagy activity . U2OS cells transiently expressing mCherry - EGFP - FAM134B ( WT ) were treated with compounds as indicated . Lysosomal mCherry - positive but GFP - negative puncta were quantified for each cell in ( H ) . For vehicle control , 53 cells were counted ( n = 53 ) , for Ionomycin , n = 56 ; for EB1089 , n = 54 ; for KN - 93 , n = 58 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( I ) FAM134B WT or knockout ( KO ) U2OS cells were treated with CAMK2 activator Ionomycin ( Iono ) , EB1089 or inhibitor KN93 for 24 hours , Western - blot was performed to analyze the proteins as indicated . ( J ) CAMK2B WT or knockdown ( KD ) U2OS cells were treated with 1 μM Thapsigargin ( Tg ) to induce ER stress for different time as indicated . Cells were collected and analyzed for proteins as indicated by Western blot . ( K ) GFP - cleavage assay . CAMK2B WT or knockdown ( KD ) U2OS cells were co - transfected with FAM134B - Flag and GFP - SEC61B , 24 hours post transfection , cells were treated with drugs as indicated and analyzed for GFP - cleavage by Western blot . Note : the levels of Flag - FAM134B , CAMK2B , GFP - SEC61B and Tubulin were analyzed by Western blot using different membranes .

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 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 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 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 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 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 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 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

Figure 3(A, B) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry-FAM134B and BAP31 upon Tg treatment. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. The colocalization was analyzed by Pearson's correlation coefficient (PCC) in (B). For Ctrl, 16 cells were counted(n=16); for Tg treatment, 18 cells were counted (n=18). ***P<0.001, one-way ANOVA, error bars indicate SEM.(C) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot. Recombinant proteins for FAM134BWT and FAM134BS151A were purified from E. coil were incubated with purified CAMK2B by IP in kinase buffer. FAM134B phosphorylation was analyzed by a specific antibody recognizing phos-Serine151 of human FAM134B.(D) FAM134B S151 phosphorylation in cells treated with CAMK2 activator (100nM EB1089 for 1h) or/and inhibitor (10μM KN93 for 2h). 293T or SKN-SH (a cell line derived from neuroblastoma) cells were treated with drugs as indicated, whole cell lysates were analyzed by phospho-FAM134B (S151) antibody.(E) FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells.(F) In vitro reconstitution of CAMK2B-FAM134B-mediated membrane fragmentation using liposome assay. Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30°C for 10min, the resultant protein mixtures were transferred to chamber coated with liposomes. Liposome fragmentation was monitored by live imaging. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT) were treated with compounds as indicated. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (H). For vehicle control, 53 cells were counted (n=53), for Ionomycin, n=56; for EB1089, n=54; for KN-93, n=58. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(I) FAM134B WT or knockout (KO) U2OS cells were treated with CAMK2 activator Ionomycin (Iono), EB1089 or inhibitor KN93 for 24 hours, Western-blot was performed to analyze the proteins as indicated.(J) CAMK2B WT or knockdown (KD) U2OS cells were treated with 1 μM Thapsigargin (Tg) to induce ER stress for different time as indicated. Cells were collected and analyzed for proteins as indicated by Western blot.(K) GFP-cleavage assay. CAMK2B WT or knockdown (KD) U2OS cells were co-transfected with FAM134B-Flag and GFP-SEC61B, 24 hours post transfection, cells were treated with drugs as indicated and analyzed for GFP-cleavage by Western blot. Note: the levels of Flag-FAM134B, CAMK2B, GFP-SEC61B and Tubulin were analyzed by Western blot using different membranes.


