Fig 2: Quantitative analyses and transcript levels of genes expressing VF epithelial structural proteins after 0.5% ECVE exposure during VF epithelial regeneration. (A) Total VF epithelial area in µm2 and P63+ basal cell density calculated as the number of P63+ cells per 10,000 µm2 in control, PG/VG and PG/VG+FN groups at different stages of epithelial recovery. All calculations were performed as blinded experiments and calculated independently by two researchers. Data presented were obtained from three biological replicates (n=3), ten images were included in the analyses for each control and experimental condition, time point and biological replicate (360 images in total). Error bars with individual data points (average values for each condition) represent ±s.e.m. of the mean. (B) Transcript levels of the genes expressing VF epithelial structural proteins, i.e. CDH1 (E-cadherin), TP63, K14 and LAMA5 in control, PG/VG and PG/VG+FN groups at different stages of VF epithelial recovery. Error bars with individual data points represent ±s.e.m. of the mean obtained from three biological and technical replicates (n=3). (C) Quantitative assessment of cell proliferation. We calculated the percentage of Ki67+ cells in VF epithelia and VF fibroblasts, in the control group and experimental groups exposed to PG/VG and PG/VG+FN at different stages of VF epithelial recovery. Error bars with individual data points (average values for each condition) represent ±s.e.m. of the mean. One-way ANOVA with Tukey’s HSD test were used to confirm statistical significance; *P=0.05.

Fig 3: Tracheal-bronchial basal cell (TBBC) isolation and characterization.a Bronchoscopic view of normal and typical TO influenced airway. b TBBCs cloned from brushing samples (non-TO n = 7, TO n = 6 biological independent samples) (c) were confirmed by immunofluorescent (IF) staining for basal cell signature markers p63/CK5, proliferation indicator Ki67, and exclusively checked for differentiating markers SCGB1A1. Scale bar, 100 µm. d Self-renewal ability was quantitatively evaluated, shown as percentage of plated cells that formed typical clones on Day 4–5 post-seeding over continuous passaging (non-TO n = 9 and TO n = 6 biologically independent experiments). Results are represented as mean ± SD, and p values are indicated to show statistical significance based on two-tailed Student’s t-tests. e Non-TO and TO-derived TBBCs from P3 to 4 culture were subjected to RNA-Seq. Volcano plot visualizing all genes according to the sequencing data, p-values (-log10P) plotted against fold changes (log2FC). Vertical dotted lines: log2FC = ±1, horizontal dotted lines: -log10(P) = 1.30103. n = 6–7/group. f Comparative expression profile was displayed by heatmap. g GO enrichment analysis of TO-derived TBBCs-upregulated genes. h, i p63, CK14, NGFR, and CK8 expression were assessed on cultured clones derived from control and TO cases. Scale bar, 100 µm. j Repsentative bright-field imaging and IF staining of fibronectin paralleled with p63 on TBBCs cultured on a feeder-free platform. Scale bar, 200 µm. h, I, j n = 3 biologically independent experiments. Source data are provided as a Source Data file.

Fig 4: CK14 siRNA receded the collective invasion in an ex vivo 3D culture assay.(A), Left showed Western blotting analysis of CK14 silencing by CK14-siRNA1 and CK14-siRNA in tumor fragments. ß-actin loading control is also shown. Representative of three independent experiments was shown. Right showed transcription levels of CK14 silencing by CK14-siRNA1 and CK14-siRNA2, relative to GAPDH, determined by quantitative RT-PCR. Error bars represent the mean ± SD of triplicate experiments. (B) Representative micrographs of the border of the SACC tumor organoids in an ex vivo 3D culture assay between CK14-siRNA1 and the control was shown. (C) Left showed the number of tumor organoids in control and CK14 siRNA group (P = 0.002). Right showed the number of tumor organoids in control and CK14 siRNA group at different time of culture. The data showed that organoids transfected with siRNA-CK14 displayed a significantly lower capability of cells to migrate compared with control at the different times of the culture (P<0.0001).

Fig 5: Cells with induced Opn5 are epithelial cells. Fluorescent images of tdTomato from Opn5Cre; Ai14 corneas (all left images) and (A) K14, (B) K12, (C) Pax6, (D) K15, (E) CD45, and (F) TCR GL3 (center images). Merged images at right also include nuclei (DAPI, blue). Scale bars: 50 µm.