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. 2022 May 23;12(5):e881. doi: 10.1002/ctm2.881

FIGURE 4.

FIGURE 4

Relationship of FL118 with DDX5, c‐Myc and survivin. (A) FL118 inhibits both survivin and c‐Myc mRNA expression. (B) FL118 inhibits both survivin and c‐Myc protein expression. MiaPaca2 and SW620 cells were treated with vehicle or FL118 for 24 h, the mRNA (A) or protein (B) expression of DDX5, survivin and c‐Myc was determined by real‐time RT‐PCR (A) or western blots (B). Each bar in A is the mean ± SD from three tests. Actin and GAPDH in (B) are internal protein loading controls. (C) DDX5 interacts with c‐Rel, c‐Myc and TCF‐4 but not Sp‐1. SW620 and Panc‐1cells were analysed by IP with (αDDX5, followed by western blots with antibodies for DDX5 (control), c‐Rel, c‐Myc, TCF4 and Sp‐1. IP with corresponding normal IgG (N‐IgG) was used as the internal control. The bottom panel in (C) is the input controls (10% of cell lysates before IP). (D) FL118 can abrogate c‐Myc from the survivin promoter c‐Myc binding site: A SimpleChIP Enzymatic Chromatin IP (ChIP) Kit was used in the ChIP assay with primers covering the c‐Myc binding site in the survivin promoter (upper panel) or with primers from the survivin intron region (lower panel, negative control). Histone 3 (H3) binding is a positive control. (E) DDX5 interacts with both CDK9 and T1 in CRC HCT‐8 cells. Cells were lysed and immunoprecipitated with CDK9 antibody or control IgG, followed by western blots with DDX5 antibody (left upper panel) or CDK9 antibody (left lower panel, control). The same cell lysates were immunoprecipitated with DDX5 antibody or control IgG, followed by western blots with T1 antibody (right upper panel) or DDX5 antibody (right lower panel, control). The input control was 10% of cell lysates before IP. (F) DDX5 interacts with both CDK9 and T1 in CRC SW620 cells. Cells were lysed and immunoprecipitated with CDK9 antibody or control IgG, followed by Western blots with DDX5 antibody (left upper panel) or CDK9 antibody (left lower panel, control). The same cell lysates were immunoprecipitated with DDX5 antibody or control IgG, followed by Western blots with T1 antibody (right upper panel) or DDX5 antibody (right lower panel, control). The input control is 10% of cell lysates before IP.