Figure 4 ( A ) Comparison of the self - interaction of FAM134B WT with mutants as indicated using co - IP . SA - G216R was constructed by simultaneously mutating S149 , S151 and S153 to alanine ( SA ) on the basis of FAM134B - G216R . ( B ) In vitro liposome fragmentation assay . Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM ( n ≧ 3 ) . ( C , D ) Measurement of the intracellular ER - scission activity of FAM134B WT , G216R , S151A - G216R and SA - G216R . GFP - tagged FAM134B proteins were expressed in U2OS cells at same levels . The autolyosomal degradation of ER fragments labelled by GFP - FAM134B were blocked by Baf A1 or DMSO . GFP - positive puncta were quantified for each cell in ( D ) . For all of the groups , at least 30 cells were included for quantification ; Scale bars , 10 μm . * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM . ( E , F ) Measurement of the ER - phagy activity of FAM134BG216R . U2OS cells transiently expressing mCherry - EGFP - FAM134B ( WT ) , mCherry - EGFP - FAM134B ( G216R ) , mCherry - EGFP - FAM134B ( S151A - G216R ) or mCherry - EGFP - FAM134B ( SA - G216R ) at same levels . Lysosomal mCherry - positive but GFP - negative puncta were quantified for each cell in ( F ) . For WT , 50 cells were counted ( n = 50 ) ; for G216R , n = 55 ; for S151A - G216R , n = 51 ; for SA - G216R , n = 52 . Scale bars , 10 μm . The scale bars in the magnification boxes are 2 μm . * P < 0 . 05 , * * * P < 0 . 001 , one - way ANOVA , error bars indicate SEM .

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Figure 4(A) Comparison of the self-interaction of FAM134B WT with mutants as indicated using co-IP. SA-G216R was constructed by simultaneously mutating S149, S151 and S153 to alanine (SA) on the basis of FAM134B-G216R.(B) In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).(C, D) Measurement of the intracellular ER-scission activity of FAM134B WT, G216R, S151A-G216R and SA-G216R. GFP-tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolyosomal degradation of ER fragments labelled by GFP-FAM134B were blocked by Baf A1 or DMSO. GFP-positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(E, F) Measurement of the ER-phagy activity of FAM134BG216R. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (G216R), mCherry-EGFP-FAM134B (S151A-G216R) or mCherry-EGFP-FAM134B (SA-G216R) at same levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (F). For WT, 50 cells were counted (n=50); for G216R, n=55; for S151A-G216R, n=51; for SA-G216R, n=52. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. *P<0.05, ***P<0.001, one-way ANOVA, error bars indicate SEM.


Figure 5 ( A , A ' ) Specific expression of FAM134B mRNA ( green ) in sensory neurons ( red ) of P4d vGlut2Cre ; R26lsl - tdTommouse . Scale bars , 100μm . The white dotted line indicates the dorsal root ganglia ( DRG ) where sensory neurons are genetically labeled with Tomato . Insets of ( a ) and ( a ' ) are high magnifications of sensory neurons showing the co - localization of FAM134B mRNA and Tomato . Scale bars , 10 μm . Cultured sensory neurons from DRGs of E14 . 5 vGlut2Cre ; R26lsl - tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP - FAM134B WT ( B ) , G216R ( C ) , S151A - G216R ( D ) , SA - G216R ( E ) , G216R - ΔLIR ( F ) respectively . Infected sensory neurons ( GFP + / Tom + ) are indicated with arrowheads . The nuclei of all cells are shown with DAPI staining . Scale bars , 20 μm . Cultured sensory neurons from DRGs of E14 . 5 vGlut2Cre ; R26lsl - tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 ( G ) Infected sensory neurons ( GFP + / Tom + ) are indicated with arrowheads . The nuclei of all cells are shown with DAPI staining . Scale bars , 20 μm . ( H ) Quantification of the viability of sensory neuron after 48h , 72h and 96h of infection . Sensory neuron viability was calculated as the percentage of transfected sensory neuron ( GFP + / Tom + ) in total sensory neurons ( Tom + ) . For each time point , four biological replicates were included for statistical analysis . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA , error bars indicate SEM . " ns " is the abbreviation for " not significant " . For all of the groups , at least 10 fields of the slides were included .

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Figure 5(A,A') Specific expression of FAM134B mRNA (green) in sensory neurons (red) of P4d vGlut2Cre;R26lsl-tdTommouse. Scale bars, 100μm. The white dotted line indicates the dorsal root ganglia (DRG) where sensory neurons are genetically labeled with Tomato. Insets of (a) and (a') are high magnifications of sensory neurons showing the co-localization of FAM134B mRNA and Tomato. Scale bars, 10 μm.Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP-FAM134B WT (B), G216R(C), S151A-G216R(D), SA-G216R(E), G216R-ΔLIR (F) respectively. Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 (G) Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.(H) Quantification of the viability of sensory neuron after 48h, 72h and 96h of infection. Sensory neuron viability was calculated as the percentage of transfected sensory neuron (GFP+/Tom+) in total sensory neurons (Tom+). For each time point, four biological replicates were included for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA, error bars indicate SEM. "ns" is the abbreviation for "not significant". For all of the groups, at least 10 fields of the slides were included.


