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: 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: 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: 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: bioRxiv

Article Title: NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1101/230706

Figure Lengend Snippet: A 53BP1-interacting partners identified by mass spectrometry in this and in previous studies. B Photobleaching FRET in U2OS cells expressing 53BP1 or 53BP1 ct and NuMA fused to GFP and mCherry (mCh). A GFP-mCh tandem construct was used as positive control, whereas coexpression of GFP and mCh was used as negative control. C Immunoprecipitation (IP) of NuMA from U2OS nuclear extracts (N.E.). Nonspecific immunoglobulins (IgGs) were used as controls. The western blots were probed for NuMA and 53BP1. Cells were exposed to IR (10 Gy, followed by 30 min recovery) or treated with H 2 O 2 (1 mM, 10 min) prior IP. Densitometric quantification of 53BP1 pull down is shown in the graph. The ratio of 53BP1 over NuMA signals in IP samples was calculated and normalized to controls. The data represent mean ± SEM. *, P < 0.05 (one sample t-test; n = 4 (IR) or 3 (H 2 O 2 )). D Western blot analysis of γH2AX and PAR in the nuclear extracts used for IP. The results confirm the induction of DSB by IR and oxidative DNA damage by H 2 O 2 . Immunoblot for H2B and Ponceau staining are shown as loading controls. E FRET measured using fluorescence lifetime imaging in nonirradiated (control) and irradiated (IR) U2OS cells. 53BP1 and NuMA were labeled with antibodies coupled to Alexa Fluor 488 (FRET donor) or with Alexa Fluor 555 (FRET acceptor), respectively. Donor fluorescence lifetime was measured in the absence or presence of acceptor and used to calculate FRET efficiencies. Data represent mean ± SEM (n = 4). F Colocalization between 53BP1 and NuMA immunostaining in S1 cells treated with vehicle (control) or with bleomycin. Mander’s M2 colocalization coefficients for 53BP1 and NuMA or for 53BP1 and DAPI are shown in the bar graphs. Bars with paler colors quantify colocalization after shifting 53BP1 images by 10 pixels. Orange bars represent colocalization between NuMA and synthetic images with low (a) or high (b) foci density, as illustrated in the inset. Values were normalized to control. *, P < 0.05 (t-test, n = 12 image frames from three experiments). Scale bar, 5 μm. G FCS analysis of GFP-53BP1, GFP, and GFP-MeCP2 diffusion in cells transfected with nontargeting (NT) or with NuMA-specific siRNA. Cells were treated with bleomycin or vehicle. *, P < 0.001 (ANOVA and Tukey; n = 50–60 cells from two independent biological replicates). H NuMA silencing verified by immunostaining in U2OS cells transfected as in (G) . Scale bar, 50 μm I NuMA silencing verified by western blot with protein extracts from U2OS cells transfected as in (G) .

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Mass Spectrometry, Expressing, Construct, Positive Control, Negative Control, Immunoprecipitation, Western Blot, Staining, Fluorescence, Imaging, Control, Irradiation, Labeling, Immunostaining, Diffusion-based Assay, Transfection

Journal: bioRxiv

Article Title: NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1101/230706

Figure Lengend Snippet: A-B Immunoprecipitation of NuMA from non-neoplastic HMT-3522 S1 (A) and malignant HMT-3522 T4-2 (B) nuclear extracts. Nonspecific IgGs were used as control. The blots were probed for NuMA and 53BP1. C 53BP1 levels in HMT-3522 S1 cells untreated (control), treated with bleomycin (20 mU/ml, 1h), or exposed to ionizing radiations (IR, 3 Gy followed by 1h recovery). Densitometry analysis of 53BP1 levels is shown in the bar graph (mean ± SEM; n = 4). D 53BP1 levels in U2OS cells after bleomycin treatment. Cells were transfected with NuMA (siNuMA) or nontargeting (siNT) siRNA. The γH2AX and NuMA immunoblots verify DNA damage induction and silencing, respectively. Densitometry analysis of 53BP1 levels is shown in the bar graph (mean ± SEM; n = 4).

