FIG 8.

SLFN5 knockdown increases expression and secretion of MMP-1 and MMP-13. (A) 786-0 cells were transiently transfected with control siRNA or SLFN5 siRNA, and gene expression for MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, MMP-14, and MMP-15 was analyzed 48 h after transfection by real-time RT-PCR using specific primers and GAPDH as an internal control. Data are expressed as the fold increase over the respective control and represent means ± standard errors of results from three experiments. Paired two-tailed t test analysis showed a two-tailed P value of 0.0363 for MMP-1 versus the respective control (*) and a P value of 0.0219 for MMP-13 versus the respective control (**). (B) 786-0 cells were transiently transfected with control siRNA or SLFN5 siRNA, and at 48 h after transfection, cells were resuspended in serum-free medium and cultured for an additional 24 h. The conditioned media were collected and concentrated, and equal amounts of proteins were resolved in parallel by SDS-PAGE and immunoblotted with antibodies against MMP-1 and MMP-13. The MMP-1 blot was also probed with anti-MMP-9 and anti-MMP-2 antibodies.