bay k8644  (MedChemExpress)

Journal: medRxiv

Article Title: Inverse correlation between NLRP3 and TMEM176B as a possible tool for colorectal cancer progression

doi: 10.1101/2024.08.30.24312793

Figure Lengend Snippet: (A-B) NLRP3 staining in formalin/PFA-fixed paraffin-embedded CRC tissue in one early-stage (TNM I) and advanced-stage (TNM III). NLRP3 is stained with 3,3’-diaminobenzidine tetrahydrochloride (DAB) chromogen (brown) and cell nuclei stained with hematoxylin (blue). The images were captured at 20x magnification. (C) Scatter plot shows the percentage of NLRP3 staining for all the CRC biopsies analyzed (n =12) grouped according to the stages (TNM I/II versus TNM III/IV). Unpaired two-tails t-test p-value is reported. (D-E) TMEM176B staining in formalin/PFA-fixed paraffin-embedded CRC tissue in one early-stage (TNM I) and advanced-stage (TNM III). NLRP3 is stained with 3,3’-diaminobenzidine tetrahydrochloride (DAB) chromogen (brown) and cell nuclei stained with hematoxylin (blue). The images were captured at 20x magnification. (F) Scatter plot shows the percentage of TMEM176B staining for all the CRC biopsies analyzed (n =12) grouped according to the stages (TNM I/II versus TNM III/IV).Unpaired two-tails t-test p-value is reported. (G) Correlation analysis for NLRP3 and TMEM176B staining in CRC biopsies. Pearson correlation coefficient (r) and p-value (p) are indicated. (H-I) NLRP3 and TMEM176B staining in formalin/PFA-fixed paraffin-embedded CRC tissue in one advanced-stage (TNM III) with focus on stromal region. NLRP3 or TMEM176B are stained with 3,3’-diaminobenzidine tetrahydrochloride (DAB) chromogen (brown) and cell nuclei stained with hematoxylin (blue). The images were captured at 20x and 100x magnification (minor rectangle in the left bottom part of the images). (J) Stacked bar graph shows NLRP3 or TMEM176B percentage of staining in the stroma. The percentage value of cells positive for staining is reported.

Article Snippet: In some experiments cells were alternatively pre-treated with the NLRP3 inhibitor MCC-950 (10 µM, Invivogen) for 30 minutes or the TMEM176B inhibitor Bay-K8644 (Sigma-Aldrich) was added in LPS-primed cells for 15 minutes.

Techniques: Staining

Journal: medRxiv

Article Title: Inverse correlation between NLRP3 and TMEM176B as a possible tool for colorectal cancer progression

doi: 10.1101/2024.08.30.24312793

Figure Lengend Snippet: A-B) Heatmap graphs show RNA expression level of NLRP3 and TMEM176B genes in fresh tumor biopsies and peripheral blood leukocytes of six CRC patients, three with TNM-II, two with TNM-III and one with TNM-IV. Values are reported as log (2exp-ΔCt). C-D) Heatmap and scatter plot graphs show plasma levels of IL-1ß and IL-18 of six CRC patients, three with TNM-II, two with TNM-III and one with TNM-IV. In heatmap values are reported as log (pg/mL). Unpaired two-tails t-test p-value is reported. (D-E) Kaplan–Meier curves for CRC patients survival according to NLRP3 expression level in TNM-I/II and TNM-III/IV stages. (F-G) Kaplan–Meier curves for CRC patients survival according to TMEM176B expression level in TNM-I/II and TNM-III/IV stages. Data and curves were obtained from the Kaplan–Meier Plotter database ( http://kmplot.com/analysis/ ). Hazard Ratio (HR) and long rank P value are reported.

Article Snippet: In some experiments cells were alternatively pre-treated with the NLRP3 inhibitor MCC-950 (10 µM, Invivogen) for 30 minutes or the TMEM176B inhibitor Bay-K8644 (Sigma-Aldrich) was added in LPS-primed cells for 15 minutes.

Techniques: RNA Expression, Expressing

Journal: medRxiv

Article Title: Inverse correlation between NLRP3 and TMEM176B as a possible tool for colorectal cancer progression

doi: 10.1101/2024.08.30.24312793

Figure Lengend Snippet: HCT-116 spheroids (1.8 x10^5) were cultured alone or with PBMC (1.8 x10^5 cells, HCT-116/PBMC ratio: 1/1) isolated from three healthy donors for 24 hours, eventually with the pre-treatment of the NLRP3 inhibitor MCC-950 (10 µM) for 30 minutes. PBMC were harvested for activated caspase-1 detection using FAM-FLICA kit and flow cytometry. IL-1ß and LDH release was measured in culture supernatants. A) A representative experiment (out of three) of flow cytometry analysis of activated caspase-1 by the use of FAM-FLICA kit. Percentage of caspase-1 positive cells is reported for PBMC alone, PBMC from co-culture assay with HCT-116 and with the pretreatment with MCC-950 (10 µM). Gate strategy is reported in Supplementary File 3 . B-D) Scatter dot plots with bars (mean) reports (B) the percentage of PBMC positive for activated caspase-1, (C) the concentration of IL-1ß and ( D) the percentage of LDH released. (E) PBMC (1.8 x10^5 cells) isolated from three healthy donors were treated or not with LPS 1 µg/mL for 24 hours and then with the TMEM176B inhibitor BayK-8664 at increasing concentrations (5, 10, 20 µM) for 15 minutes. IL-1ß release was measured in culture supernatants. (F) HCT-116 spheroids (1.8 x10^5) were cultured with PBMC isolated from three healthy donors for 24 hours, with or without the treatment with Bay-K8644 (10 µM) for 15 minutes. IL-1ß release was measured in culture supernatants. (B-D) Paired One-Way ANOVA followed by multi comparison post-test was used to compare the conditions. *: p < 0.05; **: p < 0.01. (E) Paired Two-Way ANOVA followed by multi comparison post-test was used to compare the conditions within and between the two groups. **: p < 0.01 (within the group); #: p < 0.01 (between the groups). (F) Paired t test was used to compare the conditions.

Article Snippet: In some experiments cells were alternatively pre-treated with the NLRP3 inhibitor MCC-950 (10 µM, Invivogen) for 30 minutes or the TMEM176B inhibitor Bay-K8644 (Sigma-Aldrich) was added in LPS-primed cells for 15 minutes.

Techniques: Cell Culture, Isolation, Flow Cytometry, Co-culture Assay, Concentration Assay, Comparison