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. 2021 Oct 12;12:763067. doi: 10.3389/fimmu.2021.763067

Figure 5.

Figure 5

CYP26A1 deficiency affected the polarization, phagocytic capacity and NO production capacity of Raw264.7 cells. (A) qPCR analysis of M1 phenotype genes Il6, Nos2, Tnfa, Cd86 and Il1b in CYP26A1-deficient Raw264.7 and WT cells after induced 4 h by LPS and IFN-γ (n=3). (B) ELISA was used to measure IL-6 and TNF-α secretion in supernatants of Raw264.7 cells (WT and CYP26A1−/−) treated with LPS and IFN-γ for 24 h; Mean fluorescence intensity analysis of CD86 expression levels in Raw264.7 cells (WT and CYP26A1−/−) incubation with LPS and IFN-γ for 24 h (n=3). (C) qPCR analysis of M2 markers Arg1 and Cd206 in CYP26A1 knockout Raw264.7 and WT group after induced 4 h by IL-4 and IL-13 (n=3). (D) Western blot analysis of M2 markers ARG1 and CD206 in CYP26A1 knockout Raw264.7 and WT group after induced 24 h by IL-4 and IL-13 (n=3). GAPDH was used as loading control. (E–G) Flow cytometry analysis of the phagocytic capacity of Raw264.7 (WT and CYP26A1−/−) under M0, M1 and M2 state (n=4). (H) The concentration of NO in the supernatant of RAW264.7 cells (WT and CYP26A1−/−) treated with LPS and IFN-γ at different induction time (12 h, 24 h, 36 h, 48 h, 72 h) was detected by griess reagent (n=4). Error bars represent means ± SEM; two-tailed unpaired t-test, ns, not significant, *P < 0.05, **P < 0.01, or ***P < 0.001.