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. 2021 May 17;131(10):e126089. doi: 10.1172/JCI126089

Figure 4. Pros1-deficient macrophages exhibit a proinflammatory profile regulated by the NF-κB pathway.

Figure 4

(A) Cytokine transcript levels from cultured BMDMs derived from tumor-naive control and Pros1-cKO mice measured by RT-qPCR. Relative mean expression ± SEM, n = 6–12 mice/group. *P < 0.05, ***P ≤ 0.001 (P = 0.02, 0.001, 0.03, 0.049, and 0.0003 for Tnfa, Nos2, Il-6, Il-10, and Socs3, respectively); t test. (B) ELISA measurements of TNF-α, IL-6, and IL-10 from BMDM-CM isolated from tumor-naive Pros1-cKO and control mice. Average cytokine values ± SEM are shown for 8–11 mice. ***P ≤ 0.001, ****P ≤ 0.0001 (P = 0.0001, 0.0006, and 0.001 for TNF-α, IL-6, and IL-10, respectively); t test. (C) Gas6 and MerTK expression in control and Pros1-ablated BMDMs; n = 5–6 mice/group; t test. (D and E) Representative Western blot analysis of pNF-κB and NF-κB from BMDMs at steady state (grown in 10% FBS), and (E) band intensities (n = 6/group). **P < 0.01 (=0.009). (F–I) Western blot analysis of the NF-κB pathway. (F) Representative blots of cell lysates showing pNF-κB, NF-κB, and band intensities (G) (*P = 0.04, **P = 0.01) of LPS-stimulated (100 ng/mL) serum-starved BMDMs derived from tumor-naive control and Pros1-cKO mice and of IκBα (H and I; *P = 0.05, ***P = 0.001, ****P < 0.0001). β-Actin served as loading control. Graphs indicate the mean ± SEM; n = 6; 2-way ANOVA. (J) ELISA measurements of TNF-α production by control and Pros1-cKO serum-starved and LPS-treated (500 ng/mL) BMDMs at 0, 4, and 8 hours after LPS challenge. Average ± SEM; n = 6–8 mice. *P = 0.048, ****P < 0.0001; 2-way ANOVA. NS, nonsignificant.