Fig. 4: MARC2 regulates the expression of p27 via Hippo signaling pathway.

a Volcano plot for different expression genes between HCCLM3 control and HCCLM3-MARC2 cell lines. Red plots represented the upregulated genes and blue plots represented the downregulated genes. The significant threshold were |fold change| > 2 and p < 0.05. b Heatmap representing the mRNA expression of significant different expression genes between HCCLM3 control and HCCLM3-MARC2 cells. c Significant different expression genes were filtered and enriched in KEGG signaling pathway; the Top10 signaling pathways were listed and ranked by P value. d Representative the WB images for Hippo signaling pathway in indicated cell lines. Con: Control; MAR: MARC2 overexpression; sh: MARC2 shRNA. e Expression of YAP in the indicated cells was examined by immunofluorescence. Single and merged images were taken in the indicated cells to show immunofluorescence staining of YAP (red), accompanied by the cell nucleus (blue) stained by DAPI. f Expression of SKP2 was detected by western blot in indicated cell lines. g XMU-MP-1 was used to active the YAP. 3 μM XMU-MP-1 was added to MARC2-HCCLM3 cell lines, and the expression of pYAP, YAP, and SKP2 was detected by western blot in the upper panel. SiYAP was used in shMARC2-Huh7 cell lines to suppress the Hippo signaling pathway, and the expression of YAP and SKP2 was detected by western blot in the lower panel. h To further confirmed that MARC2 regulated p27 via YAP/SKP2 axis, 3 μM XMU-MP-1 and SKP2 overexpression plasmid were added to MARC2-HCCLM3 cell lines respectively, and the expression of pYAP, YAP, SKP2, and p27 was detected by western blot in the left panel. SiYAP and siSKP2 were added to shMARC2-Huh7 cell lines respectively, and the expression of YAP, SKP2, and p27 was detected by western blot in the right panel. i Representative images for cell cycle assays in indicated cell lines. j Statistical analysis of cell cycle. k Proliferation rates were measured by CCK8 assay in the indicated cell lines at 0 h and 48 h. l Representative images of liver xenograft derived from indicated cell lines in the left panel; the statistical analysis of bioluminescence signal in the right panel. i–l Huh7 cell line was used for the experiment. *p < 0.05; **p < 0.01; ***p < 0.001, all of which were considered statistically significant.