Extended Data Fig. 5: uORF1 translation is required for ATF4 induction in s-ISR but is largely dispensable for c-ISR.
From: Plasticity of the mammalian integrated stress response
(a) Western blot analysis of the indicated proteins in cell extracts isolated from mouse ES Cells or from MEFs, treated with Tg (400 nM) for specified durations. Representative blots are shown (n = 3 independent experiments). (b) Sanger sequencing traces of genomic DNAs from WT and ΔuORF1 mouse ES Cells. Only the uORF1 region is shown. (c) Protein synthesis (incorporation of [35S]-cysteine and methionine into proteins) in ΔuORF1 mouse ES Cells treated with Tg (400 nM) for the indicated times. p-values, two-tailed Student’s t-test. Data are presented as mean ± SEM (n = 5 independent experiments). (d) Polysome profile tracings obtained by monitoring absorbance (254 nm) across the 10-50% sucrose gradients from WT or ΔuORF1 mouse ES Cells expressing control (top panel) or eIF2Bε shRNA (middle panel), or treated with Tg (400 nM, 9 h) (bottom panel). Representative experiments are shown (n = 3 independent experiments). (e) Distribution of indicated mRNAs across polysome fractions (from d) isolated from WT or ΔuORF1 mouse ES Cells expressing control or Eif2b5 shRNAs, or treated with Tg (400 nM, 9 h) was determined by RT-qPCR. Representative experiments are shown (n = 3 independent experiments).
