Figure 3 ( A ) Expression of PARP1 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell lines . ( B ) . Accumulation of PARP1 and KLF4 protein were detected in human breast cancer tissues with 183 breast invasive ductal carcinoma and 10 pairs of primary cancer and adjacent normal tissue specimens by IHC in comparison with adjacent normal breast tissues . Scale bars , 100 μm . Statistic results of KLF4 IHC staining . Normal ( n = 10 ) , tumor ( n = 183 ) ; p = 4 . 36x10 - 7 ( KLF4 ) one - way ANOVA was used for the statistical analysis . Statistic results of PARP1 IHC staining . Normal ( n = 10 ) , tumor ( n = 183 ) p = 0 . 0473 ( PARP1 ) . one - way ANOVA was used for the statistical analysis . ( E ) Statistical analysis of PARP1 staining among normal ( n = 10 ) , ER / PR positive ( ER / PR ) ( n = 68 ) , HER2 positive ( n = 62 ) , and triple negative breast cancer ( TNBC ) ( n = 48 ) . The p values were labeled in figure , one - way ANOVA was used for the statistical analysis . ( F ) Statistical analysis of KLF4 protein staining among normal ( n = 10 ) , ER / PR positive ( n = 68 ) , HER2 positive ( n = 62 ) and TNBC ( n = 47 ) . The p values were labeled in figure , one - way ANOVA was used for the statistical analysis . Elevated expression of PARP1 and KLF4 are significantly correlated in 183 breast invasive ductal carcinoma human breast cancer tissue specimens ( same batch of staining as ( B ) ) . Represents of paired IHC staining of PARP1 and KLF4 ( case 1 - 3 ) are shown ( G ) . Scale bars , 100 μm . ( H ) Statistic analysis of IHC staining indicates that PARP1 expression is positively correlated with KLF4 expression in breast cancer . n = 183 , r = 0 . 434 , p = 8 . 68x10 - 10 , Pearson correlation coefficients . ( I ) Survival analysis of KLF4 protein expression in 117 breast cancer patients . Compared to patients with low KLF4 protein expression , patients with high KLF4 levels had an inferior cumulative survival rate ( Kaplan - Meier assay , LogRank P = 0 . 012 ) ; Low , staining weak and moderate ; High , staining strong . ( J ) Survival analysis of both KLF4 and PARP1 protein expression in 66 breast cancer patients . Compared to patients with both low KLF4 and PARP1 protein expression levels , patients with both high KLF4 and PARP1 levels had lower cumulative survival rate ( Kaplan - Meier assay , LogRank P = 0 . 022 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of PARP1 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell lines.  (B). Accumulation of PARP1 and KLF4 protein were detected in human breast cancer tissues with 183 breast invasive ductal carcinoma and 10 pairs of primary cancer and adjacent normal tissue specimens by IHC in comparison with adjacent normal breast tissues. Scale bars, 100 μm. Statistic results of KLF4 IHC staining. Normal (n=10), tumor (n=183); p = 4.36x10-7 (KLF4) one-way ANOVA was used for the statistical analysis.Statistic results of PARP1 IHC staining. Normal (n=10), tumor (n=183) p = 0.0473 (PARP1). one-way ANOVA was used for the statistical analysis.(E) Statistical analysis of PARP1 staining among normal (n=10), ER/PR positive (ER/PR) (n=68), HER2 positive (n=62), and triple negative breast cancer (TNBC) (n=48). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis. (F) Statistical analysis of KLF4 protein staining among normal (n=10), ER/PR positive (n=68), HER2 positive (n=62) and TNBC (n=47). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis.  Elevated expression of PARP1 and KLF4 are significantly correlated in 183 breast invasive ductal carcinoma human breast cancer tissue specimens (same batch of staining as (B)). Represents of paired IHC staining of PARP1 and KLF4 (case 1-3) are shown (G). Scale bars, 100 μm.  (H) Statistic analysis of IHC staining indicates that PARP1 expression is positively correlated with KLF4 expression in breast cancer. n=183, r=0.434, p=8.68x10-10, Pearson correlation coefficients.  (I) Survival analysis of KLF4 protein expression in 117 breast cancer patients. Compared to patients with low KLF4 protein expression, patients with high KLF4 levels had an inferior cumulative survival rate (Kaplan-Meier assay, LogRank P=0.012); Low, staining weak and moderate; High, staining strong.  (J) Survival analysis of both KLF4 and PARP1 protein expression in 66 breast cancer patients. Compared to patients with both low KLF4 and PARP1 protein expression levels, patients with both high KLF4 and PARP1 levels had lower cumulative survival rate (Kaplan-Meier assay, LogRank P=0.022).


Figure 4 ( B ) Identification of amino acid stretch 411 - 441 on the repression domain of KLF4 as a segment mediating the interaction with PARP1 in 293T cells . . ( D ) . Identification of amino acid stretch 411 - 441 on the Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T cells . . PARylation for Flag tagged KLF4 deletion mutants were analyzed , respectively , by pulldown using PAR antibody followed by Western blotting using antibody against Flag . ( E ) Dissection of the PARylation region of KLF4 into three zinc finger motif - containing domains , Zinc 1 , Zinc 2 and Zinc 3 . While no effect was observed on Zinc 1 and Zinc 3 domains , deletion of Zinc 2 domain led to significant attenuation of KLF4 PARylation . ( G ) Identification of amino acid stretch 829 - 1014 on the C - terminal segment of PARP1 to be involved in mediating its interaction with KLF4 in 293T cells .

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

Figure 4(B) Identification of amino acid stretch 411- 441 on the repression domain of KLF4 as a segment mediating the interaction with PARP1 in 293T cells..(D). Identification of amino acid stretch 411- 441 on the Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T cells.. PARylation for Flag tagged KLF4 deletion mutants were analyzed, respectively, by pulldown using PAR antibody followed by Western blotting using antibody against Flag.(E) Dissection of the PARylation region of KLF4 into three zinc finger motif-containing domains, Zinc 1, Zinc 2 and Zinc 3. While no effect was observed on Zinc 1 and Zinc 3 domains, deletion of Zinc 2 domain led to significant attenuation of KLF4 PARylation.(G) Identification of amino acid stretch 829-1014 on the C-terminal segment of PARP1 to be involved in mediating its interaction with KLF4 in 293T cells.


Figure 5 ( B ) The effect of YYR motif mutations on KLF4 PARylation . Constructs of KLF4 - Zinc 1 - YKH / AAA mutation , KLF4 - Zinc 2 - YYR / AAA mutation and KLF4 - Zinc - 2 - Y451A mutation were co - transfected with Myc - PARP1 into 293T cells , respectively , and then pulled - down using M2 agarose followed by measuring KLF4 PARylation with anti - PAR antibody . ( C ) KLF4 - Zinc 2 - YYR / AAA mutation has only effect on KLF4 PARylation but not on other types posttranslational modifications such as protein methylation , ubiquitylation , acetylation and sumoylation in 293T cells . The FLAG - tagged wild - type or mutant KLF4 were immunoprecipitated with FLAG and detected by anti - SYM10 ( methylation ) , anti - SUMO ( sumoylation ) , Anti - ac - lysine ( acetylation ) , anti - Ub ( ubiquitination ) and anti - PAR ( PARylation ) . Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol , soluble nucleus and chromatin in MDA - MB - 231 cells . While the activation of PARP1 in response to genotoxic stress enhances chromatin recruitment of KLF4 ORC2 were the loading control for chromatid fraction . Ponceau S were the control of nuclear soluble fraction . E ) Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol , soluble nucleus and chromatin in MDA - MB - 231 cells . , inhibition of PARP1 by Olaparib attenuates the KLF4 accumulation in chromatin fraction H3 were the loading control for chromatid fraction . PARP1 were the control of nuclear soluble fraction . ( F ) KLF4 - Zinc 2 - YYR / AAA mutation remarkably reduces DNA damage - induced chromatin recruitment of ectopic expressed KLF4 in U2OS cells . ORC2 is the loading control for chromatid fraction . ( G ) . Confocal analysis of PAR and FLAG - tagged KLF4 showing that KLF4 - Zinc 2 - YYR / AAA mutation significantly reduces KLF4 PARylation in U2OS cells . Scale bars , 10 μm . ( I ) The effect of PARP1 mutations ( H909A or T824A ) on KLF4 PARylation , confirming the critical role of H909 in mediating KLF4 PARylation in 293T cells . Myc - PARP1 wildtype or mutations ( H909A or T824A ) and FLAG - KLF4 were co - transfected into 293T cells and then the FLAG - KLF4 was pulldown by anti - FLAG antibody , the PARylation modification of KLF4 was blotted with anti - PAR antibody , and the binding PARP1 was blotted with anti - myc antibody . ( J ) . Replacement of this histidine by alanine on PARP1 or replacement of the YYR motif of KLF4 by triple alanines leads to the attenuation of KLF4 PARylation . In PARP1 - / - MEFs , wild - type or mutant PARP1 ( H909A ) were co - transfected with FLAG - tagged wild - type or KLF4 - Zinc 2 - YYR / AAA mutation followed by immunoprecipitation using M2 - agarose and Western blotting by anti - PAR antibody . ( K ) KLF4 PARylation enhances its transcriptional function demonstrated by p21 promoter luciferase assay . 293T cells were co - transfected with p21 luciferase reporter plasmid with wild - type or mutant KLF4 ( YYR / AAA ) and wild - type or mutant PARP1 ( H909A ) and then submitted to luciferase assay . n = 3 , p = 4 . 74x10 - 6 ( PARP1WT vs PARP1H909A ) , Data are mean ± SEM ; one - way ANOVA was used for the statistical analysis .

