Journal: Molecular Pain

Article Title: Puerarin improves the comorbidity of chronic pain and depression by binding with Bax and reducing mitochondrial dysfunction

doi: 10.1177/17448069251335230

Figure Lengend Snippet: Effect of Pue or Bax siRNA on Bax and IL-1β levels in primary astrocytic cells. (a and b) Representative Western blotting bands and quantitative analysis for Bax and IL-1β in primary astrocytic cells of Control, siRNA, Bax siRNA, DMSO, and Pue groups. * p < 0.05 versus siRNA or DMSO group. (c and d) Representative Western blotting bands and quantitative analysis for Bax, IL-1β, and c-Fos in organotypic PAG slice of the SNI model mice with Control, siRNA and Bax siRNA groups. * p < 0.05 versus siRNA group. (e and f) Representative immunofluorescence images and quantitative analysis for Bax and GFAP in primary astrocytic cells of Control, IL-1β and IL-1β + Pue groups. Scale bar: 50 μm. (g and h) Representative Western blotting bands and quantitative analysis for Bax and GFAP in primary astrocytic cells in Control, IL-1β and IL-1β + Pue groups. Data were presented as mean ± SD. * p < 0.05 versus Control group. # p < 0.05 versus IL-1β group.

Article Snippet: Bax siRNA (sc-29213) and control siRNA (sc-44230) were purchased from Santa Cruz Biotechnology (CA, USA) and transfected with primary astrocyte cells in Control siRNA and Bax siRNA groups according to the siRNA Transfection Protocol.

Techniques: Western Blot, Control, Immunofluorescence

Journal: eLife

Article Title: Nuclear Translocation of SIRT4 Mediates Deacetylation of U2AF2 to Modulate Renal Fibrosis Through Alternative Splicing-mediated Upregulation of CCN2

doi: 10.7554/elife.98524

Figure Lengend Snippet: Figure 6. Acetylation of U2AF2 at K413 regulates CCN2 gene expression and pre-mRNA splicing. (A) The crosstalk of differences of the gene expression profiling analysis and splicing analysis. (B) The Ccn2 mRNA level in HK2 cells (n=5). (C, D) HK2 cells were transfected with Flag-U2AF2 WT, K413R, or K413Q, and Ad-SIRT4 or SIRT4 siRNA (siSIRT4) as indicated in figure. Cells lysates subjected to Co-IP with anti-Flag antibody, and western blotting using indicated antibodies. (E) Luciferase activity of Ccn2 promoter in HK2 cells transfected with Ad-SIRT4, Ad-shSIRT4, WT U2AF2, U2AF2 K413R, or U2AF2 K413Q under TGF-β1 stimulation was measured (n=5). (F) Schematics of Ccn2 alternative splicing pattern regulated by hyperacetylated U2AF2. Full line and dotted line represent normal splicing and abnormal intron retention, respectively. Grey and yellow boxes represent untranslated region (UTR) and protein coding region (CDS), respectively. The first stop codon in intron 2 or 3 was indicated with red arrow. (G, H) Quantitative real- time PCR analysis of normal or abnormal splicing isoforms of CCN2 (n=5). (I) Actinomycin D (Act D, 2 mg/mL) treatment and quantitative real-time PCR were performed to measure CCN2 mRNA levels (n=5). (J) HK2 cells were transfected with Flag-U2AF2 WT, Flag-U2AF2 WT, or Flag-U2AF2 K413R with or without Ad-SIRT4 under TGF-β1 stimulation. Cells lysates subjected to RNA Binding Protein Immunoprecipitation (RIP) with anti-U2AF2 antibody (left panel). Quantitative real-time PCR results of RIP assays (right panel). For all panels, data are presented as mean ± SD. **p< 0.01, ***p< 0.001, by Student’s t-test for B. **p<0.01, ***p<0.001 by one-way ANOVA with Bonferroni correction test for E, G, H, I, and J.

Article Snippet: The commercial siRNA Ctrl (Cat# sc- 37007, Santa Cruz), siRNA Hat1 (Cat# sc- 145898, Santa Cruz), siRNA Sirt4 (Cat# sc- 63025, Santa Cruz), siRNA Bax (Cat# sc- 29213, Santa Cruz), and siRNA Bak (Cat# sc- 29785, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Gene Expression, Transfection, Co-Immunoprecipitation Assay, Western Blot, Luciferase, Activity Assay, Alternative Splicing, Real-time Polymerase Chain Reaction, RNA Binding Assay, Immunoprecipitation

Journal: eLife

Article Title: Nuclear Translocation of SIRT4 Mediates Deacetylation of U2AF2 to Modulate Renal Fibrosis Through Alternative Splicing-mediated Upregulation of CCN2

doi: 10.7554/elife.98524

Figure Lengend Snippet: Figure 7. SIRT4 is released through BAX/BAK pore in a TGF-β1-dependent manner. (A) Organelle separation experiment and immunoblot analysis detected the expression/localization of SIRT4, Tubulin, and COX IV. (B) TECs were treated with TGF-β1 for indicated time. Organelle separation experiment and immunoblot analysis detected the localization of SIRT4, Tubulin, and COX IV. (C) Representative image of immunofluorescent staining of SIRT4 in TECs treated with TGF-β1 or not (scale bar, 50 μm). (D) TECs were treated with Tunicamycin, Cisplatin, Poly(I:C), or under serum starvation. Organelle separation experiment and immunoblot analysis detected the localization of SIRT4, Tubulin, and COX IV. (E) WT TECs incubated with Bax siRNA or Bak siRNA under TGF-β1 stimulation. Organelle separation experiment and immunoblot analysis detected the localization of SIRT4, Tubulin, and COX IV (left panels). Western blot analysis of the expression of BAX, BAK and Tubulin in TECs (middle panels). The mRNA level of Ccn2 was detected by qPCR (n=5) (right panels). (F) WT TECs were treated with MSN-125 (10 μM) or vehicle. Organelle separation experiment and immunoblot analysis detected the localization of SIRT4, Tubulin, and COX IV (left panel). The mRNA level of Ccn2 was detected by qPCR (n=5) (right panel). (G) Kidney tissues from WT or S4KO mice were treated with MSN-125 or vehicle. The mRNA level of Ccn2 was detected by qPCR in the kidney of mice (n=5). For all panels, data are presented as mean ± SD. **p<0.01, ***p<0.001 by one-way ANOVA with Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat# sc- 37007, Santa Cruz), siRNA Hat1 (Cat# sc- 145898, Santa Cruz), siRNA Sirt4 (Cat# sc- 63025, Santa Cruz), siRNA Bax (Cat# sc- 29213, Santa Cruz), and siRNA Bak (Cat# sc- 29785, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Staining, Incubation

Journal: eLife

Article Title: Nuclear Translocation of SIRT4 Mediates Deacetylation of U2AF2 to Modulate Renal Fibrosis Through Alternative Splicing-mediated Upregulation of CCN2

doi: 10.7554/elife.98524

Figure Lengend Snippet: Figure 8. ERK2 phosphorylates SIRT4 at S36 and promotes it nucleus translocation. (A) (The upper panel) Nuclear fractions were prepared from TECs pretreated with LY290042 (30 μM), SU6656 (4 μM), SP600125 (25 μM), and U0126 (20 μM) for 30 min before TGF-β1 (2 ng/ml) for 12 hr. Nuclear PCNA and cytoplasmic Tubulin were used as controls. (The bottom panel) TECs were treated with TGF-β1 (2 ng/ml) for 12 hr. Organelle separation experiment and immunoblot analysis detected the localization of SIRT4, PCNA, and Tubulin. (B) TECs were pretreated with or without U0126 (20 μM) for 30 min, then treated with TGF-β1 (2 ng/ml) for 12 hr. Immunofluorescence analyses were performed with the indicated antibodies. (C) HK2 cells were stably transfected with Flag-ERK2, Flag-ERK2 K52R, or siRNA ERK2 (left panel) or transiently transfected with HA-MEK1 Q56P and indicated Flag-tagged ERK2 proteins (right panel). The cells were treated with or without TGF-β1 (2 ng/ml) for 12 hr, and the total cell lysates, cytosol and nuclear fractions were prepared for determination of indicated proteins by western blot. (D) TECs were treated with TGF-β1, and cells lysates subjected to Co-IP with anti-SIRT4 antibody, and western blotting using indicated antibodies. (E) HK2 cells transfected with vectors expressing the indicated Flag-tagged ERK proteins were treated with or without TGF-β1 (2 ng/ml) for 12 hr. (F) SIRT4-ERK2 docking with the HDOCK server. (G) HK2 cells expressing the indicated

Article Snippet: The commercial siRNA Ctrl (Cat# sc- 37007, Santa Cruz), siRNA Hat1 (Cat# sc- 145898, Santa Cruz), siRNA Sirt4 (Cat# sc- 63025, Santa Cruz), siRNA Bax (Cat# sc- 29213, Santa Cruz), and siRNA Bak (Cat# sc- 29785, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Translocation Assay, Western Blot, Immunofluorescence, Stable Transfection, Transfection, Co-Immunoprecipitation Assay, Expressing