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Immunoprecipitation, Control, Transfection, Western Blot

Journal: bioRxiv

Article Title: NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1101/230706

Figure Lengend Snippet: A NuMA silencing and 53BP1 expression verified by western blot. Lamin B is shown as loading control. B IR-induced focal accumulation of 53BP1 in HMT-3522 S1 cells transfected with nontargeting (NT) or NuMA siRNA. Confocal images show NuMA and 53BP1 immunostaining in irradiated cells (3 Gy) left to recover for 2h. The arrowhead points to a cell retaining NuMA expression in contrast to its neighbors. Quantification of 53BP1 foci density is presented in the bar graph. *, P < 0.05 and **, P < 0.001 (ANOVA and Tukey’s post-hoc test; n = 4). The inset shows the average number of 53BP1 foci per nucleus in cells silencing (−) or retaining (+) NuMA expression within the siNuMA transfection condition. #, P < 0.05 (t-test). C Relative intensity of 53BP1 immunostaining signals at cleaved ISceI sites. Cleavage sites were identified by labeling flanking Lac arrays with GFP-Lac. *, P < 0.01 (t-test; n ≥ 40 cells from three biological replicates). D-E Accumulation of GFP-53BP1 ct at laser-microirradiated tracks in U2OS cells expressing mCherry or mCherry-NuMA (insets; D ) or transfected with nontargeting and NuMA siRNA (E) . The fractions of GFP signals at the tracks are shown in the bar graphs. * P < 0.05 and ** P < 0.0001 (t-test; n > 10 cells). F IR-induced NuMA phosphorylation at Ser395 (P-NuMA) in U2OS cells detected by immunoblot. G Localization of P-NuMA signals by immunostaining after bleomycin treatment (20 mU/ml; 1h) in cells transfected with NT or NuMA siRNA. H Accumulation of mCherry-53BP1 ct after laser-microirradiation in cells expressing GFP, GFP-NuMA, or a nonphosphorytable NuMA mutant [GFP-NuMA(S395A)]. * P < 0.05 (t-test; n > 15 cells from three biological replicates). Scale bars, 10 μm

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Expressing, Western Blot, Control, Transfection, Immunostaining, Irradiation, Labeling, Phospho-proteomics, Mutagenesis

Journal: bioRxiv

Article Title: NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1101/230706

Figure Lengend Snippet: A Detection of DSB with the neutral comet assay in HMT-3522 S1. Cells were transfected with nontargeting or NuMA siRNA, irradiated (3 Gy) and processed immediately or left to recover for 2h. Controls were mock irradiated. Average proportions of DNA in comet tails are shown in the bar graph (n = 3) B Cell cycle distribution in HMT-3522 S1 cells transfected with NuMA-specific siRNA or with nonspecific siRNA. C Enumeration of 53BP1 foci in HMT-3522 S1 cells transfected with constructs encoding NuMA specific (shNuMA) or scrambled (shControl) small hairpin RNA. Six days after transfection, cells were treated with BLM or vehicle. The number of cells with ≥30 53BP1 foci is indicated for each condition in parentheses. *, P < 0.001 (Kruskal-Wallis; n ≥ 180 cells from two biological replicates). mCherry was cotransfected with the shRNA to identify transfectants, as illustrated in the micrographs. D Representative GFP-53BP1 fluorescence images and GFP-53BP1 foci quantification (scatter plot) in U2OS cells transfected with nontargeting (NT) or NuMA siRNA. Cells were treated with vehicle, bleomycin, or MMC. Scale bar, 10 μm. *, P < 0.05 and **, P < 0.005 (ANOVA and Tukey; n > 100 cells from three biological replicates).

