Journal: PLoS ONE

Article Title: Cbp80 is needed for the expression of piRNA components and piRNAs

doi: 10.1371/journal.pone.0181743

Figure Lengend Snippet: (A, D) Knock down of Cbp80 in egg chambers expressing a dsRNA against Cbp80 (dsCbp80 ) driven by the GAL4-nos.NGT40 driver and the UAS-Dcr2. Control flies expressed a dsRNA against GFP ( dsGFP ) (B, C) Ovaries expressing specifically in the germline ( pCog-Gal4 driver) shRNAs against Cbp80 or mCherry (as control). Control flies used for RT-PCR experiments expressed also a Jupiter-mCherry fusion protein. (A-B) mRNA levels of piRNA pathway factors ( piwi , aub , Rhi , zuc and Ago3 ) and Cbp80 were measured by qRT-PCR. Fold expression levels of each mRNA in the knockdown samples relative to its expression in the corresponding control are shown for each mRNA. Total starting RNA amounts were the same in both samples. Error bars represent + /- SD of 2 controls in B and 3 control samples in A and 3 biological knockdown replicates. *p<0.05; **p<0.01; ***p<0.001. (C-D) Levels of Cbp80, Piwi, Aub, BicD, Cdk7, Clc and Tub (as loading control) were assessed by Western blotting. Ponceau staining is also shown to reveal total proteins as loading control. Levels Piwi, Aub and Cdk7 were strongly reduced upon Cbp80 reduction. On the other hand, levels of Tub, Clc and BicD were less affected. For Cbp80 knockdown samples were extracted from egg chambers showing the phenotype “d” .

Article Snippet: Western blotting was carried out with mouse anti-Piwi P3G11 (1:1,000; [ ]), mouse anti-Aub 4D10 (1:2,000; [ ]), rabbit anti-Cbp80 (1:1,000; [ ]), mouse anti-alpha tubulin primary antibodies (1:250; Developmental Studies Hybridoma Bank), mouse anti-BicD 1B11 (1:10; [ ]), mouse anti-Cdk7 (1:10 mix of 20H5 and 19E7.2 clones; [ ]) and rabbit anti-Clc (1:3000, [ ]).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining

Journal: FEBS letters

Article Title: Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism.

doi: 10.1016/s0014-5793(03)00028-0

Figure Lengend Snippet: Fig. 1. Cell cycle-dependent expression of chromosomally derived cyclin B1. NIH3T3 mouse ¢broblasts and HFF were serum-starved and re- stimulated by addition of serum to the medium. After 48 h of starvation, the 0 h time point represents cells mostly resting in G0. The following time points give results after serum restimulation. A: Cyclin B1 mRNA expression from NIH3T3 cells as measured by RT-PCR standardised relative to total RNA; averages of two experiments are given. B: FACS analyses of NIH3T3 cell populations at selected time points before and after serum addition. Cells were stained with propidium iodide and measured for DNA staining per cell. C: Expression of cyclin B1 mRNA in HFF cells with total RNA as standard quanti¢ed by RT-PCR. Averages of two experiments are given. D: Cyclin B1 protein expres- sion detected by immunoblotting. L-Actin served as a loading control.

Article Snippet: As negative control served a mixture of 0.5 Wg mouse monoclonal anti-cyclin B1 antibody (GNS1, Santa Cruz Biotechnology) and 0.5 Wg goat polyclonal anti-cyclin B2 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Western Blot, Control

Journal: FEBS letters

Article Title: Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism.

doi: 10.1016/s0014-5793(03)00028-0

Figure Lengend Snippet: Fig. 4. p300 activates transcription from the cyclin B1 gene and binds to its promoter. A: Increasing amounts of plasmids expressing the three subunits of NF-Y (A, B and C) and p300 were cotrans- fected with the cyclin B1 promoter reporter and a Renilla luciferase control plasmid in SaOS-2 cells. The total amount of DNA was held constant in all transfections by adding an irrelevant plasmid. Fire£y luciferase was measured and standardised to Renilla lucifer- ase activity. Expression from the cyclin B1 promoter without co- transfection was set at 100%. Assays from three independent experi- ments with standard deviations are shown. B: ChIP experiments with antibody directed against p300 on the cyclin B1 promoter. The antibodies used in the control lane were directed against non-DNA binding protein. The K-globin gene served as negative control pro- moter.

