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. 2023 Feb 27;42(7):e110496. doi: 10.15252/embj.2021110496

Figure 3. eIF4E reprograms the SF landscape in AML.

Figure 3

  1. WB analysis of splicing factors for Vector and eIF4E overexpressing NOMO‐1 cell lines. Myc and cyclin D1 (CCND1) served as positive controls and β‐actin as a loading control. Each β‐actin blot corresponds to the above western blots. Experiments were carried out at least three independent times, and one representative experiment is shown.
  2. Western blot analysis of untreated (Ctrl) and Ribavirin treated (Rib) Vector and eIF4E NOMO‐1 cell lines. Myc and Mcl1 served as positive controls and β‐actin as a loading control. Each β‐actin blot corresponds to the above WBs. Experiments were carried out at least three independent times, and one representative experiment is shown. Ribavirin dose was 10 μM, which is clinically achievable (Assouline et al, 2009, 2015).
  3. WB analysis of Splicing Factor levels in primary AML samples with high (AML‐H) or normal (AML‐N) eIF4E levels, as well as bone marrow mononuclear cells from healthy volunteers (Norm for normal). Numbers refer to different individuals. β‐actin was used as a loading control. (* = degraded sample).
  4. The enrichment of mRNAs and UsnRNAs in RIPs of endogenous eIF4E versus input RNAs from the nuclear fractions of NOMO‐1 cells monitored by RT–qPCR. Data were normalized to input samples and presented as a fold change. The mean, standard deviation, and P‐values (using Student t‐test) were derived from three independent experiments (each carried out in triplicate). MYC and MCL1 are known eIF4E nuclear targets and served as positive controls, while ACTB and GAPDH were used as negative controls. Student t‐test: *P < 0.05, **P < 0.01.
  5. Total levels of UsnRNA in NOMO‐1 Vector and eIF4E cells monitored by RT–qPCR. Data were normalized to Vector control and shown as a fold change. The mean and standard deviation, where no P‐values were P < 0.05 (using Student t‐test), were derived from three biological replicates each carried out in triplicate for all panels.