Ab168863

Monolayers of human small airway epithelial (SAE) cells from healthy subjects directly obtained from Lonza (Walkersville, MD, catalogue # CC-2547) were submerged cultured as previously described [22 (link), 23 (link)]. All cells are assayed and test negative for HIV-1, mycoplasma, Hepatitis-B, Hepatitis-C, bacteria, yeast and fungi upon isolation by Lonza. Cells were only used for experiments at passages 3–6 and at a confluency of approximately 70%. SAE cells were transfected by administering silencing RNA (siRNA) specific for p38, MMP9 or negative control scrambled (Scr.) (Qiagen, Gaithersburg, MD). Cells were infected with RSV at a MOI of 0.3 or treated with mock or human MMP9 protein for 24 hours. Human MMP9 (Abcam; ab168863) was activated by incubating with 2mM 4- aminophenylmercuric acetate (APMA; Sigma) at a 9:1 (MMP9:APMA) ratio for 90 minutes at 37°C and inactivated with 2mM EDTA. Active and inactive MMP9 were dialyzed twice in TBS to remove APMA or EDTA prior to treating cells or RSV. RSV was treated with various concentrations of active and inactive MMP9 prior to infecting SAE cells. TCID₅₀ assays were performed to determine the quantity of virus following MMP9 treatment. TCID₅₀ data was calculated using the method of Reed and Munch [24 ]. SAE cells were also treated with active and inactive MMP9 alone and TCID₅₀ assays were performed.

MMP activities of cultured VMSCs were evaluated by gelatin zymography. A10 VSMCs were treated for 24 h with SP-8356 in the presence of recombinant human protein CD147 (5 μg/mL, ab155636, Abcam, Cambridge, MA, USA). Conditioned media were collected and centrifuged to remove cellular debris and concentrated by Microcon centrifugal filtration (Millipore, Billerica, MA, USA). These samples were mixed with a non-reducing loading buffer without heating and loaded onto 10% SDS-PAGE gels containing 1 mg/mL gelatin (JT Baker Chemical Co., Phillipsburg, NJ, USA). Proteins were separated by electrophoresis at 125 V for 90 min. A MMP-9 recombinant protein (ab168863, Abcam, Cambridge, MA, USA) was loaded as a positive control. Following electrophoresis, the gels were rinsed twice for 30 min with Novex zymography renaturing buffer (Invitrogen, Carlsbad, CA, USA), incubated overnight with Zymogram developing buffer (Invitrogen), and stained with a Simply Blue Safe Stain (Invitrogen).

MMP activities of macrophages were evaluated via gelatin zymography. Monocyte-derived macrophages isolated from SD rats were treated with SP-8356 on the presence of CypA (200 ng/nl; ab86219, Abcam, Cambridge, MA, USA), functional CD147 antibody (ab119114, Abcam, Cambridge, MA, USA), or mock mouse IgG antibody (sc2025, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h. Conditioned media were collected, centrifuged to remove cellular debris and concentrated with a Microcon centrifugal filter device (Millipore, Billerica, MA, USA). Samples were mixed with non-reducing loading buffer without heating and loaded onto a 10% SDS gel containing 1 mg/mL gelatin (JT Baker Chemical Co., Phillipsburg, NJ, USA). Proteins were separated via electrophoresis at 125V for 90 min. An MMP-9 recombinant protein (ab168863, Abcam, Cambridge, MA, USA) was loaded as a positive control. Following electrophoresis, gels were rinsed twice for 30 min in Novex zymography renaturing buffer (Invitrogen, Carlsbad, CA, USA) and subsequently incubated with zymogram developing buffer (Invitrogen, Carlsbad, CA, USA). After overnight reaction, the gel was stained with Simply Blue Safe Stain (Invitrogen, Carlsbad, CA, USA).