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

Fig. 4. Activation of NF-κB pathway downstream of C5aR1 and C3aR1 signaling leads to sub-RPE APOE deposits in CFH(Y/Y)-iRPE and CFH(H/H)-iRPE.

Fig. 4

a, b Nuclear translocation of p65 (red) in CC-HS treated CFH(Y/Y)-iRPE2 cells shows NF-κB activation. ZO-1 (green) (N = 7 biologically independent experiments, iRPE1, 2, 3, 4, 5, 6, 8). c qRT-PCR confirms increased expression of NF-κB target genes in CC-HS treated iRPE (N = 4 independent experiments, iRPE1, 8). Genes with more than 4× expression changes and P < 0.05 are deemed significant. Exact P values are plotted in the figure, P values were determined by pairwise comparisons using two-tailed t test with 95% confidence interval and Bonferroni correction. d ELISA shows CC-HS-treated iRPE have twofold higher apical and basal secretion of IL-8, an NF-κB target cytokine (N = 3 independent experiments and N = 2 biological replicates, iRPE1, 9). 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. e–g CFH(Y/Y)-iRPE2 cells treated with sera depleted in complement factor D (CFD), C3, or C5 do not show nuclear translocation of p65 (red), ZO-1 (green) (N = 7 biologically independent replicates, iRPE1, 2, 3, 5, 6, 7, 8). h CI-HS treated iRPE derived from a patient with E391X mutation in protein NEMO demonstrate constitutive nuclear localization of p65 (red, arrowhead) ZO-1 (green), (N = 3 independent experiments, NEMO-iRPE). i, j APOE (red) positive sub-RPE deposits in NEMO (E391X)-iRPE treated with CI-HS as compared to CI-HS treatment in CFH(H/H)-iRPE5 (j) (N = 3 independent experiments, NEMO-iRPE and iRPE5. P value was 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.