G - K UAS . RNAi lines were driven with hh . Gal4 UAS . GFP for expression in posterior compartment contained ex . lacZ reporter transgene . ( G ) Control wing showing a normal ex . lacZ expression pattern , which is low at dorsal - ventral boundary but high in proximal regions of wing disc . ( H ) RNAi knock - down of α - Spectrin results in a mild up - regulation of ex . lacZ in posterior compartment . ( I ) RNAi knock - down of kibra results in a mild up - regulation of ex . lacZ in posterior compartment . ( J ) Dual RNAi knock - down of both α - spectrin kibra results in enhanced up - regulation of ex . lacZ in posterior compartment . ( K ) RNAi knock - down of hippo results in an up - regulation of ex . lacZ in posterior compartment . Scale bars , 50 μm .

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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.


A Co - IP of V5 - tagged Expanded with FLAG - tagged N - terminal region of Karst / βH - Spectrin , but not other Karst truncation constructs .

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A 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 apical of wing imaginal disc ( apical view , B ; cross section , C ) .

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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 effect of α - Spectrin RNAi . H RNAi knock - down of βH - spectrin / karst results in an overgrown wing . I Overexpression of Ex blocks effect of βH - spectrin / karst RNAi .

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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.


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

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The 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.


B 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 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 effect of α - Spectrin RNAi . K , L RNAi knock - down of Wts results in a strongly overgrown wing ( K ) expression of UAS . CrbExTM - GFP does not block effect of wts RNAi ( L ) . M , N RNAi knock - down of Ajuba results in a small wing ( M ) , expression of UAS . CrbExTM - GFP enhances effect of ajuba RNAi ( N ) . O of various wing sizes . Error bars show standard deviation .

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B 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 ) E - cadherin ( Q ) in third instar wing imaginal disc .

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P, Q Localisation of Wts-GFP (P) and E-cadherin (Q) in the third instar wing imaginal disc.


R of in ( P ) ( Q ) . No increased Wts - GFP is observed in proximal regions where are subject to stretching .

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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.


A Control egg chamber stained for DAPI to mark nuclei . B Overexpression of Yki3SA stimulates follicle proliferation ( MARCM clone , GFP positive ) . C - F Mutation of βH - spectrin / karst ( C ) or crb ( D ) does not increase follicle proliferation ( GFP negative clone ) , while mutation of α - spectrin ( E ) or β - spectrin ( F ) stimulates follicle proliferation ( GFP negative clone ) .

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A 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 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 ( GFP negative clone ) , while mutation of α - spectrin ( J ) or β - spectrin ( K ) stimulates Cut expression in posterior follicle ( 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 ( GFP negative clone ) , while mutation of α - spectrin ( O ) or β - spectrin ( P ) stimulates ex . lacZ expression in posterior follicle ( GFP negative clone ) .

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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 showing normal hexagonal packing of epithelial membranes . R Mutation of β - spectrin disrupts normal hexagonal packing , suggesting a role in maintaining tension . S Clone of β - spectrin mutant ( GFP negative ) showing differential tension at interface with wild - type ( GFP positive ) .

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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).


Basolateralα - 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 . B - E RNAi knock - down of βH - spectrin / karst ( B ) or crumbs ( C ) does not increase cellproliferation , while RNAi knock - down of β - spectrin ( D ) or α - spectrin ( E ) strongly stimulates cellproliferation . F Overexpression of Yki strongly stimulates cellproliferation . G of number of PH3 - positive mitotic . N > 13 samples . Statistical performed using two - Student ' s t - . Error bars show standard deviation . * * * P - value ≤ 0 . 001 . H - K A Hippo reporter DIAP1 - HRE - GFP is normally expressed in sporadic . Its expression is not affected by RNAi knock - down of βH - spectrin / karst ( I ) , but is up - regulated in many by RNAi knock - down of α - spectrin ( J ) or by overexpression of Yki ( K ) . Data information : Scale bars , 100 μm .

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Basolateralα-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.


A Control Caco - 2 epithelial plated a low density show strong YAP localisation . B Control Caco - 2 epithelial plated at high density show relocalisation of YAP to cytoplasm . C , D siRNA knock - down of SPTAN1 ( C ) or SPTBN1 ( D ) prevents relocalisation of YAP to cytoplasm .

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A 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 of YAP phosphorylation , of LATS1 phosphorylation .

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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 .

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G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins.


B . Immunoblot of parental wild - type ( WT ) UPF3B mutant lines showing of on right . In 3BΔ2BD , a smaller UPF3B with deletion of 20 - 155 ( UPF3BΔ20 - 155 ) is expressed . Relative Expression ( Rel Exp ) of this deletion as compared to full - UPF3B along with standard error of mean ( ) are indicated below lane 2 . UPF3B antibody recognizes antigen outside deleted region in 3BΔ2BD . HNRNPA1 is used as a loading control . ND = UPF3B not determined .

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B. 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 PTC - isoforms from same set of genes . X - axis represents fold change in 3BΔ2BD versus WT each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown .

