Fig. 2: eIF5a regulates cell cycle and autophagy.

a FACS overlay histograms showing expression of eIF5a or hypusine assessed at d3 (GC7 and Eif5a) and d4 (Dhps and Dohh). KO of cell surface marker Thy1 was included as a CRISPR control. Cell Recovery column shows the number of cells recovered at the time of harvest for each treatment paired to their control treatment (mock-treatment for GC7 and Thy1 KO for CRISPR cells, n = 4 independent experiments for GC7, 7 for Eif5a, 10 for Dhps, 6 for Dohh). Cell cycle profiles were obtained from Hoechst 33342 staining. Mean percentage of cell cycle phases was plotted, pairing positive and negative cells from individual cultures (n = 4 independent experiments for GC7, 12 for Eif5a, 7 for Dhps, 8 for Dohh). Error bars represent SD between experiments. P values were calculated using a paired two-sided t test. Lines: blue, WT or Mock; red, KO or GC7. Bars: dark blue, G0-1; light blue, S; white, G2-M. b Western blot of Eif5a-CRISPR cells sorted into eIF5a positive and negative populations by flow cytometry according to their eIF5a intensities, and detected with an eIF5a antibody (n = 5 biological replicates). Lane 1: size marker; lane 2-6: sorted eIF5a+ cells (WT); lane 7-11: sorted eIF5a- cells (KO). c Autophagic flux was quantified by measuring the level of autophagosomal membrane-bound LC3-II in cells treated with lysosomal inhibitor bafilomycin A1 (BafA1) compared to mock vehicle-treated cells (n = 3 biological samples, P values were calculated using unpaired two-sided t test) Centre line and error bars represent mean value ±SD between biological replicates. a–c OT-1 LN T cells were stimulated for 48 h with 100 nM SIINFELK peptide before CRISPR-Cas9 KO of Eif5a, Dhps or Dohh or treatment with 10 µM GC7 and were subsequently maintained in IL-2. The GC7 experiment with its controls was performed independently of the CRISPR-KO experiment.