f4 ( a ) Immunoblot of autophagy - related proteins in U2OS cells expressing a control TALEN ( U2OS - TAL2 - C1 ) or two independent subcloned TALEN cell lines targeting ATG7 ( U2OS - TAL2 - A7 , U2OS - TAL2 - A8 ) . p62 long and short refer to either long or short exposure times during film development . ( b ) U2OS cells expressing an ATG7 - TALEN ( TAL2 - ATG7 ) or control TALEN ( TAL2 - CTL ) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine - replete medium . Cells were then stained with SRB and absorbance was read at 540 nM . Graph shows average results from five clonal sub - lines for each condition . The statistical significance ( P value ) was determined by a two - tailed , paired Student ' s t - test . P0 . 05 . ( c ) Representative images of SRB - stained clones in bd ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of U2OS - TAL2 - CTL - C1 ( control ) and U2OS - TAL2 - ATG7 - A8 ( ATG7 knockout ) following 24 h glutamine deprivation . ( e ) IL - 8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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f4(a) Immunoblot of autophagy-related proteins in U2OS cells expressing a control TALEN (U2OS-TAL2-C1) or two independent subcloned TALEN cell lines targeting ATG7 (U2OS-TAL2-A7, U2OS-TAL2-A8). p62 long and short refer to either long or short exposure times during film development.(b) U2OS cells expressing an ATG7-TALEN (TAL2-ATG7) or control TALEN (TAL2-CTL) were subjected to glutamine starvation for 6 days followed by 6 days recovery in glutamine-replete medium. Cells were then stained with SRB and absorbance was read at 540 nM. Graph shows average results from five clonal sub-lines for each condition. The statistical significance (P value) was determined by a two-tailed, paired Student's t-test. P0.05. (c) Representative images of SRB-stained clones in bd) IL-8 enzyme-linked immunosorbent assay (ELISA) of U2OS-TAL2-CTL-C1 (control) and U2OS-TAL2-ATG7-A8 (ATG7 knockout) following 24 h glutamine deprivation.(e) IL-8 U2OS of U2OS parental cells starved of glutamine for the indicated times with or without BafA1. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


f5 ( a ) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE - 125132 . Scale bar , 15 μm . ( b ) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE - 125132 . ( c ) IL - 8 enzyme - linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 . ( d ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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f5(a) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm.(b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132.(c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132.(d) RT-PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


f6 ( a ) Immunoblot analysis of autophagy , ER stress - and mTOR - related proteins , and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 . ( b ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK ( JNK # 1 and JNK # 2 ) and subjected to 24 h glutamine deprivation . ( c ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of conditioned media from the experiment described in b . ( d ) Immunoblot analysis of JNK protein expression in JNK siRNA - treated cells . e ) RT - PCR analysis of IL - 8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 ( JNKi ) . ( f ) IL - 8 ELISA of conditioned medium from the experiment described in e . ( g ) IL - 8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 ( IRE - A and IRE - B ) and subjected to glutamine deprivation for 24 h . ( h ) RT - PCR analysis of IL - 8 mRNA expression from experiment described in g . ( i ) RT - PCR analysis of CHOP mRNA expression from experiment described in g . ( j ) RT - PCR analysis of IRE1 mRNA expression from experiment described in g . ( k ) Immunoblot analysis of experiment described in g showing phosphorylation of JNK . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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f6(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.(b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation.(c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b.(d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells.e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi).(f) IL-8 ELISA of conditioned medium from the experiment described in e.(g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h.(h) RT-PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT-PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT-PCR analysis of IRE1 mRNA expression from experiment described in g.(k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


f7 ( a ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) on U2OS cells starved of glutamine or treated with BPTES ( 10 μM ) for 24 h . ( b ) RT - PCR analysis of IL - 8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG . ( c ) Metabolite analysis of U2OS cells starved of glutamine , treated with BPTES or glutamine - starved co - treated with DMαKG . ( d ) IL - 8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co - treated with DMαKG . ( e ) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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f7(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.(c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG.(d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG.(e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


Figure 2Representative density plots of Ca2 + flux from conjugated OptoCAR ‑ T cells over time at 37ºC . Conjugated cells were stimulated with dimerizer addition for 0 ( F ) , 5 ( G ) , 10 ( H ) , 15 ( I ) or 30 ( J ) minutes prior to disrupting signaling by illuminating cells .

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Figure 2Representative density plots of Ca2+ flux from conjugated OptoCAR‑T cells over time at 37ºC. Conjugated cells were stimulated with dimerizer addition for 0 (F), 5 (G), 10 (H), 15 (I) or 30 (J) minutes prior to disrupting signaling by illuminating cells.


Figure 3OptoCAR ‑ T cells were conjugated with ligand - presenting cells and activated as in the upper regime shown in ( A ) . The median of ppERK intensity distribution was plotted with time , after subtraction of background intensity . Bars show mean ± SEM ( n = 3 ) . An equivalent experiment was performed as in ( B ) but following the middle regime shown in ( A ) , with sample illumination after 5 minutes for the remainder of the dataset . Indicated half - life was calculated from the time taken for the median ppERK to decrease to 50 % of maximum . Bars show mean ± SEM ( n = 4 ) . An equivalent experiment was performed as in ( B ) but following the lower regime shown in ( A ) , with sample illumination after 5 minutes for 3 minutes . Bars show mean ± SEM ( n = 3 ) .

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Figure 3OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3). An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4). An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3).


Figure 4Representative fluorescent Western blot showing the dynamics of FOS phosphorylation ( p - FOSS32 ) on light - modulated control . Conjugated OptoCAR ‑ T cells were stimulated for a defined period after dimerizer addition ( denoted above blots ) before either being left in the dark ( dark line ) or illuminated ( blue line ) . Full blot image with total protein normalization control shown in Figure EV3A . Representative fluorescent Western blot showing the dynamics of FOS expression ( FOSTotal ) on light - modulated control , from the same dataset as in ( A ) . Full blot image with total protein normalization control shown in Figure EV3B .

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Figure 4Representative fluorescent Western blot showing the dynamics of FOS phosphorylation (p-FOSS32) on light-modulated control. Conjugated OptoCAR‑T cells were stimulated for a defined period after dimerizer addition (denoted above blots) before either being left in the dark (dark line) or illuminated (blue line). Full blot image with total protein normalization control shown in Figure EV3A. Representative fluorescent Western blot showing the dynamics of FOS expression (FOSTotal) on light-modulated control, from the same dataset as in (A). Full blot image with total protein normalization control shown in Figure EV3B.


Figure 5RT - qPCR analysis of relative mRNA levels for four representative genes following OptoCAR - mediated T cell activation . OptoCAR ‑ T cell conjugates were activated in the dark ( signaling competent ) state for a period of 3 hours before light - mediated disruption of signaling ( open circles ) or maintained for a further 60 minutes in the dark ( filled circles ) . The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3 - 4 hours . Individual datasets are presented as gray lines , with bars showing mean ± SEM ( n = 3 ) . Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1 .

