Figure 5

Treatment with PR5-LL-CM01 decreases p65me2, NF-κB activity, and NF-κB target gene expression in HRECs and iCEC2 cells. (a,b) Immunoblots and quantification, indicating inhibition of p65-R30me2 in a stepwise manner with increasing concentrations of PR5-LL-CM01 in HRECs (a) or iCEC2 cells (b) overexpressing FLAG-tagged wt-p65. Lower panels show ImageJ quantification of p65-R30me2 relative to loading control (β-actin) for three independent immunoblots. *p < 0.05 vs. 0 µM PR5-LL-CM01 group. See also Supplementary Fig. S4. (c,d) NF-κB luciferase assay. wtPRMT5 overexpressing HRECs (c) or iCEC2 cells (d) were stimulated with 10 ng/ml IL-1β ± 3 µM PR5-LL-CM01 for 4 h. PRMT5 overexpression augmented NF-κB induction upon IL-1β treatment, while PR5-LL-CM01 inhibitor treatment reduced this effect. *p < 0.05 vs. IL-1β untreated group; ^#p < 0.05 vs. IL-1β-induced group; ^$p < 0.05 vs. pLV-empty + IL-1β-treated group. (e,f) Both shScramble and shPRMT5 HRECs (e) or iCEC2 cells (f) were stimulated with 10 ng/ml IL-1β for 4 h. shPRMT5 inhibited NF-κB activity upon IL-1β treatment. *p < 0.05 vs. IL-1β untreated group; ^$p < 0.05 vs. shScramble + IL-1β-treated group. n = 3–4 technical replicates. (g,i) HREC vector cells or (h,j) iCEC2 vector cells were stimulated with 10 ng/ml IL-1β ± 3 µM PR5-LL-CM01 for 4 h. PR5-LL-CM01 significantly decreased the expression of TNFA (g,h) and VEGFA (i,j) mRNA. Likewise, shPRMT5 reduced TNFA and VEGFA levels. *p < 0.05 vs. IL-1β untreated and PR5-LL-CM01 untreated group; ^#p < 0.05 vs. IL-1β-induced group, and PR5-LL-CM01 untreated group; ^$p < 0.05 vs. shScramble group. Mean ± SD, n = 3–4 biological replicates. Unpaired Student’s t-test with Welch’s correction was used for comparing two means, and one-way ANOVA with Dunnett’s post hoc tests was used when comparing more than two means.