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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 showing average fold change ( y - axis ) of PTC + PTC - isoforms from genes indicated on bottom in WT two UPF3B mutant identified in legend on top right . For each isoform , in mutant are compared to in WT ( set to 1 ) .

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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 NMD - isoforms . X - axis represents fold change in 3BΔ2BD versus WT each with control knockdown ( siNC ) . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown . F . Cumulative Distribution Function ( CDF ) plots of PTC + isoforms PTC - isoforms from same set of genes . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown . G . Cumulative Distribution Function ( CDF ) plots of NMD + isoforms NMD - isoforms . X - axis represents fold change in UPF1 knockdown ( siUPF1 ) versus control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from Kolmogorov - Smirnov ( KS ) comparing two distributions are shown .

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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.


A . Immunoblots showing of on right in input or FLAG immunoprecipitates ( IP ) from WT UPF3B mutant expressing endogenously FLAG - tagged UPF1 as indicated above each lane . presence of RNase A during FLAG - IP is indicated above each lane .

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A. 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 of ( right ) in input IP with normal rabbit IgG ( IgG - IP ) or antibody targeting EIF4A3 ( EIF4A3 - IP ) from WT UPF3B mutant .

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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 + PTC - isoforms , ( D ) NMD + NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in WT . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot . E , F . CDF plots of ( E ) PTC + PTC - isoforms , ( F ) NMD + NMD - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot . G , H . CDF plots of UPF3B - dependent ( G ) - independent ( H ) PTC + isoforms their respective PTC - isoforms . X - axis represents fold change upon UPF3A knockdown ( siUPF3A ) versus negative control knockdown ( siNC ) in 3BΔ2BD . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

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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 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 . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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 + PTC - isoform from genes indicated on bottom in WT two independent clones of 3AKO , 3BΔ2BD , 3BKO , 3DKO . Relative from each replicate are shown by circles . Error bars indicate standard errors of means .

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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.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O

B. 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).


A . Northern blots showing of β - globin reporter mRNAs in wild - type HeLa Tet - off UPF3B knockout HeLa Tet - off . β39 is a tetracycline ( Tet ) - inducible reporter with a PTC at codon 39 whose 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 . overexpressed ( OE ) in each condition are indicated on top reporter mRNA half - lives ( t1 / 2 ) along with standard errors of means are on bottom .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 of β39 NMD reporter β - GAP control in HeLa Tet - off UPF3B knockout upon transient overexpression of wild - type UPF3 or different UPF3A chimeric indicated above each lane . Below each lane , relative fold - change ( Rel FC ) indicates β39 reporter ( normalized to β - GAP control ) as compared to normalized β39 reporter in UPF3B expressing .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O

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 of EJC or HNRNPA1 in input or EIF4A3 - IP from 3DKO # 2 stably expressing different UPF3 or EGFP as a control as indicated above each lane . Relative IP of FLAG - tagged are quantified against EIF4A3 .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

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.


A , B . CDF plots of ( A ) PTC + PTC - isoforms , ( B ) NMD + NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 NMD - isoforms . X - axis represents fold change upon UPF1 knockdown ( siUPF1 ) versus negative control knockdown ( siNC ) in 3DKO # 2 . Number of transcripts in each set ( n ) p - value from KS comparing two distributions are shown on each plot .

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A , B . Western blots showing of EJC or HNRNPA1 ( control ) in input , IgG IP or EIF4A3 IP following overexpression ( OE ) of CASC3 wild - type ( WT ) EJC binding deficient ( HDAA ) mutant in ( A ) HeLa Tet - off , ( B ) 3BKO # 3 HeLa Tet - off . Ramps above lanes indicate expression of CASC3 .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O

A, 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 of EJC / UPF HNRNPA1 in input FLAG followed by EIF4A3 tandem IP from HCT116 expressing FLAG - tagged endogenous indicated above each lane . Quantifications of UPF3B CASC3 enrichment from two replicates are shown at bottom .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

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 100 nucleotide ( nt ) window from exon 3 ′ end in RIPiT - Seq replicates of MAGOH : EIF4A3 , UPF3B : EIF4A3 , CASC3 : EIF4A3 . black vertical indicates - 24 nt position .

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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 ) UPF3B : EIF4A3 EJC compared to MAGOH : EIF4A3 EJC . Each dot represents a gene where gene - level occupancy of each EJC composition was quantified at canonical position for EJC footprints . Pearson correlation coefficient is shown on top left .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 - Seq ) between genes of different NMD efficiency . Up , no , down stands for upregulation , no obvious change or downregulation of a representative PTC + isoform for each gene ( see material methods ) that is used to reflect overall NMD efficiency for each gene . In box plots , central band represents median , boxes represent interquartile range ( IQR ) , whiskers indicate maximum ( top ) minimum ( bottom ) values ( 1 . 5 - times highest or lowest IQR values , respectively ) , circles represent outliers . p - values are from Wilcoxon signed - rank comparing two indicated distributions .

