Journal: Haematologica

Article Title: Epigenetic dysregulation of eukaryotic initiation factor 3 subunit E (eIF3E) by lysine methyltransferase REIIBP confers a pro-inflammatory phenotype in t(4;14) myeloma

doi: 10.3324/haematol.2023.283467

Figure Lengend Snippet: RNA-binding protein eIF3E was dysregulated by histone methylation and participates in REIIBP oncognesis. (A) Integrative Genomics Viewer (IGV) browser of representative gene tracks from biological triplicates of eIF3E were shown for H3K4me3 and H3K27me3 histone marks in RPMI8226-vector control (VCon) and RPMI8226-REIIBP cells. CRISPR/Cas9-mediated knockdown of H3K4me3 peak using 3 different single guide RNA (sgRNA) in RPMI8226-REIIBP cells with the positions of the sgRNA indicated. β-actin is the loading control. N=2, independent repeats, representative western blots (WB) shown. (B) Left panel: the global protein synthesis rate was determined by puromycin labeling coupled with immunoblot using antibody against puromycin (12D10). Lanes 1 and 2 are mock treated, lanes 3 and 4 are labeled with 10 µg/mL of puromycin for 10 minutes and lanes 5 and 6 are labeled with puromycin and treated with 100 µM cycloheximide (CHX) for 10 minutes. Right panel: O-propargyl-puromycin (OPP)-labeling coupled with immunofluorescence in RPMI8226-VCon and RPMI8226-REIIBP. Newly synthesized proteins were stained with Alexa Flour 488 (green) and nucleus stained with DAPI (blue). Image is representative field. Scale bar, 100 µm. (C) RNA immunoprecipitation (RNA IP) was performed with eIF3E antibodies or immunoglobuolin (Ig)G control, and binding with TLR7 or BTK mRNA was determined using quantitative real time polymerase chain reaction. Asterisks represent significant differences (* P <0.05; *** P <0.001, Student’s t test). (D) The % transcript distribution of TLR7 mRNA was determined in the non-translating, 80S, low polysome and high polysome after polysome profiling comparing between RPMI8226-VCon and RPMI8226-REIIBP; 56.2% of TLR7 mRNA were associated with polysomes as compared to 64.4% in REIIBP, indicating a higher translation efficiency in REIIBP cells.

Article Snippet: Two single guide RNA (sgRNA) targeting the coding region of eIF3E were cloned into vector backbone pRP(CRISPR)-Puro-hCas9-U6 and three sgRNA targeting its H3K4me3 TSS peak (VectorBuilder, USA).

Techniques: RNA Binding Assay, Methylation, Plasmid Preparation, Control, CRISPR, Knockdown, Western Blot, Labeling, Immunofluorescence, Synthesized, Staining, RNA Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer

doi: 10.1101/2023.03.19.533333

Figure Lengend Snippet: ( A ) Oxygen tension (y-axis) as determined by an OxyLite probe in tumors from KPC mice breathing ambient air or pure oxygen (x-axis). Dark blue circles represent averages per tumor and boxplots illustrate their distribution. Light blue circles represent repeat measurments per tumor. ( B ) HIF target genes (orange) controlled directly or indirectly by BMAL2 (yellow). Indirect control involves both BMAL2’s negative influence on first (dark blue) or second tier (light blue) RP repressing HIF target genes, and positive influence on first (dark red) and second (light red) tier RP activating HIF target genes. ( C ) Fold change in cell growth relative to cells expressing non-targeting sgRNA in normoxic (21% O2) and hypoxic (1% O2) conditions ( D ) Number of migrated cells for cells expressing non-targeting or BMAL2-directed sgRNA in the indicated oxygen environment. P-values are derived from testing the indicated coefficients from a linear regression model. Significant interaction term suggests synergistic effects of BMAL2 pertubation and hypoxia on cell migration. ( E ) Media pH levels after 72 hours incubation. P-values derived from Welch’s t-test ( F ) Fold change in lactate levels in the indicated oxygen environment expressing either non-targeting or BMAL2-directed sgRNA ( G ) Western Blot for HIF1a, HIF2a,

Article Snippet: The sgRNA (small-guide RNA) to knocking-out BMAL2 as well as non-targeting (NT) sequence were purchased from GenScript (Piscataway, NJ) using pLentiGuide-Puro vector as a backbone.

Techniques: Control, Expressing, Derivative Assay, Migration, Incubation, Western Blot