Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation of keratinocytes and angiogenesis in diabetic mice. (a) Expression levels of integrin β1, K14, K10, K1, and Notch1 NICD evaluated by Western blot in the middle and at the end of wound healing. (b-f) Corresponding quantitative analysis. (g) CD31 and VEGF expression levels evaluated by Western blot. (h and i) Corresponding quantitative analysis. n = 3 in each group. ✶ P < 0.05. NICD: Notch Intracellular Domain, VEGF: Vascular endothelial growth factor, K14: Keratin 14, K10: Keratin 10, K1: Keratin 1.

Article Snippet: The source and dilution ratio of the antibody was as follows: Integrin β1 (1:1000, 26918-1-AP, Proteintech, Wuhan, China), K14 (1:1000, 22221-1-AP, Proteintech, Wuhan, China), K1 (1:800, 16848-1-AP, Proteintech, Wuhan, China), K10 (1:1000, 18343-1-AP, Proteintech, Wuhan, China), Notch 1 (Notch Intracellular Domain, NICD) (1:2000, 20687-1-AP, Proteintech, Wuhan, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000, 10494-1-AP, Proteintech, Wuhan, China), β-actin (1:5000, 20536-1-AP, Proteintech, Wuhan, China), VEGF (1:1000, 19003-1-AP, Proteintech, Wuhan, China), and CD31 (1:1000, 28083-1-AP, Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation through interaction between HIF-1α and NICD. (a) Immunofluorescence showing the expression and co-localization of HIF-1α and NICD in HaCaT cells under different conditions; bar = 100 μm. (b) Western blot showing the expression levels of HIF-1α, NICD, K14, and integrin-β1 in HaCaT cells under different conditions. (c-f) Quantitative analysis of WB. n = 3 in each group; ✶ P < 0.05. (g) Co-IP confirming the interaction between HIF-1α and NICD. HIF-1: Hypoxia-inducible factor 1, NICD: Notch intracellular domain, WB: Western blot, Co-IP: Co-immunoprecipitation, K14: Keratin 14.

Article Snippet: The source and dilution ratio of the antibody was as follows: Integrin β1 (1:1000, 26918-1-AP, Proteintech, Wuhan, China), K14 (1:1000, 22221-1-AP, Proteintech, Wuhan, China), K1 (1:800, 16848-1-AP, Proteintech, Wuhan, China), K10 (1:1000, 18343-1-AP, Proteintech, Wuhan, China), Notch 1 (Notch Intracellular Domain, NICD) (1:2000, 20687-1-AP, Proteintech, Wuhan, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2000, 10494-1-AP, Proteintech, Wuhan, China), β-actin (1:5000, 20536-1-AP, Proteintech, Wuhan, China), VEGF (1:1000, 19003-1-AP, Proteintech, Wuhan, China), and CD31 (1:1000, 28083-1-AP, Proteintech, Wuhan, China).

Techniques: Immunofluorescence, Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Advanced Science

Article Title: Spatiotemporal Adaptations‐Driven Dynamic Thra Activation Simulates a Skin Wound Healing Response

doi: 10.1002/advs.202506651

Figure Lengend Snippet: Inhibition of Thra delayed epithelialization and dermal collagen deposition. A) Phase‐contrast microscope images and schematic analysis of wound healing process after Thra inhibition treatment, with statistical analysis of the average wound rate. (Scale bars, 1 mm. N = 5, * * p < 0.01, * p < 0.05). B) Immunofluorescence images of K14 expression in the control and Thra inhibition groups, with statistical analysis of re‐epithelialization length. (Scale bars, 200 µm; N = 5, p <0.01, * p < 0.05). C) Immunofluorescence images of K14/PCNA expression in the control and Thra inhibition groups, with statistical analysis of the average number of PCNA + cells. (Scale bars, 100 µm; N = 5, * * p < 0.01, * p < 0.05). D) Masson's trichrome staining images of the control and Thra inhibition groups. (Scale bars, 100 µm). E) Statistics of the average collagen deposition. (N = 5, * * p < 0.01, * p < 0.05). F) Schematic summary of the wound healing process after Thra inhibition treatment.

Article Snippet: The following antibodies were used in the study: K14 (Boster, BM4522), VIM (BEYOTIME, AF0318), PCNA (Elabscience, E‐AB‐22001), CD31 (Servicebio, GB12063‐100), P63 (Zenbio, 381215), Thra (Affinit, AF9218), E‐cadherin (Beyotime, AF0138), GGCT (Abcam, ab198503), K16 (Affinity, AF5482), FACTIN (Invitrogen, A12379), SAA3 (Abcam, ab282730), FN1 (Santa, sc‐8422).

