Fig 1: The effect of VU‐0365114 on drug resistance in colorectal cancer cells. (A) HCT116 and HCT116‐OxaR cells were treated with various concentrations of oxaliplatin, VU‐0365114, and colchicine for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD (n = 4). Statistical significance, compared to parental HCT116 cells at each dose (*P < 0.05 and ***P < 0.001), was determined using a two‐tailed paired Student's t‐test. (B) The P‐glycoprotein (P‐gp) expression in MES‐SA, MES‐SA/Dx5, HCT116, and HCT116‐OxaR cells were examined by Western blotting. Data represent two independent experiments. (C) HCT116 and HCT116‐OxaR cells were treated with verapamil (Vera; 10 μm) or VU‐0365114 (VU; 10 μm) for 2 h. Then, an MDR assay was performed. The error bars are the mean ± SD (n = 4). Statistical significance, compared to untreated controls (**P < 0.01 and ***P < 0.001), was determined using a one‐way ANOVA with Tukey's post hoc test. Statistical significance, compared between HCT116 and HCT116‐OxaR cells (# P < 0.05), was determined using a two‐tailed paired Student's t‐test. (D) The P‐gp expression in pCMV3‐ and pCMV3‐ABCB1 (pABCB1)‐transfected HCT116, as well as HCT116 and HCT116‐OxaR cells were examined by Western blotting. Data represent two independent experiments. (E) pCMV3‐ and pABCB1‐transfected HCT116 cells were treated with verapamil (10 μm) or VU‐0365114 (10 μm) for 2 h. Then, an MDR assay was performed. The error bars are the mean ± SD (n = 3). Statistical significance, compared to untreated controls (*P < 0.05), was determined using a one‐way ANOVA with Tukey's post hoc test. Statistical significance, compared between pCMV3‐ and pABCB1‐transfected HCT116 cells (## P < 0.01), was determined using a two‐tailed paired Student's t‐test. (F) pCMV3‐ and pABCB1‐transfected HCT116 cells were treated with VU‐0365114 (10 μm), doxorubicin (Doxo; 0.25 μm), and colchicine (Col; 100 nm) for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD (n = 5). Statistical significance, compared to pCMV3‐transfected HCT116 cells at each treatment (**P < 0.01), was determined using a two‐tailed paired Student's t‐test. (G) Parental and p53‐knockout HCT116 (HCT116‐p53‐KO) cells were treated with various concentrations of 5‐fluorouracil (5‐FU), VU‐0365114, and colchicine for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD (n = 4). Statistical significance, compared to parental HCT116 cells at each dose (**P < 0.01 and ***P < 0.001), was determined using a two‐tailed paired Student's t‐test. (H) HCT116 and HCT116‐p53‐KO cells were treated with the indicated concentrations of 5‐fluorouracil (5‐FU), VU‐0365114 (VU), and colchicine for 48 h. Then, protein expressions were examined by Western blotting. Data represent two independent experiments.
Fig 2: Multi-drug resistance (MDR) activity. The activity of drug-efflux proteins MDR1/P-gp and MRP1 in wild type and CDDP-resistant ccrf-cem and A2780 cells was determined by a fluorimetric MDR assay kit (Abcam). Intracellular fluorescence was determined in 2 × 106 cells per well after 1 h of incubation. Data are representative of three independent experiments and are expressed as % of untreated cells ± SD. (A) CCRF-CEM wt treated with 0.5 μm CDDP and CCRF-CEM res treated with CDDP 2.5 μm. (B) A2780 wt treated with CDDP 0.5 μm and A2780 res treated with CDDP 2 μm.
Fig 3: Extracellular ATP upregulates activity of ABC transportersA549 or SK-HEP-1 cells were treated with either ATP of various concentrations and/or specific inhibitors of ABC transporters in the presence or absence of sunitinib. After treatment, cells were measured for either retained fluorescence dye or cell viability by MTT assay. Data is reported as mean ± standard deviation. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. (A) Extracellular ATP reduced florescence intensity of the dye (increased the efflux activity of ABCB1 and/or ABCC1). (B) Specific ABC inhibitors increased the retention of fluorescence dye. (C) Inhibitors of ABCB1 and ABCC1 reduced the viability of A549 and SK-HEP-1 cells treated by 20 μM sunitinib in the presence or absence of 1 mM extracellular ATP. Verapamil: 20 μM; MK-571: 50 μM. (D) Western blot analyses of protein levels of ABCB1 and ABCC1 in A549 or SK-HEP-1 cells treated by sunitinib in the presence or absence of 1 mM ATP. Untreated cells serve as baseline controls.
Supplier Page from Abcam for MDR Assay Kit (Fluorometric)