Figure 4Correlation of gene expression changes upon constitutive transduction of A - 431 cells with either shRNA targeting USP28 ( sh - USP28 # 1 ) or ∆ Np63 ( sh - ∆ Np63 # 2 ) relative to non - targeting control ( sh - NTC ) . The diagonal line reflects a regression build on a linear model . R : Pearsons correlation coefficient , m : slope of the linear regression modelGene set enrichment analysis ( GSEA ) of a gene set of significantly down - regulated genes in sh - ∆ Np63 # 2 transfected A - 431 cells ( " Down - regulated sh - ∆ NP63 " , Appendix Table S1 ) . The gene set was analysed in sh - ∆ Np63 # 2 ( left ) and sh - USP28 # 1 - depleted ( right ) A - 431 cells . ( N ) ES : ( normalised ) enrichment scoreGene set enrichment analysis ( GSEA ) of a gene set of significantly down - regulated genes in sh - USP28 # 1 transfected A - 431 cells ( " Down - regulated sh - USP28 " , Appendix Table S1 ) . The gene set was analysed in shUSP28 # 1 ( left ) and sh - ∆ Np63 # - depleted ( right ) A - 431 cells . ( N ) ES : ( normalised ) enrichment scoreImmunoblot of endogenous ΔNp63 and USP28 in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 and transiently transfected with exogenous ΔNp63 . ACTIN served as loading control . Representative western blot from three independent experiments . Cell growth of A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 and transiently transfected with exogenous ΔNp63 . Total cell number was measured and assessed at indicated time points . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) . p - values were calculated using two - tailed t - test statistical analysis . * p - value < 0 . 05 , * * p - value < 0 . 01Relative expression of SCC marker genes KRT5 , 14 and 19 in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 , two independent shRNA - USP28 or ΔNp63 in shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) . Relative expression of epithelial marker genes KRT10 and GPCR5A in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 and shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) . Genomic signature of primary human lung SCC samples comprising USP28 , ∆ Np63 , KRT5 , KRT14 , KRT19 , DSG3 , MKI67 , PCNA and GPRC5A . Samples were sorted dependent on relative USP28 expression ( high to low ) . n = 553 . Xena UCSC software . GSEA of consensus squamous cancer marker genes ( se Appendix Table S2 ) in A - 431 cells stably transduced with sh - ∆ Np63 # 2 or sh - NTC . ( N ) ES : ( normalised ) enrichment score GSEA of consensus squamous cancer marker genes in A - 431 cells stably transduced with sh - USP28 # 1 or sh - NTC . ( N ) ES : ( normalised ) enrichment scoreCorrelation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . n = 836 . Gepia software . Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . N = 836 . Gepia software .

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

Figure 4Correlation of gene expression changes upon constitutive transduction of A-431 cells with either shRNA targeting USP28 (sh-USP28#1) or ∆Np63 (sh-∆Np63#2) relative to non-targeting control (sh-NTC). The diagonal line reflects a regression build on a linear model. R: Pearsons correlation coefficient, m: slope of the linear regression modelGene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-∆Np63#2 transfected A-431 cells ("Down-regulated sh-∆NP63", Appendix Table S1). The gene set was analysed in sh-∆Np63#2 (left) and sh-USP28#1-depleted (right) A-431 cells. (N)ES: (normalised) enrichment scoreGene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-USP28#1 transfected A-431 cells ("Down-regulated sh-USP28", Appendix Table S1). The gene set was analysed in shUSP28#1 (left) and sh-∆Np63#-depleted (right) A-431 cells. (N)ES: (normalised) enrichment scoreImmunoblot of endogenous ΔNp63 and USP28 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. ACTIN served as loading control. Representative western blot from three independent experiments.Cell growth of A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. Total cell number was measured and assessed at indicated time points. Quantitative graph is represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis. *p-value < 0.05, **p-value < 0.01Relative expression of SCC marker genes KRT5, 14 and 19 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63, two independent shRNA-USP28 or ΔNp63 in shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).Relative expression of epithelial marker genes KRT10 and GPCR5A in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63 and shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).Genomic signature of primary human lung SCC samples comprising USP28, ∆Np63, KRT5, KRT14, KRT19, DSG3, MKI67, PCNA and GPRC5A. Samples were sorted dependent on relative USP28 expression (high to low). n=553. Xena UCSC software.GSEA of consensus squamous cancer marker genes (se Appendix Table S2) in A-431 cells stably transduced with sh-∆Np63#2 or sh-NTC. (N)ES: (normalised) enrichment score GSEA of consensus squamous cancer marker genes in A-431 cells stably transduced with sh-USP28#1 or sh-NTC. (N)ES: (normalised) enrichment scoreCorrelation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. n=836. Gepia software. Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. N=836. Gepia software.


Figure 5Representative haematoxylin and eosin ( H & E ) staining of tumour bearing animals 12 weeks post intratracheal infection . Boxes indicate highlighted tumour areas in ( C ) ( a , b , a ' and b ' ) . Scale bar = 2000µm ; nKPL = 6 and nKPLU = 5 . H = heart . Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 abundance in KPL ( n = 6 ) and KPLU ( n = 5 ) lung tumours . Scale bar : 20µm . Quantification of % tumour area ( normalised to total lung area ) in KPL ( n = 6 ) and KPLU ( n = 5 ) animals . Quantification of lung tumour numbers in KPL ( n = 6 ) and KPLU ( n = 5 ) animals . Immunoblot of endogenous LKB1 , USP28 and ∆ Np63 in KPL and KPLU tumours . ( VINCULIN as loading control ) . Western blot shown are representative of three independent experiments ( n = 3 ) . Representative immunoblot of endogenous NOTCH1 , c - MYC and c - JUN in KPL and KPLU ( ACTIN as a control ) . n = 3Kaplan - Meier survival curves comparing KPL ( n = 6 ) and KPLU ( n = 5 ) animals upon AAV intratracheal infection . The p - value was calculated using a log - rank test . Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones , resulting in KPADC and KPSCC ; ( ACTIN served as loading control ) . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) . Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences . ACTIN served as loading control . Representative haematoxylin and eosin ( H & E ) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells / animal . KPSCC sh - NTC ( n = 3 ) ; KPSCC sh - USP28 ( n = 3 ) , Scale bar = 5000µmQuantification of % tumour area ( top , normalised to total lung area ) and lung tumour numbers ( bottom ) on KPSCC sh - NTC ( n = 6 ) and KPSCC sh - USP28 ( n = 6 ) animals . Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 and GFP abundance in KPSCC sh - NTC ( n = 3 ) and KPSCC sh - USP28 ( n = 3 ) lung tumours . Scale bar = 50

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

Figure 5Representative haematoxylin and eosin (H&E) staining of tumour bearing animals 12 weeks post intratracheal infection. Boxes indicate highlighted tumour areas in (C) (a, b, a' and b'). Scale bar = 2000µm; nKPL= 6 and nKPLU = 5. H=heart.Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 abundance in KPL (n=6) and KPLU (n=5) lung tumours. Scale bar: 20µm.Quantification of % tumour area (normalised to total lung area) in KPL (n=6) and KPLU (n=5) animals. Quantification of lung tumour numbers in KPL (n=6) and KPLU (n=5) animals.Immunoblot of endogenous LKB1, USP28 and ∆Np63 in KPL and KPLU tumours. (VINCULIN as loading control). Western blot shown are representative of three independent experiments (n=3). Representative immunoblot of endogenous NOTCH1, c-MYC and c-JUN in KPL and KPLU (ACTIN as a control). n=3Kaplan-Meier survival curves comparing KPL (n=6) and KPLU (n=5) animals upon AAV intratracheal infection. The p-value was calculated using a log-rank test.Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones, resulting in KPADC and KPSCC; (ACTIN served as loading control). Quantitative graph is represented as mean ± SD of three experiments (n=3).Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences. ACTIN served as loading control.Representative haematoxylin and eosin (H&E) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells/animal. KPSCC sh-NTC (n=3); KPSCC sh-USP28 (n=3), Scale bar = 5000µmQuantification of % tumour area (top, normalised to total lung area) and lung tumour numbers (bottom) on KPSCC sh-NTC (n=6) and KPSCC sh-USP28 (n=6) animals.Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 and GFP abundance in KPSCC sh-NTC (n=3) and KPSCC sh-USP28 (n=3) lung tumours. Scale bar =50 µm