Techniques: Inhibition, Microscopy, Immunofluorescence, Expressing, Control, Staining

Journal: Advanced Science

Article Title: Spatiotemporal Adaptations‐Driven Dynamic Thra Activation Simulates a Skin Wound Healing Response

doi: 10.1002/advs.202506651

Figure Lengend Snippet: High expression level of TH promoted wound repair. A) Schematic illustration of the experimental design for hyperthyroidism and hypothyroidism models, with statistical analysis of average TT 4 and T 3 concentrations from ELISA assays. (N = 5, * * p < 0.01, * p < 0.05). B) Phase‐contrast microscope images and schematic analysis of wound healing status on PWD3 after thyroid dysfunction treatments, with statistical analysis of the average wound area rate. (Scale bars, 1 mm. N = 5, * p < 0.05). C) Immunofluorescence images of K14, PCNA/K14 and E‐cadherin/CD31 expressions in the control, hypothyroidism and hyperthyroidism groups, with statistical analysis of the average re‐epithelialization length, and the average numbers of PCNA + and CD31 + cells. (Scale bars, 100 µm; N = 5, * * p < 0.01, * p < 0.05). D) Masson's trichrome staining images of the control, hypothyroidism and hyperthyroidism groups. (Scale bars, 100 µm). E) Statistics of the average collagen deposition. (N = 5, * p < 0.05). F) Schematic summary of the wound healing process after thyroid dysfunction treatments.

Article Snippet: The following antibodies were used in the study: K14 (Boster, BM4522), VIM (BEYOTIME, AF0318), PCNA (Elabscience, E‐AB‐22001), CD31 (Servicebio, GB12063‐100), P63 (Zenbio, 381215), Thra (Affinit, AF9218), E‐cadherin (Beyotime, AF0138), GGCT (Abcam, ab198503), K16 (Affinity, AF5482), FACTIN (Invitrogen, A12379), SAA3 (Abcam, ab282730), FN1 (Santa, sc‐8422).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Microscopy, Immunofluorescence, Control, Staining

Journal: Advanced Science

Article Title: Spatiotemporal Adaptations‐Driven Dynamic Thra Activation Simulates a Skin Wound Healing Response

doi: 10.1002/advs.202506651

Figure Lengend Snippet: Thra‐regulated glutathione metabolism in the epidermis. A) Volcano Plot of the distribution of down‐ and up‐ regulated genes in the control and Thra‐inhibited wounded groups. B) KEGG analysis of pathway enriched in the Thra‐inhibited wounded group. C) iPATH analysis of the impact on metabolism pathways in the Thra‐inhibited wounded group. D) A list of the top 5 ranked genes related to glutathione metabolism according to their P‐values and p‐adjustments. E) Statistics of the expression levels of the top three ranked genes in the bulk RNA sequencing data in terms of CPM. (N = 3, * p <0.05). F) VlnPlot of the expression of Ggct. G) FeaturePlots of the expression of Ggct in HST and PWD3 skin within FB and EPI clusters. H) Spatial transcriptomics data of the expression of Ggct in HST and PWD3 skin. I) qRT‐PCR analysis of Ggct expression levels after Thra knockdown. (N = 3, ** p < 0.01) J) Immunofluorescence images of GGCT expression in the control and aThra groups. (Scale bars, 100 µm). K) GO analysis of pathways enriched in Ggct‐positive epidermal cells post‐wounding. L) Immunofluorescence images of K14 and FACTIN expressions in the control, GGCT and aThra groups, with statistical analysis of the average proportion of K14 and FACTIN in total cells. (Scale bars, 50 µm; N = 5, * * p < 0.01).

Article Snippet: The following antibodies were used in the study: K14 (Boster, BM4522), VIM (BEYOTIME, AF0318), PCNA (Elabscience, E‐AB‐22001), CD31 (Servicebio, GB12063‐100), P63 (Zenbio, 381215), Thra (Affinit, AF9218), E‐cadherin (Beyotime, AF0138), GGCT (Abcam, ab198503), K16 (Affinity, AF5482), FACTIN (Invitrogen, A12379), SAA3 (Abcam, ab282730), FN1 (Santa, sc‐8422).

