Figure 5.

Ligation of CD44 promotes cyclin D1 expression via stimulation of STAT3 acetylation. (A) Western blot analyses of H1299 cells treated with H-3 for various times as indicated. (B) H1299/HA-STAT3 cells were treated with H-3 for the time points indicated and subjected to IP followed by Western blotting. (C) H1299, HT29, and HCT116 cells were treated with or without H-3 for 4 h and subjected to IP followed by Western blotting. (D) AZ521/mock and AZ521/CD44 cells were transfected with or without plasmids encoding p300 or HDAC1 or infected by lentivirus-encoding shRNA targeting p300. Total cell lysates were prepared, immunoprecipitated with anti-STAT3, and analyzed for acetylated lysine (Ac-Lysine) by Western blotting. (E) H1299 cells ectopically expressing the Flag-tagged and HA-tagged STAT3 mutants were treated with H-3 mAb. At the time points indicated, lysates were immunoprecipitated and subjected to Western blotting. (F) AZ521/mock and AZ521/CD44 cells were transfected with plasmids encoding the Flag-tagged and HA-tagged STAT3 mutants as indicated. Nuclear extracts were subjected to tandem IP. Proteins in the first and second immunoprecipitates were analyzed by Western blotting as indicated. (G) H1299 cells were transfected with plasmids encoding HA-tagged STAT3(WT) or STAT3(K685R). Nuclear extracts were subjected to tandem IP. Proteins in the first and second immunoprecipitates were analyzed by Western blotting as indicated. Arrowheads indicate the target proteins with expected sizes. (H) AZ521/CD44 cells were transfected with plasmids encoding individual STAT3. After treatment without or with H-3 for 3 d, Western blot analysis was performed. (B, C, E, and H) The relative intensities of the bands, which were quantified by densitometry, are shown. Molecular mass is shown in kilodaltons. WB, Western blot.