Journal: Cells

Article Title: HSP90α and KLK6 Coregulate Stress-Induced Prostate Cancer Cell Motility

doi: 10.3390/cells15020166

Figure Lengend Snippet: Stress-induced protease activity leads to decreased MMP-2 activity. ( A ) MMP-2 activity in PC3 CM 24 h post-stress with PPI, phosphatase inhibitors (PhI: sodium fluoride, sodium orthovanadate, sodium pyrophosphate, and β-glycerophosphate) or protease inhibitors (PI: Bestatin, Aprotinin, Leupeptin, and E-64) as indicated by gelatin zymography, n = 2. ( B ) Gelatin zymography with 1532CPTX CM 8 h post-treatment from untreated and stressed cells with PPI, PhI, and PI n = 2. ( C ) MMP activity as measured by gelatin DQ assay in PC3 CM 24 h post-stress and ( D ) 1532CPTX CM 16 h post-stress with PPI, phosphatase inhibitors, or protease inhibitors added. ( E ) Gelatin DQ assay with individual protease inhibitors: Bestatin, Aprotinin, Leupeptin, and E-64 added to PC3 HS CM 24 h post-stress; and ( F ) 1532CPTX CM 16 h post-stress. Graphs represent combined results from 3 biologically independent experiments. Statistical significance was determined by Welch’s student t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Reagents used: Matrigel (Corning, Bedford, MA, USA, no. 354230), Halt Protease/Phosphatase Inhibitor cocktail (Thermo Scientific, Rockford, IL, USA, no. 78442), E-64 (Millipore Sigma, Minneapolis, MN, USA, no. E3132), Aprotinin (MP Biomedicals, Solon, OH, USA, no. 0219115801), Bestatin (MP Biomedicals, Solon, OH, USA, no. 0215284401), Leupeptin (VWR International, LLC, Radnor, PA, USA, no. 97063-234), Sodium Fluoride (Sigma Aldrich, St. Louis, MO, USA, S6776), Sodium Orthovanadate (Thermo Scientific Chemicals, Ward Hill, MA, USA, no. J60191.AD), Sodium Pyrophosphate (Sigma Aldrich, St. Louis, MO, USA, no. S6422), B-glycerolphosphate (Sigma Aldrich, St. Louis, MO, USA, no. G9422), Gelatin B (Sigma Aldrich, St. Louis, MO, USA, no. G9391), Bbs1 (New England Biolabs, Ipswich, MA, USA, no. R3539), and Blasticidin S hydrochloride (Thermo Fisher Scientific Chemicals, Inc., Ward Hill, MA, USA, no. AAJ67216XF)

Techniques: Activity Assay, Zymography