Figure 5

TAp73β induced the expression of cholangiocyte promoting genes (JAG1, NOTCH1, ZO-1) while repressing hepatocyte fate markers (CYP3A4, AFP, HNF4α, ALB) without affecting progenitor/stem marker (EpCAM, LGR5) expression. Hep3BTAp73β cells were treated with Doxycycline (1 µg/mL) to induce TAp73β expression for 72 h and cell lysates and RNA were obtained, then western blot and RT-PCR were performed. (A) Western blotting of cholangiocyte markers JAG1, NOTCH1, CFTR, HNF1β, ZO-1 (n = 2). (B) EpCAM progenitor/stem cell marker and hepatocyte markers, CYP3A4, AFP, HNF4α and ALB expressions were examined by RT-PCR. GAPDH was used as a housekeeping gene. Column graphs show the fold change and calculated by using the 2−∆∆Ct method. * p < 0.05, ** p < 0.001 and *** p < 0.0001. (C) Western blotting of progenitor/stem markers EpCAM, and LGR5 (n = 2). (D) Western blotting of hepatocyte markers ALB and HNF4α (n = 2). (E) Huh7 cells were transiently transfected with TAp73β and, HNF4α and ALB protein levels were studied by western blotting. (F) Doxycyline-treated and untreated Hep3BTAp73β cells were transfected with p73siRNA and scrambled siRNA, respectively. Western blot analysis for p73, HNF4α and ALB expression in control siRNA and p73 siRNA transfected Hep3BTAp73β cells with or without doxycycline (1 µg/mL) induction (n = 2). * Non-specific signals.