Journal: Communications Biology

Article Title: Lysine malonylation regulates human sperm motility

doi: 10.1038/s42003-026-09683-y

Figure Lengend Snippet: A The Kmal in total proteins of (TP) and protein fractions of the mitochondrion (Mit), head, and cytoplasm (Cyt) isolated from human sperm were examined by Western blot. The specificity of cellular components was validated by Western blot using the corresponding markers: PRM2 for sperm head, COX6B1 for mitochondrion, and ACTIN for cytoplasm. The proteins for Western blot were shown in the coomassie brilliant blue (CBB) staining image. B , C The localization of malonylated proteins within human sperm was investigated by immunofluorescence assay via laser scanning confocal microscopy (LSCM, ( B )) and super-resolution structured illumination microscopy (SIM, ( C )). All the experiments were conducted with samples from 8 normozoospermic men. The scale bar represents 5 μm.

Article Snippet: Anti-ACTIN antibody (66009-1-Ig), anti-SIRT5 antibody (67257-1-Ig), anti-acetyl-CoA carboxylase 1 (ACC1) antibody (21923-1-AP), anti-fatty acid synthase (FASN) antibody (10624-2-AP), anti-protamine 2 (PRM2) antibody (14500-1-AP), anti-GAPDHS (83290-3-RR) and anti-VDAC3 (82666-14-RR) were obtained from Proteintech Group, Inc. (Rosemont, IL, USA).

Techniques: Isolation, Western Blot, Staining, Immunofluorescence, Confocal Microscopy, Microscopy

Journal: Toxicology and applied pharmacology

Article Title: Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

doi: 10.1016/j.taap.2017.01.012

Figure Lengend Snippet: Fig. 1. Silica and ENM exposure cause LMP in macrophages. (A) BMdM were exposed to increasing doses of silica, MWCNT, TNB, and TNS and LMP measured in the cytosolic fraction at 4 h. (B) LMP over time with particle dose of 50 μg/mL. Data are presented as mean ± SEM (N = 3). *P b 0.05, **P b 0.01, ***P b 0.001 when compared to baseline.

Article Snippet: Anti-ASC, Anti-caspase-1, and anti-pro-IL-1β antibodies were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques:

Journal: Toxicology and applied pharmacology

Article Title: Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

doi: 10.1016/j.taap.2017.01.012

Figure Lengend Snippet: Fig. 2. Particle-induced LMP in macrophages is dependent on phagolysosome acidification in vitro. BMdM were exposed to silica, MWCNT, TNB, or TNS (all 50 μg/mL) with or without Bafilomycin A1 (100 nM) or CA-074-Me (10 μM) and LMP assessed after 4 h via digitonin extraction. (A) Cathepsin L activity in the cytosolic fraction. (B) NAG activity in the cytosolic fraction following silica exposure. (C) NAG activity in the cytosolic fraction following MWCNT, TNB, and TNS exposure. (D) IL-1β and (E) IL-18 levels in BMdM cell supernatants 24 h after particle exposure with LPS stimulation (20 ng/mL) and treatment with Bafilomycin A1 (100 nM) or CA-074-Me (10 μM). Data show mean ± SEM (N = 3). *P b 0.05, **P b 0.01, ***P b 0.001 indicate significance compared to particle exposures without CA-074-Me or Bafilomycin A1 treatment.

Article Snippet: Anti-ASC, Anti-caspase-1, and anti-pro-IL-1β antibodies were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: In Vitro, Extraction, Activity Assay

Journal: Toxicology and applied pharmacology

Article Title: Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

doi: 10.1016/j.taap.2017.01.012

Figure Lengend Snippet: Fig. 3. Silica exposure causes persistent NLRP3 inflammasome activity in vivo. (A) Total cell counts in the lavage fluid 7 d after silica (40 mg/kg) instillation in C57Bl/6 and caspase-1 null mice. (B) IL-1β, (C) IL-18, and (D) HMGB1 levels in the whole lavage fluid of C57Bl/6 and caspase-1 null mice at 7 d following instillation of PBS or silica (40 mg/kg). Data are presented as mean ± SEM (N = 4). (E and F) Intracellular NLRP3, ASC, and pro-caspase-1 protein levels in isolated AM from C57Bl/6 mice 7 d following silica or PBS exposure. Western Blots shown are representative of 3 separate experiments with lung lavage cells from 3 mice pooled for each lysate. (G) mRNA expression in AM isolated from C57Bl/6 mice 7 d following silica exposure relative to PBS control. Data shown as expression ratio with upper and lower quartiles and include 95% confidence intervals. *P b 0.05, **P b 0.01, ***P b 0.001 indicates significance.

