CD40 knockdown and CD40-luciferase assay in BL2 cells.

<p>(A) Schematic of the canonical CD40 – NF-|B signaling pathway in B cells. (B) RNAi perturbation of <i>CD40</i> in two distinct clones derived from BL2 cells decreases CD40 protein levels by 55% (left) and 40% (middle) compared to the BL2 parent line (black, right); (C) More CD40 on the surface of BL2 cells increases RelA (p65) phosphorylation following activation with tCD40L, as measured by Western blot, with maximum activation at 15 minutes. Results are shown for the same two shRNA lines and parental BL2 cell line as in (B). This is a representative example of multiple experiments. (D) Titration of tCD40L leads to increased luciferase activity. Each experiment was performed in triplicate. The red circle represents ∼80% maximum luciferase activity (64 ng/ml tCD40L). Luciferase activity at baseline (i.e., no tCD40L activation) was subtracted from each measurement to plot results. (E) Titration of IKK inhibitor VII leads to inhibition of luciferase activity following tCD40L activation. Each experiment was performed in duplicate. (F) The luciferase assay is robust, with Z'-factor>0.80 and >60-fold inhibition of luciferase activity without killing cells across different plates.</p

Relative IC₅₀ for two “known” and two “novel” compounds in both BL2-NFκB-Luc and Ramos-NFκB-Luc cell lines.

<p>Relative IC₅₀ is the concentration required to bring the dose-response curve to the halfway point between the top and bottom plateaus of the curve.</p

Genetic data on risk of RA and CD40 protein levels.

<p>(A) The regional association plot from analysis of Immunochip (iChip) data in 7,222 CCP+ cases and 15,870 controls. Gene location is shown along the bottom of the graph, with observed –log(P) value along the left Y-axis and recombination rate along the right Y-axis. Each SNP is plotted is a circle, with color scheme (red to white) in reference to the extent of linkage disequilibrium with the index SNP, rs4810485 (labeled as a diamond). (B) The regional association plot from analysis of iChip data and CD40 protein levels in 90 healthy control individuals. (C) A box-whisker's plot of SNP (rs4810485) and CD40 protein levels in B cells from healthy control individuals, where T = non-risk allele and G = risk allele. (D) A box-whisker's plot of SNP (rs4810485) and <i>CD40</i> mRNA levels in PBMC's from two separate collections (total of 1,441 healthy control individuals); T = non-risk allele and G = risk allele.</p

Percent maximum inhibition for two “known” and two “novel” compounds in both BL2-NFκB-Luc and Ramos-NFκB-Luc cell lines.

<p>For each compound, we calculate the maximum amount of inhibition observed at the highest concentration of drug relative to zero luciferase activity.</p

Small molecule screen of CD40-mediated NF-kB signaling in BL2 cells.

<p>(A) Results from duplicate experiments screening 1,982 compounds. Red circles are our positive control (IKK inhibitor VII); grey circles are our neutral controls (DMSO only); and blue circles are test compounds. The red dashed line indicates >2SD from the mean of the neutral controls, which defines our “hit” compounds (n = 81 compounds). (B) Dose-response curves for two compounds known to inhibit inflammation [CID = 5282230 (tranilast)] or NF-|B signaling [CID = 5282360 (4-hydroxy-estradiol)] in the BL2-NF|B-Luc cell lines. (C) Dose-response curves for two compounds not previously implicated in inflammation, NF-κB signaling, CD40 signaling, or other biological pathways related to rheumatoid arthritis: CID = 306804, [4-(1-acetyl-4-oxo-2H-3,1-benzoxazin-2-yl)phenyl] acetate; and CID = 7309015, 8-[(Z)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]-7-hydroxy-4-methylchromen-2-one. Red line = cells activated with tCD40L; black line = cells activated with either CD40 or LPS (in BL2-TLR4-NFκB-Luc cells); green line = cell toxicity, as measured by CellTiter-Glo.</p