Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: ( A ) Real-time PCR of retinal S100 profile in lean and obese Ossabaw pigs showed significant upregulation of S100A9 in pigs fed with Western diet. ( B ) Plasma samples showed elevated levels of S100A9 in obese pigs. ( C) Immunostaining showed S100A9 increase to be localized to the retinal pigmented epithelium (RPE), inner and outer plexiform layer (IPL & OPL) of the obese pigs. n = 3 eyes per group. *, P<0.05 & ***, P<0.001 vs. Lean. ( D ) Retinal lysate ran on western blot similar had a large increase in S100A9 protein, seen as a monomeric band at 13kD and a dimeric band at 26kD.

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: Double immunostaining of ( A, D ) Iba-1 for microglia and ( B, E ) S100A9 found the protein (^) to be present in the cytoplasm of microglial cells, which was amoeboid-like ( * ) in the obese Ossabaw pig retina. S100A9 was also found upregulated (↑) in the microglial cells present in the OPL of the obese pigs ( F ).

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques: Double Immunostaining

Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: pMicroglia treated with LPS for 24 hours showed the morphological transformation from ( A-D ) the ramified state (↑) to amoeboid (^) phenotype from 10ng/ml onwards. ( E ) S100A9 transcripts were upregulated in a dose-dependent manner, reflected by a significant increase in ( F ) intracellular and ( G ) secreted S100A9 protein in the culture media. *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: pMicroglia seeded at 5×10 4 cells/cm density were treated with exogenous recombinant S100A9 (0.01nM to 1000nM) in DMEM-HG on day 2 and collected after 24 hours for qPCR and western blot. ( A ) Representative phase contrast images of pMicroglia show morphological change from ramified state (↑) to amoeboid (^) phenotype treated with S100A9 at 100nM. ( B ) Real-time PCR showed IL-1β transcript increase in a dose dependent manner, with ( C ) estimated ED 50 of 89.14nM. ( D ) Western blot also showed cleaved IL-1β protein in the S100A9 (100nM) treated group. ( E ) NLRP3 and TLR4 was also increased at the RNA level, however only ( F ) NLRP3 protein was seen to be increased in western blot. *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques: Recombinant, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: Microglial cells were pre-treated with TLR4 inhibitor – TAK242 (1μM), or NLRP3 inhibitor – MCC950 (1μM) for 30 minutes prior to addition of recombinant S100A9 protein. Cells were collected after 24 hours for western blot, and cell culture supernatant was evaluated for IL-1β secretion by ELISA. ( A ) S100A9 treatment resulted in glycosylation of TLR4 receptor, seen as the 120kD band on Western blot, which was attenuated with TAK242. ( B ) Microglial cells secreted significantly high levels of IL-1β upon S100A9 stimulus but was significantly reduced after partial inhibition of the TLR4 receptor by TAK242. Similarly, ( C ) MCC950 treatment reduced the activation of NLRP3 inflammasome, as seen in reduced NLRP3 and ASC, and D ) decreased IL-1β secretion after 24 hours. The experiment was performed three times with n=3. Data are represented as mean±SEM. ***, P<0.001 compared to control; &&& , P<0.001 compared to S100A9.

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques: Recombinant, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Inhibition, Activation Assay, Control

Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: Paquinimod (Paq), a quinoline-3-carboxamide (Q compound) that inhibits S100A9 (A9) binding to TLR4, was treated 30 minutes prior to the addition of S100A9 in microglial cells. ( A-D ) Representative phase contrast images of ( B ) S100A9 treated cells after 24 hours showed furling of the cell boundaries and flattening of cell body ( ^ ), indicative of activated morphology. ( C,D ) Paquinimod (100μM or 300μM) attenuated the morphological changes without cell death. ( E ) PrestoBlue cell viability assay indicates toxicity of Paquinimod above 500uM after 24 hours of treatment. Transcript expression of ( F ) IL-1β and ( G ) S100A9 was attenuated in a dose-dependent manner with Paquinimod pre-treatment. *, P<0.05; **, P<0.01; ***, P<0.001 against control. & , P<0.05; && , P<0.01; &&& , P<0.001 against S100A9 (100nM).

