Figure 2. eIF4E S209 regulates Myc translation in CRC.
WT and 4EKI cells were analyzed for translation. (A) Polysome profiling of log-phase cells. (B) Cap-dependent luciferase reporter activities were measured 24 hr after transfection with pcDNA-LUC reporter. (C) Translation of a bi-cistronic construct pYIC encoding cap-dependent and -independent reporters was analyzed by western blotting 24 hr after transfection. (D) The cells were subjected to serum starvation for 48 hr then stimulated with 10% FBS (+) for 4 hr. Binding of indicated eIF4F components (E, G, and 4E-BP1) to a synthetic cap analog (m7GTP) or their levels in the total lysate (input) were analyzed by western blotting. (E) The indicated eIF4E targets were analyzed by western blotting. Actin is the loading control. (F) The indicated transcripts in total cellular and (G) in polysomal RNAs were analyzed by RT-PCR. (H) The ratio of mRNA in polysome and total RNA normalized to WT (100%). N = 3. (B, F-H), values are mean+s.d. (n = 3). *p<0.05, **p<0.01 (Student’s t-test, two tailed). WT vs. KI. (I) Representative images of three CRC organoids treated with eFT508 (10 μM) for 48 hr. Scale bar: 100 µM. (J) The indicated proteins in CRC organoids treated with eFT508 at 24 hr were analyzed by western blotting.
Figure 2—figure supplement 1. Schematics of pCMV-Luc reporter and pYIC reporter encoding cap-dependent translation of Myc-tagged YFP and IRES-dependent HA-tagged CFP.
