Fig.3 PKC  levels decrease in K562 overexpressing PLC  1. PKC  II and PKC  are not affected K562 cell proliferation is modulated by PLCβ1 through a.

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Fig.3 PKC  levels decrease in K562 overexpressing PLC  1. PKC  II and PKC  are not affected K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway Alessandro Poli 1, Manuela Piazzi 1, Alessia Colantoni 1, Giulia Ramazzotti 1, Alessandro Matteucci 2 and Irene Faenza 1 1 DIBINEM, Cellular Signalling Laboratory, University of Bologna, Bologna, Italy; 2 CNR - Consiglio Nazionale delle Ricerche; Institute of Molecular Genetics and IOR; Bologna, Italy Introduction Phospholipase C β1 (PLCβ1) is known to play an important role in cell proliferation. Previous studies reported an involvement of PLCβ1 in G0-G1/S transition targeting Cyclin D3 in Friend murine erythroleukemia cells (FELC). However, little has been found about its role in human models. Here, we used human erythroleukemia cells (K562) as human homologous of FELC to investigate the presence of this link in human cells and, to better understand how this mechanism could work, we decided to study the possible involvement of protein kinases C (PKC) known to be direct targets of PLC signaling and important regulators of cell proliferation. Fig.1 Transient overexpression of both PLC  1 isoforms has a positive and specific effect on cyclin D3 expression. Other cyclins and CDKs are not affected at all Fig.2 Stable overexpression of PLC  1a and PLC  1b affects cyclin D3 only in proliferating conditions leading to a prolonged S phase of the cell cycle and to a decrease in cell proliferation Fig.4 K562 cells were grown in RPMI10 % FBS and transiently transfected with DNAs encoding empty vector as control (empty vector), vectors able to overexpress wild type PLCβ1a or PLCβ1b (PLCβ1a OV and PLCβ1b OV) and catalytic inactivated PLCβ1a or PLCβ1b (His331/His378 OV). Western blotting analyses were performed with specific antibodies against PLCβ1 andPKC  Fig.2 Cells were grown in RPMI 10% FBS and stably transfected with PLC  1 or empty vector; clones were selected via G418 A) Real-time analyses of the specific and stable overexpression of PLC  1a or PLC  1b; B) Clones were seeded at a cell density of 5 × 105/ml, starved in RPMI 1640 without FBS for 24 h and then divided in two aliquots: one collected and lysed (starvation 24 h), one collected and seeded again in complete RPMI % FBS for another 24h (starvation 24h + complete RPMI % FBS 24 h). C) Clones were treated in the same way of (B) and collected in order to analyze cell cycle progression via FACS. D) Clones were seeded at a cell density of 1 × 105/ml in 6-wells plates and then counted in triplicate after 24, 48 and 72 h. Fig.1 Cells were grown in RPMI 10% FBS and transiently transfected with PLC  1a (PLC  1a, OV), PLC  1b (PLC  1b OV) or empty vector. Western blotting analyses were performed with specific antibodies against PLCβ1, cyclin D3, cyclin A, Cyclin E, cdk4 and cdk6. Fig.4 PLC  1 catalytic activity is necessary for PKC  modulation Fig.3 Cells were grown in RPMI 10% FBS and transiently transfected with PLC  1a, (PLC  1a OV), PLC  1b (PLC  1b OV) or empty vector. Western blotting analyses were performed with specific antibodies against PKC , PKC  II and PKC  Fig.5 PKC  transient silencing positively affects cyclin D3 expression and decreases cell proliferation mimicking PLC  1 overexpression Fig. 5 Cells were grown in RPMI 10% FBS and transiently transfected to silence PKC  (PKC  KD) via a specific siRNA and with a control siRNA (Scrambled); A) Western blotting analyses were performed with specific antibodies against PLCβ1, cyclin D3, cyclin A, cyclin E, cdk4 and cdk6; B) Cells were seeded at a cell density of 1 × 105/ml in 6-wells plates, transfected via siRNA and counted in triplicate after 24, 48 and 72 h. Conclusions Thanks to our data, we demonstrated a role of PLC  1 in cell proliferation also in human cell lines such as K562. The emerging feature from our study is a common pathway in which PLCβ1 can positively target cyclin D3 through PKCα and then modulate cell proliferation prolonging the S phase of the cell cycle References Suh PG, Park JI, Manzoli L, Cocco L, Peak JC, Katan M, et al. Multiple roles of phosphoinositide-specific phospholipase C isozymes. BMB Rep 2008; 41: Cocco L, Follo MY, Faenza I, Fiume R, Ramazzotti G, Weber G, et al. Physiology and pathology of nuclear phospholipase C β1. Adv Enzyme Regul 2011; 51:2-12