Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 1. IPO13, respectively, is an import receptor for nCLU and export receptor for KU70. (a–d) HeLa cells were subjected to CLSM 16 h post-transfection to co-express either DsRed2 or DsRed2-IPO13 with GFP-nCLU, Scale bar = 10 µM (a) or GFP-KU70, Scale bar = 10 µM (c) and treated ± 125 µM H2O2 for 1 h prior to imaging live. Quantitative analysis of GFP-nCLU (b) or GFP-KU70 (d) localisation was carried out using the ImageJ software on images, such as those in (a,c), to determine the nuclear-to-cytoplasmic-fluorescence ratio (Fn/c), as described in Materials and Methods. Values represent the mean ± SEM (n > 50 cells) from a single typical experiment from a series of 2 (b) or 3 (d) similar experiments. (e–i) HeLa cells were subjected to CLSM 72 h post-transfection with non-targeting or IPO13 siRNA. (e) Total cell extracts were probed by Western blotting using rabbit-anti-IPO13 (Protein Tech), with mouse-anti-actin (Abcam, Cambridge, UK) as a control and imaged using the ChemiDoc Gel Imaging System (Biorad, Hercules, CA, USA). At 16 h post-transfection, cells were transfected with either GFP-nCLU, Scale bar = 10 µM (h) or GFP-KU70, Scale bar = 10 µM (h) and treated with H2O2 as per (a,c) above. Quantitative analysis of GFP-nCLU (g) or GFP-KU70 (i) localisation was carried out as in (b,d).Values represent the mean ± SEM (n > 31 cells) from a single typical experiment from a series of 2 (g) or 3 (i) similar experiments. (j) HeLa

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Imaging, Software, Western Blot, Control

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 2. IPO13 efficiently traffics nCLU into the nucleus under oxidative stress, but IPO13-mediated nuclear export of KU70 is inhibited, as confirmed by fluorescence recovery after photo bleaching (FRAP) analysis. (a) CLSM images of HeLa cells transfected to co-express either mCherry or mCherry and IPO13 (expressed separately from the same plasmid using an IRES translation initiation site, pIRES) with GFP-nCLU and treated ± 125 µM H2O2 for 1 h, Scale bar = 10 µM. Cells were imaged prior to photobleaching (Pre) in the indicated nuclear region (dotted outline in yellow) and then monitored every 20 s for 8 min. (b) Digitised images, such as those in (a), were analysed to determine the fractional recovery of nuclear fluorescence (Frec(Fn-b)). Results shown are for a single representa- tive cell under each condition. Curves, such as those generated in (b), were used to determine the maximal recovery of nuclear fluorescence (c) and the initial rate of recovery, up to 100 s post-bleaching (Frec (Fn-b)/s−1); (d) Results represent the mean ± SEM (n > 20), typical results from 3 separate experiments. p-values represent statistical differences as determined by Student’s t-test. (e) CLSM images of HeLa cells transfected to co-express either DsRed2 or DsRed2-IPO13 with GFP-KU70 and treated ± 125 µM H2O2 for 1 h, Scale bar = 10 µM. Cells were imaged live prior to photo bleaching (Pre) in the indicated cytoplasmic region (dotted outline in blue) and then monitored every 20 s for 8 min. (f) Digitised images, such as those in (e), were analysed to determine the fractional change of nuclear fluorescence (Frec(Fn-b)). The more negative a value in this assay, the more nuclear export is occurring; therefore, when there is less loss of nuclear fluorescence, this is indicative of less export and vice versa. Results shown are for a single representative cell under each condition. Curves such as those generated in (b), were used to determine the maximal loss of nuclear fluorescence (g) and the initial rate of export, up to 100 s post-bleaching (Frec (Fn-b)/s−1); (h) Results represent the mean ± SEM (n > 20), typical results from 3 separate experiments. p-values represent statistical differences as determined by Student’s t-test.

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Plasmid Preparation, Generated

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 3. IPO13 plays a significant role in stress-induced DNA damage and repair, in part through effects on nCLU. (a) Fluorescence images of comets produced by in-gel neutral comet assay (single- cell electrophoresis) from HeLa cells ectopically expressing either GFP or GFP-tagged IPO13 after treatment without or with 50 µM H2O2 for 1 h or treatment followed by 2 h recovery in fresh media. H denotes the comet head and T denotes the comet tail (middle top panel). (b) Tail DNA content (%) was quantified using the OpenComet plugin for ImageJ to determine the percentage of DNA in the comet tail (mean ± SEM, n > 100 comets per sample). Results represent a single typical experiment from a series of 3 independent experiments. (c) Fluorescent images of comets produced as in (a) from HeLa cells transfected with either non-targeting (NT) or IPO13 siRNA after treatment as in (a) with 125 µM of H2O2. (d) Tail DNA content of pictures, such as those in (c), was quantified as in (b). (e) Fluorescent images of comets produced as in (a) from HeLa cells transfected with either NT or IPO13 siRNA that ectopically expressed either GFP or GFP-nCLU after treatment as in (a) with 125 µM H2O2. (f) Tail DNA content from images, such as those in (e), was quantified as in (b), with results representing a single typical experiment from 2 independent experiments (mean ± SEM, n > 200 comets per sample).

