Fig 1: FAM46C overexpression resulted in G2/M phase arrest (A) and a significant increase of cell apoptosis (B) as determined by flow cytometry analyses at 48 h after viral infection. ***P < 0.001 as compared with Vector virus-infected cells. (C) Protein levels of FAM46C and cell growth-related factors (PCNA, Cyclin B1 and CDK1) were evaluated by Western blotting. (D) Protein levels of FAM46C and apoptotic factors (Bcl-2, Bax and XIAP) were assessed. (E) The levels of Ras, p-MEK1/2, MEK, p-ERK1/2 and ERK1/2 were evaluated. All experiments shown were performed independently at least three times. **P < 0.01 and ***P < 0.001 as compared with Vector virus-infected cells.
Fig 2: FAM46C is critical for the anti-cell growth effects of NCTD on HCC cells. (A) MHCC-97H cells were transfected with siNC, siFAM46C-1 or siFAM46C-2. At 48 h after transfection, FAM46C protein expression was detected by Western blotting. (B,C,D) MHCC-97H cells were divided into 6 groups: siNC+DMSO, siNC+NCTD (10 μg/mL), siFAM46C-1+DMSO, siFAM46C-1+NCTD (10 μg/mL), siFAM46C-2+DMSO and siFAM46C-2+NCTD (10 μg/mL). Cell proliferation (B) was evaluated by CCK-8 assay at indicated time points. At 48 h after treatment, cell percentages in G2/M phase (C) and cell apoptosis (D) were detected by flow cytometric analysis. (E) Western blot analysis of ERK phosphorylation at 48 h after treatment. All experiments shown were performed independently at least three times. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with siNC+DMSO; ## P < 0.01, ### P < 0.001 as compared with siNC+NCTD; + P < 0.001, ++ P < 0.05,+++ P < 0.001 as compared with siFAM46C-1+NCTD; $ P < 0.001, $$ P < 0.05, $$$ P < 0.001 as compared with siFAM46C-2+NCTD.
Fig 3: Effects of NCTD on FAM46C expression. (A) RNA-sequencing data showed that NCTD significantly induced FAM46C mRNA expression. (B) SMCC-7721 and MHCC-97H cells were treated with DMSO or NCTD (10 and 20 µg/mL) for 48 h. The protein level of FAM46C was significantly induced by NCTD treatment. (C) FAM46C was down-regulated in HCC tissues (n = 191) compared to the normal tissues (n = 50) in TCGA LIHC dataset. (D) GSEA revealed that the cell apoptosis pathway was closely related with NCTD treatment. Black bars indicated individual genes in rank order. NES, normalized enrichment score. (E) GSEA was performed using TCGA LIHC dataset. The cell apoptosis pathway was strongly associated with FAM46C expression.
Fig 4: FAM46C overexpression inhibited HCC cell proliferation. (A) Expression of FAM46C in 6 HCC cell lines as determined by Western blotting. (B) Ectopic expression of FAM46C in SMCC-7721 and SK-Hep-1 cells was examined by Western blotting. (C) Ectopic expression of FAM46C significantly decreased cell proliferation as determined by CCK-8 assay. All experiments shown were performed independently at least three times. ***P < 0.001 as compared with Vector virus-infected cells.
Fig 5: Pathological changes of liver in 4 groups mice (n = 8 per group). Light microscopy of HE staining, and IHC staining of FAM46C and Ki-67 (200x magnification) in Control group, DEN group, DEN+NCTD group and DEN+FAM46C group.
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