Characterization of cathepsin L antisense clones. (A) RT-PCR of cathepsin L message levels. Semi-quantitative RT-PCR was used to determine average relative cathepsin L message levels (performed in triplicate). Actin was used as an internal control and the relative product band densities were calculated based on measured pixels per band on scanned images with Image J software. Controls are B16F10 and PC2 (empty vector); AS1–9 are putative antisense clones. S3 is a sense clone. The difference between cathepsin L message levels for control and antisense clones (except AS-2 and AS-9, *P < 0.05) were significant by Student t-test (** P < 0.001). (B) Growth rates of cathepsin L clones. Cells were seeded in 96-well culture dishes at 1 × 103 per well. At the times indicated, MTT was added (50 ul of a 5 mg/ml solution) for incubation at 37°C for 4 hours. Media was then removed and reaction product solubilized in 100 μl DMSO for 2 hours at room temperature. Absorbances were read at A570 nm wavelength. Clones were run in triplicate wells in triplicate experiments. No significant difference was noted between the clones analyzed by Student t-test. (C) Cathepsin L activity of sense and antisense cathepsin L clones. Clones S1 and S3 are sense clones. Clones AS3, AS4, AS5, AS7, and AS8 are antisense clones. Cell extracts were prepared by lysis with incubated with Z-Phe-Arg-AMC for 30 minutes, with or without cathepsin L inhibitor in triplicate. The proteolytic activity was expressed in units (1 U = 1 μmol product per minute). ANOVA analysis showed significant differences between sense and antisense cathepsin L activities (* P < 0.01). (D) Western blot analysis of cathepsin L. Controls B16F10 and PC2 (pcDNA-3 vector transfected) were compared to antisense clones AS3 and AS5 for cathepsin L levels in cell extracts by Western blot analysis. Western blot detection was with ECL (Amersham) reagents.