Journal: Nature immunology

Article Title: The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated anti-tumor immunity

doi: 10.1038/s41590-021-00888-3

Figure Lengend Snippet: a-b, Effect of TCR-engagement on MDM2 in CD8 + T cells. Human (a) and mouse (b) CD8 + T cells were stimulated with anti-CD3 and anti-CD28 mAbs. Expression of MDM2 was determined by Western blotting. One of 5 experiments is shown. c-d, Effect of MDM2 deficiency on ID8 tumor progression. ID8 tumors were inoculated into Mdm2 +/+ Cd4 -Cre and Mdm2 fl/fl Cd4 -Cre mice. Tumor volume (c) and tumor representative bioluminescence images (d) are shown. mean ± SEM, n = 9, *** p < 0.001, two-way ANOVA. e-h, Role of MDM2 deficiency in ID8 tumor infiltrating CD8 + T cells. The percentages of Annexin V + cells in CD8 + T cells (e, f) and CD8 + T cells in CD45 + immune cells (g) were analyzed by FACS. The pro- and anti-apoptotic proteins were detected in tumor infiltrating CD8 + T cells by Western blotting (h). (e-g) mean ± SEM, n = 9, * p < 0.05, ** p < 0.01, Mann Whitney test. i, Effect of MDM2 deficiency on MC38 tumor progression. MC38 tumors were inoculated into Mdm2 +/+ Cd4 -Cre and Mdm2 fl/fl Cd4 -Cre mice. Tumor volume was monitored. mean ± SEM, n = 5, **** p < 0.0001, two-way ANOVA. j-q, Role of MDM2 deficiency in MC38 tumor infiltrating CD8 + T cells. The percentages of Annexin V + cells in CD8 + T cells (j, k), CD8 + T cells in CD45 + immune cells (l, m), and granzyme B + (n, o) and IFNγ + (p, q) cells in CD8 + T cells were analyzed by FACS. mean ± SEM, n = 4 – 5, * p < 0.05, ** p < 0.01, Mann Whitney test. r, Level of p53 protein was determined in Mdm2 +/+ and Mdm2 −/− T cells by Western blotting. s-w, Role of p53 in MDM2-regulated T cell survival. Activated p53 +/+ and p53 −/− CD8 + T cells were transduced by MDM2 (MDM2 OE ) or empty vector (EV) using a retrovirus. Annexin V + CD8 + T cells (GFP positive) were determined by FACS (s). Jurkat T cells were transfected with sh Mdm2 or sh CTRL . Annexin V + Jurkat T cells were determined by FACS (t). Activated Mdm2 +/+ and Mdm2 −/− CD8 + T cells were treated with 30 μM Pifithrin α (PFTα) for 5 hours (u, v), or transduced with p53-specific interfering RNA (iRNA- p53 ) or EV using a retrovirus (w). Annexin V + CD8 + T cells (GFP positive) were determined by FACS. mean ± SEM, n = 3 – 6, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA (s, u, w) or Mann Whitney test (t).

Article Snippet: For Western blots, anti-β-actin (8H10D1, #3700, 1:1000), anti-STAT5 (D2O6Y, #94205, 1:1000), anti-STAT1 (D1K9Y, #14994, 1:1000), anti-STAT2 (D9J7L, #72604, 1:1000), anti-STAT3 (D1A5, #8768, 1:1000), anti-STAT6 (D3H4, #5397, 1:1000), anti-p53 (1C12, #2524, 1:1000), anti-PARP (46D11, 9532, 1:1000), anti-cl-Caspase 3 (5A1E, #9664, 1:1000), anti-cl-Caspase8 (D5B2, #8592, 1:1000), anti-JAK1 (6G4, #3344, 1:1000), anti-JAK2 (D2E12, 3230, 1:1000), anti-JAK3 (D7B12, #8863, 1:1000), anti-Bcl2 (D17C4, #3498, 1:1000), anti-Bcl-xL (54H6, #2764, 1:1000), anti-Bak (D4E4, #12105, 1:1000), anti-c-Cbl (D4E10, #2747, 1:1000), ant-HA-tag (6E2, #2367, 1:1000), mouse-anti-DDK (9A3, #8146, 1:1000), rabbit-anti-DDK (D6W5B, #14793, 1:1000), mouse-anti-Myc-tag (9B11, #2276, 1:1000), rabbit-anti-Myc-tag (71D10, #2278, 1:1000), mouse-IgG (G3A1, #5415), and normal rabbit-IgG (#2729) were from Cell Signaling Technology.

Techniques: Expressing, Western Blot, MANN-WHITNEY, Plasmid Preparation, Transfection, Transduction

Journal: Nature immunology

Article Title: The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated anti-tumor immunity

doi: 10.1038/s41590-021-00888-3

Figure Lengend Snippet: a-b, Effect of TCR-engagement on MDM2 in CD8 + T cells. Human (a) and mouse (b) CD8 + T cells were stimulated with anti-CD3 and anti-CD28 mAbs. Expression of MDM2 was determined by Western blotting. mean ± SEM, n = 5, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA. c-d, Protein levels of MDM2 in T cells from Mdm2 +/+ Cd4 -Cre and Mdm2 fl/fl Cd4 -Cre mice. Results are expressed as the relative mean density of MDM2 ± SEM, n = 6, ** p < 0.01, Mann Whitney test. e-j, Phenotype of Mdm2 fl/fl Cd4 -Cre mice. The representative images show the body size (e) and the lymphoid organs (f) of Mdm2 +/+ Cd4 -Cre and Mdm2 fl/fl Cd4 -Cre mice. The percentages of CD8 + T cells in T cells (g), the representative images of flow dot plots (h), PD-1 + and CD25 + cells in T cell subsets (i) in different organs, and T cells in CD45 + immune cells (j) were analyzed by FACS. k-l, Effect of MDM2 deficiency on T cell proliferation under the homeostatic stimulation. Mdm2 +/+ and Mdm2 −/− CD8 + T cells were cultured with IL-7 and IL-15 in response to PMA. Ki67 expression was analyzed by FACS. mean ± SEM, n = 5, ** p < 0.05, Mann Whitney test. m-q, Role of MDM2 deficiency in ID8 tumor infiltrating CD8 + T cells. The anti-apoptotic (Bcl2 and Bcl-xL) and pro-apoptotic (Bak, cl-caspase 8, and cl-caspase 3) proteins were detected in tumor infiltrating CD8 + T cells by Western blotting. Results are expressed as the relative mean density of specific proteins ± SEM, n = 4, * p < 0.05, Mann Whitney test. r-t, Effect of MDM2 deficiency on MC38 tumor progression and T cell proliferation. MC38 tumors were inoculated into Mdm2 +/+ Cd4 -Cre and Mdm2 fl/fl Cd4 -Cre mice. Tumor weight was measured on day 21 (r). The percentages of Ki67 + cells in tumor infiltrating CD8 + T cells were analyzed by FACS (s-t). mean ± SEM, n = 4 – 5, * p < 0.05, ** p < 0.01, Mann Whitney test. u. Protein levels of p53 in T cells isolated from p53 +/+ Cd4 -Cre and p53 fl/fl Cd4 -Cre mice. One of 6 experiments is shown v-w, Phenotype of p53 fl/fl Cd4 -Cre mice. The representative images show the body size (v) and lymphoid organs (w) of p53 +/+ Cd4 -Cre and p53 fl/fl Cd4 -Cre mice. x, T cell subset distribution in p53 +/+ Cd4 -Cre and p53 fl/fl Cd4 -Cre mice. One of 3 representative dot-plots is shown.

