Increased Ccl5 and Cxcl10 expression directly in hepatocytes with Med23 deletion in vivo and in vitro.

(A) Strategy to delete macrophages in liver and administrate with acute CCl₄ for 24 hours. (B) Representative views of F4/80 staining in the liver sections of med23^f/f and med23^Δli mice after tail intravenous injection of control liposome and clodronate liposome and then CCl₄ administration for 24 hours. (C) qRT-PCR analysis of Med23 and F4/80 in whole-liver extracts of med23^f/f and med23^Δli mice (med23^f/f-Control, n = 4; med23^Δli-Control, n = 4; med23^Δli-Clodronate, n = 7). (D, E) qRT-PCR analysis of proinflammatory cytokines and chemokines in whole-liver extracts of med23^f/f and med23^Δli mice (med23^f/f-Control, n = 4; med23^Δli-Control, n = 4; med23^Δli-Clodronate, n = 7) (D) and AML12 cells with Med23 knockdown (n = 3 per group) (E). (F) The protein level of MED23 was analyzed by western blotting in AML12 cells after CRISPR/Cas9 mediated Med23 KO. GAPDH was used as a loading control. (G) qRT-PCR analysis of Ccl5 and Cxcl10 in Med23 KO AML12 cells (n = 3 per group). (H, I) qRT-PCR analysis of proinflammatory cytokines and chemokines in AML12 cells after transient transfection (n = 3 per group). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. For underlying data, see S1 Data file. AML12, alpha mouse liver 12; Ccl, C-C motif chemokine ligand; CCl₄, carbon tetrachloride; Cxcl, C-X-C motif chemokine ligand; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Il, interleukin; KO, knockout; MED23, Mediator complex subunit 23; med23^Δli, liver-specific knockout of Med23; med23^f/f, med23-floxed; qRT-PCR, quantitative real-time PCR; shCtrl, negative control vector containing scrambled shRNA; shMed23, shRNA against Med23; Tnfα, tumor necrosis factor alpha; WT, wild-type.