Fig. 4: RNase1 modulates macrophage polarization through ALK.

a Pull-down assay and Western blot analysis of the interaction between RNase1 and ALK in BMDMs and Raw264.7 cells. b Raw264.7 sgCtrl or sgAlk cells were treated with or without RNase1 (1 μg/ml) for 15 min. Detection of ALK and RNase1 binding was performed using a Duolink assay. Scale bar, 20 μm. c HEK-293T cells were transfected with plasmids containing a control vector (Vec), wild-type ALK (ALK-WT), or a kinase-dead mutant ALK (ALK-I1250T). An immunoblot of the indicated cells treated with RNase1 (1 μg/ml) for 15 min is shown. d Western blot of BMDMs and Raw264.7 cells treated with recombinant RNase1 protein (1 μg/ml) at various time points using the indicated antibodies. e BMDMs were treated with IFN-γ (10 ng/ml) plus LPS (200 ng/ml) for various time points in the presence or absence of RNase1 (1 μg/ml). f BMDMs transfected with ALK siRNAs and treated with or without IFN-γ plus LPS in the presence or absence of RNase1 for 24 h. g, h THP-1–derived macrophages expressing ALK short hairpin RNA (shALK) were treated with IFN-γ plus LPS (g) or IL-4 (h) for 12 h in the presence or absence of RNase1. The indicated mRNA expression in these cells was measured using quantitative polymerase chain reaction and is shown as average values from six independent experiments. The error bars represent (mean ± SD) values. i, j BMDMs were pretreated with an ALKi for 2 h followed by IFN-γ plus LPS and RNase1 (1 μg/ml) for 24 h. The results of one representative experiment are shown in (i), and cumulative data from six independent experiments are presented in (j). The error bars represent mean ± SD values. k BMDMs were pretreated with various doses of crizotinib (Cri) or alectinib (Ale) for 2 h and then activated with IFN-γ plus LPS and RNase1 for 24 h (n = 6 mice per group). a–f, and k Representative results from 3 independent experiments. Statistical analysis: g, h, and j two-sided Unpaired Student’s t-test. Source data are provided as a Source Data file.