Cremophor el

For in vitro experiments, sorafenib (Selleck Chemicals, Houston, TX, USA) and fluvastatin (Sigma, St. Louis, MO, USA) were dissolved in DMSO. For in vivo experiments, sorafenib was dissolved in a Cremophor EL-ethanol solution (50:50, Sigma Cremophor EL :95% ethyl alcohol) and diluted with distilled water [7 (link)], while fluvastatin was dissolved in distilled water.

Nifuroxazide was purchased from Xiyashiji Chemical Co. Ltd (Chengdu, Sichuan, China), and was measured by ¹H-NMR, ¹³C-NMR and ESI-MS analysis. For the in vitro assays, Nifuroxazide was prepared in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mM and stored at −20 °C. Then, it was diluted in the relevant medium at a final DMSO concentration of 0.1% (v/v), and the medium with 0.1% DMSO served as a vehicle control. For in vivo studies, Nifuroxazide was dissolved in 25% (v/v) Cremophor EL/ethanol (50 : 50, Sigma Cremophor EL, 100% ethyl alcohol) and 75% ultrapure water.
DMSO, MTT, DCFH-DA, Rh123 and Cremophor EL were from Sigma Chemical Co. (St. Louis, MO, USA). Hoechst 33258 and NAC were obtained from Beyotime (Beijing, China). The primary antibodies against Stat3/p-Stat3^Tyr705, MMP-2, MMP-9, cleaved caspase-3, Bax, Bcl-2 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). FITC-CD11b-, PE-Gr1-, FITC-CD8a-, PE-CD69-, PE-CD206- and APC-F4/80-conjugated antibodies were obtained from BD Biosciences (San Diego, CA, USA). Anti-Ki-67 mouse monoclonal was purchased from Merck Millipore (Billerica, MA, USA). The Annexin V-FITC Apoptosis Detection Kit was obtained from KeyGen Biotech (Nanjing, China).

Cremophor EL, dimethyl sulfoxide (DMSO), and urethane were from Sigma-Aldrich (Steinheim, Germany); NaCl was from POCh (Gliwice, Poland). Acetylcholine chloride, (-)- phenylephrine hydrochloride, l-NAME, indomethacin, ouabain, and barium chloride (MP Biomedicals, Santa Ana, CA, USA) were dissolved in deionized water except for indomethacin (dissolved in 0.5 M NaHCO₃). SKA-31 (Abcam, Cambridge, MA, USA) was dissolved immediately before the in vivo experiments in a mixture of DMSO, Cremophor EL and saline (1:2:7) to obtain a concentration of 10 mg/mL and then it was further diluted with saline to obtain final concentrations of 1 and 3 mg/0.5 mL. Stock solutions (10 µM) of UCL1684, NS309 (Sigma-Aldrich, St.Louis, MO, USA), SKA-31 and TRAM-34 (Tocris Bioscience, Bristol, UK) were prepared in DMSO. The final concentrations of these agents were prepared by dilutions with deionized water which adjusted the final concentrations of DMSO to ≤0.1% v/v for experiments on isolated organs. None of the solvents used affected basal parameters in vivo or in vitro.

Dimethyl sulfoxide (DMSO), propidium iodide (PI), and rhodamine 123 (Rho 123) were purchased from Sigma Chemical Co. (St. Louis, MO). Cell Counting Kit-8 (CCK-8) was purchased from Dojin do Laboratory (Dojin, Japan). The primary antibodies for p-Cdc25C, p53, p-p44/42 MAPK, and cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA). The primary antibodies for anti-CD31 and anti-CD105 were purchased from BD Biosciences Co. (Franklin Lakes, NJ, USA). β-Actin and the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The TUNEL assay kit was purchased from Promega (Madison, WI, USA). The EdU kit was purchased from RiBo Biological (Guangzhou, China). The protein assay kit was purchased from Bio-Rad (Hercules, CA). All other chemicals used were of analytic grade.
For cellular experiments, #2714 was dissolved in DMSO stock solution for all in vitro assays and stored in the dark at 4 °C. The initially concentration was 40 mM, which was diluted in the cell culture medium with the final concentration of DMSO no more than 0.05% (V/V). For animal experiments in vivo, #2714 was dissolved in ultrapure water and Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 95% ethyl alcohol) mixture, and administrated via i.p. injection to the experimental animals at a dose of 10 ml/kg body weight/day.

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). Cell-Light™ EdU DNA Cell Proliferation Kit was purchased from RiboBio Co., Ltd. (Guangzhou, China). Recombinant human VEGF₁₆₅ was obtained from PeproTech Inc. Recombinant human EGF was purchased from ProSpec Company. Human basic fibroblast growth factor (bFGF), anti-CD31 antibody, and Matrigel were purchased from BD Bioscience (San Diego, CA). The primary antibodies were acquired from Cell Signaling Technology (Beverly, MA). Annexin V-FITC apoptosis detection kit was purchased from Roche (Indianapolis, IN). All of the chemicals used in this study were of analytical and culture grade. For all in vitro assays and zebrafish studies, YLT192 was dissolved in DMSO as a 40 mM stock solution and diluted in the relevant assay media. For in vivo experiments, YLT192 was dissolved in 25% (v/v) aqueous Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 100% ethyl alcohol) and dosed at 0.1 mL/10 g of body weight. For all in vitro assays, medium with 0.1% DMSO served as vehicle control.

Rats were treated with PTX as described previously [12 (link),22 (link)]. In brief, PTX (2 or 4 mg/kg per mL, prepared with saline from TAXOL^®, Bristol-Myers-Squibb, 6 mg/mL PTX in EL vehicle) or Cremophor^® vehicle (Cremophor EL-polyethoxylated castor oil and ethanol, diluted with two parts saline to one part Cremophor EL; Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally (i.p.) on four alternate days (days 0, 2, 4, and 6 of the experiment; cumulative doses of 8 or 16 mg/kg). The volume injected depended on the animal’s weight to ensure i.p. or p.o. injections of less than 2.5 mL. Oral administration of AMG9810 (30 mg/kg, Tocris Bioscience, Ellisville, MO, USA) or intrathecal administration of AMG9810 (35 μg/20 μL) was conducted on day 14 of the experiment. AMG9810,
(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl) acrylamide, is reported as a TRPV1 antagonist with high selectivity [43 (link)]. Rats were injected intrathecally with AMG9810 using a 25 µL Hamilton syringe with a 28-gauge needle. Rats showing motor impairment were excluded from further analysis.