Journal: Neurotoxicity research

Article Title: Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

doi: 10.1007/s12640-010-9172-4

Figure Lengend Snippet: MPP(+) stimulates calpain-dependent proteolysis in the absence of significant caspase activation. CGNs were incubated for 24 h in either control medium containing 25 mM KCl and 10% FBS (CONT), apoptotic medium containing 5 mM KCl without FBS (5 K), or in CONT medium containing MPP(+) (150 μM). Some cultures were co-incubated with either the calpain inhibitors, ALLN or ALLM (2.5 μM), or the pan-caspase inhibitor, BOC (20 μM). Following incubation, cells were lysed, proteins resolved by SDS-PAGE, and membranes immunoblotted (IB) with antibodies to either Fodrin (a) or PARP (b). Western blots for focal adhesion kinase (FAK) and actin are shown as corresponding loading controls. fl, full-length; p150 and p145, calpain breakdown products of fodrin; p120, caspase-generated fragment of fodrin; p85, caspase-generated fragment of PARP

Article Snippet: Some cultures were co-incubated with either the calpain inhibitors, ALLN or ALLM (2.5 μM), or the pan-caspase inhibitor, BOC (20 μM).

Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Generated

Journal: Neurotoxicity research

Article Title: Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

doi: 10.1007/s12640-010-9172-4

Figure Lengend Snippet: MPP(+) inactivates pro-caspases-9 and −3 in CGNs via calpain-dependent degradation. a CGNs were incubated for 24 h in either control medium containing 25 mM KCl and 10% FBS (CONT), CONT medium including MPP(+) (150 μM), or CONT medium containing MPP(+) (150 μM) and the calpain inhibitor ALLN (2.5 μM). Following incubation, cells were lysed, proteins separated by SDS-PAGE and membranes immunoblotted (IB) with antibodies to either pro-caspase-9 (upper panel) or pro-caspase-3 (lower panel). Western blots for actin are shown as corresponding loading controls. fl, full-length; clv, calpain-generated cleavage product of pro-caspase-3. b Caspase 3/7 activity was quantified after a 4 h incubation period for CGNs subjected to either CONT medium or apoptotic medium with 5 mM KCl and lacking FBS (5 K), in either the absence of presence of MPP(+) (150 μM). The results are expressed as the fold change in caspase activity relative to the CONT which was set at a value of 1.0. Data represent the means ± SEM (n = 3); * P < 0.01 versus 5 K alone

Article Snippet: Some cultures were co-incubated with either the calpain inhibitors, ALLN or ALLM (2.5 μM), or the pan-caspase inhibitor, BOC (20 μM).

Techniques: Incubation, SDS Page, Western Blot, Generated, Activity Assay

Journal: Neurotoxicity research

Article Title: Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

doi: 10.1007/s12640-010-9172-4

Figure Lengend Snippet: MPP(+)-induced toxicity in CGNs is significantly attenuated by a drug cocktail including glutathione and inhibitors of caspases and calpains. a CGNs were incubated for 24 h in either control medium containing 25 mM KCl and 10%FBS (CONT, upper panel) or CONT medium containing 150 μM MPP(+) (middle panel). Additionally, the lower panel shows CGNs exposed to MPP(+) and co-incubated with a pan-caspase inhibitor, BOC (20 μM), a calpain inhibitor, ALLN (10 μM), and glutathione monoethyl ester (GSH; 2 mM) (MPP + 3 INH; i.e., an inhibitor cocktail). Following incubation, cells were fixed and nuclei stained with Hoechst. Areas demarcated by the boxes were enlarged 300% to show detailed nuclear morphology. Scale bar = 10 μm. b Corresponding brightfield images show gross cell morphology including neuritic processes. Orientation identical to (a). Note healthier neurites in the lower panel (MPP + 3INH) compared to the middle panel (MPP + alone). c CGNs were treated exactly as in a. Nuclei were quantified based on their morphology as either “normal” (as seen in CONT), “condensed” (as seen in MPP+ 3 INH), or “chromatin aggregation” (as seen in MPP+ alone) (ChAgg). Data represent mean ± SEM (n = 3). d CGNs were treated as described in (a) except for 16 h. Following incubation, percent cell viability (relative to CONT) of MPP(+)-treated CGNs and CGNs exposed to MPP+ 3INH was measured by the reduction of a tetrazolium salt, MTT (see Experimental Procedures). Percent viability of CONT cells was set at 100%. Data represent the mean ± SEM (n = 5); * P < 0.01 versus MPP(+) alone. E. CGNs were treated for 24 h with either CONT medium, MPP+ alone, or MPP+ in combination with ALLN (2.5 μM). Cells possessing neurites longer than two times the cell body were quantified as viable. Data represent the mean ± SEM (n = 5); * P < 0.01 versus CONT; # P < 0.05 versus MPP+ alone