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

Figure 5(B) The effect of YYR motif mutations on KLF4 PARylation. Constructs of KLF4-Zinc 1-YKH/AAA mutation, KLF4-Zinc 2-YYR/AAA mutation and KLF4-Zinc-2-Y451A mutation were co-transfected with Myc-PARP1 into 293T cells, respectively, and then pulled-down using M2 agarose followed by measuring KLF4 PARylation with anti-PAR antibody. (C) KLF4-Zinc 2-YYR/AAA mutation has only effect on KLF4 PARylation but not on other types posttranslational modifications such as protein methylation, ubiquitylation, acetylation and sumoylation in 293T cells. The FLAG-tagged wild-type or mutant KLF4 were immunoprecipitated with FLAG and detected by anti-SYM10 (methylation), anti-SUMO (sumoylation), Anti-ac-lysine (acetylation), anti-Ub (ubiquitination) and anti-PAR (PARylation).  Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. While the activation of PARP1 in response to genotoxic stress enhances chromatin recruitment of KLF4 ORC2 were the loading control for chromatid fraction. Ponceau S were the control of nuclear soluble fraction.  E) Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. , inhibition of PARP1 by Olaparib attenuates the KLF4 accumulation in chromatin fraction H3 were the loading control for chromatid fraction. PARP1 were the control of nuclear soluble fraction.  (F) KLF4-Zinc 2-YYR/AAA mutation remarkably reduces DNA damage-induced chromatin recruitment of ectopic expressed KLF4 in U2OS cells. ORC2 is the loading control for chromatid fraction.  (G). Confocal analysis of PAR and FLAG-tagged KLF4 showing that KLF4-Zinc 2-YYR/AAA mutation significantly reduces KLF4 PARylation in U2OS cells. Scale bars, 10 μm.  (I) The effect of PARP1 mutations (H909A or T824A) on KLF4 PARylation, confirming the critical role of H909 in mediating KLF4 PARylation in 293T cells. Myc-PARP1 wildtype or mutations (H909A or T824A) and FLAG-KLF4 were co-transfected into 293T cells and then the FLAG-KLF4 was pulldown by anti-FLAG antibody, the PARylation modification of KLF4 was blotted with anti-PAR antibody, and the binding PARP1 was blotted with anti-myc antibody.  (J). Replacement of this histidine by alanine on PARP1 or replacement of the YYR motif of KLF4 by triple alanines leads to the attenuation of KLF4 PARylation. In PARP1-/- MEFs, wild-type or mutant PARP1 (H909A) were co-transfected with FLAG-tagged wild-type or KLF4-Zinc 2-YYR/AAA mutation followed by immunoprecipitation using M2-agarose and Western blotting by anti-PAR antibody.  (K) KLF4 PARylation enhances its transcriptional function demonstrated by p21 promoter luciferase assay. 293T cells were co-transfected with p21 luciferase reporter plasmid with wild-type or mutant KLF4 (YYR/AAA) and wild-type or mutant PARP1 (H909A) and then submitted to luciferase assay. n=3, p=4.74x10-6 (PARP1WT vs PARP1H909A), Data are mean ± SEM; one-way ANOVA was used for the statistical analysis.


