Figure 5. PLCβ1 related nuclear DAG increase at G2/M stimulates nuclear translocation of PKC⍺ and Cyclin B1.

(a) Cells were synchronized at G1/S and G2/M and nuclear or total samples were treated to extract lipids. The percentage of radioactivity corresponding to DAG amount was investigated. Nuclear DAG increased at G2/M. (b) Cells were treated with PMA (PMA) or U73122 (U73122) and synchronized at G2/M. Nuclear accumulation of PKC⍺ and Cyclin B1 resulted linked to DAG fluctuations during cell cycle. (c) Cells were synchronized at G0/G1, G1/S and G2/M and the localization of different PLCs was investigated. PLCβ1 was the only one mainly localized into the nucleus. (d) Immunocytochemistry was performed to further study PLCβ1 localization. (e) PLCβ1 was silenced (PLCβ1 KD) and cells were synchronized at G2/M. A Scrambled siRNA was used as control (Scrambled). PKC⍺ and Cyclin B1 had minor nuclear accumulation in PLCβ1 knock-down conditions. (f) PLCβ1 (PLCβ1 KD), PKC⍺ (PKC⍺ KD) or both the molecules (PLCβ1 KD – PKC⍺ KD) were silenced. After synchronization at G2/M phase, immunoblot analyses showed that double knock-down condition had the same effects on Cyclin B1 expression than PKC⍺ silencing alone. PLCβ1 silencing did not affect Cyclin B1. (g) PLCβ1 was silenced and cells synchronized at G2/M. Nuclear production of IP₃ decreased after silencing of PLCβ1. All the experiments were repeated at least three times.