Journal: Translational Oncology

Article Title: Overcoming the leptomeningeal seeding of medulloblastoma by targeting HSP70

doi: 10.1016/j.tranon.2026.102695

Figure Lengend Snippet: Phenotypic differences between LMS-derived seeding (S3) and non-seeding (N3) cells. (A) Morphological differences were not observed between S3 and N3 under bright field microscope. (B) The graph shows that the proliferative capacity of S3 is slower than that of N3s through High-Content Screening (HCS) analysis. (C) The trans-well migration assay reveals that there was no significant difference in the migration ability between S3 and N3 (quantification graph). (D) Representative images from the trans-well migration assay. (E) Representative images of the wound-healing assay show that N3 filled the open area faster than S3. The white area is saturated with green color due to the overlapping of S3 without filling the wound gap. (F) Quantification graph of the wound-healing assay. (G) Adhesion of S3 and N3 cells to extracellular matrix (ECM) components, including fibronectin, collagen I, collagen IV, laminin I, and fibrinogen, was quantified after 24h of incubation. (H) Adhesion values were normalized to control conditions. All data are presented as mean ± SD from n = 3 independent experiments. Statistical comparisons between two groups were performed using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell proliferation was monitored and quantified consecutively for 64 hours using a high-content screening (HCS) system (Operetta CLS, PerkinElmer).

Techniques: Derivative Assay, Microscopy, High Content Screening, Migration, Wound Healing Assay, Incubation, Control, Two Tailed Test

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 - E) hMDMs were synchronized by a 2-h serum shock. Cells were primed with LPS (0.5 µg/mL, 4 h) and activated with nigericin (20 µM, 30-240 min) at different time post synchronization (from CT12 to CT48). (A) Cell death monitored by DRAQ7 incorporation using an Opera Phenix HCS microscope. Area under the curve (AUC) values were quantified across time after synchronization (pooled data from three independent experiments), shown as mean ± SD. (B) ASC-speck-positive cells quantified at each circadian time point from three independent experiments, each performed with four experimental replicates (12 wells per condition); with ≥200 cells analyzed per condition, mean ± SD. (C) IL-1β secretion measured by ELISA in supernatants from hMDMs treated with nigericin (20 µM, 240 min) at each time point after synchronization. Data are from three independent experiments, each performed with two experimental replicates (6 wells per condition), shown as mean ± SD. (A, B, C) Right panel: Cosinor fits of raw data generated with COSINOR online software, showing acrophase and bathyphase; p values calculated by Cosinor analysis. (D) Caspase-1 activity normalized to Crystal Violet (CV) staining. Data represent four independent experiments, each performed with six technical replicates, shown as mean ± SD. Kruskal-Wallis test, p = 0.0006 (***). (E) hMDMs were collected every 4 h from 12 h to 48 h after synchronization, following LPS priming (0.5 µg/mL, 4 h). Protein expression of NLRP3, CRY1 and CRY2 were analyzed by immunoblotting; actin served as a loading control.

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: Microscopy, Enzyme-linked Immunosorbent Assay, Generated, Software, Activity Assay, Staining, Expressing, Western Blot, Control

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) Endogenous NLRP3 was immunoprecipitated from THP-1 WT or NLRP3 knockout (N3KO) cells using anti-NLRP3 antibodies. Immunoprecipitation was performed 6 h after serum shock synchronization or in unsynchronized (NS) conditions, as indicated. IgG served as a control. CRY2 co-immunoprecipitation was assessed by immunoblotting; actin was used as a loading control (n=2). (B-C) Representative proximity ligation assay (PLA) images showing CRY1- NLRP3 or CRY2-NLRP3 interactions (red) in untreated THP-1 WT cells (n=4) (B) and hMDMs (n>5) (C). Nuclei were stained with Hoechst; images were acquired on an Opera Phenix HCS microscope using a 60× objective; scale bar, 50 μm. (D) Representative PLA images (red) showing CRY2- NLRP3 interactions in hMDMs at 16 h, 28 h, and 40 h after synchronization and following LPS priming. Nuclei were stained with Hoechst. Images were acquired on an Opera Phenix HCS microscope with a 40× water objective; scale bar, 50 µm; n > 1,000 cells/condition. (E) Quantification of PLA total spot area per cell from (D). (F) Cosinor analysis of data in (E) showing circadian oscillation with acrophase and bathyphase indicated. (G) Cosinor curves for cell death, ASC speck formation, IL-1β secretion, and CRY2- NLRP3 interactions plotted together. (H) Summary table reporting acrophase, bathyphase, and cosinor p values for panels ( , B, and ).

