), 3226 (OH, str.), 2995, 2849 (C-H, str.), 1688, 1650 cm?1 (C=O, str.); [��]D23=?4.25�� (c=0.04 g/100 mL, CH3OH); 1H NMR (CD3OD, 600 MHz) �� 3.81 (1H, dddd, 3J=4.9, 3J=5.4, 3J=6.3, 3J=7.9, CH2CHN), 3.62 (1H, dd, 2J=11.1, 3J=4.9, CH2O), 3.53 (1H, dd, 2J=11.1, 3J=6.3, CH2O), 2.74 (2H, m, CH2CH2CO), 2.38 (1H, dd, 2J=14.1, 3J=5.4, CH2CHN), 2.10 (1H, dd, 2J=14.1, 3J=7.9, CH2CHN), 1.56 selleck screening library (2H, m, CH2CH2CO), 1.31 (3H, t, 3J=7.0, CH3CH2); 13C NMR (CD3OD, 150 MHz) �� 212.2 (CO), 176.7 (CON), 84.5 (COH), 65.5 (CH2OH), 54.3 (CHN), 38.7 (CH2CO), 33.1~30.8, 24.3 (CH2CH2CO), 14.4 (CH3CH2); MS (EI+) m/z=341 (M+), HRMS (EI) calculated for C19H35NO4 (M+) 341.2617 found 341.2599. PMC and analogs were dissolved in ethanol. McCoy��s 5A, FBS and antibiotics were from Pan Biotech GmbH (Aidenbach, Germany).

Phosphatase inhibitor cocktail (PhosStop) and protease inhibitor cocktail were obtained from Roche (F. Hoffman-La Roche Ltd., Basel, Switzerland). Annexin V (FITC) was from Alexis Biochemicals (Enzo Life Sciences, Inc. Farmingdale, USA). Cleaved caspase-3, cleaved caspase-9, JNK, P-JNK, p38, P-p38, Bcl-2, Bax, Bid, Bim and ��-actin antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). MAPK inhibitors SP600125 and SB203580 were from Calbiochem (La Jolla, CA, USA). Caspase inhibitors Z-VAD-FMK (general caspase inhibitor), Z-DEVD-FMK (caspase-3 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-IETD-FMK (caspase-8 inhibitor) were purchased from BD Biosciences (San Jose, CA, USA). Tris, glycine and Tween-20 were from Molekula Ltd (Shaftesbury, UK).

All other chemicals were obtained from Sigma (Darmstadt, Germany). MTT Cell Viability Asssay Cell viability upon exposure to PMC analogs was determined using an MTT (dimethyl thiazolyl diphenyl tetrazolium) assay kit (Roche, Mannheim, Germany) according to the manufacturer��s protocol. Briefly, HCT116 wt cells in 96-well plates were treated as indicated, and 10 ��l of MTT labeling reagent was added to each well, after which the plates were incubated for 4 hours. The cells were then incubated in 100 ��l of the solubilization solution for 12 hours, and the absorbance was measured with a microtiter plate reader (Bio-Rad, CA, USA) at a test wavelength of 550 nm and a reference wavelength of 650 nm. Percent viability was calculated as (OD of drug-treated sample/control OD) �� 100.

Flow Cytometry Cell death was determined by an Annexin-V affinity assay. Wt, p53?/?, and Bax ?/? HCT116 cells seeded in 12-well plates were transfected/treated as indicated, transferred to flow cytometry tubes and cells were harvested by centrifugation at 300 g for 5 minutes. Then the cells were resuspended Dacomitinib in 1 ml of cold PBS and centrifuged again at 300 g for 5 minutes. After removal of supernatant, the cells were incubated in Annexin V buffer (140 mM HEPES, 10 mM NaCl, 2,5 mM CaCl2, pH:7.4) containing 1% (v/v) Annexin V (FITC) for 15 minutes in the dark.