Fig 2: HSF1 depletion down-regulated the transcriptional level of mdr1 in drug-resistant cancer cells.a, b After A549 lung cancer cells, paclitaxel-resistant A549 cells (A549-taxolR), T47D breast cancer cells, and doxorubicin-resistant T47D cells (T47D-doxR) were transfected with a control siRNA (siCont) or siHSF1, western blotting or RT-PCR was performed. c After A549 and A549-taxolR were transfected with shRNA (shCont), shHSF1, CRISPR/Cas9-Control (Cont), or HSF1 CRISPR/Cas9 knockout (KO) plasmid, western blotting (top) or RT-PCR (middle) was performed. c, bottom Luciferase assays in A549 and A549-taxolR with CRISPR/Cas9-Control or HSF1 CRISPR/Cas9 KO plasmid were performed after transfection with a luciferase reporter construct with the mdr1 promoter. Values are presented as the mean ± SD of at least three independent experiments. Statistics calculated based on one-way ANOVA, *p < 0.05. d A549-taxolR cells were treated with indicated concentrations of paclitaxel for 24 h after transfection with siCont or siHSF1, and western blotting was performed. e A549 and A549-taxolR with CRISPR/Cas9-Control or HSF1 CRISPR/Cas9 KO plasmid were analyzed by ChIP assay to measure enrichment of mdr1 promoter sequences. IgG was used as a negative control for the HSF1 antibody. Statistics calculated based on one-way ANOVA, *p < 0.05. Values are expressed as fold change relative to the control.

Fig 3: Oestrogen-related receptors alpha (ERRa) bound to SP3 to increase the transcription of ABCB1. The ERRa was immunoprecipitated by use of its specific antibody in MG-63/Dox (A) or HOS/Dox (B) and their parental cells, respectively. The bound SP3 was measured by western blot analysis, IgG was used as the negative control; (C) MG-63/Dox cells were transfected with si-NC or si-SP3 for 24 h; MG-63/Dox cells were transfected with si-NC or si-SP3 for 12 h and then further treated with or without 1 µmol/L XCT-790 for 24 h, the mRNA (D) or protein (E) expression of P-gp was measured and quantitatively analyzed. Data are presented as means ± SD of three independent experiments. **P < 0.01 compared with control

Fig 4: BHPI inhibits protein synthesis and reduces the level of MDR1 protein in OVCAR-3 cellsA. Protein synthesis was measured using the incorporation of 35S methionine into protein at the indicated times after pretreating cells for 20 min. with either DMSO, U73122, or 2-APB (n = 4). B. Western blot indicating the MDR1 protein level at indicated time points after BHPI treatment. Concentrations: U73122, 1 µM; 2-APB, 100 µM; BHPI, 500 nM (A) or 1 µM (B) . Data is the mean ± SEM.

Fig 5: In MDR1 overexpressing cells, BHPI depletes intracellular ATP and inhibits MDR1 efflux activityA. Proposed model of the pathway by which BHPI inactivates MDR1. B., C. Intracellular ATP quantitation showing effect of BHPI on cellular ATP level after pretreating cells with either DMSO or thapsigargin (THG) (n = 6). D. Western blot analysis showing MDR1 protein level from the indicated cell lines. E., F. Rhodamine-123 (Rho-123) quantitation showing the effect of BHPI on Rho-123 concentration in the media after pretreating cells with either DMSO, U73122, or 2-APB (n = 6). Concentrations: U73122, 1 µM; 2-APB, 100 µM; BHPI, 1 µM (B, C) or 500 nM (E, F). Data is the mean ± SEM. ***P < 0.001.