Supplementary Figures 1 through 15 from PP2A Inhibition Is a Common Event in Colorectal Cancer and Its Restoration Using FTY720 Shows Promising Therapeutic Potential

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<p>PDF - 2988K, Supplementary Figure 1. Levels of immunoprecipitated PP2A from lysates of 21 CRC patients used in PP2A phosphatase assays (1D6 antibody). Supplementary Figure 2. Quantification of PP2A activity in CRC patients with normal mucosa and tumor samples (FL-309 antibody); * P < 0.05; ** P < 0.01; N: normal mucosa; T: tumor sample. Supplementary Figure 3. Levels of immunoprecipitated PP2A from lysates of 21 CRC patients used in PP2A phosphatase assays (FL-309 antibody). Supplementary Figure 4. Comparison between normal PPP2R1B sequence and silent alterations at codons 72 (A) and 391 (B) found in two CRC patients. The position altered is indicated by an arrow. Supplementary Figure 5. Western blot analysis of PPP2R2A and PPP2R5E in 21 CRC patients. Supplementary Figure 6. Quantification of PP2A activity using the 1D6 and FL-309 antibodies and western blot analysis of PP2A, CIP2A and SET in 7 CRC cell lines; * P < 0.05; ** P < 0.01; N1-2, normal tissue. Supplementary Figure 7. FTY720 treatment induces a PP2A activation. (A) Treatment with OA inhibits the FTY720-induced PP2A activity in RKO and LoVo cells. (B) Levels of immunoprecipitated PP2A (FL-309 antibody) from the RKO and LoVo lysates used in the phosphatase assays. Supplementary Figure 8. Analysis of cell viability in SW480, HT-29 and DLD-1 cells after FTY720 treatment. Supplementary Figure 9. Optical microscope images showing changes in morphology and cell viability after FTY720 treatment in RKO and DLD-1 cells. Supplementary Figure 10. Colony forming ability of HT-29 and DLD-1 cells untreated or FTY720-treated alone or in combination with okadaic acid. Supplementary Figure 11. Western blot showing PP2A, AKT and ERK1/2 phosphorylation and expression after FTY720 treatment in HT-29 and SW480 cells. Supplementary Figure 12. Western blot analysis of CIP2A, c-MYC, SET and PP2A expression after FTY720 treatment in RKO and LoVo cells. Supplementary Figure 13. MTS assays showing the effect of FTY720 and 5-FU (A), SN-38 (B) or LOHP (C) alone or in combination in DLD-1 cells. Supplementary Figure 14. MTS assays showing the effect of FTY720 and 5-FU (A), SN-38 (B) or LOHP (C) alone or in combination in HT-29 cells. Supplementary Figure 15. Effects of FTY720 on proliferation and apoptosis in a total of 5 sets of human samples, including normal mucosa and colorectal tumor. Each row represents an effect on expression of the paired control and FTY720 treated samples, and each column, a single IHC marker, including Ki-67 and cleaved caspase 3. Downregulation of expression is displayed in green, upregulation in red, and no effect in black.</p>