Journal: The Journal of Biological Chemistry

Article Title: TOX High-Mobility Group Box Family Member 4 promotes DNA double-strand break repair via nonhomologous end joining

doi: 10.1016/j.jbc.2025.110174

Figure Lengend Snippet: TOX4 mediates DNA repair via NHEJ. A , HeLa cells were treated with TOX4 siRNA and DNA-PKcs inhibitor NU7026 (10 μM) for 24 h, as indicated. The cell lysates were analyzed by immunoblotting for γ-H2AX, TOX4, and β-actin. B , corresponding densitometric analyses of γ-H2AX/β-actin, in I, were shown. C , HeLa cells were treated with TOX4 siRNA, caffeine (2 mM), and PARP inhibitor olaparib (10 μM) for 24 h, as indicated. The cell lysates were analyzed by immunoblotting for γ-H2AX, TOX4, and β-actin. D , corresponding densitometric analyses of γ-H2AX/β-actin, in K , are shown. Statistical significance was analyzed using an unpaired two-tailed Student t test (∗ p < 0.05, ∗∗∗ p < 0.001). E , NHEJ efficiency was measured using a chromosome-integrated, I-SceI-based NHEJ reporter (U2OS-EJ5) in which NHEJ repair leads to GFP expression. Cells were transfected with control or TOX4 siRNA, and DNA repair was measured by immunoblotting of GFP expression in relative to β-actin. The mean values and SDs, calculated from four independent experiments, are shown. F , an extrachromosomal NHEJ reporter was designed, as described in the section, using pEGFP-N vector linearized by EcoRI endonuclease. Upon transfection into HeLa cells, the repair of this vector resulted in GFP expression. Cells were treated with TOX4 siRNA or DNA-PKcs inhibitor NU7026, as indicated. Immunoblotting of GFP, TOX4, and β-actin is shown. G , NHEJ repair assay, as in N, was quantified for the ratio of GFP/β-actin expression. Statistical significance was determined using an unpaired two-tailed Student t test (∗∗ p < 0.01, ∗∗∗ p < 0.001). DNA-PKcs, DNA-dependent protein kinase catalytic subunit; NHEJ, nonhomologous end joining; TOX4, Thymocyte Selection–Associated High-Mobility Group Box Family Member 4.

Article Snippet: Doxorubicin (CAS: 23214-92-8), bleomycin (CAS: 11056-06-7), cisplatin (CAS: 15663-27-1), AZD7648 (CAS: 2230820-11-6), VE822 (CAS: 1232416-25-9), KU55933 (CAS: 587871-16-9), and NU7026 (CAS: 154447-35-5) were purchased from Selleckchem.

Techniques: Western Blot, Two Tailed Test, Expressing, Transfection, Control, Plasmid Preparation, Selection

Journal: Nucleic Acids Research

Article Title: Hypoosmotic stress induces R loop formation in nucleoli and ATR/ATM-dependent silencing of nucleolar transcription

