Fig 1: POR regulates RA-dependent gene expression(A) Venn diagrams showing significant DEG profiles for each line in response to the RA concentration gradient. The size of the circle is proportional to the number of DEGs indicated within each line.(B) Heatmap of hierarchical cluster analysis for genes that were defined as DEGs in at least one condition in any cell line.(C) A PCA plot of normalized counts. Each dot represents a different sample. Technical repeats (a and b) were averaged and merged (ab). All lines cluster along the PC2 axis when differentiated without RA (encircled in blue). POR-deficient lines differentiated with all RA concentrations clustered along PC2 with the CTR line differentiated with 10 μM (encircled in red). C, CTR; M, PORM; P, PORD. 0, 0.25, 1, 10: RA concentrations in μM.See also Figure S5.
Fig 2: Schematic description of the suggested mechanism(A) POR mediates the activity of CYP26s, which regulate cellular RA levels by catabolizing RA. In turn, RA regulates the expression of CYP26s, TJs, and integrin (ITGB) genes, which leads to the formation of a functional barrier.(B) When POR is malfunctioning, CYP26s are inactive, causing accumulation of cellular RA. In turn, this leads to transcriptional alterations and an inflammatory response. As a result, TJ formation is impaired, leading to a malfunctioning barrier.
Fig 3: POR is necessary for the RA-dependent development of functional TJs(A) ICC analysis of the TJ relevant markers ZO-1 (red), GLUT-1 (green), and CLDN-5 (green) for iBMECs differentiated with 10 μM RA from the healthy (POR01het), PORD, and PORM lines. White arrowheads denote gaps between cells in which ZO-1 was expressed.(B) Scatterplots of maximum (max) TEER values for the CTR, PORD, and PORM lines differentiated with 0 (vehicle [Ve]), 0.25, 1, or 10 μM RA (CTR n = 28; PORD, n = 27; PORM, n = 9). Two-way ANOVA with Tukey’s multiple comparison test; ∗∗p < 0.005, ∗∗∗p < 0.0001.(C) Quantification of the paracellular permeability of the fluorescent tracer sodium fluorescein. When iBMECs were differentiated with 10 μM RA, the PORD (n = 8) and PORM (n = 5) lines showed a significant increase in paracellular permeability compared with CTR lines (n = 9). PORM iBMECs had higher permeability compared with PORD iBMECs. Two-way ANOVA with Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0001. Bars represent 95% confidence intervals; the solid red line represents the median.See also Figure S4.
Fig 4: POR is enzymatically active in CTR and heterozygous iBMECs and severely attenuated in PORD and PORM iBMECs(A) WB analysis of POR in undifferentiated iPSCs and iBMECs. CTR, POR01het, and PORG539R (PORD) cells express POR as a 75 kDa band in both undifferentiated iPSCs and iBMECs. CTRmut1 (PORM) did not express POR, and CTRmut2 produced a shorter, 55 kDa band, as predicted in Figure S2.(B) ICC showed POR (green) expression in POR01het and POR02G539R but not in CTRmut1 iBMECs.(C) POR enzymatic activity is decreased in PORD and further reduced in the PORM lines compared with the CTR and POR01het lines. One-way ANOVA; ∗∗p < 0.005, ∗∗∗∗p < 0.0001, activity was normalized to the maximum level of each experiment. Different symbols refer to different cell lines (n = 4–10).See also Figures S2 and S3 and Table S1.
Fig 5: POR is expressed in human and mouse NVU and in iBMECs(A) POR expression in acutely purified human brain cells from the Barres lab database (Zhang et al., 2016).(B) IHC analysis of mouse cortical brain section shows that CD31 (red)-positive endothelial cells express Por (green). Nuclei were stained using DAPI (blue). White arrowheads denote the co-localization of CD31 and Por.(C) Por (green) shows a perinuclear pattern of expression.(D) Schematic illustration of iBMEC differentiation. EC, endothelial cells; bFGF, basic fibroblast growth factor; RA, all-trans retinoic acid.(E) ICC analysis confirms that CTR iBMECs express GLUT-1, ZO-1, and CLDN-5.(F) ICC analysis of iBMECs shows that POR is localized to the perinuclear area.(G) WB analysis showed expression of POR as a 75 kDa band both in undifferentiated iPSCs and iBMECs. Ponceau S staining shows total protein levels.See also Figure S1.
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