Fig. 8: Metrnl promotes FN degradation through catalyzing its K48-linked ubiquitination mediated by HECW2.

A, B Representative Western blot images and quantification analysis of FN protein levels in LX-2 cells treated with MG132 (50 μM) (A) or CQ (40 μM) (B) for 24 h (n = 3 blots). C Representative Western blot images and quantification analysis of FN in OE-Metrnl mediated LX-2 cells treated with CHX (50 μM) for indicated time under TGF-β (2 ng/mL) incubation (n = 2–3 blots). Co-IP assays of the ubiquitination of FN in OE-Metrnl overexpressing (D) or sh-Metrnl knockdown (E) LX-2 cells treated with TGF-β (2 ng/mL) for 48 h (n = 3 blots). F The mRNA expression of HECW2 in primary HSCs isolated from C57BL/6 mice and treated with TGF-β (1 and 2 ng/mL) for 48 h (n = 3 per group). G Representative Western blot images and quantification analysis of HECW2 and Metrnl in sh-Metrnl knockdown LX-2 cells treated with TGF-β (2 ng/mL) for 48 h (n = 3 blots). H The mRNA expression of HECW2 in primary HSCs isolated from Lrat-WT and Lrat-Met mice was analyzed after natural differentiation for 7 days (n = 3 per group). I Representative Western blot and quantification of HECW2 expression in the liver from mice injected with AAV-vector or AAV-Metrnl virus combined with CCl4 (n = 3 per group). J α-SMA protein expression and ubiquitinated FN level in LX-2 cells transfected with OE-Metrnl overexpression or sh-HECW2 knockdown virus, or both, under TGF-β (2 ng/mL) stimulation for 48 h (n = 2 blots). K Ubiquitination of FN in LX-2 cells co-transfected with the indicated HA-Ub plasmids and OE-Metrnl overexpression virus, after TGF-β (2 ng/mL) treatment (n = 3 blots). L ELISA-quantified FN levels in CM from LX-2 cells transfected with OE-Metrnl overexpression or sh-HECW2 knockdown virus, or both, under TGF-β (2 ng/mL) stimulation for 48 h (n = 3 per group). All data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.