akt inhibitor ii  (Merck KGaA)

Journal: Experimental Animals

Article Title: Calcium and integrin binding protein 1 (CIB1) induces myocardial fibrosis in myocardial infarction via regulating the PI3K/Akt pathway

doi: 10.1538/expanim.21-0063

Figure Lengend Snippet: The diagram of the investigation of CIB1 function in angiotensin II (Ang-II)-induced cardiac fibroblasts fibrosis in vitro . To analyze the effects of CIB1 knockdown on CFs fibrosis, the CFs were infected with Cib1 RNA-interfering lentivirus (LV-sh Cib1 ) or its negative control lentivirus (LV-shNC) followed by Ang-II treatment at 48 h post-injection. For the signaling pathway analysis, the CFs were subjected to an inhibitor of the PI3K/Akt pathway, LY294002, before Ang-II treatment.

Article Snippet: To confirm the effects of CIB1 on the PI3K/Akt pathway, LY294002 (MedChemExpres, Monmouth Junction, NJ, USA), a specific PI3K/Akt pathway inhibitor, was added to treat the CFs before Ang II treatment.

Techniques: In Vitro, Knockdown, Infection, Negative Control, Injection

Journal: Experimental Animals

Article Title: Calcium and integrin binding protein 1 (CIB1) induces myocardial fibrosis in myocardial infarction via regulating the PI3K/Akt pathway

doi: 10.1538/expanim.21-0063

Figure Lengend Snippet: CIB1 depletion represses the PI3K/Akt signaling pathway activated by Ang II to prevent cardiac fibroblasts (CFs) fibrosis. A–B. Representative images (A) and quantification (B) of phosphorylated PI3K-p85 (p-PI3K-p85) and total PI3K-p85 protein expression in Ang II-treated CFs, analyzed by western blot. C–D. Representative images (C) and quantification (D) of phosphorylated Akt (p-Akt) and total Akt protein expression in Ang II-treated CFs. E–F. Representative images (E) and quantification (F) of α-SMA and Collagen I levels in Ang II-treated CFs when CIB1 was knocked down or LY294002 (an inhibitor of the PI3K/Akt pathway) was added. Data are expressed as the mean ± SD (n=3/group). ** P <0.01 vs. Ang II.

Article Snippet: To confirm the effects of CIB1 on the PI3K/Akt pathway, LY294002 (MedChemExpres, Monmouth Junction, NJ, USA), a specific PI3K/Akt pathway inhibitor, was added to treat the CFs before Ang II treatment.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: CTLA-4 Activation of Phosphatidylinositol 3-Kinase (PI 3-K) and Protein Kinase B (PKB/AKT) Sustains T-Cell Anergy without Cell Death

doi: 10.1371/journal.pone.0003842

Figure Lengend Snippet: Pre-activated PBLs were stimulated with anti-CD3 (left panel) and anti-CD3/CTLA-4 (right panel) in the absence (upper panel) or presence of AKT inhibitor (AKT inhibitor II) (lower panel). 48 hours later, cells were stained with Annexin V-Cy5 and PI and analysed by FACS for cell death. Top panel shows CTLA-4 surface expression in these cells. Similar results were obtained from at least three other experiments.

Article Snippet: LY294002 was bought from CN Biosciences (Nottingham, UK), AKT inhibitor II from Calbiochem (Nottingham, UK) and recombinant human CD80 Ig from R&D Systems (Abingdon, UK).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CTLA-4 Activation of Phosphatidylinositol 3-Kinase (PI 3-K) and Protein Kinase B (PKB/AKT) Sustains T-Cell Anergy without Cell Death

doi: 10.1371/journal.pone.0003842

Figure Lengend Snippet: Panel A: CTLA-4 ligation induces phosphorylation of BAD at Ser-136. Upper panel: DC27.10-CTLA-4 cells were either left unstimulated (lane 1) or stimulated for 30 min with anti-CD3 (lane 2), anti-CTLA-4 (lane 3) and anti-CD3/CTLA-4 (lane 4) mAbs. In lane 5 and 6, cells were pretreated with LY 294002 (100 µM, 30 min) or AKT inhibitor II (15 µM, 30 min), respectively and then stimulated with anti-CD3/CTLA-4 antibodies. Cell lysates were immunoblotted with anti-phospho-BAD (Ser-136) antibody (lanes 1–6). Right upper panel: Histogram depiction of phospho-BAD as detected by densitometric reading. Middle panel: Similar amounts of cell lysates were immunoblotted for total BAD (lanes 1–6). Lower panel: Pre-activated PBLs were re-stimulated with anti-CD3 or anti-CD3/CTLA-4 in the absence or presence of AKT inhibitor II or LY 294002. 24 hours later, cells were washed, stained with anti-phospho-BAD (Ser 136)/anti-rabbit AlexaFluo488 antibodies and analysed by flow cytometry. Panel B: CTLA-4 ligation induces up-regulation of BcL-XL. Left panel: DC27.10-CTLA-4 cells were either left unstimulated (lane 1) or stimulated for 24 hours with anti-CD3 (lane 2), anti-CD3/CD28 (lane 3), and anti-CD3/CTLA-4 (lane 4) antibodies. Cell lysates were immunoblotted with anti-BcL-XL antibody (lanes 1–4). Right panel: Pre-activated peripheral T-cells were treated as described above and assessed for BcL-XL expression by immunoblotting with anti-BcL-XL antibody (lanes 1–4). Middle panels: Similar amounts of cell lysates were immunoblotted for actin (lanes 1–4). Panel C: CTLA-4 ligation induces up-regulation of BcL-2. Pre-activated PBLs were re-stimulated with anti-CD3, anti-CD3/CD28 or anti-CD3/CTLA-4 antibodies. 24 and 48 hours later, cells were washed, stained with anti-BcL-2/anti-rabbit AlexaFluo647 antibodies and analysed by flow cytometry. Similar results were obtained from three other experiments. Panel D: CTLA-4 induced pro-survival signaling pathways. CTLA-4 can increase cell survivial under conditions of anti-CD3/CTLA-4 induced non-responsiveness. CTLA-4-PI 3K activates PKB/AKT by phosphorylation at Thr-308 that in turn inactivates pro-apoptotic BAD by phosphorylation at Ser-136. Inhibitors of PI 3K and PKB/AKT blocked this event. Decreased active BAD induced by CTLA-4 ligation was accompanied by increased levels of BcL-XL/BcL-2 expression. BcL-XL/BcL-2 are then able to mediate their mitochondrial-dependent pro-survival effects.

Article Snippet: LY294002 was bought from CN Biosciences (Nottingham, UK), AKT inhibitor II from Calbiochem (Nottingham, UK) and recombinant human CD80 Ig from R&D Systems (Abingdon, UK).

Techniques: Ligation, Staining, Flow Cytometry, Expressing, Western Blot