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Neutral Comet Assay, Transfection, Irradiation, Construct, shRNA, Fluorescence

Journal: bioRxiv

Article Title: NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1101/230706

Figure Lengend Snippet: A Accumulation of GPF-tagged 53BP1 at laser-microirradiated tracks in U2OS cells expressing mCherry or mCherry-NuMA. The graph represents the fractions of GFP signals at the tracks. B-C Accumulation of GFP-PCNA at laser-microirradiated tracks in cells overexpressing (B) or silencing (C) NuMA. D GFP-53BP1 accumulation at laser-microirradiated tracks in cells transfected with nontargeting siRNA or with SNF2h siRNA. Scale bars, 10 μm (A-D). E Verification of SNF2h silencing by fluorescence microscopy. Scale bar, 100 μm

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Expressing, Transfection, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1101/230706

Figure Lengend Snippet: A Class switch recombination (CSR) in stimulated CH12F3-2 B cells stably transduced with scrambled, 53BP1, and NuMA-specific shRNA vectors. 53BP1 and NuMA silencing was verified by western blot. B CSR in cells nucleofected to express GFP, GFP-NuMA, or GFP-NuMA(S395A). CSR was quantified among GFP-positive cells. A representative flow cytometry analysis of cells expressing GPF and GFP-NuMA is shown on the right. C Quantification of radial chromosomes and chromosome fusions after vehicle or olaparib (0.5 μM; 24h) treatment of BRCA1-null SUM149 cells transfected with nontargeting or 53BP1 siRNA. *, P < 0.005 (ANOVA and Tukey, n ≥ 20). D Chromosomal aberrations (radials + fusions) scored in olaparib-treated SUM149 cells transfected with siRNAs, GFP, or GFP-NuMA. *, P < 0.005 (ANOVA and Tukey, n ≥ 50). #, P < 0.05 (t-test, n ≥ 25). Representative metaphase micrographs are shown. Arrowheads indicate aberrations. E Validation of 53BP1 and NuMA silencing and GFP-NuMA expression (upper band) in SUM149 cells by western blot.

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Stable Transfection, Transduction, shRNA, Western Blot, Flow Cytometry, Expressing, Transfection, Biomarker Discovery

Journal: bioRxiv

Article Title: NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

doi: 10.1101/230706

Figure Lengend Snippet: A Quantification of NHEJ activity in U2OS cells with a stably integrated NHEJ reporter constituted of the GFP coding sequence interrupted by an exogenous exon flanked by ISceI recognition sites. NHEJ reconstitutes the GFP coding sequence after ISceI cleavage. Normalized fractions of GFP-positive cells 24h after transfection with ISceI and mCherry or mCherry-NuMA are shown in the bar graph (left). The fraction of cells with mCherry fluorescence among GFP-expressing cells is shown in the cross-ruled graph whereas cell cycle distribution is shown on the right. B Quantification of repair events in U2OS-NHEJ cells transfected with NuMA or with nontargeting (NT) siRNA, and with ISceI, 48h and 24h before flow cytometry analysis, respectively. The same experiment was done in serum-starved cells to normalize cell cycle distribution. *, P < 0.05 (t-test, n = 4)

Article Snippet: The shRNA vectors targeting human NuMA were purchased from Origene (TR311065).

Techniques: Activity Assay, Stable Transfection, Sequencing, Transfection, Fluorescence, Expressing, Flow Cytometry