Figure 6 ( A ) Depletion of KLF4 directly diminishes the DNA damage - induced p21 expression , resulting in the failure of cell cycle arrest in MDA - MB - 231 cells . ( B ) Abolishment of KLF4 PARylation disrupts KLF4 - mediated p21 expression that in turn impairs DNA damage response in MDA - MB - 231 cells . ( C ) p - H3 staining analysis of KLF4 + / + , KLF4 - / - MEFs , KLF4 - / - MEF with transfection of wild - type or KLF4 - Zinc 2 - YYR / AAA mutation . n = 4 , p values were labeled in figure , one - way ANOVA assay . ( D Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci . n = 4 , p = 4 . 74x10 - 6 ( KLF4WT vs KLF4YYR / AAA ) , one - way ANOVA assay . Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci . n = 4 , p = 4 . 74x10 - 6 ( KLF4WT vs KLF4YYR / AAA ) , one - way ANOVA assay . ( E ) Summary of D . ( F ) Failure of KLF4 PARylation leads to increased aneuploidy population in U2OS cells . n = 3 , p values were labeled in figure , one - way ANOVA assay . ( G ) Effect of KLF4 PARylation on NHEJ and HR . Wild - type or KLF4 - Zinc 2 - YYR / AAA mutant KLF4 were co - transfected with I - SCE construction in U2Os - GFP - EJ5 cells ( for NHEJ assay ) or U2Os - DR - GFP ( for HR assay ) . n = 3 , p values were labeled in figure , one - way ANOVA assay . The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in KLF4 - / - MEF n = 3 , p values were labeled in figures , one - way ANOVA assay . The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA - MB - 468 n = 3 , p values were labeled in figures , one - way ANOVA assay . The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA - MB - 231 n = 3 , p values were labeled in figures , one - way ANOVA assay . ( K ) Heatmap of BRCA1 , P21 , Bax expression on U2OS cells with expression of wild - type KLF4 or KLF4 - Zinc 2 - YYR / AAA mutant as well as KLF4 - Zinc 2 ( Zinc 2 domain deletion ) mutant . No difference of BRCA1 expression was measured between expression of wild - type KLF4 or KLF4 - Zinc 2 - YYR / AAA mutant , while loss of Zinc domain causes drops of BRCA1 expression levels . ( L ) In early responsive window ( 1 - 2 hours after exposure to γ - radiation ) , while elevation of wild - type KLF4 enhances the expression levels of p21 , disruption of KLF4 PARylation diminishes KLF4 - mediated p21 accumulation . Upon DNA damage , no matter PARylation or not , elevation of both wild - type or KLF4 - PARylation - deficient mutant leads to BRCA1 accumulation suggesting the KLF4 - mediated regulation of BRCA1 is independent of PARP1 in MDA - MB - 231 cells . ( M ) KLF4 physically interacts with BRCA1 upstream promoter region measured by CHIP - PCR . ( N ) inset , top , schematic diagram of the BRCA1 promoter and relative positions of primer sets used in this study . ChIP analysis at the BRCA1 promoter using KLF4 - specific or nonspecific control IgG ( α - Gal4 ) in MDA - MB - 231 cells . Shown is the enrichment at positions of the BRCA1 locus relative to the TSS , presented as percent recovery of input . ( O ) inset , top , schematic diagram of the BRCA1 promoter cloning primer and the alignment of potential KLF4 binding motif on BRCA1 promoter with KLF4 binding motif on p21 and SLC5A6 promoter . Shown is the wild - type ( BRCA1 - WT ) or mutant ( BRCA1 - AA ) BRCA1 promoter luciferase reporter activity when co - transfect with KLF4 plasmids . KLF4 co - transfection promotes BRCA1 - WT but not BRCA1 - AA promoter reporter transcription . n = 3 , p = 0 . 018 , one - way ANOVA assay . ( P ) ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA - MB - 231 at - . 3K positions relative to the TSS in untreated and Olaparib ( 10µM for 8h ) or 5Gy radiation treat cells . No significant difference of KLF4 binds to BRCA1 promoter between untreated and Olaparib or radiation treat cells . n = 3 , p = 0 . 80 , one - way ANOVA assay . ( Q ) BRCA1 reporter assay in KLF4 - / - MEFs , KLF4 - / - MEF with transfection of wild - type ( KLF4WT ) or mutant KLF4 ( KLF4YYR / AAA , KLF4C403A and KLF4ΔZinc2 ) . The depleted the zinc domain or mutated zinc finger ( KLF4C403A and KLF4ΔZinc2 ) on KLF4 impairs the KLF4 - driven BRCA1 expression , while no effect was observed between wild - type and KLF4YYR / AAA mutant . n = 3 , p values were labeled in figure , one - way ANOVA assay . ( R ) HR analysis . U2OS - DR - GFP cells were transfected with I - SceI , BRCA1 and siBRCA1 in KLF4 - wild - type and depletion condition , respectively . GFP positive cells representing HR repair rate were measured by flow cytometry 48 - 72 hour after then . Overexpression of BRCA1 restores the HR efficiency in KLF4 knockdown cells . Data are mean ± SEM ; n = 3 , p values were labeled in figure , one - way ANOVA assay .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Depletion of KLF4 directly diminishes the DNA damage-induced p21 expression, resulting in the failure of cell cycle arrest in MDA-MB-231 cells. (B) Abolishment of KLF4 PARylation disrupts KLF4-mediated p21 expression that in turn impairs DNA damage response in MDA-MB-231 cells. (C) p-H3 staining analysis of KLF4+/+, KLF4-/- MEFs, KLF4-/- MEF with transfection of wild-type or KLF4-Zinc 2-YYR/AAA mutation. n=4, p values were labeled in figure, one-way ANOVA assay. (D Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci. n=4, p=4.74x10-6 (KLF4WT vs KLF4YYR/AAA), one-way ANOVA assay.  Abolishment of KLF4 PARylation leads to failure in removing damaged DNA as measured by 53BP1 foci. n=4, p=4.74x10-6 (KLF4WT vs KLF4YYR/AAA), one-way ANOVA assay. (E) Summary of D.  (F) Failure of KLF4 PARylation leads to increased aneuploidy population in U2OS cells. n=3, p values were labeled in figure, one-way ANOVA assay. (G) Effect of KLF4 PARylation on NHEJ and HR. Wild-type or KLF4-Zinc 2-YYR/AAA mutant KLF4 were co-transfected with I-SCE construction in U2Os-GFP-EJ5 cells (for NHEJ assay) or U2Os-DR-GFP (for HR assay). n=3, p values were labeled in figure, one-way ANOVA assay.The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in KLF4-/- MEF n=3, p values were labeled in figures, one-way ANOVA assay. The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA-MB-468 n=3, p values were labeled in figures, one-way ANOVA assay.  The effect of overexpression or knockdown KLF4 on mRNA levels of BRCA1 in MDA-MB-231 n=3, p values were labeled in figures, one-way ANOVA assay.  (K) Heatmap of BRCA1, P21, Bax expression on U2OS cells with expression of wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant as well as KLF4-Zinc 2 (Zinc 2 domain deletion) mutant. No difference of BRCA1 expression was measured between expression of wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant, while loss of Zinc domain causes drops of BRCA1 expression levels.  (L) In early responsive window (1-2 hours after exposure to γ-radiation), while elevation of wild-type KLF4 enhances the expression levels of p21, disruption of KLF4 PARylation diminishes KLF4-mediated p21 accumulation. Upon DNA damage, no matter PARylation or not, elevation of both wild-type or KLF4-PARylation-deficient mutant leads to BRCA1 accumulation suggesting the KLF4-mediated regulation of BRCA1 is independent of PARP1 in MDA-MB-231 cells. (M) KLF4 physically interacts with BRCA1 upstream promoter region measured by CHIP-PCR.(N) inset, top, schematic diagram of the BRCA1 promoter and relative positions of primer sets used in this study. ChIP analysis at the BRCA1 promoter using KLF4-specific or nonspecific control IgG (α-Gal4) in MDA-MB-231 cells. Shown is the enrichment at positions of the BRCA1 locus relative to the TSS, presented as percent recovery of input.(O) inset, top, schematic diagram of the BRCA1 promoter cloning primer and the alignment of potential KLF4 binding motif on BRCA1 promoter with KLF4 binding motif on p21 and SLC5A6 promoter. Shown is the wild-type (BRCA1-WT) or mutant (BRCA1-AA) BRCA1 promoter luciferase reporter activity when co-transfect with KLF4 plasmids. KLF4 co-transfection promotes BRCA1-WT but not BRCA1-AA promoter reporter transcription. n=3, p=0.018, one-way ANOVA assay. (P) ChIP analysis of KLF4 binding to the BRCA1 promoter in MDA-MB-231 at -.3K positions relative to the TSS in untreated and Olaparib (10µM for 8h) or 5Gy radiation treat cells. No significant difference of KLF4 binds to BRCA1 promoter between untreated and Olaparib or radiation treat cells. n=3, p=0.80, one-way ANOVA assay.  (Q) BRCA1 reporter assay in KLF4-/- MEFs, KLF4-/- MEF with transfection of wild-type (KLF4WT) or mutant KLF4 (KLF4YYR/AAA, KLF4C403A and KLF4ΔZinc2). The depleted the zinc domain or mutated zinc finger (KLF4C403A and KLF4ΔZinc2) on KLF4 impairs the KLF4-driven BRCA1 expression, while no effect was observed between wild-type and KLF4YYR/AAA mutant. n=3, p values were labeled in figure, one-way ANOVA assay.  (R) HR analysis. U2OS-DR-GFP cells were transfected with I-SceI, BRCA1 and siBRCA1 in KLF4-wild-type and depletion condition, respectively. GFP positive cells representing HR repair rate were measured by flow cytometry 48-72 hour after then. Overexpression of BRCA1 restores the HR efficiency in KLF4 knockdown cells. Data are mean ± SEM; n=3, p values were labeled in figure, one-way ANOVA assay.


