Journal: Immunity

Article Title: Mitochondrial Membrane Potential Regulates Nuclear Gene Expression in Macrophages Exposed to Prostaglandin E2

doi: 10.1016/j.immuni.2018.10.011

Figure Lengend Snippet: (A-B) RNA-seq analysis of gene expression in bone marrow macrophages (BMMs) stimulated for 6 h with: IL-4 (M(IL-4)) or PGE2 (M(−)+PGE2)(A) or M(IL-4) or M(IL-4)+PGE2 (B), compared to no stimulus controls (M(−)) (A,B). (C) Expression of CD301 and RELMα proteins, as measured by flow cytometry, in BMMs stimulated for 24 h with indicated treatments. MFI = mean fluorescence intensity. (D) Western blot of phosphorylated STAT6 (P-STAT6) at indicated times in BMMs treated as indicated. ACTB, actin loading control. (E-F) Extracellular flux analysis (EFA) of oxygen consumption rates (OCR) (E) or extracellular acidification rates (ECAR) (F) at 24 hr post stimulation. For EFA, BMMs were sequentially treated with oligomycin (Oligo), FCCP and rotenone plus antimycin A (R/A) as indicated. (G) 13C-Glucose LC-MS trace into TCA cycle metabolites from cells stimulated for 24 h with indicated treatments. (H-K) ATP concentration (H), cytoplasmic ROS (I), mitochondrial ROS (J), and Δψm as measured by TMRM incorporation (K), in BMMs stimulated as indicated for 24 h. (A-B) Significant (adjusted p value < 0.1) > 2 fold up or down regulated genes based on 3 biological replicates denoted by *. (C) Mean ± SEM from 5 biological replicates (p***<0.0005, p****<0.0001). (G) Mean ± SEM from 3 biological replicates, normalized to M(−) (p**<0.005). (H-K) Mean ± SEM from 5 biological replicates (p*<0.05, p***<0.0005, p****<0.0001). Data are representative of 2 (D) or 3 (C, E, F, H-K) independent experiments. See also Figure S1.

Article Snippet: Used fluorochrome-conjugate monoclonal antibodies included: CD301 (Milteny Biotech, clone: REA687), CD11b (Biolegend, clone: M1/70), F4/80 (Biozol, clone: BM8), TIM4 (BioLegend, clone: F31–5G3), CD45.1 (BioLegend, clone: A20).

Techniques: RNA Sequencing, Gene Expression, Expressing, Flow Cytometry, Fluorescence, Western Blot, Control, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: bioRxiv

Article Title: Rapamycin induced autophagy enhances lipid breakdown and ameliorates lipotoxicity in Atlantic salmon cells

doi: 10.1101/2024.12.11.627915

Figure Lengend Snippet: A . Representative confocal images showing the co-localisation of Plin3 (green puncta) with LD stained with BOPIPY (red) in SHK-1 cells after 72 hr treatment with lipid overload, lipid overload with RAPA, and lipid overload with RAPA and MRT treatments. Baf-A was added to all treatments to block the autophagic flux. Scale bar = 10μm, n=3. B. Quantification of lipid droplet size (40 cells were counted from 3 independent experiments). C. Quantification of co-localisation between Plin3 and lipid droplets as measured by Pearson’s correlation co-efficient. D. Representative immunoblot and E-G. Immunoblot quantification showing ATGL and MGL abundance in SHK-1 cells after 72 hr treatment with no lipid overload, lipid overload, lipid overload with RAPA, and lipid overload with RAPA and MRT treatments. The blots were normalised against total protein in (n=3). Data shown as mean +/− SD, * p < 0.05; *** p < 0.001; **** p < 0.0001, ns= non-significant.

Article Snippet: A standard immunoblotting protocol was used for rabbit polyclonal antibodies for LC3 (1:3000, PM036), SQSTM1/p62 (1:500, Ab 264313), ATGL (1:500, Proteintech, 55190-1-AP), and MGL (1:500, Proteintech, 20494-1-AP).

Techniques: Staining, Blocking Assay, Western Blot

Journal: Scientific Reports

Article Title: Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma

doi: 10.1038/srep35784

Figure Lengend Snippet: Methylation special primers were detected with MSP in all HCC cell lines but in L02 by MSP. Unmethylation special primers were only detected in L02. After treatment by DAC, methylation special primers decreased in HCC cell lines while unmethylation special primers increased ( A ). In HepG2 and SMMC-7721 cells ( B – D ), expression of LATS1 protein was suppressed. Demethylation of LATS1 promoter by DAC and knock-in LATS1 gene by plasmid transfection elevated LATS1 protein levels, which confirmed our findings that methylation of LATS1 promoter induced lost-expression of LATS1 protein in HCC. In HepG2 and SMMC-7721 cell lines, regulation of LATS1 with these methods induced significant changes in downstream signals YAP protein levels in a negatively correlative manner and pYAP protein levels in a positively correlative manner. Finally, due to methylation of LATS1 promoter and lost expression of LATS1, YAP protein could not be phosphorylated and detained in cytoplasm effectively. Excess YAP imported into nucleus and induced overexpression of MAGL.

Article Snippet: After transfer membranes were blocked with 5% nonfat milk, incubated with polyclonal antibodies for MAGL, LATS1, YAP1 or pYAP1 (Abcam, USA) respectively, and a monoclonal antibody for β-actin (Boster, China) was used as a loading control.

Techniques: Methylation, Expressing, Knock-In, Plasmid Preparation, Transfection, Over Expression

Journal: Journal of Biological Chemistry

Article Title: Deletion of Monoglyceride Lipase in Astrocytes Attenuates Lipopolysaccharide-induced Neuroinflammation

doi: 10.1074/jbc.m115.683615

Figure Lengend Snippet: FIGURE 1. MGL expression in MKOGFAP mice. A, Western blot analysis revealed absent MGL expression in lysates of primary cultivated astrocytes from MKOGFAP mice. Minor reductions were observed in lysates of MKOGFAP brains. MKOglobal brains served as control and showed no MGL expression. As controls we used floxed littermates for MKOGFAP mice and wild-type littermates for MKOglobal mice. B, MGL is expressed in primary neurons from cortex (CTX) and hippocampus (HIP) of MKOGFAP mice. Low expression was observed in primary microglia. Brain lysates of wild-type and MKOglobal mice served as positive and negative controls, respectively. C, immunofluorescence images of MGL (red) and GFAP (green) of brain cortical sections obtained from floxed control and MKOGFAP mice. Nuclei (white) were stained with DAPI. The scale bar represents 10 m.

Article Snippet: Antibodies against MGL (poly- clonal, in-house made rabbit anti-MGL serum (7), GFAP (ProSci, Flint Palace, CA), or GAPDH (Cell Signaling, Danvers, MA) and the respective horseradish peroxidase conjugated secondary antibodies were used.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining