Fig 1: Knockdown of MIST1 and SNAI1 increases adhesion and induces cells to undergo anoikis. (A) MIST1 was knocked down in the A375 and MV3 cell lines, as confirmed by western blot analysis. (B) Knockdown of MIST1 improved the adhesion ability of A375 cells (scale bar, 200 µm). Findings for MV3 were similar, data not shown. (C) Inhibition of MIST1 decreased the viability of A375 cells in suspension culture, indicating increased anoikis. Findings for MV3 were similar, data not shown. (D) SNAI1 was knocked down in the A375 and MV3 cell lines, as confirmed by western blot analysis. (E) Knockdown of SNAI1 improved the adhesion ability of A375 cells (scale bar, 200 µm). (F) Inhibition of SNAI1 decreased the viability of A375 cells in suspension culture, indicating increased anoikis. NS, no significance; *P<0.05 vs. control. shMIST1, small hairpin RNA targeting muscle intestine and stomach expression 1; Con, control.
Fig 2: MIST1 and SNAI1 disrupt cell-matrix adhesion and promote anchorage independence. (A) MIST1 was ectopically expressed in HUVEC and NHEM 2493 cell lines and confirmed by western blot analysis. (B) Overexpression of MIST1 reduced the adhesion ability of HUVEC (scale bar, 200 µm). (C) MIST1 enhanced the viability of HUVEC in suspension culture, indicating bypassing of anoikis. (D) SNAI1 was ectopically expressed in HUVEC and NHEM 2493 cell lines and confirmed by western blot analysis. (E) Overexpression of SNAI1 reduced the adhesion ability of HUVEC (scale bar, 200 µm). (F) SNAI1 enhanced the viability of HUVEC in suspension culture, indicating bypassing of anoikis. Findings for NHEM 2493 were similar, data not shown. NS, no significance; *P<0.05 vs. control. MIST1, muscle intestine and stomach expression 1; HUVEC, human umbilical vein endothelial cells; NHEM 2493, normal human epidermal melanocytes.
Fig 3: MIST1 hijacks the PTEN/AKT signaling pathway via SNAI1. (A and B) Western blot analysis indicated that overexpression of MIST1 reduced the expression of PTEN through SNAI1 and further increased p-AKT (S473) in HUVEC. Inhibitor of p-AKT (S473) LY294002 restored this change and the ability to resist anoikis. (C and D) Knockdown of MIST1 increased the expression of PTEN through SNAI1 and further reduced p-AKT (S473) in A375, as indicated by western blot analysis. Knockdown of PTEN increased p-AKT (S473) and promoted anchorage independence. *P<0.05 vs. control. shMIST1, small hairpin RNA targeting muscle intestine and stomach expression 1; HUVEC, human umbilical vein endothelial cells; p-PTEN, phosphorylated phosphatase and tensin homologue. Findings for MV3 were similar with A375, data not shown.
Fig 4: Expression of MIST1 is positively associated with SNAI1 in human normal and melanoma cells. (A and B) RNA and protein levels of MIST1 and SNAI1 in HUVEC, NHEM 2493, A375 and MV3 cell lines. (C and D) RNA and protein levels of MIST1 and SNAI1 in attached and floating cell lines. MIST1, muscle intestine and stomach expression 1; HUVEC, human umbilical vein endothelial cells; NHEM 2493, normal human epidermal melanocytes.
Fig 5: MIST1 promotes SNAI1 transcription by directly binding to its promoter region. (A) MIST1 binding motif. (B) Design of 7 chromatin regions of the SNAI1 promoter containing the MIST1 binding motif for ChIP. (C and D) ChIP performed on HUVEC and NHEM 2493 cells demonstrated that MIST1 was enriched in samples containing region 5 of the SNAI1 promoter. (E) A luciferase reporter assay determined that the -1,199 to -753 bp segment upstream of TSS site of SNAI1 combined with MIST1. TSS, transcription start site; Ctrl, control; IgG, immunoglobulin G; ChIP, chromatin immunoprecipitation; *P<0.05 vs. control; MIST1, muscle intestine and stomach expression 1; HUVEC, human umbilical vein endothelial cells; NHEM 2493, normal human epidermal melanocytes.
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