Article Snippet: For Western blots, anti-β-actin (8H10D1, #3700, 1:1000), anti-STAT5 (D2O6Y, #94205, 1:1000), anti-STAT1 (D1K9Y, #14994, 1:1000), anti-STAT2 (D9J7L, #72604, 1:1000), anti-STAT3 (D1A5, #8768, 1:1000), anti-STAT6 (D3H4, #5397, 1:1000), anti-p53 (1C12, #2524, 1:1000), anti-PARP (46D11, 9532, 1:1000), anti-cl-Caspase 3 (5A1E, #9664, 1:1000), anti-cl-Caspase8 (D5B2, #8592, 1:1000), anti-JAK1 (6G4, #3344, 1:1000), anti-JAK2 (D2E12, 3230, 1:1000), anti-JAK3 (D7B12, #8863, 1:1000), anti-Bcl2 (D17C4, #3498, 1:1000), anti-Bcl-xL (54H6, #2764, 1:1000), anti-Bak (D4E4, #12105, 1:1000), anti-c-Cbl (D4E10, #2747, 1:1000), ant-HA-tag (6E2, #2367, 1:1000), mouse-anti-DDK (9A3, #8146, 1:1000), rabbit-anti-DDK (D6W5B, #14793, 1:1000), mouse-anti-Myc-tag (9B11, #2276, 1:1000), rabbit-anti-Myc-tag (71D10, #2278, 1:1000), mouse-IgG (G3A1, #5415), and normal rabbit-IgG (#2729) were from Cell Signaling Technology.

Techniques: Expressing, Western Blot, MANN-WHITNEY, Cell Culture, Isolation

Journal: Nature immunology

Article Title: The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated anti-tumor immunity

doi: 10.1038/s41590-021-00888-3

Figure Lengend Snippet: a, APG115 structure is shown. b-e , Effect of APG115 on MDM2 and p53 expression in T cells. Human (b-c) and mouse (d-e) T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of different concentrations of APG115. MDM2 (b, d) and p53 (c, e) expression was determined by Western blotting. Results are expressed as the relative mean density of MDM2 and p53 ± SEM, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA. f-h, Effect of APG115 on p53 wild-type tumor cells. B16F10 (f), CT26 (g) and ID8 (h) cells were treated with different concentrations of APG115. Immunoblots showed MDM2, p53, and p21 expression. i, Effect of APG115 on p53 mutant tumor cells. MC38 cells were treated with different concentrations of APG115. Immunoblots showed PARP1, p53, and MDM2 expression. j, Effect of APG115 on p53-null 4T1 tumor cells. 4T1 tumor cells were treated with different concentrations of APG115. Immunoblots showed PARP1, p53, and MDM2 expression. k, Effect of APG115 on p53 −/− CD8 + T cell survival. p53 −/− CD8 + T cells were treated with APG115. Annexin V + expression was analyzed by FACS. mean ± SEM, n = 4, NS, not significant.

Article Snippet: For Western blots, anti-β-actin (8H10D1, #3700, 1:1000), anti-STAT5 (D2O6Y, #94205, 1:1000), anti-STAT1 (D1K9Y, #14994, 1:1000), anti-STAT2 (D9J7L, #72604, 1:1000), anti-STAT3 (D1A5, #8768, 1:1000), anti-STAT6 (D3H4, #5397, 1:1000), anti-p53 (1C12, #2524, 1:1000), anti-PARP (46D11, 9532, 1:1000), anti-cl-Caspase 3 (5A1E, #9664, 1:1000), anti-cl-Caspase8 (D5B2, #8592, 1:1000), anti-JAK1 (6G4, #3344, 1:1000), anti-JAK2 (D2E12, 3230, 1:1000), anti-JAK3 (D7B12, #8863, 1:1000), anti-Bcl2 (D17C4, #3498, 1:1000), anti-Bcl-xL (54H6, #2764, 1:1000), anti-Bak (D4E4, #12105, 1:1000), anti-c-Cbl (D4E10, #2747, 1:1000), ant-HA-tag (6E2, #2367, 1:1000), mouse-anti-DDK (9A3, #8146, 1:1000), rabbit-anti-DDK (D6W5B, #14793, 1:1000), mouse-anti-Myc-tag (9B11, #2276, 1:1000), rabbit-anti-Myc-tag (71D10, #2278, 1:1000), mouse-IgG (G3A1, #5415), and normal rabbit-IgG (#2729) were from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Mutagenesis

Journal: Nature immunology

Article Title: The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated anti-tumor immunity

doi: 10.1038/s41590-021-00888-3

Figure Lengend Snippet: a-b, Effect of APG115 on MDM2 expression in T cells. Human (a) and mouse (b) T cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of different concentrations of APG115. Expression of MDM2 and p53 was measured with immunoblotting. One of 3 experiments is shown. c-i, Effect of APG115 on tumor immunotherapy. Mice were inoculated with B16F10 (c-d), CT26 (e), ID8 (f-g), MC38 (h), and 4T1 (i) tumor cells and treated with APG115 or vehicle. Tumor volume and weight were monitored. mean ± SEM, c-d, n = 8; e, n = 6; f, n = 9-10; h, n = 7; i, n = 5. * p < 0.05, ** p < 0.01, **** p < 0.0001, Mann Whitney test (d) or two-way ANOVA (c, e, f, h, i). j, Effect of T cell p53 deficiency on MC38 tumor progression. p53 +/+ Cd4 -Cre or p53 fl/fl Cd4 -Cre mice were inoculated with MC38 tumor cells and treated with APG115 or vehicle. mean ± SEM, n = 7. * p < 0.05, two-way ANOVA.

Article Snippet: For Western blots, anti-β-actin (8H10D1, #3700, 1:1000), anti-STAT5 (D2O6Y, #94205, 1:1000), anti-STAT1 (D1K9Y, #14994, 1:1000), anti-STAT2 (D9J7L, #72604, 1:1000), anti-STAT3 (D1A5, #8768, 1:1000), anti-STAT6 (D3H4, #5397, 1:1000), anti-p53 (1C12, #2524, 1:1000), anti-PARP (46D11, 9532, 1:1000), anti-cl-Caspase 3 (5A1E, #9664, 1:1000), anti-cl-Caspase8 (D5B2, #8592, 1:1000), anti-JAK1 (6G4, #3344, 1:1000), anti-JAK2 (D2E12, 3230, 1:1000), anti-JAK3 (D7B12, #8863, 1:1000), anti-Bcl2 (D17C4, #3498, 1:1000), anti-Bcl-xL (54H6, #2764, 1:1000), anti-Bak (D4E4, #12105, 1:1000), anti-c-Cbl (D4E10, #2747, 1:1000), ant-HA-tag (6E2, #2367, 1:1000), mouse-anti-DDK (9A3, #8146, 1:1000), rabbit-anti-DDK (D6W5B, #14793, 1:1000), mouse-anti-Myc-tag (9B11, #2276, 1:1000), rabbit-anti-Myc-tag (71D10, #2278, 1:1000), mouse-IgG (G3A1, #5415), and normal rabbit-IgG (#2729) were from Cell Signaling Technology.

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: Nature immunology

Article Title: The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated anti-tumor immunity

doi: 10.1038/s41590-021-00888-3

Figure Lengend Snippet: a-d, Role of APG115 in CD8 + T cells. (a, b) Mouse Mdm2 +/+ Cd4 -Cre and Mdm2 fl/fl Cd4 -Cre mice CD8 + T cells were stimulated with anti-CD3 and anti-CD28 in the presence of APG115. Western blots showed STAT5 (a), Bcl-xL (b), and cl-caspase 8 (p43) (b) in T cells. 2 of 6 mice are shown. (c) Western blots showed expression of STATs and MDM2 in MC38 tumor infiltrating CD8 + T cells in mice treated with APG115. n = 6/group. (d) Western blots showed STAT5 expression in human CD8 + T cells stimulated with anti-CD3 and anti-CD28 in the presence of APG115. n = 4. e, Effect of APG115 on OT-I cell-mediated tumor regression. OT-I cells were transferred into mice bearing B16F10-OVA. Mice were treated with APG115. Tumor volume was monitored. n = 7, * p < 0.05, *** p < 0.001, **** p < 0.0001, two-way ANOVA. OVA, ovalbumin. f-j, Role of STAT5 activation in APG115-regulated T cell immunity. Activated OT-I cells were transduced with a constitutive active STAT5 (STAT5CA) and empty vector (EV) using a retrovirus. GFP positive OT-I cells were sorted and transferred into B16F10-OVA bearing mice. Tumor volume was monitored (f). The percentages of Annexin V + (g-h) or IFNγ + (i-j) in SIINFEKL-tetramer + CD8 + T cells were analyzed by FACS. one representative dot-plot is shown. n = 5 – 6, mean ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-way ANOVA (f) or one-way ANOVA (g, i). k, Role of STAT5 inhibitor in APG115-mediated T cell immunity. Activated OT-I cells were incubated with STAT5 inhibitor or DMSO, and transferred into B16F10-OVA bearing mice. Tumor volume was monitored. n = 6, mean ± SEM, * p < 0.05, two-way ANOVA. l-m, Role of STAT5 knocking down in APG115-regulated T cell immunity. Activated OT-I cells were transfected with STAT5 specific siRNA (FAM labeled with si Stat5 ) or si CTRL . FAM positive OT-I cells were sorted and transferred into B16F10-OVA bearing mice. Tumor volume was monitored (l). The percentages of IFNγ + cells in SIINFEKL-tetramer + CD8 + T cells were analyzed by FACS (m). n = 5 – 6, mean ± SEM, * p < 0.05, ** p < 0.01, two-way ANOVA (l), and one-way ANOVA (m). n-q, Effect of APG115 on anti-PD-L1 therapy. MC38 tumor bearing mice were treated with APG115, anti-PD-L1, and their combination. Tumor volume (n) and weight (o) were monitored. The percentages of IFNγ + (p-q) cells in tumor-infiltrating CD8 + T cells were analyzed by FACS. mean ± SEM, n = 5 - 7, * p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA (n), and one-way ANOVA (o-p). r-s , Effect of MDM2 and c-Cbl on p53 −/− T cell survival. Activated p53 −/− CD8 + T cells were transduced with MDM2 (MDM2 OE ) or c-Cbl (c-Cbl OE ) expressing retrovirus. The percentages of Annexin V + cells were analyzed by FACS. mean ± SEM, n = 3 - 4, * p < 0.05, ** p < 0.01, one-way ANOVA.