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques: Binding Assay, Viability Assay, Expressing, Control

Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: Microglial cells pre-treated with Paquinimod (Paq) for 30 minutes prior to S100A9 (A9) stimulus were collected after 24 hours for Western blot and ELISA. ( A ) Paquinimod reduced NLRP3 inflammasome components, including NLRP3, ASC, and Casp1 proteins. ( B ) IL-1β secretion was also significantly reduced with Paq treatment. ***, P<0.001 compared to control; &&& , P<0.001 compared to S100A9.

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

doi: 10.1101/2024.10.30.621160

Figure Lengend Snippet: Schematic depicting the mechanism of action of S100A9 signaling pathway in the Pig Retinal Microglial cells.

Article Snippet: Antibodies used were Iba-1 (1:500; Wako Pure Chemical Industries, Osaka, Japan) and S100A9 (1:50; Novus Biologicals, Littleton, CO, USA) as described in .

Techniques:

Journal: The Febs Journal

Article Title: Aberrant extracellular dopamine clearance in the prefrontal cortex exhibits ADHD ‐like behavior in NCX 3 heterozygous mice

doi: 10.1111/febs.17339

Figure Lengend Snippet: NCX3 knockdown induces aberrant dopamine intake through changes in the physical and functional coupling between DAT and CaMKII. (A) Quantitative analysis of NCX1–3 mRNA in the N27 dopaminergic cells. The analysis revealed that NCX3 mRNA levels were significantly higher in N27 dopaminergic neurons compared with the levels of other NCX isoforms ( n = 5–6 each, t ‐test, ** P < 0.01). (B) Confocal microscopy images showing triple staining of N27 cells for autophosphorylated CaMKIIα (Thr‐286) (green), DAT (red), NCX3 (blue), and merged images. White arrows indicate the colocalization between phospho‐CaMKIIα, DAT, and NCX3. Scale bars: 25 and 10 μm (enlarged) in the images. (C, D) Representative immunoblots of lysates from N27 cells probed with antibodies recognizing NCX1, NCX2, and β‐tubulin. Quantitative analyses from obtained blotting data revealed that NCX1 protein levels were significantly increased in siNCX3 N27 cells ( n = 6 per group, t ‐test, * P < 0.05). (E) Quantification of [Ca 2+ ] i in the control and siNCX3 N27 cells. The silencing of NCX3 caused an increase in basal [Ca 2+ ] i ( n = 9 per group, * P < 0.05, inter‐group comparison). (F) Representative immunoblots of lysates from N27 cells probed with antibodies recognizing DAT, phosphorylated GluA1 (Ser‐831), GluA1, autophosphorylated CaMKIIα (Thr‐286), CaMKII, phosphorylated Synapsin I (Ser‐603), Synapsin I and β‐tubulin. Representative images are shown from two independent experiments ( n = 2). (G) Quantitative analyses of DAT protein levels are presented in (G). (H) Quantitative analysis of phosphorylation, as shown in (H). CaMKIIα (Thr‐286) autophosphorylation and its downstream substrates GluA1 phosphorylation were significantly increased in siNCX3 N27 cells ( n = 6 per group, t ‐test, ** P < 0.01). (I) Representative immunoprecipitation and whole‐cell lysate input for autophosphorylated CaMKIIα (Thr‐286), along with grouped analysis of autophosphorylated CaMKIIα (Thr‐286)–DAT interaction. NCX3 knockdown promoted strong physical interaction between phospho‐CaMKIIα and DAT ( n = 6 per group, t ‐test, * P < 0.05). (J) Confocal microscopy images showing double staining of N27 cells for autophosphorylated CaMKIIα (Thr‐286) (green) and DAT (red) and merged images. NCX3 knockdown promoted strong colocalization between phospho‐CaMKIIα and DAT. Scale bars: 25 and 5 μm (enlarged) in the images. (K) Tagged DA uptake kinetics in control or siNCX3 N27 cells treated either with vehicle or KN‐93. siNCX3 cells showed suppressed dopamine intake compared to control cells, whereas CaMKII inhibition by KN‐93 partially rescued siNCX3‐induced disruption of dopamine intake ( n = 8 per group, F (2,21) = 4.46, ** P < 0.01, and * P < 0.05, inter‐group comparison). Data are expressed as the means ± standard error of the mean (SEM). CAMKII, calcium/calmodulin‐dependent protein kinase II; DA, dopamine; DAT, dopamine transporter; IB, immunoblot; IP, immunoprecipitation; NCX, sodium‐calcium exchanger.

