Extended Data Fig. 6: FOXP1 and TCF7L2 preferentially bind to the rs10871066[A] and rs10871066[G] alleles to affect the expression of PIBF1 and KLF5, respectively.
a. The effect of FOXP1 or TCF7L2 overexpression on relative luciferase activity of vectors containing rs10871066[A] or rs10871066[G] allele in HCT116 cells. The cells were first treated with plasmids containing varying doses of TCF7L2 or FOXP1-cDNA followed by transfection with luciferase plasmids of rs10871066[A] or [G] allele. The results were normalized against the group of pcDNA3.1 along with luciferase plasmids carrying rs10871066-A allele. n = 9 biologically independent samples. b. The correlations between TCF7L2 and KLF5 expression, FOXP1 and PIBF1 expression were analyzed in the GSE14333 cohort (n = 290). c-d. Scatter plots show the correlations between PIBF1 and FOXP1 expression (top), as well as the correlations between KLF5 expression with TCF7L2 expression (bottom) stratified by variant rs10871066 genotypes in our CRC samples (nAA+AG = 103, nGG = 51) (c) and TCGA CRC samples (nAA+AG = 166, nGG = 159) (d). e-f. The expression of TCF7L2 and FOXP1 in tumor and the normal tissues from our CRC samples (nTumor = 154, nNormal = 154) (e) and GSE103512 (nTumor = 57, nNormal = 38) (f). g. FOXP1 (left) suppressed tumors whereas TCF7L2 (right) promoted CRC growth based on the genome-wide CRISPRCas9-based loss-of-function screens. h-i. The effect of TCF7L2 (h) and FOXP1 (i) overexpression on the expression level of TCF7L2 and FOXP1 was validated by qRT-PCR analysis in SW480 cells (top) and HCT116 cells (bottom), respectively. n = 9 biologically independent samples per group. j-k. The effect of TCF7L2 (j) and FOXP1 (k) knockdown on the expression level of TCF7L2 and FOXP1 in CRISPR-edited SW480 (top) and HCT116 (bottom) cells was validated by qRT-PCR analysis. n = 9 biologically independent samples per group. Data were shown as the median, box limits indicate upper and lower quartiles and whiskers indicate the maximum and minimum (a, f). The P values were calculated by unpaired two-sided Student’s t-test (a, e, f). The two-sided P values and correlation coefficient were calculated by Pearson correlation analysis (b-d). Data were presented as the mean ± SD. The P values were calculated by unpaired two-sided Student’s t-test (h-k).
