Journal: Cell Metabolism
Article Title: Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation
doi: 10.1016/j.cmet.2019.05.003
Figure Lengend Snippet: The MTS of Several Mitochondrial Enzymes Exhibit an Increased Dependency on eIF5A H for Efficient Translation (A) Relative mRNA expression of indicated genes in M(IL-4) treated with 10 μM GC7 for 24 h. (B) Polysome profiles of MEFs (NIH-3T3) expressing Eif5a -shRNA for 5 days versus control MEFs. The y axis indicates absorption at 254 nm, and the x axis represents fractions separated over a 15%–55% sucrose gradient. (C) RT-PCR analysis of indicated mRNAs in ribosome fractions generated from control and Eif5a -shRNA-expressing MEFs (NIH-3T3). (D) Target sequences were cloned into the N terminus of mCherry fused to a degron (to limit its half-life and circumvent differential MTS mCherry half-life between constructs) separated by a GSGSG flexible linker to allow correct and independent folding of the introduced sequences and mCherry. These were subcloned into the MIGR1 vector and transduced into MEFs (SUCLG1 is a subunit of succinyl-CoA synthetase). (E) Representative confocal images of cloned constructs SV40 NLS-, IDH2 MTS-, and control mCherry. Scale bar, 10 μm. (F) Representative histograms of indicated constructs in MEFs (NIH-3T3) ± 10 μM GC7 for 24 h. (G) Indicated constructs were transfected with Eif5a siRNA plus a nontargeting AF647-labeled siRNA as a control for transfection into MEFs (NIH-3T3). Control cells only received nontargeting siRNA. mCherry mean fluorescence intensity (MFI) was assessed 48 h after transfection by flow cytometry. Shown are representative histograms. Histograms are gated on GFP + AF647 + cells. Bar graphs depict percent reduction of mCherry MFI between Eif5a siRNA and nontargeting siRNA: NS, nonsignificant; NR, no reduction in mCherry (n = 3/group). All data are means ± SEM (p ∗ < 0.05, p ∗∗ < 0.005, p ∗∗∗ < 0.0005). (A) Representative of two to three experiments (n = 3 per group), (B and C) of one experiment, (E) of two experiments, and (F and G) of three to four experiments.
Article Snippet: Stable transduction of the lentiviral was performed as previously described( ) using HEK293T cells, the packaging plasmids: pMDLg/pRRE (Gag/Pol), pRSV-Rev (Rev) and phCMV-VSV-G (envelope) as well as the ProFection Mammalian Transfection System Calcium Phosphate Kit (Promega).
Techniques: Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Generated, Clone Assay, Construct, Plasmid Preparation, Transfection, Labeling, Fluorescence, Flow Cytometry