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

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 occupancy ( normalized by MAGOH : EIF4A3 RIPiT - Seq ) between genes of different NMD efficiency . Up , no , down , central band , boxes , whiskers are as in G .

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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 + PTC - isoform from genes indicated on bottom in WT CASC3 - KO HCT116 . Relative from each replicate are shown by circles . Error bars indicate standard error of means .

O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

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.


viability using CellTiter - Glo of BN175 treated with indicated agents for 24 hrs 48 hrs ( n = 4 biological replicates ) . viability values are displayed as a of relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) . SM represents SM - 164 . Error bars represent SD statistical was performed with an unpaired t - , * * * * P ≤ 0 . 0001 .

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Cell 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 - II immuno - precipitation . BN175 were treated with indicated agents for 8 hrs . FADD immuno - precipitation was performed followed by western blot ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) .

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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 between Ripk1 Casp - 8 performed in BN175 upon treatment with indicated agents for 8 hrs ( n = 3 biological replicates , shown are representative ) . Scale bar represents 10 μm . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

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 of BN175 treated with indicated drugs for 6 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) Mel ( 3 . 3 µM ) . Error bars represent SD statistical was performed with an unpaired t - , * * * * P ≤ 0 . 0001 .

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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.


viability using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 Casp - 8 knockouts ( KO ) treated with indicated agents for 18 48 hrs ( n = 3 biological replicates ) . viability values are displayed as a of relative untreated control . SM represents SM - 164 . Error bars represent SD a one - way Anova was performed to compare mean value of each treatment to treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


TNF - induced - II immuno - precipitation . HT1080 were treated with indicated agents for 8 hrs . CASP - 8 immuno - precipitation was performed followed by western blot ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O

TNF-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).


measured by Celigo of PI - positive HT1080 , were treated with indicated agents for 24 hrs . ( n = 3 biological replicates ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD an unpaired t - was performed , * * * * P ≤ 0 . 0001 .

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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.


by Celigo of PI - positive HT1080 treated with 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 ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD a one - way Anova was performed , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 - II immuno - precipitation . A375 were treated with indicated agents for 6 hrs . Caspase - 8 immuno - precipitation was performed followed by western blot . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) ( n = 3 biological replicates , shown is a representative experiment ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


by Celigo of PI - positive A375 treated with indicated agents for 12 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , statistical was performed with an unpaired t - , * * * * P ≤ 0 . 0001 .

O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


viability using CellTiter - Glo of A375 treated with indicated drugs for 24 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48hrs ( n = 3 biological replicates ) . viability values are displayed as a of relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD a one - way Anova was performed to compare mean value of each treatment to treated A375 siCTRL , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


by Celigo of PI - positive MeWo ( G ) , DO4 ( H ) or SW - 872 ( I ) . were treated with indicated agents for 24 hrs ( MeWO SW - 872 ) or 12 hrs ( DO4 ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , statistical was performed with an unpaired t - , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


by Celigo of PI - positive HS - ES - 2R were treated with indicated agents for 48 hrs ( HS - ES - 2R ) DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 100 ng / ml ) SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , statistical was performed with an unpaired t - , * * * P ≤ 0 . 001 .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


by Celigo of PI - positive HS - SY - II . were treated with indicated agents for 24 hrs ( HS - SY - II ) . DMSO ( Unt ) , TNF ( 100 ng / ml ) SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , statistical was performed with an unpaired t - , * * * P ≤ 0 . 001 .

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


by IncuCyte Zoom of CellTox Green - positive MET1 ( L ) , MET2 ( M ) IC8 ( N ) . were treated with indicated agents for 40 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 6 , statistical was performed with a one - way Anova , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Measurements of tumour volume of respective cohorts ( n = 6 per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical was performed with an unpaired t - , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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

Measurements 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.


addition of SM to standard - of - care treatment ILP - TNF / Mel significantly prolonged survival compared to either modality alone ( n = 6 rats per cohort ) . Statistical was performed with a log - rank , * * P ≤ 0 . 01 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 TNF / Mel / SM is 1 . 154 . TNF / Mel 25th percentile limit 0 . 02887 75th percentile limit 0 . 7627 . TNF / Mel / SM 25th percentile limit 1 . 136 75th percentile limit 1 . 67 . TNF / Mel Min error bar - 0 . 2389 Max 0 . 7687 . TNF / SM / Mel Min error bar 1 . 12 Max 1 . 813 . Error bars represent SD . Statistical was performed with an unpaired t - , * * P ≤ 0 . 01 .

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

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 of parental BN175 drug - tolerant tumour lines isolated from ILP - TNF / Mel / SM treated ( 124 , 133 , 136 ) , treated with indicated drugs for 6 hrs ( n = 4 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O

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).


viability using CellTiter - Glo of BN175 parental 124 , 133 136 treated with indicated drugs for 24 hrs ( n = 3 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Statistical was performed with a one - way Anova , ns = not significant .

