Supplementary Figures S1-8 from Anagrelide for Gastrointestinal Stromal Tumor

Supplementary Figure S1. A box-whisker plot showing the relative KIT, ANO1, and PRKCQ (encodes for protein kinase C theta) mRNA expression in soft-tissue sarcoma samples in the MediSapiens database. Supplementary Figure S2. PDE3A (A) and KIT (B) expression in human intestinal smooth muscle tissue (magnification x400, scale bar 100 μm). Supplementary Figure S3 A, Effects of the combinations of PDE3 siRNAs and DMSO on the GIST882 and GIST48 cell lines. siPDE3B and siPDE3A/B reduced GIST882 cell viability, and none of the treatments influenced markedly GIST48 viability. SEM < 0.05 in all data points. B, Effects of PDE3 inhibitors cilostazol, milrinone, and amrinone given with DMSO on GIST882 and GIST48 cell line viability. SE < 0.05 in all data points. Supplementary Figure S4. A Sankey diagram of top SPIA pathways. A Sankey diagram showing differentially expressed miRNAs, their predicted target genes, and the biological pathways estimated to be significantly impacted by the expression change (SPIA pGFDR <0.1). The fold-change of pathways represent upregulation [2] or downregulation [-2] in the cell line 882. Supplementary Figure S5. Hematoxylin-eosin stainings to illustrate the histological responses to anagrelide, imatinib, and combination therapy in GIST xenograft models. Supplementary Figure S6. PDE3A and PDE3B are expressed in GIST xenograft models. PDE3A and PDE3B protein expression in Western blots from four GIST mouse xenograft models after treatment with imatinib, anagrelide, or both. Each column represents one mouse tumor (maximum two per mouse). The numbers of tumors available varied for the xenograft models, since some of the GIST2B, GIST882, and GIST3 model tumors shrank markedly or disappeared leaving no tissue material left for the Western blot. Low or lacking PDE3A or PDE3B expression in some treatment groups (notably anagrelide plus imatinib treatment for 36 GIST2B and GIST3) may be rather due to a high grade histological response than PDE3A or PDE3B downregulation. Supplementary Figure S7. Effects of the drug treatments on KIT signaling in GIST xenograft models. A Western plot showing the expression of KIT, phosphorylated KIT (Y703 and Y719), AKT, phosphorylated AKT, MAPK, phosphorylated MAPK, and tubulin (control). Supplementary Figure S8. Tumor PDE3A and PDE3B expression in four mouse GIST models. A, Tumor PDE3A and PDE3B mRNA levels measured with qPCR. The highest PDE3A levels and the lowest PDE3A to PDE3B ratio were found in the tumor from the GIST9 xenograft model that responded the poorest to anagrelide. The P values refer to the statistical difference between tumor PDE3A expression in the GIST9 model and the other GIST models, and the difference between tumor PDE3B expression in the GIST882 model and the other models. *P <0.5; **P < 0.01; ***P < 0.001. The bars represent the standard error of mean. The data represent mean {plus minus} s.e.m. B, Western blots showing expression of PDE3A and PDE3B in GISTs originating from the four xenograft models. Two untreated tumors were studied from each model. C, SLFN12 expression in GIST xenograft models. SLFN12 expression is increased in anagrelide treated tumors compared to the control group.