Figure 3 ( A ) Cell count - based comparison of cellular proliferation of T - ALL cells after treatment with dexamethasone ( Dexa ) . Individual and mean values of three biological replicates ± SD analyzed by two - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( B ) Flow cytometric analysis of cell death by AnnexinV staining in T - ALL cell lines upon treatment with indicated Dexa concentrations . Cell death is represented by the number of AnnexinV positive ( AnnexinV + ) , apoptotic cells as a percentage ( % ) of total cells . Mean values of n ≥ 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( C ) Expression levels of human GRα and LRH - 1 mRNA in human T - ALL cell lines were determined by probe - based real - time quantitative PCR , calculated as relative expression compared to beta - actin and used to determine the GR : LRH - 1 ratio ( right ) . Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM - C7 and GC - resistant cell lines were determined by one - way ANOVA . ( D ) Immunoblots of GR , BimEL , BimL and BimS and Tubulin from T - ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa . Representative results from n = 2 biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( E ) Immunoblots of GR , PARP ( nuclear loading control ) and Tubulin ( cytoplasmatic loading control ) from cytoplasmic ( CP ) and nucleoplasmic ( NP ) lysates of T - ALL cells treated with Dexa . Note : Nuclear extracts were more highly concentrated ( approx . 6 - fold ) than cytosolic fractions . Representative results from two biological replicates are shown . ( F ) GR activity in human T - ALL cells transfected with a control luciferase reporter plasmid ( pGL3 ) ( F ) GR responsive ( GRE ) luciferase reporter and treated with 3d2 and / or Dexa . β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO - treated pGL3 control . Individual and mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( G ) LRH - 1 activity in human T - ALL cells transfected with a control luciferase reporter plasmid ( pGL3 ) and ( G ) LRH - 1 responsive ( 5xRE ) luciferase reporter and treated with 3d2 β - galactosidase ( bGal ) was co - transfected as an internal transfection control . Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO - treated pGL3 control . Individual and mean values analyzed by two - way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM - C7 whereas technical triplicates ± SD of a representative experiment ( n = 3 biological replicates ) are shown for MOLT - 4 . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( H ) Flow cytometry - based quantification of BiFC in Jurkat cells transiently co - transfected with YFP1 - LRH - 1 and GR - YFP2 and treated for indicated time periods with Dexa . YFP + cells are shown as a percentage ( % ) of viable cells . Individual and mean values ± SD of three biological replicates analyzed by one - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0h ) was used as a solvent control . ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 3(A) Cell count-based comparison of cellular proliferation of T-ALL cells after treatment with dexamethasone (Dexa). Individual and mean values of three biological replicates ± SD analyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(B) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated Dexa concentrations. Cell death is represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells. Mean values of n≥4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(C) Expression levels of human GRα and LRH-1 mRNA in human T-ALL cell lines were determined by probe-based real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Individual and mean values of four biological replicates ± SD are shown and statistically significant differences between CEM-C7 and GC-resistant cell lines were determined by one-way ANOVA.(D) Immunoblots of GR, BimEL, BimL and BimS and Tubulin from T-ALL cells treated for 24 h with 20 µM 3d2 or 30 µM Dexa. Representative results from n=2 biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(E) Immunoblots of GR, PARP (nuclear loading control) and Tubulin (cytoplasmatic loading control) from cytoplasmic (CP) and nucleoplasmic (NP) lysates of T-ALL cells treated with Dexa. Note: Nuclear extracts were more highly concentrated (approx. 6-fold) than cytosolic fractions. Representative results from two biological replicates are shown.(F) GR activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) (F) GR responsive (GRE) luciferase reporter and treated with 3d2 and/or Dexa. β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(G) LRH-1 activity in human T-ALL cells transfected with a control luciferase reporter plasmid (pGL3) and (G) LRH-1 responsive (5xRE) luciferase reporter and treated with 3d2 β-galactosidase (bGal) was co-transfected as an internal transfection control. Luciferase reporter activity was normalized to bGal activity and calculated as relative to the DMSO-treated pGL3 control. Individual and mean values analyzed by two-way ANOVA of three biological replicates ± SD are shown for Jurkat and CEM-C7 whereas technical triplicates ± SD of a representative experiment (n=3 biological replicates) are shown for MOLT-4. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.(H) Flow cytometry-based quantification of BiFC in Jurkat cells transiently co-transfected with YFP1-LRH-1 and GR-YFP2 and treated for indicated time periods with Dexa. YFP+ cells are shown as a percentage (%) of viable cells. Individual and mean values ± SD of three biological replicates analyzed by one-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0h) was used as a solvent control. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.


Figure 4 ( A ) Daily cell counting - based comparison of cellular proliferation of T - ALL cells after ( A ) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 . ( B ) Daily cell counting - based comparison of cellular proliferation of T - ALL cells after ( B ) small hairpin RNA - mediated LRH - 1 knockdown ( shLRH - 1 ) . Individual and mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD anlyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 . ( C ) Cell cycle distribution of T - ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry . Representative results from n = 3 biological replicates are shown . Stacked bars represent the mean of technical triplicates ± SD . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 . ( D ) Simultaneous flow cytometric analysis of cell cycle distribution by EdU / DAPI staining and cell death by AnnexinV staining in MOLT - 4 cells upon treatment with 3d2 . Dead cells are represented by the number of AnnexinV positive ( AnnexinV + ) , apoptotic cells as a percentage ( % ) of total cells and cell cycle phase distribution as % of viable , AnnexinV negative ( AnnexinV - ) cells . Representative results from three biological replicates are shown . Bars represent the mean of technical triplicates ± SD and differences in AnnexinV + cells were analyzed by one - way ANOVA . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 . ( E ) Flow cytometric analysis of cell death by AnnexinV staining in T - ALL cell lines upon treatment with indicated 3d2 concentrations . Mean values of n = 4 biological replicates ± SD are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 . ( F ) Flow cytometric analysis of 3d2 - induced cell death by AnnexinV staining in wild - type ( WT ) , Bcl - 2 overexpressing ( Bcl - 2 ) and Z - VAD - FMK ( zVAD ) - treated CEM - C1 . Mean values of technical triplicates of one representative experiment ( n = 3 biological replicates ) ± SD analyzed by two - way ANOVA are shown . Data information : ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 . ( G ) Immunoblots of full - length and cleaved ( cl . ) PARP , P - p38 , c - Myc , Caspase 3 ( Casp . 3 ) , cl . Casp . 3 , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated with 40 µM 3d2 for indicated time periods . Representative results from two biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø was used as a solvent control . ns = not significant , * p < 0 . 05 , * * * p < 0 . 001 .