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Figure 5RT-qPCR analysis of relative mRNA levels for four representative genes following OptoCAR-mediated T cell activation. OptoCAR‑T cell conjugates were activated in the dark (signaling competent) state for a period of 3 hours before light-mediated disruption of signaling (open circles) or maintained for a further 60 minutes in the dark (filled circles). The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3-4 hours. Individual datasets are presented as gray lines, with bars showing mean ± SEM (n=3). Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1.


Figure 6Single pulses of varying duration were applied to OptoCAR ‑ T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling , with illumination started once the signal pulse had ended . All values have been corrected for background GFP fluorescence from non - activated OptoCAR ‑ T cells and shown relative to the output at 24 hours . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 ) Equivalent datasets as in ( B ) for the geometric mean of CD69 upregulation . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 )

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Figure 6Single pulses of varying duration were applied to OptoCAR‑T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling, with illumination started once the signal pulse had ended. All values have been corrected for background GFP fluorescence from non-activated OptoCAR‑T cells and shown relative to the output at 24 hours. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3) Equivalent datasets as in (B) for the geometric mean of CD69 upregulation. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3)


Figure 7The cRQ values from RT - qPCR of indicated cells . Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells . Conjugated OptoCARCD19 ‑ T cells were also assayed either before or after 24 hours stimulation in the dark state . Individual values from three biological replicates are shown , with mean depicted by bar plot .

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Figure 7The cRQ values from RT-qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCARCD19‑T cells were also assayed either before or after 24 hours stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot.


Figure 2Determination of maximum growth rate ( Vmax ) using a label - free cell count - based proliferation assay . Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi - log scale displaying total numbers of cells over time . Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints . Western blot for NUP58 protein expression in mutant clones ( B - E ) and control cells ( clone A and WT ) . Whole - cell lysate hybridized with customized antibody 1 ( Ab1 , top panel ) and antibody 2 ( Ab2 , bottom panel ) . Actin was used as loading control .

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Figure 2Determination of maximum growth rate (Vmax) using a label-free cell count-based proliferation assay. Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi-log scale displaying total numbers of cells over time. Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints.Western blot for NUP58 protein expression in mutant clones (B-E) and control cells (clone A and WT). Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Actin was used as loading control.


Figure 3Representation of the highest growth rates ( Vmax ) for each technical replicate for each indicated cell line . Each dot represents a technical replicate ( WT n = 35 , C1 n = 27 , C2 n = 28 , C3 n = 34 , C4 n = 32 , C5 n = 35 , C6 n = 38 ) of one independent experimental replicate performed .

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Figure 3Representation of the highest growth rates (Vmax) for each technical replicate for each indicated cell line. Each dot represents a technical replicate (WT n=35, C1 n=27, C2 n=28, C3 n=34, C4 n=32, C5 n=35, C6 n=38) of one independent experimental replicate performed.


Figure 4Western blot for NUP58 protein expression in mutant clones and control cells . Whole - cell lysate hybridized with customized antibody 1 ( Ab1 , top panel ) and antibody 2 ( Ab2 , bottom panel ) . Blots were loaded with wild - type HAP1 cell line ( WT ) , empty vector ( EV ) , non - targeting siRNA ( siNT ) and NUP58 targeting siRNA ( siNUP58 ) , followed by NUP58 mutant clones ( C1 / C2 / C3 / C4 in left panels , C5 / C6 in right panels ) . Actin was used as loading control . Asterisks indicate possible NUP58 alternative isoforms .

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Figure 4Western blot for NUP58 protein expression in mutant clones and control cells. Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Blots were loaded with wild-type HAP1 cell line (WT), empty vector (EV), non-targeting siRNA (siNT) and NUP58 targeting siRNA (siNUP58), followed by NUP58 mutant clones (C1 / C2 / C3 / C4 in left panels, C5 / C6 in right panels). Actin was used as loading control. Asterisks indicate possible NUP58 alternative isoforms.


Figure 5Cellular viability expressed as number of viable colonies following Cas9 - induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1 - KPNA4 - eGFP ( KAPs ) .

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Figure 5Cellular viability expressed as number of viable colonies following Cas9-induced gene editing of NUP58 in WT cell line and in a cell line stably overexpressing KPNB1-KPNA4-eGFP (KAPs).


Figure 6Improvement in active nuclear import upon cell passaging . Dots represent analysed cells ( WT P3 n = 16 , WT P19 n = 25 , C5 P1 n = 24 , C5 P3 n = 27 , C5 P19 n = 23 ) of one independent experimental replicate performed . NUP58 protein expression in C5 at P1 and P19 . Blots were performed using Antibody 1 ( Ab1 , top ) or Antibody 2 ( Ab2 , bottom ) . Actin was used as loading control . The band corresponding to the full - length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk . Cellular viability following Cas9 - induced gene editing of native NUP58 ( left ) or NUP85 ( right ) in WT cell line ( black circles ) and in a cell line overexpressing NUP58 mutant isoform ( grey circles ) .

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Figure 6Improvement in active nuclear import upon cell passaging. Dots represent analysed cells (WT P3 n=16, WT P19 n=25, C5 P1 n=24, C5 P3 n=27, C5 P19 n=23) of one independent experimental replicate performed.NUP58 protein expression in C5 at P1 and P19. Blots were performed using Antibody 1 (Ab1, top) or Antibody 2 (Ab2, bottom). Actin was used as loading control. The band corresponding to the full-length NUP58 is indicated by a black arrow with possible alternative isoform by an asterisk.Cellular viability following Cas9-induced gene editing of native NUP58 (left) or NUP85 (right) in WT cell line (black circles) and in a cell line overexpressing NUP58 mutant isoform (grey circles).


Figure 7Diagram at the top represents G - banding pattern of chromosome 13 and highlights chromosome locations of tested genes ( NUP58 , GRK1 , ADPRHL1 ) . Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number . Representative images of NUP58 gene - specific FISH hybridization for control HAP1 cell lines ( WT P3 - WT P19 ) and mutant C5 at P19 . Each dot represents a NUP58 locus of cluster thereof . Scale bar , 31 . 3

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Figure 7Diagram at the top represents G-banding pattern of chromosome 13 and highlights chromosome locations of tested genes (NUP58, GRK1, ADPRHL1). Graph at the bottom reports qPCR results for indicated genes preformed on the gDNA of indicated samples at the indicated passage number.Representative images of NUP58 gene-specific FISH hybridization for control HAP1 cell lines (WT P3-WT P19) and mutant C5 at P19. Each dot represents a NUP58 locus of cluster thereof. Scale bar, 31.3 μm


