Fig. 5

RGS2 suppresses the profibrotic effects in HLF-9 and NIH3T3. A, E HLF-9 (A) and NIH3T3 (E) were single cultured or co-cultured with hUC-MSCs 24 h after adding 4 ng/mL TGF-β1, treated with 8 mM PFD or an equal amount of PBS buffer for 2 h, and were then harvested for RT-PCR analysis of RGS2 and β-actin mRNA levels. Values are means ± SEM (n = 3). B, F RGS2 protein expression levels detected by Western blot in HLF-9 (B) and NIH3T3 (E) treated with hUC-MSCs and/or PFD. C, G RGS2 protein expression measured as the RGS2/β-actin protein ratio in HLF-9 (C) and NIH3T3 (G) treated with hUC-MSCs and/or PFD. Densities of bands were quantified using the ImageJ program. The results are presented as means ± SEM (n = 3). D, H HLF-9 and NIH3T3 cells were treated with TGF-β1 (4 ng/ml) firstly. Then, the Fluo-3 fluorescence of intracellular Ca²⁺ level in HLF-9 (D) and NIH3T3 (H) cells treated with PFD and/or hUC-MSCs were measured. The cells were excited at 488 nm and Ca²⁺-bound Fluo-3 emission was recorded at 530 nm. The Fluo-3 fluorescence from three separate exeachiments was plotted as means ± SEM. I, J HLF-9 (I) and NIH3T3 (J) were single cultured or co-cultured with hUC-MSCs, treated with 8 mM PFD for 24 h, and were then harvested for RT-PCR analysis of fibrosis marker mRNA levels. Values are means ± SEM (n = 3). K hUC-MSCs were treated with 4 ng/ml TGF-β1 and cultured for 24 h, treated with 8 mM PFD or an equal amount of PBS buffer for 24 h, and were then harvested for RT-PCR analysis of E-cad, FN, α-SMA mRNA levels. Values are means ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.01