towards improved death receptor targeted therapy for ... - TI Pharma

TRAIL and Hsp90 inhibition
propidium iodide (PI) staining cell cycle distribution and death cells in the sub‐G1 fraction
was determined by flow cytometry. Apoptotic events were measured by annexin V‐FITC
and PI double staining according to the manufacturer’s protocol (the Annexin V Apoptosis
Detection Kit 1; BD Pharmingen TM ). Cells stained negative for both annexin V‐FITC and PI
were living cells. Annexin V‐FITC positive cells were defined as early apoptotic cells, and
cells positive for both annexin V and PI were defined as late apoptosis. Cells that were
only PI positive are necrotic or already dead cells. All analyses were performed on a
FACScalibur flowcytometer (Becton Dickinson, Immunocytometry Systems, San Jose, CA,
USA) with an acquisition of 10,000 events using CELLQuest software package.
Western Blotting
Cells were seeded in 25‐cm 2 flasks with 1*10 6 cells in 5 ml medium. After 24 h enabling
attachment, the medium was refreshed and after 48 h, the desired concentration of the
drug(s), TRAIL, 17‐AAG or the combination were added for different time‐points. Next,
the medium was collected and centrifuged at 1,500 rpm, for 5 min at 4°C to pellet floating
cells and washed twice with ice‐cold PBS. The attached cells were washed twice with ice‐
cold PBS and were incubated together with the pelleted floating cells for 15 min with lysis
buffer from Cell Signalling Technology Inc. (Danvers, MA, USA) supplemented with fresh
0.04% protease inhibitor cocktail (PIC) and 1 mM Na2VO3 on ice. After 30 min incubation,
the cells were scraped and subjected to centrifugation at 14.000 rpm for 10 min at 4°C.
The supernatant was transferred to new vials and protein concentrations were
determined by BIO‐Rad. 30‐60 µg protein was electrophorized on 10 or 12% SDS‐PAGE.
After transfer of the proteins on polyvinylidenedifluoride (PVDF) membranes (Millipore
Immobilon TM –FL PVDF, 0.45 µm blocking was performed in Rockland blocking buffer for 1
h at room temperature (RT). Subsequently, membranes were incubated with the following
primary antibodies: anti‐Akt (#9272), anti‐p‐Akt (#9271), anti‐ERK (#9102), anti‐p‐ERK
(#9101), anti‐IκBα (#4812), anti‐p‐IκBα (#9246), anti‐RIP (#3493), anti‐caspase‐3 (#9662),
anti‐caspase‐8 (#9746), anti‐caspase‐9 (#9502), anti‐PARP (#9542) all from Cell Signalling
Technology Inc. Cathepsin B (#IM27L) was purchased from Calbiochem and β‐actin (1: 10
000) was from Sigma‐Aldrich (St. Louis, MO, USA). The primary antibodies were dissolved
(1:1000) in PBS‐Tween (0.05%) (PBS‐T): Rockland buffer (1:1) and the membranes were
incubated for 24 h at 4°C. After washing with PBS‐T (0.05%), the membranes were
incubated with the secondary antibodies (Goat‐anti‐Rabbit IRDye 800 CW and Goat‐anti‐
Mouse IRDye 680, from LI‐COR Biosciences, Lincoln, Nebraska USA) also dissolved in PBS‐
T: Rockland buffer (1:1) for 1 h at RT. After washing with PBS‐T and PBS alone to reduce
the background, the fluorescence intensity was measured with the Odyssey Infrared
Imaging System (LI‐COR Bioscience) and the measurements were evaluated with the
program Odyssey V3.0.
‐ 87 ‐