Extended Data Fig. 1: Analyses of autophagy, mTORC1 signalling and NSCs from SVZ tissues and neurospheres.
From: Autophagic lipid metabolism sustains mTORC1 activity in TSC-deficient neural stem cells
a, Brain size (upper panel) and H&E staining of sagittal sectioned brain (lower panel) of Ctrl, Tsc1GFAPcKO, 2cKO and Fip200GFAP cKO mice at P21. Five independent experiments gave similar results. b, H&E staining of sagittal sectioned brain at two positions of Ctrl, Tsc1GFAPcKO, 2cKO and Fip200GFAP cKO mice at P0. Mean ± s.e. of the number of SVZ cells in Ctrl, Tsc1GFAPcKO, 2cKO and Fip200GFAP cKO mice at P0 shown on the right. n = 5 independent experiments. c,d, The levels of p62, Fip200 and Tsc1 (c) and LC3 (d) in isolated SVZ tissue of Ctrl, Tsc1GFAPcKO, 2cKO and Fip200GFAPcKO mice treated with or without CQ for 14 d. Three independent experiments gave similar results. e, Means ± s.e. of the autophagy flux (calculated as LC3-II with CQ divided by LC3-II without CQ as shown in b). n = 3 independent experiments. f,g, Immunofluorescence of p62 (f), p4EBP1 (phosphorylated at T37/46) (g) and DAPI in SVZ of Tsc1GFAPcKO and 2cKO mice at P21. Bottom panels in g show details of p4EBP1+ cells in boxed area. h, Mean ± SE of the percentage of p4EBP1+cells in SVZ of Ctrl, Tsc1GFAP cKO, 2cKO, and FIP200GFAP cKO mice at P21. n = 4 independent experiments. i,j, Mean ± SE of the number of GFAP+Sox2+NSC (i) and the number of GFAP+Nestin+BrdU+ cells of total BrdU+ cells (j) in SVZ of Ctrl, Tsc1GFAP cKO, 2cKO, and FIP200GFAP cKO mice at P21. n = 6 independent experiments. k–m, Mean ± SE of the number (k) and size (l,m) of primary (k,l) and secondary (m) neurospheres from SVZ cells of Ctrl, Tsc1GFAP cKO, 2cKO, and FIP200GFAP cKO mice at P21. n = 4 (k), 3 (l,m) independent experiments. n, Lysates from primary neurospheres of Ctrl, Tsc1GFAPcKO, 2cKO, and FIP200GFAPcKO mice examined by immunoblotting with indicated antibodies. Three independent experiments gave similar results. o, The level of LC3 in neurospheres of Ctrl, Tsc1GFAPcKO, 2cKO, and FIP200GFAPcKO mice treated with or without BafA1. Three independent experiments gave similar results. The dotted lines indicated the boundaries of SVZ with LV (f,g). E: ependymal layer; LV: lateral ventricle; ST: striatum; SVZ: subventricular zone. Bar = 100 mm. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test (b,e,h–m).
