Kari Trumpi et al
Supplementary methods and figures
Material & Methods:
Western blot and antibodies: Colonospheres were harvested and RAS lysis buffer (15,6 mM
Hepes, 150mM NaCl, 50mM MgCl27H2O, 1% NP-40, 10%glycerol, pH 7.4) supplemented with
phosphatase and protease inhibitors was added for 30 minutes. Lysates were cleared by centrifuging
(13.200 rpm for 10 minutes at 4ºC) and the protein concentration was determined using a BCA
(bicinchoninic acid) protein assay (Pierce, Biotechnology, IL, USA). After addition of LDS sample buffer
and Reducing agent (NuPAGE®) the samples were heated for 10 minutes at 70ºC. Proteins were run
on Bis-Tris Mini Gels with SDS running buffer and 500μL antioxidant in the upper buffer chamber
(NuPAGE®) for 55 minutes at 200 V and blotted for 1,5 hours at 120 V. Membranes were blocked
with 5% skim milk in TBS-Tween for 1 hour at room temperature and incubated with primary
antibodies overnight at 4ºC. Primary antibodies were diluted in 5% skim milk in TBS-Tween at
concentrations of 1:200 (anti-ABCB1, Thermo Scientific, MS-660-P1), 1:250 (anti-CK-20, ARP, 0361054), 1:1000 (anti-cleaved-Caspase-3, Cell Signaling, 9661), and 1:20000 (anti-β-actin, Novus
Biologicals, NB600-501). Secondary antibodies (anti-rabbit, DAKO, P0448 (1:1000) and anti-mouse,
DAKO, P0447 (1:2000) were added for 1 hour. Immunoreactivity was detected using ECLTM Western
Blot Detecting Reagents (GE Healthcare, Buckinghamshire, UK) on Fuji Medical X-Ray Film (Fujifilm
Corporation, Japan).
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Kari Trumpi et al
Figures
Figure 1
Characterization of the colonosphere lines
A. Light microscopic images of a representative colonospheres of CRC29 and CRC47, bar 200um.
All colonosphere cell lines are derived from primary tumors of colorectal cancer patients.
B. Western blot analysis of the expression of ABCB1, cytokeratin 20 (CK20) and β-actin on the
colonospheres.
C. CRC29 and CRC47 colonospheres were treated either with vehicle, 2.0 μM PSC-833, 50
μg/mL Irinotecan or combination of PSC-833 and Irinotecan for 48 or 72 hours. The cells then
were incubated with Nicoletti buffer overnight and cell death was determined by FACS
analysis.
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