Journal: Molecular and Cellular Biology

Article Title: The Brd4 Extraterminal Domain Confers Transcription Activation Independent of pTEFb by Recruiting Multiple Proteins, Including NSD3 ^▿ †

doi: 10.1128/MCB.01341-10

Figure Lengend Snippet: The Brd4 ET domain-associated NSD3 is recruited to Brd4 target genes, CCND1, DCPS, and PIM2. (A to C) 293T cells stably expressing Flag-HA-NSD3 were treated with siRNA to NSD3, siNSD3, or with the control siRNA, siControl, for 72 h. ChIP was performed with anti-IgG (IgG), anti-Brd4 (Brd4), or anti-HA (NSD3) as indicated on the x axis. Immunoprecipitated DNA was quantified by real-time PCR in duplicates with primers specific to the promoter or coding regions of CCND1, PIM2, and DCPS genes, and results are presented as enrichment relative to input DNA. Each ChIP was repeated twice with reproducible results. Error bars represent standard deviations from means. (D to F) 293T/Flag-HA-NSD3 cells were treated with siRNA to Brd4, siBrd4, or with the control siRNA, siControl, for 72 h, and ChIP analysis was performed as indicated in panels A to C.

Article Snippet: Quantitative real-time PCR (RQ-PCR) was performed in an Applied Biosystems ABI 7500 Fast Sequence Detection System using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and the TaqMan Gene Expression assays (Applied Biosystems) for PIM2 (assay ID: Hs00179139_m1), CCND1 (assay ID: Hs00277039_m1), DCPS (assay ID: Hs00204009_m1), RBPL13A (assay ID: Hs03043885_g1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (assay ID: Hs99999905_m1).

Techniques: Stable Transfection, Expressing, Control, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Biochimica et biophysica acta

Article Title: Identification of Fhit as a Post-transcriptional Effector of Thymidine Kinase 1 Expression

doi: 10.1016/j.bbagrm.2017.01.005

Figure Lengend Snippet: A. Immunoblot of Vinculin, TK1, and Fhit in 293 cells 72 h post transfection with siCtrl and siFHIT. B. Immunoblot of Vinculin, TK1, and Fhit in H1299 E1 and D1 induced cells. Cells were harvested 48 h after treatment with ponasterone A. C. Immunoblot of TK1 and GAPDH in siRNA transfected 293 cells treated for indicated times with 10 μM MG132 (left panel). Relative TK1 protein expression determined by densitometric analysis of the immunoblots (right panel). D. Immunoblot of TK1 and GAPDH in siRNA transfected 293 cells treated for indicated times with 100 μg/ml cycloheximide (CHX) (left panel). Protein lysates from siFHIT cells were loaded at 2:1 ratio compared to siCtrl lysates to more accurately measure rate of degradation. Relative TK1 protein expression determined by densitometric analysis of immunoblots (right panel). E. Immunoblot of TK1 and Cyclin A2 in siRNA transfected 293 cells synchronized by thymidine/nocodazole block and released for up to 24 h. Cells pass through S and G2 phases between 16-24 h after release as determined by Cyclin A2 expression. The initial ‘0’ lanes on the two blots show siFHIT and siCtrl lanes for direct comparison to the juxtaposed ‘0’ lanes for the siCtrl and siFHIT time-course immunoblots of TK1 and Cyclin A2 expression. Error bars indicate standard deviation.

Article Snippet: 2.3. siRNAs transfections 293 cells (60-70% confluent) were transfected with siRNAs targeting FHIT and non-specific scrambled control siRNAs (Santa Cruz Biotechnology); H1299 E1 cells (80-90% confluent) were transfected with siRNAs targeting human DcpS or a non-specific control siRNA (Santa Cruz Biotechnology), using the manufacturer’s recommended protocol.

Techniques: Western Blot, Transfection, Expressing, Blocking Assay, Comparison, Standard Deviation

Journal: Biochimica et biophysica acta

Article Title: Identification of Fhit as a Post-transcriptional Effector of Thymidine Kinase 1 Expression

doi: 10.1016/j.bbagrm.2017.01.005

Figure Lengend Snippet: A. Parental H1299 cells were co-transfected with plasmids expressing firefly luciferase with the TK1 5’-UTR and Renilla luciferase, and either empty vector or plasmids expressing WT Fhit or the catalytically inactive H96N mutant. The impact of Fhit catalytic activity on expression driven by the TK1 5’-UTR was determined by dual luciferase assays. Data shown are the means ±SEM of three independent experiments (each measured in triplicate). The asterisk (*) indicates p <0.05 as determined by two-tailed Student T test. B. Extracts from DcpS positive HCT116 cells and H1299 cells used in this study were analyzed by Western blotting for DcpS and for Vinculin. C. H1299 E1 cells were transfected with control or DcpS siRNA and monitored by Western blotting for DcpS, Fhit, Vinculin and TK1.

Article Snippet: 2.3. siRNAs transfections 293 cells (60-70% confluent) were transfected with siRNAs targeting FHIT and non-specific scrambled control siRNAs (Santa Cruz Biotechnology); H1299 E1 cells (80-90% confluent) were transfected with siRNAs targeting human DcpS or a non-specific control siRNA (Santa Cruz Biotechnology), using the manufacturer’s recommended protocol.

Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Two Tailed Test, Western Blot, Control

Journal: bioRxiv

Article Title: In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme

doi: 10.1101/644492

Figure Lengend Snippet: Biochemical Assays for DcpS. ( a ) Conversion of m 7 GpppA dinucleotide to m 7 GMP. A 100 μL reaction in decapping buffer containing 50 μM m 7 GpppA and either 50 nMy DcpS (blue □) or 85 nM hDcpS (red ○) was incubated at 37 °C. 10 μL samples were withdrawn at the indicated times. The reactions were terminated by the addition of equal volume of phenol/chloroform. The relative quantity of the remaining dinucleotide m 7 GpppA, m 7 GMP, and ADP were determined by LC-MS. ( b ) Decapping of a 25mer Cap 1 RNA by yDcpS. A 30 μL reaction containing 300 ng of total E. coli RNA and 60 ng of 25mer Cap 1 RNA was incubated for 3 hours at 37 °C with 130 ng of yDcpS in 10 mM MES pH 6.5 and 1 mM EDTA. At 0 minutes a 5 μL aliquot was mixed with 2X RNA loading dye stop solution (Lane 1). Likewise 5 μL aliquots were taken at 5, 60, 120, and 180 min (Lanes 2 to 5, respectively). The RNA aliquots were analyzed by 15% TBE-Urea PAGE stained with SYBR Gold. ( c ) Capillary electrophoresis of 3’-FAM-labeled 5’-capped 25mer RNA. Top panel shows a representative example of the decapping reaction progress. The 25mer Cap 0 RNA was incubated with yDcpS for various times indicated on the left. Bottom panel shows the mobility shift for standards of 25mer RNAs containing different 5’ ends. All data were plotted relative to GeneScan™120 LIZ™ Applied Biosystems standards.

Article Snippet: Human DcpS (hDcpS) was purchased from Enzymax LLC, Lexington, KY.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Staining, Electrophoresis, Labeling, Mobility Shift