Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 −/− ESCs are impaired in ATM activation and hypersensitive to DOX treatment. (A) Phosphorylation of ATM, H2AX, and p53 in Sall4 +/− and Sall4 −/− ESCs at different time points after DOX treatment. (B and C) Sall4 +/− and Sall4 −/− ESCs were mixed, treated with DOX or mock treated, and examined for the foci formation by ATM-Ser1987p (B) or γ-H2AX (C). DNA is counterstained with DAPI (blue). Bars, 10 µm. Graphs show the change of average fluorescence intensity (AFI) determined by dividing the overall mean fluorescence intensity by the area of the cell, and values are mean ± SEM. **, P < 0.01; ***, P < 0.001 by t test throughout the figure. (D) Cell cycle G 2 /M checkpoint is impaired in Sall4 −/− ESCs after DOX treatment. (top) Mock-treated control. (bottom) 1 h after after DOX treatment. The values are means ± SEM ( n = 4). *, P < 0.05 by t test. Circles denote the percentages of cells positive for histone 3 phosphorylated at Ser10 in all cells. PI-A, propidium iodide area. (E) Inducible expression of Sall4 in Sall4 −/− ESCs rescues the activation of ATM after DOX (0.5 µM) treatment. Sall4 −/− ESCs were stably transfected with a doxycycline-inducible vector expressing ectopic Sall4 (Lenti- Sall4 ).

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Activation Assay, Phospho-proteomics, Fluorescence, Control, Expressing, Stable Transfection, Transfection, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 is relocated from the heterochromatin to DNA DSBs in ESCs after DOX treatment. (A) Sall4 is sequestered in the heterochromatin and relocated to DNA DSBs in ESCs after DNA damage. Sall4 +/− cells were examined for the foci formation of γ-H2AX and Sall4 before and after DNA DSB damage induced by DOX. Nuclei were counterstained with DAPI (blue). For intensity correlation analysis (ICA), pixels from the Sall4 channel covarying positively with the corresponding signal from the γ-H2AX or DAPI channel are denoted. Bar, 5 µm. (B) Rr analysis for the colocalization of Sall4 and γ-H2AX. Rr is the Pearson’s correlation coefficient, and a value of 1 indicates perfect colocalization. The values of Rr are means ± SEM. ***, P < 0.001 by t test. (C) Sall4 is colocalized with HP1-α foci before DNA DSB damage and is mobilized out of HP1-α foci after DNA damage. Nuclei are counterstained with DAPI (blue). Bar, 5 µm. (D) The average fluorescence intensity (AFI) of Sall4 at heterochromatin was determined by dividing the overall mean fluorescence intensity by the area of the cell, and values are means ± SEM. **, P < 0.01 by t test. (E) DNA damage disrupts the interaction between Sall4 and HP1-α in ESCs. The interaction between Sall4 and HP1-α was analyzed by coimmunoprecipitation (Co-IP) with the HP1-α antibody.

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Fluorescence, Co-Immunoprecipitation Assay

Journal: The Journal of Cell Biology

Article Title: Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

doi: 10.1083/jcb.201408106

Figure Lengend Snippet: Sall4 linking SWI/SNF-associated chromatin remodeling to MRN-dependent ATM activation. (A) Silencing of Baf60a impairs the recruitment of Sall4 to γ-H2AX foci after DNA DSB damage. Nuclei are counterstained with DAPI (blue). For intensity correlation analysis (ICA), pixels from the Sall4 channel covarying positively with the corresponding signal from the γ-H2AX channel are denoted. si-Con, control siRNA. Bar, 5 µm. (B) Inhibition of H3K14ac by CPTH2 disrupts the association of Baf60a, Sall4, and ATM with DNA DSBs in the cellular extract of Sall4 +/− ESCs treated with 0.5 µM DOX for 4 h. WNE, whole nuclear extract. (C) Inhibition of H3K14ac by CPTH2 in Sall4 +/− ESCs impaired ATM-dependent responses to DNA DSB damage. (D) The kinetics of the recruitment of Rad50, Sall4, and Balf60a to the specific DNA DSB. ChIP analysis showed the kinetics of the recruitment of Sall4, Rad50, and Baf60a to the single I-PpoI cleavage site at chromosome 3 in Sall4 +/− cells. Time denotes the minutes after the addition of 4-hydroxytamoxifen. Values are means ± SEM. (E) An illustrated model for Sall4 to function as a transducer of chromatin remodeling to ATM signaling in ESCs after DNA damage. P, phosphorylation.