O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


viability using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 Casp - 8 KO treated with indicated agents for 24 hrs ( n = 3 biological replicates ) . viability values are displayed as a of relative untreated control . DMSO ( Unt ) , SM ( 100 ng / ml ) Riboxxol ( 1 µg / ml ) . SM represents SM - 164 . Error bars represent SD a one - way Anova was performed to compare mean value of each treatment to treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Flow cytometry of tumours collected on Day 10 following treatment ( n = 3 per group ) . ILP with TNF / Mel / SM significantly increased infiltration by CD3 + lymphocytes ( B ) CD3 - CD161 + NK ( C ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

Flow 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 of tumours collected on Day 10 following treatment ( n = 3 per group ) . While ILP treatment with TNF / Mel significantly reduced Granzyme B positive NK ( D ) , numbers of Granzyme B positive NK were restored to normal upon co - treatment with SM ( TNF / Mel / SM ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

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 of tumours collected on Day 10 following treatment ( n = 3 per group ) . Importantly , ILP - mediated treatment with TNF / Mel / SM significantly increased numbers of CD3 + CD8 + cytotoxic T ( E ) .

O 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-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O

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 of tumours collected on Day 10 following treatment ( n = 3 per group ) . ILP - TNF / Mel / SM significantly upregulated Granzyme B in CD3 + CD8 + CD161 - cytoxtoxic T

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

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 cells


ILP - TNF / Mel / SM significantly reduced infiltration by CD3 + CD4 + Foxp3 - T whilst ( G ) increasing infiltrations by CD3 + CD4 + Foxp3 + T regulatory ( H ) .

O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O

ILP-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 ICOS expression was also noted ( I ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O

A non-significant increase in T regulatory cell ICOS expression was also noted (I).


Measurements of tumour volume of respective cohorts ( n = 6 per cohort ) . SM represents Birinapant . Error bars represent SD .

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

Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD.


addition of either anti - CTLA - 4 or anti - PD - 1 antibodies prolonged survival in comparison to ILP - TNF / Mel / SM alone ( n = 6 per cohort ) . SM represents Birinapant . Statistical was performed with a log - rank , * P ≤ 0 . 05 .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ILP procedure analysed by flow cytometry ( n = 3 per cohort ) . addition of anti - CTLA - 4 antibodies led to a significant increase in CD4 + Foxp3 - helper T ( C ) accompanied by a depletion of CD4 + Foxp3 + regulatory T ( D ) . SM represents Birinapant .

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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O

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 ILP procedure analysed by flow cytometry ( n = 3 per cohort ) . addition of anti - PD - 1 antibodies led to an increase in Granzyme B ( GzmB ) expression on NK ( E ) . SM represents Birinapant .

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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O

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.


Tumour volumes at time of surgery following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 6 per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical was performed with an unpaired t - , * * * * , P ≤ 0 . 0001 .

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

Tumour 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.


growth curves following surgery indicated improved control following ILP - TNF / Mel / SM ( n = 6 per cohort ) .

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

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 limb of long - term survivors following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 3 per TNF / Mel cohort n = 5 per TNF / Mel / SM cohort ) . Unless otherwise stated , SM represents Birinapant . Error bars represent S . D .

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

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 per TNF / Mel cohort n = 5 per TNF / Mel / SM cohort ) . SM represents Birinapant . Statistical was performed with a log - rank , * P ≤ 0 . 05 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Impaired wound closure in αSMA - TK mice . ( A ) for αSMA + in wound tissue sections at day 6 post - wounding . Representative are shown . Scale bar , 50 µm . Black boxes denote zoomed regions displayed in bottom panels . ( B ) of αSMA + at day 6 post - wounding . WT , N = 6 ; TK , N = 5 biological replicates . Two - non - parametric Mann - Whitney performed .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Impaired wound closure in αSMA-TK mice. (A) IHC for αSMA+ cells in wound tissue sections at day 6 post-wounding. Representative images are shown. Scale bar, 50 µm. Black boxes denote zoomed regions displayed in bottom panels. (B) Quantification of αSMA+ cells at day 6 post-wounding. WT, N=6; TK, N=5 biological replicates. Two-tailed non-parametric Mann-Whitney test performed.


( F ) Expression of ACTA2 in normal diabetic human wound tissue . - seq reads are derived from ( Davis et al . , 2020 , Data ref : Davis et al , 2020 ) . Normal , N = 3 ; diabetic , N = 4 biological replicates . Data are presented as a box whisker plot of min to max values . Unpaired t - performed .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Expression levels of ACTA2 in normal and diabetic human wound tissue. RNA-seq reads are derived from (Davis et al., 2020, Data ref: Davis et al, 2020). Normal, N=3; diabetic, N=4 biological replicates. Data are presented as a box and whisker plot of min to max values. Unpaired t-test performed.


