Fig. 7: p-EMT signalling in HCC1143 cells via transient ERK/CDK4/6 activation leading to stemness and self-renewal.

a CD104 and CD44 FACS profiles of HCC1143 cells. b TOP/FOP Flash in HCC1143 E/M and M cells, untreated (CM) or treated with Wnt3a or Wnt3a + ICG001; mean ± SEM, unpaired t test, ***P < 0.001. c IB of TCF1, CD104, E-cad, FOXC2, ALDH1, NOTCH1 in HCC1143 E/M and M cells. d E/M and M cells from HCC1143 were co-immunostained for CD104 (TRITC) and ALDH1 (FITC). e IB of p63, p73 in E/M and M cells. TAp73 and ΔNp73 mRNA fold change in Wnt3a-treated (10 min) vs untreated E/M cells; *P < 0.05; unpaired t test. f IB p-ERK, ERK, p-EGFR (Y1173), EGFR in E/M cells stimulated with 100 ng/ml of Wnt3a or EGF in 0.2%FBS/RPMI at the indicated time points. g Percent of PCNA + E/M cells untreated or treated with 100 ng/ml Wnt3a in 0.2%FBS/RPMI for 10 min, 30 min, or 1 h; mean ± SEM, one-way ANOVA, Dunnett’s multiple comparisons test; ns; **P < 0.01; ***P < 0.001. h, i HCC1143 E/M cells were untreated or treated with vehicle, 10 μM palbociclib (CDK4/6i), 1 μM PD901 (MEKi), or combination for 24 h, followed by 100 ng/ml Wnt3a in 0.2% FBS/RPMI for 10 min (except lane1). Cells were IB for h p-ERK, ERK, p-EGFR(Y1173), EGFR, p-Rb (Ser780), or i CD104, FOXC2, FOXM1. j BrdU staining (green) combined with DAPI (blue) of HCC1143 E/M cells that were treated with wnt3a for 0.5 h −/+ indicated drugs; 20 μm. The percent of BrdU+ cells (mean ± SEM); One-way ANOVA, Dunnett’s multiple comparisons test; ****P < 0.0001. k Left top: timeline of the drug treatment; HCC1143 cells were seeded and treated with vehicle, 10 μM CDK4/6i, 1 μM MEKi, or combination at Day 0; images were taken at Day 5. Left bottom: organoid images of HCC1143 cells treated as above for 5 days in organoid media; 100 μm. Middle: Organoid-forming efficiency is shown as mean ± SEM; one-way ANOVA, Dunnett’s multiple comparisons test (*P < 0.05; ***P < 0.001; ****P < 0.0001). Right: Cleaved-PARP immunoblots from organoid cultures that were treated with CDK4/6i, MEKi, or both (combo) are shown. l Left top: timeline of the drug treatment. HCC1143 cells were seeded at Day 0 and allowed to form organoids for 3 days prior to treatment with the above drugs, which were replenished on Day 5. Left bottom: organoid images on Day 7; 100 μm. Middle: Results are shown as organoid diameter (μm) in each group; mean ± SEM, one-way ANOVA, Dunnett’s multiple comparisons test; ****P < 0.0001. Right: Cleaved-PARP immunoblots from organoids that were treated with CDK4/6i, MEKi, or both (combo) are shown.