Journal: PLoS ONE

Article Title: The AP2 -Like Gene OitaAP2 Is Alternatively Spliced and Differentially Expressed in Inflorescence and Vegetative Tissues of the Orchid Orchis italica

doi: 10.1371/journal.pone.0077454

Figure Lengend Snippet: ( a ) Agarose gel electrophoresis of the PCR amplification products of the OitaAP2 cDNA with different primer pairs: lane 1, AP2F4new/AP2RealR; lane 2, AP2F4new/AP2R5; lane 3, AP2F5/APR3; lane 4, AP2RealF_iso/AP2R4; lane 5, AP2F5/AP2R2; lane 6, 100 bp DNA ladder (Thermo Scientific); lane 7, DNA Marker III (Thermo Scientific). ( b ) Schematic representation of the OitaAP2 cDNA with the relative positions of the primers used in the PCR amplifications. Black boxes represent the 5′- and 3′-UTRs; white boxes represent the exons; gray box represents the alternatively spliced region; dotted lines highlight the region corresponding to the miR172 target site with the black arrow indicating the cleavage point. ( c ) Agarose gel electrophoresis of the PCR amplification product of the OitaAP2 cDNA fragment cleaved by miR172 (lane 1); lane 2, 100 bp DNA ladder (Thermo Scientific). ( d ) Schematic representation of the genomic organization of the OitaAP2 gene. Black boxes represent the 5′- and 3′-UTRs; white boxes represent the exons; gray box represents the alternatively spliced exon; continuous lines represent introns; dotted lines indicate the two alternative splicing events; numbers indicate the length of introns (bp); question marks indicate the unknown size of intron 9.

Article Snippet: PCR amplifications were performed using the LongAmp Taq PCR Kit (New England Biolabs) following the manufacturer instructions.

Techniques: Agarose Gel Electrophoresis, Amplification, Marker

Journal: mBio

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

doi: 10.1128/mBio.03027-19

Figure Lengend Snippet: KSHV circRNAs packaged into viral particle. A recombinant KSHV virus was produced from BJAB-rKSHV.219 cells and purified on a 20% to 35% Histodenz gradient. Histodenz fractions (BF, bottom fraction; TF, top fraction; SF, supernatant fraction) were treated with 1% Triton X-100 or left untreated. (A) Experimental workflow. (B) Total numbers of infectious KSHV particles from each Histodenz fraction and the unpurified crude virus (CV) preparation. Bar graphs in panel B show means ± SD of results from three replicates. (C) LANA/ORF73 PCR to detect KSHV genome in DNA extracted from the three different Histodenz fractions with and without Triton X-100 treatment. Genomic DNA from BC1 cells was used as a positive control. (D) RT-PCR detection of KSHV circRNA (circ-vIRF4, circPAN, and circK7.3) and linear RNA (vIRF4) in Histodenz fractions with and without detergent treatment. Linear viral transcript K2/vIL6 was used as a positive control and was packaged into viral particles, while LANA/ORF73 RT-PCR served as a negative control. (E) (Right panel) Nuclear/cytoplasmic fractionation in BC1 cells and KSHV circRNA detection by RT-PCR using divergent primers. (Left panel) Schematic representation of the two differentially localized circ-vIRF4 forms (dark green arrows, DP primers; pink bars, junction-spanning TaqMan probe; red arrows, CP primers; light green bar, TaqMan probe) (top) and a Western blotting control for the fractionation assay using a nuclear marker (ORC-2) and a cytoplasmic marker (LAMP-1) (bottom). NE, nuclear extraction; CE, cytoplasmic extraction. Results are representative of at least 3 independent experiments.

Article Snippet: Semiquantitative PCR was then performed using either Q5 high-fidelity DNA polymerase (catalog no. M0491; New England BioLabs/NEB) for circRNA detection or Taq DNA polymerase with ThermoPol buffer (catalog no. M0267; NEB) for linear RNA detection, according to the protocols specified for the products.

