Journal: Nature Communications

Article Title: Optoribogenetic control of regulatory RNA molecules

doi: 10.1038/s41467-020-18673-5

Figure Lengend Snippet: a shRNA variants used to control cyclin B1 gene expression. Blue: aptamer domain; orange: siRNA domain. b Percentages of HEK293PAL cells in G 2 /M phase of the cell cycle when transfected with indicated shRNAs targeting cyclin B1. c shRNA variants used to control CDK1 gene expression. Blue: aptamer domain; green: siRNA domain. d Percentages of HEK293PAL cells in G 2 /M phase of the cell cycle when transfected with indicated shRNAs targeting CDK1. b N = 20 (SHCB1, SHCB1m) or 6 (SHCB2, SHCB3). d N = 10. b , d Each biologically independent experiment was performed in duplicates. b The identity of SHCB1 and SHCB1m was blinded and double-blinded in one experiment, each. d The identity of SHCDK1 and SHCDK1m was blinded and double-blinded in one experiment, each. b , d Wilcoxon two-sided signed-rank test was used for statistical analysis. e , Representative western blot image showing cyclin B1, CDK1 and GAPDH protein expression after transfection with the indicated shRNAs (for complete blots see Supplementary Fig. ). e , N = 4. e One biologically independent experiment was performed in duplicates, all others once. f , g Quantification of cyclin B1 and CDK1 protein levels using pixel densitometry. f , g N = 3. f , g All biologically independent experiments were performed once. f , g Cohen’s d effect size was used for statistical analysis. Values were normalized to non-transfected cells incubated in darkness (Untransfected). b , d , f , g Gray bars: cells incubated under light conditions, black bars: cells incubated under dark conditions. Values are means ± s.d. Source data for ( b , d , f , g ) are provided as source data file.

Article Snippet: 5 μg of protein per lane was loaded onto 10 or 12.5% SDS-PAGE gels and blotted in Transfer Buffer (2.5 mM Tris, 2% (w/v) glycine, 0.9 M urea) onto a nitrocellulose membrane (GE Healthcare Life Sciences) using a Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell (BioRad) for 75 min at 20 V and 30 W. Membranes were blocked with TBS-T buffer (20 mM Tris/HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20 (v/v)) containing 5% BSA (AppliChem, Western Blot grade) under agitation at room temperature for 1 h. Blots were cut according to the protein ladder (Prestained Protein Ladder—Mid-range molecular weight (10–180 kDa), abcam) in a way that all target proteins can be individually incubated with the respective primary antibody (mouse anti -cdc2 (CDK1), Cell signaling POH1, #9116 (1:1000); mouse anti -GAPDH, Santa Cruz Biotechnology sc-47724 (1:4000); goat anti- Cyclin B1, R&D Systems AF6000 (1:1000)) at 4 °C overnight or at room temperature for 1 h in TBS-T containing 5% BSA.

Techniques: shRNA, Control, Gene Expression, Transfection, Western Blot, Expressing, Incubation

Journal: Cell Discovery

Article Title: Phosphorylation of cGAS by CDK1 impairs self-DNA sensing in mitosis

doi: 10.1038/s41421-020-0162-2

Figure Lengend Snippet: a CDK1 but not AKT or CDK2 inhibitor abolishes mcGAS S291 phosphorylation. Raw264.7 cells were arrested at mitosis with nocodazole (Noc arrest) and then treated with the indicated kinase inhibitors (10 μM) for 15 min before immunoblotting or FACS analysis. b cGAS is a direct substrate of the CDK1-cyclin B complex. In vitro kinase assays were performed with recombinant CDK1-Cyclin B proteins and FLAG-cGAS immunoaffinity-purified from THP1 cells stably-expressing FLAG-cGAS in the presence or absence of RO-3306 (10 μM). The reactions were then analyzed by immunoblotting analysis with the indicated antibodies. c The CDK1-cylin B1 complex impairs cGAS activation. MITA stably-expressing HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. *** P < 0.001 (Student’s t -test, unpaired, two-tailed). Data shown are mean ± S.D. of one representative experiment performed in triplet. Data are representative of three biological repeats.

Article Snippet: The expression plasmid for cyclin B1 was purchased from Origene.

Techniques: Western Blot, In Vitro, Recombinant, Purification, Stable Transfection, Expressing, Activation Assay, Transfection, Luciferase, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: The PIM1 Kinase Is a Critical Component of a Survival Pathway Activated by Docetaxel and Promotes Survival of Docetaxel-treated Prostate Cancer Cells * S⃞

doi: 10.1074/jbc.M709479200

Figure Lengend Snippet: Independence of PIM1 expression and cell cycle arrest. A, DNA histogram analysis of RWPE-2 cells after docetaxel 10 or 100 nm treatments for 24 h. sG1, a sub-G1 cell population with less than 2 n DNA content. G1 and G2, the appearance of cells in G0/G1 or G2/M phases of the cell cycle. B, immunoblot analysis of cyclin B1 and PIM1 expression after docetaxel 10 nm (B, left) or 100 nm (B, right) treatment at various time points.

Article Snippet: The following monoclonal antibodies were used: anti-β-ACTIN (clone AC-15; Sigma), anti-PIM1 (clone 12H8; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-BCL xL (clone H-5; Santa Cruz Biotechnology), anti-phospho-STAT3 (Tyr 705 ) (clone 3E2; Cell Signaling), anti-total STAT3 (clone 84; BD Biosciences), anti-GAPDH (clone FL-335; Santa Cruz Biotechnology), anti-PRDX5 (Transduction Laboratories), and anti-human cyclin B1 (clone GNS-1; BD Biosciences).

Techniques: Expressing, Western Blot