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Fluorescence, Produced, Neutral Comet Assay, Electrophoresis, Expressing, Transfection

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 4. IPO13 contributes to nCLU-induced cell death and apoptosis. (a,b) Flow cytometric analysis for cell death in IPO13+/+ and IPO13−/−ESC transfected with GFP or GFP-nCLU and treated with H2O2 for 1 h prior to FACS analysis for percentage of cell death (PI-positive cells) within the GFP- or GFP-nCLU-transfected cell populations. (a) Representative plots of untreated and 600 µM H2O2- treated IPO13+/+ and IPO13−/−ESCs, gated to include the GFP positive populations. (b) Pooled data (n = 6 independent experiments) for % of GFP- or GFP-nCLU-expressing cells that are PI-positive (mean ± SEM) under increasing concentrations of H2O2 treatment as indicated. p-values represent statistical differences as determined by two-way ANOVA using Prism 7. (c,d) Flow cytometric analysis for apoptosis (Annexin V and/or PI staining) in IPO13+/+ and IPO13−/−ESCs treated with or without 12 µM Camptothecin (CTH) for 6 h. (c) Representative dot plots for untreated and CTH-treated conditions are typical of three independent assays. In each panel, the upper left quadrant (Q1) shows only PI-positive cells, which are necrotic. The upper right quadrant (Q2) shows cells positive for both PI and Annexin V cells. The bottom right quadrant (Q3) shows cells positive for Annexin V only, and the bottom left quadrant (Q4) shows unstained cells. (d) Pooled data (n = 3 independent experiments) for % of untreated or CTH-treated IPO13+/+ and IPO13−/−ESCs positive for Annexin V staining (Q2 + Q3) (mean ± SEM).

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Expressing, Staining

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 5. Stress disrupts the localisation of nuclear transport machinery components but not IPO13. (a,b) HeLa cells were treated ± 125 µM H2O2 for 1 h or ± 43 ◦C for 1 h prior to staining with mouse-anti-Ran (BD Biosciences) and counter-staining with DAPI, Scale bar = 10 µM. Quantitative analysis of Ran localisation (b) was carried out using the ImageJ software on images, such as those in (a), to determine the nuclear-to-cytoplasmic-fluorescence ratio (Fn/c) of Ran, as described in Materials and Methods. Values represent the mean ± SEM (n > 50 cells) from a single typical experiment from a series of 2 similar experiments. (c,d) Typical CLSM images of HeLa cells, 16 h post-transfection to express GFP or GFP-IMPα (c) and treated ± 125 µM H2O2 for 1 h prior to imaging live, Scale bar = 10 µM. Quantitative analysis of GFP or GFP- IMPα (d) was carried out as in (b). Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (e,f) Typical line fluorescence intensity histograms of GFP-IMPα (e) were measured across the nuclear envelope as indicated by the yellow line on the corresponding cell images (c). (f) Quantitative analysis of GFP-IMPα was carried out using the ImageJ software on images, such as those in (c), to determine the nuclear-envelope-to-nuclear ratio (Fne/n), as described in Materials and Methods. Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (g,h) Typical CLSM images of HeLa cells, 16 h post-transfection to express GFP or GFP-IPOβ1 (c) and treated ± 125 µM H2O2 for 1 h prior to imaging live, Scale bar = 10 µM. Quantitative analysis of GFP or GFP-IPOβ1 (d) was carried out as in (b). Values represent the mean + SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (i,j) Typical line fluorescence intensity histograms of GFP-IPOβ1 (i) were measured across the nuclear envelope, as indicated by the yellow line on the corresponding cell images (g). (j) Quantitative analysis of GFP-IPOβ1 was carried out as in (f). Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (k,l) Typical CLSM images of HeLa cells, 16 h post-transfection to express GFP or GFP-IPO7 (k) and treated ± 125 µM H2O2 for 1 h prior to imaging live, Scale bar = 10 µM. Quantitative analysis of GFP or GFP-IPO7 (l) was carried out as in (b). Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (m,n) Typical line fluorescence intensity histograms of GFP-IPO7 (m) were measured across the nuclear envelope, as indicated by the yellow line on the corresponding cell images (k). (n) Quantitative analysis of GFP-IPO7 was carried out as in (f). Values

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Staining, Software, Transfection, Imaging, Fluorescence

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 6. IPO13, unlike IPO7, continues to traffic into the nucleus under H2O2-induced oxida- tive stress. (a) CLSM images of HeLa cells transfected to express GFP-IPO7 or GFP-IPO13 and treated ± 125 µM H2O2 for 1 h. Cells were imaged prior to photobleaching (Pre) in the indicated nuclear region (dotted outline in yellow) and then monitored every 20 s for 8 min. (b) Digitised images, such as those in (a) were analysed to determine the fractional recovery of nuclear fluores- cence (Frec(Fn-b)), Scale bar = 10 µM. Results shown are for a single representative cell under each condition. Curves, such as those generated in (b), were used to determine the maximal recovery of nuclear fluorescence (c) and the time post-bleaching to reach half-maximal recovery (t1/2). (d) Results represent the mean ± SEM (n = 20); typical results from 2 separate experiments. p-values indicate statistical differences as determined by Student’s t-test.

