Anti survivin

Human renal carcinoma (Caki-1 and A498), human lung cancer (A549), and human breast cancer (MDA-MB361) were procured from American Type Culture Collection (Manassas, VA, USA). Human recombinant TRAIL, zVAD-fmk, and anti-survivin were provided by the R&D system (Minneapolis, MN, USA). MG132, PD98059, AG-490, compound C, and NVP-BEZ235 were supplied from Calbiochem (San Diego, CA, USA). Dexamethasone, cycloheximide, AR-A014418, PP242, BAY11-7082, rapamycin, and anti-actin were provided from Sigma Chemical Co. (St. Louis, MO, USA). Anti-PARP, anti-Bcl-xL, anti-DR5, anti-cIAP1, anti-caspase-8, anti-phospho-GSK3β, and anti-GSK3β were provided by Cell Signaling Technology (Beverly, MA, USA). Anti-Bim, anti-Bax, and anti-XIAP were obtained from BD Biosciences (San Jose, CA, USA). Anti-Mcl-1, anti-Bcl-2, anti-cIAP2, and anti-Cbl were purchased from Santa Cruz Biotechnology (St. Louis, MO, USA). SB203580, SP600125, and anti-c-FLIP(L) were obtained from Enzo Life Sciences (San Diego, CA, USA). Anti-DR4 were obtained from Abcam (Cambridge, MA, USA). pCMV-Myc-Cbl plasmid was a gift from Dr. S. J. Kim (CHA University, Korea). GSK3betaS9A (1016) was a gift from Scott Friedman (Addgene plasmid # 49492; http://n2t.net/addgene:49492; RRID: Addgene_49492) [36 (link)].

The HeLa cells were transfected in 6-well plates with PAMAM-NH2/asODN and controls for 72 h, and then harvested and processed for protein extraction. Then cells were incubated with RIPA lysis buffer (containing antiproteases) with gentle agitation for 30 min at 4 °C. The obtained extract was centrifuged for 30 min at 14,000 rpm at 4 °C and pellet was discarded. Proteins were quantified using Qubit® 2.0 Fluorometer. Fifteen microgram (per lane) of proteins extract was resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes using Turbo Trans-Blot, BioRad. Proteins were detected using anti-Survivin (rabbit polyclonal; R&D systems; 1:1000) and anti-α-tubulin (mouse monoclonal, SIGMA, 1:5000) antibodies. Blots were detected with the super signal West-pico chemiluminescent substrate (Thermo Scientific) and using C-DiGit Blot Scanner (LI-COR). The pixel intensity was quantified using ImageJ software (NIH).

Selleckchem supplied R428 and cisplatin (Houston, TX, USA), and R&D system supplied recombinant human recombinant TRAIL and z-VAD-fmk (Minneapolis, MN, USA). Sigma Chemical Co. provided MG132, cycloheximide, carboplatin, oxaliplatin, doxorubicin, and 5-FU (St. Louis, MO, USA). The primary antibodies were as follows: Anti-phospho-Axl (Y702) (Cell Signaling Technology, Beverly, MA, USA), anti-pro-caspase-3 (Cell Signaling Technology), anti-cleaved caspase-3 (Cell Signaling Technology), anti-PARP (Cell Signaling Technology), anti-Bcl-xL (Cell Signaling Technology), anti-DR5 (Cell Signaling Technology), anti-actin (Sigma Chemical Co.), anti-Axl (Santa Cruz Biotechnology, St. Louis, MO, USA), anti-Mcl-1 (Santa Cruz Biotechnology), anti-Bcl-2 (Santa Cruz Biotechnology), anti-cIAP2 (Santa Cruz Biotechnology), anti-Bim (BD Biosciences, San Jose, CA, USA), anti-XIAP (BD Biosciences), anti-survivin (R&D system, Minneapolis, MN, USA), anti-DR4 (Abcam, Cambridge, MA, USA), and anti-c-FLIP (Enzo Life Sciences, San Diego, CA, USA). The siRNAs were as follows: GFP (control) siRNA (Bioneer, Daejeon, Korea), Axl siRNA (Santa Cruz Biotechnology), and DR5 siRNA (Invitrogen).

Freshly isolated PBMC were stained for flow cytometry as described²² (link) using antibodies to the following human surface antigens. Cells were then fixed and permeabilized with a Cytofix-Cytoperm fixation/permeabilization kit (BD) and stained with anti-survivin (91630, R&D Systems, Minneapolis, MN, USA) and isotype control (mouse IgG1κ, R&D Systems). The cells were collected in FACSCantoII flow cytometer (BD), and the data were analyzed with FlowJo software (BD, v.10.7) and fluorescence minus one controls.

A flow cytometric analysis was performed using the FACSCalibur and LSRFortessa platforms (BD Biosciences, Tokyo, Japan). For Ki-67 and 7-amino-actinomycinD (7-AAD) staining, the FOXP3 Staining Buffer Set (eBioscience) was used. Antibodies conjugated with FITC, PerCP or APC were used. Anti-human CD14 (clone MφP9), anti-human CD45 (clone 2D1) antibodies and 7-AAD were purchased from BD Biosciences. Anti-human CD1a (clone HI149), anti-human Ki-67 (clone Ki-67) antibodies and Fixable Viability Dye (FVD520) were obtained from eBioscience. The cells were washed and stained. For intracellular staining of survivin and its splice variants, normal rabbit IgG (Santa Cruz Biotechnology, TX, USA), anti-survivin (rabbit polyclonal, R&D Systems), anti-survivin-ΔEx3 (rabbit polyclonal, Abcam), and anti-survivin-2B (rabbit polyclonal, Abcam) were labeled using the Zenon Rabbit IgG Labeling Kit (Alexa Fluor 647, Invitrogen). The FOXP3 Staining Buffer Set (eBioscience) was used for fixation and permeabilization. A survivin-WT-specific antibody was not available, therefore, the mean fluorescence intensity (MFI) of the estimated survivin-WT expression level was calculated from other MFI parameters as follows: (survivin-WT) = (survivin (total) – normal IgG) – (survivin-ΔEx3 – normal IgG) – (survivin–2B – normal IgG).