Fig 1: Effect of DTG exposure on folate transporter and receptor mRNA expression in human first trimester placental explants. Relative mRNA expression of a) SLC19A1, b) SLC46A1 and c) FOLR1 was determined in first trimester human placental explants from healthy donors, by qPCR (n=6). Placental explants were treated ex vivo with 2000 ng/mL DTG, 4000 ng/mL DTG, or vehicle control (DMSO) for 3h or 6h. For each donor, expression levels in the DTG-treated placental explants was normalized to those in the vehicle control. Each point represents relative expression in DTG-treated explants, normalized to expression in the corresponding vehicle-treated explants for a given donor. Solid line indicates mean relative mRNA expression. Vehicle control is indicated by the dotted line. Expression of SLC19A1 and FOLR1 following 3h DTG treatment at 4000 ng/mL was significantly greater than in the vehicle treated control. *, p<0•05 (Friedman test with Dunn's post hoc test).
Fig 2: Expression of folate transporters and receptor in DTG-treated folate-sufficient mouse placenta. Effect of DTG treatment on Slc19a1 (left), Slc46a1 (middle) and Folr1 (right) placental mRNA expression in a) GD 10·5 (n=15 for vehicle, n=16 for 1x-DTG treated, and n=15 for 5x-DTG treated groups), and b) GD 15·5 mouse placenta (n=40 for vehicle, n=33 for 1x-DTG+TDF/FTC treated, and n=40 for 5x-DTG+TDF/FTC treated groups). Vehicle = water; 1x-DTG = 2·5 mg/kg; 5x-DTG = 12·5 mg/kg); E/T = emtricitabine (33·3 mg/kg) + tenofovir disoproxil fumarate (50 mg/kg). Results presented as median expression normalized to the housekeeping gene Hprt1, and plotted relative to vehicle control. *, p<0·05; **, p<0·01; ****, p<0·0001 (mixed effects model to account for litter effects).
Fig 3: In vitro expression of folate transporters and Folate receptor-a in human placental cell lines. a) Relative mRNA expression of SLC19A1, SLC46A1 and FOLR1 was determined in HTR-8/SVneo, JAR and BeWo cells by qPCR, normalized to the housekeeping gene HPRT1 (n=3). b) Protein expression of RFC, PCFT and FRa, in JAR, HTR-8/SVneo and BeWo cell lines. Expression was quantified relative to ß-actin, by densitometric analysis of immunoblots (n=3). (c) Representative immunoblot demonstrating expression of RFC, PCFT, FRa and ß-actin, in JAR, HTR-8/SVneo and BeWo cell lines. Results are presented as mean relative expression ± SD.
Fig 4: Expression of folate transporters and receptor in DTG-treated folate-deficient mouse placenta. Effect of DTG treatment on Slc19a1 (left), Slc46a1 (middle) and Folr1 (right) placental mRNA expression in a) GD 10·5 (n=15 for vehicle, 16 for 1x-DTG treated, and 18 for 5x-DTG treated groups), and b) GD 15·5 mouse placenta (n=34 for vehicle, 40 for 1x-DTG + E/T treated, and 32 for 5x-DTG + E/T treated groups) under folate-deficient conditions. Vehicle = water; 1x-DTG = 2·5 mg/kg; 5x-DTG = 12·5 mg/kg); E/T = emtricitabine (33·3 mg/kg) + tenofovir disoproxil fumarate (50 mg/kg). Results presented as median expression normalized to the housekeeping gene Hprt1, and plotted relative to vehicle control. *, p<0·05; **, p<0·01 (mixed effects model to account for litter effects).
Fig 5: Relative expression of the folate transport systems in brain parenchymal cells. A Relative mRNA expression of mouse Slc19a1 (RFC), Slc46a1 (PCFT), and Folr1 (FRa) was assessed in primary cultures of mouse neurons, and primary cultures of mouse mixed glial cells. Results are presented as mean relative mRNA expression normalized to the housekeeping gene mouse cyclophilin B (n = 4). B–D Representative immunoblots from three separate experiments demonstrating protein expression of RFC, PCFT, and FRa in primary cultures of mouse neurons, and in primary cultures of mixed glial cells. HEK293 and HepG2 cells were used as positive controls for the three transport systems, with mouse kidney used as an additional positive control for FRa. Multiple protein bands for RFC and PCFT represent differential glycosylation
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