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Generated

Journal: The Journal of biological chemistry

Article Title: PC4-mediated Ku complex PARylation facilitates NHEJ-dependent DNA damage repair.

doi: 10.1016/j.jbc.2023.105032

Figure Lengend Snippet: Figure 6. PC4-mediated XRCC6 PARylation promotes XRCC6 efficient loading on DSB sites. A, representative time-lapse images showing the recruitment of XRCC6-GFP to multiphoton tracks in Huh7 cells with or without PC4 knockdown. Scale bar, 10 μm. B, quantification of XRCC6-GFP fluo- rescence intensity at DNA damage sites in A. Data were derived from three independent experiments. In each experiment, 50 different cells were investigated. C, quantification of cell death in XRCC6-GFP cells upon laser microradiation with or without PC4 knockdown. n = 3. D, quantification of XRCC6- GFP fluorescence intensity at DNA damage sites in NC, siPC4, siPARP1, or siPC4+siPARP1 group. Data were derived from three independent experiments. E, characterization of the NHEJ repair efficiency by EJ5-GFP and FACS in NC, siPC4, siPARP1, or siPC4+siPARP1 group. n = 3. F, quantification of tail moments in NC, siPC4, siPARP1, or siPC4+siPARP1 group as determined by a neutral comet assay. n = 3. G, the responses of survival factions of Huh7 cells to X-ray irradiation in NC, shPC4, siXRCC6, or shPC4+siXRCC6 group. n = 3. All graphed data were shown as means ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. DSBs, double-strand breaks; FACS, fluorescent activated cell sorting; NHEJ, nonhomologous end joining; PC4, human positive cofactor 4.

Article Snippet: NHEJ reporter plasmid EJ5-GFP (Addgene plasmid #44026) or HR reporter plasmid DR-GFP (Addgene plasmid #26475) was transfected into Huh7 cells respectively.

Techniques: Knockdown, Derivative Assay, Neutral Comet Assay, Irradiation, FACS

Journal: Nucleic Acids Research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Melanin synthesis causes γH2AX foci formation DNA strand breaks and abasic sites formation. ( A ) Immunofluorescence of B16 cells treated with PTU and tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI (blue) and γH2AX in (red). Experiment was performed with two biological replicates and a representative image is depicted. Scale bar 10 μm. ( B ) Quantitation of mean fluorescence intensity per cell of γH2AX from two biological replicates of pigmented day 7 PTU or tyrosine treated cells (shown in A). Data represented as a box plot, horizontal line represents mean and whiskers represent SEM. Ordinary one-way ANOVA was performed for multiple comparisons. Adjusted P -value: * P -value < 0.05, *** P -value < 0.001, **** P -value < 0.0001. ( C ) (Top) Cell pellet of day 7 B16 mouse melanoma cells grown at low density (100 cells/cm 2 ). Cells were left untreated for control treated with tyrosinase inhibitor 200 μM phenylthiourea (PTU) or 1mM tyrosinase substrate L-tyrosine (Tyr) for 7 days. Number of cells, mean ± SEM across three biological replicates is depicted below the image of the cell pellet. Numbers represent mean ± SEM cell counts across biological triplicates. (Bottom) Number of abasic sites in the genomic DNA was estimated by an aldehyde specific conjugation of biotin and subsequent detection using streptavidin based detection. Using standards, abasic sites per 10 5 bp is estimated. Bars represent mean ± SEM across duplicate biological experiments, each conducted in triplicates. Ordinary one-way ANOVA was performed for multiple comparisons * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001. ( D ) Single cell electrophoresis followed by comet analysis of B16 cells undergoing varying levels of pigmentation in the presence of PTU and tyrosine (alkaline comet assay). Experiment was carried out at mid phase (day 5) and late phase (day 7) of pigmentation. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed. Adjusted P values; ns non-significant, * P -value < 0.05, ** P -value < 0.01, *** P -value < 0.001, **** P -value < 0.0001. ( E ) Neutral comet assay on B16 unpigmented (day 0) and pigmented (day 7) cells. Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Student's unpaired t-test was performed. P values ns non-significant. ( F ) Single cell electrophoresis followed by comet analysis of B16 cells untreated, treated with DMSO for 24 h, melanin synthesis ( ex-cellulo L-tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM dihydroxyindole (DHI) for 24 h (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05, **** P -value < 0.00001.

Article Snippet: Polκ (ab57070), γH2AX (CST 9718), total H2AX (CST 2595), pChk1 (CST 2348P), Total Chk1 (Invitrogen PA512096), pATR (CST 2853), total ATR (CST 2790), p53 (ab38497), p21 (CST 9706), PCNA (SC56), DCT (ab74073), Tyrosinase (custom synthesized genscript), mouse anti-BrdU antibody (ab136650) and Rat anti-BrdU antibody (ab6326).