Figure 7 ( A ) KLF4 + / + , KLF4 - / - MEFs , KLF4 - / - MEF with wild - type KLF4 or KLF4 - Zinc 2 - YYR / AAA mutant were treated with 5 - 10 µM cisplatin for 48 hours followed by measuring the expression of PARP1 , p21 and Bax . Loss of PARylation on KLF4 impairs KLF4 - mediated inhibition of apoptosis in the presence of genotoxic stress . ( B ) The apoptotic response in U2OS cells with expression of wild - type and mutant KLF4 . U2Os cells with wildtype ( pLenti - tet - on - KLF4WT ) or mutant ( pLenti - tet - on - KLF4YYR / AAA ) KLF4 were incubated with 10 ng / ml doxycycline for 24h and then treated with 10uM doxorubicin ( Dox ) or cisplatin ( CDDP ) for additional 24h . While expression of wild - type KLF4 decreases Bax expression and inhibits drug - induced PARP1 cleavage , expression of PARylation - deficient KLF4 leads to failure in inhibiting genotoxic - induced Bax expression and PARP1 cleavage . ( C ) Disruption of KLF4 PARylation leads to failure in inhibiting temperature - induced senescence in BTR model ( RAS V12 - induced senescence ) . Disruption of KLF4 PARylation abolishes the KLF4 - promoted cellular transformation in MDA - MB - 231 measured by soft agar analysis . n = 3 , p values were labeled in figure , one - way ANOVA assay . Disruption of KLF4 PARylation abolishes the KLF4 - promoted cellular transformation in MCF10A measured by soft agar analysis . n = 3 , p values were labeled in figure , one - way ANOVA assay . Disruption of KLF4 PARylation abolishes the KLF4 - promoted cellular transformation in U2OS measured by soft agar analysis . n = 3 , p values were labeled in figure , one - way ANOVA assay . KLF4 sensitizes cell to Olaparib . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . ( G ) The representative of MEFs clonogenic assay . KLF4 sensitizes cell to Olaparib . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . ( G ) The representative of MEFs clonogenic assay . ( H ) is the summary of G . ( I ) Disruption of KLF4 PARylation reduces KLF4 effect on Olaparib efficacy . ( J - K ) While elevated KLF4 inhibits PARP1 inhibitor efficacy in killing MDA - MB - 231 cells , disruption of KLF4 PARylation partially rescue the KLF4 - mediated resistance to Olaparib . J ) The representative of MEFs clonogenic assay . ( K ) is the summary of J . ( L ) Elevated KLF4 expression increases resistance to Olaparib in MDA - MB - 468 ( BRCA - proficient ) cells , while disruption of KLF4 PARylation attenuates KLF4 - driven resistance to Olaparib . Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines HCC1937 cells . No significant difference between expression of wild - type KLF4 and KLF4 PARylation - deficient mutant was observed in HCC1937 cells . Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines MDA - MB - 436 cells . No significant difference between expression of wild - type KLF4 and KLF4 PARylation - deficient mutant was observed in MDA - MB - 436 cells . Modulating KLF4 by knockdown or overexpression affects cellular response of MDA - MB - 231 to Olaparib in clonogenic assay . Modulating KLF4 by knockdown or overexpression affects cellular response of MCF7 to Olaparib in clonogenic assay . Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib / Doxorubicin in MDA - MB - 231 ( Q ) Data are mean ± SEM ; one - way ANOVA was used for the statistical analysis . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . The exact p - Values were supplied in Appendix Table S4 . Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib / Cisplatin in MCF7 Data are mean ± SEM ; one - way ANOVA was used for the statistical analysis . The asterisk in the panels represents the significant difference ( p < 0 . 05 ) . The exact p - Values were supplied in Appendix Table S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) KLF4+/+, KLF4-/- MEFs, KLF4-/- MEF with wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant were treated with 5-10 µM cisplatin for 48 hours followed by measuring the expression of PARP1, p21 and Bax. Loss of PARylation on KLF4 impairs KLF4-mediated inhibition of apoptosis in the presence of genotoxic stress.  (B) The apoptotic response in U2OS cells with expression of wild-type and mutant KLF4. U2Os cells with wildtype (pLenti-tet-on-KLF4WT) or mutant (pLenti-tet-on-KLF4YYR/AAA) KLF4 were incubated with 10 ng/ml doxycycline for 24h and then treated with 10uM doxorubicin (Dox) or cisplatin (CDDP) for additional 24h. While expression of wild-type KLF4 decreases Bax expression and inhibits drug-induced PARP1 cleavage, expression of PARylation-deficient KLF4 leads to failure in inhibiting genotoxic-induced Bax expression and PARP1 cleavage.  (C) Disruption of KLF4 PARylation leads to failure in inhibiting temperature-induced senescence in BTR model (RAS V12 -induced senescence). Disruption of KLF4 PARylation abolishes the KLF4-promoted cellular transformation in MDA-MB-231 measured by soft agar analysis. n=3, p values were labeled in figure, one-way ANOVA assay. Disruption of KLF4 PARylation abolishes the KLF4-promoted cellular transformation in MCF10A measured by soft agar analysis. n=3, p values were labeled in figure, one-way ANOVA assay.  Disruption of KLF4 PARylation abolishes the KLF4-promoted cellular transformation in U2OS measured by soft agar analysis. n=3, p values were labeled in figure, one-way ANOVA assay. KLF4 sensitizes cell to Olaparib. The asterisk in the panels represents the significant difference (p<0.05). (G) The representative of MEFs clonogenic assay.KLF4 sensitizes cell to Olaparib. The asterisk in the panels represents the significant difference (p<0.05). (G) The representative of MEFs clonogenic assay. (H) is the summary of G. (I) Disruption of KLF4 PARylation reduces KLF4 effect on Olaparib efficacy.  (J-K) While elevated KLF4 inhibits PARP1 inhibitor efficacy in killing MDA-MB-231 cells, disruption of KLF4 PARylation partially rescue the KLF4-mediated resistance to Olaparib. J) The representative of MEFs clonogenic assay. (K) is the summary of J.  (L) Elevated KLF4 expression increases resistance to Olaparib in MDA-MB-468 (BRCA-proficient) cells, while disruption of KLF4 PARylation attenuates KLF4-driven resistance to Olaparib.  Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines HCC1937 cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in HCC1937 cells.  Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines MDA-MB-436 cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in MDA-MB-436 cells.  Modulating KLF4 by knockdown or overexpression affects cellular response of MDA-MB-231 to Olaparib in clonogenic assay.  Modulating KLF4 by knockdown or overexpression affects cellular response of MCF7 to Olaparib in clonogenic assay.  Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Doxorubicin in MDA-MB-231(Q) Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4.  Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Cisplatin in MCF7 Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4.


Figure 8 ( A ) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells ( 4T1 ) . 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB / c Nude mice . Drug treatment was started at 10th day . Placebo or Olaparib at the dose of 100 mg / kg was administrated daily for twelve days . Tumor volumes were measured every other day . Tumor volumes were measured every other day . n = 9 per group , p values were labeled in figure , one - way ANOVA assay . ( B & C ) The image of 4T1 xenograft tumors ( B ) and tumors were weighed and summarized ( C ) . n = 9 per group , p values were labeled in figure , one - way ANOVA assay . The combination effect when combine of Olaparib with ABT - 263 and dasatinib in suppression MDA - MB - 231 xenograft tumor growth . 8 weeks SCID / Beige female mice were injected 8x106 MDA - MB - 231 cells in matrigel ( 1 : 1 volume ) . When tumor reach to 50mm3 , 100mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) was administrated by oral gavage daily , 30mg / kg ABT - 263 ( dissolve in 5 % DMSO ) was administrated by intraperitoneal injection every other day , 50mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) or placebo ( 2 % DMSO + 30 % PEG400 + saline ) were administrated by oral gavage every other day . Mice were treated with single or combination drugs for four weeks . Tumor volumes were measured every other day ( F ) . The image of MDA - MB - 231 xenograft tumors The combination effect when combine of Olaparib with ABT - 263 and dasatinib in suppression MDA - MB - 231 xenograft tumor growth . 8 weeks SCID / Beige female mice were injected 8x106 MDA - MB - 231 cells in matrigel ( 1 : 1 volume ) . When tumor reach to 50mm3 , 100mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) was administrated by oral gavage daily , 30mg / kg ABT - 263 ( dissolve in 5 % DMSO ) was administrated by intraperitoneal injection every other day , 50mg / kg Olaparib ( dissolve in 2 % DMSO + 30 % PEG400 + saline ) or placebo ( 2 % DMSO + 30 % PEG400 + saline ) were administrated by oral gavage every other day . Mice were treated with single or combination drugs for four weeks . Tumor volumes were measured every other day ( G ) . The tumors were weighed and summarized ( H ) . Placebo ( n = 7 ) , ABT263 ( n = 9 ) , Dasatinib ( n = 8 ) , Olaparib ( n = 6 ) , Olaparib + ABT263 ( n = 9 ) , Olaparib + Dasatinib ( n = 10 ) , p values were labeled in figure , one - way ANOVA assay . ( I ) Staining of H & E , KLF4 , PARP1 , p21 and Bax in MDA - MB - 231 xenograft tumors . Scale bars , 100 μm . Combination of ABT - 263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression , but increase Bax expression in xenograft tumors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8 (A) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells (4T1). 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB/c Nude mice. Drug treatment was started at 10th day. Placebo or Olaparib at the dose of 100 mg/kg was administrated daily for twelve days. Tumor volumes were measured every other day. Tumor volumes were measured every other day. n=9 per group, p values were labeled in figure, one-way ANOVA assay.  (B & C) The image of 4T1 xenograft tumors (B) and tumors were weighed and summarized (C). n=9 per group, p values were labeled in figure, one-way ANOVA assay.  The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (F). The image of MDA-MB-231 xenograft tumors  The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (G). The tumors were weighed and summarized (H). Placebo (n=7), ABT263 (n=9), Dasatinib (n=8), Olaparib (n=6), Olaparib+ABT263 (n=9), Olaparib+Dasatinib (n=10), p values were labeled in figure, one-way ANOVA assay. (I) Staining of H & E, KLF4, PARP1, p21 and Bax in MDA-MB-231 xenograft tumors. Scale bars, 100 μm. Combination of ABT-263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression, but increase Bax expression in xenograft tumors.