Article Snippet: Differentiated N27 cells were transfected with mock or siNCX3 (sc‐44911; Santa Cruz Biotechnology, Dallas, TX, USA), and the cells were grown for 24 h after transfection before the experiment.

Techniques: Knockdown, Functional Assay, Confocal Microscopy, Staining, Western Blot, Control, Comparison, Immunoprecipitation, Double Staining, Inhibition, Disruption

Journal: International Journal of Molecular Sciences

Article Title: Low Sulfur Amino Acid, High Polyunsaturated Fatty Acid Diet Inhibits Breast Cancer Growth

doi: 10.3390/ijms24010249

Figure Lengend Snippet: Reducing sulfur amino acids (SAAs) and increasing peroxidizable polyunsaturated fatty acids (PUFAs) through diet counteracts breast cancer development in mice. ( a ) Percentage of dead TUBO cells after 24 h SAAs depletion with or without 400 μM linoleic acid (LAc). Ferrostatin-1 (FST-1) was used at 5 μM concentration and added 1 h prior treatment. Data are expressed as mean ± S.D. ( n = 4, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ( b , c ) Schematic representation of normal (ND) and low SAA-high PUFA (LSAA/HPUFA) diet composition ( b ) and experimental design of the in vivo study ( c ). ( d ) Body weight was evaluated weekly until to sacrifice (** p < 0.01, * p < 0.05). ( e ) Analysis of bio-clinical parameters after 3 weeks from TUBO cell injection in mice. Dashed lines indicate the range of normal values. Data are expressed as mean ± S.D. ( n = 5 mice ND group; n = 4 mice LSAA/HPUFA group; * p < 0.05 vs. ND). ( f ) Tumor masses were evaluated weekly until to sacrifice. ( g ) Representative photographs of explanted breast cancer masses. ( h , i ) Representative immunofluorescence images of intratumor vessels ( h ) and immune cell infiltrates ( i ) detected by using Von Willebrand and S100A9 antibody, respectively. Incubation with AlexaFluor488-conjugated phalloidin and Hoechst 33342 were used to visualize actin cytoskeleton and nuclei, respectively. White arrows in ( i ) indicate the presence of myeloid cells within the tumor mass. Scale bar: 100 μm. ( j , k ) RT-qPCR analysis of the mRNA expression of the inflammation-related genes Il1b, Il6, Tnfa and Nos2 ( j ) and epithelial to mesenchymal transition genes Cdh1, Twist1, Snail1, Snail2 and Vim ( k ). All the images reported are representative of n = 6 mice/group. Data are expressed as mean ± S.D. ( n = 9 mice/group; ** p < 0.01, * p < 0.05 vs. ND).

Article Snippet: As primary antibodies, we used anti-S100A9 (89726, Novus biologicals, Littleton, CO, USA), anti-Von Willebrand factor (A0082, Dako-Agilent, Glostrup, Denmark) and anti-p(S139)H2AX (20E3, Cell Signaling Technologies) at 1:200 dilution.

Techniques: Concentration Assay, In Vivo, Injection, Immunofluorescence, Incubation, Quantitative RT-PCR, Expressing