Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Dysregulated osteoclastogenesis is related to natural killer T cell dysfunction in rheumatoid arthritis.

doi: 10.1002/art.39244

Figure Lengend Snippet: Figure 5. Decreased phosphorylation of STAT-1 by a-galactosylceramide (aGalCer) in patients with rheumatoid arthritis (RA). A, Western blot analysis of phospho–STAT-1 expression. Monocytes were isolated from healthy control (HC) and RA patient peripheral blood mononuclear cells (PBMCs) by magnetic-activated cell sorting. PBMCs (6 3 105 cells/well) and monocytes (1 3 106 cells/well) were seeded in the upper and lower compartments, respectively, of a Transwell system with 0.4-mm pores. PBMCs in the upper compartment were incubated for 4 hours in the pres- ence of aGalCer (100 ng/ml). After stimulation, monocytes were harvested and Western blotted (top). Levels of phospho–STAT-1 were quanti- fied using an LAS3000 luminescent image analyzer (bottom). B, Flow cytometric analysis of phospho–STAT-1 expression. Freshly isolated PBMCs (1 3 106 cells/well) were incubated in the presence of aGalCer (100 ng/ml) for 24 hours, interferon-g (IFNg; 20 ng/ml) for 15 minutes, or 0.1% DMSO as control and then stained with fluorescein isothiocyanate–conjugated anti-CD14 and phycoerythrin-conjugated anti–phospho– STAT-1 monoclonal antibodies (mAb). Phospho–STAT-1 expression in the monocyte population was determined by intracellular flow cytometry. Black lines, gray lines, and dotted lines represent monocyte phospho–STAT-1 expression in the presence of IFNg, aGalCer, and 0.1% DMSO, respectively. Shaded regions indicate isotype-matched control mAb. Results are representative of 3 independent experiments.

Article Snippet: Monocytes were isolated from PBMCs at purities of .95% using CD14 MicroBeads according to the instructions of the manufacturer (Miltenyi Biotec).

Techniques: Phospho-proteomics, Western Blot, Expressing, Isolation, Control, FACS, Incubation, Staining, Bioprocessing, Flow Cytometry

Journal: bioRxiv

Article Title: Cell surface RNA expression modulates alveolar epithelial function

doi: 10.1101/2024.05.19.594844

Figure Lengend Snippet: (A) Schematic of RNA labeling with Ac 4 ManNAz and FucAz in human primary alveolar epithelial cells, and subsequent extraction and blotting. Cells were pretreated with 100 μM Ac 4 ManNAz or 200 μM FucAz for 24 h followed by 25 μM DBCO-PEG 4 -biotin. After purification, 5 μg RNA was loaded onto 1% denaturing agarose gels, subjected to electrophoresis for 35 min, and imaged on a fluorecscent gel scanner. In control experiments, glycosidases were replaced with RNase-free water while chemical inhibitors were replaced with DMSO. (B) Blotting of RNA from hPAEpC treated with 100 μAM Ac 4 ManNAz for 24 h. Following RNA purification, DBCO-biotin was added to either live hPAEpC (left; for cell surface glycoRNA labeling) or bulk RNA from hPAEpC (right, for in vitro labeling of the total glycoRNA pool). RNA was transferred onto nitrocellulose membranes, and sialoglycoRNA was visualized with streptavidin-IR800 CW (Strep) on a fluorescent scanner. RNA was stained and imaged with SYBR Gold (Sybr). (C) Blotting as in (B) for cell surface glycoRNA in hPAEpC treated with 100 μM Ac 4 ManNAz for indicated times shows de novo formation of sialoglycoRNA. (D) Blotting in hPAEpC treated with 100 μAM Ac 4 ManNAz in the presence of the sialyltransferase inhibitor P3-F Ax -Neu5Ac shows dose-dependent inhibition of glycoRNA sialylation. (E) Blotting as in (B) in the presence of neuraminidase (2 U/μL) or RNAse cocktail (1:50). (F) Blotting as in (B) in the presence of O -glycosidase (4000 U/μL), the N-glycan specific glycosidase, PNGAseF (50 U/μL) or RNAse. (G) Blotting as in (B) in the presence of oligosaccharyltransferase (OST) inhibitor NGI-1 at the given concentrations. (H) Blotting as in (B) in the presence of kifunensine (Kif), a chemical inhibitor of the N-glycan trimming enzyme α-amannosidase I, at the given concentrations. Blots (D-H) were obtained in cells fed with 100 μM Ac 4 ManNAz for 24h. (I) Blotting as in (C) in hPAEpC treated with 200 μAM FucAz for indicated times shows de novo formation of fucoglycoRNA. (J) Blotting as in (I) in the presence of O -glycosidase, PNGAseF , fucosidase (0.2U/μl) or RNAse. (K) Blotting as in (I) in the presence NGI-1 at the given concentrations. (L) Blotting as in (I) in the presence of the kifunensine (Kif) at the given concentrations. Blots (J-L) were obtained in cells fed with 200 μM FucAz for 24h. Created with Biorender.com

