Fig 2: Impact of IGFBP3 on ADSCs'epithelial cell differentiation, proliferation, and migration. Note: (A) Schematic diagram of the experimental process for Igfbp3 silencing or overexpression in ADSCs; (B) Observation of the effects of Igfbp3 silencing or overexpression on the morphology of ADSCs and the comparison with HaCaT cell morphology under an inverted microscope (scale bar = 25 μm); (C) RT-qPCR detection of changes in Krt18 and Tjp1 mRNA expression in ADSCs after Igfbp3 silencing and overexpression, and expression levels in HaCaT cells; (D) WB detection of changes in CK18 and ZO- 1 protein expression in ADSCs after Igfbp3 silencing and overexpression, and in HaCaT cells; (E) Immunofluorescence staining for CK18 and ZO- 1 protein expression in ADSCs with different Igfbp3 interventions, and in HaCaT cells (scale bar = 50 μm); (F) CCK- 8 assay detecting cell viability changes in ADSCs (with Igfbp3 interventions) and HaCaT cells at 12, 24, 36, and 48 h; (G) EDU assay detecting the proliferation capacity of ADSCs with Igfbp3 interventions, with EDU-positive cells shown in red (proliferating phase); blue indicates DAPI-stained cell nuclei (scale bar = 50 μm); (H) Transwell assay detecting the migration ability of ADSCs with Igfbp3 interventions and comparison with HaCaT cells (scale bar = 50 μm); (I) Scratch assay detecting the migration of ADSCs with Igfbp3 interventions and comparison with HaCaT cells (scale bar = 100 μm). * indicates comparison between two groups, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 3: The influence of IGFBP3 via the ITGB1/ERK signaling pathway on ADSCs epithelial cell differentiation. Note: (A) RT-qPCR was conducted to assess the changes in the expression of Igfbp3, Itgb1, Mapk3, and Mapk1 mRNA in ADSCs cells after silencing or overexpressing Igfbp3; (B) WB analysis was performed to detect the changes in the protein expression of IGFBP3, ITGB1, ERK1, ERK2, p-ERK1, and p-ERK2 in ADSCs cells after silencing or overexpressing Igfbp3; (C) Co-IP experiment was conducted to investigate the interaction between IGFBP3 and ITGB1 proteins; (D) Schematic representation of the experiment silencing Itgb1 on the basis of overexpressing Igfbp3; (E) Inverted microscopy was used to observe the effects of Itgb1 silencing on the morphology of ADSCs cells (scale bar = 25 μm); (F) RT-qPCR was performed to assess the changes in Krt18 and Tjp1 mRNA expression in ADSCs cells among different intervention groups; (G) WB analysis was carried out to determine the expression changes of CK18 and ZO- 1 proteins in ADSCs cells among different intervention groups; (H) Immunofluorescence staining was used to detect the expression changes of CK18 and ZO- 1 proteins in ADSCs cells among different intervention groups (scale bar = 25 μm); (I) EDU experiment was conducted to evaluate the proliferation ability of ADSCs cells, where EDU-positive cells are shown in red, indicating cells in the proliferative phase, while blue represents DAPI-stained cell nuclei (scale bar = 50 μm); (J) Scratch assay was performed to assess the migration of ADSCs cells (scale bar = 50 μm); (K) Transwell experiment was carried out to evaluate the migration ability of ADSCs cells (scale bar = 50 μm); (L) RT-qPCR was conducted to determine the expression changes of Mapk3 and Mapk1 mRNA; (M) WB analysis was performed to detect the changes in the protein expression of ERK1, ERK2, p-ERK1, and p-ERK2. Quantitative data in the figures are presented as Mean ± SD, and each experiment was repeated three times per group. Statistical significance is denoted as * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001 when comparing between groups

Fig 4: Analysis of key genes in STI based on single-cell and transcriptome sequencing. Note: (A) Volcano plot showing DEGs between ADSCs cells from control and STI samples, with red dots left of the dashed line indicating genes highly expressed in the STI sample, and dots on the right showing genes with low expression in the STI sample; (B) Volcano plots of DEGs from 3 control and 3 STI samples in RNA-seq, with red triangles representing upregulated genes, green triangles denoting downregulated genes, and black circles indicating non-differential genes; (C) Bubble chart (left) and circular chart (right) of GO enrichment analysis of DEGs in RNA-seq, where the color of the circles represents the significance of enrichment from blue to red, and the size of the circles indicates the number of enriched genes; (D) Bubble chart (left) and circular chart (right) of KEGG enrichment analysis of DEGs in RNA-seq, with the color of circles indicating the significance of enrichment from blue to red, and the size representing the number of enriched genes; (E) Venn diagram of the intersection between DEGs in RNA-seq, scRNA-seq ADSCs, and Genecards dataset, leading to the identification of the key gene—Igfbp3; (F) scRNA-seq analysis of expression levels of Igfbp1 gene in control and STI samples, with blue indicating upregulation; (G) scRNA-seq analysis of expression of Igfbp3 gene in ADSCs cells from control and STI samples

Fig 5: IGPA peptide hydrogel scaffold improves STI through the ITGB1/ERK signaling pathway. Note: (A) Schematic illustration of the animal experimental procedure, with 6 mice per group; (B) Therapeutic effects of various gel scaffolds on epidermal healing in mice; (C) Histological staining of mouse soft tissue with H&E, where single-headed arrows indicate non-epithelialized areas and double-headed arrows indicate the edge of granules (scale bar = 500/100 μm); (D) Results of Masson's staining in mouse soft tissue, showing interstitial collagen proteins in bluish-green; (E) TUNEL staining results of mouse soft tissue (scale bar = 100 μm); (F) Immunohistochemical staining depicting the expression changes of PCNA protein in mouse soft tissue among different experimental groups (scale bar = 100 μm); (G) Immunofluorescence staining displaying the expression changes of Ki67 protein in mouse soft tissue among different experimental groups (scale bar = 100 μm); (H) Immunofluorescence staining revealing the expression changes of CD34 and α-SMA proteins in mouse soft tissue among different experimental groups (scale bar = 100 μm); (I) Immunohistochemical staining showing the expression changes of IGFBP3, ITGB1, CK18, and ZO- 1 proteins in mouse soft tissue among different experimental groups (scale bar = 100 μm); (J) WB analysis of IGFBP3, ITGB1, ERK1, ERK2, p-ERK1, and p-ERK2 protein expressions in mouse soft tissue; (K) RT-qPCR assessment of Igfbp3, Itgb1, Mapk3, and Mapk1 mRNA expressions in mouse soft tissue. Quantitative data in the figures are expressed as Mean ± SD, with 6 mice per group. * indicates statistical significance between groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001