Techniques: Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Conjugation Assay, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Neutral Comet Assay

Journal: Nucleic Acids Research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Normal human epidermal melanocytes (NHEM) respond to pigmentation induced DNA breaks by elevating Polκ. ( A ) NHEM cells were treated with 200 μM PTU or 1mM tyrosine for 7 days for differential pigmentation. (Top) Cell pellet, (bottom) western blot analysis of cell lysates with POLK, HSC70, phosphorylated H2AX, total H2AX and beta actin antibodies. Numbers below the blot correspond to control normalized expression of the indicated protein. Experiments were performed in biological duplicates. ( B ) Immunofluorescence of NHEM treated with PTU or tyrosine with phosphorylated H2AX antibody. Nuclear DNA stained with DAPI (blue) and γH2AX in (red). Experiments were performed with two biological replicates. Scale bar 10 μm. ( C ) Quantitation of mean fluorescence intensity per cell of γH2AX from two biological replicates of NHEM treated with PTU or tyrosine (shown in B). Ordinary one-way ANOVA was performed for multiple comparisons. Adjusted P values: * P -value < 0.05, **** P -value < 0.0001. ( D ) PTU and tyrosine treated NHEM cells were subjected to single cell electrophoresis and comet analysis (alkaline comet assay). Mean tail moment distribution across each population of duplicate biological experiments with atleast 50 comets analyzed is depicted by a violin plot. Ordinary one-way ANOVA was performed. Adjusted P values: ** P -value < 0.001, **** P -value < 0.00001. ( E ) Heat map of expression (fold change) in mRNA levels of top two translesion polymerases (that were enriched in B16 microarray) (top), and a panel of known DNA replication stress response genes by qRT-PCR analysis in NHEM (Control, PTU or tyrosine treated). Data represented as mean of triplicate biological experiments. ( F ) Western blot analysis of NHEM treated with DMSO or 50 nM AZ20, a selective inhibitor of ATR kinase, for 24 h. Numbers below the blot correspond to control normalized expression of the indicated protein wrt beta-actin. Experiments were performed in biological duplicates. ( G ) mRNA levels of Polk in unpigmented B16 cells mock transfected, or with either control DNA, melanin modified DNA (plasmid DNA was incubated with L-DOPA and tyrosinase and column purified after 24 h) (Mel + DNA), DNA mixed with pre-synthesized melanin and coulmn purified [DNA+(Mel)], in-vitro melanin synthesis ( ex-cellulo l -tyrosine and tyrosinase added to cell media) for 24 h or cells treated with 1 mM DHI (DHI) for 24 h. Bars represent percent mRNA levels compared to control across biological triplicates. Ordinary one-way ANOVA was performed. Adjusted P values: * P -value < 0.05. (Inset) Western blot analysis of B16 cells transfected with only DNA (Con DNA) or melanin-modified DNA (Mel + DNA) with Polκ antibody normalized to HSC70. Experiments were performed in biological triplicates.

Article Snippet: Polκ (ab57070), γH2AX (CST 9718), total H2AX (CST 2595), pChk1 (CST 2348P), Total Chk1 (Invitrogen PA512096), pATR (CST 2853), total ATR (CST 2790), p53 (ab38497), p21 (CST 9706), PCNA (SC56), DCT (ab74073), Tyrosinase (custom synthesized genscript), mouse anti-BrdU antibody (ab136650) and Rat anti-BrdU antibody (ab6326).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Microarray, Quantitative RT-PCR, Transfection, Modification, Plasmid Preparation, Incubation, Purification, Synthesized, In Vitro

Journal: Nucleic Acids Research

Article Title: Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes

doi: 10.1093/nar/gkad704

Figure Lengend Snippet: Silencing of Polκ during pigmentation prevents replication stress response despite elevated DNA damage. ( A ) Cell pellets of control non-targeting (shNT) and Polκ silenced (shPolκ) B16 cells on day 0 (left) and day 7 (right) of pigmentation. ( B ) Immunofluorescence analysis of day 7 pigmented shNT and shPolκ cells with phosphorylated H2AX antibody (puncta labelled in green) and the nucleus is counterstained with DAPI (blue). Scale bars represent 10 μm. ( C ) Quantitation of mean fluorescence intensity of γH2AX (shown in B) from two biological replicates of shNT and shPolκ cells across day 0, 5 and 7 of pigmentation induction. Two-way ANOVA was performed for multiple comparisons. Adjusted P values * P -value < 0.05 *** P -value < 0.0005 **** P -value < 0.00001 ns non-significant. ( D ) shNT and shPolκ expressing pigmented B16 cells were subjected to single cell electrophoresis and comet analysis (alkaline comet) on days 0, 5 and 7 of pigmentation induction. Mean tail moment distribution across each population of duplicate biological experiments with at least 50 comets analyzed is depicted by a violin plot. Two-way ANOVA was performed for multiple comparisons. Adjusted P values, ns non-significant, * P -value < 0.05. ( E ) Growth curve analysis of shNT and shPolκ expressing B16 cells on days 0, 5, 6 and 7 of pigmentation. Each point represents mean ± SEM across biological triplicates. Two-way ANOVA was performed. Adjusted P values: * P -value < 0.05, *** P -value < 0.001. ( F ) Western blot analysis of DNA repair and cell cycle related proteins in shNT and shPolκ cells. Numbers below represent tubulin normalized fold changes wrt shNT. Experiments were performed in biological duplicates. ( G ) shNT and shPolκ expressing B16 cells were injected inside the flank of C57/BL6 mice and allowed to grow as tumors. The volume of the tumor was non-invasively monitored and plotted over time of biological triplicates mean ± SEM. Two-way ANOVA was performed for multiple comparisons. Adjusted P values: * P -value < 0.05. ( H ) Heat map of expression (fold change) in mRNA levels of a panel of known DNA replication stress response genes by qRT-PCR analysis in shNT (day 0-unpigmented and day 7-pigmented) and shPolκ (day 0-unpigmented and day 7-pigmented) B16 cells. ( I ) Western blot images and analysis of p-RPA2 and total RPA2 in shNT and shPolκ B16 cells (day 0 unpigmented and day 7 pigmented). Numbers below represent beta-actin normalized fold changes wrt shNT at day 0. Experiments were performed in biological triplicates. ( J ) Analysis of melanoma samples from TCGA data for mRNA expression of POLK (high, low or not detected) segregated into bar plots and proportion of mutations were plotted on y-axis. ( K ) Survival plot of melanoma patients with low or high expression of POLK from TCGA data. Analysis from Human Protein Atlas database. Paired t -test P value 0.017.

Article Snippet: Polκ (ab57070), γH2AX (CST 9718), total H2AX (CST 2595), pChk1 (CST 2348P), Total Chk1 (Invitrogen PA512096), pATR (CST 2853), total ATR (CST 2790), p53 (ab38497), p21 (CST 9706), PCNA (SC56), DCT (ab74073), Tyrosinase (custom synthesized genscript), mouse anti-BrdU antibody (ab136650) and Rat anti-BrdU antibody (ab6326).

Techniques: Immunofluorescence, Quantitation Assay, Fluorescence, Expressing, Electrophoresis, Western Blot, Injection, Quantitative RT-PCR

Journal: Aging cell

Article Title: The Redox Activity of Protein Disulphide Isomerase Functions in Non-Homologous End-Joining Repair to Prevent DNA Damage.

doi: 10.1111/acel.70079

Figure Lengend Snippet: FIGURE 1 | The redox activity of PDI is protective against DNA damage induced by etoposide. (A) Neuro-2a cells were transfected with either pcDNA empty vector (EV), PDI, or QUAD tagged with V5, or untransfected cells (UT), and treated with 13.5 μM etoposide (or vehicle DMSO only) for 30 min at 24 h post-transfection. Immunocytochemistry was performed using anti-V5 (red) and anti-γH2AX antibodies (green). Nuclei were stained with Hoechst (blue). Scale bar: 10 μm. (B) Quantification of the number of γH2AX foci in 100 cells in (A). Significantly less γH2AX foci were present in cells expressing PDI, but not QUAD, compared to untransfected and EV-expressing cells. Kruskal–Wallis test, n = 3 independent replicates, mean ± SEM. *p < 0.05. ***p < 0.001, ****p < 0.0001. (C) Western blot analyses using anti-γH2AX and anti-PDI antibodies in Neuro-2a cell lysates transfected with pcDNA EV, PDI tagged with V5, QUAD tagged with V5, or untransfected cells, following 30 min of etoposide treatment. (D) Quantification of relative band density of γH2AX in the blots in (C) using densitometry, GAPDH was used as a loading control. Significantly less γH2AX was present in cells expressing PDI, but not QUAD, following treatment with etoposide compared to UT or EV cells. One-way ANOVA fol- lowed by Tukey's multiple comparison post hoc test, n = 3 independent replicates. All values represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant. (E) Immunofluorescence images of Neuro-2a cells expressing PDI or QUAD tagged with V5 or pcDNA3.1 EV. Cells were fixed and immunostained with an anti-53BP1 antibody (green) and V5 antibody (red) following 30 min of etoposide treatment. The number of 53BP1 foci was assessed in UT, pcDNA3.1 EV, PDI, and QUAD cells. Nuclei were stained with Hoechst (blue). Scale bar: 10 μm. (F) Quantification of the aver- age number of foci per 100 cells shown in (A). Fewer 53BP1 foci were present in PDI cells compared to PDI QUAD cells, Kruskal–Wallis test, n = 3 independent replicates. ****p < 0.0001. (G) Mouse primary neurons were transduced with either PDI lentivirus tagged with V5 or untransfected cells (UT) and treated with 13.5 μM etoposide for 30 min at 24 h post-transfection. Immunocytochemistry was performed using anti-V5 (cobalt blue) and anti-γH2AX antibodies (green). Nuclei were stained with Hoechst (blue) and deep-red fluorescent Nissl stain was used for visualising neurons. Scale bar: 10 μm. (H) Quantification of the number of γH2AX foci in 30 neurons in (G). Significantly less γH2AX foci were present in neurons trans- duced with PDI compared to untransfected neurons. Kruskal–Wallis test, n = 3 independent replicates. All values represent mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Proteins were then transferred onto nitrocellulose membranes according to the manufacturer's instructions (BioRad) and blotted as above using polyclonal mouse anti- P4HB (PDI) (1:2000, Abcam, ab3672), anti- rabbit Lamin B (1:3000, Abcam, ab16048) or GAPDH (1:4000, Proteintech, 60004- Ig) antibodies.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Immunocytochemistry, Staining, Expressing, Western Blot, Control, Comparison, Immunofluorescence, Transduction

