Inhibition of Snail Family Transcriptional Repressor 2 (SNAI2) Enhances Multidrug Resistance of Hepatocellular Carcinoma Cells
Fig 3
SNAI2 knockdown promotes the efflux of Hoechst 33342 and increases ATP binding cassette (ABC) transporter and CSC gene expression of MHCCLM3 cells.
(A) Q-PCR was applied to detect mRNA levels of indicated genes in MHCCLM3 cells expressing shSNAI2/shControl shRNAs. Relative expression of indicated genes was normalized with GAPDH mRNA. (B-D) FACS was utilized to sorting cell populations of MHCCLM3 cells expressing shControl or shSNAI2 with low (black frame) and high (red frame) Hoechst 33342 staining (B), followed by Western blot for SNAI2 expression (C). Percentage of Hoechst low or high cell population was shown and compared (D). (E) Q-PCR was applied to detect mRNA levels of ABC transporter genes in MHCCLM3 cells expressing shSNAI2/shControl. Relative expression of ABC transporter genes was normalized with GAPDH mRNA. All values were represented as mean with bar as SD of three independent experiments, and the P values were measured between two linked groups. All experiments were repeated at least three times with similar results. (F) A diagram of the promoter of ABCB1 gene that drives luciferase reporter plasmid pGL3-ABCB1-luc. Empty circle and black ovals represent the transcriptional start point of ABCB1 gene and predicated binding sites of SNAI2 respectively. Sequences of predicated binding sites are shown in the text box below. (G) SMMC-7721 cells expressing shSNAI2 or shControl were transfected with luciferase reporter plasmid pGL3-ABCB1-luc and pRLSV40-Renilla. Luciferase Assay was conducted as described in Materials and Methods. (H) Western blot was applied to measure expression of indicated proteins in SMMC-7721 cells expressing shSNAI2 or shControl.