caspase 9  (R&D Systems)

Journal: Nature Communications

Article Title: NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins

doi: 10.1038/s41467-024-46322-8

Figure Lengend Snippet: Analysis of mRNA levels ( A ) quantified by RT-qPCR of IL-1β ( n = 11) (left) ( p = 0.0049; 0.0420; 0.0049; 0.0059) and IL-8 ( n = 10) (right) ( p = 0.0371; 0.0098) normalized to β-actin mRNA expression and ( D ) ELISA quantification of concentration (pg/mL) of secreted active IL-1β protein in culture supernatants of Mo isolated from healthy donors stimulated with DMSO or S1 peptide (orange), NP peptide (purple), Poly I:C (PIC, pink) and Flagellin (FLAG, khaki) ( n = 8) ( p = 0.0078; 0.0391; 0.0078; 0.0078). Western blot analysis of phosphorylated and total NFκB p65 subunit ( B ) and uncleaved versus cleaved caspase 1 ( C ) in Mo cultured in the presence of DMSO and 3 h (p65) or 16 h (caspase 1) after stimulation with SARS-CoV-2 S1, NP peptides or TLR ligands. GAPDH was included as a loading control and used for normalization purposes. Quantification of phosphorylated p65 (B; n = 5) and cleaved caspase 1 (C; n = 7) ( p = 0.0313) normalized to GAPDH are shown on the right of each panel. Statistical analyses in A , C , D were performed using a two-tailed Wilcoxon test: * p < 0.05, ** p < 0.01. Data are represented as box and whiskers with bars representing maximum and minimum values and with median highlighted as a line.

Article Snippet: Tissue slices were then stained with either a rabbit anti-human NLRC4 (abcam; 1:400 dilution), rabbit anti-human NLRP3 (Cell Signaling; 1:50 dilution) goat anti-human Caspase-1 (Bio-Techne RyD Systems; 1:100 dilution), mouse anti-human CD14 (abcam; 1:200 dilution), rabbit anti-human IFNγ (abcam; 1:100 dilution), goat anti-human IL-17a (Bio-Techne RyD Systems; 1:50 dilution) and mouse anti-human CD3 (Dako; 1:50 dilution) as primary antibodies; and donkey anti-rabbit AlexaFluor™ 488 (Invitrogen; 1:200 dilution), donkey anti-goat AlexaFluor™ 488 (Invitrogen; 1:200 dilution), and donkey anti-mouse AlexaFluor™ 647 (Invitrogen; 1:200 dilution) were used as secondary antibodies (See Supplementary Table ).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Isolation, Western Blot, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: NFκB and NLRP3/NLRC4 inflammasomes regulate differentiation, activation and functional properties of monocytes in response to distinct SARS-CoV-2 proteins

doi: 10.1038/s41467-024-46322-8

Figure Lengend Snippet: A , B Fold change in proportions (A) or raw proportions (B) of transitional (T-Mo) ( p = 0.0078; 0.0156) and non-classical (NC-Mo) Mo ( p = 0.0078; 0.0547) subsets included in cultures of pre-isolated CD14+ cells from the blood of healthy donors PB ( n = 8) ( A ) and in bulk BAL cultures from COVID-19 patients ( n = 9) ( B ) ( p = 0.0195; 0.0547) after stimulation with S1 peptide (S1) in the absence or the presence of Parthenolide (P), Z-VAD-FMK (V) and MCC950 (M). Data were normalized to baseline levels present in control DMSO conditions. For BAL samples only Parthenolide (P) and MCC950 (M) inhibitors were used. C Representative 40X magnification confocal microscopy images showing analysis of expression of Caspase-1 (red), NLRP3 or NLRC4 (green, upper and lower panels, respectively), CD14 (white) and merged images including DAPI (orange) from lung tissue from n = 3 critical COVID-19 patients. D Zoom of representative individual isolated Mo showing different low (upper images) versus high (lower images) co-distribution patterns between caspase-1 (red) and either NLRP3 or NLRC4 (green, left and right panels, respectively). Quantification of co-distribution areas per field of NLRP3 (left) ( p = 0.0234; 0.0078) or NLRC4 (right) ( p = 0.0156; 0.0078; 0.0078) with caspase-1 normalized to number of Mo in each field in the presence of DMSO (gray), S1 (orange) or NP (purple) proteins ( n = 3 patients) are shown below. E Impact of siRNA specific for NLRP3 and NLRC4 inflammasome sensors in the fold change of mean fluorescence intensity (MFI) of CD86 expression of Mo from healthy donors Mo exposed to SARS-CoV-2 S1 (left plots) ( p = 0.0078) and NP (right plots) ( p = 0.0547; 0.0547; 0.0156) proteins in classical (C-Mo, left) and transitional (T-Mo, right) in these assays ( n = 8). Data are represented as box and whiskers with bars representing maximum and minimum values and with median highlighted as a line. Statistical significance was calculated using a two-tailed Wilcoxon test: * p < 0.05, ** p < 0.01.

Article Snippet: Tissue slices were then stained with either a rabbit anti-human NLRC4 (abcam; 1:400 dilution), rabbit anti-human NLRP3 (Cell Signaling; 1:50 dilution) goat anti-human Caspase-1 (Bio-Techne RyD Systems; 1:100 dilution), mouse anti-human CD14 (abcam; 1:200 dilution), rabbit anti-human IFNγ (abcam; 1:100 dilution), goat anti-human IL-17a (Bio-Techne RyD Systems; 1:50 dilution) and mouse anti-human CD3 (Dako; 1:50 dilution) as primary antibodies; and donkey anti-rabbit AlexaFluor™ 488 (Invitrogen; 1:200 dilution), donkey anti-goat AlexaFluor™ 488 (Invitrogen; 1:200 dilution), and donkey anti-mouse AlexaFluor™ 647 (Invitrogen; 1:200 dilution) were used as secondary antibodies (See Supplementary Table ).

Techniques: Isolation, Confocal Microscopy, Expressing, Fluorescence, Two Tailed Test

Journal: Medical Science Monitor Basic Research

Article Title: Low Dose of β-Carotene Regulates Inflammation, Reduces Caspase Signaling, and Correlates with Autophagy Activation in Cardiomyoblast Cell Lines

doi: 10.12659/MSMBR.928648

Figure Lengend Snippet: Primary antibodies.

Article Snippet: 9 , Caspase 9 , Goat , AF8301 , RnD System , 1: 250.

Techniques: