Journal: Autophagy

Article Title: Internalized gap junctions are degraded by autophagy

doi: 10.4161/auto.19390

Figure Lengend Snippet: Figure 4. Pharmacological inhibition of autophagic degradation results in the accumulation of AGJ vesicles in the cytoplasm of drug-treated cells. (A) Schematic representation of autophagosome-maturation, and of representative steps blocked by drugs used in this study: (1) inhibition of cargo sequestration by 3MA and wortmannin; (2) inhibition of autophagosome/lysosome fusion by Bafilomycin A 1 . (B) Fluorescence images of drug-treated and control cells (mock) acquired approximately 21–23 h post transfection and 5 h into drug treatments are shown. Representative AGJ vesicles are marked with arrows. GJs are marked with arrowheads. Bars = 20 μm. (C) Quantitative analyses of total number of cytoplasmic AGJ vesicles (red columns) and GJ length (blue columns), and compared in percent to mock-treated cells are shown. In all drug treatments, a significant increase in the number of cytoplasmic AGJ vesicles was observed (99, 121 and 236%, marked with asterisks). GJ plaque length was not changed significantly in any of the experiments.

Article Snippet: To inhibit the phagosome-lysosome fusion step of autophagic degradation, cells were treated for 5 h in normal growth medium containing 200 nM Bafilomycin A 1 (Enzo Life Sciences/Biomol International, BML-CM110, 10 mM stock in DMSO) as described.

Techniques: Inhibition, Fluorescence, Transfection

Journal: Neural Plasticity

Article Title: Diacylglycerol Signaling Underlies Astrocytic ATP Release

doi: 10.1155/2011/537659

Figure Lengend Snippet: OAG-induced ATP release from astrocytes appears to be vesicular. (a) Incubation of cultured astrocytes with 100 nM Bafilomycin A1, an inhibitor of the vesicular H + ATPases that set up the proton gradients necessary for vesicle filling, greatly reduced OAG-induced astrocytic ATP release (bafilomycin n = 8, control n = 5; t 1 P = 0.01, t 2 P = 0.02, t 3 n = 0.004). This effect was partially washed out after 15 minutes ( t 2 bafilomycin versus t 3 wash P = 0.024). (b) Bafilomycin treatment did not affect the function of astrocytic ATP receptors, as the calcium response to 1 μ M ATP was unaffected by bafilomycin treatment (bafilomycin n = 5, control n = 5, P = 0.9). (c) The gap junction and hemichannel blocker carbenoxolone (CBX), at 50 μ M, resulted in an increase in astrocytic ATP release (control n = 14, CBX n = 5; t 2 P = 0.03, t 3 P = 0.02).

Article Snippet: Other experimental reagents included 1-Stearoyl-2-arachidonoyl-sn-glycerol (SAG, 60 mM stock in DMSO, Biomol), Suramin (stock fresh in H 2 O, Sigma, St. Louis, Min, USA), pyridoxalphosphate-6-azophenyl-2 ' ,4 ' -disulphonic acid (PPADS, stock fresh in H 2 O, Tocris, Ellisville, Min), Bafilomycin A1 (100 μ M stock in DMSO, Biomol), Carbenoxolone (80 mM stock in H 2 O, Sigma), GF 109203X (Bisindolylmaleimide I, 20 mM in DMSO, BioSource, Camarillo, Calif, USA), Calphostin C (10 mM stock in DMSO, Biomol), ATP (made fresh in NHS, Sigma), Apyrase (50 U/mL stock in H 2 O, Sigma), RHC-80267 (60 mM stock in DMSO, Biomol), R59949 (25 mM stock in DMSO, Calbiochem, San Diego, CA, USA).

Techniques: Incubation, Cell Culture

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combination therapy with protein kinase inhibitor H89 and Tetrandrine elicits enhanced synergistic antitumor efficacy