Figure 6Analysis of occurring TP63 genetic alterations in lung squamous ( LUSC ) , cervical ( CESC ) , oesophagus ( ESCA ) , head and neck ( HNSC ) and pancreatic ( PAAD ) tumours . Cbioportal . Expression of TP63 ( left ) and USP28 ( right ) in human Lung ( n = 498 ) , Cervix ( n = 254 ) , Oesophagus ( n = 96 ) and HNSC ( n = 522 ) SCC tumours and normal non - transformed tissue ( nLung = 338 nCervix = 3 , nOesophagus = 11 and nHNSC = 44 ) . In box plots , the centre line reflects the median , the cross represents the mean and the upper and lower box limits indicates the first and third quartile . Whiskers extend 1 . 5x the IQR . p - values were calculated using two - tailed t - test statistical analysis . Xena UCSC software . Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung ( nADC = 513 and nSCC = 498 ) , Cervix ( nADC = 47 and nSCC = 254 ) and Oesophagus ( nADC = 89 and nSCC = 96 ) . Blue dots : ADC ; Red dots : SCC ; R = Spearmans correlation coefficient ; m : Slope . Xena UCSC . Immunoblot of control ( sh - NTC ) and two independent shRNA targeting USP28 ( sh - USP28 # 1 and # 2 ) for ∆ Np63 , KRT14 and USP28 protein abundance in H1299 , LUDLU - 1adh , HELA , SiHa , Ca Ski , PANC - 1 and BXPC - 3 ( ACTIN as loading control ) . Cells were seeded at equal cell density and counted after five days , Brigth field images of control or sh - USP28 # 2 infected H1299 , LUDLU - 1adh , Hela , CaSKI , PANC - 1 and BXPC - 3 cells before quantification . Scale bar = 30µm Relative number of H1299 , LUDLU - 1adh , Hela , CaSKI , SiHa , PANC - 1 and BXPC - 3 sh - USP28 # 2 cells compared with sh - NTC control cells . p - values were calculated using two - tailed t - test statistical analysis . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 .

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

Figure 6Analysis of occurring TP63 genetic alterations in lung squamous (LUSC), cervical (CESC), oesophagus (ESCA), head and neck (HNSC) and pancreatic (PAAD) tumours. Cbioportal.Expression of TP63 (left) and USP28 (right) in human Lung (n=498), Cervix (n=254), Oesophagus (n=96) and HNSC (n=522) SCC tumours and normal non-transformed tissue (nLung=338 nCervix=3, nOesophagus=11 and nHNSC=44). In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR. p-values were calculated using two-tailed t-test statistical analysis. Xena UCSC software.Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung (nADC=513 and nSCC=498), Cervix (nADC=47 and nSCC=254) and Oesophagus (nADC=89 and nSCC=96). Blue dots: ADC; Red dots: SCC; R= Spearmans correlation coefficient; m: Slope. Xena UCSC.Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63, KRT14 and USP28 protein abundance in H1299, LUDLU-1adh, HELA, SiHa, Ca Ski, PANC-1 and BXPC-3 (ACTIN as loading control).Cells were seeded at equal cell density and counted after five days, Brigth field images of control or sh-USP28#2 infected H1299, LUDLU-1adh, Hela, CaSKI, PANC-1 and BXPC-3 cells before quantification. Scale bar = 30µm Relative number of H1299, LUDLU-1adh, Hela, CaSKI, SiHa, PANC-1 and BXPC-3 sh-USP28#2 cells compared with sh-NTC control cells. p-values were calculated using two-tailed t-test statistical analysis. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63.


Figure 7Immunoblot of endogenous USP28 in A - 431 cells upon treatment with warheads ( WH ) and either DMSO or indicated concentrations of AZ1 ( USP28 Inh . ) for 24 hours . USP28 Upper band : Active USP28 ; USP28 lower band : Inactive USP28 , VINCULIN as loading control . Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading control . TP63 and USP28 half maximal inhibitory protein abundance ( IC50 ) was calculated . Graphs are represented as mean ± SD ( n = 3 ) . TUBE pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours . VINCULIN served as loading control for relative Ubiquitination . Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG - 132 ( 20µM ) or DMSO . VINCULIN served as loading control . A - 431 cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . Scale bar = 250µm . n = 30 fields analysedImmunoblot of endogenous USP28 , ∆ Np63 and KRT14 in LUDLU - 1adh , H1299 , Ca Ski , Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading controlLUDLU - 1ADH , H1299 , Ca Ski , Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Scale bar for LUDLU - 1adh and H1299 = 500µm . Scale bar for Ca Ski , Hela and SiHa = 250µm . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 . Number of cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . n = 30 fields

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

Figure 7Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.) for 24 hours. USP28 Upper band: Active USP28; USP28 lower band: Inactive USP28, VINCULIN as loading control.Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control. TP63 and USP28 half maximal inhibitory protein abundance (IC50) was calculated. Graphs are represented as mean ± SD (n=3).TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours. VINCULIN served as loading control for relative Ubiquitination.Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG-132 (20µM) or DMSO. VINCULIN served as loading control.A-431 cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. Scale bar = 250µm. n= 30 fields analysedImmunoblot of endogenous USP28, ∆Np63 and KRT14 in LUDLU-1adh, H1299, Ca Ski, Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading controlLUDLU-1ADH, H1299, Ca Ski, Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Scale bar for LUDLU-1adh and H1299 = 500µm. Scale bar for Ca Ski, Hela and SiHa = 250µm. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63. Number of cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. n=30 fields analysed


Figure 8Immunofluorescence ( IF ) staining against endogenous ∆ Np63 in KP and KPL cell lines ( blue : DAPI [ staining of nuclei ] and red / magenta : ∆ Np63 ) , Scale bar = 32µm . GFP Fluorescent images of lungs treated with either PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Tumour cells are positive for GFP ( green arrows ) . n = 3 Quantification of % tumour area ( normalised to total lung area ) in control ( n = 5 ) , 125 mg / kg AZ1 ( n = 4 ) and 375 mg / kg AZ1 ( n = 4 ) treated animals . Representative staining of H & E and IHC for USP28 and ∆ Np63 of SCC tumours from mice treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Boxes indicate highlighted tumour areas . First line Scale bars = 500µm ; Lower scale bars = 20µm . n = 3 ∆ Np63 staining intensity of lung tumours treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . nControl = 33712 cells ; nAZ1 125mg / kg = 7382 cells ; nAZ1 375mg / kg = 4967 cells . Cells were analysed from three independent animals .

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Figure 8Immunofluorescence (IF) staining against endogenous ∆Np63 in KP and KPL cell lines (blue: DAPI [staining of nuclei] and red/magenta: ∆Np63), Scale bar = 32µm.GFP Fluorescent images of lungs treated with either PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Tumour cells are positive for GFP (green arrows). n=3 Quantification of % tumour area (normalised to total lung area) in control (n=5), 125 mg/kg AZ1 (n=4) and 375 mg/kg AZ1 (n=4) treated animals.Representative staining of H&E and IHC for USP28 and ∆Np63 of SCC tumours from mice treated with PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Boxes indicate highlighted tumour areas. First line Scale bars = 500µm; Lower scale bars = 20µm. n=3   ∆Np63 staining intensity of lung tumours treated with PBS/DMSO/0,1%Tween80 or indicated concentrations of AZ1. nControl = 33712 cells ; nAZ1 125mg/kg = 7382 cells; nAZ1 375mg/kg = 4967 cells. Cells were analysed from three independent animals.


