Journal: Biological chemistry

Article Title: Photoactivated inhibition of cathepsin K in a 3D tumor model

doi: 10.1515/hsz-2015-0274

Figure Lengend Snippet: (A) Taqman RT-PCR analysis of human CK gene expression in CK-1 and CK-2 clones. Data are normalized to HPRT1 and shown as average fold increase relative to EV (for 3 replicate experiments). (B) Western blot analysis of CK protein (37 kDa proenzyme and 28 kDa mature enzyme; top panel) and DDK (middle panel) expression in representative samples from PC3 prostate carcinoma cells stably transfected with empty vector (PC3-EV) or CTSK plasmids with DDK tag (PC3-CTSK); tubulin was used as loading control. (C) Immunofluorescent staining for DDK (green, left panel) indicative of CTSK expression; no primary antibody staining is shown as negative control (right panel); DAPI (blue) indicates nuclei; 40 × original magnification. (D) CK activity in CK clones. Assay was run against CK substrate Z-Gly-Pro-Arg-AMC the presence of cathepsin B inhibitor CA074. Data are shown as fold increase relative to CTSK activity in PC3-EV cells and a representative of three independent experiments. **p < 0.001; ***p < 0.0001 (values considered statistically significant).

Article Snippet: We confirmed minimal presence of CK mRNA in PC3 wild type cells (CT values > 30; data not shown) and utilized TrueORF ® Gold CK cDNA plasmids tagged with myc-DDK (OriGene) to establish stable CK-expressing clones (PC3-CK).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Staining, Negative Control, Activity Assay