Figure 1.
The knockdown or inhibition of HDAC1 downregulates IRF4 and PIM2 expression in MM cells. (A-D) RPMI 8226, MM.1S, U266 cells were transduced with shLuc (control shRNA targeting luciferase), shHDAC1 (#1, #2), or HDAC3 (#1, #2). Total RNA or whole cell lysates were extracted from transduced cells, followed by each assay. (A) RNA-seq was performed using the RNAs extracted from HDAC1-knockdown (shHDAC1 #1) or control RPMI 8226 cells. RNA-seq expression data shows as a volcano plot selected based on >20.5-fold changes (x-axis) with adjusted P < .05 (y-axis). (B) Total RNA extracted from HDAC1-knockdown RPMI 8226 cells was subjected to Q-PCR. GAPDH served as an internal control. Values represent the amount of mRNA relative to shLuc control, defined as 1. Error bars show the standard deviation (SD) of triplicates. ∗∗∗P < .001 from the control; the Tukey-Kramer multiple comparison test. (C-D) The whole cell lysates extracted were subjected to immunoblotting using the indicated antibodies. β-Actin served as a loading control. (E) RPMI 8226 cells were transduced with either HDAC1-FLAG cDNA or Empty as a control by a retrovirus. Cells were further transduced with either shHDAC1 #1 (targeting 3′ UTR of HDAC1) or shLuc. After puromycin selection, cell viability for 48 hours was assessed by the CCK-8 assay. The cell growth rate in RPMI 8226 cells induced Empty or HDAC1-FLAG cDNA with the shLuc set as 100% for control. ∗∗P < .01 significantly different from the transduced cells with Empty cDNA with shHDAC1; the Tukey-Kramer multiple comparison test. The whole cell lysates extracted were subjected to immunoblotting using indicated antibodies. β-Actin served as a loading control. (F-G) RPMI 8226 cells (F) and primary CD138-positive cells (G) were treated with LBH589, MS-275, or BG45 at the indicated concentration and time course, and whole cell lysates were then extracted from treated cells and subjected to immunoblotting using the indicated antibodies. β-Actin served as a loading control. Relative expression levels of each target, which are normalized to its loading control, are shown below for each immunoblotting image.

The knockdown or inhibition of HDAC1 downregulates IRF4 and PIM2 expression in MM cells. (A-D) RPMI 8226, MM.1S, U266 cells were transduced with shLuc (control shRNA targeting luciferase), shHDAC1 (#1, #2), or HDAC3 (#1, #2). Total RNA or whole cell lysates were extracted from transduced cells, followed by each assay. (A) RNA-seq was performed using the RNAs extracted from HDAC1-knockdown (shHDAC1 #1) or control RPMI 8226 cells. RNA-seq expression data shows as a volcano plot selected based on >20.5-fold changes (x-axis) with adjusted P < .05 (y-axis). (B) Total RNA extracted from HDAC1-knockdown RPMI 8226 cells was subjected to Q-PCR. GAPDH served as an internal control. Values represent the amount of mRNA relative to shLuc control, defined as 1. Error bars show the standard deviation (SD) of triplicates. ∗∗∗P < .001 from the control; the Tukey-Kramer multiple comparison test. (C-D) The whole cell lysates extracted were subjected to immunoblotting using the indicated antibodies. β-Actin served as a loading control. (E) RPMI 8226 cells were transduced with either HDAC1-FLAG cDNA or Empty as a control by a retrovirus. Cells were further transduced with either shHDAC1 #1 (targeting 3′ UTR of HDAC1) or shLuc. After puromycin selection, cell viability for 48 hours was assessed by the CCK-8 assay. The cell growth rate in RPMI 8226 cells induced Empty or HDAC1-FLAG cDNA with the shLuc set as 100% for control. ∗∗P < .01 significantly different from the transduced cells with Empty cDNA with shHDAC1; the Tukey-Kramer multiple comparison test. The whole cell lysates extracted were subjected to immunoblotting using indicated antibodies. β-Actin served as a loading control. (F-G) RPMI 8226 cells (F) and primary CD138-positive cells (G) were treated with LBH589, MS-275, or BG45 at the indicated concentration and time course, and whole cell lysates were then extracted from treated cells and subjected to immunoblotting using the indicated antibodies. β-Actin served as a loading control. Relative expression levels of each target, which are normalized to its loading control, are shown below for each immunoblotting image.

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