Extended Data Fig. 6. mAtg8s affect calcium fluxes and Stx17 affects mTOR and TFEB.

a, A volcano plot showing expression of calcium effectors in Hexa^KO cells. P values were calculated using Fisher’s exact test adapted for over-dispersed data (see methods) (n=3 biologically independent experiments). b,c Flow cytometry using FLUO-3AM to detect intracellular calcium in HeLa^WT or Hexa^Ko. Data, means ± SEM; (n=3 biologically independent experiments) ANOVA, Tukey’s post hoc test. d, Confocal microscopy analysis of the effects of Stx17^KO on TFEB localization, (n=3 biologically independent experiments). Scale bar 5 μm. e–g, confocal microscopy (e) and HCM (f,g) analyses of the effects of Stx17^KO on colocalization between TFEB and LAMP2. Scale bar 5 μm (e). Scale bar 10 μm (f). Data, means ± SEM; (n=3 biologically independent experiments) ANOVA, Tukey’s post hoc test. h, HCM analysis of the effects of Stx17^KO on TFEB puncta. Data, means ± SEM; (n=3 biologically independent experiments) ANOVA, Tukey’s post hoc test. i, Co-IP analysis of interactions between GFP-Stx17 and FLAG-TFEB in 293T cells (n=3 biologically independent experiments). j,k, Co-IP analysis of effects of GFP-Stx17 on FLAG-TFEB and IRGM complexes. Data, means ± SEM (n=3 biologically independent experiments) paired t-test. l,m, MS analysis showing 14-3-3 peptides those interacted with GFP or GFP-Stx17 and GFP-IRGM (n=3 biologically independent experiments). n–p, Western blot analysis and quantification of the effect of GFP-Stx17 in HeLa^WT (full media) or in Stx17^KO cells (EBSS 2h) on mTOR activity. Data, means ± SEM; (n=3 biologically independent experiments) ANOVA, Tukey’s post hoc test. q–s, Western blot analysis and quantification of pULK1 and pS6K to test the effects of GFP-Stx17 expression in WT 293T cells and cells expressing RagB^Q99L. Data, means ± SEM; (n=3 biologically independent experiments) ANOVA, Tukey’s post hoc test. t–w, Co-IP analysis of interactions between RagA and FLAG-p18 (t,u) and Raptor and FLAG-RagA (v-w) in Stx17^KO or parental HeLa cells. Data, means ± SEM of normalized intensities (n=3 biologically independent experiments) paired t-test. Uncropped blots for panels i, j, n, q, t and v are provided in Unprocessed Blots Data Extended Fig. 6 and numerical source data for panels b, f, h, k, p, o, r, s, u and w are provided in Statistical Source Data Extended Fig. 6.