(and and and and and = 3 biologically independent experiments. 1 (S6K1), which is a direct substrate of the mTORC1 kinase. Gratifyingly, when compared to the AAVS1 control sgRNA, loss of the hit genes decreased the phosphorylation of S6K1, suggesting that they likely act upstream of mTORC1 activity (Fig. 2and gene, which encodes a key component of the mitochondrial RNase P complex required for tRNA processing (Fig. 3gene (HSD17B10 dox-off cells). Gratifyingly, suppression of HSD17B10 strongly reduced mTORC1 signaling as readout by S6K1 phosphorylation (Fig. 4= 3 biologically impartial experiments. values were determined using a two-sided Students test. * 0.05 (= 3 biologically independent experiments. (and and and and and = 3 biologically impartial experiments. values were determined using a two-sided Students test. (= 3 biologically impartial experiments. (in cells lacking AMPK and HRI (AMPK and HRI TKO). (or strongly inhibits mTORC1 activity is usually consistent with IRS4, rather than IRS1/2, being the main transducer of insulin signaling in HEK293-based Estetrol cell lines, as previously reported (54). It is interesting to consider whether its tissue-specific expression might differentially modulate regulation of mTORC1 by insulin in vivo. Our focused sublibrary approach allowed us to efficiently perform many screens in different genetic backgrounds and to thus systematically define epistatic associations between hundreds of potential hit genes and core stress pathways. Finally, our study led us to concentrate on the relationship between mitochondrial stress and mTORC1. We find that two kinases, AMPK and HRI, but not GCN2, are necessary for signaling oligomycin-induced mitochondrial dysfunction to mTORC1 (Fig. 5for 45 min at 37 C with 8 g/mL polybrene and virus-containing medium. After 12 h, the culture medium was refreshed, and after 24 h, cells were selected with puromycin or blasticidin for 4 d. Pooled Genome-Wide CRISPR-Cas9 Screens. The pooled genome-wide lentiviral sgRNA library (Addgene #1000000100) was prepared and screens were performed as described in ref. 57 with slight Estetrol modifications. Briefly, 600 million HEK293T cells were transduced with the viral pool to achieve 1,000-fold library coverage after puromycin selection. After 72 h of puromycin selection, 200 million cells were passaged and expanded every 2 d until day 7 when 1 billion cells were plated into fibronectin-coated 15-cm dishes. On days 8 and 10, the Estetrol cells were washed three times with phosphate-buffered saline (PBS) supplemented with 1 mM CaCl2 and 500 M MgCl2 and placed in RPMI lacking amino acids and glucose for 3 h. After 3 h, amino acids and glucose were added for 1 h and the cells harvested. Cells were collected by washing once with ice-cold PBS, after which Accumax answer (Sigma) was added, and Estetrol plates were nutated at 4 C for 10 min. After 10 min, cells were collected into a 50-mL centrifuge tube (Celltreat) and pelleted via centrifugation at 4 C at 300 for 3 min. Subsequently, cells were fixed by resuspension in formalin for 5 min at room heat. At 5 min cells were pelleted via centrifugation at 4 C at 300 for 3 min. Finally, cells were permeabilized by resuspension in 90% methanol:PBS. After permeabilization cells were stored at ?20 C until staining. Cell Staining and FACS. A total of 500 million previously fixed and permeabilized cells were pelleted by centrifugation at 4 C at 300 for 3 min and then washed once with ice-cold PBS. Cells were then blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. For primary antibody staining, cells were incubated with primary antibody diluted (1:100) in 0.05% BSA Sema3d in PBS at room temperature for 1 h. After one wash with 0.05% BSA in PBS, cells were resuspended in Estetrol secondary antibody diluted (1:200) in 0.05% BSA in PBS and incubated for 1 h. Finally, cells were washed twice in 0.05% BSA.