Figure 1Western blot analysis of whole cell lysates from HEK 293 WT and NEDP1 KO cells reveals a loss of free NEDD8 ( indicated by asterisk ) and an accumulation of NEDD8 reactive species in the NEDP1 KO lysate . The predicted molecular weight sizes of putative , unanchored , poly - NEDD8 chains are denoted by N2 through to N5 . Unconjugated NEDD8 is denoted by N1 . NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells . Recombinant HALO - NEDP1 C163A ( CA ) conjugated to HALO - Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells . Enriched proteins were resolved by SDS - PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies . HALO - NEDP1 CA specifically enriches for NEDD8 - reactive proteins in both WT and NEDP1 KO cells , but does not enrich for Ubiquitin - modified proteins in either cell line . Components of the NEDD8 conjugation machinery are enriched in HALO - NEDP1 pulldowns from NEDP1 KO lysates . Neddylated proteins from HEK293 KO cells were enriched by HALO - NEDP1 CA pulldown , as in ( B ) but not by the NEDD8 nonbinder mutant , HALO - NEDP1 DAGC ( D29W A98K G99K C163A ) . The NEDD8 E1s , UBA3 and ULA1 , are modified in NEDP1 KO cells , as well as E2 UBE2M , and co - E3s DCNL1 and DCNL2 . Cul2 and Cul3 are hyper - neddylated in NEDP1 KO cells . CSN components , CSN5 and CSN8 , also co - precipitate in HALO - NEDP1 CA pulldowns . Western blot analysis from HEK 293 WT and NEDP1 KO cells of the components of the NEDD8 conjugation / de - conjugation pathway shows that similar levels of NEDD8 pathway components are present in both WT and NEDP1 KO cells . Apart from UBA3 , there is no detectable amount of NEDD8 - modified enzymes in whole - cell lysates from NEDP1 KO cells . Poly - NEDD8 chains can be generated by in vitro reactions ( Rxn ) . NAE ( 0 . 15 μM ) , UBE2M , and NEDD8 ( 20 μM ) were incubated on ice or incubated at 30 ° C for 3 hours and reactions were stopped by addition of LDS loading buffer . Reactions were resolved by SDS - PAGE and stained with colloidal Coomaisse . Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis . The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2 - N4 . Unconjugated NEDD8 is indicated by N1 . UBE2M modified by NEDD8 is indicated with an asterisk . Neddylated species are NEDD8 E1 dependent . WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time . Lysed cells were then processed for Western blot analysis . NEDD8 E1 inhibition results in a time - dependent decrease in the amount of Cullin and non - Cullin NEDD8 reactive bands . Neddylated species are UBE2M dependent . WT and NEDP1 KO HEK 293 cells were left untreated or treated with the indicated siRNA for 48 hours . Lysed cells were then processed for Western blot analysis . Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment ( si2M ) , but not with control siRNA ( siCTRL ) or when NEDD8 E2 UBE2F was depleted ( si2F ) . The double knockdown ( si2M / 2F ) does not further reduce NEDD8 modified proteins .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Western blot analysis of whole cell lysates from HEK 293 WT and NEDP1 KO cells reveals a loss of free NEDD8 (indicated by asterisk) and an accumulation of NEDD8 reactive species in the NEDP1 KO lysate. The predicted molecular weight sizes of putative, unanchored, poly-NEDD8 chains are denoted by N2 through to N5. Unconjugated NEDD8 is denoted by N1.NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells. Recombinant HALO-NEDP1 C163A (CA) conjugated to HALO-Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells. Enriched proteins were resolved by SDS-PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies. HALO-NEDP1 CA specifically enriches for NEDD8-reactive proteins in both WT and NEDP1 KO cells, but does not enrich for Ubiquitin-modified proteins in either cell line.Components of the NEDD8 conjugation machinery are enriched in HALO-NEDP1 pulldowns from NEDP1 KO lysates. Neddylated proteins from HEK293 KO cells were enriched by HALO-NEDP1 CA pulldown, as in (B) but not by the NEDD8 nonbinder mutant, HALO-NEDP1 DAGC (D29W A98K G99K C163A). The NEDD8 E1s, UBA3 and ULA1, are modified in NEDP1 KO cells, as well as E2 UBE2M, and co-E3s DCNL1 and DCNL2. Cul2 and Cul3 are hyper-neddylated in NEDP1 KO cells. CSN components, CSN5 and CSN8, also co-precipitate in HALO-NEDP1 CA pulldowns.Western blot analysis from HEK 293 WT and NEDP1 KO cells of the components of the NEDD8 conjugation/de-conjugation pathway shows that similar levels of NEDD8 pathway components are present in both WT and NEDP1 KO cells. Apart from UBA3, there is no detectable amount of NEDD8-modified enzymes in whole-cell lysates from NEDP1 KO cells.Poly-NEDD8 chains can be generated by in vitro reactions (Rxn). NAE (0.15 μM), UBE2M, and NEDD8 (20 μM) were incubated on ice or incubated at 30°C for 3 hours and reactions were stopped by addition of LDS loading buffer. Reactions were resolved by SDS-PAGE and stained with colloidal Coomaisse. Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis. The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2-N4. Unconjugated NEDD8 is indicated by N1. UBE2M modified by NEDD8 is indicated with an asterisk.Neddylated species are NEDD8 E1 dependent. WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time. Lysed cells were then processed for Western blot analysis. NEDD8 E1 inhibition results in a time-dependent decrease in the amount of Cullin and non-Cullin NEDD8 reactive bands.Neddylated species are UBE2M dependent. WT and NEDP1 KO HEK 293 cells were left untreated or treated with the indicated siRNA for 48 hours. Lysed cells were then processed for Western blot analysis. Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment (si2M), but not with control siRNA (siCTRL) or when NEDD8 E2 UBE2F was depleted (si2F). The double knockdown (si2M/2F) does not further reduce NEDD8 modified proteins.


Figure 2 ( Left ) Western blot analysis of large scale HALO - NEDP1 pulldowns from HEK 293 NEDP1 KO lysates . ( Right ) Diagram of mass spectrometry sample preparation . Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1 - CA pulldown . iBAQ analysis of proteins identified in A ) are plotted as the log2 value of the enrichment ratio ( mass spectrometry intensity of the HALO - NEDP1 CA pulldown over HALO - NEDP1 DAGC pulldown ) versus the log10 value of the iBAQ intensity from the HALO - NEDP1 CA pulldown . Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP - 1 in the database of ADP - ribosylated proteins , ADPriboDB ( Vivelo et al . , 2017 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates. (Right) Diagram of mass spectrometry sample preparation.Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1-CA pulldown. iBAQ analysis of proteins identified in A) are plotted as the log2 value of the enrichment ratio (mass spectrometry intensity of the HALO-NEDP1 CA pulldown over HALO-NEDP1 DAGC pulldown) versus the log10 value of the iBAQ intensity from the HALO-NEDP1 CA pulldown. Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP-1 in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).


Figure 3Non - cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa ( as indicated by the arrow ) . WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis . ( Left ) The 25 - kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells . U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis . ( Right ) The same 25 kDa NEDD8 species , which is strongly induced in WT cells , is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 ( as indicated by the arrow ) . U2OS NEDP1 KO cells are resistant to the PARP - 1 inducer , H2O2 . WT and NEDP1 U2OS cells were plated in 96 - well plates and after 24 hours were treated with the indicated concentration of H2O2 . They were assessed for viability 24 hours later by the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells . NEDP1 KO cells are re - sensitized to H2O2 , following re - expression of NEDP1 by transient transfection . U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector . 48 hours after plating , cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells . Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re - expression of NEDP1 by transient transfection . Western blot analysis from HEK 293 WT , NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re - expression , the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased . NEDP1 re - expression also increased the neddylation of each Cullin . The neddylated band of each Cullin is denoted with a star . Quantification and graph of the mean ± S . E . M . of the percentage neddylation of each Cullin in ( E ) . Deletion of Nedp1 does not lead to induction Nrf2 response genes . RNA was harvested from WT and NEDP1 KO U2OS cells , reversed transcribed to cDNA , and analysed by qPCR for Nrf2 and NQO1 expression . Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2 . WT , NEDP1 KO , and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2 . 24 hours after treatment , cell viability was measured using the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow). WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis.(Left) The 25-kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells. U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Right) The same 25 kDa NEDD8 species, which is strongly induced in WT cells, is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 (as indicated by the arrow).U2OS NEDP1 KO cells are resistant to the PARP-1 inducer, H2O2. WT and NEDP1 U2OS cells were plated in 96-well plates and after 24 hours were treated with the indicated concentration of H2O2. They were assessed for viability 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.NEDP1 KO cells are re-sensitized to H2O2, following re-expression of NEDP1 by transient transfection. U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector. 48 hours after plating, cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re-expression of NEDP1 by transient transfection. Western blot analysis from HEK 293 WT, NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re-expression, the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased. NEDP1 re-expression also increased the neddylation of each Cullin. The neddylated band of each Cullin is denoted with a star. Quantification and graph of the mean ± S.E.M. of the percentage neddylation of each Cullin in (E).Deletion of Nedp1 does not lead to induction Nrf2 response genes. RNA was harvested from WT and NEDP1 KO U2OS cells, reversed transcribed to cDNA, and analysed by qPCR for Nrf2 and NQO1 expression.Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2. WT, NEDP1 KO, and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2. 24 hours after treatment, cell viability was measured using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