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

Figure 4(A) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (A) treatment with 3d2 Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.(B) Daily cell counting-based comparison of cellular proliferation of T-ALL cells after (B) small hairpin RNA-mediated LRH-1 knockdown (shLRH-1). Individual and mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD anlyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(C) Cell cycle distribution of T-ALL cells treated for 24 h with indicated concentrations of 3d2 as measured by propidium iodide flow cytometry. Representative results from n=3 biological replicates are shown. Stacked bars represent the mean of technical triplicates ± SD. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(D) Simultaneous flow cytometric analysis of cell cycle distribution by EdU/DAPI staining and cell death by AnnexinV staining in MOLT-4 cells upon treatment with 3d2. Dead cells are represented by the number of AnnexinV positive (AnnexinV+), apoptotic cells as a percentage (%) of total cells and cell cycle phase distribution as % of viable, AnnexinV negative (AnnexinV-) cells. Representative results from three biological replicates are shown. Bars represent the mean of technical triplicates ± SD and differences in AnnexinV+ cells were analyzed by one-way ANOVA. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(E) Flow cytometric analysis of cell death by AnnexinV staining in T-ALL cell lines upon treatment with indicated 3d2 concentrations. Mean values of n=4 biological replicates ± SD are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(F) Flow cytometric analysis of 3d2-induced cell death by AnnexinV staining in wild-type (WT), Bcl-2 overexpressing (Bcl-2) and Z-VAD-FMK (zVAD)-treated CEM-C1. Mean values of technical triplicates of one representative experiment (n=3 biological replicates) ± SD analyzed by two-way ANOVA are shown. Data information: ns = not significant, * p < 0.05, *** p < 0.001.(G) Immunoblots of full-length and cleaved (cl.) PARP, P-p38, c-Myc, Caspase 3 (Casp. 3), cl. Casp. 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 for indicated time periods. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø was used as a solvent control. ns = not significant, * p < 0.05, *** p < 0.001.


Figure 6 ( A ) Immunoblots of full - length and cleaved ( cl . ) PARP , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated for 24 h with 10 µM dexamethasone ( Dexa ) and 3d2 . Representative results from three biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control . ( B ) Flow cytometric analysis of cell death by AnnexinV staining in MOLT - 4 cells treated with 3d2 , 30 µM Dexa and / or 50 µM pan caspase inhibitor Z - VAD - FMK ( zVAD ) . Individual and mean values of technical triplicates of one representative out of n = 3 biological replicates ± SD are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control . Two - way ANOVA ( B ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 . ( C ) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout ( Bim KO ) CEM - C7 cells treated with 3d2 and 30 µM Dexa . Mean values of technical triplicates of one representative of n = 4 biological replicates ± SD are shown . Data information : Two - way ANOVA C , ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 . ( D ) Immunoblots of BimEL , BimL and BimS , Bcl - 2 and Tubulin from MOLT - 4 expressing a non - ( shcontrol ) or LRH - 1 - ( shLRH - 1 ) targeting small hairpin RNA construct . ( E ) Flow cytometric analysis of cell death by AnnexinV staining in control or glucocorticoid receptor knockout ( GR KO ) MOLT - 4 cells treated with 3d2 and 30 µM Dexa . Mean values of n = 3 biological replicates ± SD are shown . Data information : Two - way ANOVA E ) ; ns = not significant , * * p < 0 . 01 , * * * p < 0 . 001 . ( F / G ) mRNA expression levels of human GR ( F ) and Bim ( G ) in shcontrol and shLRH - 1 CEM - C1 cells were determined by real - time quantitative PCR after treatment with 1 µM Dexa for indicated times . Fold induction relative to the untreated shcontrol sample ( 0h ) was calculated after normalization to beta - actin . Individual values of technical triplicates of one representative of two biological replicates ± SD are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by 0 was used as a solvent control . ( H ) Immunoblots of full - length and cleaved ( cl . ) PARP , GR and phospho - AMPK ( P - AMPK ) , c - Myc , full - length and cl . Caspase 3 , BimEL , BimL and BimS and Tubulin from MOLT - 4 cells treated with 40 µM 3d2 and / or 10 µM Dexa . Representative results from two biological replicates are shown . Data information : For all displayed experiments dimethyl sulfoxide ( DMSO ; represented by Ø ) was used as a solvent control .

