Journal: The Journal of Experimental Medicine

Article Title: A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

doi: 10.1084/jem.20112145

Figure Lengend Snippet: Knockdown of PAF complex members impairs CSR. (a) CH12 cells were retrovirally transduced with shRNAs for AID, PAF1, LEO1, CTR9, CDC73, SUPT5H, or a respective nontarget. After stimulation, IgA surface expression was monitored by flow cytometry, representative plots are shown. Numbers within the FACS plots indicate the percentage of IgA-positive cells. (b) The mean percentage (+ SD) of CSR in stimulated cells that were RV-shRNA transduced (gray bars) or LV-shRNA transduced (black bars), relative to the nontarget shRNA control (white bars) set to 100% from three independent experiments, is shown. The difference (Δ) in CSR efficiency between nontarget and target shRNA knockdown is shown below. Significance of a decrease versus nontarget is indicated by the following p-values: *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on Student’s t test. Quantitative RT-PCR for AID, PAF1, LEO1, CTR9, CDC73, and SUPT5H transcripts (c), μ germline transcript (d), α germline transcript (e), and AID transcripts from cells transduced with RV-shRNA (gray bars) or LV-shRNA (black bars) shown relative to the corresponding nontarget shRNA control (f; white bars). Results are from three independent experiments. Transcript Ct values were normalized to CD79b or HPRT mRNA abundance and are presented relative to the levels in the nontarget shRNA negative control, set to 1. *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on Student’s t test.

Article Snippet: Retroviral knockdown was done as follows: vectors containing shRNAs specific for SUPT5H, PAF1, LEO1, CTR9, CDC73, and the nontarget shRNA control were purchased from OriGene ( Table S2 ).

Techniques: Knockdown, Transduction, Expressing, Flow Cytometry, shRNA, Control, Quantitative RT-PCR, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

doi: 10.1084/jem.20112145

Figure Lengend Snippet: AID binding to Sμ is impaired after LEO1 knockdown . Unstimulated and/or stimulated CH12 cells were transduced with a lentivirus expressing shRNAs specific for AID, LEO1, or a nontarget shRNA control. AID occupancy at the Sμ switch region was ChIP analyzed using anti-AID antibodies. Normalized AID-ChIP data from three independent experiments assayed with two different primer sets is shown. For each sample, AID-ChIP values were normalized to the input control. AID-ChIP signal in cells expressing a nontarget shRNA control was set to 1. P-values, two-tailed Student’s t test.

Article Snippet: Retroviral knockdown was done as follows: vectors containing shRNAs specific for SUPT5H, PAF1, LEO1, CTR9, CDC73, and the nontarget shRNA control were purchased from OriGene ( Table S2 ).

Techniques: Binding Assay, Knockdown, Transduction, Expressing, shRNA, Control, Two Tailed Test

Journal: Aging cell

Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4.

doi: 10.1111/j.1474-9726.2010.00585.x

Figure Lengend Snippet: Fig. 2 shRNA-mediated knockdown of WRNp in NCCIT cells. NCCIT cells were transfected with plasmids encoding one of two types of shRNA against WRN, or shRNA against G9a, or one of two control plasmids (CV – empty vector, CS – vector control expressing a scrambled, nontargeting sequence, G()) – G9a-deficient clone, 31–34, 61, and 62 – WRNp-deficient clones, see Table S3). Transfected cells were selected in puromycin, and individual clones were isolated and subjected to western blotting analysis with a WRNp antibody or a c-tubulin control.

Article Snippet: An empty control plasmid (TI20003, Origene) and control nontargeting shRNA (TI30003, Origene) were utilized.

Techniques: shRNA, Knockdown, Transfection, Control, Plasmid Preparation, Expressing, Sequencing, Clone Assay, Isolation, Western Blot

Journal: Aging cell

Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4.

doi: 10.1111/j.1474-9726.2010.00585.x

Figure Lengend Snippet: Fig. 8 Expression of stem cell markers in WRNp-deficient cells. (A) Control (CV, CS) and WRNp-deficient cells (31) were treated with retinoic acid (RA) for 7 days. Cells were then harvested, and the presence of stem cell markers was analyzed by western blotting (+, cells treated with RA; ), untreated control samples). TRA-1-60, TRA-1-81, and EpCAM bands are indicated. c-tubulin served as a loading control. (B) WRNp-deficient cells (31) were treated with RA for 7 days. Cells were then transfected with Oct4 siRNA (Oi) or control siRNA (Ci). Three days after transfection, cells were harvested and the expression of stem cell markers analyzed as described above ()RA – untreated cells, +RA – RA-treated cells).

Article Snippet: An empty control plasmid (TI20003, Origene) and control nontargeting shRNA (TI30003, Origene) were utilized.

Techniques: Expressing, Control, Western Blot, Transfection

Journal: Clinical and Translational Science

Article Title: PTTG1 Levels Are Predictive of Saracatinib Sensitivity in Ovarian Cancer Cell Lines

doi: 10.1111/cts.12413

Figure Lengend Snippet: Saracatinib treatment combined with siRNA knockdown of PTTG1 decreases cytosolic FGF2 protein expression and slows cell growth. ( a ) Efficient PTTG1 knockdown is confirmed by western blot assay. ( b ) Relative cell growth after 96 h of treatment is shown in the comparison to untreated parental cells (* P‐ value < 0.05). ( c ) After siRNA transfection, ES2 cells were treated with 0 or 1μM saracatinib for 72 h. Cytosolic FGF2 concentration was normalized to nanogram FGF2 / milligram protein extract and represented in the comparison to untreated parental cells (* P‐ value < 0.05). P, parental cells; NC, negative control scrambled siRNA transfection; +T, saracatinib treatment.

Article Snippet: A nontargeting scramble siRNA construct (TR30013, Origene) was used as a negative control.

Techniques: Knockdown, Expressing, Western Blot, Comparison, Transfection, Concentration Assay, Negative Control