Journal: Aging cell

Article Title: The Redox Activity of Protein Disulphide Isomerase Functions in Non-Homologous End-Joining Repair to Prevent DNA Damage.

doi: 10.1111/acel.70079

Figure Lengend Snippet: FIGURE 2 | The redox activity of PDI prevents DNA damage induced by H2O2. (A) Neuro-2a cells transfected with PDI tagged with V5, PDI QUAD tagged with V5, or pcDNA3.1 empty vector (EV), or untransfected cells (UT), were subjected to immunocytochemistry using anti-γH2AX (green) and anti-V5 antibodies (red), following 1h treatment with 100 μM H2O2 or vehicle (PBS). Nuclei were stained with Hoechst (blue). Scale bar: 10 μm. (B) Quantification of the average number of γH2AX foci per 100 cells. Significantly fewer γH2AX foci were present in PDI cells compared to PDI QUAD cells. Kruskal–Wallis test, n = 3 independent replicates, *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001, mean ± SEM. (C) Western blot- ting using anti-γH2AX and anti-PDI antibodies of Neuro-2a cell lysates transfected with pcDNA EV, PDI tagged with V5, PDI QUAD tagged with V5, or untransfected cells, following 1 h treatment with 100 μM H2O2 or PBS. (D) Quantification of the relative band density of γH2AX in blots shown in (C) using densitometry, GAPDH was used as a loading control. Significantly less γH2AX was present in cells expressing PDI following treatment with etoposide compared to UT or EV cells. One-way ANOVA followed by Tukey's multiple comparison post hoc test, n = 3 independent replicates, mean ± SEM, **p < 0.01; ***p < 0.001; ****p < 0.0001, ns = non-significant. (E) Neuro-2a cells overexpressing PDI or PDI QUAD tagged with V5 (red) or EV were fixed and stained with anti-53BP1 (green) and V5 antibodies (red), following 1h H2O2 treatment (100 μM) or PBS. Nuclei were stained with Hoechst (blue). Scale bar: 10 μm. (F) Quantification of the average number of foci per 100 cells. Significantly fewer 53BP1 foci were present in PDI cells compared to PDI QUAD cells following H2O2 treatment, Kruskal–Wallis test, 100 cells were counted in each experiment, n = 3 independent replicates. All values represent mean ± SEM, ****p < 0.0001.

Article Snippet: Proteins were then transferred onto nitrocellulose membranes according to the manufacturer's instructions (BioRad) and blotted as above using polyclonal mouse anti- P4HB (PDI) (1:2000, Abcam, ab3672), anti- rabbit Lamin B (1:3000, Abcam, ab16048) or GAPDH (1:4000, Proteintech, 60004- Ig) antibodies.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Immunocytochemistry, Staining, Western Blot, Control, Expressing, Comparison

Journal: Aging cell

Article Title: The Redox Activity of Protein Disulphide Isomerase Functions in Non-Homologous End-Joining Repair to Prevent DNA Damage.

doi: 10.1111/acel.70079

Figure Lengend Snippet: FIGURE 3 | PDI is protective against DNA damage by NHEJ DNA repair. (A) Neuro-2a cells were either untransfected (UT, first two columns) or transfected with either empty vector (EV) (second two columns), or PDI (last four columns). At 24 h post-transfection, cells were treated with NU7441 for 1 h and 13.5 μM etoposide (or DMSO only) for 30 min. Then, cells were lysed and western blotting was performed using anti-PDI and anti-GAPDH antibodies. (B) Quantification of the blots in (A) using densitometry, GAPDH was used as a loading control. The graph depicts the relative band den- sity of γH2AX to GAPDH compared to EV-transfected cells. While γH2AX levels were significantly lower in PDI expressing cells following treatment with etoposide compared to UT or EV cells, they were significantly higher in PDI expressing cells following treatment with etoposide and NU7441 compared to PDI cells treated with etoposide only. One-way ANOVA followed by Tukey's multiple comparison post hoc test, n = 3, *p < 0.05, **p < 0.01; ***p < 0.001, ns = non-significant. (C) Representative images of neutral Comet assay of Neuro-2a cells expressing either EV or PDI, treated with 13.5 μM etoposide for 30 min and NU7441 for 1 h. (D) Quantification of Comets expressed as the Olive Tail Moment (OTM) revealed less DNA DSBs in cells expressing PDI compared to EV-expressing cells following etoposide treatment. More DNA DSBs were observed in cells expressing PDI following NU7441 treatment compared to PDI-expressing cells, n = 3, ****p < 0.0001, ns = non-significant.

