Figure 2.

Jmjd3 is essential for M2 microglia polarization depending on its demethylase activity. (a) Jmjd3 expression in the N9 microglia was knocked down by infection with pLKO-kdNC and pLKO-kdJmjd3 lentiviruses. The protein expression of Jmjd3, UTX and H3K27me3 was detected using immunoblotting. (b) Real-time PCR was performed to determine the mRNA level of Jmjd3 and UTX in the infected N9 cells. (c) Lentivirus-infected N9 cells were plated in a 96-well plate and the cell viability was measured using the MTT assay. (d–g) The Jmjd3-knockdown N9 cells were stimulated with IL-4 (10 ng/ml) for 24 h. The mRNA expression of related M2 markers was then determined using real-time PCR. (h) Jmjd3 expression was knocked down in PM cells followed by treatment with IL-4 (10 ng/ml) for 24 h. Arginase1 expression was then assessed using real-time PCR. (i and j) Lentivirus-infected N9 cells were treated with IL-4 (10 ng/ml) or LPS (100 ng/ml) for 24 h and immunoprecipitated with anti-H3K27me3 (i) and anti-Jmjd3 (j) antibodies. ChIP DNA levels were analyzed using semi-quantitative PCR and normalized to the levels of input DNA. (k) The JmjC domain was introduced into N9 cells by lentivirus (pLVX-JmjC) infection followed by treatment with M1 and M2 triggers, respectively, for 24 h. The arginase activity was then measured. (l) N9 cells were infected with pLVX-JmjC- and pLVX-JmjCm-overexpressed lentiviruses and treated with IL-4 (10 ng/ml) for 24 h. The mRNA expression of Arginase1 was then assessed using real-time PCR. (m) The H3K27me3 level in the infected N9 cells was determined using the immunoblotting assay. *P<0.05, **P<0.01, ***P<0.001, n.s, not significant