Supplementary Figures from Targeting Myddosome Signaling in Waldenström's Macroglobulinemia with the Interleukin-1 Receptor-Associated Kinase 1/4 Inhibitor R191

Supplementary Figure 1. BCWM. 1 (upper panel) and MWCL-1 cells (lower panel) were seeded in 96-well plates (20,000 cells/well) and treated with increasing concentrations (0.0, 0.15, 0.3125, 0.625, 1.25, 2.5, 5.0 ï�­M) of the indicated IRAK inhibitors for 72 hours. Cell viability was then assayed using the tetrazolium reagent WST-1, and the data presented are representative of at least 3 independent experiments. Supplementary Figure 2. MWCL-1 and BCWM.1 cells were treated with the indicated concentrations of R191 for 24 hours. They were then stained either with DAPI and analyzed by flow cytometry for cell cycle analysis (A), or they were stained with ToPRO-3 and Annexin V, and analyzed by flow cytometry for apoptosis (B). Supplementary Figure 3. Peripheral blood mononuclear cells (PBMC) from two donors were seeded in 96-well plates (50,000 cells/well) and treated with increasing concentrations (0.0, 0.0075, 0.015, 0.030, 0.075, 0.15, 0.3, 0.6, 1.25, 2.5 ï�­M) of R191 for 24 hours. Treated PBMC's were evaluated for the induction of apoptosis by staining with ToPRO-3 and Annexin V, and analyzed by flow cytometry, and the data presented are representative of 3 independent experiments. Supplementary Figure 4. (A) BCWM.1 (left) and MWCL-1 cells (right) were treated with 1.25 ï�­M R191 for 24 hours, and extracts were studies by RPPA analysis. Expression levels of selected intermediates are shown as the means {plus minus} SD for duplicate samples, and "*" indicates a p-value of <0.05 comparing the drug-treated with the vehicle-treated controls. (B) Selected data from the RPPA analysis are shown in the format of a heat map. Supplementary Figure 5. Gene set enrichment analysis (GSEA) revealed a significant effect of R191 treatment on Akt gene sets (A). GSEA revealed a significant effect of R191 treatment on a c-MYC gene set (B). Supplementary Figure 6. BCWM. 1 and MWCL-1 cells were treated with the indicated concentrations of R191 for 24 hours, and the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) was examined by qRT-PCR (A) and Western blotting (B). Supplementary Figure 7. Expression of IRAK1 and IRAK4 was achieved in BCWM.1 cells with the use of two separate shRNAs for the former and one for the latter, respectively. These cells were then exposed to vehicle or R191 at the indicated concentrations, and viability was determined as detailed above. "*" Indicates a significant difference between the viability of cells bearing a non-targeting (NT) shRNA treated with R191 and cells with either an IRAK1- or IRAK4-targeting shRNA treated with the same concentration of R191 with a p value <0.01. Supplementary Figure 8. (A) BCWM.1 and MWCL-1 cells were treated with 1.25 ï�­M R191 for 24 hours, and expression of ER-stress response genes was analyzed by qRT-PCR. Data are expressed as the means {plus minus} SD for samples assayed in triplicates, and "*" indicates p-values <0.016, "**" {less than or equal to}0.0001 indicates p-values <0.003, and "***" indicates p-values <0.0005. (B) Induction of autophagy was evaluated by Western blotting for the levels of autophagic markers LC3 I/II and p62 using β-actin as a loading control. Supplementary Figure 9. Five mice per each xenograft/treatment group were inoculated either subcutaneously (SC) with 1Ã-107 BCWM.1 GFP+/luc+ cells or intravenously (IV) with 1Ã-106 BCWM.1. Bioluminescence images of vehicle- and R191-treated mice bearing SC (A) or IV (B) xenografts were acquired at the indicated time points. Weights of the mice were determined on days 0, 7, and 14 for the IV xenografts, and on days 0, 14, and 21 for the SC xenografts, and are plotted along with the standard deviations (C). Supplementary Figure 10. Cleavage/activation of Caspase 3 and expression levels of CDK6 are shown in tissue harvested from SC xenograft tumors at the end of the in vivo experiment after the indicated treatments as analyzed by Western blotting, using β-actin as a loading control.