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Figure S1 Comparison of the Wnt Firefly luciferase activities (a) and CMV Renilla luciferase reporter activities (b) of the 82 kinase inhibitors screened in AGS cells. The direct comparison of the firefly (Wnt driven) and the renilla luciferase activity (CMV driven) shows many of the compounds showing as Wnt inhibition also to have cell death. On the other hand, there are compounds with cell death but without Wnt inhibition. It is hard to discriminate the Wnt inhibitors with the concomitant cell death, and those exhibit cell death without Wnt inhibition. However, in the screening results shown in Figure 1, the normalized Wnt reporter activity is shown which has biasness for the Wnt inhibitors without cell death.

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Figure S2 Inhibition of Wnt signaling by different groups of kinases. (a) Graph representing different classes of kinase inhibitors inhibiting Wnt signaling pathway. (b) CMGC class of kinase inhibitors show >50% inhibition of Wnt reporter activity in AGS cells at 20 μM concentration.

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Figure S3 Differential expression of kinase genes across gastric cancer transcriptome profiles GSE62254 (a), GSE35809 (b), and GSE22377 (c). The expression pattern of kinase genes across gastric tumors show consistent expression of selected genes in intestinal and diffuse subtype gastric tumors. The kinase genes expressed in intestinal subtype gastric tumors are AURKA, AURKB, CCNB1, CDC7, CDK1, CDK2, CDK5, CDK9, CHEK1, CHEK2, CHUK, CSNK2A1, CSNK2B, GSK3B, MAP2K1, PAK4, PIK3CB, PLK1, SRC and WEE1. The genes EEF2, ITPKB, KIT, LCK, PIK3CA, PIM1, PRKACA, PRKACB, PRKCB, and ROCK1 are expressed in diffuse subtype gastric tumors.

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Figure S4 Survival analysis of selected kinase genes differentially expressed in intestinal subtype gastric tumors. Kaplan–Meier survival plots show the identified intestinal kinase genes AURKA (a), CCNB1 (b), CDC7 (c), CDK2 (d), CHEK1 (e), CHEK2 (f), CHUK (g), PIK3CB (h), and WEE1 (i) to have association with the good survival of gastric cancer patients. Elevated expression of AURKB (j), CDK5 (k), CDK9 (l), CSNK2A1 (m), CSNK2B (n), GSK3B (o), MAP2K1 (p), PAK4 (q), PLK1 (r), and SRC (s) were found associated with the poor survival of gastric cancer patients.

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Figure S5 Survival outcome associated with the kinase genes expressed in diffuse subtype gastric tumors. Diffuse kinase genes EEF2 (a), ITPKB (b), KIT (c), LCK (d), PIK3CA (e), PRKACA (f), PRKACB (g), PRKCB (h), and ROCK1 (i) show association with the poor survival of gastric cancer patients. PIM1 (J), the diffuse kinase genes was found associated with the good survival in gastric cancer patients. The analysis was made from the data available at the KM plotter resource.

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Figure S6 Correlated association between the expression pattern and drug sensitivity pattern of kinase genes across gastric cancer cells. Expression of kinase genes across gastric cancer cell lines with the sensitivity pattern of corresponding kinase inhibitors obtained from Genomics of Drug Sensitivity in Cancer Database (GDSC). Expression of kinase gene KIT shows a negative correlation trend to the drug sensitivity of Motesanib across gastric cancer cell lines.

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Figure S7 Wnt/β-catenin inhibitory kinase inhibitors also have impact on cell viability. Treatment of the identified Wnt signaling pathway transcriptional inhibitors 1-Azakenpaullone (a), IKK-2 Inhibitor V (b), IKK Inhibitor VII (c), UCN-01(d), and Ro-31-8220 (e) in AGS cells for 36 h results in the inhibition of cell viability as measured by MTT assay. The assay was performed thrice with triplicates.

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Figure S8 Wnt/β-catenin inhibitory kinase inhibitors also have impact on cell viability in gastric cancer cell line. The effect of the identified Wnt signaling pathway transcriptional inhibitors was evaluated for their impact on cell viability in MKN45 cell line. The Wnt/β-catenin pathway transcriptional inhibitors IKK-2 Inhibitor V (a), IKK Inhibitor VII (b), UCN-01(c), Olomoucine II (d), SB 203580 Sulfone (e), Roscovitine, (S)-Isomer (f), Ro-31-8220 (g), and SB 239063 (h) were found to inhibit cell viability in MKN45 cells at 36 h time point as measured by MTT assay. The assay was performed twice with triplicates.

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Figure S9 Validation of the Wnt/β-catenin transcriptional inhibitory effect of the selected kinase inhibitors in gastric cancer cells. Dual-luciferase based transient Wnt reporter assay in AGS cells upon exposure to the kinase transcriptional inhibitors IKK-2 Inhibitor V (a), IKK Inhibitor VII (b), SB 239063 (c), Scytonemin Lyngbya sp.(d), and Ro-31-8220 (e) at 1 μM, 10 μM, and 20 μM concentrations. The dual-luciferase reporter assay reveals the significant inhibition of Wnt reporter activity in a dose dependent manner.

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Figure S10 Wnt/β-catenin transcriptional activity inhibiting kinase inhibition also shows the effect at the level of proteins. Immunofluorescence analysis of β-catenin (a-b) and GSK3β (c-d) protein expression upon IKK-2 Inhibitor V and IKK Inhibitor VII treatment at 1 μM and 20 μM concentrations resulted in decreased expression of β-catenin protein and increased level of GSK3β compared with the vehicle treated control AGS cells. The protein staining with Alexa-fluor 488 is shown in green and blue color shows nuclear staining with DAPI. The corrected total cell fluorescence (CTCF) for β-catenin was found to get decreased upon exposure to IKK-2 Inhibitor V (e) and IKK Inhibitor VII (f) compared with the vehicle control cells, increased CTCF for GSK3β was observed upon exposure to IKK-2 Inhibitor V (g) and IKK Inhibitor VII (h) in AGS cells.

ddr21833-sup-0011-Tables.xlsxExcel 2007 spreadsheet , 487.2 KB

Table S1 List of 82 protein kinase inhibitors (Calbiochem, Library III Cat# 539746) used for the screening against Wnt signaling pathway in AGS cells.

Table S2. List of kinase inhibitors showing more than 50% inhibition of Wnt reporter activity (normalized with CMV-renilla activity) in AGS cells at 20 μM concentration.

Table S3. List of kinase inhibitors and their primary targets with corresponding kinase genes. The primary targets were obtained from calbiochem and corresponding kinase genes from NCBI gene database.

Table S4. Kinase genes showing consistent expression pattern across gastric tumor subtypes in the mRNA profiles. Expression of 51 kinase genes in terms of their elevated expression in gastric cancer subtypes is shown. This shows the kinase genes with consistent elevated expression in intestinal and diffuse subtype gastric tumors across the gastric tumor mRNA profiles GSE15459, TCGA-STAD, GSE62254, GSE35809, and GSE22377.

Table S5. List of gastric mRNA profiles analyzed in the study with the details of the number of samples, profiling platform and other details.

Table S6. Kinase gene expression across gastric tumor profiles GSE15459 (A) and TCGA_295 (B).

Table S7. Kinase gene expression across gastric normal and tumor profiles GSE13911 (A) and GSE13861 (B).