doi: 10.1093/nar/gkz436

Figure Lengend Snippet: Hypoosmotic stress induces ATR and ATM-dependent phosphorylation of H2AX. ( A ) Human HeLa cells, untreated (control) or treated with hypoosmotic stress (OS) for 3 h were stained for γH2AX (green). The DNA was stained with DAPI (blue). Scale bar: 80 μm. ( B ) Quantification of γH2AX in control HeLa cells and cells subjected to hypoosmotic stress for the indicated time periods (0.5, 1 or 3 h). Box plots show the γH2AX fluorescence intensities. Horizontal lines represent the median. *** P < 0.0001, n.s.—not significant (unpaired t -test, n > 500). ( C ) HeLa cells were subjected to hypoosmotic stress for the indicated time periods (1 or 3 h) or treated with the indicated concentrations (5, 10 or 20 μg/ml) of the topoisomerase II poison etoposide for 1 h. WB was performed with an anti-γH2AX antibody. Throughout the figure, unmodified histone H2AX is used as a loading control in WB, and the control (C) represents untreated HeLa cells. ( D ) HeLa cells were pulse-labeled with EdU (10 μM, 30 min), subjected to hypoosmotic stress for 3 h and stained for γH2AX (green). EdU was revealed by Click Chemistry (red). The DNA was stained with DAPI (blue). Scale bar: 20 μm. ( E ) G1-, S- and G2-phase HeLa cells were subjected to hypoosmotic stress (OS) for 1 h. WB was performed with antibodies against γH2AX, cyclin B1 (a marker of G2 cells), and cyclin E (a marker of S cells). ( F ) HeLa cells were pulse-labeled with EdU (10 μM, 30 min), subjected to hypoosmotic stress (200 and 150 mOsm/l, 3 h), and stained for γH2AX. EdU was revealed by Click Chemistry. The DNA was stained with DAPI. Microscopic images were processed with CellProfiler software as follows: nuclei were segmented based on DAPI fluorescence, the cell population was divided into S-phase (EdU-positive) and non-S-phase (EdU-negative) cells and the γH2AX fluorescence intensity was measured. Box plots show the γH2AX fluorescence intensities. Horizontal lines represent the median. *** P < 0.0001, n.s.—not significant (unpaired t -test, n > 500). ( G and H ) WB analysis of γH2AX in HeLa cells pre-treated with siRNAs ( G ) or chemical compounds ( H ) to suppress the activity of either ATM (ATM kd and KU55933), ATR (ATR kd and VE821) or DNA-PKcs (PRKDC kd and NU7026) and subjected to hypoosmotic stress for 1 h. ( I and J ) HeLa cells were subjected to hypoosmotic stress for 1 or 3 h. WB was performed with antibodies against γH2AX, phospho-ATM (Ser1981), and phospho-ATR (Thr1989), phospho-CHK1 (Ser345), and phospho-CHK2 (Thr68).

Article Snippet: Hypoosmotic stress was applied by incubation the cells in 50% DMEM/50% H2O for 30 min–3 h. Hyperosmotic stress was applied by incubation in DMEM supplemented with 600 mM NaCl for 1 h. For kinase inhibition experiments, cells were treated with 20 μM KU55933 (Tocris Bioscience) for 3 h, 15 μM VE821 (Sigma) for 3 h or 50 μM NU7026 (Adooq Bioscience) for 6 h. For transcription inhibition experiments, cells were treated with 0.01 μg/ml actinomycin D (Biotium) for 3 h, or 50 μM DRB (Santa Cruz Biotechnology) for 3 h. For induction of DSBs, the cells were treated with 1–20 μg/ml etoposide (Sigma) for 1 h. I-PpoI nuclear translocation was initiated by incubation the cells with 5 μM 4-hydroxytamoxifen (4-OHT) (Sigma) for 4–16 h.

Techniques: Phospho-proteomics, Control, Staining, Fluorescence, Labeling, Marker, Software, Activity Assay

Journal: Nucleic Acids Research

Article Title: Hypoosmotic stress induces R loop formation in nucleoli and ATR/ATM-dependent silencing of nucleolar transcription

doi: 10.1093/nar/gkz436

Figure Lengend Snippet: Hypoosmotic stress-induced DDR leads to ATR/ATM-dependent silencing of rDNA transcription. ( A ) HeLa cells, untreated or exposed to hypoosmotic stress for 3 h, were simultaneously pulsed with 5-fluorouridine (FU) for 3 h. Throughout the figure, FU was revealed by immunocytochemistry. The DNA was stained with DAPI (blue). Scale bar: 15 μm. ( B ) HeLa cells, untreated or exposed to hypoosmotic stress for 0.5, 1 or 3 h, were pulsed with FU for 30 min. Box plots show the FU fluorescence intensities. Horizontal lines represent the median. ( C ) Quantification of FU fluorescence intensities in HeLa cells that were pre-treated with specific inhibitors of either ATM (KU55933), ATR (VE821) or DNA-PKcs (NU7026) and then subjected to hypoosmotic stress for 3 h. Horizontal lines represent the median. *** P < 0.0001, *not significant (unpaired t -test, n > 500). ( D ) Quantification of FU fluorescence intensities in HeLa cells with CRISPR/Cas9-based knockout of histone H2AX or RNA interference-based knockdowns of either ATM, ATR, DNA-PKcs (PRKDC) or Nbs1 that were subjected to hypoosmotic stress for 3 h. Horizontal lines represent the median. *** P < 0.0001, n.s.—not significant (unpaired t -test, n > 500). ( E ) qRT-PCR showing levels of pre-rRNA normalized to GAPDH mRNA in HeLa cells treated as in D. HeLa cells treated with Pol I inhibitor ACD were used as a negative control. The data are represented as the mean ±SD. *** P < 0.0001, n.s.—not significant (unpaired t -test, n > 500). ( F ) HeLa cells expressing homing endonuclease I-PpoI were mock-treated (−4-OHT) or treated with 4-hydroxytamoxifen (+4-OHT) for 16 to activate I-PpoI and stained for γH2AX (green) and nucleolin (red). The DNA was stained with DAPI (blue). Scale bar: 20 μm. ( G ) HeLa cells expressing homing endonuclease I-PpoI were pre-treated with specific inhibitors of either ATM (KU55933), ATR (VE821) or DNA-PKcs (NU7026), and then treated with 4-hydroxytamoxifen (4-OHT) for 4 h to activate I-PpoI and pulsed with FU for 3 h. HeLa cells expressing I-PpoI, mock-treated (−4-OHT) or incubated with 4-OHT (C), were used as controls. Box plots show the FU fluorescence intensities. Horizontal lines represent the median. *** P < 0.0001, *not significant (unpaired t -test, n > 500).

