Figure 3.

CRISPR screen and cytokine array reveal increased IL-20 expression. (A) Generation of RAW264.7 Ctsk-T2A-R647 reporter cell line stably expressing SpCas9-P2A-GFP, and analysis of Ctsk expression in the APC channel by flow cytometry on day 5 of osteoclast differentiation. The percentage of the APC⁺ population is calculated from the total GFP⁺ population and shown as boxplots. n = 16. (B) CRISPR screen for genes regulating osteoclast differentiation in the Dnmt3a KO Ctsk reporter. sgRNA-targeted genes ranked (x axis) by log2-fold change of read counts (y axis). The top right indicates genes enriched in the population with lower Ctsk reporter expression (bottom APC 5%) over higher Ctsk reporter expression (top APC 5%). These genes potentially enhance or are required for osteoclast differentiation. FDR < 0.05 (red). Cytokine receptors identified in the higher APC-expressing populations (bottom left) are not shown to due to cell fusion occurring during osteoclast differentiation. (C) Cytokine array of Dnmt3a^−/− versus WT serum. n = 13–14. Statistical significance via Wilcoxon rank sum test. FDR < 0.05 is denoted by a horizontal dotted line. (D) ELISA quantification of IL-20 from WT or Dnmt3a^−/− BMDMs (n = 10) or RAW264.7 cells (n = 12) cultured at a density of 50,000/cm² for 4 d. Error bars represent SD. *, P < 0.05; ***, P < 0.001.