Figure 2
Figure 2. Expression of class I HDACs in MM cells during bortezomib treatment. (A) MM cell lines (KMS12-BM, U266, and RPMI8226) were cultured in the absence or presence of either 4nM bortezomib (Bort), 50nM dexamethasone (Dexa), 1nM vincristine (VCR), or 100nM doxorubicin (Doxo) for 48 hours. Whole-cell lysates were subjected to immunoblotting. (B) MM cell lines were cultured in the absence or presence of bortezomib (Bort) at the indicated doses for 48 hours, or (C) cultured in the presence of 4nM bortezomib for up to 3 days. Whole-cell lysates were prepared at given time points and subjected to immunoblotting. (D) Total cellular RNA was isolated simultaneously in the experiments described in panel C and subjected to semiquantitative RT-PCR analysis for the expression of HDAC1, HDAC2, HDAC3, and GAPDH (internal control). The amplified products were visualized by ethidium bromide staining after 2% agarose gel electrophoresis. The results of suboptimal amplification cycles, 35 cycles, are shown. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values setting day 0 controls to 1.0. (E) Total cellular RNA was isolated simultaneously in the experiments described in panel C and subjected to real-time quantitative RT-PCR. The expression of HDAC1, HDAC2, and HDAC3 was normalized to that of GAPDH and quantified by the 2−ΔΔCt method. The means ± SD (bars) of 3 independent experiments are shown. (F) We cultured primary MM cells in the absence or presence of 2nM bortezomib for 48 hours, and determined the expression of HDAC1, HDAC2, HDAC3, and GAPDH (internal control) transcripts by semiquantitative RT-PCR. PCR amplification was carried out with 1 μL of cDNA solution (corresponding to 500 cells). PCR products were resolved on 2% agarose gels and visualized by staining with ethidium bromide. The results of suboptimal amplification cycles, 40 cycles, are shown. (G) The signal intensities of HDACs were quantified with a densitometer, and their means are shown as a ratio to those of GAPDH in corresponding samples. The values of individual samples isolated in the absence or presence of bortezomib are indicated as follows: patient no. 1, circles; patient no. 2, squares; patient no. 3, triangles; and patient no. 4, diamonds, respectively. Bars indicate the average values of each molecule. P values were calculated by 1-way analysis of variance (ANOVA) with the Student-Newman-Keuls multiple comparisons test. *P < .05.

Expression of class I HDACs in MM cells during bortezomib treatment. (A) MM cell lines (KMS12-BM, U266, and RPMI8226) were cultured in the absence or presence of either 4nM bortezomib (Bort), 50nM dexamethasone (Dexa), 1nM vincristine (VCR), or 100nM doxorubicin (Doxo) for 48 hours. Whole-cell lysates were subjected to immunoblotting. (B) MM cell lines were cultured in the absence or presence of bortezomib (Bort) at the indicated doses for 48 hours, or (C) cultured in the presence of 4nM bortezomib for up to 3 days. Whole-cell lysates were prepared at given time points and subjected to immunoblotting. (D) Total cellular RNA was isolated simultaneously in the experiments described in panel C and subjected to semiquantitative RT-PCR analysis for the expression of HDAC1, HDAC2, HDAC3, and GAPDH (internal control). The amplified products were visualized by ethidium bromide staining after 2% agarose gel electrophoresis. The results of suboptimal amplification cycles, 35 cycles, are shown. The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values setting day 0 controls to 1.0. (E) Total cellular RNA was isolated simultaneously in the experiments described in panel C and subjected to real-time quantitative RT-PCR. The expression of HDAC1, HDAC2, and HDAC3 was normalized to that of GAPDH and quantified by the 2−ΔΔCt method. The means ± SD (bars) of 3 independent experiments are shown. (F) We cultured primary MM cells in the absence or presence of 2nM bortezomib for 48 hours, and determined the expression of HDAC1, HDAC2, HDAC3, and GAPDH (internal control) transcripts by semiquantitative RT-PCR. PCR amplification was carried out with 1 μL of cDNA solution (corresponding to 500 cells). PCR products were resolved on 2% agarose gels and visualized by staining with ethidium bromide. The results of suboptimal amplification cycles, 40 cycles, are shown. (G) The signal intensities of HDACs were quantified with a densitometer, and their means are shown as a ratio to those of GAPDH in corresponding samples. The values of individual samples isolated in the absence or presence of bortezomib are indicated as follows: patient no. 1, circles; patient no. 2, squares; patient no. 3, triangles; and patient no. 4, diamonds, respectively. Bars indicate the average values of each molecule. P values were calculated by 1-way analysis of variance (ANOVA) with the Student-Newman-Keuls multiple comparisons test. *P < .05.

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