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 Promotes odontoblastic differentiation of progenitor cells in the dental pulp. (a) Schematic diagram of the live‐imaging in recombinant CPNE7 (rCPNE7)‐treated hDPCs. (b) Live‐cell images of hDPCs cultured in differentiation media for 7 days following 24 h of rCPNE7 treatment. Each frame was collected from a single Z plane, and recorded at 320 min intervals. Odontoblast process‐like structure elongation (white boxed area) is observed in rCPNE7‐treated hDPCs. Scale bars: 50 μm. (c) Snapshots at various points on the Z plane, which were generated in three dimensions using the Imaris software. Localizations of TAU (green sphere), F‐Actin (red), and Microtubule (blue) in hDPCs are shown. Boxed area on the left is shown at a higher magnification on the right. N, Nucleus. (d) Schematic diagram of ex vivo subcutaneous transplantation model. Stem cells were mixed with 100 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) particles alone (control) or with rCPNE7 in a poly ε‐caprolactone (PCL) spaces and transplanted subcutaneously into immunocompromised mice for 6 weeks. (e) H&E staining of the sectioned samples was analyzed histologically. Boxed areas on the left (scale bars: 200 μm) Are shown at higher magnification on the right (scale bars: 200 μm). Black arrowheads indicate newly formed mineralized tissues. The dental pulp stem cells (DPSCs), adipogenic stem cells (ADSCs), bone marrow stem cells (BMSCs), gingival fibroblasts (GFs), and periodontal ligament stem cells (PDLSCs) were mixed with 100 mg of HA/TCP particles alone (control) or with rCPNE7 in a poly ε‐caprolactone (PCL) spaces and transplanted subcutaneously into immunocompromised mice for 6 weeks. (f) Semi‐quantification of the volumes of the newly mineralized tissues. All values represented the mean ± SD of triplicate experiments. ** p < 0.01 and * p < 0.05 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Imaging, Recombinant, Cell Culture, Generated, Software, Ex Vivo, Transplantation Assay, Control, Staining

