Figure 2. SASP of senescent HSCs induces hepatocyte proliferation.

(A) Primary hepatocytes isolated from WT male livers were cocultured with HSCs, senescent HSCs (Sen-HSC), and Sen-HSC with anti–IL-6 antibody (5 μg/mL) and/or SB225002 (SB; 1 μM) as indicated for 24 hours, then stained with anti-Ki67 (red). Ki67⁺ cells were quantified by counting (n = 6). (B) Immunoblots of protein extracts from hepatocytes cocultured with HSCs or Sen-HSCs were probed with antibodies against phosphorylated (p-) YAP (Y357), YAP, p-STAT3 (Y705), STAT3, p-AKT (S473), AKT1, p-ERK1/2 (T202/Y204), ERK1/2, and β-actin. (C) Primary hepatocytes cocultured with HSCs, Sen-HSC, and Sen-HSC with Super-TDU (TDU; 50 nM), WP1066 (WP; 5 μM), and SCH772984 (SCH; 1 μM) as indicated for 24 hours and were stained with anti-Ki67 antibodies. Ki67⁺ cells were counted (n > 5). (D) Immunoblots of protein extracts from hepatocytes treated with vehicle (PBS) or IL-6 (10 ng/mL) for 24 hours were probed with indicated antibodies. (E) Hepatocytes treated with vehicle, IL-6, and IL-6 with SCH, TDU, and WP as indicated for 24 hours were stained with Ki67 antibodies and Ki67⁺ cells counted (n = 5). (F) Immunoblots of protein extracts from hepatocytes treated with vehicle or CXCL2 (10 ng/mL) for 24 hours were probed with indicated antibodies and antibodies against p-p38 (T180/Y182) and p38. (G) Hepatocytes were treated with vehicle, CXCL2, MK2206 (MK; 1 μM), SCH, or in combination as indicated for 24 hours and were stained with Ki67 antibodies. Ki67⁺ cells were counted (n = 5). (H) Immunoblots of protein extracts from vehicle-, IL-6-, CXCL2-, or IL-6+CXCL2-treated hepatocytes for 24 hours were probed with indicated antibodies. (I) Hepatocytes treated with PBS, IL-6, CXCL2, IL-6+CXCL2, or IL-6+CXCL2 together with SCH, TDU, and WP alone and in combination for 24 hours were stained with anti-Ki67 antibodies; Ki67⁺ cells were counted (n = 4). Scale bar: 50 μm. Data expressed as mean ± SD. *P < 0.033, **P < 0.002, ***P < 0.001, Student’s t test.