Fig 2: Role of OATP2B1 in rosuvastatin transport across the feto-maternal interface-OOC. (A) Schematic showing the four concentric cell culture chambers of the feto-maternal interface-OOC used to determine the role of OATP2B1 in transporting drug across the different fetal membrane chambers [AECs (blue, chamber 1), AMCs (pink, chamber 2), CTCs (yellow, chamber 3), and maternal DECs (green, chamber 4)]. Rosuvastatin (200 ng/ml) was introduced in the decidual chamber of the feto-maternal interface-OOC with either control mock siRNA or OATP2B1-knockdowned CTCs. Following incubation for 4 h, media was collected from different chambers of feto-maternal interface-OOC for mass spectrometry analysis. (B) Under control siRNA conditions, 18.45 and 1.73% of rosuvastatin propagated from the maternal DEC to the CTC chamber as well as to the AEC chamber, respectively. OATP2B1 silencing in OATP2B1 resulted in only 2.57% rosuvastatin propagation from the maternal DEC chamber to the CTC chamber, and only 0.32% of rosuvastatin propagation from the DEC chamber to the AEC chamber, and then zero to the CTC chamber. Data is represented as mean ± SEM. Rosuvastatin propagation across the feto-maternal interface -OOC was confirmed from six independent experiments.

Fig 3: Functional validation of OATP2B1 in fetal membrane and placental cells. (A) Functional activity of OATP2B1 in fetal membrane (AECs and CTCs) and placental (BeWo) cells was determined by flow cytometry. Representative histograms of zombie violet dye uptake are shown. (B) The mean fluorescence intensity of these histograms was calculated using the FlowJo software. n = 4 biological replicates. Error bars represent mean ± SEM.

Fig 4: Effect of OATP2B1 silencing on zombie violet dye uptake assay in placental BeWo cells and fetal membrane CTCs. (A) Western blot analysis and quantification of OATP2B1 in siRNA-treated BeWo cells and CTCs to determine the efficiency of knockdown. OATP2B1 was silenced in BeWo cells (70%) and CTCs (80%) using short-interfering RNA (siRNA). Non-targeted scrambled siRNA is used as control siRNA. β-Actin was used as a loading control. Representative blots are shown. Error bars represent mean ± SEM. (B) OATP2B1 siRNA-treated BeWo cells and CTCs were subjected to the zombie violet dye uptake assay. The data shows that the uptake of zombie violet dye (as indicated by the percent change in mean fluorescence intensity compared to the unstained controls) was reduced to a greater extent in OATP2B1 siRNA-treated CTCs compared to the siRNA treated BeWo cells. Representative histograms of the dye uptake determined by flow cytometry analysis are shown. The percent change in mean fluorescence intensity (MFI) in OATP2B1 knockdown CTCs and BeWo cells (**p < 0.01) compared to their respective controls was calculated using the FlowJo software. n = 4 biological replicates. Error bars represent mean ± SEM.

Fig 5: Localization and expression of OATP2B1 transporter protein in fetal membrane tissues and human placenta. (A) Bright field microscopy showing localization of OATP2B1 (brown) in human fetal membrane AECs and CTCs (left image) at a magnification of 20x (main image) and 100x (inset image) and placental syncytiotrophoblast at a magnification of 20x (main and inset image (right image). 20x and 100x images were taken in different regions of the tissue. Images are representative of three biological replicates. Scale bar = 30 µM. (B) Western blot analysis and quantification of OATP2B1 in human fetal membranes and placenta (maternal and fetal side). β-Actin is used as a loading control. Representative western blots are shown. n = 6 biological replicates. Error bars represent mean ±SEM.