Figure 4PARP - 1 activity is reduced in NEDP1 KO cells . Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time . PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells . PARP - 1 activity is rescued by re - expression of NEDP1 in NEDP1 KO cells . Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs . 72 hours after transfection , cells were treated with the indicated amount of H2O2 and processed for Western blot analysis . PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re - expression of NEDP1 . PARP - 1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO . U2OS cells were plated in 96 well plates and 24 hours later pre - treated with Olaparib ( 10 μM ) for 1 hour before treatment with the indicated concentration of H2O2 . 24 hours later , cell viability was measured using the CellTiterGlo assay . NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP - 1 inhibition in WT cells . Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment . AIF translocation to the nucleus is impaired in NEDP1 KO cells . WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 ( 600 μM ) for the indicated amount of time and then processed for immunofluorescence analysis using α - AIF antibodies and DAPI staining . PARP - 1 dependent cell death is induced in WT U2OS cells , as indicated by translocation of AIF from the mitochondria to the nucleus ( Scale bar 10 μm ) . Total nuclear AIF intensity from ( D ) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar . AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells . NEDP1 KO cells express normal levels of PARP - 1 and AIF . Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP - 1 dependent cell death are present at equal levels in both cell lines .

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Figure 4PARP-1 activity is reduced in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time. PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells.PARP-1 activity is rescued by re-expression of NEDP1 in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs. 72 hours after transfection, cells were treated with the indicated amount of H2O2 and processed for Western blot analysis. PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re-expression of NEDP1.PARP-1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO. U2OS cells were plated in 96 well plates and 24 hours later pre-treated with Olaparib (10 μM) for 1 hour before treatment with the indicated concentration of H2O2. 24 hours later, cell viability was measured using the CellTiterGlo assay. NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP-1 inhibition in WT cells. Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment.AIF translocation to the nucleus is impaired in NEDP1 KO cells. WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 (600 μM) for the indicated amount of time and then processed for immunofluorescence analysis using α-AIF antibodies and DAPI staining. PARP-1 dependent cell death is induced in WT U2OS cells, as indicated by translocation of AIF from the mitochondria to the nucleus (Scale bar 10 μm).Total nuclear AIF intensity from (D) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar. AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells.NEDP1 KO cells express normal levels of PARP-1 and AIF. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP-1 dependent cell death are present at equal levels in both cell lines.


Figure 5NEDP1 KO U2OS cells are not resistant to induction of apoptosis from combined treatment with TNF - α and cycloheximide . U2OS cells were plated in 96 well plates and incubated with cycloheximide ( 10 μg / mL ) before addition of the indicated amount of TNF - α . Cell survival was determined 24 hours later using the CellTiter - Glo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells . NEDP1 KO U2OS cells are not resistant to treatment with camptothecin ( CPT ) . WT U2OS and NEDP1 KO cells were plated in 96 - well plates and treated with the indicated amount of CPT . 48 hours after exposure cell survival was measured using the CellTiterGlo assay . Graphs represent the mean ± S . E . M . of the percent survival compared to untreated cells . NEDD8 trimers co - immunoprecipitate with endogenous PARP - 1 in NEDP1 KO cells . Lysates were prepared from U2OS NEDP1 KO cells and immunoprecipitation was performed with PARP - 1 - Trap or GFP - Trap as a negative control . Bound proteins were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies . NEDD8 trimers bind to GFP - PARP - 1 . NEDP1 U2OS KO cells were transfected with GFP or with GFP - PARP - 1 and 24 hours later cell lysates were collected . Immunoprecipitation was then performed with GFP - Trap , and bound proteins were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from NEDP1 KO cells . NEDD8 trimers form in WT U2OS cells after treatment with H2O2 and co - immunoprecipitate with endogenous PARP - 1 but not with immunoglobulin control ( IGG ) . WT U2OS cells were left untreated of treated with H2O2 ( 600 μM ) for 30 min and were harvested for immunoprecipitation and Western blot analysisNEDD8 trimers bind to GFP - PARP - 1 . WT or NEDP1 U2OS KO cells were transfected with GFP or with full - length GFP - PARP - 1 and 24 hours later treated as indicated with H2O2 ( 600 μM ) for 30 min before cell lysates were collected . Immunoprecipitation was then performed with GFP - Trap , and bound proteins were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from WT cells only after H2O2 treatment or from both treated or untreated NEDP1 KO cells . NEDD8 trimers bind to the DNA - binding domain of PARP - 1 ( Zn1 - 3 ) but not to its automodification and catalytic domain ( CAT ) . NEDP1 KO U2OS cells were transfected with GFP , GFP - PARP - 1 , GFP fused to the DNA - binding domain of PARP - 1 ( AA 1 - 336 ) or with GFP fused to the PARP - 1 automodification and catalytic domain ( AA 336 - 1014 ) . 24 hours post transfection , cell lysates were collected and immunoprecipitation with GFP - Trap was performed . Bound proteins were eluted and resolved by SDS - PAGE analysis , followed by Western blot analysis with the indicated antibodies . NEDD8 trimers specifically bind to the second zinc finger of PARP - 1 . NEDP1 KO U2OS cells were transfected with GFP , GFP - PARP - 1 , or with GFP fused to the Zn1 domain ( AA 1 - 96 ) , the Zn2 domain ( AA 97 - 215 ) , the Zn3 domain ( AA 216 - 336 ) or the Zn1 + 2 domains ( AA 1 - 215 ) of PARP - 1 . 24 hours post transfection , cell lysates were collected and immunoprecipitation with GFP - Trap was performed . Bound proteins were eluted and resolved by SDS - PAGE analysis , followed by Western blot analysis with the indicated antibodies .

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Figure 5NEDP1 KO U2OS cells are not resistant to induction of apoptosis from combined treatment with TNF-α and cycloheximide. U2OS cells were plated in 96 well plates and incubated with cycloheximide (10 μg/mL) before addition of the indicated amount of TNF-α. Cell survival was determined 24 hours later using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.NEDP1 KO U2OS cells are not resistant to treatment with camptothecin (CPT). WT U2OS and NEDP1 KO cells were plated in 96-well plates and treated with the indicated amount of CPT. 48 hours after exposure cell survival was measured using the CellTiterGlo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.NEDD8 trimers co-immunoprecipitate with endogenous PARP-1 in NEDP1 KO cells. Lysates were prepared from U2OS NEDP1 KO cells and immunoprecipitation was performed with PARP-1-Trap or GFP-Trap as a negative control. Bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.NEDD8 trimers bind to GFP-PARP-1. NEDP1 U2OS KO cells were transfected with GFP or with GFP-PARP-1 and 24 hours later cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from NEDP1 KO cells.NEDD8 trimers form in WT U2OS cells after treatment with H2O2 and co-immunoprecipitate with endogenous PARP-1 but not with immunoglobulin control (IGG). WT U2OS cells were left untreated of treated with H2O2 (600 μM) for 30 min and were harvested for immunoprecipitation and Western blot analysisNEDD8 trimers bind to GFP-PARP-1. WT or NEDP1 U2OS KO cells were transfected with GFP or with full-length GFP-PARP-1 and 24 hours later treated as indicated with H2O2 (600 μM) for 30 min before cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from WT cells only after H2O2 treatment or from both treated or untreated NEDP1 KO cells.NEDD8 trimers bind to the DNA-binding domain of PARP-1 (Zn1-3) but not to its automodification and catalytic domain (CAT). NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, GFP fused to the DNA-binding domain of PARP-1 (AA 1-336) or with GFP fused to the PARP-1 automodification and catalytic domain (AA 336-1014). 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.NEDD8 trimers specifically bind to the second zinc finger of PARP-1. NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, or with GFP fused to the Zn1 domain (AA 1-96), the Zn2 domain (AA 97-215), the Zn3 domain (AA 216-336) or the Zn1+2 domains (AA 1-215) of PARP-1. 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.