Article Snippet: Thirty micrograms of RNA in 9 μL pure water was mixed with 10 μL of RNA denaturing buffer (95% formamide, 18 mM EDTA and 0.025% SDS) and 1 μL of DBCO-PEG 4 -biotin (10 mM; Jena bioscience).

Techniques: Labeling, Extraction, Purification, Electrophoresis, Control, In Vitro, Staining, Inhibition

Journal: bioRxiv

Article Title: Cell surface RNA expression modulates alveolar epithelial function

doi: 10.1101/2024.05.19.594844

Figure Lengend Snippet: (A) Schematic of cell surface glycoRNA labeling with Cy5-hydrazide. hpAEpC were pretreated with 100 μM Ac 4 ManNAz for 24 h followed by 25 μM DBCO-Sulfo-Cy5. After purification, 3 μg RNA was loaded onto 1% denaturing agarose gels, subjected to electrophoresis for 35 min, and imaged on a fluorecscent gel scanner. In control experiments, RNA was treated with RNAse-free water instead of glycosidases while cells were treated with DMSO instead of inhibitors. RNA was stained with SYBR gold (Sybr). (B) Comparison of different DBCO-dye conjugates for in-gel detection of sialoglycoRNA in hPAEpC show clear and distinct staining with DBCO-Sulfo-Cy5 but not with DBCO AF 555 or DBCO ATTO-488. (C-D) DBCO-Sulfo-Cy5 labeled sialoglycoRNA signal is lost in hPAEpC-derived RNA in the presence of neuraminidase (2 U/μL), RNAse (1:50) and PNGaseF (50 U/μL) but not O -glycosidase (4000 U/μL). (E) DBCO-Sulfo-Cy5 labeled sialoglycoRNA signal is reduced in hPAEpC in the presence of the oligosaccharyltransferase inhibitor NGI-1 (20 μM), the α-mannosidase I inhibitor kifunensine (Kif) (15 μM), or the sialyltransferase inhibitor P3-F Ax -Neu5Ac (200 μM). (F) In-gel detection of fucoglycoRNA with DBCO-Sulfo-Cy5 in FucAz-fed hPAEpC shows reduced signal in the presence of fucosidase (0.2U/μl) or PNGaseF (50 U/μL) but not O -glycosidase (4000 U/μL). Created with Biorender.com

Article Snippet: Thirty micrograms of RNA in 9 μL pure water was mixed with 10 μL of RNA denaturing buffer (95% formamide, 18 mM EDTA and 0.025% SDS) and 1 μL of DBCO-PEG 4 -biotin (10 mM; Jena bioscience).

