Research Paper Volume 13, Issue 4 pp 5185—5196

Figure 2. The genomic binding pattern of STAT3^del in colon cancer cells. (A) Sequence alignment shows the deletion of sequence encoding amino acid residues between 400 and 411 (NNGSLSAEFKHL) in the DNA-binding domain of STAT3 in the colon cancer cells (SW480, SW620, and HCT116) using CRISPR-CAS9. (B) Representative western blot shows the total and phosphorylated levels of JAK2 and STAT3 in control and TG101348 (JAK2 inhibitor)-treated wild-type and STAT3^del SW480, SW620, and HCT116 cells. Βeta-actin is shown as the loading control. Each experiment was performed in triplicates. (C) ChIP-qPCR results show the occupancy of STAT3^del in the promoter regions of Snail, Spg20 and MMP3 genes in SW480, SW620, and HCT116 cells with wild-type STAT3 or STAT3^del. * denotes p < 0.05. Each experiment was performed in triplicate. (D) The heatmap shows the genome-wide occupancy of STAT3 and STAT3^del at all the annotated gene promoters in the SW480 cells as determined by ChIP-seq. The average STAT3 enrichment is measured based on the log2 (peak p values) in 200-bp bins and is shown within genomic regions covering 2 kb upstream and downstream of the center. (E) The volcano plot shows the comparison of different peaks (representing STAT3 genomic occupancy) between wild type and STAT3^del SW480 cells. Brown spots represent significantly altered peaks between STAT3 and STAT3^del; blue spots represent peaks without statistical significance. (F) The genome-wide distribution of STAT3 binding regions in wild-type and STAT3^del SW480 cells. The various genomic regions are represented by different colors. (G) Enrichment of STAT3 (purple) or STAT3^del (yellow) in the HNRNPAB, SDE2, STEAP1, and TARDBP genes. ChIP-seq data are shown in reads per million; y-axis floor is set to 0.5 reads per million. (H) Predicted DNA-binding motifs that are enriched in the loci bound by wild type STAT3 (top) and STAT3^del (bottom) in the SW480 cells.