( J - O ) Impaired granulation tissue formation angiogenesis in αSMA - TK mice . ( J ) Representative H & E of wound tissue sections at day 17 post - wounding . Granulation areas are marked by black lines , triangles ( open : incomplete closure , filled : closed wounds ) denote epithelial tongues . Epi , epidermis ; derm , dermis regions denoted . Scale bar : 200 μm . ( K ) Granulation tissue thickness measured on digital normalized to average values in WT control ( set to 100 % ) . NA ( not assessed ) reflects lack of granulation in αSMA - TK mice . WT , N = 5 ; TK , N = 4 biological replicates . ( L ) Immunofluorescent for CD31 ( red ) nuclei ( blue ) on wound tissue sections at day 17 post - wounding . boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( M ) of CD31 + per visual field normalized to WT mice . WT , N = 3 ; TK , N = 5 biological replicates . ( N ) for hypoxyprobe ( brown ) on wound tissue sections at day 17 post - wounding . Black boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( O ) of hypoxyprobe per visual field normalized to WT mice . WT , N = 5 ; TK , N = 6 biological replicates .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O

(J-O) Impaired granulation tissue formation and angiogenesis in αSMA-TK mice. (J) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines, triangles (open: incomplete closure, filled: closed wounds) denote the epithelial tongues. Epi, epidermis; derm, dermis regions denoted. Scale bar: 200 μm. (K) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control (set to 100%). NA (not assessed) reflects the lack of granulation in αSMA-TK mice. WT, N=5; TK, N=4 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (M) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=3; TK, N=5 biological replicates. (N) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=5; TK, N=6 biological replicates.


( D ) Representative FACS plots of digested skin from bone marrow ( BM ) transplanted αSMA - RFP mice 17 days post - wounding . ( E ) FACS of αSMA - RFP + in wounds of BM transplanted mice . αSMA - RFP with αSMA - RFP BM , N = 2 ; WT with αSMA - RFP BM , N = 3 ; αSMA - RFP with WT BM , N = 4 biological replicates . One - way ANOVA with Tukey ' s multiple comparison performed comparing WT with αSMA - RFP BM αSMA - RFP with WT BM to αSMA - RFP with αSMA - RFP BM . (

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O

(D) Representative FACS plots of digested skin from bone marrow (BM) transplanted αSMA-RFP mice 17 days post-wounding. (E) FACS quantification of αSMA-RFP+ cells in wounds of BM transplanted mice. αSMA-RFP with αSMA-RFP BM, N=2; WT with αSMA -RFP BM, N=3; αSMA-RFP with WT BM, N=4 biological replicates. One-way ANOVA with Tukey's multiple comparison test performed comparing WT with αSMA-RFP BM and αSMA-RFP with WT BM to αSMA-RFP with αSMA-RFP BM. (


( H - J ) Multispectral imaging of changes in fibroblast subsets during wound repair . ( H ) Representative ( 20x magnification ) of orthogonal wound tissue sections at day 6 post - wounding displaying αSMA , FAP , FSP1 overlaid with DAPI in wound bed . Subcutaneous ( subq ) , dermis ( derm ) , epidermis ( epi ) , scar regions are denoted . αSMA , FAP , FSP1 channel overlaid with DAPI . Scale bar , 50 µm . ( I ) of multiplex immunostaining of full thickness skin wounds performed at indicated time points . Data are presented as fractional of each image positive for indicated markers negative for CD31 . Day 0 , N = 2 ; Day 6 , N = 4 ; Day 9 , N = 3 ; Day 14 , N = 3 ; Day 21 , N = 3 ; Day 32 , N = 4 biological replicates . One - way ANOVA with Dunnett ' s multiple comparison performed . ( J ) overlap of αSMA with FAP FSP1 throughout wound healing . αSMA + denotes positive for αSMA but not FAP or FSP1 . Day 0 , N = 2 ; Day 6 , N = 4 ; Day 9 , N = 3 ; Day 14 , N = 3 ; Day 21 , N = 3 ; Day 32 , N = 4 biological replicates .

O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O 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-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-J) Multispectral imaging of changes in fibroblast subsets during wound repair. (H) Representative images (20x magnification) of orthogonal wound tissue sections at day 6 post-wounding displaying αSMA, FAP, and FSP1 overlaid with DAPI in the wound bed. Subcutaneous (subq), dermis (derm), epidermis (epi), and scar regions are denoted. Individual αSMA, FAP, and FSP1 channel images overlaid with DAPI. Scale bar, 50 µm. (I) Analysis of multiplex immunostaining of full thickness skin wounds performed at indicated time points. Data are presented as the fractional area of each image positive for the indicated markers and negative for CD31. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates. One-way ANOVA with Dunnett's multiple comparison test performed. (J) Percent overlap of αSMA with FAP and FSP1 throughout wound healing. αSMA+ denotes cells positive for αSMA but not FAP or FSP1. Day 0, N=2; Day 6, N=4; Day 9, N=3; Day 14, N=3; Day 21, N=3; Day 32, N=4 biological replicates.


