Figure 5. Col XVII-mediated suspension survival depends on laminin 5 and FAK activation.

(a,b) Bulk cancer cells (Bulk) and spheroid culture-enriched TICs (SPH) were cultured in spheroid condition for 10 h, followed by (a) western blot assay and (b) immunofluorescence followed by examination with a confocal microscope. (c) Spheroid culture of cells overexpressed with control plasmids (CTR) or S727A point-mutated STAT3 (SA) were assayed for protein levels. (d) Bulk cancer cells overexpressed with control plasmids (CTR) or S727E point-mutated STAT3 (SE) were assayed for protein levels. (e,f) Spheroid cultures of cells overexpressed with control shRNAs (CTR) or different Col17a1 shRNAs, si(1) and si(2), were assayed for (e) mRNA and protein levels, and (f) immunostaining (CCS) followed by examination with a confocal microscope. (g) Top, western blot analysis for spheroid culture-enriched TICs expressing control shRNAs (CTR) or Col17a1 shRNAs, si(1) or si(2), treated with 50 μg ml⁻¹ cycloheximide in the presence or absence of 10 μM MG132 for 6, 12, 24 and 36 h. Bottom, graphical representation of the relative band intensities of laminin 5 protein. The percentage of initial intensities of laminin 5 protein in cell extracts normalized with respect to β-tubulin amounts was plotted against time. (h) Bulk cancer cells and spheroid culture-enriched TICs were cultured under spheroid condition for 10 h, followed by western blot assay. (i) Western blot analysis for spheroid culture-enriched TICs expressing control shRNAs (CTR) or Col17a1 shRNAs, si(1) or si(2). (j) TICs (SPH) overexpressed with control plasmids (CTR), S727A point-mutated STAT3 (SA) were assayed for protein levels. (k) Bulk cells overexpressed with control plasmids (CTR), S727E point-mutated STAT3 (SE) were assayed for protein levels. Scale bar, 20 μm.