Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA‐mediated p97 depletion under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (A) (*** P < 0.001; unpaired t ‐test, n = 3, mean + SEM). Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10 μM for 6 h) under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (C) (** P < 0.01, *** P < 0.001; unpaired t ‐test, n = 3, mean + SEM). Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline‐inducible mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (E) (** P < 0.01, **** P < 0.0001; unpaired t ‐test, n = 4, mean + SEM). Model representing the processing of ubiquitinated substrate by p97 ATPase activity. Inactivation of p97 ATPase activity leads to the accumulation of ubiquitinated substrate. Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA‐mediated p97 depletion. Graph represents the quantifications of (H) (*** P < 0.001, **** P < 0.0001; two‐way ANOVA, n = 3, mean + SEM) and Western blot for efficacy of siRNA depletion of p97 (right). Western blot analysis of Flag‐RNF8 denaturing‐IP in HEK293 cells showing K48‐linked hyper‐ubiquitination of RNF8 after siRNA‐mediated p97 depletion. Western blot analysis of Strep‐p97 Co‐IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97‐WT under physiological conditions and after IR (10 Gy). Graph represents the quantifications of (K) (* P < 0.05; unpaired t ‐test, n = 2, mean + SEM). Source data are available online for this figure.

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Western Blot, Inhibition, Expressing, Variant Assay, Activity Assay, Ubiquitin Proteomics, Co-Immunoprecipitation Assay

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: Western blot analysis showing increased RNF8 protein level in HeLa cells by CHX chase after siRNA‐mediated p97 depletion. Quantification of (A) showing reduced RNF8 degradation rate after siRNA‐mediated p97 depletion. Western blot analysis of CHX chase in HEK293 cells showing reduced RNF8 degradation rate after doxycycline‐inducible mild expression of p97EQ (dominant negative ATPase inactive) variant. Western blot analysis of Flag‐RNF8 denaturing‐IP showing RNF8 K48‐Ub, K63‐Ub and total‐Ub (HA) ubiquitination pattern in soluble pool (cytosol + nucleosol) and on chromatin under physiological conditions and after IR (10 Gy). Western blot analysis showing comparison of Ub‐K48 and Ub‐K63 antibodies against different amounts (0.25, 0.5 and 1.0 μg) of purified recombinant K48‐ or K63‐Ub chains (Ub‐2 to Ub‐7). Western blot analysis showing distribution of different proteins under physiological conditions and after IR (10 Gy) in cytosol, nucleosol, loosely bound chromatin (LB Chro) and tightly bound chromatin (TB Chro) of HEK293 cells. Representative IF images showing presence of ATX3 in the nucleus of U2OS cells (scale bar: 10 μm). Nuclei are marked by white lines. Source data are available online for this figure.

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Western Blot, Expressing, Dominant Negative Mutation, Variant Assay, Ubiquitin Proteomics, Comparison, Purification, Recombinant

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: Western blot analysis of Flag‐RNF8 denaturing‐IP in HEK293 cells showing the ubiquitination pattern of RNF8‐WT and RNF8‐RING* variant, under siRNA‐mediated luciferase (siLuc) or p97‐depleted conditions (sip97). Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10 μM). Graph represents the quantifications of (B) (ns: not significant, P > 0.05, **** P < 0.0001; two‐way ANOVA, n = 2, mean + SEM). Western blot analysis of Flag‐RNF8 denaturing‐IP in HEK293 cells showing hyper‐ubiquitination of RNF8 after proteasome inhibition (MG132, 10 μM for 6 h). Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag‐RNF8‐WT and Flag‐RNF8‐RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8. Graph represents the quantifications of (E) ( ns P > 0.05, **** P < 0.0001; two‐way ANOVA, n = 3, mean + SEM). Source data are available online for this figure.

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Western Blot, Ubiquitin Proteomics, Variant Assay, Luciferase, Inhibition

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction (cytosol and nucleosol) of ∆ATX3 cell extract. Arrow represents the main RNF8 band, and asterisks represent unspecific bands. Graphs represent the quantifications of (A). RNF8 level at starting point (0 h) was shown without equalisation (left). In order to nullify the difference in RNF8 level at starting point (0 h), we equalised RNF8 level to 100% and then compared the degradation rate (right) (** P < 0.01, *** P < 0.001, **** P < 0.0001; two‐way ANOVA, n = 2, mean + SEM). Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction (cytosol and nucleosol) of ATX3‐knockdown HeLa cells. The degradation was completely blocked after simultaneous inhibition of proteasome (MG132, 10 μM). Western blot analysis of Flag‐RNF8 denaturing‐IP in HEK293 cells showing RNF8 hyper‐ubiquitination in siRNA‐mediated p97 or ATX3‐depleted conditions. Western blot analysis of Flag‐RNF8 denaturing‐IP in HeLa cells showing RNF8 hyper‐ubiquitination in ∆ATX3 condition. RNF8 hyper‐ubiquitination was suppressed by reintroduction of GFP‐ATX3‐WT but not with GFP‐ATX3‐C14A or GFP‐ATX3‐UIM*. Source data are available online for this figure.

