Cellular PKC and cdk-2 become activated by H1 and PKCα but not PKCβ is required for NEBD.

<p><b>A, B, C:</b> Activation of PKC, cdk-2 and caspase-3 in permeabilized HeLa cells by H1. Y-axis: activity in arbitrary units. The tested activity is indicated on top of each panel. The columns show the mean values of three independent experiments. The variation bars show the range between the highest and the lowest value. 1. permeabilized cells. 2. permeabilized cells+H1. 3. permeabilized cells+H1+H89. 4. permeabilized cells+H1+Roscovitine. 5. permeabilized cells+H1+zVAD-fmk. 6. H1 without cells. 7. permeabilized cells+H1+thapsigargin. 8. non-permeabilized cells. n.d. not determined. The panels show that permeabilization decrease the activity of all three enzymes and that H1 activates the activities of PKC and cdk-2, while an effect on caspase-3 is doubtful. Inhibition of PKC also reduced activity of cdk2 but not of caspase_3 despite of its inhibitor specificity shown in the supporting information. Thapsigargin pre-treatment, leading to Ca⁺⁺ depletion inhibits PKC and cdk2 implying that a Ca⁺⁺-dependent PKC is involved. <b>D.</b> PV H1-mediated NEBD is inhibited by PKCα but not PKCβ. Quantification of PI-stained chromatin of permeabilized HeLa cells to which 300 H1 per permeabilized cell were added. The bars depict 95% CI. Red, dotted line: buffer (n = 28); red line: H1 (n = 14); green dashed line: H1 using PKCα-inhibited cells (n = 19); black dashed line: H1 using PKCβ-inhibited cells. Collectively, the data show that Ca⁺⁺-dependent PKCα is required for NEBD, which is consistent with the PV-mediated activation of a Ca⁺⁺-dependent PKC. PKCα subsequently activated cdk-2, which was also shown essential for NEBD. Caspase-3 was not significantly activated by PV but its activity was however essential for NEBD.