Supplementary Figures from ADNP Is a Therapeutically Inducible Repressor of WNT Signaling in Colorectal Cancer

Suppl. Figure 1. (A) Immunostaining for ADNP and β-Catenin on serial sections of two colon cancers. (B) Staining intensities of ADNP in colon cancer cells (n = 500, 5 different tumors) with high or low nuclear β- Catenin expression. P value is t test result. (C) Immunostaining for ADNP in normal mucosa. Right panels aremagnifications of areas boxed in left panel.Scale bars, 100 μm. Suppl. Figure 2. (A-B) Immunoblotting of indicated proteins on whole cell lysates in ADNP wild-type (WT) or knockout (KO) cell lines. Numbers below immunoblots indicate fold change by densitometry. (C) Representative micrographs (left panels) and quantification (right panels) of migrated or invaded tumor cells in transwell assays for indicated ADNP WT and KO cell lines, treated with indicated siRNAs or control siRNA (siCtrl). (D) Representative proliferation kinetics based on cell quantification by impedance measurements. Data are mean, n {greater than or equal to} 3. β-Cat = β-Catenin. Suppl. Figure 3. Representative micrographs (left panels) and quantification (right panels) of migrated or invaded HCT116 or SW1222 tumor cells with transient ADNP overexpression by transfection with p4.2-hADNP compared to empty p4.2 vector for 24 h in transwell assays. Suppl. Figure 4. (A) Immunoblotting of indicated proteins on whole cell lysates of SW1222, HCT116 and primary (P-Tu) colon cancer cells. Numbers below immunoblots indicate fold change by densitometry. (B) Immunostaining of SW1222, HCT116 and primary colon cancer (P-Tu) xenografts for β-Catenin. Scale bars, 50 μm. Suppl. Figure 5. (A-B) Dual-luciferase assays for indicatedADNPwild-type (WT) or knockout (KO) colon cancer cells transfected with TOPflash reporter constructs. Treatments with PBS, ketamine or the WNT inhibitor XAV939, as indicated. (C) Representativemicrographs (left panels) and quantification (right panels) ofmigrated or invaded HCT116 or SW1222 tumor cells in transwell assays, treated with PBS or 100 μM ketamine for 24 h, as indicated.Data aremean {plus minus}SD, Pvalues are t test results, n {greater than or equal to} 3. Suppl. Figure 6. Assessment of nuclear β-catenin staining in primary human colorectal cancers. Tumors were assigned scores from 0 (no nuclear β-catenin) to 3 (most tumor cells with strong nuclear β-catenin) and accordingly categorized as β-catenin low (score 0-1) and β-catenin high (score 2-3). Scale bars, 100 μm.