Antibodies and reagents

The commercially available antibodies used in all of the experiments were as follows: anti-p38MAPK (cat. no. 144511), anti-phospho-p38 mitogen-activated protein kinase (p-p38MAPK; cat. no. 4511), anti-Notch1, and anti-c-Notch1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-AT1R (sc-1173) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. sc-20357) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-gp91^phox (clone: 53/gp91^phox) was from BD Biosciences (Bedford, MA). Anti-CD31 (ab28364) and anti-galectin-3 (ab76245) were from Abcam (Cambridge, MA). Fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (cat no. K1203) was purchased from APExBio (Houston, TA). Anti-p16-INK4A (cat. no. 10883-1-AP) and anti-homolog-1 (Hes1) were purchased from Proteintech (Chicago, IL). Antibody of p16 (cat. no. 10883-1-AP) was from Proteintech (Rosemont, IL). Antibody of CD31 (#550274) was purchased from BD Pharmingen (San Diego, CA). Rabbit pAb (ab28364) and FITC goat anti-mouse IgG (cat no. K1203) were from APExBio.

The enzyme-linked immunosorbent assay (ELISA) kits for the vascular cell adhesion molecule-1 (VCAM-1; MVC00) was from R&D Systems (Minneapolis, MN), and ADAMTS13 (cat. no. CSB-EL001301MO) was purchased from Cusabio (Houston, TX). Plasminogen activator inhibitor-1 (PAI-1, #ab197752) and von Willebrand factor (vWF, #ab208980) were purchased from Abcam. The SYBR™ Green Master Mix and RNeasy Micro Kits were purchased from Qiagen (Hilden, Germany). Nontargeting control short interfering (si)RNA (#F5129292-921) and CTSK-specific siRNA (siCTSKs: #F5129442005, #F5129442006) were purchased from Sigma-Aldrich (St. Louis, MO). The RNeasy Micro Kits and SYBRTM Green Master Mix were from Qiagen. CTSK plasmid was from Invitrogen (Carlsbad, CA). The ImmPACT™ DAB peroxidase substrate (cat. no. SK-4105) was from Vector Laboratories (Burlingame, CA). The DCTM protein assay kit (cat. no. 500-0114) was purchased from Bio-Rad Laboratories (Hercules, CA). The SuperScript III First Strand and Lipofectamine® 3000 Transfection Kits were purchased from Invitrogen. The Amersham ECL Prime Western Blotting Detection kit was from GE Healthcare (Freiburg, Germany). The nitrocellulose transfer membrane was from Amersham Bioscience (Piscataway, NJ). The 0.5 w/v% sterilized methyl cellulose 400 solution was purchased from FujiFilm Wako Pure Chemical Corp. (Osaka, Japan). The In-Fusion HD cloning kit was purchased from Takara Bio (#639648; Kusatsu, Shiga), and the CEQ8000 Genetic Analyzer was purchased from Beckman Coulter (Brea, CA). The HUVECs were purchased from Cell Applications (San Diego, CA). Endothelial growth medium (EGM)-2 SingleQuots and endothelial basal medium (EBM)-2 were from Lonza (Walkersville, MD). The In Situ Cell Death Detection Kit (TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling) was purchased from Roche Diagnostics (Lot#10131400, Mannheim, Germany).

CTSK-II (also known as a CTSK specific inhibitor; [1-(N-Benzyloxycarbonyl-leucyl)-5-(N-Boc-phenylalanyl-leucyl)carbohydrazide] (also known as CTSL inhibitor V (called CTSL-V); [Z-Phe-Tyr(OtBu)-COCHO·H₂O], CA-074Me (also known as CTSB inhibitor IV); [l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-l-isoleucyl-l-proline methyl ester), E-64d (also known as non-specific cathepsin inhibitor); [(l-3-trans-ethoxycarbonyloxirane-2-carbonyl)-l-leucine (3-methylbutyl) amide], E64 (also known as a non-specific cathepsin inhibitor); N-[N-(L-3-Trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine), and GM6001 (also known as an MMP inhibitor) were purchased from Calbiochem (San Diego, CA). Fluorescence-quenching substrate (also known as γ-secretase substrate; Nme-Asp-Gly-Cys-Gly-Val-Leu-Leu-Lys-DNP-Arg-Arg-NH2), recombinant human MMP-1 (rhMMP-1), and recombinant human membrane type 1 of MMP (rhMT1-MMP) were purchased from Enzo Life Sciences (Farmingdale, NY). DAPT (also known as the γ-secretase inhibitor IX); (3,5-difluorophenylacetyl)-l-alanyl-l-2-phenylglycine t-butyl ester] and L-685,458 (also known as γ-secretase inhibitor X); [(2R,4R,5S)-2-benzyl-5-(t-butyloxycarbonylamino)-4-hydroxy-6-phenylhexanoyl]-l-leucyl-l-phenylalanine amide) were purchased from the Peptide Institute (Osaka, Japan). Recombinant human CTSB (rhCTSB), rhCTSK, rhCTSV, and rhCTSS were purchased from R&D Systems. Anti-fluorescence quenching sealing solution with DAPI for the visualization of the cell nuclei was purchased from Beyotime (Shanghai, China). All powdered chemical inhibitors were dissolved in sterilized dimethyl sulfoxide (DMSO) and added to the cell culture medium to yield 0.2% DMSO as a final concentration.