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

Figure 6(A) Immunoblots of full-length and cleaved (cl.) PARP, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated for 24 h with 10 µM dexamethasone (Dexa) and 3d2. Representative results from three biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.(B) Flow cytometric analysis of cell death by AnnexinV staining in MOLT-4 cells treated with 3d2, 30 µM Dexa and/or 50 µM pan caspase inhibitor Z-VAD-FMK (zVAD). Individual and mean values of technical triplicates of one representative out of n=3 biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control. Two-way ANOVA (B ns = not significant, ** p < 0.01, *** p < 0.001.(C) Flow cytometric analysis of cell death by AnnexinV staining in control or Bim knockout (Bim KO) CEM-C7 cells treated with 3d2 and 30 µM Dexa. Mean values of technical triplicates of one representative of n=4 biological replicates ± SD are shown. Data information: Two-way ANOVA C, ns = not significant, ** p < 0.01, *** p < 0.001.(D) Immunoblots of BimEL, BimL and BimS, Bcl-2 and Tubulin from MOLT-4 expressing a non- (shcontrol) or LRH-1- (shLRH-1) targeting small hairpin RNA construct.(E) Flow cytometric analysis of cell death by AnnexinV staining in control or glucocorticoid receptor knockout (GR KO) MOLT-4 cells treated with 3d2 and 30 µM Dexa. Mean values of n=3 biological replicates ± SD are shown. Data information: Two-way ANOVA E); ns = not significant, ** p < 0.01, *** p < 0.001.(F/G) mRNA expression levels of human GR (F) and Bim (G) in shcontrol and shLRH-1 CEM-C1 cells were determined by real-time quantitative PCR after treatment with 1 µM Dexa for indicated times. Fold induction relative to the untreated shcontrol sample (0h) was calculated after normalization to beta-actin. Individual values of technical triplicates of one representative of two biological replicates ± SD are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by 0 was used as a solvent control.(H) Immunoblots of full-length and cleaved (cl.) PARP, GR and phospho-AMPK (P-AMPK), c-Myc, full-length and cl. Caspase 3, BimEL, BimL and BimS and Tubulin from MOLT-4 cells treated with 40 µM 3d2 and/or 10 µM Dexa. Representative results from two biological replicates are shown. Data information: For all displayed experiments dimethyl sulfoxide (DMSO; represented by Ø) was used as a solvent control.


Figure 7 ( A ) Ex vivo response of human T cell acute lymphoblastic leukemia ( T - ALL ) patient - derived xenografts ( PDX ) , co - cultured on hTERT - immortalized primary bone marrow MSCs , to treatment with indicated concentrations of 3d2 and / or dexamethasone ( Sample 1 , 3 and 4 : 1 nM ; Sample 2 : 0 . 1 nM ) . Cell viability was quantified by fluorescent live cell staining using CyQuant combined with automated image analysis and calculated as percentage ( % ) of the dimethyl sulfoxide treated control . Data information : All displayed experiments display one biological replicate . ( B ) Synergy scores ( Z - scores ) between 3d2 and dexamethasone of human T - ALL patient - derived xenografts ( PDX ) were calculated from the viability curves of PDX samples treated with increasing concentrations of 3d2 and / or dexamethasone using SynergyFinder tool . Z - scores ≥ 0 indicate additivity or synergism and < 0 antagonism . Data information : All displayed experiments display one biological replicate . ( C ) Expression levels of human GRα , LRH - 1 and Bim mRNA in human PDX were determined by real - time quantitative PCR , calculated as relative expression compared to beta - actin and used to determine the GR : LRH - 1 ratio ( right ) . Data information : All displayed experiments display one biological replicate . ( E ) In vivo leukemia progression of human T - ALL PDX transplanted into NSG mice treated for 3 weeks with vehicle ( PBS control ) , 40 mg / kg 3d2 and or 10 mg / kg Dexamethasone starting 3 days post transplantation . Engraftment was calculated as % of human vs total ( human + mouse ) CD45 positive ( CD45 + ) cells as assessed by flow cytometric analysis of peripheral blood . Data information : All displayed experiments display one biological replicate .

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

Figure 7(A) Ex vivo response of human T cell acute lymphoblastic leukemia (T-ALL) patient-derived xenografts (PDX), co-cultured on hTERT-immortalized primary bone marrow MSCs, to treatment with indicated concentrations of 3d2 and/or dexamethasone (Sample 1, 3 and 4: 1 nM; Sample 2: 0.1 nM). Cell viability was quantified by fluorescent live cell staining using CyQuant combined with automated image analysis and calculated as percentage (%) of the dimethyl sulfoxide treated control. Data information: All displayed experiments display one biological replicate.(B) Synergy scores (Z-scores) between 3d2 and dexamethasone of human T-ALL patient-derived xenografts (PDX) were calculated from the viability curves of PDX samples treated with increasing concentrations of 3d2 and/or dexamethasone using SynergyFinder tool. Z-scores ≥ 0 indicate additivity or synergism and < 0 antagonism. Data information: All displayed experiments display one biological replicate.(C) Expression levels of human GRα, LRH-1 and Bim mRNA in human PDX were determined by real-time quantitative PCR, calculated as relative expression compared to beta-actin and used to determine the GR:LRH-1 ratio (right). Data information: All displayed experiments display one biological replicate.(E) In vivo leukemia progression of human T-ALL PDX transplanted into NSG mice treated for 3 weeks with vehicle (PBS control), 40 mg/kg 3d2 and or 10 mg/kg Dexamethasone starting 3 days post transplantation. Engraftment was calculated as % of human vs total (human+mouse) CD45 positive (CD45+) cells as assessed by flow cytometric analysis of peripheral blood. Data information: All displayed experiments display one biological replicate.


Figure 1 ( a ) Immunofluorescence labeling of wildtype mouse dorsal skin samples ( Dnmt1f / f , skin from P3 ) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte - specific knockout ( Dnmt1Δ / Δep ) results in the almost absence of a specific staining in the epidermis and in hair follicles . Dashed lines indicate the dermal - epidermal border and dotted lines indicate hair follicles . ( d ) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5 - mC labeling of P3 skin . Dashed lines indicate the dermal - epidermal border and dotted lines indicate hair follicles . ( h ) Hematoxylin and Eosin ( H & E ) staining , immunolabeling for KERATIN 10 ( KRT10 ) , LORICRIN ( LOR ) and KERATIN 6 ( KRT6 ) expression of dorsal skin sections ( P5 ) of control and Dnmt1Δ / Δep mice .

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

Figure 1(a) Immunofluorescence labeling of wildtype mouse dorsal skin samples (Dnmt1f/f, skin from P3) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte-specific knockout (Dnmt1Δ/Δep) results in the almost absence of a specific staining in the epidermis and in hair follicles. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.(d) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5-mC labeling of P3 skin. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.(h) Hematoxylin and Eosin (H&E) staining, immunolabeling for KERATIN 10 (KRT10), LORICRIN (LOR) and KERATIN 6 (KRT6) expression of dorsal skin sections (P5) of control and Dnmt1Δ/Δep mice.


