Figure 5: PBX3 is a critical determinant of α2δ1⁺ HCC TICs.

(a,b) The expression of PBX3 in Hep-11 and Hep-12 cells was detected at both mRNA and protein levels using qRT–PCR (a) and western blot (b), respectively. (c,d) The expression of PBX3 was compared between α2δ1⁺ and α2δ1⁻ fractions from indicated sources at both mRNA (c) and protein levels (d). (e,f) qRT–PCR (e) and western blot (f) results showing the knockdown effects of PBX3 shRNAs on the expression of PBX3 in Hep-12 cells at both mRNA and protein levels. β-actin serves as an internal control. (g) Sorted α2δ1⁺ cells from indicated sources were infected with lentiviruses harbouring PBX3 shRNAs, followed by plating 100 cells per well into 96-well plates to assay their sphere formation potential (n=6). Data are the average±s.d. of three independent experiments. *Student’s t-test. (h) α2δ1⁻cells from indicated sources were infected with PBX3 lentivirus and assayed for PBX3 by western blot. (i) FACS-sorted α2δ1⁻ cells from indicated sources were infected with PBX3 lentivirus and assayed for their sphere formation ability (n=6). Data are the average±s.d. of three independent experiments. *Student’s t-test. (j) HE staining showing the histology of the tumours formed by α2δ1⁻cells from indicated sources after PBX3 lentivirus infection. Scale bar, 100 μm. (k) The percentage of α2δ1-positive cells were measured using flow cytometry in PBX3-lentivirus-infected Huh7 α2δ1⁻ cells compared. with vector alone control. The data are representative of three experiments. (l) The expression of stem cell-related genes in α2δ1⁻ cells overexpressing PBX3 was compared with vector alone controls using qRT–PCR. (m) The expression of PBX3 mRNA in HCC tissues was compared with adjacent normal tissues using qRT–PCR. *The numbers next to the horizontal lines are the median values (2^−ΔCt) for each group. (n,o) Kaplan–Meier curves for the DFS and OS of patients were compared between groups with high and low levels of PBX3 mRNA in HCC tissues, which were divided according to the cutoff of 2.51, the median value of PBX3 relative to GAPDH mRNA.