Article Snippet: As negative control served a mixture of 0.5 Wg mouse monoclonal anti-cyclin B1 antibody (GNS1, Santa Cruz Biotechnology) and 0.5 Wg goat polyclonal anti-cyclin B2 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Control, Plasmid Preparation, Transfection, Activity Assay, Cotransfection, Binding Assay, Negative Control

Journal: FEBS letters

Article Title: Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism.

doi: 10.1016/s0014-5793(03)00028-0

Figure Lengend Snippet: Fig. 3. Expression from a cyclin B1-promoter luciferase reporter comparing activity in G2/M versus resting cells. Mutation of the CHR leads to a loss of cell cycle regulation. Luciferase reporter plasmids carrying the human cyclin B1 upstream region together with a Renilla luciferase-expressing control plasmid were transfected into NIH3T3 ¢broblasts. Cells were synchronised by serum starva- tion for 66 h and restimulated. Ratios of luciferase activities from cell lysates 24 h after restimulation to activities in resting cells are given. Averages with standard deviations from six experiments are shown. Renilla luciferase was employed to standardise cyclin B1 ¢re- £y luciferase reporter expression.

Article Snippet: As negative control served a mixture of 0.5 Wg mouse monoclonal anti-cyclin B1 antibody (GNS1, Santa Cruz Biotechnology) and 0.5 Wg goat polyclonal anti-cyclin B2 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Control, Plasmid Preparation, Transfection

Journal: Scientific Reports

Article Title: Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein

doi: 10.1038/srep28807

Figure Lengend Snippet: ( a ) Schematic representation of full length and terminated cap transcripts. TSSs (+1) are labeled by bent arrows. AUG is the start code. ( b ) Sequence alignments of Bc2 RNAs from B. thuringiensis and B. cereus with Vc2 RNA. White letters shaded in magenta, orange, green and black denote the conserved nucleotides of A, G, and C and U, respectively. The predicted c-di-GMP binding sites were highlighted by red stars, with the consensus sequences of Bc2 RNAs depicted below the alignments. The aptamer region and expression platform were underlined by blue and brown lines, respectively.

Article Snippet: Reaction mixtures were also supplemented with c-di-GMP (Biolog Life Science Institute, Germany) at final concentrations defined for each reaction.

Techniques: Labeling, Sequencing, Binding Assay, Expressing

Journal: Scientific Reports

Article Title: Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein

doi: 10.1038/srep28807

Figure Lengend Snippet: ( a ) The relative expression levels of the 5′-UTR of cap in comparison with the complete cap gene by qPCR analyses in the BMB171 strain. ( b ) The differential expression levels of cap in the ΔBc2 and BMB171 strain by qPCR. Expression of gapdh gene was served as a negative control. ( c ) Products of in vitro transcription of DNA templates coding for the wild type (WT) and substitution mutant (A11T) riboswitches. The A11T mutant carries a single A to T mutation in the c-di-GMP binding pocket. FL and T denote full length and terminated transcripts, respectively. ( d ) The predicted secondary structures of Bc2 RNA in the absence and presence of c-di-GMP. Nucleotides predicted to bind to c-di-GMP are highlighted in red, and nucleotides predicted to form the terminator hairpin in the absence of c-di-GMP are highlighted in olive. The coding sequence of cap is boxed. Error bars depict SD of data from three independent experiments. ***P < 0.001.

Article Snippet: Reaction mixtures were also supplemented with c-di-GMP (Biolog Life Science Institute, Germany) at final concentrations defined for each reaction.

Techniques: Expressing, Negative Control, In Vitro, Mutagenesis, Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein

doi: 10.1038/srep28807

Figure Lengend Snippet: ( a ) Different intracellular c-di-GMP concentrations of the Δ 2dgc , Δ 3pde and BMB171 strains as determined by LC-MS/MS. ( b ) qPCR analyses of differential transcription profiles of cap in the Δ 2dgc and Δ 3pde mutants in comparison with the parent strain BMB171. ( c ) β-galactosidase activity analyses for B. thuringiensis strains carrying the P cap - lacZ , P cap Δ- lacZ or promoterless Pnull- lacZ transcriptional fusion plasmids. ( d ) β-galactosidase activity analyses for B. thuringiensis strains carrying either P cap - lacZ or P cap - lacZ site-mutants or promoterless Pnull- lacZ transcriptional fusion plasmids. ( e ) Model showing proposed regulation of cap transcription by Bc2 RNA. Error bars depict SD of data from three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05.

Article Snippet: Reaction mixtures were also supplemented with c-di-GMP (Biolog Life Science Institute, Germany) at final concentrations defined for each reaction.

Techniques: Liquid Chromatography with Mass Spectroscopy, Activity Assay