Figure 3
Figure 3. Effects of PKC412 and bortezomib on expression of Bim in HMC-1 cells. (A) Immunoprecipitation (IP) and Western blot (WB) were performed with HMC-1 cells exposed to PKC412 (midostaurin, 1μM) or control medium (control) at 37°C for 4 hours. IP and WB were performed as described in “Methods.” For detection of phospho-KIT (p-KIT), anti–phospho-tyr monoclonal antibody 4G10 (1:1000) was applied. As visible, midostaurin suppressed the expression of p-KIT in HMC-1.1 and HMC-1.2 cells (top panels) without affecting total KIT expression (bottom panels). (B) WB analysis of HMC-1.1 cells (left panel) and HMC-1.2 cells (right panel) exposed to control medium (control) or PKC412 (1μM) for 12 hours. WB was performed using a polyclonal anti-Bim antibody. The β-actin loading control is also shown. (C-D) Northern blot analysis of HMC-1.1 cells (C) and HMC-1.2 cells (D) exposed to control medium (control) or PKC412 (1μM) for 12 hours. Northern blotting was performed using a Bim-specific cDNA probe. Equal loading was confirmed by probing for β-actin mRNA. (E-H) Real-time PCR analysis of Bim mRNA expression in HMC-1.1 cells (E,G) and HMC-1.2 cells (F,H) exposed to control medium (0) or various concentrations of PKC412 or bortezomib as indicated for 24 hours. Bim mRNA levels are expressed as percentage of ABL mRNA and represent the mean ± SD of 8 independent experiments performed with PKC412, and mean ± SD of 7 independent experiments performed with bortezomib. *P < .05. (I-J) Cultured cord blood–derived MCs were incubated in control medium (Co) or various concentrations of PKC412 or bortezomib (as indicated) for 24 hours. Thereafter, Bim mRNA levels were determined by real-time PCR and are expressed as percentage of ABL mRNA expression. In panel J, mean ± SD values from 3 independent experiments are shown. (K) Cultured cord blood–derived MCs were incubated in control medium (left panel), PKC412 (1μM, middle), or bortezomib (1μM, right panel) for 48 hours. Thereafter, cells were examined for apoptosis by annexin V staining and flow cytometry. The percentage of apoptotic cells is also shown (control: 9%, PKC412: 12%, bortezomib: 60%).

Effects of PKC412 and bortezomib on expression of Bim in HMC-1 cells. (A) Immunoprecipitation (IP) and Western blot (WB) were performed with HMC-1 cells exposed to PKC412 (midostaurin, 1μM) or control medium (control) at 37°C for 4 hours. IP and WB were performed as described in “Methods.” For detection of phospho-KIT (p-KIT), anti–phospho-tyr monoclonal antibody 4G10 (1:1000) was applied. As visible, midostaurin suppressed the expression of p-KIT in HMC-1.1 and HMC-1.2 cells (top panels) without affecting total KIT expression (bottom panels). (B) WB analysis of HMC-1.1 cells (left panel) and HMC-1.2 cells (right panel) exposed to control medium (control) or PKC412 (1μM) for 12 hours. WB was performed using a polyclonal anti-Bim antibody. The β-actin loading control is also shown. (C-D) Northern blot analysis of HMC-1.1 cells (C) and HMC-1.2 cells (D) exposed to control medium (control) or PKC412 (1μM) for 12 hours. Northern blotting was performed using a Bim-specific cDNA probe. Equal loading was confirmed by probing for β-actin mRNA. (E-H) Real-time PCR analysis of Bim mRNA expression in HMC-1.1 cells (E,G) and HMC-1.2 cells (F,H) exposed to control medium (0) or various concentrations of PKC412 or bortezomib as indicated for 24 hours. Bim mRNA levels are expressed as percentage of ABL mRNA and represent the mean ± SD of 8 independent experiments performed with PKC412, and mean ± SD of 7 independent experiments performed with bortezomib. *P < .05. (I-J) Cultured cord blood–derived MCs were incubated in control medium (Co) or various concentrations of PKC412 or bortezomib (as indicated) for 24 hours. Thereafter, Bim mRNA levels were determined by real-time PCR and are expressed as percentage of ABL mRNA expression. In panel J, mean ± SD values from 3 independent experiments are shown. (K) Cultured cord blood–derived MCs were incubated in control medium (left panel), PKC412 (1μM, middle), or bortezomib (1μM, right panel) for 48 hours. Thereafter, cells were examined for apoptosis by annexin V staining and flow cytometry. The percentage of apoptotic cells is also shown (control: 9%, PKC412: 12%, bortezomib: 60%).

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