Journal: Journal of Advanced Research

Article Title: SIRT7 facilitates ferroptosis resistance of melanocytes via activating the SMAD3-ATF3-GPX4 signaling pathway in vitiligo

doi: 10.1016/j.jare.2025.07.020

Figure Lengend Snippet: SIRT7 de-acetylated SMAD3 to repress ATF3 transcription . (A) Transcription factor analysis of the ATF3 promoter predicted three SMAD3 binding sites. (B) Enrichment of three SMAD3 binding sites to the promoter of ATF3 by ChIP analysis in PIG1 cells. (n = 3). (C) and (D) Co-IP and immunostaining analysis of the interaction of SMAD3 and SIRT7 in PIG1 cells. (E) IP analysis of the level of acetyl-SMAD3 in PIG1 cells treated with H 2 O 2 (750 μM) for 6 h. (F) and (G) Immunoblotting and immunostaining analysis of the expression of p-SMAD3 and cells were treated with H 2 O 2 (750 μM) for 24 h with or without SIRT7 knockdown. Scale bar = 100 μm (Magnification 630 × ). (H) RT-PCR analysis of the alteration of ATF3 in PIG1 cells with the transfection of siRNA or siSMAD3 under oxidative stress. (n = 3). (I) RT-PCR analysis of the level of ATF3 in SIRT7 knockdown-PIG1 cells with H 2 O 2 (750 μM) treatment for 6 h after being transfected with siSMAD3 or siRNA for 24 h. (n = 3). (J) Enrichment of SMAD3 to the promoter of ATF3 by ChIP analysis in PIG1 cells, and the SIRT7 knockdown-PIG1 cells were treated with H 2 O 2 (750 μM) for 6 h after being transfected with siRNA or siSMAD3 for 24 h. (n = 3). Data were presented as mean ± SEM. **P < 0.01, ***P < 0.001, ns for non-significant (one-way ANOVA and two-tailed Student’s t test).

Article Snippet: For immunoprecipitation, 5–10 μL of SMAD3 antibody (9523S, Cell Signaling Technology, MA, USA), SIRT7 antibody (12994-1-AP, Proteintech, Wuhan, China) or control IgG (30000–0-AP, Proteintech) was mixed with the protein supernatant and incubated at 4°C for 2 h. Then, 25 μL of protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology, Beijing, China) was mixed with protein lysates.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Immunostaining, Western Blot, Expressing, Knockdown, Reverse Transcription Polymerase Chain Reaction, Transfection, Two Tailed Test