Techniques: Recombinant, Produced, Purification, Positive Control, Reverse Transcription Polymerase Chain Reaction, Negative Control, Fractionation, Western Blot, Marker

Journal: PLoS ONE

Article Title: Upregulation of the Cell-Cycle Regulator RGC-32 in Epstein-Barr Virus-Immortalized Cells

doi: 10.1371/journal.pone.0028638

Figure Lengend Snippet: (A) Diagram showing RGC-32 transcript variants. Black boxes represent exons and dotted lines show the parts of exons 1 and 2 that are not included in the shorter RGC-32 transcript (AF036549) that encodes a protein of 117 amino acids . The longer RGC-32 (C13ORF15) transcript (NM_014059) is predicted to encode a protein of 137 amino acids. (B) Q-PCR analysis using primers in exon 3. Transcript quantities were normalized to those of GAPDH and then expressed relative to the signal obtained in DG75 cells. Results show the mean of 3 independent experiments +/− standard deviation. (C) Non-quantitative PCR analysis of cDNA samples using primers that amplify across exon 2 to exon 4. pFLAG-RGC-32 was used as a positive control (con). Q-PCR analysis using primers that amplify across exons 2 and 3 (D) or exons 4 and 5 (E). Transcript quantities were normalized to GAPDH and results show the mean of 3 independent experiments +/− standard deviation. (F) Northern blot analysis of RGC-32 transcripts. Blots were probed with a [ 32 P]-labelled probe generated from the entire RGC-32 cDNA and then stripped and re-probed for GAPDH.

Article Snippet: For PCR across exons 2-4 of RGC-32, cDNA was amplified using Taq polymerase (New England Biolabs) according to the manufacturer's recommended conditions and 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. For RNA half-life experiments, Q-PCR was carried out using Power SYBR Green Cells-to-CT Kit (Applied Biosystems) and RGC-32 or GAPDH specific primers followed by analysis using the Relative Quantification (ddCt) method.

Techniques: Standard Deviation, Real-time Polymerase Chain Reaction, Positive Control, Northern Blot, Generated

Journal: PLoS ONE

Article Title: Upregulation of the Cell-Cycle Regulator RGC-32 in Epstein-Barr Virus-Immortalized Cells

doi: 10.1371/journal.pone.0028638

Figure Lengend Snippet: (A) Q-PCR analysis of RUNX1c mRNA levels. Transcript quantities were normalized to those of GAPDH and then expressed relative to the signal obtained in DG75 cells. Results show the mean of 3 independent experiments +/− standard deviation. (B) Western blot analysis of RUNX1c protein expression in a panel of EBV negative and positive B-cell-lines. Actin levels serve as a loading control. (C) Transient reporter assays in DG75 cells transfected with 4 µg of the RGC-32 promoter-reporter construct (pRGC-32pluc), 2 µg pRL-CMV as a transfection control and increasing amounts (2.5, 5 and 10 µg) of a RUNX1c-expressing plasmid (pBK-CMV-RUNX1c). Firefly luciferase signals were normalized to Renilla luciferase signals. Results show the mean of 3 independent experiments +/− standard deviation. RGC-32 promoter activation is expressed relative to the RUNX1-negative control. ** P value <0.01 (0.004), * p value <0.05 (0.036). (D) IB4 cells were transfected with pCEP4 or pCEP4-RUNX1c plasmids and transfected cells selected in Hygromycin B. 6 days post-transfection samples were analysed for RUNX1c protein expression by western blotting using actin as a loading control and endogenous RGC-32 mRNA expression using Q-PCR. Results show the mean of 2 independent experiments −/+ standard deviation. * P value <0.05 (0.012).

Article Snippet: For PCR across exons 2-4 of RGC-32, cDNA was amplified using Taq polymerase (New England Biolabs) according to the manufacturer's recommended conditions and 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. For RNA half-life experiments, Q-PCR was carried out using Power SYBR Green Cells-to-CT Kit (Applied Biosystems) and RGC-32 or GAPDH specific primers followed by analysis using the Relative Quantification (ddCt) method.

