Complementary Files MTALTND4 - Upregulated and downregulated genes from microarray analysis

Semi-confluent HeLa and HEK-293T cells were suspended in MiR05 mitochondrial respiratory buffer (110 mM D-sucrose, 60 mM lactobionic acid, 20 mM taurine, 20 mM HEPES, 10 mM KH2PO4, 3 mM MgCl2, 0.5 mM EGTA, BSA 1 g∙L-1 ) supplemented with 5 mM pyruvate as an external energy source to allow respiration of intact cell (Doerrier et al., 2018; Kienzle et al., 2022). Treated cells were exposed exogenously to 10 µM of synthetic MTALND4 peptide (LifeTein) for 4 hours, while water was added to control cells. Cells were then pelleted by centrifugation. The supernatant was then removed and samples were frozen. In total, samples were 40 collected for treated HeLa cells, control HeLa cells, treated HEK-293T cells and control HEK293T cells. RNA of treated and control samples was extracted using Quick-RNA™ Miniprep Kit as described by the manufacturer (Zymo Research, 2021; R1054). In brief, pelleted cells were resuspended in provided RNA lysis buffer. RNA was purified by filtration through centrifuge columns to remove genomic DNA and washed multiple times using ethanol, prep buffer and wash buffer. RNA was collected and DNase/RNase-Free Water was added. Quality and quantity of the RNA were assessed by electrophoresis on a 1% agarose gel as well as with a BioDrop µLITE spectrophotometer. Quality samples were then sent to Génome Québec for a Human Clariom S Assay via ThermoFisher Scientific (Affymetrix). Processed data from the microarray analysis was interpreted using ThermoFisher’s Transcriptome Analysis Console (TAC) Software version 4.0.2.15 (Affymetrix, Inc., Santa Clara, CA, USA). Principal component analysis (PCA) was performed and the analysis was executed using the Gene Level - SST-RMA summarization method and an ANOVA method with eBayes approach as determined by the TAC guide (ThermoFisher Scientific, 2019, Transcriptome Analysis Console 4.0.2.). Significant genes were selected by a false-discovery rate < 0.30, foldchange > 1.5 in both directions and an ANOVA p-value < 0.05, as used previously for similar analyses (Lee et al., 2015). Over and under-expressed genes were compared between HeLa and HEK-293T and significant genes common in both cell types were further analyzed.