Fig 2: Basal Cell Heterogeneity in WO Skin(A) tSNE plot for NP basal cells from two WO replicates. WO-2 was not included in this analysis given its low basal cell number. The percentage of each subpopulation per total number (1,555) of basal cells was indicated.(B) Heatmap of top 10 markers for each subcluster. A complete list of marker genes is provided in Table S6.(C) Violin plots showing expression of key marker genes. p values are from two-sided Wilcoxon rank-sum tests.(D) Gene scoring analysis using the indicated molecular signatures. p values are from two-sided Wilcoxon rank-sum tests.(E) Quantitative analysis of immunofluorescence data for Snai2 protein. Percent Snai2+ cells in the basal layer of different regions in WO skin is shown here, and representative images are shown in Figure S6F.(F) Quantitative analysis of immunofluorescence data for Fos protein. Percent Fos+ cells in the basal layer of different regions in WO skin is shown here and representative images are shown in Figure S6G.(G) RNAScope data showing spatial distribution of Col17a1 and Trp63 transcripts and K14 protein in WO skin. DAPI stains the nuclei. Scale bar: 50 µm.(H) Enlarged images of the boxed areas in (G). Zones 1–3 correspond to regions that are distal from the wound (zone 1), hyperproliferative (zone 2), and in migrating front (zone 3). Scale bar: 50 µm.(I) RNAScope data showing spatial distribution of Cdkn1a and Krt14 transcripts and K14 protein in WO skin. DAPI stains the nuclei. Scale bar: 50 µm.(J) Enlarged images of the boxed areas in (I). See legends for (H) for zone definition. Scale bar: 50 µm.(K) Quantification of fluorescence intensity for Cdkn1a and Krt14 transcripts and K14 protein in each individual cell from a representative WO skin section. The curve represents a GPR, and a 95% confidence interval is shown as shaded area.

Fig 3: Effect of CC-885 and CC-90009 on COL17A1 PTC readthrough. (A, B) HaCaT keratinocytes derived from an unaffected individual were exposed to various concentrations of CC-885 (A) or CC-90009 (B) with or without 100 μg/ml gentamicin sulfate for 48 h and cell viability was measured using the MTT assay in triplicate samples (±SD). Concentrations of CC-885 and CC-90009 are in nanomolar. (C, D) JEB01 keratinocytes derived from a JEB patient with a COL17A1 nonsense mutation (R688X/R688X) were exposed to the indicated concentrations of CC-885 (C) or CC-90009 (D) for 72 h and eRF3a, eRF3b and eRF1 levels were determined using automated capillary electrophoresis western analysis. GAPDH was used as loading control. (E, F) JEB01 keratinocytes were exposed to the indicated concentrations of CC-885 (E) or CC-90009 (F) in the presence or absence of 100 or 300 μg/ml gentamicin sulfate for 72 h and Collagen XVII levels were measured by SDS-PAGE followed by traditional western blotting. β-actin was used as loading control.

Fig 4: High COL17A1 level is associated with low proliferation and extended tumor progression in patients.The COL17A1 mRNA expression in primary breast cancer and patient clinical data were retrieved from METABRIC study. (A) Boxplot analysis of COL17A1 level in low-proliferation and high-proliferation ER+ HER2- tumors categorized by 3-gene classifier subtype. (B–C) The COL17A1 level was categorized as low and high expression comparing to median expression. Tumor-free period (B) and overall survival (C) of patients bearing tumors with high and low level of COL17A1 were analyzed. Log-rank test was used for survival analysis. Mann-Whitney U-test was used for boxplot. ***P < 0.0001.

Fig 5: The p53-dependent induction of COL17A1 in human cells and mouse tissuesA. The qPCR and western blot results of COL17A1 in p53 wild-type breast cell lines, HBC4 and HBL-100, transfected with siRNA of either EGFP or p53, with or without doxorubicin (ADR) treatment. The cells were treated with ADR for 2 hours. ADR concentrations were 1 µg/mL for HBL-100 and 2 µg/mL for HBC4. Total RNA and protein were isolated at 48 hours after ADR treatment. siRNA of EGFP was used as a control. Relative mRNA expression of COL17A1 was normalized to ACTB; n = 3. Immunoblotting results of cell lysates with antibodies against COL17 (anti-Collagen XVII), p53, and p53-S15P (p53 phosphorylated at serine 15). Cell media were blotted with anti-Collagen XVII. ß-actin is shown as a loading control. B. Immunocytochemistry of HBL-100 (40× magnification) and HBC4 (20× magnification) cells treated as described in Figure 2A. The cells were stained with DAPI (blue) and anti-Collagen XVII (green). C. Relative mRNA expression of Col17a1 normalized to Gapdh in mouse mammary tissues of p53 knockout (p53-/-) or p53 wild-type (p53+/+) mice; with or without X-ray irradiation; n = 3. The mice were sacrificed at 24 hours after X-ray. D. Immunohistochemistry results of mammary tissues staining with anti-Collagen XVII; n = 3 mice per group. Images were obtained at 10× magnification. E. The inset of Figure D, p53+/+ mice with X-ray at 40× magnification. Scale bar, 20 µm. Two-tailed Student's t-test; *P < 0.05, **P < 0.01, ***P < 0.001; NS, P = 0.05.