FIGURE 3.

G₁ entry is accelerated in Casp6 KO B cells. A, Splenic B cells were purified by anti-Thy1.2 and anti-CD11b magnetic bead negative selection. BrdU was added for 6 h. After harvesting the B cells with incorporated BrdU, samples were stained with anti-BrdU, 7AAD, and PY. From the BrdU^-7AAD^low gate (G₀-G₁ cells), the population of PY^high group was determined to be cells in G₁. The BrdU⁺ population was designated as S phase cells and the BrdU^-7AAD^high population as G₂-M phase cells. B, Percentages or cell numbers are shown for G₁ WT B cells (■) and Casp6 KO B cells (□) of CD19⁺ gated splenic cells without negative selection. C, Small resting B cells were prepared by Percoll gradient centrifugation after negative selection. Large activated cells were collected from the 50/60% interface of the gradient and small resting cells were from the 66/70% interface of the gradient. Small resting B cells were stimulated with LPS and BrdU was added for the last 6 h of culture. Harvested cells were stained with BrdU, PY, and 7AAD. Only live and single cells were gated for analysis. Closed bars (WT B cells) and open bars (Casp6 KO B cells) show percentages of G₁ cell cycle stage after LPS stimulation for the indicated times. The data are representative of more than three independent experiments.