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. 2015 Feb 19;6(6):3932–3946. doi: 10.18632/oncotarget.2846

Figure 6. Treatment of luminal breast cancer cell lines with exogenous HRG increases extracellular protease activity.

Figure 6

(A, B) HRG increases expression of proteolytic cascade proteins. Serum-starved cells were treated with HRG for 48 h, then total RNA or protein were isolated from the cells for qRT-PCR and Western blot analyses respectively (HPRT1 and α-tubulin were used as normalization and loading controls, respectively). (C) HRG increases secreted MMP-2 and MMP-9 proteolytic activities. Starved cells were treated with HRG as above and then conditioned media was concentrated and analysed for MMP-2 and MMP-9 activity by gelatin zymography (enzymatic activity is proportional to the intensity of the white bands). (D) HRG represses expression of RECK and KAI1 metastasis suppressor genes. qRT-PCR analysis was performed as for (A). HRG treatment does not substantially alter cathepsin B protein expression (E), but increases extracellular cathepsin B proteolytic activity (F). Cathepsin B expression was analyzed by Western blot analysis as for (B), with β-actin as the loading control. Enzyme activity was assayed using a fluorometric enzyme activity assay. *p = 0.01–0.001, **p = 0.001–0.0001, ***p < 0.0001 (unpaired, 2-tailed student's t-tests comparing treated to untreated control samples). FC, fold-change.