Extended Data Figure 3 (to Figure 1). SWI/SNF complex represses expression of nutrient sensor Castor1 to support mTORC1 activation.
(a) Quantification (normalized to ‘spike’) of relative p-S6 level, cell size (FSC-A) and CD71 expression in induced Treg cells transduced with indicated sgRNAs followed by stimulation with α-CD3 antibody for 3 h to measure p-S6 level, or with α-CD3/CD28 antibodies for 20 h to measure cell size (FSC-A) and CD71 expression by flow cytometry (n = 3 samples each group). (b) Imaging analysis and quantification of lysosome-associated mTOR [based on mean fluorescence intensity (MFI)] in sgNTC- or sgSmarcb1-transduced cells that were stimulated with α-CD3 antibody for 3 h, starved of amino acids (AA), and refed AA for 20 min (n > 230 cells per condition). (c) Volcano plots of expression levels of transcripts, including Castor1, in sgNTC or sgSmarcb1 (both Ametrine+)-transduced cells that were stimulated with CR for 3 h (n = 4 samples each group). (d) Castor1 mRNA expression in sgNTC- or sgSmarcb1 (both Ametrine+)-transduced cells were stimulated with α-CD3 antibody for 3 h or with α-CD3/CD28 antibodies for 20 h (n = 3 samples per group). (e) sgNTC or sgSmarcb1 (both Ametrine+)-transduced cells were left unstimulated (indicated by 0 h) or stimulated with α-CD3 antibody for 3 h or α-CD3/CD28 antibodies for 20 h. Immunoblot analysis and quantification of relative Castor1 expression (n = 3 samples each group). (f) Immunoblot analysis and quantification of relative p-S6K1 and p-S6 levels in cells transduced with empty vector or vector expressing Castor1, followed by stimulation with α-CD3/CD28 antibodies for 2 d (n = 3 samples each group). Mean ± s.e.m. (a, b, d–f). **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t-test (f); one-way ANOVA (a, b, d, e). Data are representative of one (c) or two (b), or pooled from two (d, e) or three (a, f) experiments.