Fig. 1: NMNAT1-positive cells inhibit SARM1 activation in neighboring cells through gap junction intercellular communication.

a HEK-293T cells were transfected with expression vectors encoding Flag-tagged NMNATs or an empty vector control (Vec). The cells were subsequently treated with 100 μM CZ-48 or PBS and incubated for 24 h before being lysed. Levels of cADPR and proteins were measured using a cycling assay and Western blot, respectively. b Wildtype and NMNAT1-overexpressing HEK-293T cells were mixed in the indicated ratio and treated with or without 100 μM CZ-48 for 24 h. cADPR contents were measured using the cycling assay. c Wildtype and NMNAT1-knockout HEK-293T cells were mixed in the indicated ratio and cultured for 24 h, followed by cADPR measurement. d Wildtype and NMNAT1-knockout HEK-293T cells were co-cultured in a Boyden Chamber, with one cell line in the upper chamber and another in the lower chamber, for 24 h. cADPR levels were measured in the cells in the lower chamber. e NMNAT1-KO HEK-293T cells were cultured with the conditioned medium from wildtype or NMNAT1-knockout cells for 24 h, followed by cADPR measurement. f, g HEK-293T cells expressing EGFP were co-cultured with NMNAT1-knockout HEK-293T cells in a 1:5 ratio in a 24-well plate or 6-cm dish. After 24 h, fluorescent or bright-field images were captured under a fluorescent microscope (f), and the levels of NMN, NAD, and cADPR (g) were measured by the cycling assay. Scale bar: 20 μm. h Western blot of Cx43, Cx45 and Cx46 proteins in wildtype or knockout cells generated by the CRISPR/cas9 technique. i NAD levels were measured in connexin-knockout cells or a 1:1 mixture of connexin-knockout and NMNAT1-knockout cells. All experiments were repeated at least three times, and data are presented as means ± SDs (n = 3). P values (ns, P > 0.05; ***P < 0.001; ****P < 0.0001) were determined by Student’s t test. One-way ANOVA tests were performed in a–d before t-test.