Article Snippet: Proteins were then transferred onto nitrocellulose membranes according to the manufacturer's instructions (BioRad) and blotted as above using polyclonal mouse anti- P4HB (PDI) (1:2000, Abcam, ab3672), anti- rabbit Lamin B (1:3000, Abcam, ab16048) or GAPDH (1:4000, Proteintech, 60004- Ig) antibodies.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Expressing, Comparison, Neutral Comet Assay

Journal: The FASEB Journal

Article Title: Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response

doi: 10.1096/fj.201800336R

Figure Lengend Snippet: Truncated Raf1 preferentially binds DNA-PK and alters DNA-PK phosphorylation: A) PLA for endogenous Raf1 and DNA-PK in HEK cells. Interactions between Raf1 and DNA-PK are shown in red with nuclei (blue) counterstained with DAPI. B) PLA for FLAG and DNA-PK in HEK cells expressing FLAG-tagged Raf1-fl or Raf1-tr. Interactions between FLAG-tagged Raf1-fl or Raf1-tr and DNA-PK are shown in red with nuclei (blue) counterstained with DAPI. Images are representative of experiments run in triplicate. C) Quantitation of Raf1-fl and Raf1-tr interactions with DNA-PK. A minimum of 150 cells per group were analyzed. D) Western blot of DNA-PK Ser 2056 and Thr 2609 phosphorylation, and total DNA-PK in HEK cells expressing empty vector, Raf1-fl, or Raf1-tr exposed to vehicle (−) or 0.05 mg/ml bleomycin (+) for 2 h. β-actin was used as a loading control. Blot is representative of experiments run in quadruplicate. E) Quantitation of phosphorylated DNA-PK normalized to empty vector exposed to bleomycin (Bleo). The results are shown as mean ± sem. Scale bars, 50 µm.

Article Snippet: MB-231 were also exposed to 0.05 mg/ml bleomycin with concurrent exposure to 0.5 μM DNA-PK inhibitor NU-7441 (S2638; Selleck Chemicals, Houston, TX, USA) or 25 nM MYC siRNA as previously described.

Techniques: Phospho-proteomics, Expressing, Quantitation Assay, Western Blot, Plasmid Preparation, Control

Journal: The FASEB Journal

Article Title: Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response

doi: 10.1096/fj.201800336R

Figure Lengend Snippet: Raf1-tr increases DNA damage: A) Western blot of γ-H2AX and H2AX in HEK cells expressing empty vectorRaf1-fl, or Raf1-tr exposed to vehicle (−) or 0.05 mg/ml bleomycin (+) for 2 h. β-actin was used as a loading control. Blot is representative of experiments run in triplicate. B) Quantitation of γ-H2AX normalized to empty vector exposed to bleomycin (Bleo). C) HEK cells expressing Raf-fl or Raf1-tr were irradiated with 1 Gy of radiation and allowed to recover in complete medium for 2 h. γ-H2AX immunofluorescence is shown in red with nuclei (blue) counterstained with DAPI. Scale bars, 10 µm. Images are representative of experiments run in triplicate. D) Quantitation of γ-H2AX foci. A minimum of 180 nuclei per group were analyzed. E) Western blot of RPA32 phosphorylation in HEK cells expressing empty vector, Raf1-fl, or Raf1-tr exposed to vehicle (−) or 1 µM camptothecin (+) for 1 h. Exposure to camptothecin results in a non-phosphorylated RPA32 band (lower) and a slower migrating phosphorylated band (upper). β-actin was used as a loading control. Blot is representative of experiments run in triplicate. F) Quantitation of RPA32 phosphorylation normalized to empty vector exposed to camptothecin (CPT). G) Neutral comet assay of nuclei from HEK cells expressing Raf1-fl or Raf1-tr exposed to vehicle or 0.05 mg/ml bleomycin for 30 min. Images are representative of experiments run in triplicate. H) Quantitation of DNA fragmentation by Olive tail moment (OTM). A minimum of 200 nuclei per group were analyzed. The results are shown as means ± sem. N.S., not significant.

Article Snippet: MB-231 were also exposed to 0.05 mg/ml bleomycin with concurrent exposure to 0.5 μM DNA-PK inhibitor NU-7441 (S2638; Selleck Chemicals, Houston, TX, USA) or 25 nM MYC siRNA as previously described.

Techniques: Western Blot, Expressing, Control, Quantitation Assay, Plasmid Preparation, Irradiation, Immunofluorescence, Phospho-proteomics, Neutral Comet Assay

Journal: The FASEB Journal

Article Title: Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response

doi: 10.1096/fj.201800336R

Figure Lengend Snippet: Raf1-tr increases the apoptotic response of cancer cells exposed to DNA damage. A) Measurement of Raf1-tr gene expression in colon tissue and the HCT-116 colorectal carcinoma cell line. The gene of interest was normalized to GAPDH expression and represented as fold change relative to colon tissue. B) Western blot of Raf1 in colon and HCT-116 lysate showing that Raf1-tr protein expression is absent in HCT-116 cells. Phosphorylation of Raf1-fl in HCT-116 cells results in a slower migrating species compared with normal colon. C) TUNEL stain (red) of HCT116 cells expressing Raf1-fl or Raf1-tr exposed to 0.150 mg/ml bleomycin for 72 h. Transfected cells were identified by coexpression of Raf1-fl or Raf1-tr and GFP (green) using IRES. Nuclei were counterstained with DAPI (blue). D) Quantitation of GFP-positive apoptotic cells. A minimum of 250 cells per group were analyzed. E) Measurement of Raf1-tr gene expression in MCF7 and MB-231 mammary adenocarcinoma cell lines. The gene of interest was normalized to GAPDH expression and represented as fold change relative to MCF7 cells. F) Western blot of Raf1 in MCF7 and MB-231 cell lysate demonstrating decreased Raf1-tr expression in MB-231 cells relative to MCF7 cells. G) TUNEL stain (red) of MCF7 and MB-231 cells exposed to 0.05 mg/ml bleomycin for 72 h. Nuclei were counterstained with DAPI (blue). H) Quantitation of TUNEL-positive nuclei in MCF7 cells exposed to vehicle (Veh) or bleomycin (Bleo) compared with MB-231 cells exposed to Veh alone, Bleo alone, Bleo + DNA-PK inhibitor NU7441 (0.5 μM) and Bleo + control (Ctl) or MYC siRNA (25 nM). A minimum of 250 cells per group were analyzed. The results are shown as means ± sem. N.S., not significant. Scale bars, 50 µm. Images are representative of experiments run in triplicate.