doi: 10.1186/s13046-018-0779-2

Figure Lengend Snippet: H89 in combination with tetrandrine synergistically induced concomitant apoptosis and autophagy. a FACS analysis of apoptosis following treatment with H89 (6 μM) and tetrandrine (4 μM) alone or combination on Hep3B, AGS, MDA-MB-231, and LOVO for 48 h. b Western blot analysis of PARP, caspase 9, caspase 3 and cytochrome c in the cells after treatment with H89 and/or tetrandrine for 48 h. c The cells were pretreated with z-VAD-fmk (50 μM) for 1 h, followed by treatment with H89 and/or tetrandrine for 72 h; cell viabilities were subsequently evaluated. Z-VAD, z-VAD-fmk. d Western blot analysis of the level of the autophagy-related protein LC3 in the cells after treatment with H89 and/or tetrandrine for 24 h. e Cancer cells were transiently transfected with GFP-LC3 plasmid and subsequently treated as in panel ( d ); the percentage of cells with GFP-LC3 puncta was used to quantify the percentage of autophagic cells, and 150–160 cells per condition were counted. Representative images are shown to indicate the cellular localization patterns of the GFP-LC3 fusion protein (× 40 magnification). f Fluorescence microscopy analysis of AGS cells expressing tandem mRFP-GFP-LC3 reporter treated with H89/tetrandrine, rapamycin (500 nM) and chloroquine (10 μM) for 36 h; the ratio of mRFP vs GFP puncta was used to quantify the autophagy flux. Scale bars, 5 μm. g Cell viability was determined in cells pretreated with 3-MA (2 mM) or bafilomycin A1 (100 nM) for 1 h following H89/tetrandrine combination treatment for 48 h. Baf, Bafilomycin A1. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments; * P < 0.05. All images are representative of three independent experiments

Article Snippet: The reagents used in this study are listed as follows: H89 2HCl (10 mM, dissolved in DMSO, S1582, Selleck, Houston, TX), forskolin (FSK) (10 mM, dissolved in DMSO, S2449, Selleck, Houston, TX), PKI (14–22) amide myristoylated (PKI) (0.5 mg, dissolved in DMSO, Sc471154, Santa Cruz, CA), Tetrandrine (10 mM, dissolved in DMSO, CAS:518–34-3, Shanghai Ronghe Medical, Shanghai, China), Caspase inhibitor z-VAD-fmk (10 mM, dissolved in DMSO, S7023, Selleck, Houston, TX), Bafilomycin A1 (dissolved in DMSO, S1413, Selleck, Houston, TX), Chloroquine (CQ) (C6628, Sigma-Aldrich, USA), PD98059 (Beverly, MA, USA), N-acetyl-L-cysteine (NAC) (dissolved in ddH 2 O, A0150000, Sigma-Aldrich, USA), 3-Methyladenine (3-MA) (dissolved in ddH 2 O, M9281-100MG, Sigma-Aldrich, USA), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen Carlsbad, CA), and an FITC Annexin V Apoptosis Detection Kit (556,547, BD Pharmingen).

Techniques: Western Blot, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: Lack of effect of physiological oxidative stress on N-terminal cysteine dependent proteolysis

doi: 10.64898/2026.04.20.719627

Figure Lengend Snippet: ( A ) SH-SY5Y cells were treated with the indicated concentrations of tBHP, Menadione (an oxidative stress-inducing compound), ICL670A (a cytotoxic Fe 2+ -chelator) or ionomycin (to cause Ca 2+ -dependent cell death), and viability assessed 4 hours later by MTT staining. ( B ) Cells were treated with a concentration of each compound chosen to elicit equivalent cell death to 25μM tBHP (as indicated by the dashed red line, 1.5μM ionomycin, 200μM ILC670A, 12.5μM menadione), then the accumulation of RGS5 assessed by immunoblotting. ( C ) Cells were treated with tBHP in the presence or absence of either 100 or 250μM DFO (an iron chelator) for 4 hours, and viability and ( D ) RGS5 and HIF-1α protein levels assessed as before. ( E ) Cells were treated with tBHP in the presence or absence of either DFO or the vacuolar ATPase inhibitor bafilomycin A1 (200nM) for 4 hours, and RGS5 protein accumulation assessed by immunoblotting. Levels of LC3-II were also assessed as a positive control for lysosomal inhibition. All immunoblots are representative of at least 3 independent replicates, and all data are presented as the mean ± S.D. from at least 3 independent experiments. ***P<0.001, ****P<0.0001, one/two-way ANOVA with Holm-Sidak post-hoc test.

Article Snippet: Cells were treated with tert-Butyl hydroperoxide (Sigma-Aldrich 416665, 5.5M stock in decane), 2,2-dipyridyl (D216305, 100mM stock in DMSO), MG132 (TargetMol T2154, 25mM stock in DMSO), menadione (Sigma-Aldrich M5625, 10mM stock in DMSO), ICL670A (Cayman Chemicals 16753, 200mM stock in DMSO), ionomycin (Alfa Aesar J62448.M, 1mM stock in DMSO), desferoxamine (DFO, Sigma-Aldrich D9533, 100mM stock in ddH 2 O), bafilomycin A1 (TargetMol T6740, 200μM stock in DMSO) or cycloheximide (Merck Life Sciences C4859-1ML, 10mM stock in DMSO) through dilution in the culture medium.

Techniques: Staining, Concentration Assay, Western Blot, Positive Control, Inhibition, Cell Viability Assay