Fig 1: The expression of TGM2 was regulated by circ_00081001 and miR-494-3p in MTX-resistant OS cells. a, b WB assay was employed to examine the protein level of TGM2 in U2OS/R and HOS/R cells after transfection with sh-NC, sh-circ_00081001, sh-circ_00081001 + anti-miR-NC, or sh-circ_00081001 + anti-miR-494-3p. **P < 0.01
Fig 2: Knockdown of circ_0081001 enhanced MTX sensitivity through regulating miR-494-3p and TGM2 expression in MTX-resistant OS cells. a The protein abundance of TGM2 was measured using WB analysis in U2OS/R and HOS/R cells after transfection with vector and TGM2. b–n U2OS/R and HOS/R cells were transfected with sh-NC, sh-circ_0081001, sh-circ_0081001 + anti-miR-NC, sh-circ_0081001 + anti-miR-494-3p, sh-circ_0081001 + vector, or sh-circ_0081001 + TGM2. b, c IC50 value for MTX was calculated in MTX-treated U2OS/R and HOS/R cells. d, e Cell apoptosis was measured by flow cytometry. f–i Transwell assay was utilized to test cell migration and invasion capacities. j–n The protein levels of cleaved-casp3, E-cadherin and N-cadherin were determined by WB analysis. **P < 0.01
Fig 3: Deficiency of circ_0081001 elevated MTX sensitivity of OS in vivo. U2OS/R cells transfected with sh-NC or sh circ_0081001 were inoculated subcutaneously into the nude mice. The mice were treated with MTX (5 mg/kg MTX) twice a week after injection for 1 week. a, b Tumor volume and weight were examined. c, d The expression of circ_0081001 and miR-494-3p was measured by qRT-PCR in resected tumor tissues. e The protein abundance of TGM2 was detected by WB assay in resected tumor masses. **P < 0.01
Fig 4: TGM2 was a direct target of miR-494-3p in MTX-resistant OS cells. a Starbase showed that miR-494-3p potentially targeted TGM2. b, c Dual-luciferase reporter assay was conducted to test the luciferase activity in U2OS/R and HOS/R cells co-transfected with TGM2 WT or TGM2 MUT and miR-494-3p or miR-NC. d WB assay was performed to analyze the protein expression of TGM2 in U2OS, U2OS/R, HOS, and HOS/R cells. e, f Relative miR-494-3p expression was detected using the qRT-PCR analysis in U2OS/R and HOS/R cells after transfection with miR-NC, miR-494-3p, anti-miR-NC, or anti-miR-494-3p. g, h The protein abundance of TGM2 was assessed by WB analysis in U2OS/R and HOS/R cells after transfection with miR-NC, miR-494-3p, anti-miR-NC, or anti-miR-494-3p. **P < 0.01
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