Fig. 5

Leptin transport after knockdown of CAT-1 expression. In select panels, NT denotes non-targeting and KD denotes knockdown. A Schematic of vectors that were used for generating gene silencing, including SB transposase vector (i), SB transposon/CRISPRoff vector (ii), and SB transposon/U6-sgRNA vector (iii). 3L denotes Dnmt3L. B Schematic procedure for iPSC transfection, sorting, and differentiation. After transfections, iPSCs were sorted based on BFP expression. BFP + cells were then differentiated into BMEC-like cells for the leptin transport assay. C Western blot for assessment of dCas9-based CRISPRoff protein in iPSCs with or without doxycycline treatment for 9 days. D Western blot for assessment of CAT-1 protein in iPSCs that received a non-targeting gRNA or gRNAs targeting SLC7A1. GAPDH served as the loading control. A representative image is shown, and data were quantified using 3 biological replicates (represented as mean ± standard deviation). Each sample was normalized internally to GAPDH and then to the non-targeting control. Statistical significance was calculated using the Student’s unpaired t-test. All iPSC cultures received doxycycline treatment for 9 days prior to sample collection. E qPCR assessment of SLC7A1 expression in BMEC-like cells differentiated from iPSCs that received a non-targeting gRNA or gRNAs targeting SLC7A1. Data are represented as mean ± standard deviation from 3 biological replicates. Statistical significance was calculated using the Student’s unpaired t-test. F Compiled datasets represent the basolateral chamber leptin concentration corresponding to each gRNA condition, normalized to the non-targeting control. Each data point is the mean of a single biological replicate calculated using three Transwell filters (n = 3 technical triplicates). Data are represented as mean ± standard deviation from n = 4 biological replicates. Statistical significance was calculated using the Student’s unpaired t-test. G Representative TEER measurements before and after the leptin transport assay. Each data point is the mean from a single Transwell filter (n = 3 technical measurements per filter). Data are represented as mean ± standard deviation from 3 filters per condition. Trends were confirmed across three additional biological replicates. H Representative immunostaining of endothelial or BBB markers in BMEC-like cells in both control and CAT-1 knockdown conditions. Scale bars indicate 100 μm