FIGURE 1
FROM:
Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis
Nata
a Levi
ar, Ricardo A Dewey, Emma Daley, Timothy E Bates, Derek Davies, Janko Kos, Geoffrey J Pilkington and Tamara T Lah
Figure 1.
A: Flow cytometric analysis of intracellular content of cathepsin L in human glioblastoma IPTP cells and IPTP-transfected clones. Cells were stably transfected with PreproCatL cDNA in antisense and sense orientation. Cathepsin L content was determined using monoclonal antibodies and flow cytometric analysis. The relative amounts of cathepsin L in the transfected IPTP cells are presented as the overlaid histogram and compared with the control parental IPTP cells (thick black line). The IPTP4asn clone (dark grey line) showed the lowest content of cathepsin L (shift to the left), whereas the IPTP24sn (light grey line) cells showed the highest cathepsin L protein levels (shift to the right) compared to the parental IPTP cells. IPTP cells where primary antibodies were omitted represented negative control (thin black line). B: Cathepsin L RNA quantification in IPTP parental and IPTP-transfected clones. Cathepsin L RNA content was measured by real-time PCR in the parental IPTP, and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin L RNA levels were expressed relative to GADPH levels. Significantly (29%) lower cathepsin L RNA levels in IPTP4asn cells and significantly (66%) higher cathepsin L RNA levels in IPTP24sn cells were found compared to untransfecetd IPTP cells. Bars represent mean value of two independent experiments and standard error of the mean (mean
SEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line. C: Cathepsin L protein concentration in cell lysates of selected clones of IPTP cells. Cathepsin L protein was measured by ELISA in the parental IPTP, and transfected IPTPvgfp, IPTP4asn, and IPTP24sn cell lines. Cathepsin L protein concentrations were expressed relative to total protein concentration in the cell lysates. Significantly (41%) lower cathepsin L concentrations in IPTP4asn cells and significantly (150% higher) cathepsin L concentrations in IPTP24sn cells were found compared to untransfecetd IPTP cells. Bars represent mean value of three independent experiments and standard error of the mean (meanSEM). ***Indicates statistically significant (P<.05) difference compared with the parental IPTP cell line.