Figure 2 ( c ) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ / Δep mice of different postnatal age . Cells are shown as percent of viable cells . Data are mean ± SEM , two - tailed t - test . ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , n = 3 ( P3 ) , 4 ( P5 ) and 4 ( P6 + P7 ) . Data information : Scale bars , 50 µm . ( d ) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ / Δep mice at P3 ( n = 3 ) , P5 ( n = 5 ) and P7 ( n ≥ 3 ) . Data are mean ± SEM . P values are calculated using one - way ANOVA with post hoc Tukey multiple comparison test , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . Data information : Scale bars , 50 µm .

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

Figure 2(c) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ/Δep mice of different postnatal age. Cells are shown as percent of viable cells. Data are mean ± SEM, two-tailed t-test. ns, not significant, * p≤0.05, ** p≤0.01, **** p≤0.0001, n=3 (P3), 4 (P5) and 4 (P6 + P7). Data information: Scale bars, 50 µm.(d) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ/Δep mice at P3 (n=3), P5 (n=5) and P7 (n≥3). Data are mean ± SEM. P values are calculated using one-way ANOVA with post hoc Tukey multiple comparison test, * p≤0.05, ** p≤0.01, *** p≤0.001 , **** p≤0.0001. Data information: Scale bars, 50 µm.


Figure 3 ( a ) Relative mRNA expression levels of epidermal Intracisternal A - type particle ( IAP ) during different postnatal time points ( P0 n ≥ 3 , P3 n = 3 , P5 n = 5 , P7 n ≥ 3 mice ) , which were compared using one way - ANOVA with post hoc Tukey multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 . ( c ) Relative expression of immune related genes of the epidermis from P7 of control ( n ≥ 7 ) , Dnmt1Δ / Δep , ( n ≥ 8 ) , MAVS knockout ( Mavs - / - , n = 7 ) and Dnmt1Δ / Δep Mavs - / - ( n = 6 ) mice were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

Figure 3(a) Relative mRNA expression levels of epidermal Intracisternal A-type particle (IAP) during different postnatal time points (P0 n≥3, P3 n=3, P5 n=5, P7 n≥3 mice), which were compared using one way-ANOVA with post hoc Tukey multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01.(c) Relative expression of immune related genes of the epidermis from P7 of control (n≥7), Dnmt1Δ/Δep, (n≥8), MAVS knockout (Mavs-/-, n=7) and Dnmt1Δ/Δep Mavs-/- (n=6) mice were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.


Figure 4Immortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( c , d ) PicoGreen staining of PBS - and DAC - treated keratinocytes for 48h detected micronuclei ( arrow ) , which were analyzed in a blinded manner , and compared using two - tailed t - test . Data are mean ± SEM , * p ≤ 0 . 05 ( n = 3 biological replicates ) ( d ) . Data information : Scale bars , 20 µmImmortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( e ) Co - labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes . Within enlarged sections on the right arrows point at DNA / cGAS double - positive blebs and micronuclei . ( f ) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way - ANOVA with post hoc Dunnett multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ( n = 4 biological replicates ) . Data information : Scale bars , , 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 O O O O O O O O O O O O O O O O O O O O O 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

Figure 4Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (c, d) PicoGreen staining of PBS- and DAC-treated keratinocytes for 48h detected micronuclei (arrow), which were analyzed in a blinded manner, and compared using two-tailed t-test. Data are mean ± SEM, * p≤0.05 (n=3 biological replicates) (d). Data information: Scale bars, 20 µmImmortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (e) Co-labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes. Within enlarged sections on the right arrows point at DNA/cGAS double-positive blebs and micronuclei. (f) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way-ANOVA with post hoc Dunnett multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001 (n=4 biological replicates). Data information: Scale bars, , 50 µm


Figure 5 ( c ) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f / f , Dnmt1Δ / Δep , and Dnmt1Δ / Δep Cgas - / - mice . Dashed lines indicate the dermal - epidermal border . ( d ) Quantification of CD45 - positve cells . Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ . Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . Data information : Scale bars , 100 µm ( e ) Epidermis obtained from P7 control , Dnmt1Δ / Δep , Cgas - / - and Dnmt1Δ / Δep Cgas - / - mice was analyzed for relative mRNA expression levels of immune related genes using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 . At least 7 mice per genotype were used .

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

Figure 5(c) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f/f, Dnmt1Δ/Δep, and Dnmt1Δ/Δep Cgas-/- mice. Dashed lines indicate the dermal-epidermal border. (d) Quantification of CD45-positve cells. Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ. Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ** p≤0.01, *** p≤0.001. Data information: Scale bars, 100 µm(e) Epidermis obtained from P7 control, Dnmt1Δ/Δep, Cgas-/- and Dnmt1Δ/Δep Cgas-/- mice was analyzed for relative mRNA expression levels of immune related genes using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, **** p≤0.0001. At least 7 mice per genotype were used.


Figure 152 - day - old ( A ) plants of the genotypes indicated grown in LDs . Scale bars : 5 cm . B - C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In ( B - C data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) . D - E qPCR results showing mRNA expression of CIB1 , FT in the genotypes indicated . The numbers indicate independent transgenic lines . The transcript levels of CIB1 or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed . F . 25 - day - old plants ( F ) of the genotypes indicated grown in LDs . Scale bars : 5 cm . , G - H . The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In G - H ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) . , I - J . qPCR results showing mRNA expression of CO , FT in the genotypes indicated . The numbers indicate independent transgenic lines . The transcript levels of CO or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed .

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

Figure 152-day-old (A) plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B-C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B-C data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).D-E qPCR results showing mRNA expression of CIB1, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CIB1 or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.F. 25-day-old plants (F) of the genotypes indicated grown in LDs. Scale bars: 5 cm. , G-H. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In G-H), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test)., I-J. qPCR results showing mRNA expression of CO, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CO or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


Figure 2Immunoblots showing CIB1 ( A ) protein levels in WT and co - 1 backgrounds ( A ) over the course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - actin antibody ( ACT ) . B . Immunoblots showing CO ( B ) protein levels in WT and cibh backgrounds ( B ) over the course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - actin antibody ( ACT ) . The relative levels of CIB1 in WT and co - 1 backgrounds over the course of 20 hours under LDs were calculated Data information : Error bars represent the standard deviation of three biological replicates of the Western blots . D . The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information : Error bars represent the standard deviation of three biological replicates of the Western blots .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O 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 2Immunoblots showing CIB1 (A) protein levels in WT and co-1 backgrounds (A) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).B. Immunoblots showing CO (B) protein levels in WT and cibh backgrounds (B) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).The relative levels of CIB1 in WT and co-1 backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.D. The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.


Figure 3A . Fluorescent microscopy images showing that CIB1 co - localizes with CO in nuclear speckles . Scale bars : 2 μm . B . BiFC assays to show in vivo protein interactions . Epidermal cells of N . benthamiana leaves were co - transformated with cCFP - CO and nYFP - CIB1 , with cCFP - CO and nYFP , or with cCFP and nYFP - CIB1 . BF , bright field . Merge , overlay of the YFP and bright field images . Scale bars : 50 μm . Data information : For each experiment , two or more biological replicates were performed . C . Split - LUC assay showing that CIB1 interacts with CO . Leaf epidermal cells of N . benthamiana were co - transformed with CO - nLUC and cLUC - CIB1 or cLUC or nLUC with cLUC - CIB or cLUC . Data information : For each experiment , two or more biological replicates were performed . D . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 , 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag seedlings grown in LDs . Data information : For each experiment , two or more biological replicates were performed . F . In vitro pull - down assays showing the interaction between CIBC and CO , or CIBC and COΔN and the lack of interaction between CIB1C and COΔC . His - TF tagged CO , COΔN , COΔC or His - TF tagged UVR8 were mixed with GST - CIB1C purified from E . coli , and His antibody was used for the in vitro pull - down assay . The products were analyzed by immunoblots probed with the anti - CIB1C antibody or the anti - His antibody ( UVR8 , CO , COΔC , COΔN ) . Data information : For each experiment , two or more biological replicates were performed .

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

Figure 3A. Fluorescent microscopy images showing that CIB1 co-localizes with CO in nuclear speckles. Scale bars: 2 μm.B. BiFC assays to show in vivo protein interactions. Epidermal cells of N. benthamiana leaves were co-transformated with cCFP-CO and nYFP-CIB1, with cCFP-CO and nYFP, or with cCFP and nYFP-CIB1. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bars: 50 μm. Data information: For each experiment, two or more biological replicates were performed.C. Split-LUC assay showing that CIB1 interacts with CO. Leaf epidermal cells of N. benthamiana were co-transformed with CO-nLUC and cLUC-CIB1 or cLUC or nLUC with cLUC-CIB or cLUC. Data information: For each experiment, two or more biological replicates were performed.D. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1, 35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag seedlings grown in LDs. Data information: For each experiment, two or more biological replicates were performed.F. In vitro pull-down assays showing the interaction between CIBC and CO, or CIBC and COΔN and the lack of interaction between CIB1C and COΔC. His-TF tagged CO, COΔN, COΔC or His-TF tagged UVR8 were mixed with GST-CIB1C purified from E. coli, and His antibody was used for the in vitro pull-down assay. The products were analyzed by immunoblots probed with the anti-CIB1C antibody or the anti-His antibody (UVR8, CO, COΔC, COΔN). Data information: For each experiment, two or more biological replicates were performed.


Figure 4A . 28 - day - old plants of the genotypes indicated grown in LDs . Scale bars : 5 cm . B , C . The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In ( B , C ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) . D . Immunoblots showing Myc - CIB1 and CO - Flag protein levels in the genotypes indicated . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - Flag antibody . E . qPCR results showing mRNA expression of FT in the genotypes indicated . The transcript levels in Col are set to 1 . 0 . Samples were collected from 7 - day - old seedlings of the genotypes indicated at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed . G . Box plots ( the minimum , first quartile , median , third quartile , and maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in plants expressing different reportors and effectors . The relative LUC activities normalized to the REN activity are shown . The LUC / REN in plants expressing ProFT alone is set to 1 . 0 . Data information : data are presented as mean ± SD , n = 4 . The letters " a " to " g " indicate statistically significant differences determined by Tukey ' s HSD test ( P ≤ 0 . 05 ) . Two biological replicates were performed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 28-day-old plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B, C. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B, C), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).D. Immunoblots showing Myc-CIB1 and CO-Flag protein levels in the genotypes indicated. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-Flag antibody.E. qPCR results showing mRNA expression of FT in the genotypes indicated. The transcript levels in Col are set to 1.0. Samples were collected from 7-day-old seedlings of the genotypes indicated at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.G. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The relative LUC activities normalized to the REN activity are shown. The LUC/REN in plants expressing ProFT alone is set to 1.0. Data information: data are presented as mean ± SD, n=4. The letters "a" to "g" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.


Figure 5A . Fluorescent microscopy images showing that CRY2 , CIB1 and CO co - localized to the nucleus . Scale bars : 2 μB . Co - IP assays showing that CRY2 , CIB1 and CO form a protein complex . Samples were prepared from 10 - day - old WT or 35S : Myc - CIB1 / 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) , and samples were collected at ZT16 . C . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) . Samples were collected at the times indicated over the course of one day . E . Representative results of the ChIP - qPCR assays . Chromatin fragments ( ~ 500 bp ) were prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag seedlings , immunoprecipitated by the anti - CRY2 , anti - Flag ( CO - Flag ) , anti - Myc ( Myc - CIB1 ) antibody or anti - preimmune IgG , and the precipitated DNA was analyzed by qPCR using the primer pairs indicated . The IP / input ratio of anti - preimmune is set to 1 . 0 . Data information : the relative IP / input ratios are shown with their standard deviation ( n = 3 ) . Two biological replicates were performed . F . Box plots ( the minimum , first quartile , median , third quartile , and maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in plants expressing different reportors and effectors . The LUC / REN in plants expressing ProFT alone is set to 1 . 0 . Immunoblots were done to show GFP - CRY2W374A , Myc - CIB1 and Myc - CO protein levels in the transient Dual - LUC assay . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , and with the anti - GFP antibody . Data information : the relative LUC activities normalized to the REN activity are shown ( LUC / REN ) with their standard deviation ( n = 6 ) . The letters " a " to " f " indicate statistically significant differences determined by Tukey ' s HSD test ( P ≤ 0 . 05 ) . Two biological replicates were performed .

