Fig. 5
PTDEs upregulate ROS in recipient cancer cells by enhancing SMAD3 signaling. a, b Huh-7 cells were cultured with TGF-β (5 ng/ml) with or without SIS3 (3 µM) pretreatment for 6 h. a The ROS level was measured. b The relative mRNA levels of indicated genes were measured using qRT-PCR. c Huh-7 cells were cultured on normal plates and fibronectin-coated plates and treated with TGF-β (5 ng/ml) for 6 h. Adhesive cells were counted. d Naive detached Huh-7 cells were incubated with PTDEs for 6 h, and the expression of SMAD3 and p-SMAD3 was detected. e Huh-7 cells were incubated with PTDEs in the presence or absence of SIS3 (3 µM) pretreatment. Adhesive cells were counted. f SMAD3 protein was not detectable in SMAD3−/− Huh-7 cells. g Wild-type and SMAD3−/− Huh-7 cells were cultured on a 96-well plate for 2 h, and adhesive cells were counted in presence or absence of TGF-β. h Wild-type and SMAD3−/− Huh-7 cells were incubated with PTDEs for 6 h, and the mRNA level of SMAD3 was measured by qRT-PCR. i Huh-7 cells were treated with cycloheximide (CHX; 50 µg/ml) and/or PTDEs for 6 h. SMAD3 expression was detected. j, k Huh-7 cells were cultured on a 96-well plate and treated with PTDEs from naive or SMAD3−/− Huh-7 cells for 6 h. j SMAD3 expression was detected. k The mRNA level of SMAD3 were measured by qRT-PCR. l The existence of SMAD3 mRNA and protein in PTDEs was evaluated by reverse transcription PCR and western blotting, respectively. m The expression of FLAG and SMAD3 was detected in FLAG-SMAD3 stably transfected Huh-7 cells by western blotting. n, o Huh-7 cells were incubated with PTDEs from naive or FLAG-SMAD3 Huh-7 cells for 6 h. n The existence of FLAG-SMAD3 mRNA product was evaluated by reverse transcription PCR. o The FLAG-SMAD3 fusion protein was detected by immunofluorescence. a–c, e, g, h, k Data are expressed as means ± SD of at least three independent experiments. Statistical analysis was performed using the unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001