Figure 1A - O UAS . RNAi lines were driven with eyeless . Gal4gmr . Gal4 for expression during eye development or nubbin - Gal4 for expression during wing development . ( A , B ) Control adult Drosophilaeye ( A ) and wing ( B ) . ( C , D ) α - spectrin RNAi results in overgrowth of the eye ( C ) and wing ( D ) . ( E , F ) βH - spectrin / karst RNAi results in overgrowth of the eye ( E ) and wing ( F ) . ( G , H ) β - spectrin RNAi does not affect eye size ( G ) or wing size ( H ) . ( I , J ) kibra RNAi results in overgrowth of the eye ( I ) and wing ( J ) . ( K , L ) α - spectrin , kibra double RNAi results in stronger overgrowth of the eye ( K ) and wing ( L ) . ( M , N ) βH - spectrin / wing , kibra double RNAi results in stronger overgrowth of the eye ( M ) and wing ( N ) . ( O ) Quantification of female wing sizes by pixel area , 5 wings per genotype were measured . Error bars show standard deviation . P - RThe eyeless FLP MARCM system was used to generate clonally mutant flyeyes . eyes mutant eyes ( Q ) overgrow slightly compared to controls ( P ) , while α - spectrin mutant eyes expressing α - spectrin RNAi ( R ) overgrow strongly compared to controls ( P ) . Data information : Scale bars , 250 μm .

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Figure 1A-O UAS.RNAi lines were driven with eyeless.Gal4gmr.Gal4 for expression during eye development or nubbin-Gal4 for expression during wing development. (A, B) Control adult Drosophilaeye (A) and wing (B). (C, D) α-spectrin RNAi results in overgrowth of the eye (C) and wing (D). (E, F) βH-spectrin/karst RNAi results in overgrowth of the eye (E) and wing (F). (G, H) β-spectrin RNAi does not affect eye size (G) or wing size (H). (I, J) kibra RNAi results in overgrowth of the eye (I) and wing (J). (K, L) α-spectrin, kibra double RNAi results in stronger overgrowth of the eye (K) and wing (L). (M, N) βH-spectrin/wing, kibra double RNAi results in stronger overgrowth of the eye (M) and wing (N). (O) Quantification of female wing sizes by pixel area, 5 wings per genotype were measured. Error bars show standard deviation.P-RThe eyeless FLP MARCM system was used to generate clonally mutant flyeyes. eyes mutant eyes (Q) overgrow slightly compared to controls (P), while α-spectrin mutant eyes expressing α-spectrin RNAi (R) overgrow strongly compared to controls (P).Data information: Scale bars, 250 μm.


Figure 2A - F The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS . RNAi targeting the Spectrin cytoskeleton . Pupalretinas were examined at 42 - 46 h after puparium formation ( APF ) . Cellmembranes were marked with Dlg staining . ( A ) Control pupalretina showing cone cells surrounded by interommatidial cells . ( B ) kibra mutant pupalretina displaying additional interommatidial cells . ( C ) Pupalretina expressing α - spectrin RNAi showing additional interommatidial cells . ( D ) kibra mutant pupalretinas expressing α - spectrin RNAi showing many additional interommatidial cells . ( E , F ) Clones of α - spectrin mutant cells ( GFP negative ) ( E ) and βH - spectrin / karst mutant cells ( GFP negative ) ( F ) show extra interommatidial cells . Scale bars , 20 μm . G - K UAS . RNAi lines were driven with hh . Gal4 UAS . GFP for expression in the posterior compartment and contained the ex . lacZ reporter transgene . ( G ) Control wing showing a normal ex . lacZ expression pattern , which is low at the dorsal - ventral boundary but high in the proximal regions of the wing disc . ( H ) RNAi knock - down of α - Spectrin results in a mild up - regulation of ex . lacZ in the posterior compartment . ( I ) RNAi knock - down of kibra results in a mild up - regulation of ex . lacZ in the posterior compartment . ( J ) Dual RNAi knock - down of both α - spectrin and kibra results in enhanced up - regulation of ex . lacZ in the posterior compartment . ( K ) RNAi knock - down of hippo results in an up - regulation of ex . lacZ in the posterior compartment . Scale bars , 50 μm .

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

Figure 2A-F The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS.RNAi targeting the Spectrin cytoskeleton. Pupalretinas were examined at 42-46 h after puparium formation (APF). Cellmembranes were marked with Dlg staining. (A) Control pupalretina showing cone cells surrounded by interommatidial cells. (B) kibra mutant pupalretina displaying additional interommatidial cells. (C) Pupalretina expressing α-spectrin RNAi showing additional interommatidial cells. (D) kibra mutant pupalretinas expressing α-spectrin RNAi showing many additional interommatidial cells. (E, F) Clones of α-spectrin mutant cells (GFP negative) (E) and βH-spectrin/karst mutant cells (GFP negative) (F) show extra interommatidial cells. Scale bars, 20 μm.G-K UAS.RNAi lines were driven with hh.Gal4 UAS.GFP for expression in the posterior compartment and contained the ex.lacZ reporter transgene. (G) Control wing showing a normal ex.lacZ expression pattern, which is low at the dorsal-ventral boundary but high in the proximal regions of the wing disc. (H) RNAi knock-down of α-Spectrin results in a mild up-regulation of ex.lacZ in the posterior compartment. (I) RNAi knock-down of kibra results in a mild up-regulation of ex.lacZ in the posterior compartment. (J) Dual RNAi knock-down of both α-spectrin and kibra results in enhanced up-regulation of ex.lacZ in the posterior compartment. (K) RNAi knock-down of hippo results in an up-regulation of ex.lacZ in the posterior compartment. Scale bars, 50 μm.


Figure 3A Co - IP of V5 - tagged Expanded with the FLAG - tagged N - terminal region of Karst / βH - Spectrin , but not other Karst truncation constructs . B , C Expanded co - localises with Karst at the apical membrane of wing imaginal disc cells ( apical view , B ; cross section , C ) . D Control nubbin . Gal4 expressing wing . E Overexpression of Ex activates Hippo signalling to produce a small wing . F RNAi knock - down of α - Spectrin results in an overgrown wing . G Overexpression of Ex blocks the effect of α - Spectrin RNAi . H RNAi knock - down of βH - spectrin / karst results in an overgrown wing . I Overexpression of Ex blocks the effect of βH - spectrin / karst RNAi .

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 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

Figure 3A Co-IP of V5-tagged Expanded with the FLAG-tagged N-terminal region of Karst/βH-Spectrin, but not other Karst truncation constructs.B, C Expanded co-localises with Karst at the apical membrane of wing imaginal disc cells (apical view, B; cross section, C).D Control nubbin.Gal4 expressing wing.E Overexpression of Ex activates Hippo signalling to produce a small wing.F RNAi knock-down of α-Spectrin results in an overgrown wing.G Overexpression of Ex blocks the effect of α-Spectrin RNAi.H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.I Overexpression of Ex blocks the effect of βH-spectrin/karst RNAi.