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Western Blot, Knockdown, Inhibition, Ubiquitin Proteomics

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: Western blot analysis of CHX chase kinetics showing accelerated RNF8 degradation in soluble fraction (cytoplasm + nucleoplasm) of siATX3‐depleted HeLa cells. Graph represents the quantifications of (A) (**** P < 0.0001; two‐way ANOVA, n = 3, mean + SEM). ATX3 and RNF8 mRNA expression level analysed by quantitative PCR after indicated siRNA treatment in U2OS cells. The experiment was performed in quadruplet ( n = 1) and columns represent the mean + SEM. Representative IF micrographs of U2OS cells showing recruitment of p97 or ATX3 to UV‐A micro‐laser‐induced DNA damage tracks. Scale bar: 10 μm. Representative IF images showing the UV‐A micro‐laser‐induced DNA damage tracks in U2OS cells. Endogenous RNF8 and γ‐H2AX signal at damage tracks after 30 min and 5 h of damage induction under indicated siRNA‐depleted conditions. Scale bar: 10 μm. Quantification of (E) at 30 min time point. Graph represents the average intensity of the RNF8 signal (*** P < 0.001; unpaired t ‐test, n = 3, mean + SEM, in average, at least 70 nuclei per condition and experiment). Quantification of (E) at 5‐h time point. Graph represents the average intensity of the RNF8 signal (* P < 0.05, *** P < 0.001; unpaired t ‐test, n = 3, mean + SEM, in average, at least 70 nuclei per condition and experiment). Quantification of endogenous RNF8 signal intensity in HeLa cells at UV‐A micro‐laser‐induced DNA damage tracks 30 min and 5 h after damage induction under indicated siRNA‐depleted conditions. A second, commercially available (Proteintech) RNF8 antibody was used. Graph represents the average intensity of RNF8 signal ( ns P > 0.05, ** P < 0.01, *** P < 0.001; unpaired t ‐test, n = 1, mean + SEM, more than 50 nuclei were analysed per condition and experiment). Representative IF micrographs of HeLa cells showing Flag‐RNF8 signal intensity at UV‐A micro‐laser‐induced DNA damage tracks 30 min after damage induction under indicated siRNA‐depleted conditions. Scale bar: 10 μm. Quantification of (I). Graph represents the average intensity of RNF8 signal (*** P < 0.001; unpaired t ‐test, n = 1, mean + SEM, more than 50 nuclei were analysed per condition and experiment). Graph represents the recruitment kinetics of GFP‐RNF8 at sites of two‐photon laser‐induced DNA damage spot in living U2OS cells under indicated conditions ( ns P > 0.05, ** P < 0.01; unpaired t ‐test on area under curve, n = 2, for siNpl4 n = 1, mean + SEM, in average, at least five nuclei per condition and experiment). Graph represents the recruitment kinetics of GFP‐RNF8 at sites of two‐photon laser‐induced DNA damage spot in living U2OS cells under indicated conditions (** P < 0.01; unpaired t ‐test on area under curve, n = 3, mean + SEM, in average, at least five nuclei per condition and experiment). Western blot analysis showing depletion efficiency of indicated siRNAs in U2OS cells. Graph represents the quantification of GFP (RNF8) foci in fixed U2OS cells after 30 min of IR (2 Gy) treatment, under indicated conditions (* P < 0.05, **** P < 0.0001; unpaired t ‐test, n = 2, +SEM). Source data are available online for this figure.

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm + nucleoplasm) of HeLa cells, expressing DOX‐inducible GFP‐ATX3‐C14A variant as compared to GFP‐ATX3‐WT. Endogenous ATX3 was depleted with siRNA targeting 3′UTR region of ATXN3. Graph represents the quantifications of (A). In order to nullify the difference in RNF8 level at starting point (0 h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0 h) was also shown without equalisation (* P < 0.05, ** P < 0.01; two‐way ANOVA, n = 2, mean + SEM). Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm + nucleoplasm) of HeLa cells, expressing DOX‐inducible ATX3‐UIM* variant as compared to ATX3‐WT. Endogenous ATX3 was depleted with siRNA ATX3_3′UTR. Graph represents the quantifications of (C). In order to nullify the difference in RNF8 level at starting point (0 h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0 h) was also shown without equalisation ( ns P > 0.05, * P < 0.05, ** P < 0.01, **** P < 0.0001; two‐way ANOVA, n = 2, mean + SEM). Western blot analysis showing endogenous RNF8 protein level in soluble fraction (cytosol and nucleosol) of HeLa cells under indicated conditions. RNF8 level was significantly reduced after siRNA‐mediated ATX3 depletion and then was significantly rescued by DOX‐inducible expression of GFP‐ATX3‐WT but not with GFP‐ATX3‐VBM. Graphs represent the quantifications of (E) ( ns P > 0.05, * P < 0.05, ** P < 0.01; unpaired t ‐test, n = 3, mean + SEM). Representative IF images showing γ‐H2AX‐positive micronuclei (white arrows) under physiological conditions with indicated siRNA‐mediated depletion. Scale bar: 10 μm. Graph represents quantification of (G), showing percentage of cells with γ‐H2AX‐positive micronuclei in U2OS cells (* P < 0.05, ** P < 0.01, *** P < 0.001; unpaired t ‐test, n = 3, mean + SEM, at least 200 randomly selected nuclei were counted per condition per experiment). Source data are available online for this figure.

