Fig. 3

Endogenous CTSK can active endogenous MMP-9 in triple negative metastatic breast cancer cells MDA-MB-231. a Immunoblot of CTSK from total cell lysates of MDA-MB-231 cells transduced with scrambled mirRNA sequence (WT) or a mirRNA sequence targeting CTSK (CTSK KD). GAPDH band was used as the loading control. b qRT-PCR of CTSK mRNA levels in MDA-MB-231 WT and MDA-MB-231 CTSK-KD. CTSK mRNA levels were normalized to RRS18 N and the data is represented as the mean ± SD, n = 3. c CTSK activity was measured in conditioned media (pH 5.0) and total cell lysate (pH 5.0) from MDA-MB-231 WT and MDA-MB-231 CTSK-KD. Data is represented as the mean ± SD, n = 5. d MMP-9 activity as measured in the conditioned media from MDA-MB-231 WT and MDA-MB-231 CTSK-KD. Media was acidified to pH 5.5 or pH 7.4 and incubated for 1 h at 37°C, afterwards the pH was adjusted to 7.4 and MMP-9 activity was measured using a fluorescently quenched substrate for MMP-9 (Mca-RPPGFSAFK(Dnp)). Data is represented as the mean ± SD, n = 5. e qRT-PCR of MMP-9 mRNA levels in MDA-MB-231 WT and MDA-MB-231 CTSK-KD. MMP-9 mRNA levels were normalized to RRS18 N and data is represented as mean ± SD, n = 3. f Immunoblot of proMMP-9 from total cell lysates of MDA-MB-231 WT and MDA-MB-231 CTSK-KD. GAPDH band was used as loading control.*P < 0.05, **P < 0.01 and ***P < 0.005.