Legends to SupplementarySUPPLIMENTARY Figures
Figure S1: Wnt signaling regulates SATB1 (A, Left panel) Relative mRNA expression levels of SATB1 in indicated colorectal cancer cell lines were determined by Taqman-qRT-PCR as described in ‘Materials and Methods’. Actin was used as endogenous control. Data was normalized with that of primary cell line CRL1790. Error bars represent standard deviation calculated from triplicates.(Right panel) Immunoblot for expression of SATB1 using equal number of cells. Actin was used as loading control.
(B) Expression of SATB1 at transcript level in CRL1790 cells under BIO (1 µM) treatment for 24 h determined by qPCR. Figure represents data from two independent experiments plotted separately. The error bars represent standard deviation from triplicates.(C) Relative mRNA levels of SATB1 under depletion of TCF7L2 and β-catenin in HCT116 cells. Expression levels were normalized with that of the control cells. (D) Immunoblot showing expression of SATB1, AXIN2 and TCF7 in control and TCF7L2 knockdown HCT-15 cells. Actin was used as endogenous control. (E) Immunoblot showing expression of SATB1 and TCF7 in control and β-catenin knockdown HCT-15 cells. (F)Left panel: Immunoblot showing decrease in expression of SATB1 under β-catenin depletion in SW620 metastatic colorectal cells and upon treatment of Wnt signaling inhibitor Pyrvinium Pamoate (PP, 100 nM), Right panel: Immunoblot showing decrease in expression of SATB1 and β-catenin in COLO201 cells upon degradation of β-catenin by treatment with PP. (G) Immunoblot showing expression of SATB1 upon treatment with Wnt antagonistic DKK1 in MDA-MB231 breast cancer cells. Cells were treated with 100ng DKK1 for 48 h.
Figure S2: Regulation of SATB1 by TCF7L2/β-catenin signaling (A) Immunoblot showing decrease in expression of β-catenin upon β-catenin knockdown used for SATB1 promoter reporter assay in Figure 2E. Actin used as endogenous control. (B) ChIP-qPCR showing relative enrichment of β-catenin and H3K4(me)3 and not of H3K27(me)3 on Satb1 promoter in control cells. In β-catenin knockdown cells, the occupancy profile for these three is reversed.
Figure S3: Expression of SATB1 correlates with aggressive phenotype of colorectal cancer cell lines. (A)Immunoblot for SATB1 and TCF7L2 expression in colorectal cancer tissue samples (T) in comparison with adjacent non-cancerous tissue (N) from the same patient determined by western blotting. Actin was used as endogenous control.Expression of TCF7L2 correlates with SATB1 expression. (B) Decreased expression of SATB1 in sh1 and sh2 HCT-15 stable cells in comparison with control cells determined by immunoblotting (upper panel) and by qPCR (lower panel). Actin was used as loading control for immunoblotting and qPCR. (C) SATB1 depletion reduced cell growth of SATB1 knockdown cells in comparison with control cells as determined by colony assay. The assay was performed in four different 60mm dishes for control and for shSATB1 cells. (D) Proliferation assay in control HCT-15 and shSATB1 knockdown stable cells using non-radioactive proliferation assay kit. Depletion of SATB1 reduced the proliferative potential of SATB1 knockdown cells in comparison with control cells. (E) Depletion of SATB1 reduced the migratory potential of SATB1 knockdown cells in comparison with control cells. Phase contrast images of HCT-15 control cells and shSATB1 cells subject to wound healing assay in serum free condition. Images were taken at 0 h and 24 h after wounding. Graphical representation of distance migrated by control cells and shSATB1 cells (right panel). (F) HCT-15 and SATB1 knockdown cells were plated on soft agar and grown for 14 days. Representative images are shown.** P<0.01 using Student’s paired t-test.
Figure S4:SATB1 modulates Wnt signaling (A) Left panel- Immunoblot showing expression of SATB1, β-catenin, DVL2, DVL3 and TCF7L2 in control cells and in siSATB1-HCT116 cells. Right panel - Immunoblot showing expression of TCF7L1 (TCF3) in control HCT116 and shSATB1 HCT116 cells. Depletion of SATB1 reduces the expression of TCF7L1. Actin was used as endogenous control. (B) SATB1 knockout cells (KO) were generated using TALEN technology.However, after transfection of the TALEN construct the cells were not subjected to selection of clones (as described in Sanjana et al. 2012) and hence the polyclonal mixture of cells yielded only partial depletion in SATB1 levels. Expression of SATB1, AXIN2 and TCF7 in SATB1 knockout and under shRNA mediated stable knockdown of SATB1 was monitored by immunoblot analysis (Upper panel). Lower panel represents immunoblot showing expression of TCF7L2 and SATB1 under SATB1 knockdown and knockout conditions. (C)Immunoblot for expression of TCF7L2(Left panel)in primary colorectal cell line (CRL1790), Type A (SW1116), Type B (SW480), Type C (HCT-15, HT-29, DLD1, COLO320), Type D and metastatic cell lines (COLO201, COLO205, COLO741 and T84). Expression of TCF7L2 correlates with expression of SATB1 and aggressive phenotype of colorectal cancers. Tubulin was used as endogenous control. Right panel- Immunoblot depicting expression of TCF7L2 and SATB1 in CRL1790 and SW620. Actin was used as endogenous control.
Figure S5: Effect of SATB1 knockdown and SATB1-DN overexpression (A) Relative expression levels of cancer-associated genes and various effectors of Wnt signaling pathway under SATB1 knockdown condition in HCT-15 CRC and upon overexpression in SW480 CRC were evaluated by quantitative PCRs using Taqman low-density arrays (TLDA). 18S RNA and Actin were used as endogenous controls. The box provides the key for all bars. The y-axis represents foldchange in gene expression normalized with the controls (Bars 1 and 3 respectively). Names of genes are indicated on the left side of each graph. Error bar represents the standard deviation calculated from triplicates. (B)Relative mRNA expression levels of TCF7L2 under SATB1 knockdown condition in indicated colorectal cancer cell lines were determined by Taqman-qPCR. Actin was used as endogenous control. (C)Immunoblot showing decrease in expression of TCF7L2 and AXIN2 upon ectopic expression of SATB1-DN (1-204) in HCT-15 cells. Actin was used as endogenous control.
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