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Western Blot, Expressing, Variant Assay

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA‐depleted conditions, before and after IR (4 Gy) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; two‐way ANOVA, n = 3, mean + SEM). Colony formation assay (CFA) showing the IR sensitivity of HeLa cells under siRNA‐mediated p97 or ATX3‐depleted conditions ( n = 3, mean ± SD). CFA showing the IR sensitivity of HeLa‐WT cells compared to HeLa∆ATX3 cells ( n = 3, mean ± SD). CFA showing the rescue of cell survival in IR‐treated, ATX3‐depleted (siRNA), HeLa cells, after DOX‐inducible expression of ATX3‐WT but not with ATX3‐C14A or ATX3‐UIM* variant ( ns P > 0.05, ** P < 0.01, **** P < 0.0001; two‐way ANOVA, n = 3, mean ± SD). CFA showing the rescue of cell survival in IR‐treated, ATX3‐depleted (siRNA), HeLa cells, after DOX‐inducible expression of ATX3‐WT but not with ATX3‐VBM variant ( ns P > 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001; two‐way ANOVA, n = 3, mean ± SD). Representative IF micrographs in U2OS cells showing IRIF formation of GFP‐RNF8‐WT, 53BP1 and γ‐H2AX (first panel) and GFP‐RNF8‐∆RING, 53BP1 and γ‐H2AX (second panel) under indicated siRNA‐depleted or p97‐inhibited (CB5083) conditions. Scale bar: 10 μm. Quantification of (F) for GFP‐RNF8‐WT. Graph represents the percentage of cells with increased GFP‐RNF8‐WT foci (white bars) or percentage of cells with > 5 53BP1 foci (grey bars) (*** P < 0.001, **** P < 0.0001; two‐way ANOVA, n = 2, mean + SEM, at least 100 nuclei per condition and experiment). Quantification of (F) for GFP‐RNF8‐∆RING. Graph represents the percentage of cells with increased GFP‐RNF8‐∆RING foci (white bars) or percentage of cells with > 5 53BP1 foci (grey bars) ( ns P > 0.05, * P < 0.01, ** P < 0.01, **** P < 0.0001; two‐way ANOVA, n = 2, mean + SEM, at least 100 nuclei per condition and experiment). Representative IF images and quantification showing endogenous K63‐Ub and γ‐H2AX at UV‐A micro‐laser‐induced DNA damage tracks in U2OS cells under indicated siRNA‐depleted conditions. Scale bar: 10 μm. Quantification of (I). Graph represents the average intensity of the K63‐Ub ( ns P > 0.05, ** P < 0.01, *** P < 0.001; unpaired t ‐test, n = 2, mean + SEM, more than 50 nuclei were analysed).

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Neutral Comet Assay, Colony Assay, Expressing, Variant Assay

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet: GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA‐depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001; one‐way ANOVA, n = 3, mean + SEM). GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA‐depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments ( ns P > 0.05, ** P < 0.01; one‐way ANOVA, n = 3, mean + SEM). CFA showing the IR sensitivity of 53BP1‐proficient and 53BP1‐deficient MCF7 cells under indicated siRNA‐depleted conditions. Data were collected from two individual experiments with triplicates ( ns P > 0.05; two‐way ANOVA, n = 2, mean ± SEM). CFA showing the IR sensitivity of BRCA2‐proficient and BRCA2‐deficient DLD1 cells under indicated siRNA‐depleted conditions. Data were collected from two individual experiments with triplicates (** P < 0.01, *** P < 0.001; two‐way ANOVA, n = 2, mean ± SEM). Representative IF images showing Rad51 foci in S‐phase (EdU‐positive) U2OS nucleus after 5 h of IR (2 Gy) exposure. Scale bar: 10 μm. Graph represents the quantification of (E) showing average number of Rad51 foci per S‐phase (EdU‐positive) nucleus ( ns P > 0.05, **** P < 0.0001; unpaired t ‐test, n = 2, mean + SEM, at least 50 nuclei per condition and experiment). CFA showing the rescue of cell survival in IR‐treated, ATX3‐depleted (siRNA), HeLa cells, after co‐depletion (siRNA) of RNF8 ( ns P > 0.05, * P < 0.05, *** P < 0.001; two‐way ANOVA, n = 3, mean ± SEM). Model of the p97–ATX3 complex in the regulation of RNF8 homeostasis under physiological condition (soluble environment) and in RNF8 chromatin extraction at sites of DSBs to promote NHEJ. Source data are available online for this figure.

