Journal: International Journal of Molecular Sciences

Article Title: RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis

doi: 10.3390/ijms20143535

Figure Lengend Snippet: Induction of DRAK1 by RNase L activation and type I IFN signaling. DU145 and PC3 cells were transfected with Poly I:C (2 µg/mL) or 2–5A (10 µM) for indicated times and ( A , B ) mRNA expression of DRAK1 and DRAK2 were analyzed by qPCR, ( C , D ) protein expression was determined by immunoblot analysis. ( E , F ) DRAK1 and DRAK2 expression on immunoblots in HT1080 and U3A (STAT1-defective) cells, ( G ) mRNA levels in indicated cells were compared to untreated cells and analyzed by qPCR. Inset shows immunoblot analysis of RNase L KO cells ( H ) DRAK1 mRNA levels in PC3 levels transfected with Poly I:C (2 µg/mL), interferon (1000 U/mL) or combined treatment for 6 h, ( I ) Schematic of DRAK1 promoter region showing IFN-stimulated response element (ISRE) sequences (in green) and Octamer-Binding Protein-2 (Oct-2) binding sites (in red). DRAK1 promoter driven luciferase activity normalized to renilla luciferase in PC3 treated with Poly I:C (2 µg/mL) or 2–5A (10 µM) or interferon (1000 U/mL). Values shown are mean ± s.d of triplicate assays. * p < 0.01, ** p < 0.001, *** p < 0.0001, n.s: not significant, WT: Wild Type.

Article Snippet: DRAK1 and DRAK2 targeting siRNA and control siRNA were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Transfection, Expressing, Western Blot, Binding Assay, Luciferase, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis

doi: 10.3390/ijms20143535

Figure Lengend Snippet: Nuclear localization of DRAK1 and induction of apoptosis ( A ) Fluorescence microscopy of DRAK-GFP or GFP vector expression and bright field images at indicated times in PC3 cells (scale bar, 10 µm, representative images are shown). PC3 cells were transfected with vector or DRAK1 construct and cell viability was determined at indicated times by ( B ) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and normalized to control, ( C ) uptake of Propidium Iodide (PI) by dying cells as measured by flow cytometry after staining with PI, ( D ) trypan blue dye exclusion assay normalized to control cells. Data shown as % apoptotic cells. PC3 cells were transfected with siRNA targeting DRAK1, DRAK2 or scrambled control siRNA and knock down and JNK phosphorylation and caspase 3 cleavage was determined on immunoblots following transfection with Poly I:C ( E , F ), and ( G , H ) cell viability was analyzed after transfection with 2–5A (10 µM) by MTT assay and % apoptotic cells was determined by trypan blue exclusion assay. Values shown are mean ± s.d of triplicate assays. * p < 0.01, ** p < 0.001, ns: not significant.

Article Snippet: DRAK1 and DRAK2 targeting siRNA and control siRNA were obtained from Santa Cruz Biotechnology.

Techniques: Fluorescence, Microscopy, Plasmid Preparation, Expressing, Transfection, Construct, MTT Assay, Control, Flow Cytometry, Staining, Exclusion Assay, Knockdown, Phospho-proteomics, Western Blot, Trypan Blue Exclusion Assay