Additional file 3: Figure S3. of TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression

TSG101 depletion leads to increased levels of MMP-9 mRNA in U2OS cells. (A) Depletion of TSG101 by siRNA. Total cell lysates of cells transfected with control (con) or TSG101 (TSG#1) siRNA were analyzed by western blot using the indicated antibodies. (B) and (C) Secretion and expression of MMP-9 in TSG101-depleted cells. Cells transfected with control (con) or TSG101 (TSG#1) siRNA were incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. MMPs in conditioned media (B) and cell lysates (C) were measured using gelatin zymography. (D) MMP-9 mRNA expression in PMA-treated cells. Subconfluent cells were serum starved for 16 h and subsequently incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. Expression levels of MMP-9 and GAPDH mRNAs were analyzed by RT-PCR. The ratio of MMP-9 mRNA level relative to the GAPDH mRNA level is expressed as arbitrary units. MMP-9 mRNA level in non-treated cells is set to 1.0. (E) MMP-9 mRNA expression in TSG101-depleted cells. Cells transfected with control (con) or TSG101 (TSG#1) siRNA were incubated in fresh serum-free medium containing or not 200 nM PMA for 7 h. The ratio of MMP-9 mRNA level relative to the GAPDH mRNA level is expressed as arbitrary units. MMP-9 mRNA level in the cells transfected with control (con) siRNA is set to 1.0 individually in non- and PMA-treated cells. The blots and gels shown are representative of three independent experiments. The results shown are means ± S.D. of three independent experiments. **, p < 0.005, by a Student’s t-test. (PDF 592 kb