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. 2021 Dec 15;12:7293. doi: 10.1038/s41467-021-27488-x

Fig. 3. AMD cellular phenotypes in CFH(Y/Y)-iRPE and CFH(H/H)-iRPE are induced by C3a–C3aR1 and C5a–C5aR1 signaling.

Fig. 3

a Western blot of membrane and cytoplasmic fractions confirms the presence of complement receptor 3a (C3aR1) and complement receptor 5a (C5aR1) in iRPE membranes. Liver and A549 cell line cell lysates served as positive controls. Na+/K+ ATPase was used as a loading control (N = 3 biologically independent replicates, iRPE1, 5, 8). b, c Predominant apical localization of C3aR1 (red) (b) and C5aR1 (red) (c) in CFH(H/H)-iRPE5 cells. ZO-1 (green) marks apical tight junctions (N = 4 biologically independent replicates, iRPE1, 5, 8, 9). d–g Western blots (d, f) and their quantification (e, g) demonstrate 7-d-fold upregulation of pAKT (d, e) and two- to threefold upregulation of pERK (f, g) in CC-HS treated iRPE cells, compared to CI-HS-treated iRPE cells (N = 6 biologically independent replicates and n = 2 technical replicates, iRPE1, 2, 3, 4, 5, 7 for e, g). P values were determined by multiple pairwise comparisons using two-tailed t test with 95% confidence interval and Bonferroni correction. The horizontal lines in the boxplots indicate the median, the boxes indicate the first and third quartiles, and the whiskers indicate 5th and 95th percentile. h TER measurements after 48 h of CI-HS or CC-HS treatment on apical only, basal only, and combined apical/basal sides of iRPE. (N = 3 independent experiments and n = 2 technical replicates; iRPE6, 8). P values were determined by pairwise comparisons using two-tailed t test with 95% confidence interval and Bonferroni correction. The horizontal lines in the boxplots indicate the median, the boxes indicate the first and third quartiles, and the whiskers indicate 5th and 95th percentile. i, j APOE-positive sub-RPE deposits (i) and TER measurement (j) across iRPE monolayers treated with CI-HS, C3, or C5-depleted sera, CC-HS and receptor blockers for C3aR1 (PMX53 10 μM) and C5aR1 (compstatin 10 μM), CC-HS + vehicle (vehicle). N = 4 independent experiments for (i). N = 3 biologically independent replicates and N = 4 independent experiments, iRPE1, 5, 6, for (j); additional iRPE samples in Supplementary Fig. 3t, u). P values were determined by pairwise comparisons using two-tailed t test with 95% confidence interval and Bonferroni correction. The horizontal lines in the boxplots indicate the median, the boxes indicate the first and third quartiles, and the whiskers indicate 5th and 95th percentile.