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: Reporter Assay, Extraction

Journal: The EMBO Journal

Article Title: The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8

doi: 10.15252/embj.2019102361

Figure Lengend Snippet:

Article Snippet: Human: cervical carcinoma HeLa cells (female) , ATCC , Cat# CCL‐2, RRID:CVCL_0030.

Techniques: CRISPR

Journal: PLoS ONE

Article Title: CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B

doi: 10.1371/journal.pone.0239411

Figure Lengend Snippet: (A) HEK293T clonal cells lipofected with PX459.ATP7B vector were cultivated for 48 hours using cell culture medium containing the indicated concentrations of Cu. Cellular metabolic activities of cells were determined by MTT assay. HEK293T ATP7B WT cells served as control (circle). Four ATP7B homozygous (rectangle), and three heterozygous (triangle) cell clones are shown. The survival of cells is given as percentage relative to cells that received no Cu treatment (100%). Mean ± SE of four experiments is shown. * indicate significance (p < 0.05). (B) Sanger sequencing of HEK293T cell clone #1 after CRISPR/Cas9-mediated ATP7B KO revealed a deletion of one cytosine nucleotide at position 1184 (black rectangle), three nucleotides upstream the PAM region (red rectangle). Forward (top) and reverse (bottom) sequence analysis is depicted.

Article Snippet: A total of 12 ssODN repair templates were designed with homologous genomic flanking sequences varying in nucleotide (NT) arm length (30, 40, 50 or 60 NT´s) centered around the targeted CRISPR/Cas9 cleavage site. ssODN repair templates contained 2 to 3 silent CRISPR/Cas9 blocking mutations (ssODN_2M, ssODN_3M) or no blocking mutation (ssODN_C) (PAGEpurified, IDT, Coralville, IA, USA; and ).

Techniques: Plasmid Preparation, Cell Culture, MTT Assay, Clone Assay, Sequencing, CRISPR

Journal: PLoS ONE

Article Title: CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B

doi: 10.1371/journal.pone.0239411

Figure Lengend Snippet: ATP7B KO (HEK293TΔC) cells exhibit a point mutation in form of a cytosine nucleotide deletion (red hyphen). Cas9 cuts three nucleotides (red arrow) upstream the PAM region (blue). Repair ssODNs contain silent blocking mutations at positions 1, 2 and 3 (ssODN_3M) or at positions 2 and 3 (ssODN_2M).

Article Snippet: A total of 12 ssODN repair templates were designed with homologous genomic flanking sequences varying in nucleotide (NT) arm length (30, 40, 50 or 60 NT´s) centered around the targeted CRISPR/Cas9 cleavage site. ssODN repair templates contained 2 to 3 silent CRISPR/Cas9 blocking mutations (ssODN_2M, ssODN_3M) or no blocking mutation (ssODN_C) (PAGEpurified, IDT, Coralville, IA, USA; and ).

Techniques: Mutagenesis, Blocking Assay

Journal: PLoS ONE

Article Title: CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B

doi: 10.1371/journal.pone.0239411

Figure Lengend Snippet: ( A ) Percentage of CRISPR/Cas9 activity in HEK293TΔC cells after Cu selection, comprising homozygous and heterozygous gene edited cell clones, and cell clones indicated sequence deletions. ( B ) Percentage of CRISPR/Cas9 repair efficiency in HEK293TΔC cells after Cu selection, comprising homozygous and heterozygous gene edited cells. (C) Percentage of CRISPR/Cas9 repair efficiency with regard to ssODN applications.

Article Snippet: A total of 12 ssODN repair templates were designed with homologous genomic flanking sequences varying in nucleotide (NT) arm length (30, 40, 50 or 60 NT´s) centered around the targeted CRISPR/Cas9 cleavage site. ssODN repair templates contained 2 to 3 silent CRISPR/Cas9 blocking mutations (ssODN_2M, ssODN_3M) or no blocking mutation (ssODN_C) (PAGEpurified, IDT, Coralville, IA, USA; and ).

Techniques: CRISPR, Activity Assay, Selection, Clone Assay, Sequencing