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A B Supplementary Figure 1 Supplementary Figure 1. Lenalidomide-sensitive wild-type (wt) and lenalidomide-resistant (R10R) ANBL-6 and U266 cells were infected.

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Presentation on theme: "A B Supplementary Figure 1 Supplementary Figure 1. Lenalidomide-sensitive wild-type (wt) and lenalidomide-resistant (R10R) ANBL-6 and U266 cells were infected."— Presentation transcript:

1 A B Supplementary Figure 1 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.

2 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.

3 A B Supplementary Figure 3 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 students 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.

4 A B Supplementary Figure 4 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 students 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.

5 Supplementary Figure 5 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 ( 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. A B

6 Supplementary Figure 6 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 For example, the posterior probability of cooperative effect was found to be 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.


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