Journal: Nature protocols

Article Title: Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites

doi: 10.1038/nprot.2017.104

Figure Lengend Snippet: Protein markers for subcellular compartments and the corresponding antibodies.

Article Snippet: Suitable antibodies to use are detailed in . table ft1 table-wrap mode="anchored" t5 caption a7 Protein Apparent molecular weight (kDa) Subcellular compartment Reagent Dilution CS 45 Mitochondrial matrix CST anti-CS antibody (cat. no. 14309) 1/1000 RPS6KB1 50–80 Cytosol CST anti-RPS6KB1 antibody (cat. no. 2708) 1/1000 CALR 60 Endoplasmic reticulum CST anti-CALR antibody (cat. no. 12238) 1/1000 GOLGA1 90 Golgi complex CST anti-GOLGA1 antibody (cat. no. 13192) 1/1000 LAMP2 100–140 Lysosome SCBT anti-LAMP2 antibody (cat. no. sc-18822) 1/1000 CTSC 15–20 (mature form) Lysosome SCBT anti-CTSC antibody (cat. no. sc-74590) 1/500 LMNA 75 Nucleus SCBT anti-LMNA antibody (cat. no. sc-20680) 1/1000 CAT 60 Peroxisome CST anti-CAT antibody (cat. no. 12980) 1/1000 Open in a separate window CRITICAL: We have found that catalase (CAT) is a more faithful marker for peroxisomes than PEX19, which we used previously to assess peroxisomal contamination 21 .

Techniques: Molecular Weight

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 stimulates AT II cell proliferation and reduces AT II cell apoptosis in LPS induced lung injury. C57BL/6 J mice were intra-tracheal given N.S or LPS 10 mg/kg for 24 h, with or without LXA4 1μg intraperitoneal injection per mouse. Immunofluorescence staining of lung specimens was shot by fluorescence microscope and calculated by positive goals compared to DAPI. a - b : co-dying of SP-C and PCNA (× 200, × 400). c - d : co-dying of SP-C and TUNEL (× 200, × 400), scar bar = 50 μm. Data were presented with means ± SD; n = 4; *** P < 0.001, **** P < 0.0001

Article Snippet: HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors.

Techniques: Injection, Immunofluorescence, Staining, Fluorescence, Microscopy, TUNEL Assay

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 alleviates apoptosis in LPS-induced lung injury. C57BL/6 J mice were intra-tracheal given N.S or LPS 10 mg/kg for 24 h, with or without LXA4 1μg per mouse intraperitoneal injection. Immunofluorescence staining of lung specimens was shot by fluorescence microscope and calculated by positive goals compared to DAPI. a - b : Immunofluorescence staining of cleaved-caspase-3, scar bar = 50 μm. c - d : WB of cleaved-caspase-3. Data were presented with means ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001; n = 3

Article Snippet: HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors.

Techniques: Injection, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 stimulates HAT II cell proliferation and reduces HAT II cell apoptosis in vitro. HAT II cells were cultured as mentioned in the methods. a and b : apoptosis of HAT II after the stimulation of LPS and LXA4. c : Proliferation of HAT II after the stimulation of LPS and LXA4. Data were presented with means ±SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 4 for each culture condition, repeated using cells from 4 donors

Article Snippet: HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors.

Techniques: In Vitro, Cell Culture

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 inhibits the epithelial-mesenchymal transition (EMT) in LPS-induced lung injury. C57BL/6 J mice were intra-tracheal given N.S or LPS 10 mg/kg (for 24 h, 48 h or 72 h), with or without LXA4 1μg per mouse intraperitoneal injection. Immunofluorescence staining of lung specimens was shot by fluorescence microscope and calculated by positive goals compared to DAPI. a - h : Immunofluorescence staining of marker of EMT (× 400): E-cadherin( a - b ), N-cadherin( c - d ), α-SMA( e - f ) and Vimentin( g - h ). i and j : co-dying of SP-C and α-SMA. scar bar = 50 μm. All of the data was conducted in triplicate. Data were presented with means ± SD. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors.