Article Snippet: For Western blots, anti-β-actin (8H10D1, #3700, 1:1000), anti-STAT5 (D2O6Y, #94205, 1:1000), anti-STAT1 (D1K9Y, #14994, 1:1000), anti-STAT2 (D9J7L, #72604, 1:1000), anti-STAT3 (D1A5, #8768, 1:1000), anti-STAT6 (D3H4, #5397, 1:1000), anti-p53 (1C12, #2524, 1:1000), anti-PARP (46D11, 9532, 1:1000), anti-cl-Caspase 3 (5A1E, #9664, 1:1000), anti-cl-Caspase8 (D5B2, #8592, 1:1000), anti-JAK1 (6G4, #3344, 1:1000), anti-JAK2 (D2E12, 3230, 1:1000), anti-JAK3 (D7B12, #8863, 1:1000), anti-Bcl2 (D17C4, #3498, 1:1000), anti-Bcl-xL (54H6, #2764, 1:1000), anti-Bak (D4E4, #12105, 1:1000), anti-c-Cbl (D4E10, #2747, 1:1000), ant-HA-tag (6E2, #2367, 1:1000), mouse-anti-DDK (9A3, #8146, 1:1000), rabbit-anti-DDK (D6W5B, #14793, 1:1000), mouse-anti-Myc-tag (9B11, #2276, 1:1000), rabbit-anti-Myc-tag (71D10, #2278, 1:1000), mouse-IgG (G3A1, #5415), and normal rabbit-IgG (#2729) were from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Activation Assay, Transduction, Plasmid Preparation, Incubation, Transfection, Labeling

Journal: Nature immunology

Article Title: The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated anti-tumor immunity

doi: 10.1038/s41590-021-00888-3

Figure Lengend Snippet: a-e, Role of APG115 in CD8 + T cells. (a, c) Mouse Mdm2 +/+ Cd4 -Cre and Mdm2 fl/fl Cd4 -Cre CD8 + T cells were stimulated with anti-CD3 and anti-CD28 in the presence of APG115. Western blots showed STAT5 (a), Bcl-xL (b), and cl-caspase 8 (c) in T cells. mean ± SEM, n = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA, (d) Western blots showed expression of STATs and MDM2 in MC38 tumor infiltrating CD8 + T cells in mice treated with APG115. mean ± SEM, n = 6, *** p < 0.001, one-way ANOVA. (e) Western blots showed STAT5 expression in human CD8 + T cells stimulated with anti-CD3 and anti-CD28 in the presence of APG115. mean ± SEM, n = 4, * p < 0.05, Mann Whitney test. f-h, Role of APG115 in CD8 + T cells. Mouse CD8 + T cells were stimulated with anti-CD3 and anti-CD28 in the presence of APG115. IFNγ + (f), TNFα + (g), and IL-2 + (h) cells in CD8 + T cells were analyzed by FACS. mean ± SEM, n = 4, * p < 0.05, Mann Whitney test. i. Effect of STAT5CA expression on MDM2-mediated T cell survival. Activated Mdm2 +/+ and Mdm2 −/− CD8 + cells were transduced with STAT5CA or vector expressing retrovirus, and treated with APG115. The percentages of Annexin V + cells in Mdm2 +/+ or Mdm2 −/− CD8 + T cells were determined by FACS. mean ± SEM, n = 4, * p < 0.05, **** p < 0.0001, one-way ANOVA. j. Effect of STAT5 knock down in OT-I-mediated tumor killing. B16F10-OVA cells were cultured with STAT5 deficiency OT-I T cells for 24 hours. Tumor cell apoptosis was determined by flow cytometry analysis. Results are shown as the percentages of 7AAD + tumor cells. mean ± SEM, n = 4, *** p < 0.001, one-way ANOVA. k, Role of APG115 on MDM2, c-Cbl, and STAT5 expression in p53 −/− T cells. p53 −/− CD8 + T cells were treated with APG115. MDM2, c-Cbl, and STAT5 levels were analyzed by Western Blotting. One of 3 experiments is shown.

Article Snippet: For Western blots, anti-β-actin (8H10D1, #3700, 1:1000), anti-STAT5 (D2O6Y, #94205, 1:1000), anti-STAT1 (D1K9Y, #14994, 1:1000), anti-STAT2 (D9J7L, #72604, 1:1000), anti-STAT3 (D1A5, #8768, 1:1000), anti-STAT6 (D3H4, #5397, 1:1000), anti-p53 (1C12, #2524, 1:1000), anti-PARP (46D11, 9532, 1:1000), anti-cl-Caspase 3 (5A1E, #9664, 1:1000), anti-cl-Caspase8 (D5B2, #8592, 1:1000), anti-JAK1 (6G4, #3344, 1:1000), anti-JAK2 (D2E12, 3230, 1:1000), anti-JAK3 (D7B12, #8863, 1:1000), anti-Bcl2 (D17C4, #3498, 1:1000), anti-Bcl-xL (54H6, #2764, 1:1000), anti-Bak (D4E4, #12105, 1:1000), anti-c-Cbl (D4E10, #2747, 1:1000), ant-HA-tag (6E2, #2367, 1:1000), mouse-anti-DDK (9A3, #8146, 1:1000), rabbit-anti-DDK (D6W5B, #14793, 1:1000), mouse-anti-Myc-tag (9B11, #2276, 1:1000), rabbit-anti-Myc-tag (71D10, #2278, 1:1000), mouse-IgG (G3A1, #5415), and normal rabbit-IgG (#2729) were from Cell Signaling Technology.

Techniques: Western Blot, Expressing, MANN-WHITNEY, Transduction, Plasmid Preparation, Knockdown, Cell Culture, Flow Cytometry

Journal: PLoS ONE

Article Title: Exclusive Association of p53 Mutation with Super-High Methylation of Tumor Suppressor Genes in the p53 Pathway in a Unique Gastric Cancer Phenotype

doi: 10.1371/journal.pone.0139902

Figure Lengend Snippet: Epigenetic conversion in the p53 pathway.

Article Snippet: Total protein was extracted from cell lines that were epigenetically treated with/without chemotherapeutic treatment, and was subjected to Western blotting analysis using the following antibodies: mouse anti-p53 monoclonal antibody (1C12, dilution of 1:1000, Cell Signaling Technology, Inc.) or mouse anti-β-actin IgG 2a monoclonal antibody (dilution of 1:10000, Sigma-Aldrich).

Techniques:

Journal: PLoS ONE

Article Title: Exclusive Association of p53 Mutation with Super-High Methylation of Tumor Suppressor Genes in the p53 Pathway in a Unique Gastric Cancer Phenotype

doi: 10.1371/journal.pone.0139902

Figure Lengend Snippet: Distribution of clinical and pathological factors for correlation with gene & methylation status and univariable prognostic analysis in 163 pStageII/III gastric cancer with gastrectomy and subsequent S-1 treatment.

Article Snippet: Total protein was extracted from cell lines that were epigenetically treated with/without chemotherapeutic treatment, and was subjected to Western blotting analysis using the following antibodies: mouse anti-p53 monoclonal antibody (1C12, dilution of 1:1000, Cell Signaling Technology, Inc.) or mouse anti-β-actin IgG 2a monoclonal antibody (dilution of 1:10000, Sigma-Aldrich).

Techniques: Methylation, Mutagenesis

Journal: PLoS ONE

Article Title: Exclusive Association of p53 Mutation with Super-High Methylation of Tumor Suppressor Genes in the p53 Pathway in a Unique Gastric Cancer Phenotype

doi: 10.1371/journal.pone.0139902

Figure Lengend Snippet: (A) According to p53 gene mutation status and (B) according to the p53 aberration group.

Article Snippet: Total protein was extracted from cell lines that were epigenetically treated with/without chemotherapeutic treatment, and was subjected to Western blotting analysis using the following antibodies: mouse anti-p53 monoclonal antibody (1C12, dilution of 1:1000, Cell Signaling Technology, Inc.) or mouse anti-β-actin IgG 2a monoclonal antibody (dilution of 1:10000, Sigma-Aldrich).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Exclusive Association of p53 Mutation with Super-High Methylation of Tumor Suppressor Genes in the p53 Pathway in a Unique Gastric Cancer Phenotype

doi: 10.1371/journal.pone.0139902

Figure Lengend Snippet: (A) Methylation of the indicated genes was analyzed using Q-MSP and TAqMeth values in tumors with p53 wild type were compared with those with p53 mutation. The threshold value for determination that a gene was methylated was determined as the maximum TaqMeth value of p53 wild type. (B) Tumors with p53 wild type or p53 mutation were compared in terms of the presence of super-high methylation of the indicated genes. Super-high methylation was defined that at least one gene showed higher TaqMeth value than each threshold values among three genes.

Article Snippet: Total protein was extracted from cell lines that were epigenetically treated with/without chemotherapeutic treatment, and was subjected to Western blotting analysis using the following antibodies: mouse anti-p53 monoclonal antibody (1C12, dilution of 1:1000, Cell Signaling Technology, Inc.) or mouse anti-β-actin IgG 2a monoclonal antibody (dilution of 1:10000, Sigma-Aldrich).

Techniques: Methylation, Mutagenesis

Journal: PLoS ONE

Article Title: Exclusive Association of p53 Mutation with Super-High Methylation of Tumor Suppressor Genes in the p53 Pathway in a Unique Gastric Cancer Phenotype

doi: 10.1371/journal.pone.0139902

Figure Lengend Snippet: Clinicopathological characters of recurrent tumors of pStage II/III gastric cancer in the p53 aberrant group (A) and the p53 non-aberrant group (B).

Article Snippet: Total protein was extracted from cell lines that were epigenetically treated with/without chemotherapeutic treatment, and was subjected to Western blotting analysis using the following antibodies: mouse anti-p53 monoclonal antibody (1C12, dilution of 1:1000, Cell Signaling Technology, Inc.) or mouse anti-β-actin IgG 2a monoclonal antibody (dilution of 1:10000, Sigma-Aldrich).

Techniques:

Journal: PLoS ONE

Article Title: Exclusive Association of p53 Mutation with Super-High Methylation of Tumor Suppressor Genes in the p53 Pathway in a Unique Gastric Cancer Phenotype

doi: 10.1371/journal.pone.0139902

Figure Lengend Snippet: (A) Survival curves for intestinal type were compared with those of diffuse type gastric cancer with pStage II/III. (B) Cancer stage distribution according to the p53 aberration group and histological findings.

Article Snippet: Total protein was extracted from cell lines that were epigenetically treated with/without chemotherapeutic treatment, and was subjected to Western blotting analysis using the following antibodies: mouse anti-p53 monoclonal antibody (1C12, dilution of 1:1000, Cell Signaling Technology, Inc.) or mouse anti-β-actin IgG 2a monoclonal antibody (dilution of 1:10000, Sigma-Aldrich).

Techniques:

Journal: PLoS ONE

Article Title: Exclusive Association of p53 Mutation with Super-High Methylation of Tumor Suppressor Genes in the p53 Pathway in a Unique Gastric Cancer Phenotype

doi: 10.1371/journal.pone.0139902

Figure Lengend Snippet: NUGC4 and KATOIII cells were treated with the demethylating agent, 5-aza-2’-deoxycytidine (5-aza-dC) in the presence or absence of the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), and in the presence or absence of the chemotherapeutic reagent, CDDP. 1A and 5A, 1 and 5 μM 5-aza-dC; T, TSA. Subsequently the cells were analyzed for: (A) p53 protein expression by Western Blotting. (B) A dual reporter assay to confirm p53 transcription activity. The dashed line indicates the optimal cut-off value (0.15) for determination of p53 activity. (C) Cell apoptosis was assayed using a Nexin Assay. A representative image is shown. Data are shown as the percentage of early and late apoptotic cells. *P<0.05, **<0.001, ***<0.0001.

Article Snippet: Total protein was extracted from cell lines that were epigenetically treated with/without chemotherapeutic treatment, and was subjected to Western blotting analysis using the following antibodies: mouse anti-p53 monoclonal antibody (1C12, dilution of 1:1000, Cell Signaling Technology, Inc.) or mouse anti-β-actin IgG 2a monoclonal antibody (dilution of 1:10000, Sigma-Aldrich).