( L ) Representative H & E of wound tissue sections at day 17 post - wounding . Granulation areas are marked by black lines . Scale bar , 200 μm . ( M ) Granulation tissue thickness measured on digital normalized to average values in WT control . WT , N = 3 ; FAP - TK , N = 3 ; WT , N = 17 , FSP1 - TK , N = 9 biological replicates . ( N ) Immunofluorescent for CD31 ( red ) nuclei on wound tissue sections at day 17 post - wounding . boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( O ) of CD31 + per visual field normalized to WT mice . WT , N = 4 ; FAP - TK , N = 4 ; WT , N = 3 ; FSP1 - TK , N = 3 biological replicates . ( P ) for hypoxyprobe ( brown ) on wound tissue sections at day 17 post - wounding . Black boxes denote zoomed regions depicted in right panels . Scale bar , 100 μm . ( Q ) of hypoxyprobe per visual field normalized to WT mice . WT , N = 3 ; FAP - TK , N = 3 ; WT , N = 3 ; FSP1 - TK , N = 4 biological replicates . ( R )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

(L) Representative H&E images of wound tissue sections at day 17 post-wounding. Granulation areas are marked by black lines. Scale bar, 200 μm. (M) Granulation tissue thickness measured on digital images and normalized to the average values in the WT control. WT, N=3; FAP-TK, N=3; WT, N=17, FSP1-TK, N=9 biological replicates. (N) Immunofluorescent staining for CD31 (red) and nuclei on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (O) Quantification of CD31+ cells per visual field normalized to WT mice. WT, N=4; FAP-TK, N=4; WT, N=3; FSP1-TK, N=3 biological replicates. (P) IHC for hypoxyprobe (brown) on wound tissue sections at day 17 post-wounding. Black boxes denote zoomed regions depicted in right panels. Scale bar, 100 μm. (Q) Quantification of hypoxyprobe area per visual field normalized to WT mice. WT, N=3; FAP-TK, N=3; WT, N=3; FSP1-TK, N=4 biological replicates. (R) W


( C ) Enrichment plot of genes in αSMA C1 ( left panel ) . Violin plots for genes enriched in αSMA C1 ( right panels ) . ( D ) Enrichment plot of genes in αSMA C2 ( left panel ) . Violin plots for genes enriched in αSMA C2 ( right panels ) . ( E ) Enrichment plot of genes in αSMA C3 ( left panel ) . Violin plots of enriched genes in αSMA C3 cluster ( right panels ) . Raw scRNA - seq data are derived from ( Haensel et al . , 2020 , Data ref : Haensel et al 2020 ) .

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

(C) Enrichment plot of genes in αSMA C1 (left panel). Violin plots for genes enriched in αSMA C1 (right panels). (D) Enrichment plot of genes in αSMA C2 (left panel). Violin plots for genes enriched in αSMA C2 (right panels). (E) Enrichment plot of genes in αSMA C3 (left panel). Violin plots of enriched genes in the αSMA C3 cluster (right panels). Raw scRNA-seq data are derived from (Haensel et al., 2020, Data ref: Haensel et al 2020). Da


( A ) for collagen I on wound tissue sections at day 17 post - wounding ( brown stain ) . Black boxes denote zoomed regions depicted in bottom panels . Scale bar : 50 μm . ( B ) of collagen I positive normalized to average in WT controls . WT , N = 5 ; TK , N = 5 biological replicates . ( C ) Polarized of picrosirius red stained wound tissues at day 17 post - wounding . Red : collagen . boxes denote zoomed regions depicted in bottom panels . Scale bar , 50 µm . ( D ) of collagen fiber ( picrosirius red ) normalized to average in WT controls . WT , N = 3 ; TK , N = 3 biological replicates . ( E -

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

(A) IHC for collagen I on wound tissue sections at day 17 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 50 μm. (B) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=5; TK, N=5 biological replicates. (C) Polarized light images of picrosirius red stained wound tissues at day 17 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (D) Quantification of collagen fiber (picrosirius red) percent area normalized to the average area in WT controls. WT, N=3; TK, N=3 biological replicates. (E-


( E - H ) Reduced collagen deposition in Col1α1cKO wounds . ( E ) for collagen I on wound tissue sections at day 14 post - wounding ( brown stain ) . Black boxes denote zoomed regions depicted in bottom panels . Scale bar : 25 μm . ( F ) of collagen I positive normalized to average in WT controls . WT , N = 7 ; Col1α1cKO , N = 4 biological replicates . ( G ) Polarized of picrosirius red stained wound tissues at day 14 post - wounding . Red : collagen . boxes denote zoomed regions depicted in bottom panels . Scale bar , 50 µm . ( H ) of collagen fiber fiber width normalized to average width , respectively , in WT controls . WT , N = 4 ; Col1α1cKO , N = 5 biological replicates .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O

(E-H) Reduced collagen deposition in Col1α1cKO wounds. (E) IHC for collagen I on wound tissue sections at day 14 post-wounding (brown stain). Black boxes denote zoomed regions depicted in bottom panels. Scale bar: 25 μm. (F) Quantification of collagen I positive area normalized to the average area in WT controls. WT, N=7; Col1α1cKO, N=4 biological replicates. (G) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in bottom panels. Scale bar, 50 µm. (H) Quantification of collagen fiber percent area and fiber width normalized to the average area and width, respectively, in WT controls. WT, N=4; Col1α1cKO, N=5 biological replicates.