Figure 4The apical α - βHSpectrincytoskeleton may be mechanosensory in the wing imaginal discA Schematic diagram of central compression and circumferencial stretching in the third instarwing pouch . B - D Third instarwing imaginal disc stained for beta - galactosidase expressed from the expanded . lacZ reporter gene ( B ) , Crb ( C ) and Kst - YFP ( D ) . Quantification of line intensity shown below . Note inverse correlation of Crb and Kst - YFP with ex . lacZ . Scale bars , 50 μm . E , F Zoom of Kst - YFP cells under compression ( E ) and under stretch ( F ) . Scale bars , 10 μm . G Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling .

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

Figure 4The apical α-βHSpectrincytoskeleton may be mechanosensory in the wing imaginal discA Schematic diagram of central compression and circumferencial stretching in the third instarwing pouch.B-D Third instarwing imaginal disc stained for beta-galactosidase expressed from the expanded.lacZ reporter gene (B), Crb (C) and Kst-YFP (D). Quantification of line intensity shown below. Note inverse correlation of Crb and Kst-YFP with ex.lacZ. Scale bars, 50 μm.E, F Zoom of Kst-YFP cells under compression (E) and under stretch (F). Scale bars, 10 μm.G Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling.


Figure 5B Control nub . Gal4 wing . C , D Expression of UAS . CrbExTM - GFP with Gal4 . Gal4 ( C ) or UAS . Wts with nub . Gal4 ( D ) results in a small wing . E RNAi knock - down of α - Spectrin results in an overgrown wing . F , G Expression of UAS . CrbExTM - GFP ( F ) or UAS . Wts ( G ) blocks the effect of α - Spectrin RNAi . H RNAi knock - down of βH - spectrin / karst results in an overgrown wing . I , J Expression of UAS . CrbExTM - GFP ( I ) or UAS . Wts ( J ) blocks the effect of α - Spectrin RNAi . K , L RNAi knock - down of Wts results in a strongly overgrown wing ( K ) and expression of UAS . CrbExTM - GFP does not block the effect of wts RNAi ( L ) . M , N RNAi knock - down of Ajuba results in a small wing ( M ) , and expression of UAS . CrbExTM - GFP enhances the effect of ajuba RNAi ( N ) . O Quantification of various wing sizes . Error bars show standard deviation . P , Q Localisation of Wts - GFP ( P ) and E - cadherin ( Q ) in the third instar wing imaginal disc . R Quantification of line intensity in ( P ) and ( Q ) . No increased Wts - GFP intensity is observed in proximal regions where cells are subject to stretching .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 5B Control nub.Gal4 wing.C, D Expression of UAS.CrbExTM-GFP with Gal4.Gal4 (C) or UAS.Wts with nub.Gal4 (D) results in a small wing.E RNAi knock-down of α-Spectrin results in an overgrown wing.F, G Expression of UAS.CrbExTM-GFP (F) or UAS.Wts (G) blocks the effect of α-Spectrin RNAi.H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.I, J Expression of UAS.CrbExTM-GFP (I) or UAS.Wts (J) blocks the effect of α-Spectrin RNAi.K, L RNAi knock-down of Wts results in a strongly overgrown wing (K) and expression of UAS.CrbExTM-GFP does not block the effect of wts RNAi (L).M, N RNAi knock-down of Ajuba results in a small wing (M), and expression of UAS.CrbExTM-GFP enhances the effect of ajuba RNAi (N).O Quantification of various wing sizes. Error bars show standard deviation.P, Q Localisation of Wts-GFP (P) and E-cadherin (Q) in the third instar wing imaginal disc.R Quantification of line intensity in (P) and (Q). No increased Wts-GFP intensity is observed in proximal regions where cells are subject to stretching.


Figure 6A Control egg chamber stained for DAPI to mark nuclei . B Overexpression of Yki3SA stimulates follicle cell proliferation ( MARCM clone , GFP positive ) . C - F Mutation of βH - spectrin / karst ( C ) or crb ( D ) does not increase follicle cell proliferation ( GFP negative clone ) , while mutation of α - spectrin ( E ) or β - spectrin ( F ) stimulates follicle cell proliferation ( GFP negative clone ) . G Control egg chamber stained for Cut , a marker of proliferating cells that is normally down - regulated after stage 6 of oogenesis . H - K Mutation of βH - spectrin / karst ( H ) or crb ( I ) does not increase Cut expression in follicle cells ( GFP negative clone ) , while mutation of α - spectrin ( J ) or β - spectrin ( K ) stimulates Cut expression in posterior follicle cells ( GFP negative clone ) . L Control egg chamber showing ex . lacZ reporter gene expression . M - P Mutation of βH - spectrin / karst ( M ) or crb ( N ) does not increase ex . lacZ expression in follicle cells ( GFP negative clone ) , while mutation of α - spectrin ( O ) or β - spectrin ( P ) stimulates ex . lacZ expression in posterior follicle cells ( GFP negative clone ) . Q Wild - type follicle cells showing normal hexagonal packing of epithelial membranes . R Mutation of β - spectrin disrupts the normal hexagonal packing , suggesting a role in maintaining membrane tension . S Clone of β - spectrin mutant cells ( GFP negative ) showing differential membrane tension at the interface with wild - type cells ( GFP positive ) .

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

Figure 6A Control egg chamber stained for DAPI to mark nuclei.B Overexpression of Yki3SA stimulates follicle cell proliferation (MARCM clone, GFP positive).C-F Mutation of βH-spectrin/karst (C) or crb (D) does not increase follicle cell proliferation (GFP negative clone), while mutation of α-spectrin (E) or β-spectrin (F) stimulates follicle cell proliferation (GFP negative clone).G Control egg chamber stained for Cut, a marker of proliferating cells that is normally down-regulated after stage 6 of oogenesis.H-K Mutation of βH-spectrin/karst (H) or crb (I) does not increase Cut expression in follicle cells (GFP negative clone), while mutation of α-spectrin (J) or β-spectrin (K) stimulates Cut expression in posterior follicle cells (GFP negative clone).L Control egg chamber showing ex.lacZ reporter gene expression.M-P Mutation of βH-spectrin/karst (M) or crb (N) does not increase ex.lacZ expression in follicle cells (GFP negative clone), while mutation of α-spectrin (O) or β-spectrin (P) stimulates ex.lacZ expression in posterior follicle cells (GFP negative clone).Q Wild-type follicle cells showing normal hexagonal packing of epithelial membranes.R Mutation of β-spectrin disrupts the normal hexagonal packing, suggesting a role in maintaining membrane tension.S Clone of β-spectrin mutant cells (GFP negative) showing differential membrane tension at the interface with wild-type cells (GFP positive).


