Journal: Cells

Article Title: Perturbation of Cellular Redox Homeostasis Dictates Divergent Effects of Polybutyl Cyanoacrylate (PBCA) Nanoparticles on Autophagy

doi: 10.3390/cells10123432

Figure Lengend Snippet: PBCA-mediated potentiation of Torin1-induced autophagy is dependent on the p38–MK2 pathway and JNK1/2. RPE-1 cells with inducible expression of LDHB-mKeima were induced for 2 d, washed, and pre-treated with SB203580 (SB203, 3 µM), JNK-IN-8 (IN-8, 3 µM), U0126 (10 µM), GSH (10 mM), SB202190 (SB202, 3 µM), BIRB 796 (BIRB, 10 µM), or PF-3644022 (PF, 2.5 µM) before addition of Torin1 (50 nM) and PBCA (6.25 µg/mL). The incubation was continued for 4 h before cell lysates were prepared for immunoblotting ( a ), and the relative amounts of free mKeima were quantified ( b ). The data were normalized to the respective Torin1-treated sample for each inhibitor. The graph shows mean values ± SEM quantified from at least three independent experiments. The asterisks denote the statistical significances compared to Torin1 + PBCA alone. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Solid line demarks samples from two different gels, whereas the dashed lines are introduced for visual purposes only (i.e., the membranes were not cut).

Article Snippet: Torin1 was purchased from R&D Systems (Minneapolis, MN, USA), Bafilomycin A1 (BafA1) from Enzo Life Sciences (Farmingdale, NY, USA), JNK-IN-8 from Calbiochem (Millipore, Merck, Darmstadt, Germany), BIRB 796 from Axon Medchem BV (Groningen, The Netherlands), U0126 and PF-3644022 from Tocris Bioscience (Bio-Techne Ltd., Abingdon, UK), l -[ 14 C]valine from Perkin Elmer (Waltham, MA, USA), and MRT68921 HCl and SAR405 from Selleckchem (Houston, TX, USA).

Techniques: Expressing, Incubation, Western Blot