Techniques: Histone Deacetylase Assay, Expressing, Western Blot, Reporter Assay, Activity Assay

Journal: PLoS ONE

Article Title: Lipocalin 2 Enhances Migration and Resistance against Cisplatin in Endometrial Carcinoma Cells

doi: 10.1371/journal.pone.0155220

Figure Lengend Snippet: a; WST-1 assay to measure cisplatin (CDDP) sensitivity in HHUA, RL95-2 and HEC1B cells. Decreases in the viability of LCN2-silenced cells (LCN2 shRNA-1 and 2) were significantly greater than those in the control cells (Control) in both HHUA and RL95-2. Decreases in the viability of LCN2-overexpressing cells (LCN2 cDNA) were significantly smaller than those in the control cells (Control) in HEC1B. *; significantly lower than the control (P<0.05). b; Apostrand assay. Apoptosis was significantly enhanced in LCN2-silenced HHUA. *; P<0.05. c; WST-1 assay. The difference in viability between both cells under the CDDP treatment was canceled by the addition of DFO. *; P<0.05, N.S.; not significant. d; Western blotting under the CDDP treatment. No apparent change in pAkt expression was observed. The strong expression of p53 and p21 was observed in LCN2-silenced HHUA under the CDDP treatment. e; WST-1 assay under the CDDP treatment. Neither wortmannin nor U0126 affected the viability of control HHUA, and slightly increased the viability of LCN2-silenced HHUA.

Article Snippet: Proteins extracted from 1 ml of culture supernatant and proteins extracted from cultured cells were subjected to a Western blot analysis, as described previously [ ], using antibodies against human LCN2 (rat-monoclonal, clone # 220310, R & D systems, Minneapolis, MN), phospho-Akt (pAkt) (Ser473) (rabbit monoclonal, D9E, Cell Signaling Technology, Danvers, MA), Akt (rabbit monoclonal, C67E7, Cell Signaling Technology), phospho-MAPK (pMAPK) (Thr202/Tyr204) (rabbit monoclonal, D13.14.4E, Cell Signaling Technology), MAPK (rabbit monoclonal, 137F5, Cell Signaling Technology), p53 (mouse monoclonal, 1C12, Cell Signaling Technology), p21 (mouse monoclonal, EA10, Abcam, Cambridge, UK) and β-actin (mouse monoclonal, AC-15, BioMakor, Rehovot, Israel) as primary antibodies.

Techniques: WST-1 Assay, shRNA, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Lipocalin 2 Enhances Migration and Resistance against Cisplatin in Endometrial Carcinoma Cells

doi: 10.1371/journal.pone.0155220

Figure Lengend Snippet: a; Cell viability of HHUA Control cells treated with CDDP and/or DFO. (WST-1 assay) *: The viability of cells treated with CDDP and DFO was significantly lower than that treated with each drug at 72 hours. (P<0.05) b; Cell viability of HHUA LCN2 shRNA-1 cells treated with CDDP and/or DFO. (WST-1 assay) N.S.: The viability of cells was not different between the treatment with both drugs and that with single drug. c; The expression of pAkt, Akt, p21 and p53 in HHUA Control and LCN2 shRNA-1 cells at 24, 48 and 72 hours of CDDP/DFO treatment (Western blotting). The density of each band was measured by densitometry. ACTB (beta-actin) was used as internal control. Graphs indicated the ratio of density compared with ACTB.

Article Snippet: Proteins extracted from 1 ml of culture supernatant and proteins extracted from cultured cells were subjected to a Western blot analysis, as described previously [ ], using antibodies against human LCN2 (rat-monoclonal, clone # 220310, R & D systems, Minneapolis, MN), phospho-Akt (pAkt) (Ser473) (rabbit monoclonal, D9E, Cell Signaling Technology, Danvers, MA), Akt (rabbit monoclonal, C67E7, Cell Signaling Technology), phospho-MAPK (pMAPK) (Thr202/Tyr204) (rabbit monoclonal, D13.14.4E, Cell Signaling Technology), MAPK (rabbit monoclonal, 137F5, Cell Signaling Technology), p53 (mouse monoclonal, 1C12, Cell Signaling Technology), p21 (mouse monoclonal, EA10, Abcam, Cambridge, UK) and β-actin (mouse monoclonal, AC-15, BioMakor, Rehovot, Israel) as primary antibodies.

Techniques: Control, WST-1 Assay, shRNA, Expressing, Western Blot

Journal: Cell death & disease

Article Title: Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

doi: 10.1038/s41419-023-06310-0

Figure Lengend Snippet: Fig. 2 Reintroduction of p53 suppressed the DOXO-induced formation of nuclear actin filaments in p53 knockdown cells. MCF-7 cells expressing p53 shRNA were cotransfected with nAC-GFP expression vector and HA-p53 expression vector and subsequently treated with DOXO (1 μg/mL) for 16 h. a Cell lysates were subjected to immunoblot analysis with antibodies against HA, p53, and α-tubulin as a loading control. b Confocal images of nAC-GFP (gray/green), HA-p53 stained using HA antibody (magenta), and DNA stained using DAPI (blue) are shown. The Z stack projections of 30 central plane images acquired at 0.1 μm intervals were obtained. The scale bar is 10 μm. c For each treatment, the nuclear area occupied by actin filaments was measured. The horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values, respectively (d). The cells with non-uniform nAC-GFP localization in ≥1% of their area were classified as nuclear actin filament-containing cells, whereas those with non-uniform nAC-GFP localization in <1% were classified as nuclear actin filament-free cells (d). N ≥150 for each treatment. Asterisks, p < 0.005.

Article Snippet: Anti-p53 mouse monoclonal (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA, or 1C12; Cell Signaling Technology, Danvers, MA, USA), anti-Flag mouse monoclonal (M2; Sigma-Aldrich, St. Louis, MO, USA), anti-caspase 1 mouse monoclonal (14F468; Santa Cruz Biotechnology, Dallas, TX, USA), anti-γH2AX rabbit polyclonal (2577; Cell Signaling Technology, Danvers, MA, USA), anti-H2AX rabbit polyclonal (GeneTex, Inc., Irvine, CA, USA), and anti-α-tubulin mouse monoclonal (DM1A; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used for immunoblot analysis.

