Fig 2: 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 3: P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and von Willebrand factor (vWF) are expressed by capillary-like tube structures formed by early and mid-gestation primary human fetal brain endothelial cells (hfBECs). Representative immunofluorescence images of P-gp (A,D,G) BCRP (J,M,P) and vWF (B,E,H,K,N,Q) in early gestation (A–C, J–L), and mid-gestation (D–F, M–O) hfBECs and in the adult endothelial cell line hCMEC/D3 (as positive control) (G–I, P–R). Arrows indicate P-gp, BCRP and vWF staining within capillary-like tube structures. Expression of vWF (red) confirmed the endothelial cell phenotype. P-gp and BCRP (green) and vWF (red) staining co-localize (yellow) inside the capillary-like tube structures. Insets (bottom right) in (I,R) represent PBS as the negative control. Sections were counter-stained with DAPI (blue; a nuclear marker). n = 4/gestational age (representative images presented). Scale bars = 90 um.

Fig 4: P-gp and BCRP protein expression in the plasma membrane 4 h or 24 h after treatment with LPS (b-e), PolyI:C (f-i) or ssRNA (j-m) and their respective controls, measured using Western blot. Representative images of P-gp, BCRP and Na+/K+-ATPase (loading control) bands were cropped and are shown in A. N = 4–5/group. Dose: 106 pg/mL. Statistical analysis: unpaired Student’s t-test. Data are presented as mean ± SEM. *p < .05 and ***p < .001

Fig 5: P-gp and BCRP function 4 h or 24 h after treatment with the pro-inflammatory factors IL-6 (a-d), TNF-a (e-h) or IFN-? (i-l), measured using the Ca-AM and Ce6 assays. Dose: 101–104 pg/mL. N = 6–8/group. Values are expressed as a percentage of controls (vehicle). Data are presented as mean ± SEM. Statistical analysis: Kruskal-Wallis test, followed by Dunn’s multiple comparison test. *p < .05, **p < .01 and ***p < .001