Fig 1: Activation of GPR120 signaling reduces the intracellular accumulation of epirubicin and upregulates the expression of ABC transporters. a, MCF-7 cells transfected with shRNA targeting GPR120 or with negative control vector were pretreated with GW9508 for 24 h before the addition of 5 µg/ml epirubicin. Two hours after the addition of epirubicin, fluorescence intensity of intracellular epirubicin was evaluated by flow cytometry. Relative epirubicin fluorescence intensity was presented. b, MCF-7 cells were pretreated with 50 µM AH7614 for 30 min before the addition of GW9508. After 24 h, the cells were further treated with 5 µg/ml epirubicin for 2 h. Fluorescence intensity of intracellular epirubicin was evaluated by flow cytometry. Relative epirubicin fluorescence intensity was presented. c, MCF-7 cells transfected with shRNA targeting GPR120 or with negative control vector were treated with GW9508 for 24 h, and the cells were harvested for the analysis of ABC transporters (ABCB1, ABCC1, and ABCG2) mRNA expression. d, MCF-7 cells were pretreated with 50 µM AH7614 for 30 min before the addition of GW9508. After a 24 h stimulation, the cells were harvested for the analysis of ABC transporters (ABCB1, ABCC1, and ABCG2) mRNA expression. e and f, MCF-7 cells were simultaneously treated with GW9508 and 5 µM PGP-4008, an inhibitor of ABCB1, or 25 µM MK-571, an inhibitor of ABCC1, or 5 µM fumitremorgin C, an inhibitor of ABCG2, or the combination of the three inhibitors. After 24 h treatment, the cells were further treated with 5 µg/ml epirubicin for 2 h. Fluorescence intensity of intracellular epirubicin was evaluated by flow cytometry. Relative epirubicin fluorescence intensity was presented. Values were displayed with mean ± SEM. Statistical analysis was carried out by one-way ANOVA. *P < .05, **P < .01.
Fig 2: GPR120 expression is correlated with ABCC1, ABCG2 and FASN expression in breast cancer tissues. a, Correlation analysis between GPR120 expression and ABCC1, ABCG2, and FASN expression at mRNA levels or FFA levels in human breast cancer tissues. b, Proposed mechanism by which GPR120 activation promotes the development of chemoresistance in breast cancer.
Fig 3: Biological verification of the results of proteomic and N-glycoproteomic study. (A,B) Representative images of the Transwell migration (A) and invasion assay (B) of A549 and A549/DDP cells, and the quantitative number of migration and invasion cells. (C,D) WB assay of LRRC8A (C) and MRP1 (D) in ACM and DCM groups. (E) The amount of Pt in A549 and A549/DDP cells measured by ICP-MS. Error bars represent the mean ± SD; ** p < 0.01, **** p < 0.0001, Student’s t-test.
Fig 4: PVT1 knockdown sensitized T24/DR cells to DOX and DDPDOX (A) and DDP (B) dose-response curves and IC50s in T24 and T24/DR cells. MDR1 and MRP1 expression in T24/DR cells (C) and in BUC patients treated with DOX and DDP (D) *P<0.05 vs NC.
Fig 5: Akt/NF-?B pathway is involved in the regulation of ABC transporters expression by GPR120. a, MCF-7 cells were pretreated with 50 µM AH7614 for 30 min or transfected with GPR120-siRNA 48 h in advance. Next, the cells were further treated with GW9508 for the indicated time (15, 30, or 60 min). Levels of signaling molecules were evaluated by western blotting. b, MCF-7 cells were transiently co-transfected with 2.5 µg of reporter constructs and 100 ng of the Renilla luciferase vector pRL-TK. Dual-Luciferase reporter assays were carried out following treatment with GW9508 for 1 h. c, MCF-7 cells were treated with GW9508 for 1 h. ChIP assay was performed, and p65 recruitment to the ABCC1 and ABCG2 regulatory regions was examined by real-time PCR. d, MCF-7 cells were simultaneously treated with GW9508 and BAY11–7082 or MK2206. After 24 h, the cells were harvested for the analysis of ABCC1 and ABCG2 mRNA expression. e and f, MCF-7 cells were simultaneously treated with GW9508 and BAY11–7082 or MK2206. After 24 h, the cells were further treated with 5 µg/ml epirubicin for 2 h. Fluorescence intensity of intracellular epirubicin was evaluated by flow cytometry. Relative epirubicin fluorescence intensity is presented. Values were displayed with mean ± SEM. Statistical analysis was carried out by one-way ANOVA. *P < .05, **P < .01.
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