Supplementary Figure Legends.
Supplementary Figure 1. Lovastatin and 17-AAG induce apoptosis in RPMI-8226
cells. Annexin V, propidium iodide (PI) flow cytometric experiments were performed in
RPMI-8226 cells treated with 17-AAG (0.05-0.25 M) and/or lovastatin (10 M) for 48
hours. The percentages of cells in the early apoptotic (Annexin V positive, PI negative
(Ann+ PI-)) and late apoptotic/necrotic (Annexin V positive, PI positive (Ann+ PI+))
fractions are shown (n =3). The * denotes p<0.05 per unpaired two-tailed t-test and
compares lovastatin + 17-AAG treatments to 17-AAG alone (using total Annexin V
positive values). Flow cytometry was performed with a Becton Dickinson FACScan.
Cellquest software (Becton Dickinson) was used for acquisition (Cellquest V3.3) and
analysis (Cellquest Pro V4.0) of data. Forward scatter (FSC) and orthogonal scatter
(SSC) were collected using linear amplification. Annexin V FITC and propidium iodide
fluorescence were collected using log amplification. 10,000 events were collected in
listmode. A bitmap gate was placed around the cell population on the basis of forward
and orthogonal light scatter to eliminate small debris and aggregates. The bitmap was
large enough so that apoptotic cells were not eliminated. Cells satisfying the bitmap
gate were analyzed using quadrant statistics in an Annexin V FITC versus propidium
iodide dual parameter histogram.
Supplementary Figure 2. Effects of caspase inhibitors in ALMC-2 cells. ALMC-2
cells were incubated for 48 hours in the presence or absence of 17-AAG (0.5 M),
lovastatin (10 M), caspase-2 inhibitor (50 M VD-VAD, C2i), caspase-4 inhibitor (50 M
Y-VAD, C4i), or pan-caspase inhibitor (50 M VAD, Pan-i). Immunoblots depicting
PARP, calnexin, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, and -
tubulin are shown. *Denotes the PARP cleavage product while ** denotes the calnexin
cleavage product. -Tubulin is shown as a loading control.
Supplementary Figure 3. Semi-quantitative RT-PCR analysis of caspase 4 mRNA
levels in myeloma/amyloid cell lines. Total RNA was isolated from each cell line,
cDNA was prepared, and PCR was performed using caspase 4-specific primers. PCR
samples were separated on an agarose gel, stained with ethidium bromide and
visualized on a UV transilluminator. -actin was used as a control.
Supplementary Figure 4. 17-AAG abrogates lovastatin-induced effects on
monoclonal protein trafficking in U266 cells. U266 cells were incubated for 48 hours
in the presence or absence of 10 M lovastatin (Lov) in combination with 0.05-1 M 17AAG (AAG). Intracellular lambda light chain levels were determined via ELISA and data
are expressed as a percentage of control (normalized to total cellular protein content
mean, +/- SD, n=3). The * denotes p<0.05 per unpaired two-tailed t-test and compares
17-AAG-treated cells to control cells. The ** denotes p<0.05 per unpaired two-tailed ttest and compares lovastatin + 17-AAG- treated cells to lovastatin alone.
Supplementary Figure 5. Effects of lovastatin and 17-AAG on light chain secretion.
RPMI-8226 cells were incubated for 48 hours in the presence or absence of 10 M
lovastatin (Lov) and/or varying concentrations of 17-AAG (AAG). Secreted lambda light
chain levels were determined via ELISA and data are expressed as a percentage of
control (normalized to total cellular protein content; mean +/- SD, n=3). The # denotes
p<0.05 per unpaired two-tailed t-test and compares lovastatin-treated cells to control
cells. The * denotes p<0.05 per unpaired two-tailed t-test and compares 17-AAG-treated
cells to control cells. The ** denotes p<0.05 per unpaired two-tailed t-test and compares
lovastatin + 17-AAG- treated cells to lovastatin alone.
