atg5 d5f5u rabbit mab  (Cell Signaling Technology Inc)

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: Impaired SIRT7 expression and activity in vitiligo melanocytes under oxidative stress. (A) The expression of Ac-lysine and Ac-histone on PIG1 and PIG3V cells after the treatment of H 2 O 2 (750 μM) for 24 h (mean ± SEM is shown, n = 3). (B) Cell viability of PIG1 and PIG3V cells. The cells were treated with H 2 O 2 (750 μM) for 24 h and pretreated with or without Fer-1 (5 μM) or TSA (5 μM), or NAM (100 nM) for 6 h (mean ± SEM is shown, n = 3). PIG1 and PIG3V cells were treated with H 2 O 2 (750 μM) for 24 h. (C) NAD + level (mean ± SEM is shown, n = 3). (D) Relative mRNA level of SIRT1-SIRT7 (mean ± SEM is shown, n = 3). (E) Relative protein expression of SIRT7 was analyzed (mean ± SEM is shown, n = 3). (F) Western blot results of p53 and Ac-p53. Relative expression of Ac-p53/p53 was determined by Image J software (mean ± SEM is shown, n = 3). (G) Relative protein expression of Ac-H3K18 was analyzed (mean ± SEM is shown, n = 3). (H) The differentially-expression of SIRT7 in healthy skin versus perifocal skin of vitiligo based on single-cell RNA sequencing (scRNA-seq) data. (I) Representative images of melanocytes for SIRT7 in healthy skin and perilesional skin of vitiligo (mean ± SD is shown, n = 5). Melanocytes were stained with Melan-A (red) antibody. Nuclei were counterstained with DAPI (blue). Scale bar = 100 μm (Magnification the left 200 ×; right 400 × ). *P < 0.05, **P < 0.01, ***P < 0.001, ns for non-significant (one-way ANOVA and two-tailed Student’s t test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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: Expressing, Activity Assay, Western Blot, Software, Single Cell, RNA Sequencing, Staining, Two Tailed Test

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 inhibited ferroptosis in melanocytes under oxidative stress. (A) and (B) Enrichment analysis of GSEA and heatmap of representative ferroptotic genes between SIRT7 and ferroptosis pathway in PIG1 cell line. (C) The cell death ratio of PIG1 with the transfection of siRNA or si-SIRT7, and the PIG1 cells were treated with H 2 O 2 (750 μM) for 24 h with the pretreatment of Fer-1 (5 μM). The SIRT7 knockdown or control PIG1 cells were treated with or without H 2 O 2 (750 μM) for 24 h. (n = 3). (D) , (E) and (F) The levels of lipid ROS, MDA, and Fe 2+ were analyzed in PIG1 cells. (n = 3). (G) and (H) The alterations of GPX4 mRNA and protein were analyzed in PIG1 cells. (I) Cellular GSH level was analyzed in PIG1 cells. (n = 3). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

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: Transfection, Knockdown, Control

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 inhibited ferroptosis via the regulation of ATF3-GPX4 signaling . (A) RT-PCR analysis of the alteration of GPX4 in PIG1 cells with the transfection of siRNA or siATF3 under oxidative stress. (n = 3). PIG1 cells were stimulated with H 2 O 2 (750 μM) with or without SIRT7 knockdown. (B) Volcano map of differentially-expressed genes. (C) and (D) RT-PCR and immunoblotting analysis of the level of ATF3. PIG1 cells were treated with H 2 O 2 (750 μM) for 24 h after being transfected with siRNA for 24 h. (E) , (F) and (G) Cell death ratio, lipid ROS levels, and Fe 2+ contents were analyzed. (n = 3). PIG1 cells of SIRT7 knockdown were treated with H 2 O 2 (750 μM) for 6 h after being transfected with siRNA for 48 h. (H) RT-PCR analysis of the level of GPX4. (n = 3). (I) The detection of GSH level. (n = 3). (J) Transcription factor analysis of the GPX4 promoter predicted the ATF3 binding site. (K) Enrichment of ATF3 binding site to the promoter of GPX4 by ChIP analysis in PIG1 cells. (n = 3). (L) Enrichment of ATF3 to the promoter of GPX4 by ChIP analysis, and the PIG1 cells were treated with H 2 O 2 (750 μM) for 6 h with or without SIRT7 knockdown. (n = 3). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (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: Reverse Transcription Polymerase Chain Reaction, Transfection, Knockdown, Western Blot, Binding Assay, Two Tailed Test

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

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: Melanocytic-specific Sirt7 knockout accelerated depigmentation of tail skin in a mouse model of vitiligo . (A) Diagram of the principle of vitiligo induction. (B) Representative whole-mount tail epidermis immunofluorescence staining images in female Sirt7 fl/fl control mice, Sirt7 fl/fl control-Vitiligo mice, and Sirt7 MCKO-Vitiligo mice induced for 30 days. Nuclei were stained with Hoechst (blue). CD8 + T cells were counterstained with CD8α (green) antibody. Melanocytes were stained with Melan-A (red) antibody. (Magnification 100 ×, scale bar = 200 μm) (n = 3). (C) The representative mouse tail images and ImageJ analysis images in female Sirt7 fl/fl control mice, Sirt7 fl/fl control-Vitiligo mice, and Sirt7 MCKO-Vitiligo mice induced for 30, 60 and 90 days. (n = 3). (D) Statistical analysis of the tail pigmentation percentages in female Sirt7 fl/fl control mice, Sirt7 fl/fl control - Vitiligo mice, and Sirt7 MCKO-Vitiligo mice induced for 30, 60 and 90 days. (n = 3). (E) Representative whole-mount tail epidermis immunofluorescence staining images in female Sirt7 fl/fl control mice, Sirt7 fl/fl control-Vitiligo mice, and Sirt7 MCKO-Vitiligo mice induced for 60 days. (n = 3). (F) Immunofluorescence images of GPX4 expression within melanocytes in female Sirt7 fl/fl control-Vitiligo mice and Sirt7 MCKO-Vitiligo mice. Nuclei (blue), Melanocytes (green), GPX4 (purple). (Magnification 200 ×, scale bar = 20 μm). (n = 3). Data were presented as mean ± SD (one-way ANOVA and two-tailed Student’s t test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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: Knock-Out, Immunofluorescence, Staining, Control, Expressing, Two Tailed Test