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

Figure 5A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μB. Co-IP assays showing that CRY2, CIB1 and CO form a protein complex. Samples were prepared from 10-day-old WT or 35S:Myc-CIB1/35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D), and samples were collected at ZT16.C. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D). Samples were collected at the times indicated over the course of one day.E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0. Data information: the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed.F. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The LUC/REN in plants expressing ProFT alone is set to 1.0. Immunoblots were done to show GFP-CRY2W374A, Myc-CIB1 and Myc-CO protein levels in the transient Dual-LUC assay. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, and with the anti-GFP antibody. Data information: the relative LUC activities normalized to the REN activity are shown (LUC/REN) with their standard deviation (n =6). The letters "a" to "f" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.


Figure 1MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes . mRNA was isolated from each time point and PROM2 expression was quantified by qPCR . Shown is an average of three independent experiments with standard deviation ( n = 3 experiments per time point ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer - 1 , followed by 60 minutes with either DMSO , 5uM RSL3 or RSL3 and fer - 1 . mRNA was isolated and PROM2 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes . mRNA was isolated from each time point and PROM2 expression was quantified by qPCR . Shown is an average of three independent experiments with standard deviation ( n = 3 experiments per time point ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer - 1 , followed by 60 minutes with either EtOH , 25uM 4HNE or RSL3 and fer - 1 . mRNA was isolated and PROM2 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . n = 3 experiments per group . Exact p ‑ values are reported in the Appendix Table S1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either DMSO, 5uM RSL3 or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either EtOH, 25uM 4HNE or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. n=3 experiments per group. Exact p‑values are reported in the Appendix Table S1.


Figure 2MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes . Isolated protein was assessed by immunoprecipitating phospho - p38 ( Thr180 / Tyr182 ) and immunoblotting for 4HNE and phospho - p38 ( Thr180 / Tyr182 ) . Shown is one replicate of three independent experiments ( n = 3 ) . MCF10A cells were pretreated for 15 minutes with either DMSO , EtOH , or 10uM BIRB , followed by 60 minutes with either DMSO , EtOH , 25uM 4HNE , 5uM RSL3 , 4HNE and BIRB or RSL3 and BIRB . Isolated protein was assessed by immunoblotting for prominin2 , phospho - p38 ( Thr180 / Tyr182 ) and total p38 . Shown is one replicate of three independent experiments ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes. Isolated protein was assessed by immunoprecipitating phospho-p38 (Thr180/Tyr182) and immunoblotting for 4HNE and phospho-p38 (Thr180/Tyr182). Shown is one replicate of three independent experiments (n=3).MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-p38 (Thr180/Tyr182) and total p38. Shown is one replicate of three independent experiments (n=3).


Figure 3HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO , EtOH , DMSO , 25uM 4HNE or 5uM RSL3 . Isolated protein was assessed by immunoblotting for prominin2 , phospho - HSF1 ( Ser326 ) , total HSF1 , phospho - p38 ( Thr180 / Tyr182 ) , total p38 , and β - actin expression . Shown is one replicate of three independent experiments ( n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO, EtOH, DMSO, 25uM 4HNE or 5uM RSL3. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).


Figure 4MCF10A cells were treated for 60 minutes with either DMSO , 25uM 4HNE , 5uM RSL3 , 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11 . mRNA was isolated and PROM2 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were treated for 60 minutes with either DMSO , 25uM 4HNE , 5uM RSL3 , 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11 . mRNA was isolated and HSF1 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were treated for 60 minutes with either EtOH , DMSO , 25uM 4HNE , 5uM RSL3 , 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11 . Isolated protein was assessed by immunoblotting for prominin2 , phospho ‑ HSF1 ( Ser326 ) , total HSF1 , phospho - p38 ( Thr180 / Tyr182 ) , total p38 , and β - actin expression . Shown is one replicate of three independent experiments ( n = 3 ) .

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

Figure 4MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.MCF10A cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. Isolated protein was assessed by immunoblotting for prominin2, phospho‑HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).


Figure 5Hs578t cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11 . Cells were assessed for viability after 24 hours . Absorbance was normalized to DMSO control . Shown are three independent replicates with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . SF295 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11 . Cells were assessed for viability after 24 hours . Absorbance was normalized to DMSO control . Shown are three independent replicates with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . NCI H1975 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11 . Cells were assessed for viability after 24 hours . Absorbance was normalized to DMSO control . Shown are three independent replicates with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Hs578t cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. SF295 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. NCI H1975 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.


Figure 6Twenty - four hours after the last treatment , tumors were excised , frozen and sectioned . Each tumor was stained with Abs specific for either 4HNE , prominin2 or HSF1 . Images were taken at 20x magnification . Higher ‑ magnification images ( 1 . 5x ) are shown to the right of its corresponding inset ( white box ) . Scale bar is 50 microns . Shown is one representative image for each condition .

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Figure 6Twenty-four hours after the last treatment, tumors were excised, frozen and sectioned. Each tumor was stained with Abs specific for either 4HNE, prominin2 or HSF1. Images were taken at 20x magnification. Higher‑magnification images (1.5x) are shown to the right of its corresponding inset (white box). Scale bar is 50 microns. Shown is one representative image for each condition.


