Fig. 1: The Nox2 subunit Ncf1 facilitates neurosphere formation in a non-cell autonomous manner by regulating Igfbp2 secretion and activity.

a–c Non-adherent cultures of primary postnatal forebrain NSCs were established from WT and Ncf1^–/– mice and 2^nd passage NSCs were grown for 11 days. a Photomicrographs of NSCs after 11 days in culture. b Quantification of neurospheres ≥60 µm in diameter (n = 5 donors). c Quantification of total cells in each NSC culture (n = 10 donors). d–f Monolayer cultures of 2^nd passage neonatal NSCs were grown for 3 days with L-VNIO (100 µM) or vehicle. NSCs were pulsed with EdU (5 µM) for 4 h before fixation. d Photomicrographs of EdU (Red) and TUNEL (Green) labeling. e Quantification of EdU⁺ NSCs. f Quantification of cell death (n = 6 donors). g Photomicrographs of NSC cultures 5 days after plating 2^nd passage of Ncf1^–/– Tomato⁺ NSCs, in either direct coculture or transwell insert coculture with Ncf1^+/+ Tomato^– NSCs. h Quantification of neurospheres ≥60 µm in diameter (n = 8, 12, 10, and 6 donors in WT, coculture, transwell coculture, and Ncf1^–/– groups, respectively). i Western blot analysis and quantification comparing Igfbp2 levels in the conditioned medium 5 days after plating (n = 5 donors). The samples were derived from the same experiment and blots were processed in parallel. kD = kilodalton. Two-tailed Mann–Whitney U test (b, c, i) and Kruskal–Wallis (see Source data for full details) followed by Benjamini–Hochberg FDR multiple comparison posttest (e, f, h). Error bars indicate s.e.m. Scale: 200 µm. Source data are provided as a Source data file.