Supplementary Figure 1
A
B
Supplementary Figure 1. Lenalidomide-sensitive wild-type (wt) and
lenalidomide-resistant (R10R) ANBL-6 and U266 cells were infected with lentiviral
particles containing shRNA constructs targeted to a non-specific scrambled
sequence (.scr), or shRNAs targeting CD44 (.shCD44). Initial cell surface
knockdown of CD44 was analyzed by FACS utilizing Alexa Fluor® 488 conjugated
CD44 antibody shown in part (A). Following selection with puromycin, whole
protein lysates were then subjected to Western blot analysis to look at relative
CD44 knockdown levels shown in part (B). Following knockdown validation, cells
were treated with lenalidomide on immobilized HA-coated plates, and viability was
determined as shown in Figure 4.
Supplementary Figure 2
Supplementary Figure 2. FACS layout representative of the separation of
myeloma cells into fractions that are CD44-High or CD44-Low. Following
fractionation, cells were treated with lenalidomide, and viability was determined as
shown in Figure 4.
Supplementary Figure 3
A
B
Supplementary Figure 3. Lenalidomide-resistant, IL-6 dependent ANBL-6/R10R
and KAS-6/R10R (A), and their lenalidomide-sensitive counterparts ANBL-6wt and
KAS-6wt (B), were treated with the anti-IL-6 antibody, siltuximab (1-10 µg/mL),
with and without the presence of 10 µM lenalidomide for 72 hrs and analyzed for
cellular viability. The student’s paired t-test was used to determine statistical
significance, where “*” denotes p<0.05. Cellular viability measurements were
performed using the WST-1 assay, and all data points were normalized to the
vehicle control, which was set at 100%. Mean viability values are provided from
three independent experiments, along with the +/- S.D.
Supplementary Figure 4
A
B
Supplementary Figure 4. Lenalidomide-resistant KAS-6/R10R and U266/R10R
(A), and their lenalidomide-sensitive counterparts KAS-6wt and U266 (B), were
treated treated with the β-catenin antagonist FH535 (1-10 µM), with and without
the presence of 10 µM lenalidomide for 72 hrs and analyzed for cellular viability.
The student’s paired t-test was used to determine statistical significance, where “*”
denotes p<0.05. Cellular viability measurements were performed using the WST1 assay, and all data points were normalized to the vehicle control, which was set
at 100%. Mean viability values are provided from three independent experiments,
along with the +/- S.D
.
Supplementary Figure 5
A
B
Supplementary Figure 5. (A) Lenalidomide-sensitive KAS-6wt cells were
treated with increasing concentrations of ATRA at various time points, and the
relative lymphoid enhancer factor/T-cell factor (LEF/TCF) promoter activity was
determined. (B) Relative LEF/TCF reporter activity was determined by FACS
analysis in lenalidomide-sensitive KAS-6wt cells following exposure to increasing
concentrations of lenalidomide (0.01-10 µM) with and without the presence of
ATRA (1-10 µM). All values are relative to the vehicle control, which was arbitrarily
set at 1.
0.7
0.6
0.5
0.4
0.3
0.2
0.0
0.1
Pr(min(muF,muE)>muC|data)
0.8
0.9
1.0
Supplementary Figure 6
D7
D9
D12
D14
D18
D16
D21
D23
D25
D28
Time
Supplementary Figure 6. Analysis of cooperative effects of lenolidomide and
ATRA on tumor volume. In terms of the tumor volume, from Day 16 afterward, the
posterior probability of cooperative effect was found to be greater than 0.9952. For
example, the posterior probability of cooperative effect was found to be 0.9981 at
Day 24, which suggests that there is very low chance (20 in 10,000), that the
combination does not have exhibit a cooperative effect (as defined in Statistical
section). The cooperative effect reached peak at Day 21.