Screening and identification of MSX1’s downstream effector genes associated with promoting HBx degradation.

(A) Huh7 cells were transfected with pMSX1-Flag and HA-tagged HBx-expressing plasmid (pHBx-HA) or its control plasmid at a transfection ratio of 1:1. At 60 h post transfection, cells were treated with MG132 for additional 12 h. Interaction between MSX1 and HBx was analyzed using Co-IP assay. (B) Schematic representation of pMSX1-Flag and its serial deletion mutants. Deleted region within MSX1 is represented by dashed lines, and numbers denote amino acid positions. (C) Western blot was performed to analyze the effects of MSX1 and its deletion mutants on HBx expression in Huh7 cells. (D) Huh7 cells were transfected with pMSX1-Flag or pCtrl in triplicates and then subjected to transcriptome sequencing analysis. Volcano plot (left) and heat map (right) were presented with top 10 upregulated DEGs indicated. (E) RT-qrtPCR assay was performed on pMSX-Flag- or pCtrl-transfected Huh7 cells to determine the mRNA expression levels of indicated genes. (F) 5 of the 7 confirmed MSX1-upregulated genes belong to heat shock protein (HSP) families. (G) Huh7 cells were transfected with pHBx-Flag and an increasing amount of Flag-tagged MSX1, HSPA6, DNAJA4, DNAJB1, HSPA1B, VGF, CRYAB, or RRAD overexpression plasmid at a transection ratio of 1:0, 1:0.5, 1:1, 1:2. Western blot was utilized to analyze the effects of exogenously expressed genes on HBx. (C and H) HBx protein levels were quantified using densitometry scanning and signal levels in control group were normalized as 1. Group means and SEMs of normalized data were presented and significances calculated using unpaired two-tailed t test. **, P < 0.01; ***, P < 0.001.