Figure 7Basolateralα - β Spectrins are required to restrict cellproliferation in theDrosophila intestinal epitheliumA Control myo1A . Gal4UAS . GFP adult midgut stained for phospho - histone H3 to mark mitotic stem cells . B - E RNAi knock - down of βH - spectrin / karst ( B ) or crumbs ( C ) does not increase stem cellproliferation , while RNAi knock - down of β - spectrin ( D ) or α - spectrin ( E ) strongly stimulates stem cellproliferation . F Overexpression of Yki strongly stimulates stem cellproliferation . G Quantification of the number of PH3 - positive mitotic cells . N > 13 samples . Statistical analysis performed using the two - tailed Student ' s t - test . Error bars show standard deviation . * * * P - value ≤ 0 . 001 . H - K A Hippo reporter DIAP1 - HRE - GFP is normally expressed in sporadic stem cells . Its expression is not affected by RNAi knock - down of βH - spectrin / karst ( I ) , but is up - regulated in many stem cells by RNAi knock - down of α - spectrin ( J ) or by overexpression of Yki ( K ) . Data information : Scale bars , 100 μm .

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

Figure 7Basolateralα-β Spectrins are required to restrict cellproliferation in theDrosophila intestinal epitheliumA Control myo1A.Gal4UAS.GFP adult midgut stained for phospho-histone H3 to mark mitotic stem cells.B-E RNAi knock-down of βH-spectrin/karst (B) or crumbs (C) does not increase stem cellproliferation, while RNAi knock-down of β-spectrin (D) or α-spectrin (E) strongly stimulates stem cellproliferation.F Overexpression of Yki strongly stimulates stem cellproliferation.G Quantification of the number of PH3-positive mitotic cells. N > 13 samples. Statistical analysis performed using the two-tailed Student's t-test. Error bars show standard deviation. ***P-value ≤ 0.001.H-K A Hippo reporter DIAP1-HRE-GFP is normally expressed in sporadic stem cells. Its expression is not affected by RNAi knock-down of βH-spectrin/karst (I), but is up-regulated in many stem cells by RNAi knock-down of α-spectrin (J) or by overexpression of Yki (K).Data information: Scale bars, 100 μm.


Figure 8A Control Caco - 2 epithelial cells plated a low density show strong nuclear YAP localisation . B Control Caco - 2 epithelial cells plated at high density show relocalisation of YAP to the cytoplasm . C , D siRNA knock - down of SPTAN1 ( C ) or SPTBN1 ( D ) prevents relocalisation of YAP to the cytoplasm . E Quantification of YAP localisation in ( B - D ) . F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal levels of YAP phosphorylation , and of LATS1 phosphorylation . G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins .

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

Figure 8A Control Caco-2 epithelial cells plated a low density show strong nuclear YAP localisation.B Control Caco-2 epithelial cells plated at high density show relocalisation of YAP to the cytoplasm.C, D siRNA knock-down of SPTAN1 (C) or SPTBN1 (D) prevents relocalisation of YAP to the cytoplasm.E Quantification of YAP localisation in (B-D).F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal levels of YAP phosphorylation, and of LATS1 phosphorylation.G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins.


Figure 1B . Immunoblot of parental wild - type ( WT ) and UPF3B mutant cell lines showing levels of proteins on the right . In 3BΔ2BD , a smaller UPF3B protein with deletion of amino acids 20 - 155 ( UPF3BΔ20 - 155 ) is expressed . Relative Expression ( Rel Exp ) of this deletion protein as compared to the full - length UPF3B along with standard error of mean ( SEM ) are indicated below lane 2 . UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD . HNRNPA1 is used as a loading control . ND = UPF3B levels not determined . C . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms and PTC - isoforms from the same set of genes . X - axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . D . Bar plots from isoform specific RT - qPCR analysis showing average fold change ( y - axis ) of PTC + and PTC - isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right . For each isoform , levels in mutant cells are compared to the levels in WT cells ( set to 1 ) . E . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms and NMD - isoforms . X - axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . F . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms and PTC - isoforms from same set of genes . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown . G . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms and NMD - isoforms . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from Kolmogorov - Smirnov ( KS ) test comparing the two distributions are shown .

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

Figure 1B. Immunoblot of parental wild-type (WT) and UPF3B mutant cell lines showing levels of proteins on the right. In 3BΔ2BD, a smaller UPF3B protein with deletion of amino acids 20-155 (UPF3BΔ20-155) is expressed. Relative Expression (Rel Exp) of this deletion protein as compared to the full-length UPF3B along with standard error of mean (SEM) are indicated below lane 2. UPF3B antibody recognizes antigen outside the deleted region in 3BΔ2BD. HNRNPA1 is used as a loading control. ND = UPF3B levels not determined.C. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from the same set of genes. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.D. Bar plots from isoform specific RT-qPCR analysis showing average fold change (y-axis) of PTC+ and PTC- isoforms from genes indicated on the bottom in WT and the two UPF3B mutant cells identified in the legend on the top right. For each isoform, levels in mutant cells are compared to the levels in WT cells (set to 1).E. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in 3BΔ2BD versus WT cells each with control knockdown (siNC). Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. F. Cumulative Distribution Function (CDF) plots of PTC+ isoforms and PTC- isoforms from same set of genes. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown. G. Cumulative Distribution Function (CDF) plots of NMD+ isoforms and NMD- isoforms. X-axis represents fold change in UPF1 knockdown (siUPF1) versus control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from Kolmogorov-Smirnov (KS) test comparing the two distributions are shown.


Figure 2A . Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates ( IP ) from WT and UPF3B mutant cells expressing endogenously FLAG - tagged UPF1 protein as indicated above each lane . The presence of RNase A during FLAG - IP is indicated above each lane . B . Immunoblots showing levels of proteins ( right ) in input and IP with normal rabbit IgG ( IgG - IP ) or antibody targeting EIF4A3 ( EIF4A3 - IP ) from WT and UPF3B mutant cells . C , D . CDF plots of ( C ) PTC + and PTC - isoforms , ( D ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in WT cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . E , F . CDF plots of ( E ) PTC + and PTC - isoforms , ( F ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . G , H . CDF plots of UPF3B - dependent ( G ) and - independent ( H ) PTC + isoforms and their respective PTC - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . I . CDF plots of UPF3B - dependent and UPF3B - independent NMD + isoforms as compared to NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . J . Bar plot showing average fold change as measured by isoform specific RT - qPCR of PTC + and PTC - isoform from genes indicated on the bottom in WT and two independent clones of 3AKO , 3BΔ2BD , 3BKO , and 3DKO cells . Relative levels from each replicate are shown by white circles . Error bars indicate standard errors of means .

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

Figure 2A. Immunoblots showing levels of proteins on the right in input or FLAG immunoprecipitates (IP) from WT and UPF3B mutant cells expressing endogenously FLAG-tagged UPF1 protein as indicated above each lane. The presence of RNase A during FLAG-IP is indicated above each lane.B. Immunoblots showing levels of proteins (right) in input and IP with normal rabbit IgG (IgG-IP) or antibody targeting EIF4A3 (EIF4A3-IP) from WT and UPF3B mutant cells.C, D. CDF plots of (C) PTC+ and PTC-isoforms, (D) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in WT cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. E, F. CDF plots of (E) PTC+ and PTC-isoforms, (F) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot. G, H. CDF plots of UPF3B-dependent (G) and -independent (H) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.I. CDF plots of UPF3B-dependent and UPF3B-independent NMD+ isoforms as compared to NMD- isoforms. X-axis represents fold change upon UPF3A knockdown (siUPF3A) versus negative control knockdown (siNC) in 3BΔ2BD cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.J. Bar plot showing average fold change as measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and two independent clones of 3AKO, 3BΔ2BD, 3BKO, and 3DKO cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard errors of means.