Figure 1Kaplan Meier survival curve of N2 , odr - 3 ( n2150 ) and odr - 3 ( n2046 ) worms on partial lawn of P . aeruginosa at 25 ° C . Kaplan Meier survival curve of N2 , odr - 3 ( n2150 ) and odr - 3 ( n2046 ) worms on full lawn of P . aeruginosa at 25 ° C .

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Figure 1Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046) worms on partial lawn of P. aeruginosa at 25°C.   Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046)   worms on full lawn of P. aeruginosa at 25°C.


Figure 2Chemotactic response of N2 worms to varying dilutions of 1 - undecene . n ≥ 3 assays . Error bars indicate SEM . ( i ) Schematic of setup used to record the movement of 5 worms each under control and 1 - undecene odor exposure . ( ii ) Movement trajectories of N2 worms on E . coli OP50 lawn exposed to 1 - undecene odor for 30 min and 60 min , each color represents the trajectory of single worm . Scale bar = 10 mm .

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Figure 2Chemotactic response of N2 worms to varying dilutions of 1-undecene. n ≥ 3 assays. Error bars indicate SEM.(i) Schematic of setup used to record the movement of 5 worms each under control and 1-undecene odor exposure. (ii) Movement trajectories of N2 worms on E. coli OP50 lawn exposed to 1-undecene odor for 30 min and 60 min, each color represents the trajectory of single worm. Scale bar = 10 mm.


Figure 3Average calcium responses of AWB : : GCaMP3 worms exposed to 1 - undecene at indicated concentrations . Each worm was recorded for 180 s . Worms were under stimulus between 11 s - 130 s , window shown in grey , and stimulus withdrawal beyond 130 s . Shaded regions around the curves represent SEM . n = 3 . Two dashed box represents 10 s time window after stimulus addition and stimulus removal respectively .

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Figure 3Average calcium responses of AWB::GCaMP3 worms exposed to 1-undecene at indicated concentrations. Each worm was recorded for 180 s. Worms were under stimulus between 11 s-130 s, window shown in grey, and stimulus withdrawal beyond 130 s. Shaded regions around the curves represent SEM. n = 3. Two dashed box represents 10 s time window after stimulus addition and stimulus removal respectively.


Figure 4Real time PCR analysis of irg - 1 , irg - 2 and irg - 3 genes in N2 , odr - 3 ( n2150 ) and odr - 3 ( n2046 ) worms exposed to 1 - undecene odor upon respective naive worms . n = 3 . * P ≤ 0 . 05 , * * P ≤ 0 . 01 as determined by two - tailed unpaired t - test . Error bars indicate SEM . Real time PCR analysis of irg - 1 , irg - 2 and irg - 3 genes in N2 , lim - 4 ( ky403 ) and lim - 4 ( yz12 ) worms exposed to 1 - undecene odor upon respective naive worms . n = 3 . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 as determined by two - tailed unpaired t - test . Error bars indicate SEM . Kaplan Meier survival curve on the full lawn of P . aeruginosa for N2 ( naive ) worms and N2 worms pre exposed to 1 - undecene odor . Survival assay was performed at 20 ° C .

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

Figure 4Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, odr-3(n2150) and odr-3(n2046) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM. Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, lim-4(ky403) and lim-4(yz12) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 as determined by two-tailed unpaired t-test. Error bars indicate SEM.Kaplan Meier survival curve on the full lawn of P. aeruginosa for N2 (naive) worms and N2 worms pre exposed to 1-undecene odor. Survival assay was performed at 20°C.


Figure 1B . Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non - targeting siRNA ( siNT ) or silenced with the indicated oligos . 300 ng / mL MMC was added for 24 h . Each dot represents the number of PLA events in a single cell , from two to seven biological replicates ( siNT - MMC ( n = 6 ) , siNT + MMC ( n = 7 ) , siDPP9 - MMC ( n = 4 ) , siDPP9 + MMC ( n = 5 ) , siBRCA2 - MMC ( n = 2 ) , siBRCA2 + MMC ( n = 3 ) , siFLNA - MMC ( n = 5 ) , siFLNA + MMC ( n = 6 ) , NgtCntrl - MMC ( n = 3 ) , NgtCntrl + MMC ( n = 6 ) ) . The number of foci are shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * * * = p ≤ 0 . 0001 ) . C . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells . Exposure of cells to MMC triggers more PLA events ( white ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 2889071D . Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S - Phase . Each dot represents the number of PLA events in a single cell , from three biological replicates . Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS ( 250 ng / mL for 30 min ) . Cells were sampled prior to NCS treatment , after NCS treatment and with 1 . 5 h and 3 h of recovery after NCS treatment . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * * = p ≤ 0 . 0001 ) . E . Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells . Exposure of cells to NCS triggers more PLA events ( white ) , which increase in time . Cells in S - Phase show a greater fold - increase in the number of PLA events in comparison to asynchronous cells . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Antibodies as in ( C ) . F . Co - immunoprecipitation assays showing binding of BRCA2 and DPP9 - SWT . HEK293 DPP9 KO + DPP9WT cells , were treated with 1µg / mL Dox ( 24 h ) to induce the expression of DPP9 - FLAG . DNA damage was induced with 300 nM MMC treatment for 24h . Control cells do not express DPP9 ( - Dox ) . Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting ( anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - FLAG RRID : AB _ 262044 ) .

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

Figure 1B. Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non-targeting siRNA (siNT) or silenced with the indicated oligos. 300 ng / mL MMC was added for 24 h. Each dot represents the number of PLA events in a single cell, from two to seven biological replicates (siNT-MMC (n = 6), siNT+MMC (n = 7), siDPP9-MMC (n = 4), siDPP9+MMC (n = 5), siBRCA2-MMC (n = 2), siBRCA2+MMC (n = 3), siFLNA-MMC (n = 5), siFLNA+MMC (n = 6), NgtCntrl-MMC (n = 3), NgtCntrl+MMC (n = 6)). The number of foci are shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, **** = p ≤ 0.0001). C. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells. Exposure of cells to MMC triggers more PLA events (white). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071D. Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S-Phase. Each dot represents the number of PLA events in a single cell, from three biological replicates. Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS (250 ng / mL for 30 min). Cells were sampled prior to NCS treatment, after NCS treatment and with 1.5 h and 3 h of recovery after NCS treatment. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001). E. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells. Exposure of cells to NCS triggers more PLA events (white), which increase in time. Cells in S-Phase show a greater fold-increase in the number of PLA events in comparison to asynchronous cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Antibodies as in (C).F. Co-immunoprecipitation assays showing binding of BRCA2 and DPP9-SWT. HEK293 DPP9 KO + DPP9WT cells, were treated with 1µg/mL Dox (24 h) to induce the expression of DPP9-FLAG. DNA damage was induced with 300 nM MMC treatment for 24h. Control cells do not express DPP9 (- Dox). Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting (anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-FLAG RRID:AB_262044).


