Figure 5.
PKT16-induced PCD in CLL is mediated by heterotrimeric Gαi proteins and modulated by cAMP levels and PKA activity. (A) Cell death was measured in CLL cells preincubated with the vehicle (Control) or the protein G inhibitor suramin (100 µM, 20 minutes) and untreated (−) or treated with PKT16 (100 µM, 6 hours) or venetoclax (4 nM, 24 hours). After Annexin-V/PI costaining, the percentages of Annexin-V positive cells were recorded and graphed as a mean ± SD (n = 5). (B) Cell death was measured in CLL cells preincubated with the vehicle (Control) or the GP-antagonist 2A (protein Gαq inhibitor, 30 µM, 2 hours), gallein (protein G βγ dimer inhibitor, 30 µM, 2 hours), or forskolin (protein Gαi inhibitor, FSK, 100 µM, 2 hours) and treated with PKT16 (100 µM, 6 hours) or venetoclax (4 nM, 24 hours). After Annexin-V/PI colabeling, the percentages of Annexin-V-positive cells were recorded, graphed, and depicted as a ratio of PCD relative to B cells preincubated with the vehicle and treated with PKT16 or venetoclax (considered as the maximum of PCD induced for each drug, set at 1.0). Data are expressed as a mean ± SD (n = 6). (C) cAMP levels were evaluated by HTRF in CLL cells left untreated or treated with PKT16 at the indicated concentration and preincubated with the vehicle or the protein G inhibitor suramin (100 µM, 20 minutes). Data are expressed as a mean ± SD (n = 6). (D) PKA activity was evaluated by fluorescence in CLL cells treated with PKT16 (6 hours) at the indicated doses. Results are expressed as a ratio of PKA activity relative to untreated B cells (considered as a 100% of cellular PKA activity). Data are expressed as a mean ± SD (n = 6). (E) CLL cells were preincubated or not with FSK (100 µM, 2 hours) or suramine (100 µM, 20 minutes) and treated with PKT16 (100 µM, 6 hours) before PLCγ1-Y783 phosphorylation assessment by immunoblot. Equal loading was confirmed by whole PLCγ1 detection. The OD PLCγ1-Y783/PLCγ1 ratio depicted in the graph illustrates the PLCγ1-Y783 inhibition immunodetected in PKT16-treated CLL cells coincubated with FSK or suramin. Data are expressed as a mean ± SD (n = 3). The statistical significance in the figure was calculated by the Student t test.

PKT16-induced PCD in CLL is mediated by heterotrimeric Gαi proteins and modulated by cAMP levels and PKA activity. (A) Cell death was measured in CLL cells preincubated with the vehicle (Control) or the protein G inhibitor suramin (100 µM, 20 minutes) and untreated (−) or treated with PKT16 (100 µM, 6 hours) or venetoclax (4 nM, 24 hours). After Annexin-V/PI costaining, the percentages of Annexin-V positive cells were recorded and graphed as a mean ± SD (n = 5). (B) Cell death was measured in CLL cells preincubated with the vehicle (Control) or the GP-antagonist 2A (protein Gαq inhibitor, 30 µM, 2 hours), gallein (protein G βγ dimer inhibitor, 30 µM, 2 hours), or forskolin (protein Gαi inhibitor, FSK, 100 µM, 2 hours) and treated with PKT16 (100 µM, 6 hours) or venetoclax (4 nM, 24 hours). After Annexin-V/PI colabeling, the percentages of Annexin-V-positive cells were recorded, graphed, and depicted as a ratio of PCD relative to B cells preincubated with the vehicle and treated with PKT16 or venetoclax (considered as the maximum of PCD induced for each drug, set at 1.0). Data are expressed as a mean ± SD (n = 6). (C) cAMP levels were evaluated by HTRF in CLL cells left untreated or treated with PKT16 at the indicated concentration and preincubated with the vehicle or the protein G inhibitor suramin (100 µM, 20 minutes). Data are expressed as a mean ± SD (n = 6). (D) PKA activity was evaluated by fluorescence in CLL cells treated with PKT16 (6 hours) at the indicated doses. Results are expressed as a ratio of PKA activity relative to untreated B cells (considered as a 100% of cellular PKA activity). Data are expressed as a mean ± SD (n = 6). (E) CLL cells were preincubated or not with FSK (100 µM, 2 hours) or suramine (100 µM, 20 minutes) and treated with PKT16 (100 µM, 6 hours) before PLCγ1-Y783 phosphorylation assessment by immunoblot. Equal loading was confirmed by whole PLCγ1 detection. The OD PLCγ1-Y783/PLCγ1 ratio depicted in the graph illustrates the PLCγ1-Y783 inhibition immunodetected in PKT16-treated CLL cells coincubated with FSK or suramin. Data are expressed as a mean ± SD (n = 3). The statistical significance in the figure was calculated by the Student t test.

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