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Additional file 1.

Additional file 5: Supplementary Table 1. Complete list of antibodies used in this study.

Additional file 3: Supplementary Figure 3. Che-1 phosphorylation is required for its pro-proliferative ability. A: WB analysis with the indicated antibodies showing the transfection efficiency of the experiment described in Fig. 3A. B, C and D: Cell proliferation analysis (left) and relative WB analysis (right) of KMS27, HeLa and 293 T cells transiently transfected with Myc-Che-1, Myc-Che-1 3S or control vector (pCS2MT). Bar plot shows the average number of cells observed in these experiments (n = 5). E: Cell number-normalized total RNA quantification of the indicated cell line transiently transfected with Myc-Che-1 wt, 3S mutant or control vector. Error bars represent the SD of triplicate experiments (n = 3). F: Nuclear extracts from HCT116 cells transiently transfected with Myc-Che-1 wt or 3S and subjected to IP with H3 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. G: Cell proliferation analysis of KMS27 cells transiently transfected with the indicated peptides. Bar plot shows the average number of cells observed in these experiments (n = 3). Statistical significance is indicated by asterisks as follow: *P

Additional file 4: Supplementary Figure 4. CK2 phosphorylates Che-1. A: Nuclear extracts from HCT116 cells were subjected to IP with CK2 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. B and C: Representative WB analyses of total cell extracts showing the transfection efficiency of the experiments described in Fig. 5B and C. D: The panel shows the MS/MS spectrum of the phosphopeptide YLVDGTKPNAGSEEISSEDDELVEEK identifying the phosphorylation of Che-1 residues S320 and S321. E: Representative WB analyses of total cell extracts showing the transfection efficiency of the experiment shown in Fig. 5D. F: Nuclear extracts from HCT116 cells treated or not with 80 μM TBB for 4 h and then subjected to IP with Che-1 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. G: WB of the IP experiments used for densitometryc analysis shown in Fig. 5E.

Additional file 2: Supplementary Figure 2. Che-1 is highly phosphorylated. A: Two different replicates of 2D- experiments of total cell extracts from HeLa cells treated or not with λ-PP related to Fig. 2B. B: Two different replicates of 2D-Gel electrophoresis (top) and representative WB (bottom) of HeLa cells transiently transfected with Myc-Che-1 or Myc-Che-1 3S, related to Fig. 2D.

Additional file 5: Figure S1. High level workflow for ICI biomarker validation and combination. The N biomarkers were individually tested for each dataset, this test serving as a primary validation of the proposed performance. Mean accuracy was computed for each classifier in all available datasets. Combinatorial analysis was carried out with majority voting and Generalized Linear Models. The E[X] formula stands as an example of the model to create when estimating the a0â ŚaN linear factors.

Tables S1. Primers used for resequencing of B16 neoantigens. Table S2. B16 selected neoantigens. Table S3. Summary results of RNAseq of in vitro and in vivo MC38 cells. Table S4. M285 selected peptides. Binding affinity prediction of M285 neoantigens according to NetMHC-4 and RNAseq values. Table S5. U11 selected peptides. Binding affinity prediction of U11 neoantigens according to NetMHC-4 and RNAseq values. (DOCX 116 kb)

Additional file 1. Methods.

Figure S1. Representative gating strategy for the identification of neoantigen specific immune responses. Figure S2. Comparison of CEA specific immune responses induced by the M1 vaccine vector vs. the full length CEA protein delivered by DNA-EP. Figure S3. Comparison in peripheral blood by IFN-Îł ICS analysis for the Reps1 neoantigen and cognate WT peptide. Figure S4. M2 specific memory T cells. Figure S5. M3 specific T cells. Figure S6. Immuno modulators prevent MC38 tumor growth. (PPTX 1226 kb)

Additional file 2. Representative examples of immunohistochemical expression of TAZ and YAP in gastric cancer. Two cases are presented with combined nuclear expression of both TAZ and YAP (Aâ D).

Additional file 1. Baseline characteristics of gastric cancer (GC) patients included in this study (N = 83).

Additional file 1. First-line chemotherapy regimens and schedules (NÂ =Â 86).

Additional file 3. Multivariate Cox regression model for progression-free survival (PFS) evaluating three different molecular signatures (N = 83).

Different mRNAs associated to eEF1BÎł. (DOC 42 kb)

A. Chromatin immunoprecipitation was performed in hSH-SY5Y cells using anti-eEF1Bγ rabbit polyclonal antibodies or no-Ab as a control. Immunoprecipitates from each sample were analyzed by PCR performed with primers specific for the human Che-1 promoter and for the human TP53 promoter. The thymidine kinase human promoter was amplified as a negative control. A sample representing linear amplification of the total input chromatin (input) was included in the PCR as a control. B. Co-localization of endogenous Che-1, performed with the anti-Che-1 rat polyclonal antibody (green), and the mitochondrial marker Tom20 (red), in hSH-SY5Y cells. Extensive co-localization (yellow) between Che-1 and Tom20 is visualized by the merged-color image. The boxed area represents a high magnification image of co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 10 μm. C. Western blot analysis of hSH-SY5Y whole-cell lysate and mitochondrial enriched fraction. The quality of mitochondrial-enriched fractions was monitored using anti-HSP60 monoclonal antibodies and anti-Tom20 rabbit polyclonal antibodies. D. Quantitative real time PCR (qPCR) analysis of the eEF1Bγ and Che-1 mRNAs in hSH-SY5Y cells (siRNA-Control and siRNA-eEF1Bγ). The gene expression ratio between eEF1Bγ and GAPDH and between Che-1 and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. E. Representative Western blot of hSH-SY5Y whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. (PDF 199 kb)

Oligos used in the present study. (DOC 56 kb)

A. RIP assay output was analyzed by semi-quantitative RT-PCR with specific primers to validate some of mRNAs co-immunoprecipitated with eEF1Bγ and they are listed in Table S2. B. The myc-eEF1Bγ and MS2-GFP fusion proteins were expressed in HeLa cells with the report mRNA carrying both the MS2 binding site and the indicated 3′ UTR. C. RIP assay-eEF1Bγ mRNAs (Additional file 2: Table S2) analyzed by quantitative real time PCR (qPCR) in HeLa whole-cell lysates treated with siRNA as shown. The gene expression ratio between mRNAs and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. D. Representative western blot of HeLa whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. Densitometric analysis represents the mean ± S.D. of 3 independent experiments (right panel). (PDF 208 kb)