Article Snippet: Anti-ASC, Anti-caspase-1, and anti-pro-IL-1β antibodies were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Activity Assay, In Vivo, Isolation, Western Blot, Expressing, Control

Journal: Toxicology and applied pharmacology

Article Title: Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity.

doi: 10.1016/j.taap.2017.01.012

Figure Lengend Snippet: Fig. 6. LMP and resultant cathepsin B activity drives persistent NLRP3 inflammasome activity in AM ex vivo following particle exposure. AM were isolated from C57BL/6 mice 7 d following silica (40 mg/kg), MWCNT, TNB, and/or TNS (2 mg/kg) exposure and cultured ex vivo with/without LPS (20 ng/mL) stimulation and CA-074-Me (10 μM) or Bafilomycin A1 (100 nM) when designated in figure legend. Cytokine levels were measured in cell supernatants after 24 h of ex vivo culture. (A) IL-1β levels in supernatants of AM cultured from silica or PBS exposed mice (7 d). (B) IL-18 levels in supernatants of AM cultured from silica or PBS exposed mice (7 d). (C) HMGB1 levels in cell supernatants after 24 h from AM isolated and cultured from silica or PBS exposed mice (7 d). (D) IL-1β levels in supernatants of AM cultured from silica or PBS exposed mice (56 d). (E) IL-18 levels in supernatants of AM cultured from silica or PBS exposed mice (56 d). (F) IL-1β levels in supernatants of AM from MWCNT, TNB, and TNS or vehicle exposed mice (7 d). (G) HMGB1 levels in supernatants of AM from MWCNT and TNB or vehicle-exposed mice (7 d, N = 3). Data for all other experiments are shown as mean ± SEM (N = 4). *P b 0.05. **P b 0.01. ***P b 0.001 indicate significant reduction with inhibitor treatment.

Article Snippet: Anti-ASC, Anti-caspase-1, and anti-pro-IL-1β antibodies were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Activity Assay, Ex Vivo, Isolation, Cell Culture

Journal: The FASEB Journal

Article Title: Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock–induced oxidation

doi: 10.1096/fj.201600257R

Figure Lengend Snippet: Primers

Article Snippet: The primary antibodies were as follows: TRPM8, 1:1000 (ab109308; Abcam); heat shock protein A1 (HSPA1), 1:500 (sc-1060; Santa Cruz Biotechnology, Dallas, TX, USA); HSPA2, 1:2000 (ab83204; Abcam, Cambridge, United Kingdom); heat shock factor 1-C5 (HSF1-C5), 1:500 (sc-17756; Santa Cruz Biotechnology); HSF2, 1:500 (sc-13056; Santa-Cruz); CIRP, 1:200 (sc-161012; Santa Cruz Biotechnology); monoclonal antibody Ki 67 (MKI67), 1:500 (Ab15580; Abcam); SOD1, 1:500 (sc-11407; Santa Cruz Biotechnology); SOD2, 1:500 (sc-133254; Santa Cruz Biotechnology); GPx4, 1:200 (sc-50497; Santa-Cruz); GPx5, 1:200 (sc-54826; Santa-Cruz Biotechnology); β-actin, 1:1000 (A-2228; Sigma-Aldrich); protamine 2 (Prm2) 1:200 (sc-23102; Santa-Cruz Biotechnology); phospho-mammalian target of rapamycin (p-MTOR) (Ser 2448 ), 1:200 (sc-101738; Santa-Cruz Biotechnology); and mTOR, 1:200 (sc-8319; Santa-Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction, Variant Assay

Journal: The FASEB Journal

Article Title: Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock–induced oxidation

doi: 10.1096/fj.201600257R

Figure Lengend Snippet: The TRPM8-dependence of gene expression in mouse germ cells subjected to cold shock with a 3 d recovery period

Article Snippet: The primary antibodies were as follows: TRPM8, 1:1000 (ab109308; Abcam); heat shock protein A1 (HSPA1), 1:500 (sc-1060; Santa Cruz Biotechnology, Dallas, TX, USA); HSPA2, 1:2000 (ab83204; Abcam, Cambridge, United Kingdom); heat shock factor 1-C5 (HSF1-C5), 1:500 (sc-17756; Santa Cruz Biotechnology); HSF2, 1:500 (sc-13056; Santa-Cruz); CIRP, 1:200 (sc-161012; Santa Cruz Biotechnology); monoclonal antibody Ki 67 (MKI67), 1:500 (Ab15580; Abcam); SOD1, 1:500 (sc-11407; Santa Cruz Biotechnology); SOD2, 1:500 (sc-133254; Santa Cruz Biotechnology); GPx4, 1:200 (sc-50497; Santa-Cruz); GPx5, 1:200 (sc-54826; Santa-Cruz Biotechnology); β-actin, 1:1000 (A-2228; Sigma-Aldrich); protamine 2 (Prm2) 1:200 (sc-23102; Santa-Cruz Biotechnology); phospho-mammalian target of rapamycin (p-MTOR) (Ser 2448 ), 1:200 (sc-101738; Santa-Cruz Biotechnology); and mTOR, 1:200 (sc-8319; Santa-Cruz Biotechnology).

Techniques: Gene Expression