Acetic acid

Spot urine was collected and 2 μL of each urine sample, and BSA standards at 0, 1, 3, and 10 μg, were separated by SDS‐PAGE on a NuPAGE Novex 4–12% Bis‐Tris gel (Thermo Fisher Scientific). After electrophoresis, the gel was immersed in fixing solution (50% (v/v) methanol (Fisher Scientific), 10% (v/v) acetic acid (Acros)) for 1 h, incubated for 20 min with agitation in staining solution (0.1% (w/v) Comassie brilliant blue R‐250 (Bio‐Rad), 50% (v/v) methanol, 10% (v/v) acetic acid), and finally incubated several times in destaining solution (40% (v/v) methanol, 10% (v/v) acetic acid) until destained.
Urinary albumin content in spot urine was assessed using Albuwell M indirect ELISA (Exocell) and urinary creatinine content was measured, using Creatinine Companion kit (Exocell). Both measurements were performed according to manufacturer′s instructions. Albumin:creatinine ratio was calculated by dividing μg albumin/mL urine with mg creatinine/mL urine, getting the ratio μg albumin/mg creatinine.

Lyophilized papain (~30,000 USP units/mg) was purchased from Himedia, India. Sodium acetate (anhydrous, ≥99%), trichloroacetic acid (≥99%), and 2,4,6-Tris(2-pyridyl)-s-triazine (≥98%) were obtained from the Sigma-Aldrich group (Schnelldorf, Germany). Citric acid (99%), potassium sulfate (≥99%), copper sulfate (≥99%) sulfuric acid (≥99%), phenolphthalein (≥98%), methanol (≥98%), glucose (≥99%), phenol (≥99%), and sodium hydroxide (≥99%) were purchased from Reanal (Budapest, Hungary). Ultrasil P3-11 was purchased from Ecolab-Hygiene Kft (Budapest, Hungary). Ferric chloride (≥99%), sulfuric acid (≥99.9%), amyl alcohol (≥99.9%), ascorbic acid (99.7%), bacteriological agar powder, and soybean casein digestive medium were procured from Merck (Darmstadt, Germany). Acrylamide (≥99%), sodium-dodecyl sulfate (≥99%), ammonium persulfate (≥99%), Tetramethylethylenediamine (≥99%), tris(hydroxymethyl)aminomethane (≥99%), glycine (≥99%), ethyl alcohol (≥99%), coomassie blue stain (≥99%), acetic acid (≥99%), isopropanol (≥99%), glycerol (≥99%), 2-βmercaptoethanol (≥99%), and bromophenol blue (≥99%) were purchased from BIO-RAD (BIO-RAD, USA). Milli-Q ultrapure deionized water (18.2 MΩ·cm; Merck-Millipore, Molsheim, France) was used throughout the experiment.

Fetal bovine serum (FBS) and trypsin (0.25% w/v) were purchased from Hyclone (Thermo Fisher Scientific Inc. Rockford, IL). Topotecan (TOPO) was purchased from 21st Century Global E-Commerce Network (East Sussex, UK). Dimethyl sulfoxide (DMSO), sulforhodamine B (SRB), TRIS buffer, acetic acid, ECL western blotting substrate for chemiluminescence were obtained from Bio-Rad (Hercules CA, United States ). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RNase A were purchased from Sigma-Aldrich Inc (St. Louis, MO). Mouse anti-human antibodies (MMP-9, MMP-1, Ang-2, VEGF, Maspin and c-Fos were purchased from Santa Cruz Biotechnology (uPA; H77A10, MMP-1; SB12e, Ang-2; F-1, VEGF; JH121, Maspin; E10 Santa Cruz, CA) and Cell Signaling Technology (PAI-1; D9C4, uPAR; D7X2N, MMP-9; D6O3H, c-Fos; 9F6, Danvers, MA). Goat anti-Rabbit IgG (H = L) Secondary antibody, HRP, was purchased from Cell Signaling Technology; 345,897 (Danvers, MA). β-actin was purchased from Sigma-Aldrich; A5316. All glass and plasticware were purchased from VWR (Radnor, PA).