Journal: Cell Death & Disease

Article Title: Targeting POLRMT by IMT1 inhibits colorectal cancer cell growth

doi: 10.1038/s41419-024-07023-8

Figure Lengend Snippet: The primary human colon cancer cells, pCan1, underwent treatment with IMT1 (1 μM). Subsequently, they were cultivated for the designated duration, and the assessments including the Caspase-3 activity ( A ) and the Caspase-9 activity ( B ), expression of apoptosis-related proteins ( C ), and cytosol cytochrome C contents (ELISA assays, D ) were performed. Cell apoptosis was tested through nuclear TUNEL staining assays ( E ). Preceding IMT1 (1 μM) treatment for the specified period, pCan1 cells underwent pretreatment with zDEVD-fmk (50 μM) or zVAD-fmk (50 μM) for 1 h. Subsequent assessments included the evaluation of apoptosis via nuclear TUNEL staining assays ( F ) and the determination of cell death through Trypan blue staining ( G ). Other primary colon cancer cells (“pCan-2/pCan-3”) or immortalized colon cancer cells (HCT116) were exposed to IMT1 (1 μM) for a designated duration and assessments included the Caspase-3 activity ( H ) and nuclear TUNEL staining ( I ). “Veh” represents the vehicle control (0.1% DMSO). The data were presented as mean ± standard deviation (SD, n = 5), with *indicating P < 0.05 compared to the “Veh” treatment ( A – I ) and # indicating P < 0.05 compared to the IMT1 only treatment ( F , G ). The experiments were repeated five times, yielding consistent results. The scale bar is set at 100 μm.

Article Snippet: The pan-caspase inhibitor (zVAD-fmk) and the specific caspase-9 inhibitor zDEVD-fmk were procured from Calbiochem (Darmstadt, Germany).

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Control, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: P. aeruginosa triggers human NLRP1 inflammasome activation in corneal and nasal epithelial cells. (A) Cell lysis (LDH) and IL-1β/IL-18 release evaluation in pHCECs and pHNECs upon P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. When specified, the pan Caspase inhibitor (Z-VAD, 20 µM), Caspase-1 inhibitor (Z-YVAD, 20 µM), Caspase-3/7 inhibitor (Z-DEVD, 20 µM), and Caspase-8 inhibitor (Z-IETD, 20 µM) were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. (B) Immunoblotting examination of NLRP1, NLRP3, and Tubulin in resting, PAO1-exposed as in A or LPS-primed pHCECs and pHNECs or in pHCECs and pHNECs genetically invalidated for NLRP1 using CRISPR-Cas9. PMA (100 µg/ml)- or LPS (100 ng/ml)-primed THP1 monocytic cell line was used as a positive control for NLRP3 expression. Immunoblots show lysates from one experiment performed three times. (C) Florescence microscopy and associated quantifications of ASC-GFP specks in A549 NLRP1+/ASC-GFP and A549 NLRP1−/ASC-GFP reporter cell lines exposed to P. aeruginosa (PAO1, 1.10 5 bacteria) for 24 h. ASC-GFP (green) pictures were taken in the dish after the infection. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 10 µm. ASC complex percentage was performed by determining the ratios of cells positive for ASC speckles on the total nuclei (Hoechst). At least 10 fields from each experiment were analyzed. Values are expressed as mean ± SEM. ***P ≤ 0.001, one-way ANOVA. (D) Immunoblotting characterization of genetic invalidation of NLRP1 in pHCECs and pHNECs population using CRISPR-Cas9 and microscopy visualization of plasma membrane permeabilization (PI incorporation, orange) in pHCECs co-cultured with PAO1 (1.10 5 bacteria) for 24 h. (E) sgRNA CD8 (SgCD8) was used as control and served as WT cells during subsequent experiments described in E. Images shown are from one experiment and are representative of n = 3 independent experiments; scale bars, 20 µm. Cell lysis (LDH), IL-18 release, and CFU evaluation in WT (SgCD8, D) or NLRP1 -deficient pHCECs and pHNECs, upon VbP (15 µM) treatment or P. aeruginosa (PAO1, 1.10 5 bacteria) co-culture for 24 h. For CFU analysis 1 × 10 4 (MOI 1) or 1 × 10 5 (MOI 10) bacteria were used. ***P ≤ 0.001, two-way ANOVA with multiple comparisons. Values are expressed as mean ± SEM. Graphs show one experiment performed in triplicates at least three times. Source data are available for this figure: .

Article Snippet: Caspase-3/7 inhibitor ZDEVD-FMK 20 µM , S7312 , Selleck.

Techniques: Activation Assay, Lysis, Bacteria, Co-Culture Assay, Western Blot, CRISPR, Positive Control, Expressing, Microscopy, Infection, Membrane, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption

doi: 10.1084/jem.20230104

Figure Lengend Snippet: List of reagents used in the study

Article Snippet: Caspase-3/7 inhibitor ZDEVD-FMK 20 µM , S7312 , Selleck.

Techniques: Protease Inhibitor, Staining, Marker, CyQUANT Assay, Mutagenesis, Membrane, Recombinant