Figure 1 ( A ) Picrosirius red ( PSR ) staining of collagens ( red ) in PDAC tissue following surgical resection . Scale bar represents 200 µm . ( B ) Quantification of PSR in low - grade ( 1 - 2 ) and high - grade ( 3 ) tumors indicated in panel A as percentage of area . n = 15 primary PDAC samples . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Quantification of tumor cell percentage as scored by pathologists in low - and high - grade PDAC indicated in panel A . n = 15 primary PDAC samples . Data information : data are represented as mean ± SD , Student ' s t - test . ( D ) PSR and α - SMA staining of indicated PDAC cell lines which were injected in NSG mice . Scale bar represents 200 µm for PSR and 100 µm for α - SMA images . ( E ) Quantification of stainings indicated in panel D . Three tumor ROIs of a tumor derived from epithelial cell lines ( Capan - 2 and AsPC - 1 ) and mesenchymal cell lines ( MIA PaCa - 2 and PANC - 1 ) are displayed and averages were pooled for analysis . Data information : data are represented as mean ± SD , Student ' s t - test . ( F ) IHC staining of α - SMA in 3D organotypic co - cultures of PS - 1 cells and indicated PDAC cell lines . Scale bar represents 100 µm .

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Figure 1(A) Picrosirius red (PSR) staining of collagens (red) in PDAC tissue following surgical resection. Scale bar represents 200 µm. (B) Quantification of PSR in low-grade (1-2) and high-grade (3) tumors indicated in panel A as percentage of area. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.(C) Quantification of tumor cell percentage as scored by pathologists in low- and high-grade PDAC indicated in panel A. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.(D) PSR and α-SMA staining of indicated PDAC cell lines which were injected in NSG mice. Scale bar represents 200 µm for PSR and 100 µm for α-SMA images. (E) Quantification of stainings indicated in panel D. Three tumor ROIs of a tumor derived from epithelial cell lines (Capan-2 and AsPC-1) and mesenchymal cell lines (MIA PaCa-2 and PANC-1) are displayed and averages were pooled for analysis. Data information: data are represented as mean±SD, Student's t-test.(F) IHC staining of α-SMA in 3D organotypic co-cultures of PS-1 cells and indicated PDAC cell lines. Scale bar represents 100 µm.


Figure 2 ( A ) Brightfield images of PS - 1 cells which were exposed for 72 hrs to CM from control ( CM of PS - 1 cells ) , epithelial ( blue ) or mesenchymal ( red ) cell lines . Scale bar represents 100 µm . ( B ) Quantification of bead - calibrated cell count of PS - 1 cells after treatment using flowcytometry . n = 6 per group , 3 biological replicates for each cell line . ( C ) EdU assay performed after treatment showing percentage of PS - 1 cells in G0 / G1 phase , G2 / M phase and S phase . n = 6 per group , 3 biological replicates for each cell line . .

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Figure 2(A) Brightfield images of PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) cell lines. Scale bar represents 100 µm.(B) Quantification of bead-calibrated cell count of PS-1 cells after treatment using flowcytometry. n=6 per group, 3 biological replicates for each cell line.(C) EdU assay performed after treatment showing percentage of PS-1 cells in G0/G1 phase, G2/M phase and S phase. n=6 per group, 3 biological replicates for each cell line..


Figure 3 ( A ) Gene expression level of stromal activation markers COL1A1 , ACTA2 , SPARC , FN1 and FAP in PS - 1 cells which were exposed for 72 hrs to CM from control ( CM of PS - 1 cells ) , epithelial ( blue ) or mesenchymal ( red ) PDAC cell lines using qPCR . Values were normalized to control and n = 6 per group , 3 biological replicates for each cell line . Data information : data are represented as mean ± SD , Student ' s t - test . ( B ) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS - 1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m . Values were Log2 transformed and normalized to Suit - 2 expression . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Brightfield images of PS - 1 cells exposed to 10 days of treatment with 1 and 10 µM all - trans retinoic acid ( ATRA ) . Scale bar represents 100 µm . ( D ) Gene expression levels of COL1A1 and ACTA2 in PS - 1 cells after treatment using qPCR , values were normalized to control . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test . ( E ) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control ( CM of primary CAFs ) or mesenchymal PDAC cell lines . Scale bar represents 100 µm . ( F ) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR , values were normalized to control . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test .

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Figure 3(A) Gene expression level of stromal activation markers COL1A1, ACTA2, SPARC, FN1 and FAP in PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) PDAC cell lines using qPCR. Values were normalized to control and n=6 per group, 3 biological replicates for each cell line. Data information: data are represented as mean±SD, Student's t-test.(B) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS-1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m. Values were Log2 transformed and normalized to Suit-2 expression. Data information: data are represented as mean±SD, Student's t-test.(C) Brightfield images of PS-1 cells exposed to 10 days of treatment with 1 and 10 µM all-trans retinoic acid (ATRA). Scale bar represents 100 µm.(D) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.(E) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control (CM of primary CAFs) or mesenchymal PDAC cell lines. Scale bar represents 100 µm.(F) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.


Figure 4 ( A ) Brightfield images of PS - 1 cells after 72 hrs of treatment with unfiltered , < 100 kDa or < 30 kDa filtered CM of indicated cell lines . Scale bar represents 100 µm . ( B ) Gene expression levels of COL1A1 and ACTA2 in PS - 1 cells after treatment using qPCR . Data was normalized against control CM . Statistical significance was tested against unfiltered CM samples . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Graphical representation of 64 cyto - and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward - phase protein array . Significantly differential expression of proteins ( triangles ) and genes ( green triangles ) are identified between epithelial and mesenchymal cell lines . Dotted line indicates P = 0 . 05 . Data information : data are represented as mean ± SD , Student ' s t - test . ( D ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or epithelial PDAC cells supplemented with recombinant human CSF - 1 . Scale bar represents 100 µm . ( E ) Gene expression levels of COL1A1 in PS - 1 cells after treatment using qPCR . n = 3 biological replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test . ( F ) PS - 1 cells were starved and stimulated with 500 ng / ml rh - CSF - 1 at indicated time points . Activation of pathways involved in CSF1R signaling was analyzed using western blot . Samples were derived from the same experiment and blots were processed in parallel .

