SUPPLEMENTARY INFORMATION
Targeting β-tubulin:CCT-β complexes incurs Hsp90 and VCP-related
protein degradation and induces ER stress-related cell apoptosis via
triggering capacitative Ca2+-entry, mitochondrial perturbation and
caspase over-activation
Yuan-Feng Lin, Ya-Fei Lee, and Po-Huang Liang
Supplementary Materials and Methods
Pull-down assay. Cell lysates (1 mg) were diluted in binding buffer (20 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 5 mM CaCl2, and 2 mM MgCl2) and pre-incubated with 30 μl
of Ni-NTA resin (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) for
30 minutes at 4ºC in order to avoid non-specific binding of proteins throughout the
assay. Pre-cleaned cell lysates were then resuspended in 1-ml binding buffer and
incubated with recombinant CCT- (100 μg) for 2 hours at 4ºC with gentle agitation,
followed by incubating with 30 l of Ni-NTA resin for another 1 hour at 4ºC. After
centrifugation, the pellets were washed with binding buffer for three times and
subjected to SDS-PAGE analysis.
Immunoprecipitation. Cell lysates (0.5 mg) were diluted in 1-ml cell lysis buffer and
pre-incubated with 20 μl of protein A-agarose (SantaCruz Biotechnology, CA, USA)
to avoid non-specific binding of proteins. The pre-cleaned cell lysates were then
incubated with anti--tubulin antibody (2 g) for 2 hours at 4 ºC with gentle agitation
prior to the addition of 20 l of protein A-agarose (StantaCruz). After centrifugation,
the pellets were washed with cell lysis buffer for four times and then subjected to
Western blot analysis using CCT- specific antibody (StantaCruz).
Microarray analysis. Transcriptional profiling results of CCT- were obtained from
a microarray (Accession no.: GSE23209) in GEO DataSets on NCBI website. The
relative mRNA expression levels of each untreated cells were normalized by their
median and presented as log2 value.
Supplementary Figure, Table and Movie Legends
Supplementary Figure 1: Protein expression levels of -tubulin and CCT- in panel of
cancer or normal transformed cell lines. Cell lysates derived from the employed cell
lines were subjected to Western blot analysis using -tubulin or CCT--specific
antibody. The values in the upper figures denote the density of each protein band.
Each column of bottom histogram represents the relative density of respective CCT-
or -tubulin protein band in comparison with the CCT- or -tubulin level in
HEK-293 cells.
Supplementary Figure 2: Paclitaxel (PTX)-induced G2M arrest and staurosporine
(STS)-caused subG0 accumulation in HEK-293 cells with enforced expression of
wild-type or C354S mutant -tubulin. (a, b) Cells were treated with PTX at 10 nM (a)
or STS (1 M) for 24 hours. Cellular SubG0 accumulation (M1) and G2M arrest (M2)
were assessed by PI-based Flow-cytometric analysis. X and Y axis represents
fluorescent intensity of PI and cell counts, respectively. The results are presented as %
of total cells.
Supplementary Figure 3: Pull-down assay of recombinant CCT-. (a) Recombinant
His-tagged CCT- was generated by E. coli expression system and purified by
Ni-NTA affinity column. The eluted proteins (Elute) were analyzed by SDS-PAGE
with coomassie blue staining. (b) HEK-293 cell lysates were incubated with
recombinant CCT-. The immunoprecipitation (IP) was performed by using -tubulin
antibody followed by protein A-agarose precipitation. The levels of CCT- were
detected by immunoblotting (IB) with CCT--specific antibody. IgH and IgL denote
heavy chain (H) and light chain (L) of immunoglobulin (Ig). (c) Cells lysates derived
from HEK-293, MES-SA and MES-SA/Dx5 cells were incubated with recombinant
CCT- for 2 hours at 4 ºC. The CCT--interacting proteins were then precipitated by
Ni-NTA beads and subjected to SDS-PAGE analysis. Protein bands A, B and C from
HEK-293 cell lysates were minced and referred to mass spectrometric analysis.
Supplementary Figure 4: The mRNA levels of -actin, -tubulin, XIAP and caspase-7
in I-Trp-treated HEK-293 cells. Cells were treated with I-Trp at 5 μM for indicated
time periods. The mRNA levels of -actin, -tubulin, XIAP and caspase-7 were
analyzed by RT-PCR using their paired primers. GAPDH presented in Fig. 4e was
used as an internal control.
Supplementary Figure 5: The effects of DTL on I-Trp-elevated mRNA levels of VCP,
Hsp90 and CCT in HEK-293 cells. Cells were pre-treated with DTL at 50 μM for 30
min prior to the treatment with I-Trp at 5 μM for 4 hours. The mRNA levels of VCP,
Hsp90 and CCT were analyzed by RT-PCR using their paired primers. GAPDH
presented in Fig. 5e was used as an internal control.
Supplementary Figure 6: CCT- levels in cancer cells with chemoresistance against
Hsp90 inhibitor, Tanespimycin, are comparable or even higher than that in
Tanespimycin-sensitive cancer cells. The mRNA levels of CCT- were analyzed from
microarray dataset GSE23209 and presented as log2 values.
Supplementary Table 1: DNA sequences of paired primers used in this study.
Supplementary Movie 1: A rapid intracellular Ca2+ elevation evoked by I-Trp.
Supplementary Movie 2: Iodoacetamide failed to evoke intracellular Ca2+ elevation.