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. 2020 Nov 2;9:e60151. doi: 10.7554/eLife.60151

Figure 3. eIF4E S209 regulates CRC growth and the ISR in vivo.

Paired HCT116 WT and 4EKI cells were characterized in vitro and in vivo. (A) Reactome Pathway analysis of genes elevated in WT cells (3-fold or more) visualized by ClueGO. (B) RT-PCR of indicated transcripts in total and (C) in polysomal RNAs. (D) The indicated transcripts were analyzed by RT-PCR in HCT116 cells transfected with the indicated siRNAs for 24 hr. N = 3. (E–K) Xenograft tumors. (E) The growth curve of WT and KI (KI1 and KI2) cells (four million) injected s.c. into different flanks of nude mice by day 23. (F) Representative images of WT and KI (KI1) xenografts on day 23. (G) Representative IF/IHC of the indicated makers in randomly chosen WT and KI tumors. Scale bar: 50 µm and (H) quantification of markers in (G) using 400x fields. (H) Representative PLA of eIF4E-4E-BP1 binding, scale bar: 25 µm, and (J) quantification (dots/cell). (K) The indicated transcripts were detected by RT-PCR. WT values were set at 100%. (B-D, F, H, J-K), values represent mean+s.d. (n = 3 or as indicated). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test, two tailed). WT vs. KI. +p<0.05, ++p<0.01, (one-way ANOVA with TUKEY post-hoc test), or scrambled vs. specific siRNAs.

Figure 3.

Figure 3—figure supplement 1. eIF4E S209 regulates the ISR in vitro and in vivo.

Figure 3—figure supplement 1.

(A) Scatter plot of microarray data from log-phase cells. Significantly elevated genes in WT (red) (89) or 4E KI cells (blue) (Croft et al., 2014), 3-fold or more, *p<0.05. (B) Reactome pathway analysis of genes elevated in WT cells from (A). (C) Reactome pathway analysis of genes elevated in 4EKI visualized by ClueGO. (D) Reactome pathway analysis of genes elevated in 4EKI cells from (A). (E) Indicated transcripts in translation, (F) cell death and (G) other Myc metabolic targets (nucleotide synthesis, fatty acid, and glucose metabolism) were analyzed by RT-PCR. (H) Enlarged p-GCN2 and p-eIF2a IF in the indicated xenografts. Scale bar: 25 µM. (I) Representative p-S6 IF in the indicated xenografts. Scale bar: 50 µM. (J) Indicated transcripts involved in cell death were analyzed in xenografts by RT-PCR. (K) GEPIA expression analysis of indicated ISR targets in TCGA COAD and READ cohorts with matched TCGA normal and GTEX data. (E-G, J), values represent mean+s.d. (n = 3). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test, two tailed).