Supplementary Figure 4: ATF4 and MYC cooperatively regulate EIF4EBP1.
From: ATF4 couples MYC-dependent translational activity to bioenergetic demands during tumour progression
a. qPCR showing expression of mRNAs in wild type and ATF4 -/-, MycER MEFs normalized to 18s RNA. b. qPCR showing expression of mRNAs in control and ATF4 knockdown DLD-1, MycER cells normalized to 18s RNA. a,b Three independent experiments, error bars represent mean ± SD, two tailed student t-test. c. Representative immunoblot of ATF4 -/-, MycER MEFs pretreated with indicated drugs for 2 hours prior to MYC activation. Rapamycin (Rapa) 200nM. Representative immunoblot from three independent experiments showing similar results. d. Clonogenic survival of ATF4 -/-, MycER MEFs after activation of MYC in the absence or presence of Rapamycin (200nM). Graph from there independent experiments, error bars represent mean ± SD, two tailed student t-test. e.35S incorporation in ATF4 -/-, MycER MEFs following MYC activation. Representative autoradiograph is shown. Graph data from three independent experiments, normalized to β-actin. Error bars represent mean ± SD, two tailed student t-test. f. ATF4-/-, MycER MEFs were pretreated with 5mM 4-Phenylbutyric acid (4PBA) for 2hrs followed by MYC activation. Representative western blot from two independent experiments is shown. g. Annexin V staining of cells transfected with the shown siRNAs, n=2, error bars represent mean ± SEM. Unprocessed scans of blots are shown in Supplementary Fig 7.