Figure 1 . Entry of MLV pseudotyped with SARS - CoV - 2 S , SARS - CoV - 2 Sfur / mut and SARS - CoV S in VeroE6 cells . Entry of MLV pseudotyped with SARS - CoV - 2 S or SARS - CoV - 2 Sfur / mut in BHK cells transiently transfected with hACE2 . The experiments were carried out in triplicate with two independent pseudovirus preparations and a representative experiment is shown . . Western blot analysis of SARS - CoV - 2 S - MLV , SARS - CoV - 2 Sfur / mut - MLV and SARS - CoV S - MLV pseudovirions using an anti - SARS - CoV S2 antibody .

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Figure 1.Entry of MLV pseudotyped with SARS-CoV-2 S, SARS-CoV-2 Sfur/mut and SARS-CoV S in VeroE6 cells.Entry of MLV pseudotyped with SARS-CoV-2 S or SARS-CoV-2 Sfur/mut in BHK cells transiently transfected with hACE2. The experiments were carried out in triplicate with two independent pseudovirus preparations and a representative experiment is shown.. Western blot analysis of SARS-CoV-2 S-MLV, SARS-CoV-2 Sfur/mut-MLV and SARS-CoV S-MLV pseudovirions using an anti-SARS-CoV S2 antibody.


Figure 2 . Biolayer interferometry binding analysis of the hACE2 ectodomain to immobilized SARS - CoV - 2 SB ( B ) or SARS - CoV

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Figure 2.Biolayer interferometry binding analysis of the hACE2 ectodomain to immobilized SARS-CoV-2 SB (B) or SARS-CoV SB


Figure 3 . Two orthogonal views of the closed SARS - CoV - 2 S trimer cryoEM map . C . Atomic model of the closed SARS - CoV - 2 S trimer in the same orientation as in panel A . D - E . Two orthogonal views of the partially open SARS - CoV - 2 S trimer cryoEM map ( one SB domain is open ) . F . Atomic model of the closed SARS - CoV - 2 S trimer in the same orientation as in panel D . The glycans were omitted for clarity .

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Figure 3.Two orthogonal views of the closed SARS-CoV-2 S trimer cryoEM map. C. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel A.D-E. Two orthogonal views of the partially open SARS-CoV-2 S trimer cryoEM map (one SB domain is open). F. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel D. The glycans were omitted for clarity.


Figure 5 . Entry of SARS - CoV - 2 and SARS - CoV S pseudotyped viruses is potently inhibited by four SARS - CoV S mouse polyclonal immune plasma .

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Figure 5.Entry of SARS-CoV-2 and SARS-CoV S pseudotyped viruses is potently inhibited by four SARS-CoV S mouse polyclonal immune plasma.


Figure 1 ( A ) Western blot showing the protein expression of ASCL1 and / or NEUROD1 in NCI - H69 , NCI - H82 , NCI - H146 , NCI - H510A , NCI - H526 , SHP - 77 and DMS - 53 SCLC cells ( lanes 4 - 10 ) ; A549 and NCI - H460 NSCLC cells and a IMR - 90 HFL cell lines ( lanes 1 - 3 ) . Tubulin ( α - Tub ) is shown as loading control . Each blot is representative from at least three independent experiments .

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Figure 1(A) Western blot showing the protein expression of ASCL1 and/or NEUROD1 in NCI-H69, NCI-H82, NCI-H146, NCI-H510A, NCI-H526, SHP-77 and DMS-53 SCLC cells (lanes 4-10); A549 and NCI-H460 NSCLC cells and a IMR-90 HFL cell lines (lanes 1-3). Tubulin (α-Tub) is shown as loading control. Each blot is representative from at least three independent experiments.


Figure 2 ( A - E ) ChIP - seq experiments indicates the localization of ( A ) ASCL1 , ( B ) NEUROD1 and ( E ) RNAPII . ATAC - seq ( C ) and ( D ) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t = 0 ( blue ) and t = 4h ( green ) after lurbinectedin treatment ; - 2 . 0 and + 2 . 0 kb to TSS coordinates of all hg19 genes .

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Figure 2(A-E) ChIP-seq experiments indicates the localization of (A) ASCL1, (B) NEUROD1 and (E) RNAPII. ATAC-seq (C) and (D) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t=0 (blue) and t=4h (green) after lurbinectedin treatment; -2.0 and +2.0 kb to TSS coordinates of all hg19 genes.


