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. 2021 Mar 3;11:644608. doi: 10.3389/fonc.2021.644608

Figure 3.

Figure 3

Plinabulin induces M1-macrophage proliferation and pro-inflammatory cytokine release. (A) Experimental outline of macrophage generation from healthy donor PBMCs, CTV labeling and treatment with plinabulin or controls prior to analysis by flow cytometry and multiplex cytokine analysis. (B) Histograms showing CTV expression, i.e., proliferation of CD86+ (left) or CD206+ (right) human macrophages treated for 48 h with plinabulin (1,000 or 200 nM), IL-4 (25 ng/mL), LPS (25 ng/mL), and IFN-γ (50 ng/mL), combination or untreated. (C) Quantification of CTV signal as gMFI in CD86+ (left) or CD206+ (right) human macrophages treated for 48 h with plinabulin or control treatments. (D) Percentage of AnnexinV+ cells (left) and gMFI of AnnexinV (right) in human macrophages treated for 48 h with plinabulin or control conditions. (E) Quantification of pro-inflammatory cytokinesIL-1β IL-6 and IL12p40 in the supernatant of human macrophages from four healthy donors treated for 0, 24, or 48 h with plinabulin (top) or LPS (25 ng/mL), and IFN-γ (50 ng/mL), combination treatment (bottom). (F) Quantification of iNOS mRNA expression by qPCR in human macrophages after 4 or 8 h of treatment with plinabulin or LPS (25 ng/mL), and IFN-γ (50 ng/mL), combination. (C–F) Statistical significance was determined by one-way Anova with multiple comparisons to control group (untreated cells for C,D; 0 h for E,F). P-values indicated on the graphs: *p < 0.05, **p < 0.01, ****p < 0.0001. Error bars show SD. (C,D) Data are derived from two independent experiments with samples pooled from two donors. Each symbol represents a pooled sample.