PTEN differentially regulates expressions of ICAM-1 and VCAM-1 through PI3K/Akt/GSK-3β/GATA-6 signaling pathways in TNF-α-activated human endothelial cells  Konstantin Tsoyi, Hwa Jin Jang, Irina Tsoy Nizamutdinova, Kyungok Park, Young Min Kim, Hye Jung Kim, Han Geuk Seo, Jae Heun Lee, Ki Churl Chang  Atherosclerosis  Volume 213, Issue 1, Pages 115-121 (November 2010) DOI: 10.1016/j.atherosclerosis.2010.07.061 Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Effect of PTEN on the expression of p-Akt, VCAM-1 and ICAM-1 in TNF-α stimulated HUVECs. Cells were transfected with pcDNA3-PTEN or pcDNA3 (EV) as described in Section 2 treated with TNF-α (10ng/mL) for 1h (upper) or 6h (lower). (B) pCMV5-HA-DN-Akt (upper) or CA-Akt-HA (lower) and then treated with TNF-α for 6h. (C) Cells were transfected with pcDNA3-PTEN with or without CA-Akt-HA. After incubation cells were stimulated with TNF-α for 6h. After incubation cells were subjected to Western blot. Data are presented as mean±SD of three independent experiments. Significance compared with control, **P<0.01; significance compared with TNF-α+EV, #P<0.05, ##P<0.01 significance compared with PTEN+TNF-α ††P<0.01. Atherosclerosis 2010 213, 115-121DOI: (10.1016/j.atherosclerosis.2010.07.061) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Effect of siPTEN on the expression of p-Akt and adhesion molecules induced by TNF-α in endothelial cells. Cells were transfected with scramble siRNA (ssiRNA) or siPTEN at final concentration 100nM. After transfection cells were stimulated with TNF-α for 1h (upper) and 6h (lower). Then cells were harvested and subjected to Western blot. Data are presented as mean±SD of three independent experiments. Significance compared with control, **P<0.01; significance compared with TNF-α+ssiRNA, ##P<0.01. Atherosclerosis 2010 213, 115-121DOI: (10.1016/j.atherosclerosis.2010.07.061) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Involvement of GATA but not IRF binding site in PTEN mediated VCAM-1 inhibitory effect in TNF-α-stimulated HUVECs. (A) Cells were transfected or co-trasfected with wt-VCAM-1 (wt-ICAM-1), wt-VCAM-1+pcDNA3 (wt-ICAM-1+pcDNA3) or wt-VCAM-1+pcDNA3-PTEN (wt-ICAM-1+pcDNA3-PTEN), then incubated with TNF-α (10ng/mL) for 3h. (B) Cells were transfected or co-trasfected with VCAM-1-luciferase with mutated IRF-1 site (mIRF-1), mIRF-1+pcDNA3 or mIRF-1+pcDNA3-PTEN or with mutated GATA site (mGATA), mGATA+pcDNA3 or mGATA+pcDNA3-PTEN, then incubated with TNF-α for 3h. After incubation, cells were lysed for Luciferase assay as described in Section 2 were transfected with EV, pcDNA3-PTEN, ssiRNA or siPTEN and stimulated with TNF-α after incubation mRNA was quantified as described in Section 2. Data are presented as mean±SD of three independent experiments. Significance compared with control, **P<0.01; significance compared with TNF-α+EV, ##P<0.01. Atherosclerosis 2010 213, 115-121DOI: (10.1016/j.atherosclerosis.2010.07.061) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 PTEN modulates GATA-6 but not GATA-2 or IRF-1 binding to VCAM-1 promoter induced by TNF-α in HUVECs. (A) Cells were transfected with PTEN or siPTEN as described before, then cells were treated with TNF-α (10ng/mL) and after 6h the cells were cross-linked with formaldehyde. Soluble chromatin was subjected to ChIP assay as described in Section 2. The experiment shown is representative of at least three experiments. (B) pCMV5-HA-Akt or pCMV5 was transefected and incubated for 16h then cells were collected and VCAM-1, ICAM-1, β-actin by Western blot (upper) or ChIP assay (lower) as described in Section 2. TNF-α was used as positive control. After incubation cells were stimulated for 6h by TNF-α and the subjected to ChIP assay as described Section 2. The results are representative of at least three independent experiments. Atherosclerosis 2010 213, 115-121DOI: (10.1016/j.atherosclerosis.2010.07.061) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Akt regulates GATA-6 through GSK-3β in TNF-α-treated endothelial cells (A) Cells were treated by TNF-α (10ng/mL) for the indicated time periods (5, 15, 30, 60min). Cells were transfected with DN-Akt-HA or EV as in Fig.1, then stimulated with TNF-α for 1h. After stimulation cells were subjected to immunoprecipitation and immunoblotting as described in Section 2. (B, C and D) Cells were transfected with wild type (WT) or Ser9-muated (Ser9-mut) GSK-3β vectors. Sixteen hour after transfection cells were stimulated with TNF-α for 6h, which were subjected to Western blot (B), ChIP assay (C) and IP (D). Data are presented as mean±SD of three independent experiments. Significance compared with control, **P<0.01; significance compared with TNF-α alone, ##P<0.01. Atherosclerosis 2010 213, 115-121DOI: (10.1016/j.atherosclerosis.2010.07.061) Copyright © 2010 Elsevier Ireland Ltd Terms and Conditions