( J - M ) Reepithelization vascularization in WT Col1α1cKO mice . ( J ) Representative of keratin 5 ( Krt5 , red ) stained wounds at day 14 post - wounding . boxes denote zoomed regions depicted in right panels . Scale bar , 500 µm . ( K ) of epidermal thickness . WT , N = 6 ; Col1α1cKO , N = 5 biological replicates . ( L ) Immunofluorescent for CD31 ( red ) nuclei ( blue ) on wound tissue sections at day 14 post - wounding . boxes denote zoomed regions depicted in right panels . Scale bar , 20 μm . ( M ) of CD31 + per visual field normalized to WT mice . WT , N = 6 ; Col1α1cKO , N = 5 biological replicates .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(J-M) Reepithelization and vascularization in WT and Col1α1cKO mice. (J) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (K) Quantification of epidermal thickness. WT, N=6; Col1α1cKO, N=5 biological replicates. (L) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (M) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Col1α1cKO, N=5 biological replicates.


( C ) IF for β1 integrin ( red ) αSMA ( green ) on wound tissue sections at day 17 post - wounding . boxes denote zoomed regions depicted in right panels . Scale bar : 20 μm .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) IF for β1 integrin (red) and αSMA (green) on wound tissue sections at day 17 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar: 20 μm.


( F - K ) Reepithelization , granulation tissue formation , vascularization in WT Itgb1cKO mice . ( F ) Representative of keratin 5 ( Krt5 , red ) stained wounds at day 14 post - wounding . boxes denote zoomed regions depicted in right panels . Scale bar , 500 µm . ( G ) of epidermal thickness . WT , N = 4 ; Itgb1cKO , N = 6 biological replicates . ( H ) Immunofluorescent for CD31 ( red ) nuclei ( blue ) on wound tissue sections at day 14 post - wounding . boxes denote zoomed regions depicted in right panels . Scale bar , 20 μm . ( I ) of CD31 + per visual field normalized to WT mice . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates . ( J ) Polarized of picrosirius red stained wound tissues at day 14 post - wounding . Red : collagen . boxes denote zoomed regions depicted in right panels . Scale bar , 50 µm . ( K ) of collagen fiber normalized to average in WT controls . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates . ( L ) of collagen fiber width normalized to average width in WT controls . WT , N = 6 ; Itgb1cKO , N = 6 biological replicates .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR O O O O O O O

(F-K) Reepithelization, granulation tissue formation, and vascularization in WT and Itgb1cKO mice. (F) Representative images of keratin 5 (Krt5, red) stained wounds at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 500 µm. (G) Quantification of epidermal thickness. WT, N=4; Itgb1cKO, N=6 biological replicates. (H) Immunofluorescent staining for CD31 (red) and nuclei (blue) on wound tissue sections at day 14 post-wounding. White boxes denote zoomed regions depicted in right panels. Scale bar, 20 μm. (I) Quantification of CD31+ area per visual field normalized to WT mice. WT, N=6; Itgb1cKO, N=6 biological replicates. (J) Polarized light images of picrosirius red stained wound tissues at day 14 post-wounding. Red: collagen fibers. White boxes denote zoomed regions depicted in right panels. Scale bar, 50 µm. (K) Quantification of collagen fiber percent area normalized to the average area in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. (L) Quantification of collagen fiber width normalized to the average width in WT controls. WT, N=6; Itgb1cKO, N=6 biological replicates. D


Neurosphere assays were performed using mouse E14 . 5 neural progenitors transduced with a retroviral vector expressing

Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1


XTT assays were performed using mouse E14 . 5 neural progenitors transduced with a retroviral vector expressing

XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a retroviral vector expressing Ttyh1


Neurosphere assays were performed using mouse E14 . 5 neural progenitors transduced with a short hairpin specific to Ttyh1 ( shTtyh1 )

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

Neurosphere assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)


XTT assays were performed using mouse E14 . 5 neural progenitors transduced with a short hairpin specific to Ttyh1 ( shTtyh1 )

XTT assays were performed using mouse E14.5 primary neural progenitors transduced with a short hairpin RNA specific to Ttyh1 (shTtyh1)


Ttyh1 - expressing in E15 . 5 embryonic brains that were intraventricularly electroporated at E13 . 5 were immunostained using an anti - GFP ( reporter gene ) Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained ( blue ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . of ( E )

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ttyh1-expressing cells in E15.5 embryonic brains that were intraventricularly electroporated at E13.5 were immunostained using an anti-GFP primary (reporter gene) and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. Quantification of (E)


Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) Sox2 ( red ) antibodies , Alexa Fluor 488 - 555 - conjugated secondary antibodies . Scale bar : 50 μm .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm.


