Fig. 6: ChP barrier components and permeability are diurnally regulated.

a TRAP candidates associated with barrier function. FPKM for each individual mouse is shown at 9 a.m. (orange circles) and 9 p.m. (blue squares) for those transcripts associated with RPL10A (solid) and those in the supernatant (open). N = 3 biologically independent samples (LV ChP pooled from 3 animals per sample). Data are presented as mean values ± standard deviation (SD). b RT-qPCR analysis of the expression of barrier components Itgb8, Slca8, Chd3, Abcf3, Chmp1b, and Snx12 in LV ChP every 3 h. Itgb8, Slca8, Chd3, Abcf3, and Snx12 showed significant rhythmicity by RAIN analysis. N = 4 biologically independent animals at each time across 2 independent experiments. c Experimental design for barrier permeability assay using intravenous (i.v) injection of horseradish peroxidase (HRP) followed by transmission electron microscopy. d Example of HRP-filled vesicles (green circles) taken up during the 7-min incubation period within the peri-junctional area (yellow shading). Vesicles outside of the quantified region are circled in brown. e Quantification of total number of HRP-filled vesicles in the peri-junctional area. Each point represents one junction, data combined from three animals at each timepoint. Welch’s two-tailed unpaired t test, N = 3 biologically independent animals per time in one experiment. f Example of the apical-basal distances calculated for HRP-filled vesicles in the peri-junctional area. Quantification of the (apical-basal)/apical ratio (0 = basal (blue); 1 = apical (red) for HRP-filled vesicles in the peri-junctional area at 9 a.m. (orange) and 9 p.m. (blue). Kolmogorov–Smirnov test, N = 3 biologically independent animals per time in one experiment. g Example of tight junctions at apico-lateral surface of ChP epithelial cells. Dotted box indicates junction area and bracket indicates width. Scale bars: top = 1 μm; bottom zoom = 500 nm. h Quantification of distances between each cell membrane within the tight junction. Each point is the average of 5 distances per junction for 10 junctions in a single animal. *p < 0.05; Welch’s two-tailed unpaired t test, N = 3 biologically independent animals per time in one experiment. Data are presented as mean values ± standard error of the mean (SEM). Male mice were analyzed. Source data are provided as a Source Data file (Source Data).