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 ‐null mice molars demonstrate pathologic dentin‐pulp complex phenotype. (a) Light microscopic images of 6‐month‐old WT and Cpne7 −/− mice molars. Thinner coronal dentin (red arrowheads) through which the underlying pulp chambers are seen in Cpne7 −/− mice. Mx, Maxilla; Mn, Mandible. (b) Micro‐computed tomographic images of WT and Cpne7 −/− mice mandibles manifest thinner coronal dentin (red arrowheads) in Cpne7 −/− mice. Quantitative analysis of coronal dentin thickness in WT and Cpne7 −/− mice molars. (c) 3D reconstruction of pulp spaces and quantitative analysis of pulp space volumes in WT and Cpne7 −/− mice molars. (d) Histological analysis of mandibular first molars of WT and Cpne7 −/− mice at 6 months by H&E staining. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; green arrowhead, entrapped cells; red arrow, cells getting entrapped. (e) Cell counting analysis at the dentin‐pulp interfaces of WT and Cpne7 −/− mice molars. (f) TUNEL staining of dental pulp cells of WT and Cpne7 −/− mice at 6 months. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, pathologic dentin; black arrow, TUNEL‐positive cells. (g) Quantitative analysis of the number of TUNEL‐positive cells in WT and Cpne7 −/− mice molars. (h) Transmission electron microscopic images of dentin‐pulp interfaces in the WT and Cpne7 −/− mice molars. Normal nuclear‐shaped hDPCs (white dotted line) and nuclear‐deformed hDPCs (red dotted line) are observed in WT and Cpne7 −/− mice molars, respectively. D, dentin; Op, odontoblast process; N, nucleus; M, mineralized tissue; Cp, cytoplasmic process. (i) Semi‐quantification of nuclear deformation in WT and Cpne7 −/− odontoblasts. All values represented the mean ± SD of triplicate experiments. ** p < 0.01 and * p < 0.05 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Staining, Cell Counting, TUNEL Assay, Transmission Assay, Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 deletion leads to premature aging of the mouse molar pulp environment. (a) Histological analysis of mandibular first molars of WT and Cpne7 −/− mice at 1 year by H&E staining. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; green arrowhead, entrapped cells; red arrow, cells getting entrapped. (b) Senescence‐associated β‐galactosidase (SA‐ β gal) activity (black arrowhead) is observed in 6‐month‐old WT and Cpne7 −/− mice molar by light microscope. Quantitative analysis of the number of SA‐β gal positive cells in WT and Cpne7 −/− mice molars. Scale bars: 20 μm. Boxed areas are shown at higher magnification. D, Dentin; P, Dental pulp; PD, Pathologic dentin. (c) Human DPCs were transfected with shControl (shCon) or shCpne7 vectors for 48 h and cultured in differentiation medium for up to 21 days. Representative images of SA‐β gal activity in Control, shCon, or shCpne7 groups. (d) The hDPCs (passage 3) were treated with 1 mM H 2 O 2 or H 2 O 2 + rCPNE7 for 30 min or 1 h. The hDPCs (passage 22) were used as positive control. SA‐β gal activity (red arrowhead) is observed in H 2 O 2 only or with rCPNE7‐treated hDPCs. (e) Semi‐quantification of SA‐β gal activity was analyzed. (f) The hDPCs (passage 3) were treated with 1 mM H 2 O 2 or H 2 O 2 + rCPNE7 for 72 h. SA‐β gal activity is observed in H 2 O 2 only or with rCPNE7‐treated hDPCs. (g) Quantitative analysis of the number of SA‐β gal positive cells. (h) Histological analysis of mandibular first molars of WT and Cpne7 TG mice at 23 months by H&E staining. Scale bars: 100 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm. D, Dentin; TD, Tertiary dentin; P, Dental pulp; PD, Pathologic dentin. (i) Quantitative analysis of the pulp space volumes in WT and Cpne7 TG mice molars at 23 months. All values represented the mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Staining, Activity Assay, Light Microscopy, Transfection, Cell Culture, Control, Positive Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 modulates intracellular reactive oxygen species (ROS) level in dental pulp cells. (a) Human DPCs were transfected with shCon or shCpne7; 48 h later, hDPCs were treated with 50 μM or 100 μM H 2 O 2 for 24 h and WST‐8 cell viability assay was performed. Oxidative stress‐induced cytotoxicity was analyzed by WST‐8 cell viability assay in hDPCs. (b) Control, shCon, or shCpne7 group incubated with DCFDA for 1 h and microplate analysis of green fluorescent DCFDA signals. (c) DCFDA signal was analyzed in 6‐month‐old WT and Cpne7 −/− mice molar by confocal microscopy. Scale bars: 50 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin (d) Semi‐quantification of DCFDA signal was analyzed in 6‐month‐old WT and Cpne7 −/− mice molar. (e) Live cell imaging of base intracellular ROS level in hDPCs through DCFDA staining. (f) Live cell imaging of intracellular ROS level by rCPNE7 treatment 15 min after H 2 O 2 treatment in hDPCs through DCFDA staining. Messenger RNA level of Cpne7 was analyzed by RT‐PCR at 30 min or 1 h after H 2 O 2 100 μM, 500 μM, or 1 mM treatment in hDPCs, respectively. (g) DCFDA fluorescence intensity was analyzed. (h) Light microscopic images of hDPCs treated with H 2 O 2 only or with rCPNE7. (i) Cytotoxicity was analyzed by WST‐8 cell viability assay in hDPCs treated with H 2 O 2 only or with rCPNE7. All values represented the mean ± SD of triplicate experiments. * p < 0.05 and ** p < 0.01 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Transfection, Viability Assay, Control, Incubation, Confocal Microscopy, Live Cell Imaging, Staining, Reverse Transcription Polymerase Chain Reaction, Fluorescence

