Bulk RNA sequencing of mouse embryonic fibroblasts in autophagy processes

Autophagy, a catabolic process to remove unnecessary or dysfunctional cells, is triggered by various signals including nutrient starvation. Depending on the type of the nutrient deficiency, diverse sensing mechanisms and pathways are used for autophagy, suggesting subsequent nutrient dependent transcriptional regulation. Still, however, our knowledge about nutrient specific transcriptional regulation during autophagy is limited. To understand nutrient type dependent transcriptional mechanisms during autophagy, we performed single cell RNA sequencing (scRNAseq) for the mouse embryonic fibroblasts (MEFs) before and after applying glucose- (GS) as well as amino acid starvation (AAS). Trajectory analysis using scRNAseq identified sequential induction of potential transcriptional regulators for each deficiency condition. Gene regulatory rules inferred using TENET newly identified CCAAT/enhancer binding protein ? (C/EBP?) regulates autophagy processes specifically to AAS condition. Strikingly, knockdown of C/EBP? attenuated the autophagic process only in the AAS condition. Cell biological and biochemical studies validated that C/EBP? plays a switching role for ATF4 to activate autophagy genes under AAS, but not under GS. Together, our data identified C/EBP? as a previously unidentified key regulator under amino acid starvation-induced autophagy. Overall design: MEF cells (MEFs) were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS) and antibiotics in a humidified incubator with 5 % CO2. MEFs used in the study was regularly tested for mycoplasma contamination. For glucose/amino acid starvation, cells were washed with PBS, then incubated with glucose/amino acid-free DMEM supplemented with 10 % dialyzed FBS. Transfection was performed with Lipofectamine 3000 (Invitrogen). 500000 of MEFs were plated on culture dish containing high glucose DMEM (Cat. SH30243.01) with FBS and were stabilized for one day. After stabilization, the cells were washed with either glucose starvation media (Cat. LM001-56) or amino acid starvation media (Cat. LM001-90) containing dialyzed FBS. Subsequently, the normal media was changed with appropriate starvation media and cells were incubated in the cell incubator for different duration of time according to starvation signal (amino acid starvation and glucose starvation 3, 6 and 12 hours). At the end of starvation, MEFs were collected using trypsin EDTA and resuspended in freezing media (10% DMSO + 90% dialyzed FBS). The control sample (0 hour starvation) was collected and resuspended in freezing media composed of 90% FBS instead of dialyzed FBS. Vials containing each sample were stored in a deep freezer before proceeding to sequencing.