Figure 3B . Immunoblot showing levels of EJC and UPF proteins ( on the right ) in input or FLAG - IP samples from 3DKO # 2 cells stably expressing different FLAG - tagged proteins indicated above each lane . D . Immunoblot showing EJC / UPF proteins ( on the right ) in input or FLAG - IP samples from 3DKO # 2 cells stably expressing FLAG - tagged proteins given above each lane ( hUPF3B = human UPF3B ; hUPF3B - h3AC = human UPF3B protein with C - terminal domain from human UPF3A ; hUPF3B - m3AC = human UPF3B protein with C - terminal domain from mouse UPF3A ) .

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

Figure 3B. Immunoblot showing levels of EJC and UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing different FLAG-tagged proteins indicated above each lane. D. Immunoblot showing EJC/UPF proteins (on the right) in input or FLAG-IP samples from 3DKO#2 cells stably expressing FLAG-tagged proteins given above each lane (hUPF3B = human UPF3B; hUPF3B-h3AC = human UPF3B protein with C-terminal domain from human UPF3A; hUPF3B-m3AC = human UPF3B protein with C-terminal domain from mouse UPF3A).


Figure 4A . Northern blots showing levels of β - globin reporter mRNAs in wild - type HeLa Tet - off cells and UPF3B knockout HeLa Tet - off cells . β39 is a tetracycline ( Tet ) - inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut - off ( chase ) as indicated above each lane . β - GAP is a stable , constitutively - expressed , longer β - globin mRNA used as transfection control . Proteins overexpressed ( OE ) in each condition are indicated on top and reporter mRNA half - lives ( t1 / 2 ) along with standard errors of means are on the bottom . C . Northern blot showing steady - state levels of β39 NMD reporter and β - GAP control in HeLa Tet - off UPF3B knockout cells upon transient overexpression of wild - type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane . Below each lane , relative fold - change ( Rel FC ) indicates β39 reporter levels ( normalized to β - GAP control ) as compared to the normalized β39 reporter levels in UPF3B expressing cells . D . Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3 - IP from 3DKO # 2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane . Relative IP of FLAG - tagged proteins are quantified against EIF4A3 .

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 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 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

Figure 4A. Northern blots showing levels of β-globin reporter mRNAs in wild-type HeLa Tet-off cells and UPF3B knockout HeLa Tet-off cells. β39 is a tetracycline (Tet)-inducible reporter with a PTC at codon 39 whose levels are shown at different timepoints after transcriptional shut-off (chase) as indicated above each lane. β-GAP is a stable, constitutively-expressed, longer β-globin mRNA used as transfection control. Proteins overexpressed (OE) in each condition are indicated on top and reporter mRNA half-lives (t1/2) along with standard errors of means are on the bottom.C. Northern blot showing steady-state levels of β39 NMD reporter and β-GAP control in HeLa Tet-off UPF3B knockout cells upon transient overexpression of wild-type UPF3 proteins or different UPF3A chimeric proteins indicated above each lane. Below each lane, relative fold-change (Rel FC) indicates β39 reporter levels (normalized to β-GAP control) as compared to the normalized β39 reporter levels in UPF3B expressing cells.D. Immunoblot showing levels of EJC proteins or HNRNPA1 in input or EIF4A3-IP from 3DKO#2 cells stably expressing different UPF3 proteins or EGFP as a control as indicated above each lane. Relative IP of FLAG-tagged proteins are quantified against EIF4A3.


Figure 5A , B . CDF plots of ( A ) PTC + and PTC - isoforms , ( B ) NMD + and NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . D , E . CDF plots of UPF3A / 3B - independent ( UPF3A / 3B - indep . , D ) and - dependent ( UPF3A / 3B - dep . , E ) PTC + isoforms and their respective PTC - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot . F . CDF plots of UPF3A / 3B - dependent ( UPF3A / 3B - dep . ) , UPF3A / 3B - independent ( UPF3A / 3B - indep . ) NMD + isoforms and NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 cells . Number of transcripts in each set ( n ) and p - value from KS test comparing the two distributions are shown on each plot .

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

Figure 5A, B. CDF plots of (A) PTC+ and PTC-isoforms, (B) NMD+ and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.D, E. CDF plots of UPF3A/3B-independent (UPF3A/3B-indep., D) and -dependent (UPF3A/3B-dep., E) PTC+ isoforms and their respective PTC- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.F. CDF plots of UPF3A/3B-dependent (UPF3A/3B-dep.), UPF3A/3B-independent (UPF3A/3B-indep.) NMD+ isoforms and NMD- isoforms. X-axis represents fold change upon UPF1 knockdown (siUPF1) versus negative control knockdown (siNC) in 3DKO#2 cells. Number of transcripts in each set (n) and p-value from KS test comparing the two distributions are shown on each plot.


Figure 6A , B . Western blots showing levels of EJC proteins or HNRNPA1 ( control ) in input , IgG IP or EIF4A3 IP following overexpression ( OE ) of CASC3 wild - type ( WT ) and EJC binding deficient ( HDAA ) mutant proteins in ( A ) HeLa Tet - off cells , and ( B ) 3BKO # 3 HeLa Tet - off cells . Ramps above lanes indicate expression levels of the CASC3 proteins . C . Western blots showing levels of EJC / UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG - tagged endogenous protein indicated above each lane . Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom . D . Meta - exon plot showing read distributions within the 100 nucleotide ( nt ) window from the exon 3 ′ end in RIPiT - Seq replicates of MAGOH : EIF4A3 , UPF3B : EIF4A3 , and CASC3 : EIF4A3 . The black vertical line indicates the - 24 nt position . F . Scatter plot comparing log2 - transformed fold change in occupancy of CASC3 : EIF4A3 EJC as compared to MAGOH : EIF4A3 EJC ( x - axis ) and UPF3B : EIF4A3 EJC compared to MAGOH : EIF4A3 EJC . Each dot represents a gene where gene - level occupancy of each EJC composition was quantified at the canonical position for EJC footprints . Pearson correlation coefficient is shown on the top left . G . Box plots comparing occupancy of a specific EJC composition on mRNA ( indicated on y - axis ; normalized by gene expression from RNA - Seq ) between genes of different NMD efficiency . Up , no , and down stands for upregulation , no obvious change or downregulation of a representative PTC + isoform for each gene ( see material and methods ) that is used to reflect overall NMD efficiency for each gene . In the box plots , central band represents the median , boxes represent the interquartile range ( IQR ) , whiskers indicate maximum ( top ) and minimum ( bottom ) values ( 1 . 5 - times the highest or lowest IQR values , respectively ) , and circles represent the outliers . p - values are from Wilcoxon signed - rank test comparing the two indicated distributions . H . Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy ( normalized by MAGOH : EIF4A3 RIPiT - Seq ) between genes of different NMD efficiency . Up , no , down , central band , boxes , and whiskers are as in G . I . Bar plots showing fold changes measured by isoform specific RT - qPCR of PTC + and PTC - isoform from genes indicated on the bottom in WT and CASC3 - KO HCT116 cells . Relative levels from each replicate are shown by white circles . Error bars indicate standard error of means .

