Fig 2: (A) MDR cancer cells were derived from parent cancer cell lines by incrementally increasing the Dox concentration in growth media. (I) TRAMP-C2 (TC2) prostate parent cancer cell line. (II) B16-F10 (B16) melanoma parent cancer cell line. (III) 4T1-Luc2 (4T1) breast parent cancer cell line. (IV) TC2-MDR cancer cell line. (V) B16-MDR cancer cell line. (VI) 4T1-MDR cancer cell line. Scale: 200 µm (B) Western blot shows the increased expression of P-gp in MDR cancer cells compared to parent cancer cell lines. (C) TC2-Parent, TC2-MDR, B16-Parent, and B16-MDR stained with Anti-P-gp-Alexa Fluor 488 antibody conjugate reveals the increased expression of P-gp in MDR-derived cancer cell lines, as well as membrane localization of P-gp.

Fig 3: (A) Competitive experiments performed at 37 °C with P-gp substrate Rh123. IMQ leads to an increase in Rh123 accumulation in MDR-derived cancer cells under active transport conditions. Data representative of triplicate experiments. (B) Uptake experiments performed at 4 °C with P-gp substrate, Rhodamine 123. IMQ leads to an increase in Rh123 accumulation in MDR-derived cancer cells under passive diffusion conditions. Data representative of triplicate experiments. p-values were calculated between parent vs. MDR cancer cell type with * p < 0.01, ** p < 0.001 and NS: Not significant.

Fig 4: P-gp and BCRP distribution within subcellular fractions of early and mid-gestation hfBECs. (A) Representative western blot images showing specific markers in subcellular fractions of second trimester hfBECs: plasma membrane (PM, lane 1), cytosol (Cyt, lane 2), organelles (Orgs, lane 3), and nuclear (Nucl, lane 4) fractions. Sodium/potassium ATPase (Na+/K+-ATPase; plasma membrane marker), early endosome antigen 1 (EEA1; cytosol marker), prolyl 4-hydroxylase 3 (P4H3; endoplasmic reticulum (ER) marker) and histone3 (H3; nuclear marker), n = 3. (B) Representative western blot images and (C–F) densitometric analysis of plasma membrane, cytosolic, organelle (ER) and nuclear P-gp expression and (G–J) densitometric analysis of plasma membrane, cytosolic, organelle (ER) and nuclear BCRP expression, normalized to Na+/K+-ATPase (plasma membrane loading control) or ERK2 (loading control for cytosol, organelle and nucleus), in early and mid-gestation hfBECs. Data are expressed as mean ± SD. n = 6/group. Statistical differences were tested using an unpaired t-test. * p = 0.05.

Fig 5: Developmental expression and function of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in early and mid-gestation primary human fetal brain endothelial cells (hfBECs). (A) Representative images of western blots (B) densitometric analysis of total P-gp and (C) BCRP protein comparing early and mid-gestation hfBECs. ERK2 is used as loading control. (D) Relative ABCB1 (encoding P-gp) and (E) ABCG2 (encoding BCRP) mRNA expression in early and mid-gestation hfBECs. Data are normalized by the geometric mean of TOP1 and YWHAZ (reference genes), (F) calcein-AM (P-gp substrate) and (G) Ce6 (BCRP substrate) accumulation in early and mid-gestation hfBECs. Data are expressed as mean ± SD. n = 6/gp. Statistical differences were conducted using an unpaired t-test. * p = 0.05.