Supplementary Fig. 1: Additional figure panels describing the primary rpS6 screen and the secondary mTOR translocation screen.

a, rpS6 phosphorylation is a robust and sensitive readout for mTORC1 activation using leucine and various known inhibitors of the mTORC1 pathway. Hs68 cells were starved of serum and amino acids for 2.5 h and restimulated as indicated. Images were quantified by CellProfiler, and the integrated cytoplasmic intensity of phospho-rpS6 was used as a measure of mTORC1 activation. The mean of two independent experiments is shown. b, Screen quality assessed by quantification of siRNA-mediated changes in integrated single-cell rpS6 phosphorylation immunofluorescence intensities for all siControl, siRHEB and siTSC2 wells in the primary screen (n = 384 wells each). Each point represents the average of triplicates for the same well position, normalized by the plate median and the plate s.d. c, The strongest single siRNA effect in each pool of siRNAs dominates the pool behaviour in the primary screen (left). The value of the strongest single siRNA effect is plotted on the x axis against the primary screen value on the y axis. The rpS6 phosphorylation effect of the average of the second and third strongest single siRNA effects is also plotted on the x axis against the primary screen Z-score on the y axis (right). This provides an independent validation that the effect of the siRNA knockdown is specific. d, Pathway analysis (Ingenuity) shows statistical enrichment and the number of deconvolution hits in a subset of canonical pathways (red and green for negative and positive regulators, respectively). Only siRNAs selected as unbiased top hits (988 genes) were used in the analysis. P values were calculated by the right-tailed Fisher’s exact test. e, Image-based correlation analysis shows cycloheximide-stimulated and RagC-dependent colocalization of mTORC1 and Lamp2. Error bars are ±s.d. of the population average; a two-tailed Student’s t-test was used for n = 3 independent experiments.