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

Figure 6A, B. Western blots showing levels of EJC proteins or HNRNPA1 (control) in input, IgG IP or EIF4A3 IP following overexpression (OE) of CASC3 wild-type (WT) and EJC binding deficient (HDAA) mutant proteins in (A) HeLa Tet-off cells, and (B) 3BKO#3 HeLa Tet-off cells. Ramps above lanes indicate expression levels of the CASC3 proteins.C. Western blots showing levels of EJC/UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG-tagged endogenous protein indicated above each lane. Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom.D. Meta-exon plot showing read distributions within the 100 nucleotide (nt) window from the exon 3′ end in RIPiT-Seq replicates of MAGOH:EIF4A3, UPF3B:EIF4A3, and CASC3:EIF4A3. The black vertical line indicates the -24 nt position.F. Scatter plot comparing log2-transformed fold change in occupancy of CASC3:EIF4A3 EJC as compared to MAGOH:EIF4A3 EJC (x-axis) and UPF3B:EIF4A3 EJC compared to MAGOH:EIF4A3 EJC. Each dot represents a gene where gene-level occupancy of each EJC composition was quantified at the canonical position for EJC footprints. Pearson correlation coefficient is shown on the top left.G. Box plots comparing occupancy of a specific EJC composition on mRNA (indicated on y-axis; normalized by gene expression from RNA-Seq) between genes of different NMD efficiency. Up, no, and down stands for upregulation, no obvious change or downregulation of a representative PTC+ isoform for each gene (see material and methods) that is used to reflect overall NMD efficiency for each gene. In the box plots, central band represents the median, boxes represent the interquartile range (IQR), whiskers indicate maximum (top) and minimum (bottom) values (1.5-times the highest or lowest IQR values, respectively), and circles represent the outliers. p-values are from Wilcoxon signed-rank test comparing the two indicated distributions.H. Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy (normalized by MAGOH:EIF4A3 RIPiT-Seq) between genes of different NMD efficiency. Up, no, down, central band, boxes, and whiskers are as in G.I. Bar plots showing fold changes measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and CASC3-KO HCT116 cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard error of means.


Figure 1Cell viability analysis using CellTiter - Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs ( n = 4 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . SM represents SM - 164 . Error bars represent SD and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . TNF - induced complex - II immuno - precipitation . BN175 cells were treated with the indicated agents for 8 hrs . FADD immuno - precipitation was performed followed by western blot analysis ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . Proximity ligation assay between Ripk1 and Casp - 8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs ( n = 3 biological replicates , shown are representative images ) . Scale bar represents 10 μm . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . Error bars represent SD and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . Cell viability analysis using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 and Casp - 8 knockouts ( KO ) cells treated with the indicated agents for 18 and 48 hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Cell viability analysis using CellTiter-Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs (n = 4 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). SM represents SM-164. Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.TNF-induced complex-II immuno-precipitation. BN175 cells were treated with the indicated agents for 8 hrs. FADD immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).Proximity ligation assay between Ripk1 and Casp-8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs (n = 3 biological replicates, shown are representative images). Scale bar represents 10 μm. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 knockouts (KO) cells treated with the indicated agents for 18 and 48 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


Figure 2TNF - induced complex - II immuno - precipitation . HT1080 cells were treated with the indicated agents for 8 hrs . CASP - 8 immuno - precipitation was performed followed by western blot analysis ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . Cell death analysis measured by Celigo of PI - positive HT1080 cells , cells were treated with the indicated agents for 24 hrs . ( n = 3 biological replicates ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and an unpaired t - test was performed , * * * * P ≤ 0 . 0001 . Cell death analysis by Celigo of PI - positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed , * * * * P ≤ 0 . 0001 . TNF - induced complex - II immuno - precipitation . A375 cells were treated with the indicated agents for 6 hrs . Caspase - 8 immuno - precipitation was performed followed by western blot analysis . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) ( n = 3 biological replicates , shown is a representative experiment ) . Cell death analysis by Celigo of PI - positive A375 treated with the indicated agents for 12 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . Cell viability analysis using CellTiter - Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL , * * * * P ≤ 0 . 0001 . Cell death analysis by Celigo of PI - positive MeWo ( G ) , DO4 ( H ) or SW - 872 ( I ) cells . Cells were treated with the indicated agents for 24 hrs ( MeWO and SW - 872 ) or 12 hrs ( DO4 ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . Cell death analysis by Celigo of PI - positive HS - ES - 2R Cells were treated with the indicated agents for 48 hrs ( HS - ES - 2R ) DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * P ≤ 0 . 001 . Cell death analysis by Celigo of PI - positive HS - SY - II cells . Cells were treated with the indicated agents for 24 hrs ( HS - SY - II ) . DMSO ( Unt ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * P ≤ 0 . 001 . Cell Death analysis by IncuCyte Zoom of CellTox Green - positive MET1 ( L ) , MET2 ( M ) and IC8 ( N ) cells . Cells were treated with the indicated agents for 40 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 6 , and statistical analysis was performed with a one - way Anova , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2TNF-induced complex-II immuno-precipitation. HT1080 cells were treated with the indicated agents for 8 hrs. CASP-8 immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml).Cell death analysis measured by Celigo of PI-positive HT1080 cells, cells were treated with the indicated agents for 24 hrs. (n = 3 biological replicates). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and an unpaired t-test was performed, **** P ≤ 0.0001.Cell death analysis by Celigo of PI-positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed, **** P ≤ 0.0001.TNF-induced complex-II immuno-precipitation. A375 cells were treated with the indicated agents for 6 hrs. Caspase-8 immuno-precipitation was performed followed by western blot analysis. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) (n = 3 biological replicates, shown is a representative experiment).Cell death analysis by Celigo of PI-positive A375 treated with the indicated agents for 12 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.Cell viability analysis using CellTiter-Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL, **** P ≤ 0.0001.Cell death analysis by Celigo of PI-positive MeWo (G), DO4 (H) or SW-872 (I) cells. Cells were treated with the indicated agents for 24 hrs (MeWO and SW-872) or 12 hrs (DO4). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.Cell death analysis by Celigo of PI-positive HS-ES-2R Cells were treated with the indicated agents for 48 hrs (HS-ES-2R) DMSO (Unt), zVAD (10 µM), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.Cell death analysis by Celigo of PI-positive HS-SY-II cells. Cells were treated with the indicated agents for 24 hrs (HS-SY-II). DMSO (Unt), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.Cell Death analysis by IncuCyte Zoom of CellTox Green-positive MET1 (L), MET2 (M) and IC8 (N) cells. Cells were treated with the indicated agents for 40 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=6, and statistical analysis was performed with a one-way Anova, *** P ≤ 0.001, **** P ≤ 0.0001.


