Fig. 2.

Knocking down of Lgr5 expression decreases Lgr5⁺ cells’ stem cell properties and increases liver fibrosis. a Lgr5 was knocked down by Ad-Lgr5 shRNA. Lgr5⁺ liver stem cells were isolated and infected with Ad-Lgr5 shRNA, or nonsense control shRNA Ad-Ctrl shRNA, western blot, and qRT-PCR were used to analyze the proteins level (left) and the mRNA level (right) of Lgr5. Actin was used as a loading control. b Lgr5⁺ liver stem cells failed to form an organoid when Lgr5 was downregulated by Ad-Lgr5 shRNA. Lgr5⁺ liver stem cells were isolated and infected with Ad-Lgr5 shRNA or Ad-Ctrl shRNA, and cultured in stem cells medium in Matrigel, and the number and sizes of the liver organoid were measured at day 14. b–e Ad-Lgr5 shRNA increased CCL4-induced liver fibrosis. As shown in Supplementary Fig. 10a, Lgr5-GFP mice infected with Ad-Lgr5 shRNA or Ad-Ctrl shRNA were administered with CCL4 (2 ml/kg i.p.) twice/week for 6 weeks, respectively. The representative histology of H&E and Sirius Red, and the quantification of positive-staining areas is measured by Image J software (c). Serum levels of ALT and AST were measured (d, e). For a, d, e, triplicates for each condition were analyzed. The results are shown as mean ± s.d. of three independent experiments. *p < 0.05. For b, the results are shown as mean ± s.d. of three independent experiments. **p < 0.01. For c, scale bars, 200 μm, the results are shown as mean ± s.d. of five independent sections taken randomly per mice and a total of 50 tissue specimens in each group (n = 10 mice), *p < 0.05