Techniques: Injection, Immunofluorescence, Staining, Fluorescence, Microscopy, Marker

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 inhibits TGF-β 1 induced EMT in primary HAT II cells. HAT II cells were incubated with or without TGF-β 1 10 ng/ml for 48 h with or without LXA4 0.1 nM, 1 nM, 10 nM and 100 nM. a - g : the mRNA expression of CDH-1, SP-C, AQP-5, CDH-2, α-SMA and fibronectin. h - k : the protein expression level of E-cadherin, N-cadherin and α-SMA. n = 4 for each culture condition, repeated using cells from 4 donors. Data were presented with means ±SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors.

Techniques: Incubation, Expressing

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 inhibits TGF-β 1 -induced EMT in primary AT II cells through activation of LXA4 receptor (ALX). HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors. Data were presented with means ±SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors.

Techniques: Activation Assay, Incubation

Journal: Respiratory Research

Article Title: Lipoxin A 4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

doi: 10.1186/s12931-019-1158-z

Figure Lengend Snippet: LXA4 reduces TGF-β 1 -induced EMT in HAT II cells partly through the SMAD and PI3K/AKT signaling pathways. HAT II cells were pre-incubated with 10 μM Sis3 (a specific Smad3 inhibitor), and 10 μM LY294002(PI3Kinhibitor) for 30 min prior to TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - h : Sis3 and LY294002 treatment abolished the inhibition of LXA4 on the EMT in AT II cells. i - k : LXA4 inhibited TGF-β 1 -induced the phosphorylation of AKT and Smad in primary AT II cells. n = 4 for each culture condition, repeated using cells from 4 donors. Data were presented with means ±SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: HAT II cells were pre-incubated with or without BOC-2 10μΜ or BML-111 10μΜ 30 min before TGF-β 1 10 ng/ml for 48 h with or without LXA4 100 nM. a - d : the effect of LXA4 on EMT was promoted by the pre-incubation of AT II cells with BML-111 (the LXA4 receptor agonist). e - h : the effect of LXA4 on EMT was abrogated by the pre-incubation of AT II cells with BOC-2(the LXA4 receptor antagonist). n = 4 for each culture condition, repeated using cells from 4 donors.

Techniques: Incubation, Inhibition

Journal: Glia

Article Title: TREM2 triggers microglial density and age‐related neuronal loss

doi: 10.1002/glia.23563

Figure Lengend Snippet: Less production of nitric oxide and reactive oxygen species and reduced signs of age‐related oxidation in TREM2 KO mice. (a) The pathways of the production of nitric oxide (NO) by inducible NO synthase (iNOS) and reactive oxygen species (ROS) by NADPH oxidase are less activated in 24 months old TREM2 knock‐out (KO) animals as revealed by RNA‐seq. Significantly down‐regulated genes are shown in red and bold. Protein names corresponding to differentially expressed (DE) genes are shown. Ingenuity pathway analysis was performed on significantly DE genes (FC > 0.5, FDR < 0.05). (b) Semi‐quantitative real‐time PCR of oxidative stress markers in half brain of 3, 12, and 24 months old TREM2 wild‐type (wt) and KO. At 24 months of age, transcription of Inos , Cyba , and Cybb was lower in TREM2 KO mice. Gapdh was used as internal loading control. (c) Quantification of 4‐hydroxynonenal (4‐Hne) staining intensity per area in the hippocampus ( left) and substantia nigra (SN) pars reticulata ( right ) showed less (hippocampus) or no (SN) age‐related increase of oxidized lipids in 12 and 24 months old TREM2 KO mice. (d) Relative malondialdehyde (MDA) concentrations of 3 and 12 months old TREM2 KO mice were lower in the hippocampus ( left ) and midbrain ( right ). (e,f) Analysis of oxidative stress response in primary microglia obtained from TREM2 wt and KO mice. Quantification of levels of superoxide production after necrotic neural debris feeding (e) or inactivated bacteria treatment (f) as determined by DHE staining intensity showed increased superoxide production in primary wt microglia. The increase was completely (e) or partially (f) antagonized by superoxide dismutase 1 (SOD1) or trolox. In primary TREM2 KO microglia, no (e) or less (f) significant increase in superoxide production was observed after treatment. Data are presented as mean + SEM or mean ± SEM ( n = 6–12 per group (b–d) and n = 4 independent culture preparations (e,f). * p ≤ .05, ** p ≤ .01, *** p ≤ .001, n.s. not significant)

