Paclitaxel

Chemotherapeutic sensitivity was measured using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were plated at 3 × 10⁴ cells per well in 96-well plates and incubated with 1, 10, or 50 nM paclitaxel (Cayman Chemical) for 24 or 48 h. Following treatment, 10 μl of 12 mM MTT was added to each well and cells were placed back in the incubator at 37 °C for 4 h. The formazan was solubilized by adding 100 ul solubilization solution (40% DMSO, 16% SDS, 2% acetic acid) followed by another incubation at 37 °C for 1 h. Spectrometric absorbance at 570 nm was measured with a microplate reader. Each assay included four technical replicates and was repeated at least three times. paclitaxel effects at each dose, and differences in paclitaxel response between cell lines were analyzed using a two factor mixed design ANOVA using SPSS version 19 software (Chicago, IL). A Bonferroni test was used to adjust for multiple pairwise comparisons. Mean differences were considered statistically significant at p < 0.05.

Parental and DIAPH3-silenced or over-expressing cell lines, such as DU145, Human Mammary Epithelial (HMEC), or U87, have been described5 (link). Paclitaxel (Cayman Chemicals, Sigma), epothilone B (Cayman Chemicals), and docetaxel (LC Laboratories). Cholera toxin B (CTxB, Sigma, Life Technologies). β-octylglucopyranoside and cacodylate buffer (Sigma), Protein A/G Agarose (Santa Cruz Biotechnology), and Oregon Green-488 Paclitaxel (Invitrogen). Antibodies: Ac-tubulin (Abcam, Cell Signaling), GFP (Genscript), Cleaved PARP, E-cadherin, β-catenin, (Cell Signaling), α-tubulin (Cell Signaling, Genscript), β-tubulin and tubulin βIII (Genscript), DIAPH3 (HNH3.1, a generous gift of Dr. Henry Higgs, Dartmouth Medical School).

In the SRB colorimetric assay, 3000 cells/well were seeded in a 96-well plate. Cells were cultured overnight and incubated or treated with drugs for the indicated times. At the indicated time, 10% trichloroacetic acid (TCA, Sigma-Aldrich, St. Louis, MO, USA) was used to fix the cells at 4 °C for 1 h. Then, the cells were washed with distilled water and air dried. Subsequently, cells were stained with 0.057% SRB (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min and washed with 1% acetic acid (J.T. Baker, Center Valley, PA, USA). Finally, 10 mM Tris Base (J.T. Baker, Center Valley, PA, USA) was used to dissolve the SRB, and the absorbance value at a wavelength of 510 nm was determined using an ELISA reader. Cisplatin and 5-fluorouracil were obtained from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin and paclitaxel were obtained from Cayman chemical (Ann Arbor, MI, USA).

Immortalized human muscle cell (7304.1) shPAB and control stable cell lines were generated as previously described^{12 (link)}. In brief, PABPN1 knockdown (shPAB) was generated after lentivirus transduction with a shRNA to PABPN1. The control cell line was transduced with scrambled shRNA^{12 (link)}. Myoblast cells were maintained in growth medium (F10 medium supplemented with 15% FCS, 1 ng/ml bFGF, 10 ng/ml EGF and 0.4 ug/ml Dexamethasone). Differentiation (cell fusion) was conducted in DMEM supplemented with 2% horse-serum for four days. Stable actin-eGFP overexpressing cells were generated with a lentivirus transduction in control or shPAB cell cultures. The actin-eGFP was subcloned into a lentivirus expression cassette. Actin-eGFP expression was driven by the CMV promoter. All cultures were mycoplasma-free. Microtubule destabilization or stabilization treatments were performed using nocodazole (250 nM, Cayman Chemical) or paclitaxel (25 nM, Cayman Chemical), respectively for 2 h.

The H. coubaril plant was collected in the metropolitan area of Bucaramanga, Santander, Colombia (7.11° N, 73.11° W), after an ornamental pruning. A specimen was collected and identified at the herbarium of Pontificia Universidad Javeriana and classified with voucher number: HPUJ-30302. Six-hundred and twenty grams of the leaves of the plant were subjected to Soxhlet extraction with hexane (three days), followed by dichloromethane (three days) and then ethanol (eight days), thereby obtaining three extracts with different polarities [38 ]. Stock solutions of the extracts, fractions, and the isolated compound caryophyllene oxide (OXC) were prepared at a concentration of 10 mg/mL, and of the positive control Paclitaxel (Cayman Chemical, Ann Arbor, MI, USA) at a concentration of 10 μg/mL in dimethylsulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA). These stock solutions were used for all tests. Cultured cells were maintained as follows: prostate adenocarcinoma (PC-3) (ATCC^®, Manassas, VA, USA, CRL1435™) and lung fibroblasts (MRC-5) (ATCC^®, Manassas, VA, USA, CCL-171™) in supplemented EMEM (Lonza, CH, Walkersville, MD, USA) medium with 10% (v/v) Fetal Bovine Serum (Biowest, Riverside, MO, USA), 2 mM L-glutamine, and 5000 IU/mL penicillin and 5 mg/mL streptomycin (Lonza, CH, Walkersville, MD, USA). Incubation was performed at 37 °C and 5% CO₂.