Fig 2: All discernable domains of SAV1 are required for MST2 activation.(A) Immunoblots of lysates of 293FT cells co-transfected with the indicated Myc-MST2 and HA-SAV1 or HA-RASSF1A plasmids. (B) Immunoblots of lysates of 293FT cells co-transfected with Myc-SAV1 and the indicated FLAG-MST2 plasmids. (C) Immunoblots of lysates of 293FT cells co-transfected with Myc-MST2 and the indicated FLAG-SAV1 plasmids. (D) Immunoblots of cell lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated Myc-SAV1 plasmids. (E) Total MST2 and pT180 blots of the auto-kinase reactions by the indicated MST2-SAV1 complexes at the indicated time points (left panel). The kinetic profiles of T180 autophosphorylation of MST2-FL/SAV1-?N198 (red line) and MST2-FL/SAV1-?N268 (black line) are shown on the right. The relative anti-pT180 intensities were normalized against MST2-FL without PP2A phosphatase treatment (100%). Data are representative of at least two independent experiments. (F) Immunoblots of lysates of 293FT cells co-transfected with FLAG-MST2 and the indicated N-terminal truncation of Myc-SAV1 constructs. (G) 293FT cells were co-transfected with FLAG-MST2 and Myc-SAV1 plasmids. The total cell lysates were blotted with the indicated antibodies. (H) 293FT cells were co-transfected with the indicated plasmids encoding FLAG-SLMAP, HA-MST2 and Myc-SAV1. Anti-FLAG IP and the total cell lysates (input) were blotted with the indicated antibodies.

Fig 3: Effect of PP2Ac silencing on the levels of a4/PP2Ac complex and cell migration. (a) representative immunoblots of PP2Ac and a4 in IEC-TRPC1L1 cells transfected with siPP2Ac or C-siRNA for 48 h. (b) levels of a4/PP2Ac complex as measured by IP assay in cells described in (a). (c) summarized data of cell migration after wounding in cells described in (a) Values are means ± SE from 6 dishes. *p < 0.05 compared with controls and cells transfected with C-siRNA

Fig 4: RNASeq and protein analysis of PP2A and downstream target proteins in CRC cells(A and B) RNASeq analysis of FOLFOX-HT29 cells. Cells were treated with 20 µM of NSC49L and 1 nM of TRAIL either alone or in combination for 16 h. Total RNA was isolated by TRIzol reagent and processed for RNASeq analysis. Ward clustering algorithm with Euclidean distance measurement was used for heat map analysis.(C) Correlation of PP2A expression level with AKT1(S473P) and p21 levels in CRC cell lines. To determine a correlation of PP2A with AKT1(S473P) and p21, we performed Western bots of these proteins using the whole-cell lysate of different CRC cell lines, including FOLFOX-resistant HCT116 and HT29 cells.(D and E) Protein levels of AKT1, p53, p21, DR5, caspase 3, caspase 8, and PARP1 in HCT116, FOLFOX-CHT116, HT29, and FOLFOX-HT29 cell lines, respectively, treated with NSC49L and TRAIL either alone or in combination for 24 h. The blots are representative of three experiments. See also Figures S2 and S3.

Fig 5: PPP2CA/B expression is lower in FOLFOX-non-responsive (NR) versus responsive (R) CRC tumors, which negatively correlated with the expression of AKT1, mTOR, 4EBP1, and CDKN1AData were analyzed from the NCBI GEO accession number GSE72970. Number of samples in the NR and R groups was 61 and 63, respectively. The p value is shown at the top of each graph.