Figure 6

MMP13 knockdown in murine melanocytes and the human melanoma cell line A375 reduces proliferation. A, left: Realtime-PCR of Mmp13 expression in melan-a Hm cells transgenic for a control pRetroSuper vector (ctrl.) compared to melan-a Hm cells expressing pRS-shMmp13. A, right, top: Western blot displaying the decrease of the pro-form of MMP13 after knockdown in murine melan-a Hm cells. Note that only the human, but not the murine active form is recognized by the antibody (also see D). A, right, bottom: Zymography of unconcentrated supernatant, showing a decreased MMP13 band after knockdown. B: Starved melan a Hm pRS and Hm pRS-shMmp13 cells were stimulated with EGF, and cell proliferation was measured after 2, 4, 6, 8 and 10 days. C, left: Appearance of cell pellets of EGF-stimulated Hm cells either transfected with the control vector or the pRS-shMmp13 vector. C, middle panel: Quantification of melanin in EGF-stimulated Hm cells either transfected with the control vector or the pRS-shMmp13 vector. C, right: Realtime PCR analysis of tyrosinase transcript levels in control Hm cells and Hm cells expressing pRS-shMmp13. β-actin was used as reference gene. D, left: Realtime-PCR of MMP13 expression in A375 cells transfected with siRNA against MMP13 or an siRNA control, 72 h after transfection. D, right: Western blot of MMP13, showing the full-length (60 kDa) and the active (48 kDa) form of the protein in A375 cells treated with control or MMP13-specific siRNA. E: BrdU incorporation of A375 cells transfected with control siRNA or MMP13-specific siRNA. The right bar shows the BrdU incorporation of A375 cells, treated with MMP13-specific siRNA, but in presence of the 48 h-conditioned supernatant from control siRNA-transfected cells. BrdU incorporation of the control cells is used as reference and is set as 100%. The assay was performed three times independently. *: p < 0.05 (Student's t test, paired, two-tailed)