Figure 62D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α - NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point ( pI ) of 6 . 592D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST - Zn1 + 2 followed by Western blot analysis with α - NEDD8 antibody . Some of the 25 - kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 ( denoted by white triangle ) , indicating the species is most likely an unanchored NEDD8 trimer . However , the majority of NEDD8 trimer migrates to multiple spots of lower pI ( indicated in the brackets ) , which suggests that NEDD8 trimers undergo a post - translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer . Overexpression of HDAC1 or HDAC2 decreases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . Cells were also co - transfected , as indicated , with empty FLAG vector , HDAC1 - FLAG , or HDAC2 - FLAG . 24 hours post transfection , cells were harvested and immunoprecipitation was performed on the lysates with GFP - Trap . Immunoprecipitated proteins were resolved by SDS - PAGE followed by Western blot analysis with the indicated antibodies . HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . 24 hours later , cells were left untreated or treated with the HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . Cells were harvested and immunoprecipitation was performed on the lysates with GFP - Trap . Immunoprecipitated proteins were resolved by SDS - PAGE followed by Western blot analysis with the indicated antibodies . HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . Cells were harvested and lysates were incubated with recombinant Zn1 - GFP or Zn2 - GFP ( 25 nM ) for 1 hour followed by immunoprecipitation with GFP - Trap . Immunoprecipitated proteins were resolved by SDS - PAGE followed by Western blot analysis with the indicated antibodies . Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment . U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors . 48 post - transfection cells , were treated with H2O2 ( 600 μM ) for the indicated amount of time and harvested directly in sample loading buffer . Lysates were resolved by SDS - PAGE and processed for Western blot analysis with the indicated antibodies .

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Figure 62D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.592D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST-Zn1+2 followed by Western blot analysis with α-NEDD8 antibody. Some of the 25-kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post-translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer.Overexpression of HDAC1 or HDAC2 decreases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). Cells were also co-transfected, as indicated, with empty FLAG vector, HDAC1-FLAG, or HDAC2-FLAG. 24 hours post transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.


Figure 7In vitro de - neddylation by NEDP1 is sensitive to oxidation . Poly - NEDD8 reactions were prepared with NAE , UBE2M , and NEDD8 as in Figure 1E . NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 ( 3 μM ) . NEDD8 reactions were incubated with GST - NEDP1 at 30 ° C for the indicated amount of time under reducing ( 50 μM DTT ) or oxidizing conditions ( 50 μM DTT + 150 μM H2O2 ) . Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS - PAGE before processing for Western blot analysis with the indicated antibodies . In vitro neddylation reactions with UBE2M are insensitive to H2O2 . Poly - NEDD8 reactions were prepared with NAE , UBE2M , and NEDD8 as in Figure 1E . Before reactions were started , increasing concentrations of H2O2 ( as indicated ) were added to the NEDD8 reactions . Subsequently the reactions were incubated at 30 ° C for the indicated amount of time . Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS - PAGE before processing for Western blot analysis with the indicated antibodies .

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Figure 7In vitro de-neddylation by NEDP1 is sensitive to oxidation. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST-NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H2O2). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.In vitro neddylation reactions with UBE2M are insensitive to H2O2. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. Before reactions were started, increasing concentrations of H2O2 (as indicated) were added to the NEDD8 reactions. Subsequently the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.


Figure 2Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2 - ( I401Q ) mutant compared to that of wild - type Nrxn1 LNS2SS2 - . Replicate numbers are shown in E . Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 - 3 LNS2SS2 - and LNS2SS2 - ( I401Q ) , plotted on a logarithmic scale . Error bars represent SEM and replicate numbers are indicated in bars . Significance values were calculated using Welch ' s t - test , * P < 0 . 05 , * * P < 0 . 01 .

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Figure 2Representative BLI traces showing decreased binding of Nxph1 to the Nrxn1 LNS2SS2- (I401Q) mutant compared to that of wild-type Nrxn1 LNS2SS2-. Replicate numbers are shown in E.Comparison of binding affinities for Nxph1 binding to mouse Nrxn1-3 LNS2SS2- and LNS2SS2- (I401Q), plotted on a logarithmic scale. Error bars represent SEM and replicate numbers are indicated in bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01.


Figure 3Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A + and Nrxn1 LNS2SS2AB + compared to that of Nrxn1 LNS2SS2 - . Replicate numbers are shown in E . Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice - insert containing Nrxn1 LNS2 with the I401Q mutation ; error bars represent SEM and replicate numbers are indicated in or above bars . Significance values were calculated using Welch ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 .

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Figure 3Representative BLI traces showing increased binding of Nxph1 to Nrxn1 LNS2SS2A+ and Nrxn1 LNS2SS2AB+ compared to that of Nrxn1 LNS2SS2-. Replicate numbers are shown in E.Comparison of binding affinities for Nxph1 binding to mouse Nrxn1 LNS2 splice variants and splice-insert containing Nrxn1 LNS2 with the I401Q mutation; error bars represent SEM and replicate numbers are indicated in or above bars. Significance values were calculated using Welch's t-test, *P < 0.05, **P < 0.01, ****P < 0.0001.


Figure 4Representative fluorescence polarization binding curves are shown for LNS2SS2 - and LNS2SS2A + interacting with Nxph1 with or without Ca2 + added . Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2 - and Nrxn1 LNS2SS2A + with or without Ca2 + added ; replicate numbers are indicated in bars . Error bars represent SEM , and significance values were calculated using Welch ' s t - test . ns , not significant .

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Figure 4Representative fluorescence polarization binding curves are shown for LNS2SS2- and LNS2SS2A+ interacting with Nxph1 with or without Ca2+ added.Comparison of binding affinities of Nxph1 for Nrxn1 LNS2SS2- and Nrxn1 LNS2SS2A+ with or without Ca2+ added; replicate numbers are indicated in bars. Error bars represent SEM, and significance values were calculated using Welch's t-test. ns, not significant.


Figure 5Comparison of binding affinities of Nxph1 binding to different Nrxn1 - 3 LNS2 isoforms . Comparison of binding affinities for Nxph3 binding to Nrxn1 - 3 LNS2SS2 - .

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Figure 5Comparison of binding affinities of Nxph1 binding to different Nrxn1-3 LNS2 isoforms.Comparison of binding affinities for Nxph3 binding to Nrxn1-3 LNS2SS2-.


Figure 1Representative current traces from oocytes expressing wild - type ( WT ) and double cysteine mutant NMDARs before and after DTE treatment . For each mutant , traces are normalized ( in height , not in current amplitude ) to the maximal response obtained after DTE treatment on wild - type ( WT ) receptorsSummary of the DTE - induced potentiation of WT and double cysteine mutantsImmunoblots from Xenopus oocytes expressing either wt or mutant subunits . M1 indicates the GluN1 monomer ( ~ 110 kDa ) ; M2 the GluN2B monomer ( ~ 180 kDa ) ; D1 / 1 the GluN1 homodimer ( ~ 220 kDa ) ; and D1 / 2 the GluN1 / GluN2B heterodimer ( ~ 290 kDa ) . Lower panels : immunoblots in reducing conditions ( + β - mercaptoethanol ) . N . I . , non - injected

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Figure 1Representative current traces from oocytes expressing wild-type (WT) and double cysteine mutant NMDARs before and after DTE treatment. For each mutant, traces are normalized (in height, not in current amplitude) to the maximal response obtained after DTE treatment on wild-type (WT) receptorsSummary of the DTE-induced potentiation of WT and double cysteine mutantsImmunoblots from Xenopus oocytes expressing either wt or mutant subunits. M1 indicates the GluN1 monomer (~110 kDa); M2 the GluN2B monomer (~180 kDa); D1/1 the GluN1 homodimer (~220 kDa); and D1/2 the GluN1/GluN2B heterodimer (~290 kDa). Lower panels: immunoblots in reducing conditions (+ β-mercaptoethanol). N.I., non-injected oocytes


Figure 2Assessment of receptor channel activity using MK - 801 inhibition kinetics . Representative current traces from oocytes expressing either wild - type ( WT ) or GluN1 - E698C / GluN2B - L795C mutant receptors in response to 10 nM MK - 801 during agonist application . Responses were scaled to the current amplitude obtained before MK - 801 application . Inset , mono - exponential fits of MK - 801 wash - in and wash - out . Note the strikingly faster kinetics in mutant receptors , both at the onset and offset of MK - 801Relative MK - 801 inhibition on - and off - rate constants ( kon and koff ) . All values were normalized to the value obtained for WT GluN1 / GluN2B

