Effects of HDAC1 overexpression on bortezomib-induced apoptosis in RPMI 8226 cells in vivo. (A) RPMI 8226 cells were transfected with CSII-DsRed (mock) or CSII-DsRed-HDAC1 (HDAC1) vector. DsRed⁺ cells were collected using a FACSAria flow cytometer and seeded 1 cell/well in a 96-well plate; single-cell clones were then obtained. Each subline was analyzed by a flow cytometer, and representative histogram plots of whole cells are shown. (B) Whole-cell lysates were subjected to immunoblotting. The signal intensities of HDAC1 were quantified, normalized to those of the corresponding GAPDH, and shown as relative values. (C) NOD/SCID mice were inoculated subcutaneously with 3 × 10⁷ cells of RPMI 8226 sublines in the right thigh. The following treatments were started at day 0 when tumors were measurable: bortezomib intravenously twice a week, romidepsin intraperitoneally every other day, and vehicle (0.9% NaCl) alone at the same schedule. Caliper measurements of the longest perpendicular tumor diameters were performed on alternate days to estimate the tumor volume (mm³) using the following formula: 4/3π × (width/2)² × (length/2). Mice inoculated with mock clones were treated with vehicle alone (mock; ○; n = 5), 0.5 mg/kg bortezomib (mock + Bort; □; n = 4), or 0.5 mg/kg bortezomib and 0.25 mg/kg romidepsin (HDAC1/Romid + Bort; ▵; n = 4). Mice inoculated with HDAC1 clones were treated with vehicle alone (HDAC1; ●; n = 4), 0.5 mg/kg bortezomib (HDAC1 + Bort; ■; n = 3), or 0.5 mg/kg bortezomib and 0.25 mg/kg romidepsin (HDAC1/Romid + Bort; ▲; n = 4). (D) Representative photographs of inoculated NOD/SCID mice at day 21 are shown (original magnification, ×2). Arrowheads indicate inoculated tumors. (E) The y-axis shows the tumor volume in inoculated mice at day 21. The means ± SD (bars) are shown. P values were calculated by 1-way ANOVA with the Student-Newman-Keuls multiple comparisons test.