Fig. 3.

Regulatory role of Orm1 on the proliferation of mouse regenerating HPCs and human hepatic cells. (a) Schematic overview of loss-of-function experimental procedures. Orm1 expression at transcript levels in (b) whole-liver tissue and (c) serum protein levels. (d) IHC staining and (e) gene expression of cell proliferation marker Ki-67 in whole-liver tissue at 48 h post-PH. The average percentage of Ki-67 positive cells presented in (d) was quantified from five randomly selected areas in three slides from each mouse (n = 2–4). Scale bar, 50 μm. siCtl, control siRNA; siOrm1, Orm1 siRNA. The quantitative data were presented as the mean plus standard error (n = 4). (f) Data mining of Orm1 expression in human tissues obtained from the freely available FANTOM5 database. Reference RNA, commercially obtained universal human reference total RNA. Others, other human hepatic non-parenchymal cells, primary cells, tissues and cancer cell lines. (g) The gene expression of Orm1 was examined in human liver cancer cell lines FLC4, FLC7, and HepG2, human hepatic stellate cell line LX2, and immortalized human liver endothelial cell line M1 using real-time RT-PCR. Gene expression was normalized to that of GAPDH. Effect of Orm1 knockdown on (h) gene expression of Orm1 and cyclin D1 and (i) cell proliferation of human hepatic cell line FLC4 in the in the absence [ORM1(−)] and presence [ORM1(+)] of 25 ng/mL recombinant human Orm1. The quantitative data were presented as the mean plus the standard deviation of three replicates. *P < 0.05 assessed using two-tailed Student's t-test, Mann-Whitney U test or ANOVA with post-hoc Tukey HSD Calculator for multiple comparison.