Figure 2B . Pull - down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N - terminal1 - 40 fragment immobilized on HA - beads . The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21 - 40HA for interaction with DPP9 . Representative data of three technical replicates is shown . Anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 . C . Quantification of PLAs showing fewer MMC - induced DPP9 - BRCA2 PLA events in HeLa cells treated with 10µM 1G244 . Each dot represents the number of PLA events in a single cell , from three biological replicates . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 ) . D . Representative images of DPP9 - BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9 . Control cells were mock treated with DMSO . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 2889071F . Pull - down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N - terminus . While each protein can bind to the BRCA2 N - terminal peptide , in the presence of DPP9 , the PALB2 - BRCA2 binding is negatively affected . Representative data of three technical replicates is shown . Anti - PALB2 : RRID : AB _ 890607 , anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

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

Figure 2B. Pull-down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N-terminal1-40 fragment immobilized on HA-beads. The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21-40HA for interaction with DPP9. Representative data of three technical replicates is shown. Anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.C. Quantification of PLAs showing fewer MMC-induced DPP9-BRCA2 PLA events in HeLa cells treated with 10µM 1G244. Each dot represents the number of PLA events in a single cell, from three biological replicates. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001). D. Representative images of DPP9-BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9. Control cells were mock treated with DMSO. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071F. Pull-down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N-terminus. While each protein can bind to the BRCA2 N-terminal peptide, in the presence of DPP9, the PALB2-BRCA2 binding is negatively affected. Representative data of three technical replicates is shown. Anti-PALB2: RRID:AB_890607, anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


Figure 4A - B . Representative Western Blots and accompanying graph from more than three biological replicates show that MMC ( 300 nM , 24 h ) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells , which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 ( 100µM ) , in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 ( siDPP9 ) . RAD51 stability is not altered in HeLa DPP9 KD cells . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test . ( * = p ≤ 0 . 03 ) . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - Vinculin : RRID : AB _ 477629 , anti - Tubulin : RRID : AB _ 628412 , anti - RAD51 : RRID : AB _ 1142428 . C - D . Representative Western Blots and graph summarizing results of more than three biological replicates . CHX chase assays show that the MMC - induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9 - SS729A cells overexpressing an inactive DPP9 - S mutant . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . Cells that were overexpressing the active variant ( DPP9 - SWT ) show similar levels of MMC - induced BRCA2 degradation . RAD51 stability is not altered by MMC and is similar in all cell lines . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown images originate from one representative cycloheximide ( CHX ) chase assay . The ratios of BRCA2 to Vinculin are defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 , * * = p ≤ 0 . 002 ) . Antibodies as described in ( A - B ) . F - G . Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2 ∆ MP3 - 1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21 - 1000 . Cells were co - transfected with a GFP expressing plasmid , as transfection and loading control . Control cells were transfected with GFP only ( U . T ) . Image originates from one representative experiment . BRCA2 - FLAG signals are related to the transfection and loading control GFP . BRCA2 levels in relation to GFP were defined as 100 % at time 0 h . Mean ± SEM , data were analysed by a paired two - tailed t - test ( * = p ≤ 0 . 03 ) . Anti - FLAG : RRID : AB _ 262044 , anti - GFP : RRID : AB _ 641123 .

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

Figure 4A-B. Representative Western Blots and accompanying graph from more than three biological replicates show that MMC (300 nM, 24 h) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells, which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 (100µM), in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 (siDPP9). RAD51 stability is not altered in HeLa DPP9 KD cells. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test. (*= p ≤ 0.03). Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-Vinculin: RRID:AB_477629, anti-Tubulin: RRID:AB_628412, anti-RAD51: RRID:AB_1142428.C-D. Representative Western Blots and graph summarizing results of more than three biological replicates. CHX chase assays show that the MMC-induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9-SS729A cells overexpressing an inactive DPP9-S mutant. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. Cells that were overexpressing the active variant (DPP9-SWT) show similar levels of MMC-induced BRCA2 degradation. RAD51 stability is not altered by MMC and is similar in all cell lines. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03, **= p ≤ 0.002). Antibodies as described in (A-B).F-G. Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2∆MP3-1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21-1000. Cells were co-transfected with a GFP expressing plasmid, as transfection and loading control. Control cells were transfected with GFP only (U.T). Image originates from one representative experiment. BRCA2-FLAG signals are related to the transfection and loading control GFP. BRCA2 levels in relation to GFP were defined as 100 % at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03). Anti-FLAG: RRID:AB_262044, anti-GFP: RRID:AB_641123.


Figure 5A . Representative immunofluorescence images of γH2AX signals ( white , RRID : AB _ 309864 ) in gki MEF DPP9S729A , showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin ( NCS ) , quantification in ( B ) . MEF DPP9S729A and control MEF cells were treated with 250 ng / mL NCS for 30 min and allowed to recover for the indicated time - points . γH2AX signals at time 0 , reflect 30 min of NCS and no recovery time . Nuclei are shown in blue ( DAPI ) . Scale bar 10 µm . B . Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in ( A ) . Signals from more than 1700 cells were quantified per condition per experiment . Mean ± SEM from six biological replicates , each in technical duplicates . Data were analysed by an unpaired two - way ANOVA with Sidak ' s multiple comparison test ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 ) .

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

Figure 5A. Representative immunofluorescence images of γH2AX signals (white, RRID:AB_309864) in gki MEF DPP9S729A, showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin (NCS), quantification in (B). MEF DPP9S729A and control MEF cells were treated with 250 ng/mL NCS for 30 min and allowed to recover for the indicated time-points. γH2AX signals at time 0, reflect 30 min of NCS and no recovery time. Nuclei are shown in blue (DAPI). Scale bar 10 µm. B. Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in (A). Signals from more than 1700 cells were quantified per condition per experiment. Mean ± SEM from six biological replicates, each in technical duplicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01).


Figure 6C . Representative PLA images of BRCA2 - PALB2 PLA experiments showing more PLA events ( white ) in HeLa cells silenced for DPP9 ( siDPP9 ) with respect to cells treated with non - targeting siRNA ( siNT ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - PALB2 : RRID : AB _ 890607 , anti - BRCA2 : RRID : AB _ 2259370 . D . Quantification of PLAs experiments showing more BRCA2 - PALB2 PLA events in cells silenced for DPP9 ( siDPP9 ) in comparison to non - targeting controls ( siNT ) . Each dot represents the number of PLA events in a single cell , from four biological replicates . The technical control samples ( NgtCntrl ) omitted the BRCA2 antibody . The number of foci is shown based on their cellular localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . E . Representative PLA images of γH2Ax - BRCA2 PLA experiment showing a reduction in the number of PLA events ( white ) between γH2Ax and BRCA2 in HeLa DPP9 KD cells . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains the nucleus . Scale bar 10 µm . Anti - γH2Ax : RRID : AB _ 2118009 , anti - BRCA2 : RRID : AB _ 2259370 F . Quantification of PLAs showing fewer MMC - induced γH2Ax - BRCA2 PLA events in HeLa DPP9 KD cells , in comparison to HeLa WT cells . Each dot represents the number of PLA events in a single cell , from three biological replicates . The technical control samples ( NgtCntrl ) omitted the γH2Ax antibody . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) .

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

Figure 6C. Representative PLA images of BRCA2-PALB2 PLA experiments showing more PLA events (white) in HeLa cells silenced for DPP9 (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.D. Quantification of PLAs experiments showing more BRCA2-PALB2 PLA events in cells silenced for DPP9 (siDPP9) in comparison to non-targeting controls (siNT). Each dot represents the number of PLA events in a single cell, from four biological replicates. The technical control samples (NgtCntrl) omitted the BRCA2 antibody. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).E. Representative PLA images of γH2Ax-BRCA2 PLA experiment showing a reduction in the number of PLA events (white) between γH2Ax and BRCA2 in HeLa DPP9 KD cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-γH2Ax: RRID:AB_2118009, anti-BRCA2: RRID:AB_2259370 F. Quantification of PLAs showing fewer MMC-induced γH2Ax-BRCA2 PLA events in HeLa DPP9 KD cells, in comparison to HeLa WT cells. Each dot represents the number of PLA events in a single cell, from three biological replicates. The technical control samples (NgtCntrl) omitted the γH2Ax antibody. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


Figure 7A . Representative immunofluorescence images showing that re - expression of DPP9 - SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9 - SWT cells . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 . B . Graph showing the number of RAD51 foci following induction of DPP9 - SWT expression , compared to uninduced HEK293 DPP9 KO + DPP9WT cells ( - Dox ) . Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9 - SS729A did not result in more RAD51 foci . Each dot represents the number of RAD51 foci in a single cell , from three biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . Representative images ( C , and summarizing graph ( D showing the number of RAD51 foci per nucleus in HeLa WT cells ( C , D ) Where stated , cells were treated with control siRNA ( siNT ) or silenced for BRCA2 , and transiently transfected with the BRCA21 ‑ 3418 or BRCA23 - 3418 constructs . Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells ( C , D ) . Each dot represents the number of RAD51 foci in a single cell , from three ( D ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; ; anti - BRCA2 : RRID : AB _ 2259370 . Representative images , E ) and summarizing graph F ) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells ( E , F ) . In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2 ∆ MP3 - 3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21 - 3418 ( E , F ) . Each dot represents the number of RAD51 foci in a single cell , from four ( F ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison test . Shown are Mean ± SEM ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in white , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; anti - BRCA2 : RRID : AB _ 2259370 .

