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Fig. 4 | Clinical Epigenetics

Fig. 4

From: Epigenetic silencing of TMEM176A activates ERK signaling in human hepatocellular carcinoma

Fig. 4

TMEM176A inhibits HCC cells invasion, migration, and the ERK signaling pathway. a Growth curves represent cell viability evaluated by MTT assay in the control group, siTMEM176A group, and siTMEM176A plus SCH772984 treatment group. siTMEM176A: siRNA knockdown of TMEM176A. ***P < 0.001. b The migration assays show migration cells before and after restoration of TMEM176A expression in SNU449 and LM3 cells as well as in the control group, siTMEM176A group, and siTMEM176A plus SCH772984 treatment group. The number of cells migrating to the lower chamber is presented by bar diagram. Each experiment was repeated for three times. **P < 0.01, ***P < 0.001. c The invasion assays show invasive cells before and after restoration of TMEM176A expression in SNU449 and LM3 cells as well as in the control group, siTMEM176A knockdown group, and siTMEM176A plus SCH772984 treatment group. The invasion cell number is presented by bar diagram. Each experiment was repeated three times. **P < 0.01, ***P < 0.001. d The expression levels of TMEM176A, MMP2, and MMP9 were detected by Western blot. e LM3 cells were transfected with pcDNA3.1-TMEM176A-Flag or Vector-Flag. Immunoprecipitation was performed using an anti-Flag antibody or Rabbit IgG antibody. The bands specific to TMEM176A, as pointed out by the numbers (bands 1–3), were subjected to mass spectrometry. The TMEM176A (band 2) is indicated by the red arrow. LM3 cells were transfected with pcDNA3.1-TMEM176A-Flag or Vector-Flag. Immunoblots showing SAR1A in the anti-Flag-TMEM176A immunoprecipitates. Input: cell lysis of TMEM176A re-expressed LM3 cells; IgG: negative control. f Western blots show the levels of TMEM176A, SAR1A, ERK1/2, and p-ERK1/2 in SNU449, LM3, SNU387, and SNU475 cells. β-actin: internal control. -: no serum stimulation. +: serum stimulation. g Western blots show the levels of TMEM176A, SAR1A, ERK1/2, and p-ERK1/2 in the control group and siSAR1A knockdown group. SiSAR1ANC: SiRNA for SAR1A negative control; SiSAR1A1: SiRNA for SAR1A set1. β-actin: internal control. -: no serum stimulation. +: serum stimulation

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