Journal: bioRxiv

Article Title: Acid stress modulates metabolo-inflammatory pathways in oral epithelial cells

doi: 10.64898/2026.03.16.711383

Figure Lengend Snippet: Schematic representation of the experimental design to investigate the impact of acid stress on oral epithelial cells (OECs) and Toll-like receptor (TLR) agonist stimulation. pH Conditioning : OECs (80% confluence) were cultured in acidified (pH:=:3.0) or complete growth media (pH:=:8.0) for 24h. Morphometric Analysis ( a ): Brightfield micrographs and a machine-learning based image analysis pipeline were used to assess changes in cellular morphology. TLR Agonist Challenge : Cell cultures were subjected to either 100 ng/mL flagellin (TLR5 agonist) or 1 mg/mL Pam3CSK4 (TLR2/1 agonist) for 2-, 6-, or 24h. Molecular profiling ( b ): was conducted on OEC RNA collected after 6h TLR agonist challenge using NanoString® nCounter® technology followed by pathway analysis using the Gene Ontology knowledgebase. A TGF- β ELISA ( c ) was performed on OEC supernatants collected 2-, 6-, and 24h post TLR agonist stimulation. Figure made with Biorender.com

Article Snippet: Low-passage mixed-donor, Primary Human Oral Epithelial Cells (Cat# 36063-01, Celprogen, Inc., Torrance, USA), were thawed from liquid nitrogen, plated on poly-L-lysine-coated T-75 cell culture flasks (Sigma-Aldrich Co., St. Louis, USA; Corning Inc., Durham, USA) and maintained in Human OEC Culture Complete Growth Media (Celprogen, Inc.) containing serum and antibiotics at 37°C and 5% CO 2 for two passages prior to experimentation according to manufacturer recommendations.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCF FBXL2 E3 Ligase *

doi: 10.1074/jbc.M115.645549

Figure Lengend Snippet: FBXL2 triggers NALP3 degradation. A, MLE cells were transfected with increasing amounts of FBXL2 plasmid for 24–48 h. Cells were collected and assayed for V5 (FBXL2), NALP3, and β-actin by immunoblotting. B, MLE cells were transfected with several FBXL2 lentiviral shRNAs (4 μg). Human primary macrophages were also transfected with several siRNAs to FBXL2 (500 nm) for gene silencing. Shown are protein levels of FBXL2 after transfection of shRNAs or siRNAs and corresponding levels of NALP3 protein. Compared with a control RNA, despite a modest reduction in FBXL2 levels, increased NALP3 levels in the shRNA or siRNA groups were observed. C, U937 cells were lysed, and FBXL2 proteins were coimmunoprecipitated (IP) followed by NALP3, FBXL2, or NALP6 immunoblotting (IB, top panel). In addition, Beas2B cells were transfected with NALP3-V5 prior to IP for V5 and then, NALP3 and various F box proteins were immunoblotted (bottom blot). IgG was used as a control in all IP experiments, and inputs are the IBs of 10% of the cell lysates before performing IP. Note that an upper IgG chain band was detected in the FBXL7 immunoblot. D, in vitro ubiquitination assays showing that NALP3 is a substrate for FBXL2-mediated ubiquitination. E, A549 cells were incubated with or without LPS (200 ng/ml) for 16 h, and cells were lysed. FBXL2 protein was coimmunoprecipitated; first IP for FBXL2 followed by NALP3, FBXL2, or β-actin immunoblotting. F, the relative association between FBXL2 and NALP3 after LPS was quantified using densitometric analysis of the bands in E. The binding capacity between NALP3 and FBXL2 is reduced by ∼45–50% after LPS exposure. Data are representative of at least two to three independent experiments.

Article Snippet: Human primary alveolar macrophages from the fluid were cultured in culture medium (Celprogen) as described previously ( 9 ).

