Journal: iScience

Article Title: Immunometabolic adaptation in monocytes underpins functional changes during pregnancy

doi: 10.1016/j.isci.2024.109779

Figure Lengend Snippet:

Article Snippet: CD163 human antibody (clone GHI/61.1) , Miltenyi , Cat#130-123-249; RRID: AB_2819455.

Techniques: Recombinant, Adhesive, Sequencing, Modification, DC Protein Assay, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Gene Expression, Software, Saline

Journal: Nature Communications

Article Title: Extracellular Ca 2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

doi: 10.1038/ncomms2339

Figure Lengend Snippet: ( a ) Immunoblot (IB) analysis demonstrating the expression of GPRC6A (left panel) and CaSR (right) in human monocytes. In both blots, lanes 1, 2 and 3 represent three different healthy donors. ( b ) Influence of Calhex231 and NPS2143 on the IL-1β secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( c ) IB analysis showing the active form of caspase-1 (p20) in supernatants collected from monocytes stimulated with increased ex[Ca 2+ ]+LPS in the absence or presence of Calhex231 after 16 h of culture. One representative blot out of three experiments performed is shown. ( d ) Ex[Ca 2+ ]-induced IL-1β secretion of CD11b+ mononuclear cells isolated from bone marrow and of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice ( n =5). ( e ) Influence of CaSR and GPRC6A inactivation by transfection with siRNA on IL-1β secretion of THP-1 cells in response to ex[Ca 2+ ]+LPS ( n =3). IBs show inhibition of protein expression of GPRC6A and CaSR in cells transfected with the respective siRNA compared with control siRNA. ( f ) IL-1β secretion of monocytes in response to increasing concentration of Al 3+ with or without LPS ( n =3). ( g ) IL-1β secretion of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice in response to 0.9mM Al 3+ plus LPS ( n =3). ( h ) IL-1β secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with 0.9 mM Al 3+ plus LPS, for 16 h ( n =3). ( i ) IL-1β release of monocytes stimulated with different GPCR ligands at the indicated concentration alone (white bars) or with GPCR ligands +LPS (black bars) for 16 h. ( j ) IL-1β release from monocytes stimulated with ex[Ca 2+ ]+LPS in the presence of dimethyl sulfoxide (control) or with the phosphoinositide-specific PLC inhibitor U-73122 (5 μM) in dimethyl sulfoxide ( n =3). ( k ) PLC activity in monocytes stimulated for 16 h with LPS alone or with ex[Ca 2+ ]+LPS ( n =5). ( l ) For [Ca 2+ ] i imaging, monocytes were loaded with the Ca 2+ -sensitive indicator dye Fura-2-AM and subsequently stimulated with ex[Ca 2+ ] (1.7 mM). The increase in [Ca 2+ ] i was inhibited by NPS2143. One representative experiment out of three is shown. ( m ) IL-1β release from monocytes stimulated with ex[Ca 2+ ]+LPS in the presence of increasing concentrations of BAPTA-AM ( n =3). * P <0.05, ** P <0.01 and *** P <0.001, always in comparison with the negative control. Statistical analysis was performed using the two-tailed Student's t -test. All error bars show mean±s.e.m.

Article Snippet: BAY 61-3606, Calhex231, NPS2143, rabbit-polyclonal anti-IL-1β, rabbit-polyclonal anti-CaSR and anti-GPRC6A Abs and peroxidase-conjugated goat-anti-rabbit secondary Ab were obtained from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Isolation, Transfection, Inhibition, Control, Concentration Assay, Activity Assay, Imaging, Comparison, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: Extracellular Ca 2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

doi: 10.1038/ncomms2339

Figure Lengend Snippet: ( a ) IL-1α release from unprimed (white bars) and LPS-primed (black bars) monocytes in response to stimulation with ex[Ca 2+ ] ( n =3). ( b ) Influence of the specific inhibitors Calhex231 and NPS2143 on IL-1α secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( c ) ex[Ca 2+ ]-induced IL-1α secretion of CD11b+ mononuclear cells isolated from peripheral blood and of peritoneal macrophages from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice. ( d ) IL-1α secretion of THP-1 cells (control), NLRP3-deficient THP-1 cells (THP1defNLRP3) and ASC-deficient THP-1 cells (THP1defASC) stimulated with increased ex[Ca 2+ ] concentration and LPS, after 16 h of culture ( n =3). ( e ) Secretion of TNF from LPS-stimulated peritoneal macrophages or from macrophages stimulated with ex[Ca 2+ ]+LPS from GPRC6A +/+ (wt) or GPRC6A −/− (ko) mice ( n =3). ( f ) Influence of the specific inhibitor NPS2143 on IL-6 secretion of monocytes stimulated with increased ex[Ca 2+ ]+LPS ( n =3). ( g ) Prostaglandin E2 (PGE 2 ) release from unprimed (white bars) and LPS-primed (black bars) monocytes in response to stimulation with ex[Ca 2+ ] ( n =3). ( h ) Influence of 7-BIO-induced monocyte necrosis on the Ca 2+ concentration in the supernatant ( n =3). ( i ) IL-1β release from LPS-primed CD14+ monocytes cultured alone (white bars) or co-cultured with necrotic autologous CD4+T cells (black bars) in the absence or presence of the specific inhibitor Calhex231 ( n =3). ( j ) mIL-1β secretion of LPS-primed monocytes from GPRC6A +/+ (wt) and GPRC6A −/− (ko) mice cultured alone (LPS) or co-cultured with necrotic autologous CD4+ T cells (Nc+LPS, n =9). All bars show mean±s.e.m. Statistical analysis was performed using the two-tailed Student’s t -test. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: BAY 61-3606, Calhex231, NPS2143, rabbit-polyclonal anti-IL-1β, rabbit-polyclonal anti-CaSR and anti-GPRC6A Abs and peroxidase-conjugated goat-anti-rabbit secondary Ab were obtained from Santa Cruz Biotechnology.

Techniques: Isolation, Control, Concentration Assay, Cell Culture, Two Tailed Test