Ribosome profiles during differentiation of SBDS-knockdown cells. (A) Undifferentiated control and SBDS-knockdown K562 cells were lyzed using TMK100 buffer with cycloheximide. Equal amounts of RNA were layered on 5% to 45% sucrose gradients. Absorbance of each fraction at A254 was done by the Brandel Density Gradient Fractionator. The 40S, 60S, 80S, and polysomes peaks are seen on the graphs. A representative diagram of 3 independent experiments is shown. (B) SBDS-knockdown and control K562 cells were induced to undergo erythroid differentiation with hemin. After 72 hours, cells were lyzed and analyzed for ribosome profile as described in panel A. A representative diagram of 2 independent experiments is shown. Arrows are pointed to the markedly reduced polysomes in SBDS-knockdown cells. (C) The ribosomal profiles of undifferentiated control and SBDS-knockdown K562 cells were analyzed by sucrose gradient density centrifugation under conditions that allowed dissociation of the 40S and 60S subunits. Cells were harvested using TMK100 lysis buffer with cycloheximide and EDTA. Equal amounts of RNA were layered on 5% to 35% sucrose gradients. Absorbance of each fraction at A254 was done by the Brandel Density Gradient Fractionator. The ratios between 60S and 40S subunits were calculated by measuring area under the curve, using Adobe Illustrator, and are depicted under the curves. A representative diagram of 2 independent experiments is shown. (D) Ribosome profiles of differentiated K562 cells after 48 hours of hemin induction under EDTA dissociating conditions as described in panel C. A representative diagram of 2 independent experiments is shown.