Techniques: Transfection, Plasmid Preparation, Western Blot, shRNA, In Vitro, Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCF FBXL2 E3 Ligase *

doi: 10.1074/jbc.M115.645549

Figure Lengend Snippet: An FBXO3 inhibitor reduces NALP3 abundance, thereby decreasing cytokine release. A, U937 cells were treated with BC-1215 at different concentrations for 16 h. Cells were collected for FBXL2 and β-actin immunoblotting. B, U937 and THP1 cells (3 × 106 cells each) were incubated with LPS (200 ng/ml) or BC-1215 (8 μg/ml) for 16 h. Cells were collected and lysed for NALP3, NALP6 (negative control), and GAPDH/β-actin immunoblotting. C and D, U937 cells (3 × 106 cells) were primed with LPS (200 ng/ml) in combination with different concentrations of BC-1215 for 16 h as indicated (C) or primed with LPS (200 ng/ml for 16 h) and then exposed to BC-1215 (4 μg/ml) in fresh culture medium for the indicated periods of time (D). Cells were collected to measure NALP3 and β-actin protein (top panels) and mRNA levels (bottom panels). Box plots of the -fold increase of steady-state NALP3 mRNA are shown. Data represent four independent experiments. The p values were determined by a Kruskal-Wallis test. E, U937 cells were incubated in LPS (200 ng/ml) with or without BC-1215 (4 μg/ml for 16 h). Cells were then exposed to CHX (40 μg/ml) at different time points for a half-life study. Immunoblotting for NALP3 and GAPDH (loading control) was performed. Densitometric plots of adjusted NALP3 protein decay over time under different conditions were best fitted. The half-life of LPS-primed NALP3 protein was reduced with BC-1215 treatment comparable with native conditions. Data are mean ± S.E. of two independent experiments. F, MLE cells were transfected either with WT or point mutant NALP3 plasmids for 48 h. LPS (40 μg/ml) was then added to the medium with or without BC-1215 (20 μg/ml) for 18 h. Cells were collected and lysed for NALP3 (V5-tagged) and β-actin (loading control) immunoblotting. Levels of K689R, the point mutant for a putative ubiquitin acceptor site within the NALP3 protein, did not decrease with BC-1215 compared with WT or mutant NALP3. Data are representative of two independent experiments. G, THP1 and K562 cells (3 × 106 cells each) were incubated with LPS (200 ng/ml) with or without BC-1215 (8 μg/ml) for 20 h and then pulsed with ATP (5 mm for 20 min). Culture medium was collected for immunoblotting of pro-IL-1β, IL-1β, pro-IL-18, and IL-18. H, THP1 cells (3 × 106 cells each) were incubated with LPS (200 ng/ml) for 20 h and different time periods of BC-1215 (8 μg/ml), as indicated, in the same volume of culture medium. Cells were then exposed to ATP (5 mm for 20 min). Culture medium was collected for immunoblotting of pro-IL-1β and IL-1β. The ratio of IL-1β versus pro-IL-1β by densitometry is shown (bottom panel). Data represent mean ± S.D. of two independent experiments. The p value was determined by nonparametric test for trend. I, primary human alveolar macrophages were obtained from a healthy volunteer. Human alveolar macrophages (3 × 105 cells) were incubated with BC-1215 (4 μg/ml) for 2 h and then exposed to LPS (100 ng/ml) for 16 h. Cells were lysed and collected for immunoblotting for NALP3 and β actin. J, primary human alveolar macrophages (3 × 105 cells) from the same subject as in I were incubated with BC-1215 (4 μg/ml) for 2 h and then exposed to LPS (100 ng/ml) for 16 h. ATP (5 mm) was added for 15 min before collecting cell culture supernatants for ELISA. Data represent the mean ± S.D. of duplicate measurements.

Article Snippet: Human primary alveolar macrophages from the fluid were cultured in culture medium (Celprogen) as described previously ( 9 ).

Techniques: Western Blot, Incubation, Negative Control, Transfection, Mutagenesis, Cell Culture, Enzyme-linked Immunosorbent Assay