Journal: Science Advances

Article Title: Regulation of eIF4E guides a unique translational program to control erythroid maturation

doi: 10.1126/sciadv.add3942

Figure Lengend Snippet: ( A ) HUDEP-2 cells transduced with retrovirus encoding eIF4E [pMSCV-eIF4E-IRES-GFP (green fluorescent protein)] or The RNAi Consortium (TRC) control (pMSCV-TRC-IRES-GFP) followed by GFP sorting, precursor expansion, and differentiation. ( B ) Representative flow cytometry plots comparing HUDEP-WT and HUDEP-eIF4E cells 4 days after induction of maturation. ( C to E ) Quantitation of flow cytometry–defined differences in maturation between WT and eIF4E HUDEP-2 cells by (C) CD71 and FSC, (D) CD34 and CD105, and (E) cKit. * P < 0.05, N = 3. ( F ) Representative Wright-Giemsa–stained cytospins of day 0 and +4 HUDEP-WT and HUDEP-eIF4E cells displaying relative homogeneity in day 0 of both conditions (top) and day +4 (bottom) showing marked anisocytosis in HUDEP-WT cells compared to HUDEP-eIF4E cells. Green arrowheads denote erythroblasts showing nuclear condensation and size restriction. Magnification, ×40. Black line indicates size marker of 100 μm. ( G ) Representative day +4 cell pellet of WT versus eIF4E HUDEP-2 cells showing impaired hemoglobinization evidenced by the pale color of the pellet. Statistical analysis between populations using paired t test compared to HUDEP-WT. N.S., not significant.

Article Snippet: Thawed cells were plated on retronectin-coated wells in CD34 + expansion media [StemSpan SFEM II, hSCF (100 ng/ml; Peprotech), Flt3 ligand (100 ng/ml; Peprotech), Thrombopoietin (TPO) (50 ng/ml; Peprotech), low-density lipoprotein (10 μg/ml; STEMCELL Technologies), UM171 (35 nM; Benchchem), and penicillin/streptomycin (Thermo Fisher Scientific)] for 48 hours of expansion before retroviral transduction with pMSCV-IRES-GFP or pMSCV-eIF4E-IRES-GFP as described above.

Techniques: Transduction, Flow Cytometry, Quantitation Assay, Staining, Marker

Journal: Science Advances

Article Title: Regulation of eIF4E guides a unique translational program to control erythroid maturation

doi: 10.1126/sciadv.add3942

Figure Lengend Snippet: ( A ) HUDEP-2 WT and eIF4E cells nucleofected with RNP containing Cas9 with crRNA:tracrRNA duplex specific to either noncoding negative control, PTPN6, or Igf2bp1. Single-cell clones were generated and expanded before induction of maturation. ( B ) Representative flow cytometry plots of CD71 versus FSC and CD34 versus CD105 expression demonstrating the reversal of erythroid maturation in HUDEP-eIF4E (negative CRISPR control) cells with simultaneous knockdown of PTPN6 (4E PTPN6 1 or 4E PTPN6 2) or Igf2bp1 (4E Igf2bp1 1 or Igf2bp1 2) in comparison to HUDEP-WT (negative CRISPR control). ( C ) Partial rescue of CD71 + erythroid maturation phases dependent on PTPN6 knockdown. * P < 0.05, N = 6. ( D ) Partial rescue of CD71 + erythroid maturation phases dependent on Igf2bp1 knockdown. * P < 0.05, N = 6. ( E ) Partial rescue of CD34 + , CD105 + and CD34 − , CD105 + populations with knockdown of PTPN6 or Igf2bp1. * P < 0.05, N = 6. Statistical analysis between populations calculated using paired t test compared to HUDEP-4E negative control (4E Ng Cnt).

Article Snippet: Thawed cells were plated on retronectin-coated wells in CD34 + expansion media [StemSpan SFEM II, hSCF (100 ng/ml; Peprotech), Flt3 ligand (100 ng/ml; Peprotech), Thrombopoietin (TPO) (50 ng/ml; Peprotech), low-density lipoprotein (10 μg/ml; STEMCELL Technologies), UM171 (35 nM; Benchchem), and penicillin/streptomycin (Thermo Fisher Scientific)] for 48 hours of expansion before retroviral transduction with pMSCV-IRES-GFP or pMSCV-eIF4E-IRES-GFP as described above.

Techniques: Negative Control, Clone Assay, Generated, Flow Cytometry, Expressing, CRISPR