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

Figure 7A. Representative immunofluorescence images showing that re-expression of DPP9-SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9-SWT cells. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428. B. Graph showing the number of RAD51 foci following induction of DPP9-SWT expression, compared to uninduced HEK293 DPP9 KO + DPP9WT cells (-Dox). Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9-SS729A did not result in more RAD51 foci. Each dot represents the number of RAD51 foci in a single cell, from three biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).Representative images (C, and summarizing graph (D showing the number of RAD51 foci per nucleus in HeLa WT cells (C, D) Where stated, cells were treated with control siRNA (siNT) or silenced for BRCA2, and transiently transfected with the BRCA21‑3418 or BRCA23-3418 constructs. Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells (C, D). Each dot represents the number of RAD51 foci in a single cell, from three (D) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; ; anti-BRCA2: RRID:AB_2259370.Representative images , E) and summarizing graph F) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells (E, F). In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2∆MP3-3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21-3418 (E, F). Each dot represents the number of RAD51 foci in a single cell, from four (F) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; anti-BRCA2: RRID:AB_2259370.


figf1a , Phase partitioning of Apg8 . Wild - type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning ( see Methods ) . b , MALDI mass spectrum of His6 - Apg8 . Purified His6 - Apg8 ( predominantly Apg8FG - X and a small amount of Apg8FG ) was extracted with 0 . 4 % β - octylglycoside from a polyacrylamide gel and desalted with ZipTipC4 ( Millipore ) before mass measurement . The principal MH + ion signal at m / z 15 , 003 is shifted in mass by about + 635 from the MH + value corresponding to His6 - Apg8FG . c , MS / MS spectra of the C - terminal peptides of Apg8FG - X ( top ) and Apg8FG ( bottom ) . C - terminal fragments were generated from Apg8FG - X and Apg8FG ( see Methods ) . Each fragment that was observed only as non - 18O - labelled signals was subjected to MS / MS . The MS / MS spectra , deconvoluted with MaxEnt3 , were interpreted by SeqMS software26 . Arrows show the sequences from the N and C terminus based on y ″ m and bl ions , respectively ( where m and l denote positions counted from the C and N terminus ) that were produced by cleavage of peptide bonds during MS / MS . Amino acids shown in three - letter code denote immonium ions . The nomenclature of these ions is as in previous work28 .

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

figf1a, Phase partitioning of Apg8. Wild-type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning (see Methods).b, MALDI mass spectrum of His6-Apg8. Purified His6-Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4(Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6-Apg8FG.c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.


figf2d , Thioester formation between Apg8 and Apg7 . Cell extracts of Δapg7Δapg8 cells expressing Apg7 - Myc and / or HA - Apg8 from 2µ plasmids were immunoprecipitated with anti - HA antibody , and immunoblotted ( IB ) with anti - Myc and anti - HA antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg7 conjugate . b , Interaction between Apg8 and Apg7 . Δapg7Δapg8 cells were transformed with APG7 - Myc and / or APG8 on 2µ plasmids . Cell lysates were immunoprecipitated with anti - Myc antibody , and immunoblotted with anti - Myc or anti - Apg8 antibody . Apg8 co - immunoprecipitated with Apg7 - Myc . c , Effect of Apg8 C - terminal processing on the Apg8 - Apg7 interaction . Δapg4 cells co - expressing Apg7 - Myc and HA - Apg8 ( FGR , FG and F ) from 2µ plasmids . FGR , wild - type Apg8 ; FG , Apg8 lacking C - terminal arginine ; F , Apg8 lacking C - terminal glycine and arginine . Cell lysates were immunoprecipitated with anti - HA antibody , and immunoblotted with anti - HA and anti - Myc antibody .

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

figf2d, Thioester formation between Apg8 and Apg7. Cell extracts of Δapg7Δapg8 cells expressing Apg7-Myc and/or HA-Apg8 from 2µ plasmids were immunoprecipitated with anti-HA antibody, and immunoblotted (IB) with anti-Myc and anti-HA antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg7 conjugate.b, Interaction between Apg8 and Apg7. Δapg7Δapg8 cells were transformed with APG7-Myc and/or APG8 on 2µ plasmids. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc or anti-Apg8 antibody. Apg8 co-immunoprecipitated with Apg7-Myc.c, Effect of Apg8 C-terminal processing on the Apg8-Apg7 interaction. Δapg4 cells co-expressing Apg7-Myc and HA-Apg8 (FGR, FG and F) from 2µ plasmids. FGR, wild-type Apg8; FG, Apg8 lacking C-terminal arginine; F, Apg8 lacking C-terminal glycine and arginine. Cell lysates were immunoprecipitated with anti-HA antibody, and immunoblotted with anti-HA and anti-Myc antibody.


figf3b , Interaction between Apg8 and Apg3 . Lysates of Δapg3 cells expressing APG3 and / or Myc - APG8 from 2µ plasmids were immunoprecipitated with anti - Apg3 antibody , and immunoblotted with anti - Apg3 and anti - Myc antibody . c , Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3 . Lysates of Δapg3 cells expressing Myc - APG8 and either wild - type ( wt ) APG3 or mutant APG3 ( C234S and C234A ) from 2µ plasmids were immunoprecipitated with anti - Myc antibody , and immunoblotted with anti - Myc and anti - Apg3 antibodies . d , Thioester formation between Apg8 and Apg3 . Δapg3 , Δapg7 and Δapg10 cells were transformed with both Myc - APG8 and Apg3 , or only Apg3 , on 2µ plasmids . Cell extracts were immunoprecipitated with anti - Myc antibody , and subjected to immunoblot ( IB ) using anti - Myc and anti - Apg3 antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg3 conjugate ; diamond , possible Myc - Apg8 dimer .

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figf3b, Interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing APG3 and/or Myc-APG8 from 2µ plasmids were immunoprecipitated with anti-Apg3 antibody, and immunoblotted with anti-Apg3 and anti-Myc antibody.c, Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing Myc-APG8 and either wild-type (wt) APG3 or mutant APG3 (C234S and C234A) from 2µ plasmids were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc and anti-Apg3 antibodies.d, Thioester formation between Apg8 and Apg3. Δapg3, Δapg7 and Δapg10 cells were transformed with both Myc-APG8 and Apg3, or only Apg3, on 2µ plasmids. Cell extracts were immunoprecipitated with anti-Myc antibody, and subjected to immunoblot (IB) using anti-Myc and anti-Apg3 antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg3 conjugate; diamond, possible Myc-Apg8 dimer.


figf4a , Apg7 and Apg3 are necessary for attachment of Apg8 to PE . Δapg cell lysates were subjected to SDS - PAGE containing 6 M urea , and immunoblotted with anti - Apg8 antibody . b , Autophagic activity in apg3 mutants . Wild - type , Δapg3 and apg3C243A cells were grown in rich medium ( open bar ) and subjected to nitrogen starvation for 4 h ( filled bar ) . The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity . The apg3C243A mutant showed defective autophagy .

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figf4a, Apg7 and Apg3 are necessary for attachment of Apg8 to PE. Δapg cell lysates were subjected to SDS-PAGE containing 6 M urea, and immunoblotted with anti-Apg8 antibody.b, Autophagic activity in apg3 mutants. Wild-type, Δapg3 and apg3C243A cells were grown in rich medium (open bar) and subjected to nitrogen starvation for 4 h (filled bar). The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity. The apg3C243A mutant showed defective autophagy.


