Journal: Advanced Science

Article Title: Autophagosomes Defeat Ferroptosis by Decreasing Generation and Increasing Discharge of Free Fe 2+ in Skin Repair Cells to Accelerate Diabetic Wound Healing

doi: 10.1002/advs.202300414

Figure Lengend Snippet: SAPs inhibited ferroptosis by accelerating Exos’ release for discharging free Fe 2+ from HG‐HDFs. a) Quantitative statistics demonstrating the concentrations of Exos and Fe 2+ in the Exos derived from HG‐HDFs stimulated with or without SAPs ( n = 3 per group). b) Schematic diagram and fluorescent images indicating the internalization of Exos containing Fe 2+ (derived from HG‐HDFs stimulated with or without SAPs) by HUVECs for 2 h. Exos were co‐stained with green PKH67 and red ferro‐orange ( n = 3 per group). c) qRT‐PCR analysis indicating the gene expression level of CHMP6 and CHMP5 in HG‐HDFs with or without SAPs ( n = 3 per group, all mRNA levels are normalized to loading control, GAPDH). d,e) Western blotting analysis (d) and corresponding quantitative band intensities (e) displaying the protein expression level of CHMP6 and CHMP5 in HG‐HDFs cultured with SAPs ( n = 3 per group, all proteins levels are normalized to loading control, GAPDH). f,g) Corresponding fluorescent images (f) and quantitative analysis (g) showing Alix positive HG‐HDFs treated with different groups (SAPs, SAPs+NC, SAPs+si6, SAPs+si5) ( n = 3 per group). h) Representative Western blotting images showing the expression of biomarkers (CD9, CD63, Alix) on Exos secreted by HG‐HDFs treated with different groups ( n = 3 per group). i) Quantitative statistics of the concentration of Exos derived from HG‐HDFs treated with different groups demonstrated by NTA analysis ( n = 3 per group). j) Quantitative analysis of Fe 2+ concentration inside Exos of HG‐HDFs treated with different groups measured by ICP‐MS ( n = 3 per group). k,l) Representing fluorescent images (k) and quantitative analysis (l) indicating intracellular Fe 2+ concentration in HG‐HDFs treated with different groups ( n = 3 per group). Data represent mean ± SD. Statistical significance was calculated by Students t ‐test with Welchs correction for (a) and (c), or one‐way ANOVA with Tukey's significant difference multiple comparisons for (e), (g), (i), (j), and (l). Significant differences between SAPs − or 0 m m groups with other groups are indicated as **p < 0.01, *** p < 0.001. Significant differences between SAPs+NC group and other groups are indicated as p < 0.001. Scale bar,50 µm for (b), 100 µm for (f) and (k).

Article Snippet: Then, electrophoresis was performed at 120 V for 90 min. After electrophoresis, proteins were transferred onto PVDF membrane by electroblot and blocked by 5% milk for 1 h. After that, PVDF membranes were co‐incubated with primary antibodies including LC3b (1:1000), GPX4 (1:1000, Abclonal, A1933), FTH1 (1:1000, CST, 4393), HASP5 (1:1000, CST, 3177), p‐elf2 α (1:1000, Proteintech, 28740‐1‐AP), elf2 α (1:1000, Proteintech, 11170‐1‐AP), ATF4 (1:1000, Proteintech, 10835‐1‐AP), DDIT3 (1:1000, CST, 2895T), CHMP5 (1:1000, Abcam, ab96273), CHMP6 (1:1000, Abclonal, A4975), Alix (Abclonal, A2215), CD63 (Abclonal, A19023), CD9 (Abclonal, A19027), and GAPDH (1:10 000, Abcam, ab181602).

Techniques: Derivative Assay, Staining, Quantitative RT-PCR, Expressing, Western Blot, Cell Culture, Concentration Assay