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Figure 2Assessment of receptor channel activity using MK-801 inhibition kinetics. Representative current traces from oocytes expressing either wild-type (WT) or GluN1-E698C/GluN2B-L795C mutant receptors in response to 10 nM MK-801 during agonist application. Responses were scaled to the current amplitude obtained before MK-801 application. Inset, mono-exponential fits of MK-801 wash-in and wash-out. Note the strikingly faster kinetics in mutant receptors, both at the onset and offset of MK-801Relative MK-801 inhibition on- and off-rate constants (kon and koff). All values were normalized to the value obtained for WT GluN1/GluN2B receptors


Figure 3Zinc , ifenprodil and pH dose - response curves of wild - type ( WT ) GluN1 / GluN2B receptors and mutant GluN1 - E698C / GluN2B - L795C receptors . For comparison , zinc and ifenprodil dose - response curves of GluN1 / GluN2B receptors lacking the whole GluN2B NTD ( GluN1 WT / GluN2B - delNTD ) are also shown ( dashed lines ; data from Rachline et al , 2005 ) Spermine ( 200 µM , pH 6 . 3 ) potentiation of WT GluN1 / GluN2B and mutant GluN1 - E698C / GluN - L795C receptors . Spermine ( 200 µM , pH 6 . 5 ) sensitivity is also shown for WT GluN1 / GluN2B receptors and receptors lacking either the GluN1 ( GluN1 - delNTD / GluN2B WT ) or GluN2B ( GluN1 WT / GluN2B - delNTD ) NTD ( data from Mony et al , 2011 ) Glutamate and glycine dose - response curves of WT GluN1 / GluN2B receptors and mutant GluN1 - E698C / GluN2B - L795C

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Figure 3Zinc, ifenprodil and pH dose-response curves of wild-type (WT) GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors. For comparison, zinc and ifenprodil dose-response curves of GluN1/GluN2B receptors lacking the whole GluN2B NTD (GluN1 WT/GluN2B-delNTD) are also shown (dashed lines; data from Rachline et al, 2005)Spermine (200 µM, pH 6.3) potentiation of WT GluN1/GluN2B and mutant GluN1-E698C/GluN-L795C receptors. Spermine (200 µM, pH 6.5) sensitivity is also shown for WT GluN1/GluN2B receptors and receptors lacking either the GluN1 (GluN1-delNTD/GluN2B WT) or GluN2B (GluN1 WT/GluN2B-delNTD) NTD (data from Mony et al, 2011)Glutamate and glycine dose-response curves of WT GluN1/GluN2B receptors and mutant GluN1-E698C/GluN2B-L795C receptors


Figure 5MK - 801 inhibition kinetics . On - rate constants ( kon ) of inhibition by 10 nM MK - 801 on wild - type ( WT ) and mutant receptors . All values are normalized to that obtained for WT GluN1 / GluN2B

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Figure 5MK-801 inhibition kinetics. On-rate constants (kon) of inhibition by 10 nM MK-801 on wild-type (WT) and mutant receptors. All values are normalized to that obtained for WT GluN1/GluN2B receptors


Figure 6Representative recordings of patches containing one single wild - type ( WT ) or GluN1 - E698C / GluN2B - L795C receptor . The bottom trace is an expanded view . C , closed channel ; O , open channelSingle channel properties ( equilibrium channel open probability , mean open time , mean closed time ) of the WT and super - active GluN1 - E698C / GluN2B - L795C receptorsClosed ( left ) and open ( right ) time histograms for WT ( upper panels ) or super - active GluN1 - E698C / GluN2B - L795C ( lower panels ) receptors . Closed time histograms were best fit with 5 exponentials , whereas open time histograms were best fit with 2 exponentials ( see Online Methods and Appendix Fig S8 ) . Smooth lines are associated exponential

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Figure 6Representative recordings of patches containing one single wild-type (WT) or GluN1-E698C/GluN2B-L795C receptor. The bottom trace is an expanded view. C, closed channel; O, open channelSingle channel properties (equilibrium channel open probability, mean open time, mean closed time) of the WT and super-active GluN1-E698C/GluN2B-L795C receptorsClosed (left) and open (right) time histograms for WT (upper panels) or super-active GluN1-E698C/GluN2B-L795C (lower panels) receptors. Closed time histograms were best fit with 5 exponentials, whereas open time histograms were best fit with 2 exponentials (see Online Methods and Appendix Fig S8). Smooth lines are associated exponential fits


Figure 1 ( A ) H1 - Hela cells were mock infected or infected with poliovirus ( MOI = 50 pfu / cell ) and cells were lysed at the indicated hours post - infection ( h . p . i . ) . Three independent experiments were measured for quantification of p62 levels , and the blot shown is representative . ( B ) H1 - Hela cells were mock infected or infected with poliovirus ( MOI = 50 pfu / cell ) and cells were lysed at 6 h . p . i . Cycloheximide ( Cy . ) was added to mock infected cells at a final concentration of 50 µg / mL , for 4 hours . Cells were then pre - treated with 0 . 1 µM bafilomycin A1 ( Baf . A1 ) for 14 hours prior to infection and kept under treatment throughout infection . MG132 was used at a final concentration of 20 µM and added to the media at the time of infection . The blot shown is representative of three independent experiments .

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Figure 1(A) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at the indicated hours post-infection (h.p.i.). Three independent experiments were measured for quantification of p62 levels, and the blot shown is representative.(B) H1-Hela cells were mock infected or infected with poliovirus (MOI = 50 pfu/cell) and cells were lysed at 6 h.p.i. Cycloheximide (Cy.) was added to mock infected cells at a final concentration of 50 µg/mL, for 4 hours. Cells were then pre-treated with 0.1 µM bafilomycin A1 (Baf.A1) for 14 hours prior to infection and kept under treatment throughout infection. MG132 was used at a final concentration of 20 µM and added to the media at the time of infection. The blot shown is representative of three independent experiments.


Figure 2 ( A ) Cells were transfected with scrambled ( SCR ) or anti - LC3 ( LC3 ) siRNAs , and 48 h later infected with PV at an MOI of 50 pfu / cell , or mock infected . Cells were collected at 6 h . p . i . , and immunoblots were performed on lysates for p62 , GAPDH , and LC3 . ( B ) Cells were treated with 10 mM 3 - MA for 12 h , then infected with PV at an MOI of 50 pfu / cell , or mock infected . Cells were collected at 6 h . p . i . and lysates were used for immunoblots .

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Figure 2(A) Cells were transfected with scrambled (SCR) or anti-LC3 (LC3) siRNAs, and 48 h later infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i., and immunoblots were performed on lysates for p62, GAPDH, and LC3.(B) Cells were treated with 10 mM 3-MA for 12 h, then infected with PV at an MOI of 50 pfu/cell, or mock infected. Cells were collected at 6 h.p.i. and lysates were used for immunoblots.


Figure 3Triplicate samples of H1 - Hela cells were infected with PV at an MOI of 0 . 1 pfu / cell ( A ) or MOI of 50 pfu / cell ( B ) . Cells were pre - treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection . Cell - associated virus was collected at the indicated times post - infection , and virus titers were determined by plaque assay . Parallel infections were collected for Western blots of LC3 ( A ) or p62 ( B ) In ( B ) cell - associated virus was collected at 6 h . p . i . for plaque assay . ( C ) Infections were performed as in ( A ) , then 10 µg / mL each of E64d and Pepstatin A ( Pep . A ) were added to the media at the time of infection . Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH . ( D ) Infection at an MOI of 50 pfu / cell , with E64 / Pep . A as in ( C ) .

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Figure 3Triplicate samples of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell (A) or MOI of 50 pfu/cell (B). Cells were pre-treated with 20 µM leupeptin for 14 hours prior to infection and kept under treatment throughout infection. Cell-associated virus was collected at the indicated times post-infection, and virus titers were determined by plaque assay. Parallel infections were collected for Western blots of LC3 (A) or p62 (B) In (B) cell-associated virus was collected at 6 h.p.i. for plaque assay.(C) Infections were performed as in (A), then 10 µg/mL each of E64d and Pepstatin A (Pep.A) were added to the media at the time of infection. Inhibition of lysosomal degradation was confirmed by immunoblot for LC3 and GAPDH. (D) Infection at an MOI of 50 pfu/cell, with E64/Pep.A as in (C).