RPS6 and infection: We first utilised primary mouse embryo fibroblasts (MEFs) to establish RNAi tools to knockdown Tsc1 and Tsc2. Infection with lentiviruses expressing shRNA-Tsc1 or shRNA-Tsc2 efficiently reduced TSC1 or TSC2 protein abundance, increased phosphorylation of ribosomal protein S6 (Ser240/244) and 4E-BP1 (Thr37/46), indicative of mTORC1 activation, and caused accumulation of HIF-1α and the HIF-1α inducible protein GLUT1 (Supplementary Fig. 1a), mimicking previously published effects of knockout of Tsc1 or Tsc2 in MEFs23–25.