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: Immunoprecipitation, Knock-Out, Control, Western Blot, Proximity Ligation Assay, Staining, Microscopy

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) Immunoblot analysis of NLRP3, CRY1, CRY2, and GSDMD in hMDMs (n=6) treated with LPS (0.5 µg/mL, 4 h) with or without nigericin (10 µM, 1 h). Actin served as a loading control. (B) Representative PLA images (red) showing CRY1-NLRP3 or CRY2-NLRP3 interactions in hMDMs (N=6) 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× objective; scale bar, 50 µm. (C-D) Quantification of PLA total spot area per cell for CRY1-NLRP3 (C) and CRY2-NLRP3 (D). ≥500 cells per condition; mean ± SEM; Mann-Whitney test, ****p < 0.0001. (E and F) Pooled Cosinor fits of CRY2-NLRP3 PLA signal over time from the five donors shown in Figure S5, either untreated (E) or treated with nigericin (F). (G) Lollipop plots showing MESOR values of CRY2-NLRP3 interactions corresponding to the cosinor fits in (E-F). Blue indicates untreated cells; pink indicates nigericin-treated cells.

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: Western Blot, Control, Staining, Microscopy, MANN-WHITNEY

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

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) hMDMs were synchronized as in Figure S5, primed with LPS (0.5 µg/mL, 4 h), and treated or not with MCC950 (150 nM, 30 min pretreatment) before stimulation with nigericin (20 µM). Cell death was monitored over time by DRAQ7 incorporation using an Opera Phenix HCS microscope. Area under the curve (AUC) quantification of cell-death time courses from (Figure S8A). Data represent n = 4 independent experiments with 6 replicates each; two-way repeated-measures ANOVA with time and treatment as factors and individual donors, ns = not significant, *p < 0.03, ***p = 0.0002. (B) Cosinor analysis of AUC data from (A), showing acrophase, bathyphase, and p-values. (C) IL-1β secretion measured by ELISA in supernatants from hMDMs at each time point after synchronization. The untreated condition corresponds to the same samples shown in and is displayed here for direct comparison with MCC950-treated cells. Data are from three independent experiments, each performed with two experimental replicates (6 wells per condition), shown as mean ± SD. (D) Cosinor analysis of IL-1β secretion data from (C), showing acrophase, bathyphase, and p-values. (E) Experimental timeline for U937 cells expressing doxycycline-inducible NLRP3 WT or CAPS variants used in panels (F-J). (F-I) IL-1β secretion measured by ELISA in culture supernatants from synchronized U937 cells collected at 24 h, 30 h, 36 h, 42 h, and 48 h post-synchronization. Cells expressing WT NLRP3 (F, G), M701T (F, H), or Q703K (F, I) were treated or not with MCC950. (F) Left panel: IL-1β quantification across time. Middle panel: summary table reporting two-way ANOVA results. Right panel: Q-Q plot assessing normality of the IL-1β data. (G-I) IL-1β secretion profiles for each NLRP3 genotype treated or left untreated with MCC950, along with the corresponding two-way ANOVA tables. (J) Summary tables of three-way ANOVA results for panels (G-I).

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: Microscopy, Enzyme-linked Immunosorbent Assay, Comparison, Expressing