Techniques: Knockdown, Expressing, shRNA, Plasmid Preparation, Western Blot, Control, Staining

Journal: Cell death & disease

Article Title: Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

doi: 10.1038/s41419-023-06310-0

Figure Lengend Snippet: Fig. 1 Knockdown of p53 promotes nuclear actin filament formation in nAC-GFP-expressing MCF-7 cells by treatment with DNA- damaging agents. MCF-7 cells expressing control (a–c) or p53 shRNA (a–d) were transfected with nAC-GFP expression vector and subsequently treated with or without DOXO (1 μg/mL) or VP16 (100 μM) for 16 h. a Confocal images of nAC-GFP (gray/green) and DNA stained using DAPI (blue). The Z stack projections of 30 central plane images acquired at 0.1 μm intervals were obtained. The scale bar is 10 μm. b, c For each treatment, the nuclear area occupied by actin filaments was measured. The horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values, respectively (b). The cells with non-uniform nAC-GFP localization in ≥1% of their area were classified as nuclear actin filament-containing cells, whereas those with non-uniform nAC-GFP localization in <1% were classified as nuclear actin filament-free cells (c). N ≥60 (b, c) for each treatment. Asterisks, p < 0.005. d The cells treated with DOXO for 16 h were incubated in the presence of mycalolide B (1 μM) for 20 min. The disappearance of the nuclear actin filament was observed using a time- lapse confocal microscope for 20 min.

Article Snippet: Anti-p53 mouse monoclonal (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA, or 1C12; Cell Signaling Technology, Danvers, MA, USA), anti-Flag mouse monoclonal (M2; Sigma-Aldrich, St. Louis, MO, USA), anti-caspase 1 mouse monoclonal (14F468; Santa Cruz Biotechnology, Dallas, TX, USA), anti-γH2AX rabbit polyclonal (2577; Cell Signaling Technology, Danvers, MA, USA), anti-H2AX rabbit polyclonal (GeneTex, Inc., Irvine, CA, USA), and anti-α-tubulin mouse monoclonal (DM1A; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used for immunoblot analysis.

Techniques: Knockdown, Expressing, Control, shRNA, Transfection, Plasmid Preparation, Staining, Incubation, Microscopy

Journal: Cell death & disease

Article Title: Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

doi: 10.1038/s41419-023-06310-0

Figure Lengend Snippet: Fig. 3 Caspase inhibitors promote the DOXO-induced formation of nuclear actin filaments in MCF-7 cells. a–c, e–h MCF-7 cells (a–c, e, f) or MCF-7 cells expressing p53 shRNA (g, h) were transfected with nAC-GFP expression vector and subsequently treated with or without DOXO (1 μg/mL) and pan-caspase inhibitor Q-VD-OPh (100 μM) (a–c) or caspase-1 inhibitor Z-YVAD-FMK (100 μM) (e, f) for 16 h. g, h The cells were cotransfected with Flag-tagged caspase-1 (F-CASP1) expression vector with nAC-GFP expression vector. a Confocal images of nAC-GFP (gray/ green) and DNA stained using DAPI (blue) are shown. The Z stack projections of 30 central plane images acquired at 0.1 μm intervals were obtained. The scale bar is 10 μm. b, c, e–h For each treatment, the nuclear area occupied by actin filaments was measured. The horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values, respectively (b, e, g). The cells with non-uniform nAC-GFP localization in ≥1% of their area were classified as nuclear actin filament-containing cells, whereas those with non- uniform nAC-GFP localization in <1% were classified as nuclear actin filament-free cells (c, f, h). N ≥89 (b, c), N ≥75 (e, f), and N ≥80 (g, h) for each treatment. d The expression of CASP1-encoding caspase-1 in the MCF-7 cells expressing control and p53 shRNA was evaluated by quantitative real-time PCR. Each bar represents the mean ± S.D.; n = 3. Asterisks, p < 0.005; double asterisks, p < 0.05.

Article Snippet: Anti-p53 mouse monoclonal (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA, or 1C12; Cell Signaling Technology, Danvers, MA, USA), anti-Flag mouse monoclonal (M2; Sigma-Aldrich, St. Louis, MO, USA), anti-caspase 1 mouse monoclonal (14F468; Santa Cruz Biotechnology, Dallas, TX, USA), anti-γH2AX rabbit polyclonal (2577; Cell Signaling Technology, Danvers, MA, USA), anti-H2AX rabbit polyclonal (GeneTex, Inc., Irvine, CA, USA), and anti-α-tubulin mouse monoclonal (DM1A; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used for immunoblot analysis.

Techniques: Expressing, shRNA, Transfection, Plasmid Preparation, Staining, Control, Real-time Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

doi: 10.1038/s41419-023-06310-0

Figure Lengend Snippet: Fig. 4 Depletion of p53 induces nuclear actin filament formation in YFP-nβ-actin-expressing cells upon treatment with DOXO. MCF-7 cells expressing control or p53 shRNA were treated with or without DOXO (1 μg/mL) for 16 h. The cells were transfected with YFP-nβ-actin WT or S14C mutant expression vectors before treatment with DOXO. Confocal images of YFP-nβ-actin WT or S14C (green), F-actin stained using phalloidin (magenta), and DNA stained using DAPI (blue) are shown. The scale bar is 10 μm. Arrowheads show the transfected cells. Arrows indicate non-transfected cells.

Article Snippet: Anti-p53 mouse monoclonal (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA, or 1C12; Cell Signaling Technology, Danvers, MA, USA), anti-Flag mouse monoclonal (M2; Sigma-Aldrich, St. Louis, MO, USA), anti-caspase 1 mouse monoclonal (14F468; Santa Cruz Biotechnology, Dallas, TX, USA), anti-γH2AX rabbit polyclonal (2577; Cell Signaling Technology, Danvers, MA, USA), anti-H2AX rabbit polyclonal (GeneTex, Inc., Irvine, CA, USA), and anti-α-tubulin mouse monoclonal (DM1A; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used for immunoblot analysis.

Techniques: Expressing, Control, shRNA, Transfection, Mutagenesis, Staining

Journal: Cell death & disease

Article Title: Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

doi: 10.1038/s41419-023-06310-0

Figure Lengend Snippet: Fig. 5 Expression of nLifeact-GFP induces structural changes of chromatin in DOXO-treated p53 knockdown MCF-7 cells. MCF-7 cells expressing control (b) or p53 shRNA (a–e) were transfected with nAC-GFP (a, c, e) or nLifeact-GFP (b–e) expression vectors and subsequently treated with or without DOXO (1 μg/mL) for 16 h. a–c Confocal images of nAC-GFP or nLifeact-GFP (gray/green), F-actin stained using phalloidin (magenta), and DNA stained using DAPI (blue) are shown. a–c, e The scale bars are 10 μm. a The arrows show the transfected cells. The arrowheads show the non-transfected cells. d Line plots of nAC-GFP or nLifeact-GFP and DAPI fluorescence intensity (denoted by yellow lines) in (c). The intensity values were normalized to the maximum value of each fluorescence. e Images of 2D hologram (gray) and fluorescence visualized nuclear actin with nLifeact-GFP or nAC-GFP (green). The arrowhead shows the position of the nuclear actin bundle from nLifeact-GFP.