Figure 3 ( A - D ) Chip - seq genome track of ASCL1 / NEUROD1 , RNAPII , and H3K27Ac on ASCL1 , BCL2 ; INSM1 and MYB ( hg19 ) , in untreated ( dark blue ) and in lurbinectedin treated ( green ) conditions respectively ; to be noticed the location of CGG rich motifs ( lower part of each panel ) which parallel the presence of lurbinectedin ( red ) .

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Figure 3(A-D) Chip-seq genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1 and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red).


Figure 4 ( a ) Western blot showing RNAPII across the panel of ; A549 and NCI - H460 NSCLC cells ; IMR - 90 HFL cells ; NCI - H146 , NCI - H82 , DMS - 53 , NCI - H510A , NCI - H526 and SHP - 77 SCLC cells before ( - ) and after ( + ) treatment with lurbinectedin . α - Tubulin ( α - Tub ) is shown as loading control ; lower panel , Histogram showing the average of RNAPII before ( dark blue ) and after ( light Blue ) lurbinectedin treatment in arbitrary units ( a . u . ) normalized against α - Tub . Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák ' s multiple comparisons test ( n = 3 biological replicas ) . * * * * P ≤ 0 . 0001 . ( B Immunofluorescence of RNAPII ( red ) before ( Ctrl ) and after lurbinectedin treatment . DAPI ( blue ) was used to stain nuclei ; scale bar is 65 μm . ( D ) Affinity purification of Bio - lur fractions containing elongating RNAPII Ser2 ( lane 4 ) . Input ( lanes 1 - 2 ) represents 10 % of the starting material . Respective molecular weights are shown on the right ( kDa ) . ( E ) Upper panel : GST - pulldown of ubiquitinated proteins showing hypo - ( IIA ) , - hyper ( II0 ) phosphorylated RNAPII ( lanes 4 - 5 upper panel ) and Ser2 Phosphorylated RNAPII ( Ser2P ) ( lanes 4 - 5 lower panel ) after lurbinectedin treatment . Empty beads ( Mock ) ( lane 3 ) as a negative control ; Lower Panel : GST - pulldown of ubiquitinated proteins Ser2P ) ( lanes 4 - 5 ) . Representative smear of successful ubiquitinated proteins pulldown ( Lanes 4 and 5 ) as compared to input ( lanes 1 and 2 ) . Empty beads ( Mock ) ( lane 3 ) as a negative control . Each Western blot is representative of three independent experiments .

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Figure 4(a) Western blot showing RNAPII across the panel of; A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526 and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin. α-Tubulin (α-Tub) is shown as loading control; lower panel, Histogram showing the average of RNAPII before (dark blue) and after (light Blue) lurbinectedin treatment in arbitrary units (a.u.) normalized against α-Tub. Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák's multiple comparisons test (n=3 biological replicas). ****P ≤0.0001.(B Immunofluorescence of RNAPII (red) before (Ctrl) and after lurbinectedin treatment. DAPI (blue) was used to stain nuclei; scale bar is 65 μm.(D) Affinity purification of Bio-lur fractions containing elongating RNAPII Ser2 (lane 4). Input (lanes 1-2) represents 10% of the starting material. Respective molecular weights are shown on the right (kDa).(E) Upper panel: GST-pulldown of ubiquitinated proteins showing hypo- (IIA), -hyper (II0) phosphorylated RNAPII (lanes 4-5 upper panel) and Ser2 Phosphorylated RNAPII (Ser2P) (lanes 4-5 lower panel) after lurbinectedin treatment. Empty beads (Mock) (lane 3) as a negative control; Lower Panel: GST-pulldown of ubiquitinated proteins Ser2P) (lanes 4-5). Representative smear of successful ubiquitinated proteins pulldown (Lanes 4 and 5) as compared to input (lanes 1 and 2). Empty beads (Mock) (lane 3) as a negative control. Each Western blot is representative of three independent experiments.