Journal: Advanced Science

Article Title: Autophagosomes Defeat Ferroptosis by Decreasing Generation and Increasing Discharge of Free Fe 2+ in Skin Repair Cells to Accelerate Diabetic Wound Healing

doi: 10.1002/advs.202300414

Figure Lengend Snippet: Gel‐SAPs promoted diabetic wound healing in vivo by inhibiting the ferroptosis of skin repair cells. a) Timeline of wound treatment process. b–d) Representative images (b) closure traces (c) and quantitative analysis (d) of wound closure at days 0, 3, 7, and 14 treated with PBS, SAPs, Gel, and Gel‐SAPs ( n = 6 per group). e,f) Immunofluorescence staining images (f) and quantification of α ‐SMA (e) (red fluorescence) at wound sites with the treatment of PBS, SAPs, Gel, and Gel‐SAPs for day 7 ( n = 6 per group). g,i) Immunofluorescence staining images (i) and quantification of COL1A1 (g) (red fluorescence) at wound sites with the treatment of PBS, SAPs, Gel, and Gel‐SAPs for day 7 ( n = 6 per group). h,l) Immunofluorescence staining images (l) and quantification of CD31 (h) (red fluorescence) at wound sites with the treatment of PBS, SAPs, Gel, and Gel‐SAPs for day 7 ( n = 6 per group). j,m) Representative fluorescent images (m) and quantitative analysis (j) of FTH1 level in PBS, SAPs, Gel, and Gel‐SAPs groups for day 7. ( n = 6 per group). k,n,o) Representative Western blotting images (k) and quantitative band intensities (n,o) indicating the expression level of CHMP6 and CHMP5 in PBS, SAPs, Gel, and Gel‐SAPs groups. ( n = 6 per group, all proteins levels are normalized to loading control, GAPDH). The cell nuclei were dyed blue fluorescence with 4',6‐diamidino‐2‐phenylindole (DAPI) for (f), (i), (l), and (m). Results are representative of at least three independent experiments. Data represent mean ± SD. Statistical significance was calculated by one‐way ANOVA with Tukey's significant difference multiple comparisons for (d), (e), (g), (h), (j), (n), and (o). Significant differences between PBS groups and other group are indicated as ***p < 0.001. Significant differences between Gel‐SAPs group and other groups are indicated as # p < 0.05, p < 0.001 compared with other groups. NS, not significant. Scale bar, 100 µm for (f) and (m), 50 µm for (i) and (l).

Article Snippet: Then, electrophoresis was performed at 120 V for 90 min. After electrophoresis, proteins were transferred onto PVDF membrane by electroblot and blocked by 5% milk for 1 h. After that, PVDF membranes were co‐incubated with primary antibodies including LC3b (1:1000), GPX4 (1:1000, Abclonal, A1933), FTH1 (1:1000, CST, 4393), HASP5 (1:1000, CST, 3177), p‐elf2 α (1:1000, Proteintech, 28740‐1‐AP), elf2 α (1:1000, Proteintech, 11170‐1‐AP), ATF4 (1:1000, Proteintech, 10835‐1‐AP), DDIT3 (1:1000, CST, 2895T), CHMP5 (1:1000, Abcam, ab96273), CHMP6 (1:1000, Abclonal, A4975), Alix (Abclonal, A2215), CD63 (Abclonal, A19023), CD9 (Abclonal, A19027), and GAPDH (1:10 000, Abcam, ab181602).

Techniques: In Vivo, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing

Journal: Advanced Science

Article Title: Autophagosomes Defeat Ferroptosis by Decreasing Generation and Increasing Discharge of Free Fe 2+ in Skin Repair Cells to Accelerate Diabetic Wound Healing

doi: 10.1002/advs.202300414

Figure Lengend Snippet: Underlying mechanisms of SAPs to inhibiting ferroptosis of HG‐impaired skin repair cells. i) SAPs downregulated the production of free Fe 2+ by reducing ER stress. ii) SAPs promoted the secretion of Exos to discharge Fe 2+ by upregulating the expression of CHMP5/CHMP6.

Article Snippet: Then, electrophoresis was performed at 120 V for 90 min. After electrophoresis, proteins were transferred onto PVDF membrane by electroblot and blocked by 5% milk for 1 h. After that, PVDF membranes were co‐incubated with primary antibodies including LC3b (1:1000), GPX4 (1:1000, Abclonal, A1933), FTH1 (1:1000, CST, 4393), HASP5 (1:1000, CST, 3177), p‐elf2 α (1:1000, Proteintech, 28740‐1‐AP), elf2 α (1:1000, Proteintech, 11170‐1‐AP), ATF4 (1:1000, Proteintech, 10835‐1‐AP), DDIT3 (1:1000, CST, 2895T), CHMP5 (1:1000, Abcam, ab96273), CHMP6 (1:1000, Abclonal, A4975), Alix (Abclonal, A2215), CD63 (Abclonal, A19023), CD9 (Abclonal, A19027), and GAPDH (1:10 000, Abcam, ab181602).

Techniques: Expressing