Journal:

Article Title: The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

doi:

Figure Lengend Snippet: The C-terminal substitution mutant hsp70AAAA inhibits JNK activation without blocking its rate of dephosphorylation. IMR90 lung fibroblast cells (20 to 30 population doublings) were infected with an adenovirus expressing tTA (AdCMV-tTA) and an adenovirus encoding a tetracycline-regulated expression cassette encoding full-length hsp70 (+Hsp70) or hsp70 with the C-terminal four amino acids EEVD replaced with AAAA (+Hsp70-4AA). Control cells received the same total multiplicity of infection of the hsp70-encoding virus but without the Ad CMV-tTA virus (control). The infected cells were heat shocked for 30 min at 45°C and then incubated at 37°C with the protein kinase inhibitor staurosporine to block new JNK phosphorylation. Extracts were prepared from non-heat-shocked cells (Con.), heat-shocked cells (0), and heat-shocked cells that were incubated with staurosporine for 20 or 40 min at 37°C after the heat shock. Shown is an immunoblot of cytosolic extracts probed with an anti-phospho-JNK antibody.

Article Snippet: JNK activation was measured by Western blotting using a phosphorylation state-specific anti-JNK antibody (Promega Corp., Madison, Wis.).

Techniques: Mutagenesis, Activation Assay, Blocking Assay, De-Phosphorylation Assay, Infection, Expressing, Incubation, Western Blot

Journal: International Journal of Endocrinology

Article Title: β -Endorphin Mediates the Development and Instability of Atherosclerotic Plaques

doi: 10.1155/2020/4139093

Figure Lengend Snippet: Effects of β -endorphin on the migration, proliferation, apoptosis, and signal transduction in HASMCs. (a) Migration was determined with the scratch assay in HASMCs incubated in serum-free SmGM-2 and the indicated concentrations of PDGF-BB or β -endorphin. Photos were taken 0 h and 24 h after stimulation. Representative results are shown from eight independent experiments. Graph shows quantification of β -endorphin-induced cell migration ( n = 8). ∗ p < 0.05, † p < 0.0005, ‡ p < 0.0001 vs. 0 pM β -endorphin + 0 ng/mL PDGF-BB. (b) Proliferation of HASMCs was determined with the WST-8 assay following a 48 h incubation in 5% FBS-SmGM-2 with the indicated concentrations of β -endorphin ( n = 4). (c) HASMCs treated with β -endorphin for 48 h were stained to detect apoptotic cells (green) using the TUNEL method. Nuclei were stained using DAPI (blue). The graph indicates the percentage of apoptotic cells in three independent experiments. Scale bar = 100 μ m. (d–k). HASMCs were incubated in 5% FBS-SmGM-2 with β -endorphin (0, 10, and 100 pM) for 24 h. The effects of β -endorphin on intracellular signals were assessed with immunoblot analyses. Densitometric data for each molecule after normalization to α -tubulin (p-ERK1/2, n = 3; p-Akt, n = 4; Bcl-2, n = 4; p-JNK, n = 4; p-p38, n = 3; p-NF- κ B, n = 4; Bax, n = 4; cleaved caspase-3, n = 3). (b–k) ∗ p < 0.05, § p < 0.005, p < 0.01, # p < 0.001, p < 0.0001 vs. 0 pM β -endorphin.

Article Snippet: Aliquots of protein extracts derived from THP-1 monocytes, THP-1-derived macrophages, HASMCs, and HUVECs were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then immunoblotted with antibodies raised against the following proteins: CD36, CD68, ACAT-1, ICAM-1, VCAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ABCA1, phosphorylated nuclear factor- κ B (p-NF- κ B), phosphorylated c-jun N-terminal kinase (p-JNK), α -tubulin, arginase-1 (GeneTex, Irvine, CA, USA), E-selectin, MARCO (Bioss, Woburn, MA, USA), peroxisome proliferator-activated receptor- γ (PPAR- γ ; Signalway Antibody, College Park, MD, USA), phosphorylated Akt, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated p38 (p-p38), Bax (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (Abcam, Cambridge, UK), cleaved caspase-3 (R&D Systems, Minneapolis, MN, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Acris-OriGene Technologies, Herford, Germany), and β -actin (Sigma, St. Louis, MO, USA) [ , – ].

Techniques: Migration, Transduction, Wound Healing Assay, Incubation, Staining, TUNEL Assay, Western Blot