Article Snippet: After permeabilization with 0.4% Triton X-100, the cells were blocked in 3% BSA and then labeled with rabbit or mouse antibodies against Sall4 ab29112 (Abcam) or sc-101147, respectively; pATM (Ser 1981; 200–301-400; ROCKLAND); γ-H2AX; Baf60a; and Rad50 overnight at 4°C.

Techniques: Activation Assay, Control, Inhibition, Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Localization and retention of p90 ribosomal S6 kinase 1 in the nucleus: implications for its function

doi: 10.1091/mbc.E11-07-0658

Figure Lengend Snippet: RSK1 phosphorylation by PDK1 is required for its nuclear translocation. (A) Schematic of phosphorylation sites on rat RSK1. (B) Top two rows, serum-starved HeLa cells were treated with and without EGF (100 nM) for 10 min and the localization of total RSK1 was monitored with anti-RSK1 antibody; 4′,6-diamidino-2-phenylindole staining shows nuclei. Bottom four rows, the PDK1 inhibitor BX795 inhibits nuclear translocation of RSK1. HeLa cells were treated as described above with and without overnight preincubation with 0.5 μM BX795. The localization of active RSK1 was monitored with anti–phospho-T573-RSK antibody. (C) BX795 inhibits phosphorylation of RSK1 on S221 and S732. Cells were treated as in B. After immunoprecipitation with anti-RSK1 antibody, Western analyses of the immune complexes were performed with anti–phospho RSK antibodies. Scale bar, 10 μm. Representative data from three independent experiments are shown.

Article Snippet: Anti–phospho-RSK1/2 (S221) from R&D Systems (Minneapolis, MN), anti–phospho-RSK (T359/S363) from Cell Signaling (Beverly, MA), anti–phospho-RSK (S380) from Epitomics (Burlingame, CA), anti–phospho-RSK (T573) from Cell Signaling, and anti–phospho-RSK S732 raised by Rockland (Gilbertsville, PA) were used.

Techniques: Phospho-proteomics, Translocation Assay, Staining, Immunoprecipitation, Western Blot

Journal: Journal of Dental Sciences

Article Title: Overexpression of sprouty 1 protein in human oral squamous cell carcinogenesis

doi: 10.1016/j.jds.2020.07.013

Figure Lengend Snippet: Western blot analyses: phosphor-ERK and total-ERK in human oral cancer cell lines as compared with a primary culture of normal oral mucosa (HOK). Upregulation of phosphor-ERK (normalized to total-ERK) in OECM1 and Ca922 cell lines as compared with HOK, and a slightly decreased expression in SAS as compared with HOK. Results were quantified using densitometric analysis, normalized to the level of β-actin, and expressed as a fold change relative to the normal oral mucosa. Bars represent means ± standard deviation of the mean (∗ P < 0.05). A representative result of three independent experiments is shown.

Article Snippet: Further, samples were analyzed using 10% SDS-PAGE (Sigma-Aldrich) gels, and the proteins were transmitted onto a PVDF membrane (Sigma-Aldrich) using Bio-Rad's transblot with primary antibodies against phosphor-ERK (Boster Biological Technology, CA, USA; Cat. No. P00104; 1:1000) and total-ERK (Boster Biological Technology; Cat. No. P00104; 1:1000), with species specificity for human tissues and an observed molecular weight of 42–44 kDa; and β-actin (Sigma-Aldrich; 1:1000), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich; 1:5000).

Techniques: Western Blot, Expressing, Standard Deviation