Fig 1: OLA1 regulates oral cancer metastasis through Smad2/Smad4 mediated EMT pathway. a mRNA fold change level of EMT markers between UM-1 siR-OLA1 cell and siR-NC cell. mRNA fold change level of EMT marker in UM-2 OLA1 knock down cells (b) and UM-1 OLA1 overexpression cells (c). d mRNA fold change level of TGFß/SMDA2 pathway markers between UM-1 siR-OLA1 cells and siR-NC cells. mRNA fold change level of TGFß/SMDA2 pathway markers in UM-2 OLA1 knock down cells (e) and UM-1 OLA1 overexpression cells (f). g Protein level of EMT marker in UM-1 OLA1 overexpression cells and UM-2 OLA1 knock down cells. h Protein level of TGFß/SMDA2 pathway markers in UM-1 OLA1 overexpression cells and UM-2 OLA1 knock down cells. i Western blotting assay showed SMAD2 was silenced by siR-SMAD2 in UM-2 shOLA1 cells. j Western blotting assay showed that silenced SMAD2 decreased OLA1 expression in UM-2 cells. k Wound healing assay showed SMAD2 silenced could rescue enhanced metastasis induced by knocking down OLA1 in UM-2 cells (n = 3). l Transwell assay to examine the effect of OLA1 on metastasis by smad2 in UM-2 cells (n = 3). vs. Control, *P < 0.05, **P < 0.01, ***P < 0.001
Fig 2: Dysregulation of OLA1 affected the ability of metastasis in oral cancer cells. a Wound healing assay to examine the effect of down regulating OLA1 on UM-1 and UM-2 cells (n = 3). b Transwell assay to investigate the impact of downregulating OLA1 on metastatic ability in UM-1 and UM-2 cells (n = 3). c Construction of stable cell lines with UM-1 (YFP-OLA1OE) plasmid or lentiviral vector in UM-2 (GFP-shOLA1). Top panel: Fluorescent photos of indicated cells. Bottom panel: OLA1 expression in relatived cells. d Morphology of cells (200X) after OLA1 plasmid transfection or TGFß1 (1 ng/mL) induction. e Wound healing assay to examine the effect of upregulating OLA1 on UM-1 and downregulating OLA1 on UM-2 cells (n = 3). f Transwell assay to examine the effect of upregulating OLA1 on UM-1 and downregulating OLA1 on UM-2 cells (n = 3). vs. Control, *P < 0.05, **P < 0.01, ***P < 0.001
Fig 3: Reduced OLA1 expression is related to paclitaxel-induced cancer cell metastasis. a The OLA1 expression in UM-1 cell treated with 100 nM PTX for 0–8 days. b and c Cell viability assay showed that OLA1 overexpression enhanced the PTX sensitivity in UM-1 cells (n = 3). d Cell flow cytometry was used to determine the effect of overexpression of OLA1 on paclitaxel-induced (20 μM) cell necrosis and apoptosis (n = 2). e Cell flow cytometry showed the effect of OLA1 on paclitaxel-induced (20 μM) cell necrosis and apoptosis through SMAD2 (n = 2). f Mechanism of OLA1 inhibits metastasis of OSCC. vs. Control, *P < 0.05, **P < 0.01, ***P < 0.001
Fig 4: The endogenous level of OLA1 in OSCC and oral cell lines. a The average expression level of OLA1 in patients HNSC in TCGA and GTEx oral cancer dataset. T = Tumor, N = Normal, num = Numbers. b OLA1 RPKM in OSCC from GEO140707, N = 3 paried tissues. c OLA1 expression in clinical OSCC (Red arrow) and the adjacent tissues (Black arrow) n = 5. mRNA (d) and protein (e) level in five oral cell lines. f OLA1 expression in OLA1-siRNA cells. Top panel: Fluorescent photos of cells transfected with OLA1 siRNA for 24 h. Middle panel: OLA1 protein expression in indicated cells. Bottom panel: OLA1 mRNA expression in indicated cells. g and h Cell proliferation curve was drawn in indicated cells. vs. Control, *P < 0.05, ns = no significance
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