Fig. 4.

HBXIP-enhanced acetylation of HOXB13 stabilizes HOXB13 in the facilitation of TAM resistance. a Immunoblotting analysis of HOXB13 in MCF-7 cells treated with the indicated concentrations of leupeptin for 36 h (lower panel). The upper panel is the quantification of the intensity relative to β-actin. b Immunoblotting analysis of HOXB13 in BT474 cells cultured with serum-supplemented or serum-free media for the indicated time courses (lower panel). The upper panel is the quantification of the intensity relative to β-actin. c Immunoblotting analysis of HOXB13 in MCF-7 cells cultured with serum-supplemented or serum-free media for 48 h along with DMSO or TSA (1 μM) (lower panel). Before that, the cells were transiently transfected with pCMV or pCMV-HBXIP (1.5 μg). The protein level of HBXIP was determined by the anti-Flag antibody. The upper panel is the quantification of the intensity relative to β-actin. d Immunoblotting analysis of GFP-HOXB13 in MCF-7 cells time-dependently treated with 100 μg/ml CHX after being transiently transfected with GFP-HOXB13-WT or GFP-HOXB13-K277R (lower panel). The protein level of GFP-HOXB13 was determined by the anti-GFP antibody. The upper panel is the quantification of the intensity relative to β-actin. e Cell viability assay with MCF-7 cells treated with the indicated concentrations of TAM after being transiently transfected with the displayed plasmids. Error bars represent ± SD. *P < 0.05 and ***P < 0.001 (GFP-HOXB13-WT compared with GFP-HOXB13-K277R) by two-tailed Student’s t test. f A colony photograph of MCF-7 cells treated with DMSO or TAM (1 μM) after being transiently transfected with the displayed plasmids. All experiments were repeated at least three times. Error bars represent ± SD. *P < 0.05 and ***P < 0.001 by two-tailed Student’s t test