Bcl xl

Cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris-HCL pH 8, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, 1 mM EDTA) supplemented with a mix of protease and phosphatase inhibitors (Thermo Fisher Scientific). Equal amount of protein extracts was resolved on SDS-PAGE gels and blotted on nitrocellulose membranes. Membranes were exposed overnight at 4 °C to PARP (#9524, rabbit, IgG, CST), Caspase-3 (#9662, rabbit, IgG, CST), cleaved Caspase-3 (Asp175) (#9661, rabbit, IgG, CST), Bcl-2 (sc-7382, mouse, IgG, Santa Cruz), Bcl-xL (sc-8392, mouse, IgG, Santa Cruz), PI3K (p110α) (#4249, C73F8, rabbit, IgG, CST), phospho-AKT (Ser473 XP) (#4060, D9E, rabbit, IgG, CST), AKT (#9272, rabbit, IgG, CST), PTEN (#9559, 138G6, rabbit, IgG, CST), phospho-JNK (Thr183/Tyr185) (#4668, 81E11, rabbit, IgG, CST), JNK (#9258, 56G8, rabbit, IgG, CST), phospho-ERK (sc-7383, E-4, mouse, IgG2_a, Santa Cruz), ERK (sc-94, rabbit, IgG, Santa Cruz), p21 (#2946, DCS60, mouse IgG2a, CST), and β-actin (#3700, 8H10D10, mouse IgG2_b, CST). Primary antibodies were detected using specific secondary HRP-conjugated antibodies (Thermo Fisher Scientific) and chemiluminescence signals were revealed using Amersham imager 600 (GE Healthcare). Protein expression levels were measured by densitometry analysis using ImageJ software.

PEG-CdTe QDs and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, USA). DOX was obtained from Sigma (Wuhan, China). 1-(3-dimethylamino propyl)-3-ethyl carbon diimine hydrochloride (EDC) and N-hydroxysuccinide (sulfo-NHS) were offered by Sigma(Shanghai,China). Roswell Park Memorial Institute medium (RPMI) 1640 and fetal bovine serum (FBS) were bought from Gibco Chemical Co. (Carlsbad, CA, USA). Pierce™ bicinchoninic acid (BCA) protein assay kits, Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kits, and hematoxylin–eosin kits were provided by KeyGen Biotech Co., Ltd. (Jiangsu, China). Cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies. Inc. (Nanjing, China). Monoclonal antibodies for CXCR4, SURVIVIN, Bcl-xl, cleaved caspase-3/9, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were made in Santa Cruz Biotechnology Inc. (CA, USA). All reagents were analytically pure.

Preparations of Zeylenone (Zey) and mPEG-PLGA-loaded Zeylenone nanomicelles were described previously [8 (link)]. Zey used for the in vitro studies was stored as 150 mM solutions in DMSO at −20 °C and further diluted to desired working concentrations before use (DMSO concentration <1%). The mPEG-PLGA-loaded Zeylenone nanomicelles used for the in vivo studies were stored in a dry container at room temperature.
RPMI-1640, DMEM, and fetal bovine serum (FBS) were purchased from Corning Inc. (Corning, NY, USA). MTT, Hoechst 33258, and fluorescent dye JC-1 were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against Bcl-2, Bcl-xl, MMP-9, and MMP-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Bax, p-AKT, AKT, p-mTOR, pro-caspase3, p-ERK, ERK, and GAPDH, as well as all secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). An Annexin V-FITC/PI kit and Matrigel matrix were obtained from BD Biosciences (San Jose, CA, USA).

3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was purchased from USB Corporation (USA). Pen strep, RPMI 1640, High Glucose DMEM, and Heat inactivated Fetal Bovine Serum (FBS), 5,5′,6,6′-tetrachloro-1,1′,3,3′- tetraethyl benzimidazolyl carbocyanine iodide (JC-1), and 5-(and-6)-chloromethyl-2′,7′-dichloro dihydrofluorescein diacetate (CM-H₂DCFDA) were obtained from Invitrogen (Carlsbad, CA, USA). Caspase-3, Caspase-8, Caspase-9 colorimetric assay kits were procured from Biovision (Milpitas, CA, USA). The antibodies against Bcl2, Bcl-xl, Bax, Bad, β-Actin, and PARP, Alkaline phosphatase/Horseradish peroxidase conjugated secondary antibodies, and enhanced chemiluminescence kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Andrographolide was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in DMSO and kept at 4 °C at a concentration of 50 mM. AnnexinV-FITC Apoptosis Detection Kit was purchased from BD Pharmingen (Pharmingen, USA). Caspases fluoremetric assay kit was purchased from Chemicon International Corporation (USA). Ac-DEVD-CHO (caspase-3 inhibitor), and Ac-LEHD-CHO (caspase-9 inhibitor) were from Calbiochem (La Jolla, USA). Primary antibodies (Bcl-2, Bcl-xL, Bax, Apaf-1, cytochrome c, β-actin and COX IV) and polyclonal secondary antibody were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, USA). The fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and antibiotics were purchased from Gibco BRL (Grand Island, USA). Plastic wares for cell culture were procured from NUNC (Roskilde, Denmark). Other chemicals including 4, 6-diamidino-2-phenylindole (DAPI), 3[4-dimethylthiazol-2-71]-2-5-diphenyl tetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, USA).

Human colorectal cancer HCT116 and HT-29 cell lines were purchased from the American Type Culture Collection. Cells were maintained in 10% fetal bovine serum (FBS)-supplemented RPMI 1640 medium (GIBCO, Grand Island, NY, USA) and 1% penicillin-streptomycin (GIBCO) at 37°C in a humidified incubator containing 5% CO₂. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), zVAD and all of the other chemical reagents were purchased from Sigma Chemical (St. Louis, MO, USA). Antibodies against various proteins were obtained from the following sources: PARP (Poly-ADP-ribose polymerase), Mcl-1, Bcl-2, Bcl-XL, survivin, cytochrome c, Bax, Bak, Bim, anti-mouse and anti-rabbit IgGs were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Phospho-Akt (Ser473), phospho-GSK-3β, Akt, phospho-p44/42 MAPK (1/2 Erk) (Thr202/Tyr204), p44/42 MAPK (1/2 Erk), caspase8, caspase 9, γH2AX, p21 and acetyl-α-tubulin were obtained from Cell signaling (Danvers, MA, USA)., Actin was obtained from Chemicon (Billerica, MA, USA). Acetyl-histone H3 and GPADH were from Millipore (Billerica, MA, USA). Caspase 3 and was obtained from IMGENEX (San Diego, CA, USA).