Figure 1A ) GFP tagged Syb protein expressed in Hh - receiving cells co - localizes with endogenous immuno - labelled Ptc along the apico - basal axis ( 3D reconstruction ) . Vesicles containing Syb and Ptc are visualized at lateral and basal planes ( arrows ) , and can be detected along cytonemes marked with Syb - GFP ( arrowhead ) . B ) Expression of the GFP tagged Syt1 also co - localizes with inmmuno - labelled endogenous Ptc at vesicles ( lateral plane , arrows point at vesicles with co - localization ) and decorates the cytoneme membranes ( basal plane , arrowheads indicate cytonemes ) . C ) GRASP experiment shows exocytic events in Hh - receiving cells at basal membrane contacts . Hh - receiving cells express the GFP barrel 1 - 10 tagged to Syb and Hh - producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4 . Note the fluorescent discrete punctae ( arrows ) along Hh producing cells cytonemes ( stabilized with Ihog - RFP ) crossing the reception area . D ) Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP . Note a strong localization in clusters close to the basal membrane in the 3D reconstruction ( left panel ) and in confocal lateral and basal planes , including cytonemes ( right panels , arrows and arrowhead indicate vesicle - like structures along cytoenemes ) .

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Figure 1A) GFP tagged Syb protein expressed in Hh-receiving cells co-localizes with endogenous immuno-labelled Ptc along the apico-basal axis (3D reconstruction). Vesicles containing Syb and Ptc are visualized at lateral and basal planes (arrows), and can be detected along cytonemes marked with Syb-GFP (arrowhead).B) Expression of the GFP tagged Syt1 also co-localizes with inmmuno-labelled endogenous Ptc at vesicles (lateral plane, arrows point at vesicles with co-localization) and decorates the cytoneme membranes (basal plane, arrowheads indicate cytonemes).C) GRASP experiment shows exocytic events in Hh-receiving cells at basal membrane contacts. Hh-receiving cells express the GFP barrel 1-10 tagged to Syb and Hh-producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4. Note the fluorescent discrete punctae (arrows) along Hh producing cells cytonemes (stabilized with Ihog-RFP) crossing the reception area.D) Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP. Note a strong localization in clusters close to the basal membrane in the 3D reconstruction (left panel) and in confocal lateral and basal planes, including cytonemes (right panels, arrows and arrowhead indicate vesicle-like structures along cytoenemes).


Figure 2A ) Ptc protein distribution in a wing disc ( 3D reconstitution ) under down - regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment ( D ) , keeping the ventral compartment ( V ) as a WT internal control . Note the basal accumulation of endogenous Ptc ( arrowheads ) when knocking down Syb or Syt1 . B ) Confocal images of immuno - labelled endogenous Ptc and cytonemes stabilized with Ihog - RFP protruding from Hh producing cells in wild type receiving cells ( left panel ) , or B ' ) when either blocking exocytosis by down - regulating Syb ( middle panel ) or B ' ' ) blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin , Shibire ( right panel ) in the Hh - receiving cells . Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization and processing after Hh reception ; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae ( arrowheads ) , and endocytosis inhibition leads to Ptc accumulation at the plasma membrane ( arrows ) . C ) Confocal apical ( left ) and basal ( right ) images of a shits mutant wing disc expressing Syb RNAi dorsally ( D ) to also block exocytosis , keeping the ventral compartment ( V ) as an internal control where just endocytosis is inhibited . After dorsal Syb RNAi induction , endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature . Note the dorsal reduction of Ptc at plasma membrane after blocking exocytosis . D ) Quantification of Ptc - GFP levels ( Yac construct ) along the wing disc apico - basal axis , integrating both fluorescence intensity and signal area ( Integrated Density ) . The graph on the left shows values for wild type situations in purple ( average in blue ) , while Syb RNAi treatment values are shown in green ( average in orange ) . a clear shift of higher values towards the basal side of the discs after inhibition of Syb function . The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side ( - 1 = basal , + 1 = apical ) of discs after RNAi expression for different SNARE proteins and the wild type . Central horizontal lines show median values of N = 7 - 16 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . A basal association is particularly noticeable for Syb , Syx1A and α - Snap down - regulation . Right panel is a scheme illustrating the wild type and RNAi - treated patterns of quantified Ptc - GFP levels distribution along the wing disc apico - basal axis .