Figure 7D , Affinity capture of proteins by OC43p and OC43 - SCR bound magnetic beads . Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies . OC43p - SCR - coated and uncoated beads ( Beads ) were used to control non - specific binding . Spinal cords were isolated from male ( M ) and female ( F ) rats ( n = 2 / group ) . Input protein lysates ( right side , in duplicates ) served as loading controls . Approximate molecular weights ( in kDa ) are indicated by arrows .

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Figure 7D, Affinity capture of proteins by OC43p and OC43-SCR bound magnetic beads. Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies. OC43p-SCR-coated and uncoated beads (Beads) were used to control non-specific binding. Spinal cords were isolated from male (M) and female (F) rats (n=2/group). Input protein lysates (right side, in duplicates) served as loading controls. Approximate molecular weights (in kDa) are indicated by arrows.


f1a , b , Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells ( a ) ( n = 5 / 4 ) and for autophagy proteins in primary intestinal epithelial cell scrapings ( b ) ( n = 3 ) . c , Transmission electron microscopy ( TEM ) of crypts . Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr ; IEC hypomorphic Paneth cells . d , e , Crypt showing GFP - LC3 punctae ( d ) , quantified in e ( n = 10 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . Scale bar , 5 µm . f , g , Immunoblot of silenced MODE - K cells ( f ) and primary intestinal epithelial cell scrapings ( g ) for the PERK - eIF2α branch ( n = 3 ) . j , GFP - LC3 punctae per crypt after treatment with tamoxifen , and vehicle or salubrinal ( n = 10 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) . k , Enteritis histology score after salubrinal and tamoxifen co - treatment ( n = 12 / 14 / 13 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent three ( a , f , g ) or two ( c , e , h , i ) independent experiments . * P 0 . 05 , * * * P 0 . 001 .

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f1a, b, Immunoblot for LC3 conversion in isolated primary intestinal epithelial cells (a) (n = 5/4) and for autophagy proteins in primary intestinal epithelial cell scrapings (b) (n = 3).c, Transmission electron microscopy (TEM) of crypts. Note autophagic vacuoles in various stages of evolution in Xbp1 Dgr;IEC hypomorphic Paneth cells.d, e, Crypt showing GFP-LC3 punctae (d), quantified in e (n = 10; unpaired Student's t-test; mean ± s.e.m.). Scale bar, 5 µm.f, g, Immunoblot of silenced MODE-K cells (f) and primary intestinal epithelial cell scrapings (g) for the PERK-eIF2α branch (n = 3).j, GFP-LC3 punctae per crypt after treatment with tamoxifen, and vehicle or salubrinal (n = 10; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).k, Enteritis histology score after salubrinal and tamoxifen co-treatment (n = 12/14/13; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent three (a, f, g) or two (c, e, h, i) independent experiments. *P 0.05, ***P 0.001.


f2a , TEM of crypts of Lieberkühn ( n = 2 ) . Scale bars , 2 µm . b , Representative haematoxylin and eosin stainings . Note transmural inflammation extending through muscularis propria ( white arrow ) into serosa ( black arrow ) in Atg7 / Xbp1Dgr ; IEC and Atg16l1 / Xbp1Dgr ; IEC mice scored in c and e . Scale bars , 50 µm , except for lower two panels ( 10 µm ) . c , Enteritis histology score ( n = 26 / 12 / 18 / 27 ; 10 - 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . e , Enteritis histology score ( n = 11 ; 18 weeks ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . NS , not significant . d , CryptTEM in indicated genotypes ( n = 2 ) . Scale bars , 2 µm . f , Xbp1 mRNA splicing ( Xbp1u , unspliced ; Xbp1s , spliced ) of crypts ; densitometry in g ( n = 7 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . h , GRP78 ( green ) immunofluorescence , white arrows indicate GRP78 + crypts ( DAPI , blue ; n = 5 ) . Scale bars , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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f2a, TEM of crypts of Lieberkühn (n = 2). Scale bars, 2 µm.b, Representative haematoxylin and eosin stainings. Note transmural inflammation extending through muscularis propria (white arrow) into serosa (black arrow) in Atg7/Xbp1Dgr;IEC and Atg16l1/Xbp1Dgr;IEC mice scored in c and e. Scale bars, 50 µm, except for lower two panels (10 µm). c, Enteritis histology score (n = 26/12/18/27; 10-18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).e, Enteritis histology score (n = 11; 18 weeks; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). NS, not significant.d, CryptTEM in indicated genotypes (n = 2). Scale bars, 2 µm.f, Xbp1 mRNA splicing (Xbp1u, unspliced; Xbp1s, spliced) of crypts; densitometry in g (n = 7; unpaired Student's t-test; mean ± s.e.m.).h, GRP78 (green) immunofluorescence, white arrows indicate GRP78+ crypts (DAPI, blue; n = 5). Scale bars, 10 µm. *P 0.05, **P 0.01, ***P 0.001.


f3a , shCtrl or shXbp1 MODE - K cells were co - silenced for Atg16l1 ( siAtg16l1 ) or with scrambled siRNA ( siCtrl ) , and analysed by flow cytometry for annexin V and propidium iodide ( PI ; n = 3 ; one - way ANOVA with post - hoc Bonferroni ; mean ± s . e . m . ) . b , Immunoblot of primary intestinal epithelial cell scrapings ( n = 3 ) . c , Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE - K cells after TNF stimulation . d , TUNEL + intestinal epithelial cells ( IECs ) per 100 crypts after BAY11 - 7082 or vehicle treatment ( n = 3 / 4 / 4 ; one - way ANOVA with post - hoc Holm ' s - corrected unpaired Student ' s t - test ; mean ± s . e . m . ) . e , Enteritis histology score of mice treated with BAY11 - 7082 or vehicle ( n = 10 / 10 / 9 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . f , Immunoblot of intestinal epithelial cell scrapings for ( p - ) IRE1α ; after IRE1α ; immunoprecipitation ( IP ) . beta - actin , loading control of whole lysates . g , Immunoblot of primary intestinal epithelial cell scrapings ( n = 4 ) . h , i , Enteritis histology score of indicated genotypes ( h , n = 15 / 16 / 14 / 15 ; i , n = 5 / 10 / 12 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . j , Enteritis histology score of specific pathogen - free ( SPF ) and germ free ( GF ) housed mice ( n = 10 / 9 / 7 / 7 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . k , Representative images of p - IκBα immunoreactivity under conditions as in j ( n = 4 ) . Scale bars , 20 µm . Results represent four ( f ) , three ( c ) or two ( a , b ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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f3a, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI; n = 3; one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.).b, Immunoblot of primary intestinal epithelial cell scrapings (n = 3).c, Immunoblot of cytoplasmic extracts from shCtrl or shXbp1 MODE-K cells after TNF stimulation.d, TUNEL+ intestinal epithelial cells (IECs) per 100 crypts after BAY11-7082 or vehicle treatment (n = 3/4/4; one-way ANOVA with post-hoc Holm's-corrected unpaired Student's t-test; mean ± s.e.m.). e, Enteritis histology score of mice treated with BAY11-7082 or vehicle (n = 10/10/9; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).f, Immunoblot of intestinal epithelial cell scrapings for (p-)IRE1α; after IRE1α; immunoprecipitation (IP). beta-actin, loading control of whole lysates.g, Immunoblot of primary intestinal epithelial cell scrapings (n = 4).h, i, Enteritis histology score of indicated genotypes (h, n = 15/16/14/15; i, n = 5/10/12; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test).j, Enteritis histology score of specific pathogen-free (SPF) and germ free (GF) housed mice (n = 10/9/7/7; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). k, Representative images of p-IκBα immunoreactivity under conditions as in j (n = 4). Scale bars, 20 µm. Results represent four (f), three (c) or two (a, b) independent experiments. *P  0.05, **P  0.01, ***P  0.001.