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Figure 4(A) Brightfield images of PS-1 cells after 72 hrs of treatment with unfiltered, <100 kDa or <30 kDa filtered CM of indicated cell lines. Scale bar represents 100 µm.(B) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR. Data was normalized against control CM. Statistical significance was tested against unfiltered CM samples. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.(C) Graphical representation of 64 cyto- and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward-phase protein array. Significantly differential expression of proteins (triangles) and genes (green triangles) are identified between epithelial and mesenchymal cell lines. Dotted line indicates P=0.05. Data information: data are represented as mean±SD, Student's t-test.(D) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or epithelial PDAC cells supplemented with recombinant human CSF-1. Scale bar represents 100 µm.(E) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. n=3 biological replicates per group. Data information: data are represented as mean±SD, Student's t-test.(F) PS-1 cells were starved and stimulated with 500 ng/ml rh-CSF-1 at indicated time points. Activation of pathways involved in CSF1R signaling was analyzed using western blot. Samples were derived from the same experiment and blots were processed in parallel.


Figure 5 ( A ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or mesenchymal PDAC cell lines supplemented with anti - CSF - 1 . Scale bar represents 100 µm . ( B ) Gene expression levels of COL1A1 in PS - 1 cells after treatment using qPCR . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Brightfield images of PS - 1 cells after 72 hrs of treatment with PANC - 1 CM of pLKOctrl or shCSF1 ( # 560 - 562 ) . Scale bar represents 100 µm . ( D ) Correlation graph of CSF1 expression in PANC - 1 pLKOctrl or shCSF1 ( # 560 - 562 ) and how these various CM affect COL1A1 expression in PS - 1 cells using qPCR , values were normalized for RPLPO expression . n = 3 biological replicates for all individual experiments , error bars indicated SD . R2 and P - value were analyzed with linear regression . ( E ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of Capan - 2 cells with control vector or Capan - 2 cells overexpressing CSF1 . Scale bar represents 100 µm . ( F ) Gene expression level of CSF1 in Capan - 2 cells after transduction with control vector or overexpressing CSF1 . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test . ( G ) Gene expression levels of COL1A1 in PS - 1 cells after treatment indicated using qPCR . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test .

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Figure 5(A) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or mesenchymal PDAC cell lines supplemented with anti-CSF-1. Scale bar represents 100 µm.(B) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.(C) Brightfield images of PS-1 cells after 72 hrs of treatment with PANC-1 CM of pLKOctrl or shCSF1 (#560-562). Scale bar represents 100 µm.(D) Correlation graph of CSF1 expression in PANC-1 pLKOctrl or shCSF1 (#560-562) and how these various CM affect COL1A1 expression in PS-1 cells using qPCR, values were normalized for RPLPO expression. n=3 biological replicates for all individual experiments, error bars indicated SD. R2 and P-value were analyzed with linear regression.(E) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of Capan-2 cells with control vector or Capan-2 cells overexpressing CSF1. Scale bar represents 100 µm.(F) Gene expression level of CSF1 in Capan-2 cells after transduction with control vector or overexpressing CSF1. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.(G) Gene expression levels of COL1A1 in PS-1 cells after treatment indicated using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.


Figure 6 ( A ) IHC staining of CSF - 1 in PDAC tissue following surgical resection representative image of CSF - 1 expression in grade 1 and grade 3 PDAC is depicted . Scale bar represents 100 µm . ( B ) For each tumor of panel A , CSF - 1 expression in ductal adenocarcinoma cells was quantified . Student ' s t - test . Shown is mean ± SD . n = 15 primary PDAC samples . ( C ) A validation cohort of PDAC patients ( n = 21 ) that underwent direct resection was stained for CSF - 1 ( scale bar represents 100 µm ) , PSR ( scale bar represents 500 µm ) and α - SMA ( scale bar represents 200 µm ) . OK8 is low in CSF - 1 expression and high in PSR and α - SMA expression , while the opposite accounts for OK7 . ( D ) Correlation of % of PSR in the tumor and CSF - 1 expressing tumor cells , as indicated in panel C . R2 and P - value were analyzed with linear regression . n = 21 primary PDAC samples ( E ) Correlation of % of α - SMA in the tumor and CSF - 1 expressing tumor cells , as indicated in panel C , R2 and P - value were analyzed with linear regression . n = 21 primary PDAC samples

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Figure 6(A) IHC staining of CSF-1 in PDAC tissue following surgical resection representative image of CSF-1 expression in grade 1 and grade 3 PDAC is depicted. Scale bar represents 100 µm. (B) For each tumor of panel A, CSF-1 expression in ductal adenocarcinoma cells was quantified. Student's t-test. Shown is mean±SD. n=15 primary PDAC samples.(C) A validation cohort of PDAC patients (n=21) that underwent direct resection was stained for CSF-1 (scale bar represents 100 µm), PSR (scale bar represents 500 µm) and α-SMA (scale bar represents 200 µm). OK8 is low in CSF-1 expression and high in PSR and α-SMA expression, while the opposite accounts for OK7. (D) Correlation of % of PSR in the tumor and CSF-1 expressing tumor cells, as indicated in panel C. R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples (E) Correlation of % of α-SMA in the tumor and CSF-1 expressing tumor cells, as indicated in panel C, R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples


Figure 1D . Immunoblot detection of the indicated proteins in whole cell extracts ( WCE ) , PrimPol immunoprecipitates ( IP ) , and non - precipitated flow - through fractions ( FT ) . IgG shows the presence of anti - PrimPol antibody in the IP fractions ( lanes 4 - 6 ) .

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Figure 1D. Immunoblot detection of the indicated proteins in whole cell extracts (WCE), PrimPol immunoprecipitates (IP), and non-precipitated flow-through fractions (FT). IgG shows the presence of anti-PrimPol antibody in the IP fractions (lanes 4-6).