Figure 5. Senescent hepatocytes secrete Shh to promote liver fibrosis and inflammation.

(A) Huh7 cells were treated with Palbociclib to induce senescence; conditioned media (CM) was harvested and added to LX2 cells for 48h. (B) CM from senescent Huh7 cells upregulates expression of proliferative and fibrogenic markers in LX2 cells. (C) Primary hepatocytes were freshly isolated from mice treated with AAV8-TBG-eGFP-P16 (P16) or its control vector, AAV8-TBG-eGFP (GFP) and cultured for 24h to obtain CM. (D) CM from P16-overexpressing hepatocytes upregulated expression of proliferative and fibrogenic markers in LX2 cells. (E) GSEA of RNA seq data from P16 over-expressing hepatocytes reveals enrichment for the Hedgehog ligand biogenesis gene signature. (E) q-PCR and (F) immunostaining confirm that Shh is upregulated in the P16-overexpressing hepatocytes. (G) Shh protein expression in livers of WD-CCl₄ mice treated with vorapaxar or its vehicle. (H) CM harvested from GFP control or P16-overexpressing hepatocytes was added to the Shh-Light II cells and luciferase activity was evaluated. Results shown as MEAN +/− SEM (n=4 assays/group, *p<0.05 vs GFP or ctrl CM; n= 10 mice/group, *p<0.05 vs WDCCl4+Veh).