Journal: Frontiers in Molecular Neuroscience

Article Title: The Role of Non-canonical and Canonical Inflammasomes in Inflammaging

doi: 10.3389/fnmol.2022.774014

Figure Lengend Snippet: Anti-ASC inhibits non-canonical inflammasome activation in the cortex of aged mice. Mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. (A) Representative immunoblot of cortical protein lysates of young (3 months), aged (18 months) and anti-ASC-treated aged mice (mAb) blotted for caspase-8 (B) and caspase-11 (C) . Data presented as mean ± SEM. 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody. N = 6 per group. * p < 0.05. β-actin was used as a protein loading control and internal standard.

Article Snippet: Briefly, cortical lysates were resolved in 4–20% Criterion TGX Stain-Free precasted gels (Bio-Rad, Hercules, CA, United States), using antibodies (1:1,000 dilution) to NLRP1 (Novus Biologicals, Littleton, CO, United States), caspase-1 (Novus Biologicals, Littleton, CO, United States), ASC (Santa Cruz, Dallas, TX, United States), IL-1β (Cell Signaling Technology, Danvers, MA, United States), caspase-8 (Novus Biologicals, Littleton, CO, United States), caspase-11 (Novus Biologicals, Littleton, CO, United States), and β-actin (Sigma-Aldrich, St. Louis, MO, United States).

Techniques: Activation Assay, Saline, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: The Role of Non-canonical and Canonical Inflammasomes in Inflammaging

doi: 10.3389/fnmol.2022.774014

Figure Lengend Snippet: The non-canonical NLRP1-ASC-caspase-8 inflammasome forms in the cortex of aged mice. Mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. Cortical protein lysates of aged and young mice were co-immunoprecipitated (IP) with anti-ASC and blotted for ASC, caspase-8, NLRP1 and caspase-1 indicating protein-protein interactions among these proteins. 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody.

Article Snippet: Briefly, cortical lysates were resolved in 4–20% Criterion TGX Stain-Free precasted gels (Bio-Rad, Hercules, CA, United States), using antibodies (1:1,000 dilution) to NLRP1 (Novus Biologicals, Littleton, CO, United States), caspase-1 (Novus Biologicals, Littleton, CO, United States), ASC (Santa Cruz, Dallas, TX, United States), IL-1β (Cell Signaling Technology, Danvers, MA, United States), caspase-8 (Novus Biologicals, Littleton, CO, United States), caspase-11 (Novus Biologicals, Littleton, CO, United States), and β-actin (Sigma-Aldrich, St. Louis, MO, United States).

Techniques: Saline, Immunoprecipitation

Journal: Frontiers in Molecular Neuroscience

Article Title: The Role of Non-canonical and Canonical Inflammasomes in Inflammaging

doi: 10.3389/fnmol.2022.774014

Figure Lengend Snippet: Inflammasome proteins are elevated in cortical neurons of aged mice. Aged mice were treated with anti-ASC (10 mg/kg) and saline control (i.p.) and sacrificed 3 days later. Merged image of frozen cortical sections of young and aged mice were double stained with the neuronal marker NeuN (green) and inflammasome proteins NLRP1, ASC, caspase-1, and caspase-8 (red). 3 m, 3 months; Sal, Saline; mAb, Monoclonal antibody. Scale bar: 10 μm.

Article Snippet: Briefly, cortical lysates were resolved in 4–20% Criterion TGX Stain-Free precasted gels (Bio-Rad, Hercules, CA, United States), using antibodies (1:1,000 dilution) to NLRP1 (Novus Biologicals, Littleton, CO, United States), caspase-1 (Novus Biologicals, Littleton, CO, United States), ASC (Santa Cruz, Dallas, TX, United States), IL-1β (Cell Signaling Technology, Danvers, MA, United States), caspase-8 (Novus Biologicals, Littleton, CO, United States), caspase-11 (Novus Biologicals, Littleton, CO, United States), and β-actin (Sigma-Aldrich, St. Louis, MO, United States).

Techniques: Saline, Staining, Marker

Journal: The Journal of Biological Chemistry

Article Title: The long non-coding RNA HOTAIR enhances pancreatic cancer resistance to TNF-related apoptosis-inducing ligand

doi: 10.1074/jbc.M117.786830

Figure Lengend Snippet: Overexpression of HOTAIR attenuates TRA-8-induced apoptosis in sensitive pancreatic cancer cells. A and B, BxPC3 (A) and MiaPaCa-2 (B) cells were infected with lentiviruses carrying control vector or HOTAIR cDNA (HOTAIR), and stable clones were selected by puromycin. Panels Aa and Ba, HOTAIR expression, as determined by qRT-PCR and normalized by β-actin expression (n = 3, ***, p < 0.001). Panels Ab and Bb, TRA-8-induced apoptosis. BxPC3 and MiaPaCa-2 cells with HOTAIR overexpression and their control vector cells were seeded into 6-well plates at 2 × 105 per well. After culturing for 24 h, cells were exposed to TRA-8 (1 μg/ml) for 24 h, and apoptosis was determined by flow cytometry using Annexin PE and 7 AAD staining kit. TRA-8-induced apoptosis is shown in the hatched bars (n = 3, ***, p < 0.001). Panels Ac and Bc, Western blot analysis of the expression of caspase-8 (Casp8). The expression of β-actin was used as a loading control. Representative blots from three independent experiments are shown.

Article Snippet: Antibodies were purchased as follows: rabbit anti-DR5 antibody (ProSci, no. 2019, lot number 5355–1502), mouse anti-caspase 8 (BIOSOURCE no. AHZ0502, lot number 22363–01S), mouse anti-β-actin (Sigma, no. A5541–2MI, lot number 014M4759), rabbit anti-EZH2 (Cell Signaling Technology, no. D2C9, lot number 7), and mouse anti-trimethyl-histone H3 (lysine 27) (Active Motif, no. 61017, lot number 23115012).

Techniques: Over Expression, Infection, Plasmid Preparation, Clone Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Western Blot