Article Snippet: Cells were pre‐incubated with superoxide dismutase‐1 (SOD‐1; 40 μg/ml; Serva, Germany) or trolox (80 μM; Cayman, Germany) as indicated before the treatment with inactivated bacteria.

Techniques: Knock-Out, RNA Sequencing, Real-time Polymerase Chain Reaction, Control, Staining, Bacteria

Journal: Antioxidants

Article Title: OR2AT4, an Ectopic Olfactory Receptor, Suppresses Oxidative Stress-Induced Senescence in Human Keratinocytes

doi: 10.3390/antiox11112180

Figure Lengend Snippet: OR2AT4 activation by sandalore mediates the CAMKKβ/AMPK/mTORC1/autophagy axis. Expression of total and phosphorylated ( A ) CAMKKβ and ( B ) AMPK by immunoblot analysis. ( C ) Representative images of MDC-stained HaCaT cells. Scale bar, 20 µm (left panel). Quantification of the MDC fluorescence intensity in HaCaT cells (right panel). Fluorescence was quantified using the VICTOR ™ X Multilabel Plate Reader. ( D ) The expression of LC3I and LC3II by immunoblot analysis (left panel). Bar graphs represent the ratio of the LC3II to LC3I within each sample (right panel). ( E ) Expression of the total and phosphorylated mTORC1 and 70S6K by immunoblot analysis. ( F ) Expression of the total and phosphorylated AMPK in the OR2AT4 -knockdown HaCaT cells. The relative protein expression of p-CAMKKβ/CAMMKβ, p-AMPK/AMPK, p-mTORC1/TORC1, and p-p70S6K/p70S6K were analyzed using ImageLab software. Data are means ± SEM. * p < 0.05, ** p <0.01, and *** p < 0.001. Student’s t -test was performed for comparisons between groups.

Article Snippet: The primary antibodies used for immunoblotting were as follows: OR2AT4 (ab105828, 36 kDa; Abcam, Cambridge, MA, USA), GNAL (sc-48345, 46 kDa; Santa Cruz Biotechnology, Santa Cruz, CA, USA), adenylate cyclase 3 (ADCY3) (sc-588, 130 kDa; Santa Cruz Biotechnology), p21/CIP1/CDKN1A (NBP2-294563, 21 kDa; Novus, St. Louis, MO, USA), phosphorylated calcium/calmodulin-dependent protein kinase kinase (p-CaMKKβ) (12818S, 50 kDa; Cell Signaling Technology, Danvers, MA, USA), CaMKKβ (sc-50341, 50 kDa; Santa Cruz Biotechnology), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) (44-1150G, 62 kDa; Invitrogen, Carlsbad, CA, USA), AMPK (sc-25792, 62 kDa; Santa Cruz Biotechnology), LC3 antibody (sc-0376404, 15 kDa; Santa Cruz Biotechnology), phosphorylated mammalian target of rapamycin complex 1 (p-mTORC1) antibody (ab63552, 289 kDa; Abcam), mTORC1 antibody (2983S, 289 kDa; Cell Signaling Technology), phosphorylated p70 ribosomal S6 kinase (p-p70S6K) antibody (2211S, 80 kDa; Cell Signaling Technology), p70S6K antibody (2217S, 80 kDa; Cell Signaling Technology), and β-actin (sc-47778, 43 kDa; Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Western Blot, Staining, Fluorescence, Knockdown, Software