American Society of Hematology

Figure 5

$Figure 5. PKA substrates mediate the PGE2 proangiogenic signal. (A) Knockdown efficiency of representative targets. HMVECs were transiently transfected with scrambled siRNA (CN) or siRNA targeting indicated genes (KD) and allowed to recover for 72 hours. Cell lysates were collected and analyzed by immunoblotting with the use of the corresponding Abs. (B) Quantitation of HMVEC tube formation. HMVECs transfected with siRNA corresponding to the indicated gene or scramble siRNA for 72 hours were seeded into growth factor–reduced Matrigel in the presence of vehicle DMSO or PGE2 (100nM) for 6 hours, followed by counting the branching points, as described. The branching points of the tubular network of cells transfected with scramble siRNA and treated with DMSO were set to 100. All other branching point counts are presented as the fraction of this control value. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, compared with vehicle in the control group; ##P < .01, compared with corresponding treatment in the control group. (C) Representative images depicting tube formation of HMVECs transfected with siRNA targeting GSK3β (siGSK3β) or scrambled siRNA (siCon) after treatment, or not, with PGE2 (100nM) for 6 hours. (D) PGE2 promotes the dose-dependent GSK3β phosphorylation. HMVECs were treated with PGE2 (in nM) for 5 minutes (left) or LiCl (in mM) for 5 minutes (right). Cell lysates were analyzed by Western blotting with the use of phospho-Ser9 or total GSK3β Abs. (E) LiCl promotes the HMVEC tube formation. HMVECs were subjected to tube formation assay in the presence of LiCl or NaCl (both in mM) for 6 hours, followed by measurement of branching points of the tubular network, as described. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, **P < .01, compared with NaCl group. (F) Knockdown of PKA Cγ attenuates PGE2-induced GSK3β phosphorylation. HMVECs transfected with siCon or siPKA that targeted individual catalytic subunits were stimulated, or not, with PGE2 (100nM) for 5 minutes, and cell lysates were subjected to Western blot analysis with the use of anti–phospho-Ser9 (top) or total (bottom) GSK3β Abs.$

PKA substrates mediate the PGE2 proangiogenic signal. (A) Knockdown efficiency of representative targets. HMVECs were transiently transfected with scrambled siRNA (CN) or siRNA targeting indicated genes (KD) and allowed to recover for 72 hours. Cell lysates were collected and analyzed by immunoblotting with the use of the corresponding Abs. (B) Quantitation of HMVEC tube formation. HMVECs transfected with siRNA corresponding to the indicated gene or scramble siRNA for 72 hours were seeded into growth factor–reduced Matrigel in the presence of vehicle DMSO or PGE2 (100nM) for 6 hours, followed by counting the branching points, as described. The branching points of the tubular network of cells transfected with scramble siRNA and treated with DMSO were set to 100. All other branching point counts are presented as the fraction of this control value. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, compared with vehicle in the control group; ##P < .01, compared with corresponding treatment in the control group. (C) Representative images depicting tube formation of HMVECs transfected with siRNA targeting GSK3β (siGSK3β) or scrambled siRNA (siCon) after treatment, or not, with PGE2 (100nM) for 6 hours. (D) PGE2 promotes the dose-dependent GSK3β phosphorylation. HMVECs were treated with PGE2 (in nM) for 5 minutes (left) or LiCl (in mM) for 5 minutes (right). Cell lysates were analyzed by Western blotting with the use of phospho-Ser9 or total GSK3β Abs. (E) LiCl promotes the HMVEC tube formation. HMVECs were subjected to tube formation assay in the presence of LiCl or NaCl (both in mM) for 6 hours, followed by measurement of branching points of the tubular network, as described. Each point represents the mean ± SEM of values obtained from 3 experiments performed in triplicate. *P < .05, **P < .01, compared with NaCl group. (F) Knockdown of PKA Cγ attenuates PGE2-induced GSK3β phosphorylation. HMVECs transfected with siCon or siPKA that targeted individual catalytic subunits were stimulated, or not, with PGE2 (100nM) for 5 minutes, and cell lysates were subjected to Western blot analysis with the use of anti–phospho-Ser9 (top) or total (bottom) GSK3β Abs.

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