Figure 4. Col XVII upregulated by S727-phosphorylated STAT3 mediates the suspension-survival ability of enriched TICs.

(a) Top, western blot for spheroid culture of days 0, 5 and 15. Bottom, the schematic representation of CCS cell preparation for investigating gene expression profiles and subsequent data analysis. (b) Upregulated genes in CCS-derived TICs were distributed in cancer and cell death categories through Gene Ontology (GO) and pathway analysis. (c–d) Bulk cancer cells (Bulk) and spheroid culture-enriched TICs (SPH) were cultured under spheroid condition for 10 h, followed by (c) western blot, and (d) immunostaining followed by examination with a confocal microscope (CCS). (e) Cells overexpressed with control plasmids (CTR) or S727A point-mutated STAT3 (SA) were assayed for mRNA and protein levels. (f) Cells overexpressed with control plasmids (CTR) or S727E point-mutated STAT3 (SE) were assayed for mRNA and protein levels. (g) Spheroid cultures or (h) bulk cancer cells expressing S727E point-mutated STAT3 were overexpressed with control shRNAs (CTR) or different Col17a1 shRNAs, si(1) and si(2). Cells were cultured under spheroid condition for 24 h, followed by TUNEL assay. Top, western blot of Col17a1 knockdown efficiency. Bottom, quantification of TUNEL-positive cells. (i) Genomic organization of the region flanking the promoter region of Col17a1 (top panel) and the schematic representation of the pEZX-PG04-Col17a1 reporter construct. Transcription start site, TSS. (j) Left, ChIP analysis of bulk cancer cells expressing S727E point-mutated STAT3. The chromatin was incubated either without antibodies, with an anti-FLAG antibody or with an isotype IgG antibody. Fragments of the eighth binding site in the Col17a1 promoter (C8) were amplified by PCR. Right, quantification of DNA binding with quantitative RT–PCR. Input, 4% of total input lysate. (k) Spheroid culture-enriched TICs (SPH) expressing S727A point-mutated STAT3 were employed to analyse the role of S727 phosphorylation in activating the Col17a1 promoter. (l) Mutational analysis of the eighth binding site in the Col17a1 promoter. Reporter constructs containing WT and box 8 mutations were employed to analyse the importance of this site in mediating activation by STAT3. The results are mean±s.d. of three independent experiments. Asterisks indicate significant differences (*P<0.05, **P<0.01 versus CTR) determined by Student's t-test and one-way ANOVA. Scale bar, 20 μm.