Article Snippet: Hypoosmotic stress was applied by incubation the cells in 50% DMEM/50% H2O for 30 min–3 h. Hyperosmotic stress was applied by incubation in DMEM supplemented with 600 mM NaCl for 1 h. For kinase inhibition experiments, cells were treated with 20 μM KU55933 (Tocris Bioscience) for 3 h, 15 μM VE821 (Sigma) for 3 h or 50 μM NU7026 (Adooq Bioscience) for 6 h. For transcription inhibition experiments, cells were treated with 0.01 μg/ml actinomycin D (Biotium) for 3 h, or 50 μM DRB (Santa Cruz Biotechnology) for 3 h. For induction of DSBs, the cells were treated with 1–20 μg/ml etoposide (Sigma) for 1 h. I-PpoI nuclear translocation was initiated by incubation the cells with 5 μM 4-hydroxytamoxifen (4-OHT) (Sigma) for 4–16 h.

Techniques: Immunocytochemistry, Staining, Fluorescence, CRISPR, Knock-Out, Quantitative RT-PCR, Negative Control, Expressing, Incubation

Journal: Scientific Reports

Article Title: HERC2 regulates RPA2 by mediating ATR-induced Ser33 phosphorylation and ubiquitin-dependent degradation

doi: 10.1038/s41598-019-50812-x

Figure Lengend Snippet: Effect of suppression of HERC2 E3 ligase activity on RPA2 Ser33 phosphorylation and ubiquitination. ( a ) Wild type and HERC2 ΔE3/ΔE3 HCT116 cells were treated with 0.2 mM HU for the indicated time and subjected to immunoblotting with the indicated antibodies. HERC2 (N) : the antibody to an epitope 1781–1974 of HERC2. ( b ) HCT116-HERC2 ΔE3/ΔE3 cells were incubated with or without 10 μM ATR inhibitor VE821, 10 μM ATM inhibitor Ku55933, or 10 μM DNA-PK inhibitor Nu7026 for 2 h as indicated and subjected to immunoblotting with the indicated antibodies. ( c ) Wild type and HERC2 ΔE3/ΔE3 HCT116 cells were treated or not with MG132 and 5 μM ATR inhibitor for 12 h as indicated and subjected to immunoprecipitation in denature condition with control IgG or anti-RPA2 antibody followed by immunoblotting (left panels) or to direct immunoblotting (right panels).

Article Snippet: Chemical agents used in the present study were hydroxyurea (HU) (Sigma-Aldrich), mitomycin C (MMC) (Sigma-Aldrich), irinotecan hydrochloride (CPT) (Sigma-Aldrich), aphidicolin (APH) (Sigma-Aldrich), MG132 (Calbiochem), the ATR inhibitor VE821 (Toronto Research Chemicals), the ATM inhibitor Ku55933 (TOCRIS Bioscience), and the DNA-PK inhibitor Nu7026 (ChemScene).

Techniques: Activity Assay, Phospho-proteomics, Ubiquitin Proteomics, Western Blot, Incubation, Immunoprecipitation, Control

Journal: EMBO Molecular Medicine

Article Title: The evolving genetic landscape of telomere biology disorder dyskeratosis congenita

doi: 10.1038/s44321-024-00118-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: DNA-PK inhibitor NU7026 , STEMCELL Technologies , 74172.

Techniques: Recombinant, FLAG-tag, Sequencing, In Situ, Software