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

Journal: Nature metabolism

Article Title: DNA methylation mediates HbA1c-associated complications development in Type 1 diabetes

doi: 10.1038/s42255-020-0231-8

Figure Lengend Snippet: a , Genomic location of the depicted region at/near TXNIP . b , Manhattan plots of association between mean-DCCT HbA1c and DNAme at the covered CpGs. Multiple linear regression models adjusting for covariates were applied to each CpG across all the samples (two-sided tests based on t-statistic, n=499). X-axis represents CpG location and Y-axis represents –log(p). Two HbA1c-assoc regions are highlighted with pink background. c , Heatmap showing the 15 chromatin states (top) and 18 states (bottom). Colors are shown in the legends ( panel j ). d , Ribbon plots representing the genomic interactions identified by IHEC PCHi-C data. Red represents the interactions involved in PIRs containing TXNIP 3’UTR. Blue represents interactions containing TXNIP promoter. Height of each ribbon represents the average CHiCAGO score (Y-axis) of the corresponding interaction across 5 major blood cells including monocytes, neutrophils, CD4 + T-cells, CD8 + T-cells and B-cells. e , Annotations of the RefSeq genes in the depicted region. f , Pearson correlation of DNAme at 33 CpGs in TXNIP and its 25kb flanking region. The associations of DNAme with mean-DCCT HbA1c at each CpG are obtained by the same linear regression model (n=499) indicated in panel b and shown as a Manhattan plot in the upper panel. HbA1c-assoc CpGs (FDR < 15%) are labeled in red font with the most significant cg19693031 underlined. Pair-wise correlations of DNAme of these CpGs were analyzed by Pearson correlation with coefficients shown as heatmap. Blue/red represents negative/positive correlation, respectively. The majority of the CpGs depict positive correlations including all 9 HbA1c-assoc CpGs (in red), while 5 CpGs (in blue) showed little to negative correlation with other CpGs. g , Ribbon plot representing the association of DNAme at cg19693031with expression of its nearby genes (including TXNIP ) in monocytes. Multiple linear regression models adjusting for covariates based were applied to DNAme at cg19693031and expression of each nearby gene within 500 bp distance (n=1202 monocytes) to identify DNAme-assoc genes with nominal p < 0.05 (two-sided tests based on t-statistic). Blue indicates negative association at FDR < 0.05, while grey indicates associations with p < 0.05. h , In-vitro DNA hypomethylation at 3 CpGs in the 3’UTR of TXNIP (including cg19693031) induced by high glucose (HG) treatment of human primary bone marrow CD34 + cells. DNAme was measured by amplicon-seq. i , Upregulation of TXNIP expression induced by HG treatment of human primary bone marrow CD34 + cells. Cells were cultured in medium (see ) containing 25mmol/L glucose (control), or same medium with the addition of 20mmol/L glucose (45mmol/L total) for 72 hours (HG treatment). RT-PCRs were performed in triplicate, and the data shown represent means of triplicates from one experiment. j , Color codes of chromatin states in the heatmaps for 15 and 18 chromatin states shown in panel c.

Article Snippet: Human primary bone marrow CD34 + cells (1M-101C, Lonza, Switzerland,) were cultured in growth medium (#09605, STEMCELL Technologies, MA), STEMSPAN II plus StemSpanTM CD34+ Expansion Supplement (#02691, STEMCELL Technologies, MA) containing 25mmol/L glucose (control), or same medium with the addition of 20mmol/L glucose for 72 hours (HG treatment).

Techniques: Labeling, Expressing, In Vitro, Amplification, Cell Culture, Control