Fig. 3.

STAT3 is methylated by SET9 and demethylated by LSD1. (A) Cells were transfected transiently with pcDNA3.1-SET9. After 48 h, the cells were treated with IL-6 for 4 h and total cell lysates were analyzed by Western blot. A4, STAT3-null cells; WT, K140A and K140R, A4 cells expressing normal levels of STAT3 proteins. (B) Cells were transfected transiently with pcDNA3.1-SET9. After 48 h, the cells were treated with IL-6 for 4 h and total RNAs were analyzed by Northern blot. (C) Cells were transfected transiently with pcDNA3.1-LSD1. After 48 h, the cells were treated with IL-6 for 4 h and total cell lysates were analyzed by Western blot. A4, STAT3-nullcells; WT, K140A and K140R, A4 cells expressing normal levels of STAT3 proteins. (D) Cells were transfected transiently with pcDNA3.1-LSD1. After 48 h, the cells were treated with IL-6 as above for 4 h and total RNAs were analyzed by Northern blot. (E) Western analyses for SET9 and LSD1. A4 cells were infected with retroviral constructs and stable pools of cells were selected with G418. DLD1, parental cells; A4, STAT3-null cells; WT, K140A, and K140R, A4 cells expressing normal levels of STAT3 proteins. The cells were treated with IL-6 for 4 h and total cell lysates were analyzed by Western blot. (F) STAT3 was immunoprecipitated from whole-cell extracts of the above cells by using anti-Flag M2 beads. Western analyses were performed to detect SET9 and LSD1. (G) A4 cells expressing wild-type STAT3 were transfected transiently with siRNAs directed against SET9 or LSD1 and, 24 h later, the cells were transfected again with the same siRNAs. After an additional 24 h, some of the cells were transfected with siRNA-resistant SET9 or LSD1 cDNAs. After another 24 h, some of the cells were treated with IL-6 for 4 h more and analyzed for SOCS3 expression by Northern blot and some were left untreated and analyzed for SET9 and LSD1 expression by Western blot.