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Figure 2A) Ptc protein distribution in a wing disc (3D reconstitution) under down-regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment (D), keeping the ventral compartment (V) as a WT internal control. Note the basal accumulation of endogenous Ptc (arrowheads) when knocking down Syb or Syt1.B) Confocal images of immuno-labelled endogenous Ptc and cytonemes stabilized with Ihog-RFP protruding from Hh producing cells in wild type receiving cells (left panel), or B') when either blocking exocytosis by down-regulating Syb (middle panel) or B'') blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin, Shibire (right panel) in the Hh-receiving cells. Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization and processing after Hh reception; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae (arrowheads), and endocytosis inhibition leads to Ptc accumulation at the plasma membrane (arrows).C) Confocal apical (left) and basal (right) images of a shits mutant wing disc expressing Syb RNAi dorsally (D) to also block exocytosis, keeping the ventral compartment (V) as an internal control where just endocytosis is inhibited. After dorsal Syb RNAi induction, endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature. Note the dorsal reduction of Ptc at plasma membrane after blocking exocytosis.D) Quantification of Ptc-GFP levels (Yac construct) along the wing disc apico-basal axis, integrating both fluorescence intensity and signal area (Integrated Density). The graph on the left shows values for wild type situations in purple (average in blue), while Syb RNAi treatment values are shown in green (average in orange). a clear shift of higher values towards the basal side of the discs after inhibition of Syb function. The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side (-1=basal, +1=apical) of discs after RNAi expression for different SNARE proteins and the wild type. Central horizontal lines show median values of N=7-16, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. A basal association is particularly noticeable for Syb, Syx1A and α-Snap down-regulation. Right panel is a scheme illustrating the wild type and RNAi-treated patterns of quantified Ptc-GFP levels distribution along the wing disc apico-basal axis.


Figure 3A ) Electron Microscopy imaging of wing discs expressing the UAS - Ptc - GFP construct for 24 hours in receiving cells using Ptc - Gal4 ; TubGal80ts . Left panel shows areas with anti - GFP immuno - labelling ( marked in blue ) throughout the apico / basal axis of the wing disc epithelium a ) anti - GFP gold - labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation . b ) Ptc immuno - gold labelling is present in multivesicular bodies ( MVBs ) subapically ( top right panels ) . b1 ) subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes . c ) Ptc label is also present in MVBs close to the basal membrane ( lower right panels ) , c1 ) basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles ( ILV ) . B ) Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5 . The top panel is a schematic representation of the localized expression . The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections . that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc , compared to the wild type control on the ventral side . accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc , compared to the wild type control on the ventral side . C ) Digital 3D reconstruction ( left ) and apical and basal confocal sections ( right ) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire .

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Figure 3A) Electron Microscopy imaging of wing discs expressing the UAS-Ptc-GFP construct for 24 hours in receiving cells using Ptc-Gal4; TubGal80ts. Left panel shows areas with anti-GFP immuno-labelling (marked in blue) throughout the apico/basal axis of the wing disc epithelium a) anti-GFP gold-labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation. b) Ptc immuno-gold labelling is present in multivesicular bodies (MVBs) subapically (top right panels). b1) subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes. c) Ptc label is also present in MVBs close to the basal membrane (lower right panels), c1) basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles (ILV).B) Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5. The top panel is a schematic representation of the localized expression. The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections. that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side.accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side. C) Digital 3D reconstruction (left) and apical and basal confocal sections (right) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire.


Figure 4A ) 3D reconstructions of immuno - labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components . All RNAis ' expression result in Ptc accumulation in large structures , located apically under Hrs ( ESCRT - 0 component ) inhibition , and basally under inhibition of the ESCRT components I ( Tsg101 ) , II ( Vps22 ) and III ( Shrub ) . Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation . B ) Detailed confocal imaging of immuno - labelled endogenous Ptc localization after clonal Tsg101 down - regulation . The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells ( arrows ) , since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh receptionC ) Ptc accumulation in these punctated structures ( arrows ) is also visualized on receiving cytonemes stabilized with the co - receptor Ihog - RFP and after Tsg101 inhibition . D ) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog - RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions ( arrow ) .

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Figure 4A) 3D reconstructions of immuno-labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components. All RNAis' expression result in Ptc accumulation in large structures, located apically under Hrs (ESCRT-0 component) inhibition, and basally under inhibition of the ESCRT components I (Tsg101), II (Vps22) and III (Shrub). Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation.B) Detailed confocal imaging of immuno-labelled endogenous Ptc localization after clonal Tsg101 down-regulation. The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells (arrows), since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh receptionC) Ptc accumulation in these punctated structures (arrows) is also visualized on receiving cytonemes stabilized with the co-receptor Ihog-RFP and after Tsg101 inhibition.D) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog-RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions (arrow).