Journal: bioRxiv
Article Title: CRY-NLRP3 complexes define a circadian checkpoint controlling inflammasome activation
doi: 10.64898/2026.02.26.708242
Figure Lengend Snippet: (A-G) hMDMs were differentiated with M-CSF (7 d) and pretreated for 48 h with DMSO or CRY stabilizers KL001 (5 µM) (A,C,E,F) or KL044 (5 µM) (B,D,E,G), then primed with LPS (0.5 µg/mL, 4 h) and activated with nigericin (20 µM). (A,B) Time course of cell death measured by DRAQ7 incorporation (Opera Phenix). Left: kinetic curves of DRAQ7 signal over time. Middle: quantification of cell death as area under the curve (AUC) (GraphPad Prism), shown as mean ± SD. Upper: final point from paired individual values from six independent experiments, each represented by three pooled technical replicates (18 total points). Statistical comparisons were performed using a paired Mann-Whitney test (*p < 0.03, **p < 0.005). (C, D) Caspase-1 activity normalized to CV staining. Left: pooled quantification shown as mean ± SD from six independent experiments, each performed with two technical replicates (12 total values). Lower: paired individual values from the six independent experiments. Statistical comparisons were performed using a paired Mann-Whitney test (**p < 0.005, ****p < 0.0001). (E) Representative confocal images of ASC specks (green) in hMDMs preteated with DMSO, KL001 (5 µM) or KL044 (5 µM), primed with LPS (0.5 µg/mL, 4 h) and left untreated or activated with nigericin (20 µM, 1.5 h). Nuclei were stained with Hoechst; images were acquired on an Opera Phenix HCS microscope using a 20× air objective; scale bar, 50 µm; arrows indicate ASC specks. (F, G) Quantification of ASC-speck-positive shown as mean ± SD from three independent experiments, each performed with three experimental replicates (9 wells per condition) with ≥200 cells analyzed per condition. Statistical comparisons were performed using a paired Mann-Whitney test (**p = 0.0068, ****p < 0.0001). (H) hMDMs were pre-treated with DMSO or KL001 (5 µM), primed with LPS (0.5 µg/mL, 4 h) and treated with nigericin (20 µM) for 3 h. Levels of GSDMD, caspase-1, and IL-1β were analyzed by immunoblotting in cell lysates and corresponding culture supernatants (n = 2). (I) IL-1β secretion measured by ELISA in supernatants from hMDMs pretreated with KL001 (5 µM), primed with LPS (0.5 µg/mL, 4 h) and treated with nigericin (20 µM, 3 h). Top: pooled quantification from six measurements obtained across four independent experiments, shown as mean ± SD. Bottom: paired individual values from the four independent experiments. Statistical comparisons were performed using an unpaired Mann-Whitney test. (J) Representative PLA images (red) showing CRY1- NLRP3 or CRY2-NLRP3 interactions in hMDMs pretreated with DMSO, KL001 (5 µM), or KL044 (5 µM), primed with LPS (0.5 µg/mL, 4 h) and left untreated or activated with nigericin (20 µM, 1 h). Nuclei were stained with Hoechst; images were acquired on an Opera Phenix HCS microscope using a 40× water objective; scale bar, 50 µm. (K-L) Quantification of PLA total spot area per cell for CRY2-NLRP3 after KL001 treatment (K) or KL044 treatment (L), as shown in (J). Left: representative experiment showing three wells per condition with ≥1,000 cells analyzed per well. Right: paired summary of three independent experiments. Data are shown as paired dots with mean ± SD. Statistical significance was determined using a paired Mann-Whitney test (****p < 0.0001).
Article Snippet: Nuclei were counterstained with Hoechst (Invitrogen, H3570) for 30 min prior to image acquisition using a Revvity Opera Phenix Plus microscope (Revvity) equipped with a 60× water-immersion objective at the Cell Imaging Platform of the CRCL (PIC, CRCL, Lyon).
Techniques: MANN-WHITNEY, Activity Assay, Staining, Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay
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