f4a , Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days ( n = 3 ) . b , Enteritis histology score for experiment as in a ( n = 4 ; median shown ; Mann - Whitney U - test ) . c , Representative images of EYFP - Rosa26 / D6 - cre + / - reporter mice and EYFP - Rosa26 ( controls ) . Co - localization of Defa6 Cre - driven EYFP expression ( yellow ) with lysozyme - expressing Paneth cells ( red ; n = 3 ) . DAPI , blue ; scale bars , 50 µm . d , Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr ; PC and wild - type controls ( n = 2 ) . e , f , Representative confocal images of lysozyme ( green ) expressing Paneth cells ( e ) with quantification of crypts with indicated number of lysozyme + granulated dots in f ( n = 5 ; unpaired Student ' s t - test ; mean ± s . e . m . ) . DAPI , blue ; scale bars , 10 µm . g , Immunohistochemistry for p - eIF2α ( n = 3 ) . Scale bars , 20 µm . h , Representative haematoxylin and eosin images of Xbp1Dgr ; PC and wild - type mice scored in i . Scale bars , 50 µm . i , Enteritis scoring in Xbp1fl / fl ( WTfl ) , Defa6 - cre + ( WTcre ) and Defa6 - cre + ; Xbp1fl / fl ( Xbp1Dgr ; PC ) mice ( n = 21 / 26 / 29 ; median shown ; Kruskal - Wallis with post - hoc Holm ' s - corrected Mann - Whitney U - test ) . Results represent two ( b , d ) independent experiments . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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f4a, Immunoblot of primary intestinal epithelial cell scrapings from mice treated with or without rapamycin for 14 consecutive days (n = 3). b, Enteritis histology score for experiment as in a (n = 4; median shown; Mann-Whitney U-test).c, Representative images of EYFP-Rosa26/D6-cre+/- reporter mice and EYFP-Rosa26 (controls). Co-localization of Defa6 Cre-driven EYFP expression (yellow) with lysozyme-expressing Paneth cells (red; n = 3). DAPI, blue; scale bars, 50 µm.d, Immunoblots of crypt intestinal epithelial cells from Xbp1Dgr;PC and wild-type controls (n = 2).e, f, Representative confocal images of lysozyme (green) expressing Paneth cells (e) with quantification of crypts with indicated number of lysozyme+ granulated dots in f (n = 5; unpaired Student's t-test; mean ± s.e.m.). DAPI, blue; scale bars, 10 µm.g, Immunohistochemistry for p-eIF2α (n = 3). Scale bars, 20 µm.h, Representative haematoxylin and eosin images of Xbp1Dgr;PC and wild-type mice scored in i. Scale bars, 50 µm. i, Enteritis scoring in Xbp1fl/fl (WTfl), Defa6-cre+ (WTcre) and Defa6-cre+;Xbp1fl/fl (Xbp1Dgr;PC) mice (n = 21/26/29; median shown; Kruskal-Wallis with post-hoc Holm's-corrected Mann-Whitney U-test). Results represent two (b, d) independent experiments. *P 0.05, **P 0.01, ***P 0.001.


Figure 1D . BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N . benthamiana leaves . Images were taken at 60 hpi . Scale bar = 20 μm . E . Co - IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM . N . benthamiana leaves were agroinfiltrated and treated with 2 mM 3 - MA at 48 hpi , and then for IP with anti - Flag beads 12 h later .

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Figure 1D. BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N. benthamiana leaves. Images were taken at 60 hpi. Scale bar = 20 μm.E. Co-IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM. N. benthamiana leaves were agroinfiltrated and treated with 2 mM 3-MA at 48 hpi, and then for IP with anti-Flag beads 12 h later.


Figure 2Confocal microscopy of YFP - ATG8e - labeled autophagic bodies in root cells of Col - 0 / YFP - ATG8e and VISP1OE / YFP - ATG8e plants . 7 - day - old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark . Scale bar = 20 μm . Puncta numbers of YFP - ATG8e - labeled autophagic bodies per 10 cells in panel A . Data points represent means of three biological repeats . Error bars indicate SD . * * * p < 0 . 001 ( Student ' s t - test ) . Western blotting analysis of accumulation of ATG8a and ATG8a - PE in leaves of 4 - week - old Col - 0 , VISP1OE , VISP1mUIM / OE and VISP1mARM / OE plants . Representative TEM images of autophagic structures in inoculated leaves of Col - 0 and VISP1OE plants treated with mock buffer or CMV - 2blm infection at 4 dpi . The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling . Magenta arrowheads indicate autophagic bodies inside the vacuoles . Scale bar = 2 μm . Cp , chloroplast ; CW , cell wall ; V , vacuole . Autophagic structure numbers per 10 cells in panel F . Error bars indicate SD . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) .

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Figure 2Confocal microscopy of YFP-ATG8e-labeled autophagic bodies in root cells of Col-0/YFP-ATG8e and VISP1OE/YFP-ATG8e plants. 7-day-old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark. Scale bar = 20 μm.   Puncta numbers of YFP-ATG8e-labeled autophagic bodies per 10 cells in panel A. Data points represent means of three biological repeats. Error bars indicate SD. ***p < 0.001 (Student's t-test).   Western blotting analysis of accumulation of ATG8a and ATG8a-PE in leaves of 4-week-old Col-0, VISP1OE, VISP1mUIM/OE and VISP1mARM/OE plants.Representative TEM images of autophagic structures in inoculated leaves of Col-0 and VISP1OE plants treated with mock buffer or CMV-2blm infection at 4 dpi. The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling. Magenta arrowheads indicate autophagic bodies inside the vacuoles. Scale bar = 2 μm. Cp, chloroplast; CW, cell wall; V, vacuole.   Autophagic structure numbers per 10 cells in panel F. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).


Figure 3Long - term symptoms of Col - 0 , VISP1OE1 , VISP1OE2 , rdr6 - 15 , and sgs3 - 1 plants inoculated with CMV - 2blm at 50 dpi . Scale bar = 2 cm . Northern blotting analyzing accumulation of genomic RNAs ( gRNAs ) in systemically leaves of Col - 0 , OE1 ( VISP1OE1 ) , OE2 ( VISP1OE2 ) , rdr6 - 15 , and sgs3 - 1 plants infected by CMV - 2blm at 14 dpi . Methylene blue - stained rRNA were used as loading controls . Northern blotting analyzing accumulation of RNA3 - derived vsiRNAs in the same samples as shown in panel D .

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Figure 3Long-term symptoms of Col-0, VISP1OE1, VISP1OE2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.Northern blotting analyzing accumulation of genomic RNAs (gRNAs) in systemically leaves of Col-0, OE1 (VISP1OE1), OE2 (VISP1OE2), rdr6-15, and sgs3-1 plants infected by CMV-2blm at 14 dpi. Methylene blue-stained rRNA were used as loading controls.   Northern blotting analyzing accumulation of RNA3-derived   vsiRNAs in the same samples as shown in panel D.


Figure 4Long - term systemic symptoms of Col - 0 , visp1 - 1 , visp1 - 2 , rdr6 - 15 , and sgs3 - 1 plants inoculated with CMV - 2blm at 50 dpi . Scale bar = 2 cm . Northern blotting detecting accumulation of genomic RNAs and RNA3 - derived vsiRNAs in systemically infected leaves at 14 dpi . Relative accumulation ( RA ) of gRNA3 was calculated from band densities . Northern blotting analyzing accumulation of RNA3 - derived vsiRNAs in the same samples as shown in panel D . Bottom values represent relative accumulation ( RA ) of RNA3 - derived vsiRNAs .

