American Society of Hematology

Figure 5

$Figure 5. Effects of HDAC1 overexpression on bortezomib-induced apoptosis in MM cells in vitro. (A) MM cell lines were lentivirally transfected with CSII-DsRed (mock) or CSII-DsRed-HDAC1 (HDAC1) vector. Whole-cell lysates were prepared from DsRed+ cells collected using a FACSAria flow cytometer and subjected to immunoblotting. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values with mock-transfected controls setting to 1.0. (B) MM cell lines transfected with mock or HDAC1 vector were cultured in the absence or presence of 2nM bortezomib. After 48 hours, MM cells were harvested, stained with annexin-V/APC, and subjected to flow cytometric analysis. The y-axis shows the proportion of annexin-V positivity in the DsRed+ fraction. The means ± SD (bars) of 3 independent experiments are shown. P values were calculated by 1-way ANOVA with the Student-Newman-Keuls multiple comparisons test. *P < .05 against the mock. (C) RPMI 8226 cells transfected with mock or HDAC1 vector were cultured in the absence or presence of 2nM bortezomib. Total numbers of DsRed+ cells were calculated by flow cytometer at the indicated time points. P values were calculated by 1-way ANOVA with the Student-Newman-Keuls multiple comparisons test. *P < .05 against the mock + Bort. (D) After 48 hours, whole-cell lysates were prepared from DsRed+ cells collected using a FACSAria flow cytometer and subjected to immunoblotting. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values with untreated mock-transfected controls setting to 1.0.$

Effects of HDAC1 overexpression on bortezomib-induced apoptosis in MM cells in vitro. (A) MM cell lines were lentivirally transfected with CSII-DsRed (mock) or CSII-DsRed-HDAC1 (HDAC1) vector. Whole-cell lysates were prepared from DsRed⁺ cells collected using a FACSAria flow cytometer and subjected to immunoblotting. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values with mock-transfected controls setting to 1.0. (B) MM cell lines transfected with mock or HDAC1 vector were cultured in the absence or presence of 2nM bortezomib. After 48 hours, MM cells were harvested, stained with annexin-V/APC, and subjected to flow cytometric analysis. The y-axis shows the proportion of annexin-V positivity in the DsRed⁺ fraction. The means ± SD (bars) of 3 independent experiments are shown. P values were calculated by 1-way ANOVA with the Student-Newman-Keuls multiple comparisons test. *P < .05 against the mock. (C) RPMI 8226 cells transfected with mock or HDAC1 vector were cultured in the absence or presence of 2nM bortezomib. Total numbers of DsRed⁺ cells were calculated by flow cytometer at the indicated time points. P values were calculated by 1-way ANOVA with the Student-Newman-Keuls multiple comparisons test. *P < .05 against the mock + Bort. (D) After 48 hours, whole-cell lysates were prepared from DsRed⁺ cells collected using a FACSAria flow cytometer and subjected to immunoblotting. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values with untreated mock-transfected controls setting to 1.0.

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