Fig. 3

The Asp-125 residue of SOX9 is required for SOX9-induced YAP nuclear translocation. a Map of the HMG domain of the SOX9 protein (upper). Huh-7 cells were transfected with different truncations of the SOX9-HMG domain (Δ94–126, Δ120–144, Δ142–176, Δ176–210, Δ94–210). FLAG-IP was performed with the cell extracts, followed by immunoblotting analysis. b MST assay to determine the binding affinity between the YAP protein and nNLS-containing fragment of SOX9 (aa 94–126). c Nuclear and cytoplasmic fractions were separated and followed by immunoblot assay to access the expression level of YAP in Huh-7 cells transfected with FLAG-SOX9 or its truncations (Δ94–126, Δ176–210). d Nuclear and cytoplasmic fractions were separated and followed by immunoblot assay to access the expression level of YAP in Huh-7 cells transfected with V5-YAP upon CaM inhibitor CDZ treatment for 24 h (right); The 8×GTIIC reporter activity in Huh-7 cells infected with lenti-YAP, lenti-S127A, or control lentivirus, followed by treatment with CDZ or DMSO for 24 h (right). Data are represented as mean ± SD, ***P < 0.001. e Schematic showing the nNLS mutant sequence (nNLS-mu, upper); Immunoblot assay was conducted to evaluate the expression level of YAP in the cytoplasmic and nuclear fractions of Huh-7 cells transfected with FLAG-SOX9 or FLAG-nNLS-mu (lower). f The 8×GTIIC reporter activity in Huh-7 cells transfected with SOX9, nNLS-mu, or control plasmid for 24 h (left). Data are represented as mean ± SD, ***P < 0.001; qPCR analysis of CTGF, CYR61, and AREG expression levels in Huh-7 cells transfected with SOX9, nNLS-mu or control plasmid for 24 h or 48 h (right). Data are represented as mean ± SD, *P < 0.05, **P < 0.01. g Putative binding model of residues 121–135 of YAP (colored in teal) and the SOX9-HMG domain (PDB ID 4EUW, colored in wheat). The key residues forming hydrogen bonds are marked and represented as sticks. Hydrogen bonds are depicted as yellow dashed lines. The predicted structure of YAP (121–135) was downloaded from the AlphaFold Protein Structure Database (AF-P46937-F1, https://alphafold.ebi.ac.uk/entry/P46937). More details of generating the docking model figure are described in Methods section. h Huh-7 cells were co-transfected with the V5-YAP plasmid and FLAG-tagged SOX9 or SOX9 with mutations (K106A, V114A, or D125A). Immunoprecipitation was performed on cell extracts using anti-FLAG beads (left) or anti-V5 beads (right), followed by immunoblot analysis. i Immunoblot analysis of the cytoplasmic and nuclear fractions of YAP in SOX9^KO cells with the overexpression of SOX9 and SOX9-D125A for 24 h (left); The 8×GTIIC reporter activity of SOX9^KO cells treated as described (right). Data are represented as mean ± SD, **P < 0.01, ***P < 0.001. j qPCR analysis of CYR61 and AREG expression in Huh-7 cells with DOX-inducible EGFP-YAP and mCherry fusion proteins Data are represented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. k The proliferation of Huh-7 cells with DOX-inducible co-expression of EGFP-YAP and mCherry fusion proteins. Data are represented as mean ± SD, ***P < 0.001