Supplementary Figure 6. Lambda light chain mRNA levels are not altered
following treatment with lovastatin and/or 17-AAG. RPMI-8226 and U266 cells were
incubated for 24 hours in the presence or absence of 10 M lovastatin (Lov) and/or 0.5
M 17-AAG (AAG). Real-time PCR was performed using primers for RPMI-8226- and
U266-specific lambda light chain. Data were normalized to -actin levels and are
expressed as relative to control untreated cells (mean + SD, n=3). Unpaired two-tailed ttesting did not reveal statistically significant differences between treated and untreated
cells.
Supplementary Figure 7. Knockdown of HSP90. A) Immunoblot analysis of
transfected RPMI-8226 cells demonstrates knock-down of HSP90 and upregulation of
HSP70. -tubulin is shown as a loading control. These gels are representative of two
independent experiments. B) Cell count and viability. At the conclusion of the incubation
period, cells were stained with Trypan blue. Viable and nonviable cells were counted
using a BioRad TC10 automated cell counter. These data represent one independent
experiment.
Supplementary Figure 8. Three-dimensional rendering of lambda light chain
localization. Three-dimensional renderings of the confocal data sets (from
supplementary figure 16) were performed using Imaris software (Bitplane, Ag).
Representations of untreated (Con), lovastatin-treated (Lov), 17-AAG-treated (AAG) and
drug combination-treated (Lov + AAG) cells are shown. The blue represents nuclear
DAPI staining; green, PDI ER staining; and red, lambda light chain staining. The Imaris
Spot function was used to locate individual vesicles based on size and intensity. The
vesicles are represented by red spheres centered on the vesicle location.
Supplementary Figure 9. Agents which impair Rab geranylgeranylation induce
upregulation of the UPR. RPMI-8226 cells were incubated for 48 hours in the presence
or absence of 10 M lovastatin (Lov), 10 M FTI-277 (FTI), 10 M GGTI-2133 (GGTI), or
5 mM 3-PEHPC. Real-time PCR was performed using primers for PERK, ATF6, IRE1,
and GADD153. Data were normalized to -actin levels and are expressed as relative to
control untreated cells (mean + SD, n=3). Data are representative of two independent
experiments.
Supplemental Figure 10. Effects of lovastatin and 17-AAG on the regulation of
UPR mediators. RPMI-8226 (R), U226 (U), H929 (H), and ALMC-2 (A) cells were
incubated for 48 hrs in the presence or absence of lovastatin (Lov, 10 M) and/or 17AAG (AAG, 0.5 M). Whole cell lysate was obtained and immunoblot analysis was
performed for ATF6, IRE1 and -tubulin.
Supplemental Figure 11. 17-AAG suppresses lovastatin and brefeldin-A induced
splicing of XBP-1. Cells were incubated for 48 hrs (A) or 24 hrs (B) in the presence or
absence of lovastatin (Lov, 10 M) or brefeldin A (BFA, 0.5 M) and/or 17-AAG (AAG,
0.5 M). Total RNA was isolated, cDNA prepared, and PCR performed using XBP-1specific primers as described by Yan et al.1 PCR products were separated on a 2%
agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator. The
upper band represents unspliced XBP-1 and the lower band represents spliced XBP-1.
Gels are representative of two independent experiments.
Supplemental Figure 12. 4-PBA augments lovastatin-induced changes in UPR
mediators. RPMI-8226 cells were treated for 48 hrs with lovastatin (Lov, 10 uM) and 4PBA (PBA, 0.5 mM). Cells were lysed and immunoblot analysis performed for IRE1,
PERK, and -tubulin.
Supplemental Figure 13. Effects of 4-PBA on lovastatin and 17-AAG-induced
cytotoxicity. RPMI-8226 cells were incubated for 48 hours in the presence or absence
of 10 mM lovastatin (Lov) and/or 0.5 mM 4-PBA. MTT assays were performed. Data are
expressed as a percentage of control (mean +/- SD, n=4). The * denotes p<0.05 per
unpaired two-tailed t-test and compares the lovastatin + 4-PBA treated cells to lovastatin
alone.
1.
Yan Y, Gao YY, Liu BQ, Niu XF, Zhuang Y, Wang HQ. Resveratrol-induced
cytotoxicity in human Burkitt's lymphoma cells is coupled to the unfolded protein
response. BMC Cancer 2010; 10: 445.