Extended Data Fig. 5: Beclin 1 C137 palmitoylation regulates PI3KC3-C1 assembly.

a, BECN1-KD H4 cells expressing mCherry-tagged WT Beclin 1 or C137S mutant were analysed for the localization of mCherry-Beclin 1 and TGN46. Pearson’s coefficient was calculated from 20 cells that expressing mCherry-Beclin 1 of one representative experiment out of n = 3 independent experiments and was shown on the right as box plot with whiskers from minimum to maximum. b, c, BECN1-KD (b) or DHHC5-WT/KD (c) H4-GFP-LC3 cells expressing mCherry-tagged WT Beclin 1 or C137S mutant were analysed for the localization of mCherry-Beclin 1 and GFP-LC3 without or with Torin-1 (200 nM, 8 h) using Operetta CLS High Content Analysis System. mCherry⁺GFP⁺ puncta and GFP⁺ puncta were identified and quantified for each cell automatically. Ratio of mCherry⁺GFP⁺/GFP⁺ was calculated for the determination of Beclin 1 recruitment to autophagosomes. d, HEK293T cells were co-transfected with HA-Beclin 1 and Flag-tagged WT or mutant Beclin 1 as indicated for 24 h. The interaction of HA-Beclin 1 and Flag-Beclin 1 was detected by IP-IB assay. e, HEK293T cells were co-transfected with expression vectors of HA-DHHC5 and Flag-tagged WT or mutant Beclin 1 as indicated for 24 h. The interaction of HA-DHHC5 and Flag-Beclin 1 was detected by IP-IB assay. f, Quantification of proteins in Flag-IPs in Fig. 4b. g, BECN1-KD H4 cells expressing WT murine Beclin 1 (mBeclin 1) or C135S mutant mBeclin 1 were subjected to IP-IB of mBeclin 1/VPS34 complex. Quantification of indicated proteins in Flag-IPs is shown below. h, Quantification of proteins in ATG14L and UVRAG-IPs in Fig. 4c. Similar results were observed in n = 3 independent experiments (d, e). Quantification of indicated proteins is relative to Beclin 1-WT control for each independent experiment (f-h). Bars represent mean ± s.e.m. of n = 3 independent experiments (f-h). The box plot with whiskers from minimum to maximum represents 100–200 cells analysed for one of n = 3 independent samples per condition (b, c). Boxes represent quartiles, center lines denote medians, and whiskers extend to most extreme values within 1.5 × interquartile range (a-c). Unpaired two-tailed t-tests (a-c).

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