Techniques: Standard Deviation, Western Blot, Expressing, Transfection, Construct, Plasmid Preparation, Luciferase, Activation Assay, Negative Control

Journal: PLoS ONE

Article Title: Upregulation of the Cell-Cycle Regulator RGC-32 in Epstein-Barr Virus-Immortalized Cells

doi: 10.1371/journal.pone.0028638

Figure Lengend Snippet: (A) Mutu I, Mutu III and IB4 cells were treated in parallel with 50 or 100 µM MG132 and harvested after 2 or 6 hrs for Western blot analysis. Blots were probed in sections for RGC-32 and actin. IB4 blots were also probed for p53 as a control for MG132-mediated protein stabilization through proteasomal inhibition. (B) Mutu I and III cells were fractionated into cytoplasmic (C) and nuclear (N) protein extracts and analysed by Western blotting for nuclear (Spt16) and cytoplasmic (actin) marker proteins to confirm purity of the fractions. RGC-32 (C) and GAPDH (D) transcript levels were quantified by Q- PCR in cytoplasmic (C) and nuclear (N) RNA extracts prepared in parallel to protein extracts.

Article Snippet: For PCR across exons 2-4 of RGC-32, cDNA was amplified using Taq polymerase (New England Biolabs) according to the manufacturer's recommended conditions and 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. For RNA half-life experiments, Q-PCR was carried out using Power SYBR Green Cells-to-CT Kit (Applied Biosystems) and RGC-32 or GAPDH specific primers followed by analysis using the Relative Quantification (ddCt) method.

Techniques: Western Blot, Inhibition, Marker

Journal: PLoS ONE

Article Title: Upregulation of the Cell-Cycle Regulator RGC-32 in Epstein-Barr Virus-Immortalized Cells

doi: 10.1371/journal.pone.0028638

Figure Lengend Snippet: Cell-lines were treated with Actinomycin D and samples analysed at the times indicated. RGC-32 mRNA levels were deteremined by Q-PCR and normalized to those of the stable control message GAPDH. Results are expressed relative to the level detected at time 0 and show the mean +/− standard deviation of 4 independent experiments for Akata (A) or two independent experiments for Mutu I (B) and Mutu III cells (C). The half-life values indicated were calculated using non-linear regression analysis.

Article Snippet: For PCR across exons 2-4 of RGC-32, cDNA was amplified using Taq polymerase (New England Biolabs) according to the manufacturer's recommended conditions and 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. For RNA half-life experiments, Q-PCR was carried out using Power SYBR Green Cells-to-CT Kit (Applied Biosystems) and RGC-32 or GAPDH specific primers followed by analysis using the Relative Quantification (ddCt) method.

Techniques: Standard Deviation

Journal: PLoS ONE

Article Title: Upregulation of the Cell-Cycle Regulator RGC-32 in Epstein-Barr Virus-Immortalized Cells

doi: 10.1371/journal.pone.0028638

Figure Lengend Snippet: Cytoplasmic extracts were sedimented on sucrose density gradients and 0.5 ml or 1 ml fractions collected with continuous monitoring of absorbance at 254 nm (upper panels in A and B). 80S monosome peaks are indicated by arrows. Transcript levels in each fraction were determined using Q-PCR and specific primers to RGC-32 (black bars), GAPDH (open bars) and actin (grey bars). Transcript levels are expressed as a percentage of the total transcript levels detected across the gradient (nt indicates fractions that were not tested). (A) Parallel analysis of Akata (latency I, no RGC-32 protein expression) and LCL#3 (latency III, RGC-32 protein expressed) polysomes. (B) Parallel analysis of Mutu I (latency I, no RGC-32 protein expression) and Mutu III (latency III, RGC-32 protein expressed) polysomes.

Article Snippet: For PCR across exons 2-4 of RGC-32, cDNA was amplified using Taq polymerase (New England Biolabs) according to the manufacturer's recommended conditions and 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. For RNA half-life experiments, Q-PCR was carried out using Power SYBR Green Cells-to-CT Kit (Applied Biosystems) and RGC-32 or GAPDH specific primers followed by analysis using the Relative Quantification (ddCt) method.

Techniques: Expressing