Figure 1

Effect of metalloprotease inhibitors on the expression and secretion of GP1, and cytotoxicity on LASV GPC-expressing HEK-293T/17 cells. Cells were transfected for 12 hours, followed by addition of metalloprotease inhibitors. (A) Supernatants were collected at 42 - 45 hours post transfection, cleared by centrifugation, and equal volumes from each reaction were resolved on SDS-PAGE, blotted, and probed with anti-GP1 mAb L52-74-7A. (B) GP1 secretion was detected by western blot analysis as early as 12 hours post transfection, as shown in a longer exposure (lane 17B). Control pcDNA vector was also overexposed in the same blot (lane 18B). (C) Intracellular expression of GPC at the time of harvest (lane 13C), and the corresponding pcDNA vector control (lane 15C), were compared with the same conditions at 12 hours post transfection (lanes 17C, 18C). (D) Relative levels of secreted GP1 were determined from triplicate experiments by densitometry scanning of exposed x-ray films. Data was normalized against GPC+DMSO (= 1) and plotted as mean ± SD, N = 3. Data analysis using ANOVA did not reveal statistically significant differences between any of the conditions containing metalloprotease inhibitors and untreated GPC expressing controls (p > 0.05). (E) MTT cytotoxicity assay from triplicate experiments, plotted as mean A570 nm ± SD, N = 3. ANOVA established a statistically very significant difference (p < 0.001) between MMP-2 inhibitor I and the GPC+DMSO control only. The metalloprotease inhibitors and experimental designations outlined on the x-axis in E. apply to D., and correspond to the numeration in A., B, and C. Positions for secreted and intracellular GP1 (42 KDa), and unprocessed GPC (75 KDa) in cell extracts are noted.