Figure 1.
Overexpression of CD9 in HT1080 cells does not alter the expression of other tetraspanins.
(A) The cell surface expression of CD9 on wild-type HT1080 cells was assessed using flow cytometric analysis after binding a monoclonal antibody specific for CD9 (mAb7, shaded histogram). A non-specific isotype-matched antibody (IgG) was used as a negative control (unshaded histogram). (B, C) Total RNA was collected from Mock- and CD9-HT1080 cells, reverse transcribed to cDNA, and probed using primers specific for integral tetraspanins using qRT-PCR. The fold change in mRNA expression of tetraspanins was calculated from resulting cycle threshold values normalized to cyclophilin D. (D) Flow cytometry was used to assess the cell surface expression of key tetraspanins on the cell surface of Mock- (left panels) and CD9- (right panels) HT1080 cells **, p<0.001.
Figure 2.
CD9-HT1080 cells have stable mRNA expression of integrins though α2 and β1 are reduced at the cell surface.
(A) The fold change in mRNA expression of integrins was compared between Mock- and CD9-HT1080 cells using qRT-PCR. (B) Cell surface expression of the same integrin subunits (shaded histograms) was evaluated by flow cytometry. A non-specific isotype-matched antibody (IgG) was used as a negative control (unshaded histograms).
Figure 3.
The invasive phenotype of HT1080 cells is increased upon CD9 overexpression.
A matrigel invasion assay was used to assess the invasive properties of Mock- and CD9-HT1080 cells. Cells were allowed to invade through matrigel and adhere to the bottom of a cell culture insert as detailed in the Materials and methods section. (A) A representative image of stained cells after 20 hours of invasion though matrigel. (B) Invasion through matrigel-coated inserts was compared to cells that invaded through uncoated inserts and results shown are normalized to Mock-HT1080 cell invasion *, p<0.05.
Figure 4.
MMP-9 expression and release are greatly enhanced in CD9-HT1080 cells.
(A) Fold changes in MMP and TIMP mRNA expression for Mock- and CD9-HT1080 cells were calculated from cycle threshold values using qRT-PCR. (B, C) Specific ELISA kits were used to measure the concentration (ng/ml) of pro- and active- MMP-1 and MMP-9 in the cleared supernatant of Mock- and CD9-HT1080 cells. (D) A representative gelatin zymogram of Mock- and CD9- HT1080 cell conditioned media. The absence of Coomassie staining at 92 kDa indicates the presence of pro-MMP-9 and at 72 kDa represents pro-MMP-2. (E) Quantification of the relative band intensity was calculated by densitometry analysis as described in the Materials and methods section *, p<0.05; **, p<0.001.
Figure 5.
Silencing MMP-9 in CD9-HT1080 cells is sufficient to suppress the invasive phenotype.
CD9-HT1080 cells were transiently transfected with short-interfering RNA directed to MMP-9 (CD9+ MMP-9 siRNA). Likewise, Mock- and CD9- HT1080 cells were transfected with a scrambled siRNA as a negative control (Mock+Ctr siRNA or CD9+ Ctr siRNA). (A) qRT-PCR analysis was used to measure changes in MMP-9 mRNA levels among Ctr and MMP-9 siRNA transfected cells. (B) Changes in release of MMP-9 (ng/ml) in to the cleared culture supernatant were examined using a MMP-9 specific ELISA kit. (C) Pro-MMP-9 levels in the supernatants of transiently transfected cells was measured using gelatin zymography and (D) quantified by densitometry. (E) A representative image of cells invading though matrigel after transfection with siRNA. (F) Percent cell invasion through matrigel-coated inserts after 20 hours was quantified and the results were normalized to Mock+Ctr siRNA treated cells *, p<0.05; **, p<0.001.
Figure 6.
Characterization of the second extracellular loop (EC2) deletion mutant of CD9 in HT1080 cells.
Upon transfection with the EC2 deletion mutant in HT1080 cells, mRNA expression was measured using primer pairs specific for the nucleotides coding the EC2 (A) and TM1 (B) regions of CD9. (C) Flow cytometric analysis of Mock-, CD9-, and Δ6-HT1080 cells is indicated by each row, and each column indicates CD9 EC2 (mAb7), CD9 TM1 (Rap2), or CD151 binding (shaded histograms) **, p<0.001.
Figure 7.
An increase in MMP-9 expression and release and subsequent cell invasion requires the second extracellular loop (EC2) of CD9.
(A) MMP-9 mRNA expression was measured in Mock-, CD9-, and Δ6-HT1080 cells using qRT-PCR (B,C) Release of pro-MMP-9 and pro-MMP-2 was measured by gelatin zymography and quantified using Image J. (D,E) A representative picture of cell invasion through matrigel coated inserts and relative quantification of cell invasion *, p<0.05; **, p<0.001.