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Figure 4Long-term systemic symptoms of Col-0, visp1-1, visp1-2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.Northern blotting detecting accumulation of genomic RNAs and RNA3-derived vsiRNAs in systemically infected leaves at 14 dpi. Relative accumulation (RA) of gRNA3 was calculated from band densities.   Northern blotting analyzing accumulation of RNA3-derived vsiRNAs in the same samples as shown in panel D. Bottom values represent relative accumulation (RA) of RNA3-derived vsiRNAs.


Figure 5Co - immunoprecipitation analyses of the interactions between SGS3 , SGS3ΔCC , or SGS3CC and VISP1 or VISP1mARM . N . benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3 - MA at 48 hpi , and collected for IP with anti - Flag beads 12 h later . GST pull - down analysis of the in vitro interactions of SGS3CC with VISP1 , VISP1mARM or VISP1mUIM . GST - VISP1 , GST - VISP1mARM , GST - VISP1mUIM or GST were incubated with SGS3CC - His for IP with anti - GST beads , and detected by Western blotting with GST or His antibodies .

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Figure 5Co-immunoprecipitation analyses of the interactions between SGS3, SGS3ΔCC, or SGS3CC and VISP1 or VISP1mARM. N. benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3-MA at 48 hpi, and collected for IP with anti-Flag beads 12 h later.GST pull-down analysis of the in vitro interactions of SGS3CC with VISP1, VISP1mARM or VISP1mUIM. GST- VISP1, GST- VISP1mARM, GST- VISP1mUIM or GST were incubated with SGS3CC-His for IP with anti-GST beads, and detected by Western blotting with GST or His antibodies.


Figure 6Confocal analysis of co - localization of CFP - NbATG8f - labelled autophagic bodies with the VISP1 - YN and SGS3 - YC bodies in N . benthamiana leaves . The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi . Scale bar = 20 μm . Confocal analysis of effect of VISP1 - Flag VISP1mARM - Flag , and VISP1mUIM - Flag on RDR6 - YN and SGS3 - YC formed bodies in N . benthamiana leaves at 60 hpi . EV : empty vector . Scale bar = 20 μm . Numbers of RDR6 - YN and SGS3 - YC formed bodies per 10 cells in E were used for quantification . Data points represent means of three biological repeats . Error bars indicate SD . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) . Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2 - week - old Col - 0 , VISP1OE , visp1 , and sgs3 - 1 Arabidopsis plants with anti - SGS3 antibodies . Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2 - week - old Col - 0 , VISP1OE , VISP1mUIM / OE , and VISP1mARM / OE Arabidopsis plants . Western blotting detecting accumulation of the endogenous SGS3 protein in 2 - week - old VISP1 - overexpressing Col - 0 , atg5 - 1 , and atg7 - 1 mutants .

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Figure 6Confocal analysis of co-localization of CFP-NbATG8f-labelled autophagic bodies with the VISP1-YN and SGS3-YC bodies in N. benthamiana leaves. The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi. Scale bar = 20 μm.Confocal analysis of effect of VISP1-Flag VISP1mARM-Flag, and VISP1mUIM-Flag on RDR6-YN and SGS3-YC formed bodies in N. benthamiana leaves at 60 hpi. EV: empty vector. Scale bar = 20 μm.   Numbers of RDR6-YN and SGS3-YC formed bodies per 10 cells in E were used for quantification. Data points represent means of three biological repeats. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).   Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, visp1, and sgs3-1 Arabidopsis plants with anti-SGS3 antibodies.   Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, VISP1mUIM/OE, and VISP1mARM/OE Arabidopsis plants.   Western blotting detecting accumulation of the endogenous SGS3 protein in 2-week-old VISP1-overexpressing Col-0, atg5-1, and atg7-1 mutants.


Figure 7Developmental phenotypes of rosette leaves of 5 - week - old Col - 0 , visp1 - 1 , visp1 - 2 , VISP1OE1 , VISP1OE2 , and sgs3 - 1 seedlings . Arrowheads indicate the 8th and 9th rosette leaves from the top leaves . Scale bar = 2 cm . The length / width ratios of the 8th and 9th rosette leaves of the seedlings shown in A . Every dot represents data from the length / width ratio of one leave ( n = 20 ) . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) . Northern blotting detecting accumulation of TAS1 - TasiR255 , TAS2 - TasiR1511 , TAS3 - TasiR5 ′ D8 , miR173 , and miR390 in leaves of Arabidopsis plants shown in panel A . Values represent relative accumulation ( RA ) of siRNAs and values in Col - 0 plants were set as 1 . rRNA served as loading controls .

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Figure 7Developmental phenotypes of rosette leaves of 5-week-old Col-0, visp1-1, visp1-2, VISP1OE1, VISP1OE2, and sgs3-1 seedlings. Arrowheads indicate the 8th and 9th rosette leaves from the top leaves. Scale bar = 2 cm.   The length/width ratios of the 8th and 9th rosette leaves of the seedlings shown in A. Every dot represents data from the length/width ratio of one leave (n = 20). Letters indicate significant   differences (ANOVA, P < 0.05).Northern blotting detecting accumulation of TAS1-TasiR255, TAS2-TasiR1511, TAS3-TasiR5′D8, miR173, and miR390 in leaves of Arabidopsis plants shown in panel A. Values represent relative accumulation (RA) of siRNAs and values in Col-0 plants were set as 1. rRNA served as loading controls.


Figure 1 ( G ) t - SNE ( top ) and violin plots ( bottom ) showing the expression of selected signature genes in different cell clusters . ( J ) Representative images of immunofluorescence of CCSC marker gene ( LGR5 ) and non - CCSC cluster signature genes in the xenografts harvested at different time points . Cluster T1 ( KRT19 ) , Cluster T2 ( HSPA1B ) , Cluster T3 ( Ki67 ) , Cluster T4 ( AGR2 ) , Cluster T5 ( ODC1 ) , and Cluster T6 ( ANXA1 ) were immunostained . Scale bar , 60 μm . ( K ) Quantification of ( J ) showing the dynamic change of cell clusters during the progression of CD133 + LGR5 + CCSC - formed xenografts . Error bars represent SEM of three xenografts . Three sections of each xenograft and 3 fields of each section were quantified .

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

Figure 1(G) t-SNE (top) and violin plots (bottom) showing the expression of selected signature genes in different cell clusters.(J) Representative images of immunofluorescence of CCSC marker gene (LGR5) and non-CCSC cluster signature genes in the xenografts harvested at different time points. Cluster T1 (KRT19), Cluster T2 (HSPA1B), Cluster T3 (Ki67), Cluster T4 (AGR2), Cluster T5 (ODC1), and Cluster T6 (ANXA1) were immunostained. Scale bar, 60 μm.(K) Quantification of (J) showing the dynamic change of cell clusters during the progression of CD133+LGR5+ CCSC-formed xenografts. Error bars represent SEM of three xenografts. Three sections of each xenograft and 3 fields of each section were quantified.


Figure 2 ( A ) Representative images of CD133 and LGR5 immunofluorescence in CCSC sphere cells bearing a Notch - GFP reporter . Scale bar , 60 μm . ( B ) Representative images of CD133 and LGR5 immunofluorescence in dividing CCSCs carrying a Notch - GFP reporter . Scale bar , 10 μm . ( C ) Representative time - lapse images of a Notch - GFP reporter during asymmetric division of CCSCs . Scale bar , 30 μm . ( G ) t - SNE ( up ) and Violin plot ( bottom ) showing the expression of representative marker genes in each cluster .

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Figure 2(A) Representative images of CD133 and LGR5 immunofluorescence in CCSC sphere cells bearing a Notch-GFP reporter. Scale bar, 60 μm. (B) Representative images of CD133 and LGR5 immunofluorescence in dividing CCSCs carrying a Notch-GFP reporter. Scale bar, 10 μm. (C) Representative time-lapse images of a Notch-GFP reporter during asymmetric division of CCSCs. Scale bar, 30 μm.(G) t-SNE (up) and Violin plot (bottom) showing the expression of representative marker genes in each cluster.