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. 2024 Aug 28;15:7458. doi: 10.1038/s41467-024-51901-w

Fig. 2. eIF5A is required to sustain elevated protein synthesis rates during cellular senescence.

Fig. 2

a Experimental setup for the CRISPR-Cas9 screen on protein synthesis rates. b CRISPR screen results, with sgRNAs sorted by enrichment in proliferating (top) and senescent cells (bottom). Z-score and Z-ratio calculations are detailed in the methods. c Western blots from BJ-Ras-ER (top) and A549 (bottom) cells with eIF5A-targeting sgRNAs, representative of 3 independent experiments. d OP-Puro incorporation in proliferating (top) and senescent BJ-Ras-ER cells (bottom) with control or eIF5A sgRNAs. Data show mean ± SD from n = 3 biologically independent experiments. p-values via two-tailed unpaired t test. NS, not significant; **P  <  0.01. e BrdU incorporation in BJ-Ras-ER cells post 12 days of 4OHT treatment, transduced with eIF5A-targeting sgRNAs. Data are mean ± SD from n = 3 biologically independent experiments. p-values via two-tailed unpaired t test. NS, not significant; ***P  <  0.001. f β-galactosidase-positive cell quantification after 12 days of 4OHT treatment, transduced with eIF5A-targeting sgRNAs. Mean ± SD from n = 6 biologically independent experiments. p-values via two-tailed unpaired t test. NS is not significant. Scale bars, 50 μm. g qRT-PCR mRNA quantification after 12 days of 4OHT treatment in BJ-Ras-ER cells with eIF5A sgRNAs. Data are mean ± SD from n = 3 biologically independent experiments. p-values via two-tailed unpaired t test. NS is not significant. h Experimental design for transfecting two siRNAs targeting eIF5A in BJ-Ras-ER cells. siRNAs were transfected on day 8 post 4-OHT treatment and analyzed on day 12. i Western blots of BJ-Ras-ER cells transfected with siRNAs against eIF5A, collected on day 12 post 4-OHT treatment, representative of 3 independent experiments. j OP-Puro incorporation quantification in proliferating and senescent BJ-Ras-ER cells transfected with siRNA control or eIF5A siRNAs. Data are mean ± SD from n = 3 biologically independent experiments. p-values via two-tailed unpaired t test. NS, not significant; **P < 0.01. k BrdU incorporation in BJ-Ras-ER cells with eIF5A-targeting siRNAs. Data are mean ± SD from n = 3 biologically independent experiments. p-values via two-tailed unpaired t test. NS is not significant. Source data and exact p-values are provided in the Source Data file.