Techniques: Labeling, Purification, Electrophoresis, Control, Staining, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Cell surface RNA expression modulates alveolar epithelial function

doi: 10.1101/2024.05.19.594844

Figure Lengend Snippet: (A) Schematic of cell surface glycoRNA labeling with Cy5-hydrazide. hPAEpC were subjected to mild periodate oxidation with 1 mM NaIO 4 for 15 minutes, and the reaction was quenched by 1mM glycerol followed by the addition of 25 μM Cy5-hydrazide for 15 minutes. After purification, 3 μg RNA was loaded onto 1% denaturing agarose gel, electrophoresed for 35 minutes, and imaged on a fluorescent gel scanner. All subsequent gels were treated in this manner. In control experiments, cells were treated with PBS instead of NaIO 4 or with DMSO instead of inhibitors while RNA was treated with RNAse-free water instead of glycosidases. RNA was stained with SYBR gold (Sybr). (B) Cy5-hydrazide labeled sialoglycoRNA signal is lost in hPAEpC-derived RNA in the presence of neuraminidase (2 U/μL). (C) Cy5-hydrazide labeled sialoglycoRNA signal is lost in hPAEpC-derived RNA in the presence of PNGAseF (50 U/μL) or RNAse (1:50) but not O -glycosidase (4000 U/μL). (D) Cy5-hydrazide labeled sialoglycoRNA signal is reduced in hPAEpC-derived RNA in the presence of the oligosaccharyltransferase inhibitor NGI-1 (20 μM), the α-mannosidase I inhibitor kifunensine (Kif) (15 μM), or the sialyltransferase inhibitor P3-F Ax -Neu5Ac (200 μM). Created with Biorender.com

Article Snippet: Thirty micrograms of RNA in 9 μL pure water was mixed with 10 μL of RNA denaturing buffer (95% formamide, 18 mM EDTA and 0.025% SDS) and 1 μL of DBCO-PEG 4 -biotin (10 mM; Jena bioscience).

Techniques: Labeling, Purification, Agarose Gel Electrophoresis, Control, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Cell surface RNA expression modulates alveolar epithelial function

doi: 10.1101/2024.05.19.594844

Figure Lengend Snippet: (A) Schematic of cell surface glycoRNA labeling with lectins. RNA was isolated from the plasma membrane of hPAEpC, stained with biotinylated lectins (20 μg/ml) and detected via Streptavidin infrared dye. After purification, 3 μg RNA was loaded onto 1% denaturing agarose gel, electrophoresed for 35 minutes, and imaged on a fluorescent gel scanner. All subsequent gels were treated in this manner. In control experiments, RNA was treated with PBS instead of lectins. RNA was stained with SYBR gold (sybr). (B) In-gel imaging of hpAEpC sialoglycoRNA shows positive staining for wheat germ agglutinin (WGA) that is reduced or lost in the presence of neuraminidase (2 U/μL) or RNAse (1:50). (C) In-gel imaging of hpAEpC sialoglycoRNA shows positive staining for Maackia amurensis agglutinin (MAA) that is lost in the presence of neuraminidase (2 U/μL) or RNAse (1:50). (D) In-gel imaging of hpAEpC sialoglycoRNA shows positive staining for Sambucus nigra agglutinin (SNA) that is lost in the presence of neuraminidase (2 U/μL) or RNAse (1:50). (E) In-gel imaging of hpAEpC fucoglycoRNA shows positive staining for Ulex europaeus agglutinin (UEA-1) that is lost in the presence of fucosidase (0.2 U/μL) or RNAse (1:50). Created with Biorender.com

Article Snippet: Thirty micrograms of RNA in 9 μL pure water was mixed with 10 μL of RNA denaturing buffer (95% formamide, 18 mM EDTA and 0.025% SDS) and 1 μL of DBCO-PEG 4 -biotin (10 mM; Jena bioscience).

Techniques: Labeling, Isolation, Membrane, Staining, Purification, Agarose Gel Electrophoresis, Control, Imaging