Double - immunolabeling of E15 . 5 brain sections electroporated in utero with Ttyh1 - expressing plasmid at E13 . 5 using anti - GFP ( green ) Sox2 ( red ) antibodies , Alexa Fluor 488 - 555 - conjugated secondary antibodies . Scale bar : 50 μm . of ( G ) ( n = 3 ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Double-immunolabeling of E15.5 brain sections electroporated in utero with Ttyh1-expressing plasmid at E13.5 using anti-GFP (green) and Sox2 (red) antibodies, and Alexa Fluor 488- and 555-conjugated secondary antibodies. Scale bar: 50 μm. Quantification of (G) (n = 3).


Immunolabeling of E14 . 5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1 - resistant Ttyh1 ( Ttyh1res ) expression vector at E13 . 5 using anti - GFP Alexa Fluor 488 - conjugated secondary antibodies . DAPI counterstain is shown in blue . Scale bar : 100 μm . of ( I )

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunolabeling of E14.5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1-resistant Ttyh1 (Ttyh1res) expression vector at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. The DAPI nuclear counterstain is shown in blue. Scale bar: 100 μm. Quantification of (I)


Two days after E14 . 5 neural progenitor were transduced with retroviral vectors expressing ( A ) Ttyh1 mRNA expression of each indicated Notch target gene were measured by

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

Two days after E14.5 primary neural progenitor cells were transduced with retroviral vectors expressing (A) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by qPCR


Two days after E14 . 5 neural progenitor were transduced with shTtyh1 , mRNA expression of each indicated Notch target gene were measured by

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

Two days after E14.5 primary neural progenitor cells were transduced with shTtyh1, mRNA expression levels of each indicated Notch target gene were measured by qPCR


Effects of dominant negative MAML1 ( dnMAML1 ) on Ttyh1 - enhanced Notch target gene expression were tested by qPCR using E14 . 5 neural progenitors at 2 days posttransduction ( n = 4 ) .

O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Effects of dominant negative MAML1 (dnMAML1) on Ttyh1-enhanced Notch target gene expression were tested by qPCR using E14.5 primary neural progenitors at 2 days posttransduction (n = 4).


dnMAML1 plasmid was introduced with Ttyh1 vector into embryonic brains at E13 . 5 localization was analyzed at E15 . 5 by anti - GFP immunostaining . GFP DAPI signals are shown in green blue , respectively . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . E of GFP + positions in ( D ) ( n = 3 ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. E Quantification of GFP+ cell positions in (D) (n = 3).


NICD plasmid was electroporated with Ttyh1 knockdown vector ( shTtyh1 ) into embryonic brains at E13 . 5 localization was analyzed at E14 . 5 by anti - GFP immunostaining . Scale bar : 100 μm . G of GFP + positions in ( F ) ( n = 3 ) .

B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The NICD plasmid was electroporated with the Ttyh1 knockdown vector (shTtyh1) into embryonic brains at E13.5 and cell localization was analyzed at E14.5 by anti-GFP immunostaining. Scale bar: 100 μm. G Quantification of GFP+ cell positions in (F) (n = 3).


Effects of Ttyh1 expression on generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor were analyzed by Western blotting using an anti - NICD antibody . of band

O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O

Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. Quantification of band intensities


Effects of Ttyh1 knockdown ( C ) on generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor were analyzed by Western blotting using an anti - NICD antibody . ( B , D ) of band

O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

Effects of Ttyh1 knockdown (C) on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. (B, D) Quantification of band intensities


Ttyh1 - transduced E14 . 5 neural progenitors were incubated with DAPT for 2 days , then were harvested lysates were used for luciferase ( n = 3 ) .

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ttyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3).


Effects of Ttyh1 - knockdown on γ - secretase activity was also assessed using E14 . 5 neural progenitor

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Effects of Ttyh1-knockdown on γ-secretase activity was also assessed using E14.5 neural progenitor cells


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in presence or absence of DAPT , samples were prepared for Western blotting using anti - NICD

B-CONTROLLED_VAR O O O O 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-MEASURED_VAR

Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibody


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in presence or absence of DAPT , samples were prepared for qPCR for Notch target genes ( I )

B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for qPCR for Notch target genes (I)


indicated plasmids were electroporated into E13 . 5 brains , brain samples were harvested at E15 . 5 for anti - GFP immunolabeling . GFP DAPI signals are shown in green blue , respectively . of GFP + localization in ( J ) dnPS1 , dominant negative presenilin 1 ; VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. Quantification of GFP+ cell localization in (J) dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


Effects of ion selectivity - perturbing R371Q mutation in Ttyh1 E14 . 5 neural progenitor were transduced with retroviral vectors expressing each indicated gene , 2 days posttransduction , were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effects of the ion selectivity-perturbing R371Q mutation in Ttyh1 E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).


Effects of ion selectivity - perturbing R371Q mutation in other Ttyh family members on Notch - dependent transcription . E14 . 5 neural progenitor were transduced with retroviral vectors expressing each indicated gene , 2 days posttransduction , were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

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

Effects of the ion selectivity-perturbing R371Q mutation in other Ttyh family members on Notch-dependent transcription. E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).