Journal: Nutrients

Article Title: Vitamin C: A Novel Regulator of Neutrophil Extracellular Trap Formation

doi: 10.3390/nu5083131

Figure Lengend Snippet: Autophagy signaling is induced in Vitamin C deficient neutrophils. ( A ) Real time QPCR for ATG3, ATG5, ATG6, ATG7, and ATG8 mRNA from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice, ( N = 6 for each group, * p < 0.05). ( B ) Representative Western blot for expression of LC3B-I and LC3B-II from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice. Densitometry of LC3B-II/actin from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice ( N = 6 for each group, * p < 0.05). ( C ) Representative Western blot for expression of p62 and actin from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice. Densitometry of normalized p62 expression from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice ( N = 6 for each group, ns p = 0.3).

Article Snippet: Purified rabbit polyclonal antibodies to LC3B (L7543, Sigma-Aldrich), cleaved caspase-3 (#9661, Cell Signaling), caspase-3 (#9662, Cell Signaling), p62/SQSTM1 (NBP1-48320, Novus Biologicals), NFκB p65 (sc-109, Santa Cruz Biotechnology), Lamin B (sc-6216, Santa Cruz Biotechnology), and actin (sc-1616, Santa Cruz Biotechnology) were used in this study.

Techniques: Western Blot, Expressing

Journal: Respiratory Research

Article Title: Autophagy regulates the effects of ADSC-derived small extracellular vesicles on acute lung injury

doi: 10.1186/s12931-022-02073-y

Figure Lengend Snippet: List of primers for RT-PCR

Article Snippet: Anti-microtubule-associated protein 1-light chain 3 (LC3) B (3868), anti-Beclin-1 (3495), anti-p62 (88588S), anti-GAPDH (2118) and anti-rabbit IgG (7074) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Sequencing

Journal: Frontiers in immunology

Article Title: Activator-Mediated Pyruvate Kinase M2 Activation Contributes to Endotoxin Tolerance by Promoting Mitochondrial Biogenesis.

doi: 10.3389/fimmu.2020.595316

Figure Lengend Snippet: FIGURE 2 | Mitochondrial biogenesis is induced in PKM2-activated macrophages. (A) Mitochondrial mass was assessed by measuring the uptake of NAO by using flow cytometry (n=3). (B) The autophagy level of RAW264.7 cells after stimulation with TEPP-46 was assessed by measuring the protein expression of p62 and LC3 using Western blotting (n=3). (C) The microstructure of RAW264.7 cells stimulated with TEPP-46 was measured using transmission electron microscopy (TEM) (n=3). (D) mtDNA copy number was determined by measuring MTND1 relative to B2M using RT-PCR in RAW264.7 cells transducted with a control siRNA (con siRNA) or siPKM2 (n=4). (E) Western blot analysis of mtTFA in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 for different times (n=3). (F) The mitochondrial OCR was measured in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 by using a Seahorse XF96e Extracellular Flux analyzer. Dashed vertical lines indicate the addition of 1 mM oligomycin (Oligo), 0.5 mM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 mM rotenone plus 1 mM antimycin A (Rot/Ant) (n=3). (G–I) Quantitative analysis of basal respiration, ATP-linked respiration and maximal respiration in control or PKM2 knockdown RAW264.7 cells were stimulated with TEPP-46 for different times (n=3). *p < 0.05.

Article Snippet: We used antibodies against PKM2 (Cell Signaling, #4053, 1:1,000), PKM1 (Cell Signaling, #7067, 1:1,000), PGC-1a (Cell Signaling, #2178, 1:1,000), PGC-1b (Abcam, ab176328, 1:1,000), p62 (Cell Signaling, #16177, 1:1,000), LC3 (Abcam, ab192890, 1:1,000), mtTFA (Abcam, ab252432, 1:1,000), NRF1 (Abcam, ab221792, 1:1,000), NRF2 (Abcam, ab137550, 1:1,000), p-AMPK (Cell Signaling, #4186, 1:500), AMPK (Cell Signaling, #4150, 1:1,000), SIRT1 (Abcam, ab189494, 1:1,000), p-Akt (Abcam, ab38449, 1:500), Akt (Abcam, ab8805, 1:500), p-PI3K (Cell Signaling, #17366, 1:1,000), PI3K (Cell Signaling, #4255, 1:1,000), and GAPDH (Santa Cruz, sc365062, 1:1,000).

Techniques: Cytometry, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown