Journal: NAR Molecular Medicine

Article Title: Human pneumovirus induces IFN-dependent expression of the immune-responsive gene 1 and is inhibited by 4-octyl itaconate in human macrophages

doi: 10.1093/narmme/ugag017

Figure Lengend Snippet: 4OI reduces expression of ATP-dependent citrate lyase required for HMPV replication. ( A ) 4OI reduces ATP-dependent citrate lyase ( ACLY) expression. MDMs were treated with 4OI (250 µM) or DMSO prior to infection with HMPV for 24 h. Expression levels of ACLY, FASN , and SCD1 mRNA were quantified relative to untreated, uninfected MDMs via qRT-PCR ( n ≥ 4). ( B ) ACLY inhibition reduces HMPV levels. MDMs were pretreated with 10 or 20 µM of the ACLY inhibitor BMS-303141 or DMSO prior to infection with HMPV for 24 h. HMPV N-gene mRNA was determined by qRT-PCR ( n = 4). Protein expression of HMPV N and GAPDH was analysed via immunoblotting of whole cell lysates ( n = 2). Protein levels were quantified by normalizing of band intensities against GAPDH and expressed as fold change compared to siNTC-transfected, infected MDMs. Multiple comparisons were analysed by a paired one-way ANOVA with Tukey post-hoc test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001 and ns, not significant. ( C ) HMPV stimulates increased level of neutral lipids in infected MDMs. MDMs were left uninfected (−) or infected with a MOI 1 of GFP-expressing recombinant HMPV for 24 h. HCS LipidTOX™ Deep Red neutral lipid stain was used to monitor lipids by confocal microscopy. Left panels: Representative images for each treatment showing HCS LipidTOX™ Deep Red (magenta) and HMPV (green). Right panel: Quantification of the LipidTOX™ signal per area. Signal was quantified from z-stacks of six fields of view per condition using the 20× numerical aperture yielding to ~2000 cells per condition and normalized to the area. Scalebar is adjusted to 100 µm ( n = 1). The data were assessed for normality and then compared with a non-parametric, two-tailed Mann–Whitney test. Significance was ranked as * P < .05; ** P < .01; *** P < .001; **** P < .0001, and ns, not significant.

Article Snippet: Inhibition with S-Ruxolitinib (Merck) or GSK 872 (GlaxoSmithKline) was performed 1 h before infection with HMPV, whereas BMS-303141 (MedChemExpress) was preincubated for 2 h. The IFNAR neutralizing antibody (nIFNAR, clone MMHAR-2) was purchased from PBL; 5 or 10 μg/ml were incubated for 30 min before infection with HMPV.

Techniques: Expressing, Infection, Quantitative RT-PCR, Inhibition, Western Blot, Transfection, Recombinant, Staining, Confocal Microscopy, Two Tailed Test, MANN-WHITNEY

Journal: Circulation

Article Title: A Glycolytic Switch is Required for Transdifferentiation to Endothelial Lineage

doi: 10.1161/CIRCULATIONAHA.118.035741

Figure Lengend Snippet: A, ACL specific inhibitors BMS303141 and SB204990 decreased transdifferentiation yield. (n=3) B, ACL siRNA impaired transdifferentiation efficiency. (n=3) C, ACL knockdown reduced the effect of PolyI:C to induce glycolysis as reflected by ECAR measured by Seahorse assay. (n=3) D, LDHA inhibitor reduces the nuclear level of ACL induced by PolyI:C. (n=3) E. Schematic model of the control of glycolytic switch in transdifferentiation. Innate immune signaling induces a glycolytic switch and glucose is used to generate lactate or citrate, the latter is transported to the nucleus. There, it will be converted into acetyl-coA by ACL, and to support histone acetyl-coA and transdifferentiation. (*, 0.01

Article Snippet: Reagents FBPase-1 inhibitor was purchased from Alfa Aescar, (2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) was purchased from Calbiochem, 2-Deoxy-D-glucose (2-DG), Sodium Oxamate, Sodium dichloroacetate (DCA), Dispase I and Nile Red were purchased from Sigma, CPI613, Anacardic Acid (ANAC), BMS303141, SB204990, 8-Br-cAMP and SB431542 were purchased from Tocris Bioscience.

Techniques: Knockdown, Control