Fig. 5: Neudesin suppresses erythrophagocytosis and expression of FcγRs through ERK activation in bone marrow-derived macrophages.

a Scheme of experiments to measure phagocytosis of bone marrow-derived macrophages (BMDMs) in vitro. At 1 h after adding fluorescent baits, cells were collected and analyzed by flow cytometry to detect phagocytosis by BMDMs (n = 9). b Percentages of induced F4/80⁺ macrophages from WT and neudesin KO bone marrow cells (n = 9, P = 0.5997 by unpaired t-test). c Percentage of phagocytosis by BMDMs against RBCs, apoptotic thymocytes, and killed E. coli. *P < 0.05 by unpaired t-test (n ≧ 8, RBCs: P = 0.0164, apoptotic thymocytes: P = 0.2423, and E. coli: P = 0.3560 by unpaired t-test). d Pre-treatment with recombinant neudesin (100 ng/mL) for 2 days into the culture medium inhibited enhanced erythrophagocytosis by neudesin KO BMDMs (n = 6 each group, Control:WT vs. Control:KO: P = 0.0231, Control:WT vs. + neudesin:WT: P = 0.9998, Control:WT vs. + neudesin:KO: P = 0.9994, Control:KO vs. + neudesin:WT: P = 0.0192, Control:KO vs. + neudesin:KO: P = 0.0297, and + neudesin:WT vs. + neudesin:KO: P = 0.997 by a 2-way analysis of variance followed by Sidak’s post-test). e Mean fluorescence intensity (MFI) of surface macrophage receptors on BMDMs induced from WT and neudesin KO bone marrow cells were measured by flow cytometry. The effects of pre-treatment with recombinant neudesin (100 ng/mL) were also investigated (n = 6 each group, Fcgr1; Control:WT vs. Control:KO: P = 0.0001, Control:WT vs. + neudesin:WT: P = 0.371, Control:WT vs. + neudesin:KO: P = 0.972, Control:KO vs. + neudesin:WT: P < 0.0001, Control:KO vs. + neudesin:KO: P = 0.0003, and + neudesin:WT vs. + neudesin:KO: P = 0.19, Fcgr2/3; Control:WT vs. Control:KO: P = 0.0234, Control:WT vs. + neudesin:WT: P < 0.0001, Control:WT vs. + neudesin:KO: P = 0.0014, Control:KO vs. + neudesin:WT: P < 0.0001, Control:KO vs. + neudesin:KO: P < 0.0001, and + neudesin:WT vs. + neudesin:KO: P = 0.1137, and Fcgr4; Control:WT vs. Control:KO: P = 0.9866, Control:WT vs. + neudesin:WT: P = 0.3411, Control:WT vs. + neudesin:KO: P = 0.9993, Control:KO vs. + neudesin:WT: P = 0.2027, Control:KO vs. + neudesin:KO: P = 0.9966, and + neudesin:WT vs. + neudesin:KO: P = 0.2835 by a 2-way analysis of variance followed by Sidak’s post-test). f Cell lysates from WT and neudesin KO BMDMs were extracted 30 min after incubation with recombinant neudesin (100 ng/mL). Phosphorylated ERK1/2, ERK1/2, phosphorylated AKT, AKT, phosphorylated CREB, CREB, and β-Actin were detected by western blotting. g Relative expression of p-ERK/ERK, p-AKT/AKT, and p-CREB/CREB was determined (n = 4, p-ERK/ERK Control: P = 0.0435, p-ERK/ERK +neudesin: P = 0.3870, p-AKT/AKT Control: P = 0.8851, p-AKT/ AKT +neudesin: P = 0.3878, p-CREB/CREB Control: P = 0.6681, p-CREB/CREB +neudesin: P = 0.8863 by a 2-way analysis of variance followed by Sidak’s post-test)). h Pre-treatment with MEK inhibitor U0126 (100 nM) for 2 days into the culture medium enhanced erythrophagocytosis by WT, but not neudesin KO BMDMs (n = 6 each group, Control:WT vs. Control:KO: P = 0.0457, Control:WT vs. + U0216:WT: P = 0.0238, Control:WT vs. + U0216:KO: P = 0.0336, Control:KO vs. + U0216:WT: P = 0.9894, Control:KO vs. + U0216:KO: P = 0.9988, + U0216:WT vs. + U0216:KO: P = 0.9984 by a 2-way analysis of variance followed by Sidak’s post-test). i Pre-treatment with MEK inhibitor U0126 (100 nM) for 2 days into the culture medium enhanced surface expression of FcγR1 on WT, but not neudesin KO BMDMs. (n = 6 each group, Control:WT vs. Control:KO: P = 0.0019, Control:WT vs. + U0216:WT: P = 0.0034, Control:WT vs. + U0216:KO: P = 0.0004, Control:KO vs. + U0216:WT: P > 0.9999, Control:KO vs. + U0216:KO: P = 0.9897, + U0216:WT vs. + U0216:KO: P = 0.9446 by a 2-way analysis of variance followed by Sidak’s post-test)*P < 0.05, and ***P < 0.001 by a 2-way analysis of variance followed by Sidak’s post-test. Each symbol represents an individual mouse. Data are shown as means ± SD of three or more experiments.