Article Snippet: MB-231 were also exposed to 0.05 mg/ml bleomycin with concurrent exposure to 0.5 μM DNA-PK inhibitor NU-7441 (S2638; Selleck Chemicals, Houston, TX, USA) or 25 nM MYC siRNA as previously described.

Techniques: Gene Expression, Expressing, Western Blot, Phospho-proteomics, TUNEL Assay, Staining, Transfection, Quantitation Assay, Control

Journal: Nature communications

Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

doi: 10.1038/s41467-019-13667-4

Figure Lengend Snippet: Fig. 4 ATAD5 promotes generation of single-stranded DNA-associated breaks in response to replication stress. a U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 or 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. b, c U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 3 h before fixation. The fixed cells were stained with an anti-pRPA2 S4/S8 antibody. b Representative images of chromatin-bound pRPA2 S4/S8. Scale bar: 20 μm. c The intensity of chromatin-bound pRPA2 S4/S8 staining was quantified from ~20,000 cells. Error bars represent standard deviation of the mean (n = 3). Statistical analysis: t test; *p < 0.05. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for 3 or 6 h. Chromatin-bound proteins were separated by SDS-PAGE and subjected for immunoblotting with indicated antibodies. e U2OS cells transfected with a combination of ATAD5 siRNA and a DNA vector expressing ATAD5-myc under the Noco-APH condition were treated with 2 mM HU for 6 h. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. f U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU or 1 μM CDC7 inhibitor (CDC7i, PHA-76941) for the indicated times. Then, chromatin-bound proteins were fractionated and subjected for immunoblotting. g HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min prior to treatment with 2 mM HU as indicated. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. h U2OS-TetOn-ATAD5 cells treated with doxycyclinfor 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected for immunoblotting. i, j U2OS cells transfected with siRNAs or treated with a RAD51 inhibitor (B02, 10, 20, 40 μM) at the time of release from aphidicolin under the Noco-APH condition were treated with 2 mM HU as indicated. Chromatin-bound proteins were fractionated and subjected for immunoblotting.

Article Snippet: U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073).

Techniques: Transfection, Western Blot, Staining, Standard Deviation, Expressing, SDS Page, Plasmid Preparation, Labeling

Journal: Nature communications

Article Title: ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

doi: 10.1038/s41467-019-13667-4

Figure Lengend Snippet: Fig. 5 ATAD5 promotes generation of MUS81-mediated single-stranded DNA-associated breaks in response to replication stress. a, b U2OS cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for analysis by pulsed field gel electrophoresis. a Representative data from three independent experiments. Asterisk (*) indicates a DNA break. b DNA breaks were quantified and displayed (N = 4). c U2OS or HeLa cells transfected with ATAD5 siRNA under the Noco-APH condition were treated with 2 mM HU for 6 h before being collected for a neutral COMET assay. The tail moment was calculated from ~200 cells and plotted. d U2OS cells expressing ATAD5AID were pre-treated with auxin and treated with 2 mM HU for another 6 h before being collected for a neutral COMET assay. Two independent experiments were performed, and one representative result is displayed. e U2OS-TetOn-ATAD5 cells treated with doxycycline for 24 h before entering the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. f U2OS cells transfected with siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h. Chromatin-bound proteins were fractionated and subjected to immunoblotting. g U2OS cells transfected with a combination of ATAD5 and MUS81 siRNAs under the Noco-APH condition were treated with 2 mM HU for 6 h before collection for the neutral COMET assay. The tail moment was calculated from ~300 cells and plotted. h HEK293T cells transfected with ATAD5 siRNA for 48 h were labeled with 10 μM EdU for 20 min, washed, and treated with 2 mM HU for the indicated times. Samples were processed for iPOND, and captured proteins were separated by SDS-PAGE and immunoblotted. b–e, g Error bars represent standard deviation of the mean. Statistical analysis: two-tailed Student’s t test; *p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001.

Article Snippet: U2OS cells expressing osTIR1-9Myc were generated by retroviral infection of pBabe Blast osTIR1-9Myc from Andrew Holland (Addgene, plasmid # 80073).

Techniques: Transfection, Nucleic Acid Electrophoresis, Neutral Comet Assay, Expressing, Western Blot, Labeling, SDS Page, Standard Deviation, Two Tailed Test