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 deletion leads to DNA damage accumulation in dental pulp cells. (a–c) Heat map construction for hierarchical clustering and a list of downregulated or upregulated genes in rCPNE7‐treated hDPCs. (d) Human DPCs were transfected with shCon or shCpne7 for 48 h. Timeless and Chd6 mRNA levels were evaluated by qPCR. (e) Representative immunofluorescence images of γ‐H2AX (red) and semi‐quantification in 6‐month‐old WT and Cpne7 −/− mice molar ( n = 3). DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 20 μm. Boxed areas are shown at higher magnification. D, Dentin; Od, Odontoblasts; P, Dental pulp. (f) Semi‐quantification of γ‐H2AX (red) signal was analyzed. (g, h) Representative immunofluorescence images of γ‐H2AX (red) and ROS (green) in hDPCs ( n = 3). DAPI (blue) was counterstained to indicate the nucleus. Semi‐quantification of γ‐H2AX (red) and ROS (green) signal was analyzed in hDPCs. All values represented the mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Transfection, Immunofluorescence, Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 is involved in the repair process of oxidative stress‐induced DNA damage in dental pulp cells. (a) Schematic diagram of the microirradiation model in Cpne7 ‐GFP transfected hDPCs. (b) Expression level of CPNE7 and its localization (white arrowheads) before and after microirradiation in hDPCs. (c) Time course images of CPNE7 recruitment (white arrowheads) to the sites of DNA damage in hDPCs. (d) Semi‐quantification of GFP‐tagged CPNE7 signal at each time point. (e) Schematic diagram of the microirradiation model in hDPCs. (f) The recruitment of endogenous CPNE7 (green) was detected 10 and 30 min after DNA damage induction and co‐localized with γ‐H2AX (red). DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 10 μm. Boxed areas are shown at higher magnification. (g) Representative immunofluorescence images of γ‐H2AX (white arrow) in H 2 O 2 only or with rCPNE7‐treated hDPCs. DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 20 μm. Boxed areas are shown at higher magnification. (h, i) The hDPCs were treated with 5 μM, 100 μM, 1 mM, or 2 mM of H 2 O 2 only or w H 2 O 2 + rCPNE7 for 24 h, respectively. Gamma‐H2AX protein levels were evaluated by western blot analysis and semi‐quantified. (j, k) Neutral comet assay of hDPCs, treated for 24 h with H 2 O 2 only or H 2 O 2 + rCPNE7. Comet images of the hDPCs (green) were observed under a confocal microscope and semi‐quantified. Scale bars: 20 μm. Boxed areas are shown at higher magnification. All values represented the mean ± SD of triplicate experiments. * p < 0.05 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Transfection, Expressing, Immunofluorescence, Western Blot, Neutral Comet Assay, Microscopy, Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: CPNE7‐derived functional peptide rescues the pathologic dentin‐pulp complex phenotypes in Cpne 7 −/− mice molar. (a) Schematic diagram of the intraperitoneal CPNE7‐derived functional peptide (CPNE7‐DP) injection into WT and Cpne 7 −/− mice. (b) Histological analysis of dental pulp responses with or without intraperitoneal CPNE7‐DP injection in WT and Cpne7 −/− mice 3 months after, by H&E staining. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm. D, dentin; P, pulp; PD, pathologic dentin. (c) Representative immunofluorescence images of γ‐H2AX (red) with or without intraperitoneal CPNE7‐DP injection into 6‐month‐old Cpne7 −/− mice molar. DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 20 μm. Boxed areas were shown at higher magnification. D, Dentin; Od, Odontoblasts; P, Dental pulp. (d) Semi‐quantification of γ‐H2AX (red) signal was analyzed. (e) Schematic diagram of DNA damage rescue model in shCon or shCpne7‐transfected hDPCs. (f, g) CPNE7, γ‐H2AX, and P21 protein levels were evaluated by western blot analysis and semi‐quantified. (h) Histological analysis of dental pulp responses at the cavity preparation sites with or without CPNE7‐DP treatment in WT and Cpne7 −/− mice 3 weeks after H&E staining. *, Defected area; D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; green arrowhead, entrapped cells; red arrow, odontoblast‐like cells. (i) Schematic diagram of the indirect pulp capping model. Defect areas were restored with glass ionomer (GI) cement in WT, Cpne7 −/− , and Cpne7 TG mice at 8 months. (j) Histological analysis of the defected areas after 4 weeks was performed by H&E staining. Scale bars: 500 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. *, Defected area; D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; ND, Newly‐formed physiologic dentin. * p < 0.05 and # p < 0.0001 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Derivative Assay, Functional Assay, Injection, Staining, Immunofluorescence, Transfection, Western Blot, 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: 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