Article Snippet: Anti-p53 mouse monoclonal (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA, or 1C12; Cell Signaling Technology, Danvers, MA, USA), anti-Flag mouse monoclonal (M2; Sigma-Aldrich, St. Louis, MO, USA), anti-caspase 1 mouse monoclonal (14F468; Santa Cruz Biotechnology, Dallas, TX, USA), anti-γH2AX rabbit polyclonal (2577; Cell Signaling Technology, Danvers, MA, USA), anti-H2AX rabbit polyclonal (GeneTex, Inc., Irvine, CA, USA), and anti-α-tubulin mouse monoclonal (DM1A; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used for immunoblot analysis.

Techniques: Expressing, Knockdown, Control, shRNA, Transfection, Staining

Journal: Cell death & disease

Article Title: Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

doi: 10.1038/s41419-023-06310-0

Figure Lengend Snippet: Fig. 6 Expression of nLifeact-GFP suppresses RNA synthesis in p53 knockdown MCF-7 cells treated with DNA-damage agent. MCF-7 cells expressing p53 shRNA were transfected with GFP, nAC-GFP, or nLifeact-GFP expression vectors and subsequently treated with DOXO (1 μg/mL) (a, b) or VP16 (100 μM) (c–e) for 16 h. c, d The cells were incubated with 1 mM EU and then labeled with Alexa Fluor 594 azide by Click-iT reaction after fixation. a, c Confocal images of GFP, nAC-GFP, and nLifeact-GFP (gray), acetylated histone H3 at K9 (H3ac; magenta), and EU (magenta). The nuclei of the transfected cells are enclosed by a dotted line. The scale bar is 10 μm. b, d The intensity values of fluorescence of acetylated histone H3 or EU in the cells expressing GFP, nAC-GFP, or nLifeact-GFP. The horizontal line represents the median and the upper and lower whiskers represent the maximum and minimum values, respectively. N ≥36 and N ≥31 for each experiment. Each bar represents the mean ± S.D. Asterisks, p < 0.005; double asterisks, p < 0.05; dagger, p = 0.0594. e Confocal images of GFP, nAC-GFP or nLifeact-GFP (gray/green), γH2AX (red), or DAPI (blue). The scale bar is 10 μm. f The intensity values of fluorescence of γH2AX in the cells expressing GFP, nAC-GFP, or nLifeact-GFP. The horizontal line represents the median and the upper and lower whiskers represent the maximum and minimum values, respectively. N ≥24 for each experiment. Each bar represents the mean ± S.D. Asterisks, p < 0.01; double asterisks, p < 0.05.

Article Snippet: Anti-p53 mouse monoclonal (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA, or 1C12; Cell Signaling Technology, Danvers, MA, USA), anti-Flag mouse monoclonal (M2; Sigma-Aldrich, St. Louis, MO, USA), anti-caspase 1 mouse monoclonal (14F468; Santa Cruz Biotechnology, Dallas, TX, USA), anti-γH2AX rabbit polyclonal (2577; Cell Signaling Technology, Danvers, MA, USA), anti-H2AX rabbit polyclonal (GeneTex, Inc., Irvine, CA, USA), and anti-α-tubulin mouse monoclonal (DM1A; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were used for immunoblot analysis.

Techniques: Expressing, Knockdown, shRNA, Transfection, Incubation, Labeling

Journal: Scientific Reports

Article Title: Double deletion of tetraspanins CD9 and CD81 in mice leads to a syndrome resembling accelerated aging

doi: 10.1038/s41598-018-23338-x

Figure Lengend Snippet: Double knockdown of CD9/CD81 results in reduced SIRT1 expression, thereby increasing apoptosis and decreasing cell proliferation. ( a ) siRNA knockdown of CD9 and CD81 in epithelial cells downregulated the expression of Foxo3a, but not that of Klotho, ATR and SIRT6. Reciprocally, expression of p53 and p21 was upregulated. ( b ) SA-β-gal staining of DKD epithelial cells (n = 4). ( c ) Proliferation assay (n = 4). ( d ) Apoptosis assay (n = 4). ( e ) ICC image of DKD epithelial cells. ( f ) Electron microscopy of knockdown epithelial cells. Scale bar, 50 µm for ( b ) and ( e ) and 5 µm for ( f ). Bars represent means ± SD; **P < 0.01 versus si-cont.

Article Snippet: The following primary Abs were used: mouse anti-human CD9 (MM2/57; Invitrogen), mouse anti-human CD81 (JS64; BECKMAN COULTER), rat anti-mouse CD9 (KMC8; BD Bioscience), hamster anti-mouse CD81 (Eat2; AbD SeroTec), mouse anti-human SIRT1 (1F3; Cell Signaling Technology [CST]), mouse anti-mouse SIRT1 (19A7AB4; Abcam), rabbit anti-human SIRT6 (EPR5079(N); Abcam), rabbit anti-mouse SIRT6 (D8D12; CST), rabbit anti-FOXO3a (CST), mouse anti-p53 (1C12; CST), rabbit anti-p21 (12D1; CST), rabbit anti-p21 (EPR18021; Abcam), mouse anti-p16 (D25; CST), rabbit anti-Klotho (Abcam), mouse anti-WRN (8H3; CST), rabbit anti-ATR (CST), NF-κB (E379; Abcam), rabbit anti-phospho-NF-κB p65 (93H1; CST), rabbit anti-acetyl-NF-κB p65 (D2S3J; CST), rabbit anti-IκB (CST), and rabbit anti-β-actin (13E5; CST).

Techniques: Knockdown, Expressing, Staining, Proliferation Assay, Apoptosis Assay, Electron Microscopy