Techniques: Control, Expressing, RNA Sequencing, Quantitative RT-PCR, Knockdown, Immunofluorescence

Journal: Advanced Science

Article Title: Spatiotemporal Adaptations‐Driven Dynamic Thra Activation Simulates a Skin Wound Healing Response

doi: 10.1002/advs.202506651

Figure Lengend Snippet: Thra‐activated SAA3 upregulated at the dermal wound edge. A) Pie chart of the genes in the top two signaling pathways in Thra‐positive cells in the FB post‐wounding. B) VlnPlot and FeaturePlots of the expression of Saa3. C) Spatial transcriptomics data of the expression of Saa3 in HST and PWD3 skin, with statistical analysis of expression level of Saa3. (N = 3, * p < 0.05) D) Immunofluorescence images of SAA3 expression in the control and aThra groups. (Scale bars, 200 µm; N = 5) E) Schematic summary of the wound healing process after SAA3 treatment. F) Immunofluorescence images of PCNA/K14 expression in the control and SAA3 groups, with statistical analysis of the average re‐epithelialization length and the average number of PCNA + cells. (Scale bars, 200 µm; N = 5, * * p < 0.01, ns: no significance).

Article Snippet: The following antibodies were used in the study: K14 (Boster, BM4522), VIM (BEYOTIME, AF0318), PCNA (Elabscience, E‐AB‐22001), CD31 (Servicebio, GB12063‐100), P63 (Zenbio, 381215), Thra (Affinit, AF9218), E‐cadherin (Beyotime, AF0138), GGCT (Abcam, ab198503), K16 (Affinity, AF5482), FACTIN (Invitrogen, A12379), SAA3 (Abcam, ab282730), FN1 (Santa, sc‐8422).

Techniques: Protein-Protein interactions, Expressing, Immunofluorescence, Control

Journal: Advanced Science

Article Title: Spatiotemporal Adaptations‐Driven Dynamic Thra Activation Simulates a Skin Wound Healing Response

doi: 10.1002/advs.202506651

Figure Lengend Snippet: SAA3 favored dermal FN1 protein functions. A) GO analysis of pathways enriched in Saa3‐positive dermal cells post‐wounding. B) Immunofluorescence images of CD31/K14 expression in the control and SAA3 groups. (Scale bars, 200 µm; N = 5) C) MF analysis of pathways enriched in Saa3‐positive dermal cells post‐wounding; Venn Plot of the six most strongly related genes. D) VlnPlots of the expression of the six most strongly related genes. E) Protein docking of the binding status of SAA3 and FN1. F) Immunofluorescence images of SAA3, FN1 and FN1/SAA3 expressions post‐wounding. (Scale bars, 100 µm; Statistics of gray value traces).

Article Snippet: The following antibodies were used in the study: K14 (Boster, BM4522), VIM (BEYOTIME, AF0318), PCNA (Elabscience, E‐AB‐22001), CD31 (Servicebio, GB12063‐100), P63 (Zenbio, 381215), Thra (Affinit, AF9218), E‐cadherin (Beyotime, AF0138), GGCT (Abcam, ab198503), K16 (Affinity, AF5482), FACTIN (Invitrogen, A12379), SAA3 (Abcam, ab282730), FN1 (Santa, sc‐8422).

Techniques: Immunofluorescence, Expressing, Control, Binding Assay

Journal: Advanced Science

Article Title: Spatiotemporal Adaptations‐Driven Dynamic Thra Activation Simulates a Skin Wound Healing Response

doi: 10.1002/advs.202506651

Figure Lengend Snippet: GGCT, SAA3, and TH regulatory networks demonstrated in mouse skin organoid models. A) Epithelial scratch assay results of the migratory ability of cells in different treatment groups, with statistical analysis of the average scratch area. (Scale bars, 200 µm; N = 3, * * p < 0.01, * p < 0.05, ns: no significance) B) Schematic illustration of the experimental design for mouse skin organoids. C) Immunofluorescence images of VIM/K14, PCNA/K14, Ecad/P63, and CD31/K14 expressions on the sixth day of cultured skin organoids, with statistical analysis of the average number of PCNA + cells in dermis, the average number of P63 + cells in epidermis and the average number of CD31 + cells. (Scale bars, 50 µm; N = 3, * * p < 0.01, * p < 0.05, ns: no significance).

Article Snippet: The following antibodies were used in the study: K14 (Boster, BM4522), VIM (BEYOTIME, AF0318), PCNA (Elabscience, E‐AB‐22001), CD31 (Servicebio, GB12063‐100), P63 (Zenbio, 381215), Thra (Affinit, AF9218), E‐cadherin (Beyotime, AF0138), GGCT (Abcam, ab198503), K16 (Affinity, AF5482), FACTIN (Invitrogen, A12379), SAA3 (Abcam, ab282730), FN1 (Santa, sc‐8422).

Techniques: Wound Healing Assay, Immunofluorescence, Cell Culture