Fig 1: Effect of PTEN-knockdown (KD) on Akt phosphorylation and cytokine production. BEAS-2B cells were incubated with random oligonucleotide (RO; 100 nM) or short interference (si) RNA against PTEN (100 nM) for 48 h. The levels of PTEN were determined by RT-qPCR (A) and Western blot analysis (B). The ratio of p-Akt/total-Akt as well as PTEN/β-actin were also determined (n = 3). Effects of PTEN knockdown on cytokine production determined by cytokine array (C–E) or by ELISA for IL-6 (F), CXCL8 (G), CCL5 (H) and CCL2 (I). J: effect of stimulation with IL-1β (0, 0.01, 0.1, and 1 ng/ml) on CXCL8 release for 24 h in PTEN-KD cells. An impact of PTEN KD on gene expression IL-6 (K), CXCL8 (L), matrix metalloproteinase-9 (MMP-9; M), tranforming growth factor-β (TGF-β; N), MUC5AC (O) and MUC5B (P), all which were corrected with the gene expression of a housekeeping gene GNB2L1 (n = 3). All values are mean values ± SE of 3-6 separate experiments. *P < 0.05, **P < 0.01, compared with the values of RO-treated group; ††P < 0.01, compared with the values of nontreatment RO group; ‡P < 0.05, ‡‡P < 0.01, compared with the value of nontreatment PTEN-KD group. §§P < 0.01, between the RO group and PTEN-KD group.
Fig 2: The effect of CSE on PTEN and PI3K signaling after short- and long-term exposure in BEAS-2B cells. A: after 16 h pretreatment with or without 100 μM l-buthionine-sulfoximine (BSO), BEAS-2B cells were cultured in the presence or absence of the 3% CSE for 24 h, and then PTEN protein expression (corrected with β-actin) and Akt phosphorylation (corrected with total Akt) were determined by Western blot analysis (n = 3). B: effect of N-acetyl cysteine (10 mM, 30 min before CSE stimulation) on CSE (3%) induced reduction of PTEN and elevation of Akt phosphorylation (n = 5). C: effect of 3% CSE on PTEN (top) or p-Akt/total-Akt (bottom) up to 60 min in the presence or absence of 100 μM BSO. D: time-dependent effect of 3% CSE on PTEN (top) and p-Akt/total-Akt (bottom) at 6, 12, 18 and 24 h after stimulation in the presence or absence of BSO pretreatment. All values are mean values ± SE of at least 3 experiments. *P < 0.05, **P < 0.01, compared with the values of nontreatment group. †P < 0.05, ††P < 0.01, between the 2 groups.
Fig 3: Relative Expression of miR-21-5p and miR-107 for the Absence of PTEN Protein Expression. A) Relative expression of miR-21-5p for absence of PTEN expression B) Relative expression of miR-107 for absence of PTEN expression
Fig 4: Analysis of mRNA and protein levels of genes, regulated by miR-21, miR-17 and miR-155 in murine lymphosarcoma RLS40 cells after treatment with µ-oligonucleotides. (a) Relative expression of KRAS, TIMP3, PDCD4, and ADAM17 mRNAs 72 h after transfection of cells with µ-oligonucleotides alone and in combination, measured by qPCR. (b) Relative level of PTEN protein measured by Western blot analysis and relative expression of PTEN mRNA measured by qPCR 72 h after mono- and combinative treatment with µ-oligonucleotides. (c) Relative level of STAT3 protein measured by Western blot analysis and relative expression of STAT3 mRNA measured by qPCR 72 h after mono- and combinative treatment with µ-oligonucleotides. The numbers above the Western blot image correspond to the following experimental groups: 1—Control (intact RLS40 cells); 2—RLS40 cells incubated with Lipofectamine2000TM; 3, 4, 5, and 6—cells incubated with singular oligonucleotides µ-Scr-ON, µ-21-ON, µ-17-ON, and µ-155-ON, respectively (150 nM); 7—cells incubates with the µ-21-ON/µ-17-ON pair (75 nM of each ON); 8—cells incubated with the triple combination µ-21-ON/µ-17-ON/µ-155-ON (50 nM of each ON); 9—cells treated with pair µ-21-ON/µ-155-ON (75 nM of each ON). (d) Relative level of MMP9 protein measured by flow cytometry analysis and relative expression of MMP9 mRNA measured by qPCR 72 h after mono- and combinative treatment with µ-oligonucleotides. The level of STAT3 and PTEN protein was normalized to the level of GAPDH protein. Expression of mRNAs was normalized to the expression of housekeeping genes HPRT1 and GAPDH.
Fig 5: PTEN activity in BEAS2B cells and peripheral lung from COPD. A: protein extracts of BEAS-2B cells were immunoprecipitated for PTEN and detected by Western blot analysis. WCE, whole cell extracts. B: BEAS-2B cells were treated with or without [nontreated (NT)] 2 mM hydrogen peroxide (H2O2) for 24 h, and PTEN was immunopurified and IP-PTEN activity measured. C: IP-PTEN was directly treated with 1 mM S-nitrosothiol (SNO), and its activity was measured. †P < 0.05. D: correlation of PTEN activity levels and the %FEV1.
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