Figure 3Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . The addition of SM to the standard - of - care treatment ILP - TNF / Mel significantly prolonged survival compared to either modality alone ( n = 6 rats per cohort ) . Statistical analysis was performed with a log - rank test , * * P ≤ 0 . 01 . Median TNF / Mel is 0 . 4472 and TNF / Mel / SM is 1 . 154 . TNF / Mel 25th percentile limit 0 . 02887 and 75th percentile limit 0 . 7627 . TNF / Mel / SM 25th percentile limit 1 . 136 and 75th percentile limit 1 . 67 . TNF / Mel Min error bar - 0 . 2389 and Max 0 . 7687 . TNF / SM / Mel Min error bar 1 . 12 and Max 1 . 813 . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * P ≤ 0 . 01 . DEVDase activity assay of parental BN175 cells and drug - tolerant tumour cell lines isolated from ILP - TNF / Mel / SM treated animals ( 124 , 133 , 136 ) , treated with the indicated drugs for 6 hrs ( n = 4 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . Cell viability analysis using CellTiter - Glo of BN175 parental and 124 , 133 and 136 cells treated with the indicated drugs for 24 hrs ( n = 3 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Statistical analysis was performed with a one - way Anova , ns = not significant . Cell viability analysis using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 and Casp - 8 KO cells treated with the indicated agents for 24 hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , SM ( 100 ng / ml ) and Riboxxol ( 1 µg / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

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

Figure 3Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, * P ≤ 0.05, *** P ≤ 0.001.The addition of SM to the standard-of-care treatment ILP-TNF/Mel significantly prolonged survival compared to either modality alone (n = 6 rats per cohort). Statistical analysis was performed with a log-rank test, ** P ≤ 0.01.Median TNF/Mel is 0.4472 and TNF/Mel/SM is 1.154. TNF/Mel 25th percentile limit 0.02887 and 75th percentile limit 0.7627. TNF/Mel/SM 25th percentile limit 1.136 and 75th percentile limit 1.67. TNF/Mel Min error bar -0.2389 and Max 0.7687. TNF/SM/Mel Min error bar 1.12 and Max 1.813. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ** P ≤ 0.01.DEVDase activity assay of parental BN175 cells and drug-tolerant tumour cell lines isolated from ILP-TNF/Mel/SM treated animals (124, 133, 136), treated with the indicated drugs for 6 hrs (n = 4 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml).Cell viability analysis using CellTiter-Glo of BN175 parental and 124, 133 and 136 cells treated with the indicated drugs for 24 hrs (n = 3 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Statistical analysis was performed with a one-way Anova, ns=not significant.Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 KO cells treated with the indicated agents for 24 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), SM (100 ng/ml) and Riboxxol (1 µg/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


Figure 4Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP with TNF / Mel / SM significantly increased infiltration by CD3 + lymphocytes ( B ) and CD3 - CD161 + NK cells ( C ) . Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . While ILP treatment with TNF / Mel significantly reduced Granzyme B positive NK cells ( D ) , the numbers of Granzyme B positive NK cells were restored to normal levels upon co - treatment with SM ( TNF / Mel / SM ) . Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . Importantly , ILP - mediated treatment with TNF / Mel / SM significantly increased the numbers of CD3 + CD8 + cytotoxic T cells ( E ) . Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP - TNF / Mel / SM significantly upregulated Granzyme B in CD3 + CD8 + CD161 - cytoxtoxic T cellsILP - TNF / Mel / SM significantly reduced infiltration by CD3 + CD4 + Foxp3 - T cells whilst ( G ) increasing infiltrations by CD3 + CD4 + Foxp3 + T regulatory cells ( H ) . A non - significant increase in T regulatory cell ICOS expression was also noted ( I ) .

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 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 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

Figure 4Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP with TNF/Mel/SM significantly increased infiltration by CD3+ lymphocytes (B) and CD3-CD161+ NK cells (C).Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). While ILP treatment with TNF/Mel significantly reduced Granzyme B positive NK cells (D), the numbers of Granzyme B positive NK cells were restored to normal levels upon co-treatment with SM (TNF/Mel/SM).Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). Importantly, ILP-mediated treatment with TNF/Mel/SM significantly increased the numbers of CD3+CD8+ cytotoxic T cells (E).Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP-TNF/Mel/SM significantly upregulated Granzyme B in CD3+CD8+CD161- cytoxtoxic T cellsILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H).A non-significant increase in T regulatory cell ICOS expression was also noted (I).


Figure 5Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . The addition of either anti - CTLA - 4 or anti - PD - 1 antibodies prolonged survival in comparison to ILP - TNF / Mel / SM alone ( n = 6 animals per cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 . Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - CTLA - 4 antibodies led to a significant increase in CD4 + Foxp3 - helper T cells ( C ) accompanied by a depletion of CD4 + Foxp3 + regulatory T cells ( D ) . SM represents Birinapant . Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - PD - 1 antibodies led to an increase in Granzyme B ( GzmB ) expression on NK cells ( E ) . SM represents Birinapant .

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 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 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

Figure 5Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD.The addition of either anti-CTLA-4 or anti-PD-1 antibodies prolonged survival in comparison to ILP-TNF/Mel/SM alone (n = 6 animals per cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-CTLA-4 antibodies led to a significant increase in CD4+ Foxp3- helper T cells (C) accompanied by a depletion of CD4+ Foxp3+ regulatory T cells (D). SM represents Birinapant.Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-PD-1 antibodies led to an increase in Granzyme B (GzmB) expression on NK cells (E). SM represents Birinapant.


Figure 6Tumour volumes at the time of surgery following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * * * , P ≤ 0 . 0001 . Individual growth curves following surgery indicated improved control following ILP - TNF / Mel / SM ( n = 6 animals per cohort ) . Tumour volume on re - challenge with BN175 sarcoma on contralateral limb of long - term survivors following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . Unless otherwise stated , SM represents Birinapant . Error bars represent S . D . ILP - TNF / Mel / SM significantly delayed growth leading to prolonged survival on re - challenge ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 .

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

Figure 6Tumour volumes at the time of surgery following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ****, P ≤ 0.0001.Individual growth curves following surgery indicated improved control following ILP-TNF/Mel/SM (n=6 animals per cohort).Tumour volume on re-challenge with BN175 sarcoma on contralateral limb of long-term survivors following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). Unless otherwise stated, SM represents Birinapant. Error bars represent S.D.ILP-TNF/Mel/SM significantly delayed growth leading to prolonged survival on re-challenge (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.