Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus MicroRNAs Induce Metabolic Transformation of Infected Cells

doi: 10.1371/journal.ppat.1004400

Figure Lengend Snippet: A. Oxygen consumption rate (OCR) in cells expressing a non-targeting control vector or the KSHV miRNA cluster was measured using the Seahorse XF24 Analyser. Cells were seeded at a density of 4×10 4 cells per well and the assay was performed according to the manufacturer's Mito stress protocol. Uncoupled, maximal and non-mitochondrial respiration was determined after the addition of 5 µM oligomycin, 1 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 2 µM antimycin-A. The bar graph presents the average base line OCR in 3 independent experiments relative to non-targeting control OCR (Mean+SEM, n = 3). B. Lactate levels in the control and miR-LEC culture media. Equal numbers of cells were grown for 24 hours and lactate levels in the media were measured using the MBL Lactate Colorimetric assay kit. The bar graph presents the average ratio between control and miR-LEC from 3 independent experiments (Mean+SEM, n = 3). C. Glucose uptake into control and miR-LEC cell. Control and miR-LEC were incubated with 30 µM of the fluorescent glucose analogue 6-NBDG for 20 minutes prior to analysis by fluorescence-activated cell sorter (FACS). The histogram displays one representative experiment with control shown in black and miR-LEC shown in grey. The bar graph presents the average ratio between the control and miR-LEC from 3 independent experiments (Mean+SEM, n = 3). D. GLUT1 protein expression, as measured by Western blotting, in control or miR-LEC. Values indicate the relative signal of the GLUT1 antibody normalized to α-tubulin as measured using the Odyssey. E. HIF1α protein expression, as measured by Western blotting, in LEC expressing the viral miRNA cluster or the control vector. The bars show relative values of HIF1α antibody intensity normalized to α-tubulin (Mean+SEM, n = 3). F. Expression of the HIF1α target genes VEGF and ADM . mRNA levels were determined by quantitative real-time PCR (qRT-PCR). Tubulin beta ( TUBB ) levels were used for normalization. In all panels statistical significance denoted by *P<.05; **P<.01; ***P<.001.

Article Snippet: Detection of the mature KSHV miRNAs was performed using the kshv-miR LNA PCR primer sets (Exiqon).

Techniques: Expressing, Plasmid Preparation, Colorimetric Assay, Incubation, Fluorescence, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus MicroRNAs Induce Metabolic Transformation of Infected Cells

doi: 10.1371/journal.ppat.1004400

Figure Lengend Snippet: A. Mitochondrial volume in miR-LEC. Cells were loaded with 5 µM Calcein-AM and 5 nM MitoTracker Deep Red FM and Z-series of images were. Maximal projections of images were used to quantify the area of green (Calcein) and red (MitoTracker Deep Red) signals as previously described . Representative single-plane images of the mitochondrial structure are shown on the left panel. The bar graph on the right presents the average relative mitochondrial volume in miR-LEC compared to control cells (Mean±SEM, n = 5). B. Mitochondrial DNA (mtDNA) copy number in cells expressing the viral miRNA cluster relative to control cells. qPCR was carried out as described in . C. Expression levels of the 5 OXPHOS complexes as measured by Western blotting analysis using the MitoProfile Total OXPHOS Human WB Antibody Cocktail in miR-LEC and the control cells. In all panels statistical significance denoted by *P<.05; **P<.01; ***P<.001. D. Expression of COXIV and TFAM in cells expressing the KSHV miRNA cluster relative to control cells. mRNA levels were determined by qRT-PCR. TUBB levels were used for normalization.

Article Snippet: Detection of the mature KSHV miRNAs was performed using the kshv-miR LNA PCR primer sets (Exiqon).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus MicroRNAs Induce Metabolic Transformation of Infected Cells

doi: 10.1371/journal.ppat.1004400

Figure Lengend Snippet: A. Relative mRNA levels of EGLN2 and HSPA9 in miR-LEC compared to control cells. mRNA levels were determined by qRT-PCR. TUBB levels were used for normalization. B. Protein levels of EGLN2 and HSPA9 in miR-LEC. Top right panel: protein expression, as measured by Western blotting, in miR-LEC and the control cells. C. Relative mRNA levels of EGLN2 and HSPA9 in LEC expressing the individual KSHV miRNAs. mRNA levels were determined by qRT-PCR. TUBB levels were used for normalization. D. Reporter assay indicating the sensitivity of the EGLN2 or HSPA9 3′UTRs to targeting by the individual KSHV miRNAs. Firefly expression was normalized to Renilla expression to give the relative light units (RLU), which are shown relative to the non-targeting control. In all panels statistical significance denoted by *P<.05; **P<.01; ***P<.001.

Article Snippet: Detection of the mature KSHV miRNAs was performed using the kshv-miR LNA PCR primer sets (Exiqon).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Reporter Assay

Journal: PLoS Pathogens

Article Title: Kaposi's Sarcoma Herpesvirus MicroRNAs Induce Metabolic Transformation of Infected Cells

doi: 10.1371/journal.ppat.1004400

Figure Lengend Snippet: A–B. 7500 cells expressing the KSHV miRNA cluster or the control vector (A) and cells expressing the non-targeting control or shEGLN2 (B) were plated in 96 well plates. Cells were fixed after 30 minutes, 24, 48 and 72 hours using 10% Trichloroacetic acid, stained with Sulforhodamine B, and plates were read at 564 nm. Optical density indicates the amount of proteins in the different wells. C. Expression of COXIV in BCBL1 cells treated with in indicated concentration of Resveratrol. mRNA levels were determined by qRT-PCR. TUBB levels were used for normalization. D. Expression of the 5 OXPHOS complexes, as measured by Western blotting analysis using the MitoProfile Total OXPHOS Human WB Antibody Cocktail in BCBL1 cells treated with the indicated concentration of Resveratrol. Values indicate the relative signal of the different antibodies normalized to HSP90 as measured using the ImageQuant software. E. BCBL1 cells were treated with the indicated concentration of Resveratrol for 48 hours. Viability was determined using the Muse Count & Viability Assay Kit on the Muse cell analyzer (Merck Millipore). F. Relative mRNA levels of the KSHV lytic genes ORF65 and K8.1 in BCBL1 cells treated with Resveratrol or TPA for 48 hours. mRNA levels were determined by qRT-PCR. TUBB levels were used for normalization. G. Relative KSHV DNA copy number in 293T cells infected using the growth media of BCBL1 cell treated with the incubated concentration of Resveratrol. H. LEC were infected with rKSHV.219 virus and selected as previously described . Cells were treated with Resveratrol and analyzed by Flow cytometer after 72 hours. The numbers denote the percentage of RFP positive cells, which reflects lytic cells.

Article Snippet: Detection of the mature KSHV miRNAs was performed using the kshv-miR LNA PCR primer sets (Exiqon).

Techniques: Expressing, Plasmid Preparation, Staining, Concentration Assay, Quantitative RT-PCR, Western Blot, Software, Viability Assay, Infection, Incubation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: c-Myc is a novel Leishmania virulence factor by proxy that targets the host miRNA system and is essential for survival in human macrophages

doi: 10.1074/jbc.RA118.002462

Figure Lengend Snippet: Commercial primers used in RT–qPCR

Article Snippet: miR-148b-3p , Exiqon , microRNA LNA TM PCR primer sets , 204047.

Techniques:

Journal: Non-Coding RNA

Article Title: miRNA–mRNA Conflux Regulating Immunity and Oxidative Stress Pathways in the Midgut of Blood-Fed Anopheles stephensi

doi: 10.3390/ncrna1030222

Figure Lengend Snippet: Tissue specific profiling of miRNAs by RT-PCR. Expression profiling of eight miRNAs, ( A ) miR-34; ( B ) miR -1174; ( C ) miR-277; ( D ) miR-219; ( E ) miR-309, ( F ) miR-989; ( G ) miR-210 and ( H ) miR-285 in ovary, midgut and carcass tissue of sugar fed (SF), female mosquito at 42 h (BF 42 h) and 5 days (BF 5d) post blood-feeding and mosquitoes at 5 days post Plasmodium infection (iBF 5d). Fold change was calculated using 2 −ΔΔC T method. SF was used as a calibrator, with its fold change taken as 1. Values are mean ± s.e.m of three biological replicates profiled in triplicate reactions. Data was statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05 was considered significant ( ** p < 0.01, *** p < 0.001).

Article Snippet: The mature miRNA sequence was used to synthesize custom miRNA LNA PCR primer sets (Exiqon).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection

Journal: Non-Coding RNA

Article Title: miRNA–mRNA Conflux Regulating Immunity and Oxidative Stress Pathways in the Midgut of Blood-Fed Anopheles stephensi

doi: 10.3390/ncrna1030222

Figure Lengend Snippet: Knockdown of miRNA expression by antagomirs. Knock-down of ( A ) miR-34; ( B ) miR-219 and ( C ) miR-277 expression in cells transfected with 50 pmol and 100 pmol of antagomirs. MicroRNA expression was profiled by RT-PCR at 48 h and 72 h post-transfection in control, scrambled RNA and antagomir transfected cells. Control transfected cells were used as a calibrator, in which expression (%) of miRNA was taken as 100; ( D ) Knock-down of miR-989 by nano-injecting miR-989 specific antagomir in female mosquito. MicroRNA expression was profiled in midgut tissue of PBS, scrambled and antagomir injected female mosquito taking PBS injected mosquitoes as calibrator. Expression (%) of miRNA in PBS injected midgut tissue was taken as 100. Values are mean ± s.e.m. of three biological replicates profiled in triplicate reactions. Data was statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05 was considered significant ( ** p < 0.01, *** p < 0.001).

Article Snippet: The mature miRNA sequence was used to synthesize custom miRNA LNA PCR primer sets (Exiqon).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Injection

Journal: Non-Coding RNA

Article Title: miRNA–mRNA Conflux Regulating Immunity and Oxidative Stress Pathways in the Midgut of Blood-Fed Anopheles stephensi

doi: 10.3390/ncrna1030222

Figure Lengend Snippet: Validation of targets by miRNAs loss of function strategy: Expression profiling of mRNA target(s) of ( A – D ) miR-34; ( E ) miR-219 and ( F ) miR-277 was carried out by RT-PCR in control, scrambled and antagomir transfected cells. Expression (%) of mRNA was compared with miRNA expression at ( i ) 48 h and ( ii ) 72 h post-transfection; ( G ) MiRNA-989 target expression was profiled by RT-PCR in midgut of control, scrambled and miR-989 specific antagomir injected female mosquitoes. Expression (%) of miRNA and mRNA was compared at 24 h post blood-feeding. Expression (%) of mRNAs in control was taken as 100. Values are mean ± s.e.m of three biological replicates profiled in triplicate reaction. Data were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05 was considered significant ( ** p < 0.01, *** p < 0.001). ns = No significant down-regulation.

Article Snippet: The mature miRNA sequence was used to synthesize custom miRNA LNA PCR primer sets (Exiqon).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Injection