Journal: Cell Death & Disease

Article Title: TAGLN2 induces resistance signature ISGs by activating AKT-YBX1 signal with dual pathways and mediates the IFN-related DNA damage resistance in gastric cancer

doi: 10.1038/s41419-024-07000-1

Figure Lengend Snippet: A The IC 50 of Cisplatin, Oxaliplatin, Capecitabine and 5-FU were calculated in BGC-823 cells with TAGLN2 downregulation using Cell Counting Kit-8. TAGLN2 was abbreviated as TAG2. B The effect of X-ray irradiation on cell viability and clonogenic survival in both TAGLN2 downregulation (sh TAG2 ) and control (sh NC ) BGC-823 cells irradiated at doses of 0, 4, and 6 Gy X-rays. * P < 0.05, ** P < 0.01, *** P < 0.001, treatment vs. before treatment. ^ P < 0.05, ^^ P < 0.01, ^^^ P < 0.001, sh TAG2 vs. sh NC at 4 Gy or 6 Gy, respectively. C Relative mRNA levels of TAGLN2 and the panel of IFN-related genes prior to and after 0, 4, and 6 Gy X-ray treatment were evaluated by quantitative RT‒PCR analysis in both sh TAG2 and sh NC BGC-823 cells. D Hallmark pathway analysis by gene set enrichment analysis (GSEA) using a stomach adenocarcinoma (STAD) dataset from The Cancer Genome Atlas (TCGA). E Overexpression of TAGLN2 induces the accumulation of cytosolic ssDNA as evaluated by BrdU-γH2AX double labeling. Cells were stained for the primary BrdU antibody (red) and phospho-histone H2AX (green). F Representative images of tumors in different groups harvested at 5 h after one dose radiation (IR1-5 h) or 24 h after four doses of radiation (IR4-24 h) were analyzed by immunofluorescence with the antibody against phospho-histone H2AX (red). Slides were mounted with VectaShield antifade mounting medium with DAPI (blue). Images were taken using a 63× objective on a Zeiss LSM780 confocal microscope.

Article Snippet: Samples were blocked with 10% FBS in PBS for 1 h and incubated with the primary antibodies BrdU and phospho-histone H2AX (S139) (R&D Systems, Cats#MAB7225 and AF2288) for 3 h and with secondary antibodies for 1.5 h at room temperature.

Techniques: Cell Counting, Irradiation, Control, Over Expression, Labeling, Staining, Immunofluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: TAGLN2 induces resistance signature ISGs by activating AKT-YBX1 signal with dual pathways and mediates the IFN-related DNA damage resistance in gastric cancer

doi: 10.1038/s41419-024-07000-1

Figure Lengend Snippet: A ~ C Multiplex immunofluorescence of TMA was performed using the Opal 7-color Manual IHC Kit and VECTASHIELD ® HardSet Antifade Mounting Medium. The multiplex antibody panel was optimized as follows: TAGLN2, Opal 520 (yellow); CK, Opal 570 (green); YBX1, Opal 620 (red). The TMA was counterstained with DAPI (blue) and incubated with an antifluorescence quencher. Expression and spatial distribution of TAGLN2 or YBX1 in tissues and the correlation with patient clinical data. The DAPI channel was used to identify individual cells. A tissue segmentation algorithm combined with CK staining was applied to define tumoral and stromal areas. The scale bar is 200 μm. D Fisetin or MK2206 inhibited the accumulation of cytosolic ssDNA induced by overexpression of TAGLN2 by BrdU-γH2AX double labeling. HGC-27 cells stably transfected with TAGLN2 were prelabeled with BrdU and subsequently treated with 1 μg/ml Cisplatin with or without 10 μM Fisetin or 200 nM MK2206 in the medium. Cells were stained for DNA (DAPI, blue), the primary BrdU antibody (red) and phospho-histone H2AX (green). E Relative mRNA levels of the panel of IFN-related genes with or without 10 μM Fisetin or 200 nM MK2206 in the medium after 6 Gy X-ray treatment were evaluated by quantitative RT‒PCR analysis. The cytotoxicity induced by MK2206 from 16.25 nM to 13 μM ( F ) or MK2206 (200 nM) and Cisplatin (0.4 μg/ml) combination on tumor cells was detected ( G ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Samples were blocked with 10% FBS in PBS for 1 h and incubated with the primary antibodies BrdU and phospho-histone H2AX (S139) (R&D Systems, Cats#MAB7225 and AF2288) for 3 h and with secondary antibodies for 1.5 h at room temperature.

