Fig. 4

SHH signalling activation was responsible for stemness maintenance in PARD3-overexpressing CD133⁺ TICs. (A) CD133⁺ and CD133⁻ cells were sorted from PARD3-overexpressing Hepa1-6 cells and subjected to RNA-seq analysis. (B) PARD3 activation induced perturbation of gene expression in CD133⁺ cells compared with CD133⁻ cells. (C) Significant signalling pathways activated in CD133⁺ PARD3-overexpressing cells, as revealed by functional enrichment analysis. D&E. SHH signalling activity in different subclusters of cancer cells, as determined by single-cell sequencing. F. GSEA results showing that SHH signalling was activated in CD133⁺ PARD3-overexpressing cells. G. Relative expression of Gli1, PTCH1 and SHH in CD133⁺ and CD133⁻ PARD3-overexpressing cells. H. Gli1 protein expression was increased in CD133⁺ PARD3-overexpressing cells. I. Quantification of the CD133⁺ population within PARD3-overexpressing and wild-type Hepa1-6 cells treated with GANT58 (n = 3). J. In vitro limiting dilution assay showing the tumour sphere formation ability of PARD3 OE/wild-type CD133⁺ cells with or without GANT58 treatment. K. The interaction between aPKC and PARD3 was increased upon PARD3 overexpression, as determined by co-IP. L. The phosphorylation of aPKC was increased upon PARD3 overexpression. M. Gli1 binding to the Sox2 promoter was attenuated by ZIP. N. Quantification of the CD133⁺ population within PARD3-overexpressing Hepa1-6 cells with or without inhibition of aPKC signalling by ZIP. O. In vitro tumour sphere formation ability of PARD3 OE/wild-type CD133⁺ cells treated with ZIP, as determined by an in vitro limiting dilution assay. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not statistically significant