Fig 2: Blocking SP1 or 12LOX attenuates SKOV3-R tumor growth and reverses metastasis in vivo. a SKOV3-R cells (1 × 106) were subcutaneously injected in nude mice, tumor reached ~ 300 mm3 at Day 30, then the mice were treated with PBS or DDP (1 mg/kg) or DDP (1 mg/kg) plus Bai (30 mg/kg) or MA (0.5 mg/kg) once every 2 days. The tumor volume was assessed until Day 36. b SKOV3-R cells (1 × 106) were subcutaneously injected in nude mice. The mice were treated with PBS or DDP (1 mg/kg) plus Bai (30 mg/kg) or MA (0.5 mg/kg) every 2 days in the third week. Mice were sacrificed for assessment of liver metastasis at Day 21. The overall view of the livers, H&E staining and the metastasis numbers in live lesions (right panel) were determined. Data in (b) are expressed as mean ± SEM for 5 mice. c IHC of SP1, 12LOX, MRP1 and Snail in liver tissues in four groups including control (PBS), DDP, DDP plus MA, DDP plus Bai. IOD against control group in above groups were depicted in fold change. d The correlation between SP1, 12LOX, COX1, CYP2J2 with MRPs, Slug and Snail. Pearson correlation coefficient was used to analyze the correlation between MRPs and EMT markers with SP1, 12LOX, COX1, and CYP2J2 using the clinical database GSE13876. e Schematic diagram of SP1-driven chemoresistance and metastasis. Data are expressed as mean ± SEM for 5 mice. *P < 0.5, **P < 0.01, ***P < 0.001, ****P < 0.0001; DDP + MA vs. DDP; #P < 0.05, DDP + Bai vs. DDP

Fig 3: MRP1 activity and expression in Calu-3 cells and EpiArway™ cultured under ALI conditions for 21 d. Left: the apical-to-basolateral (AB) and basolateral-to-apical (BA) fluxes of 3H-estrone-3-sulphate (0.5 µM, 3 µCi/mL) were monitored both in the absence (none) and in the presence of 50µM MK-571, as indicated. Data obtained were employed to calculate the efflux (ER) and uptake (UR) ratio, as defined in Methods. Bars represent the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs. none. Right: the expression of the transporter was evaluated by means of confocal laser scanning microscopy. Green signal: MRP1 immunolabeling; red signal: nuclear staining with propidium iodide. A single XY scan is shown, along with the XZ section of the plane (top). Scale bar = 10 µm.

Fig 4: Effects of different theaflavin monomers on the mRNA and protein levels of MRP1, MRP2, MRP3, P-gp and BCRP in Caco-2 cells. (A) Effect of theaflavins on mRNA levels of efflux transporters. (B) Effect of theaflavins on protein levels of efflux transporters. Ctrl: Caco-2 cells were incubated with HBSS; TF, TF3G, TF3’G and TFDG: Caco-2 cells were incubated with TF, TF3G, TF3’G and TFDG. Results are expressed as means ± SD (N = 3). Different small letters indicate significant difference between different treatments at p<.05.

Fig 5: HOTAIR was upregulated in DDP-resistant OS tissues and cells, and HOTAIR knockdown decreased the DDP resistance of Saos2/DDP and MG-63/DDP cells. (A, B) The expression of HOTAIR in DDP-sensitive (n = 20) and DDP-resistant (n = 20) OS tissues, Saos2/DDP and MG-63/DDP cells (n = 3), and their matched controls was measured by qPCR. Next, HOTAIR siRNA was transfected into Saos2/DDP and MG-63/DDP cells; following transfection for 48 h, (C) the interference efficiencies were detected with qPCR (n = 3). (D, E) The IC50 values of DDP (n = 3) and (F, G) the protein levels of MDR1, ABCB1, ABCC1, ABCG2, MRP5, and LRP1 (n = 3) were detected by CCK-8 and western blotting. *P < 0.05, **P < 0.01. DDP: cisplatin; OS: osteosarcoma; qPCR: quantitative polymerase chain reaction.