Techniques: Multiplex Assay, Immunofluorescence, Incubation, Expressing, Staining, Over Expression, Labeling, Stable Transfection, Transfection

Journal: Clinical and Translational Medicine

Article Title: Interleukin enhancer‐binding factor 2 promotes cell proliferation and DNA damage response in metastatic melanoma

doi: 10.1002/ctm2.608

Figure Lengend Snippet: ILF2 controls DNA damage response in metastatic melanoma cell lines. (A and B) Representative images and quantification of the number of nuclei per cell in M‐204 (A) and IM‐0223 (B) cells transduced with the ILF2 shRNA (sh‐ILF2) or non‐silencing shRNA (sh‐Ctrl). The nuclei were stained with DAPI (blue) and F‐actin was stained with Texas‐Red‐X phalloidin (red). Scale bar = 10 μm. (C and D) Drug sensitivity assays for melanoma cell lines with sh‐ILF2 (C) or ILF2‐OV (D) that were treated with different concentrations of TMZ for 3 days and compared to their respective controls. (E–J) Representative images at day 1 and day 13 (E, F, H and I) and quantification of spheroids area (G, J) of melanoma cell lines with ILF2 knockdown (sh‐ILF2) or ILF2 overexpression (ILF2‐OV) treated with 600 μM TMZ for 24 h starting at day 4. Scale bar = 50 μm. (K) Western blot and the quantification of ILF2 and γ‐H2AX in melanoma cells treated with 600 μM TMZ for the indicated time (1, 6, 12 and 24 h). β‐actin was used as the loading sample control. Data represent the mean ± SD. ns : not significant, ** p < .01 and **** p < .0001

Article Snippet: For quantification of γ‐H2AX foci, DP‐0574 ILF2‐OV and EV cells were stained with rabbit anti‐human γ‐H2AX primary Ab (1:50, Novus Biologicals, Littleton, CO) and CyTM3 goat anti‐rabbit IgG secondary Ab (1:600).

Techniques: Transduction, shRNA, Staining, Knockdown, Over Expression, Western Blot, Control

Journal: Clinical and Translational Medicine

Article Title: Interleukin enhancer‐binding factor 2 promotes cell proliferation and DNA damage response in metastatic melanoma

doi: 10.1002/ctm2.608

Figure Lengend Snippet: ILF2 regulates the ATM pathway by recruiting U2AF2. (A‐C) Drug sensitivity assays in U2AF2 knockdown melanoma cell lines treated with different concentrations of TMZ for 72 h. (D and E) Western blot and the quantification of U2AF2 and γ‐H2AX in IM‐0223 (D) and M‐204 (E) melanoma cells with U2AF2 knockdown. (F and G) Homologous recombination efficiency assay on U2AF2 knockdown DP‐0574 (F) and M‐204 (G) melanoma cells. (H) Western blot and the quantification of U2AF2, RAD50, ATM and p‐ATM in U2AF2 knockdown cells. (I and J) The RT‐qPCR assay analysing the mRNA expression of U2AF2 , RAD50 and ATM in melanoma cells transfected with si‐Ctrl or si‐U2AF2 (I), or ILF2 , RAD50 and ATM in melanoma cells transfected with sh‐Ctrl or sh‐ILF2 (J). (K) Western blot and the quantification of U2AF2, ILF2 and RAD50 in melanoma cells. β‐actin was used as the loading sample control. (L) Proposed schematic model for the function of ILF2 in regulating the ATM pathway in metastatic melanoma. Data represent the mean ± SD. * p < .05, ** p < .01, *** p < .001 and **** p < .0001

Article Snippet: For quantification of γ‐H2AX foci, DP‐0574 ILF2‐OV and EV cells were stained with rabbit anti‐human γ‐H2AX primary Ab (1:50, Novus Biologicals, Littleton, CO) and CyTM3 goat anti‐rabbit IgG secondary Ab (1:600).

Techniques: Knockdown, Western Blot, Homologous Recombination, Quantitative RT-PCR, Expressing, Transfection, Control

Journal: Experimental gerontology

Article Title: Correlations between biomarkers of senescent cell accumulation at the systemic, tissue and cellular levels in elderly patients.

doi: 10.1016/j.exger.2023.112176

Figure Lengend Snippet: Fig. 1. Linear regression graph, where the dependent variable is the expression level of H2AX in FBs, the independent variable is the expression level of p21 in FBs.

Article Snippet: The samples were then incubated with rabbit primary antibodies to p21 (2947S, Cell Signaling) or gamma-H2AX (AF2288, R&D) at +4 ◦C overnight.

Techniques: Expressing