Article Snippet: Some cultures were co-incubated with either the calpain inhibitors, ALLN or ALLM (2.5 μM), or the pan-caspase inhibitor, BOC (20 μM).

Techniques: Incubation, Staining

Journal: Neurotoxicity research

Article Title: Calpain Plays a Central Role in 1-Methyl-4-phenylpyridinium (MPP(+))-Induced Neurotoxicity in Cerebellar Granule Neurons

doi: 10.1007/s12640-010-9172-4

Figure Lengend Snippet: The cell cycle protein, Cyclin E, is processed by calpain to an active low molecular weight fragment in MPP(+)-treated CGNs. CGNs were incubated for either 12 or 24 h in control medium containing 25 mM KCl and 10% FBS (c), or control medium containing 150 μM MPP(+) (M). Alternatively, CGNs were co-incubated with MPP(+) and either a pan-caspase inhibitor, BOC (B; 20 μM), a calpain inhibitor, ALLN (A; 10 μM), glutathione (G; 2 mM), or all three inhibitors together (3INH). To compare to a classical apoptotic stimulus, cells were incubated for 24 h in medium containing 5 mM KCl minus FBS (5 K). Following incubation, cells were lysed, proteins resolved by SDS-PAGE, and membranes immunoblotted (IB) with antibodies to Cyclin E (a and b). Western blots for actin are shown as corresponding loading controls. fl, full-length; NS non-specific band, LMWF low-molecular weight fragment produced by calpain

Article Snippet: Some cultures were co-incubated with either the calpain inhibitors, ALLN or ALLM (2.5 μM), or the pan-caspase inhibitor, BOC (20 μM).

Techniques: Molecular Weight, Incubation, SDS Page, Western Blot, Produced

Journal: ACS Central Science

Article Title: Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay

doi: 10.1021/acscentsci.1c00910

Figure Lengend Snippet: Determined Enzymatic and Cellular IC 50 Values in Inhibiting SARS-CoV-2 M Pro for Different Inhibitors

Article Snippet: We purchased HEK293T/17 cells from ATCC; DMEM with high glucose with GlutaMAX supplement, fetal bovine serum, 0.25% trypsin-EDTA, phenol red, puromycin, Lipofectamine 3000, and dimethyl sulfoxide from Thermo Fisher Scientific; linear polyethylenimine MW 25000 from Polysciences; RealTime-Glo annexin V apoptosis and a necrosis assay kit from Promega; an EndoFree plasmid DNA midi kit from Omega Biotek; antimycin a from Sigma-Aldrich; GC376 from Selleck Chem; boceprevir, calpeptin, MG-132, telaprevir, and carmofur from MedChemExpress; ebselen from TCI; calpain inhibitors II and XII from Santa Cruz Biotechnology; MG-115 From Abcam; tideglusib, disulfiram, and PX-12 from Cayman Chemical; chloroquine diphosphate from Alfa Aesar; hydroxychloroquine sulfate from Acros Organics; and a fluorogenic M Pro substrate DABCYL-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-EDANS termed as Sub3 from Bachem.

Techniques: