disulfiram  (MedChemExpress)

Journal: Cell Death & Disease

Article Title: A MIF-p38-GSDMD inflammatory loop in keratinocytes underlies UVB-induced cutaneous lupus

doi: 10.1038/s41419-026-08443-4

Figure Lengend Snippet: a Schematic of experimental design to generate lupus-prone keratinocytes via transfection of HaCaT cells with endogenous nucleic acids (eNAs). b Protein levels and quantification of MIF and CD74 in eNAs-transfected HaCaT cells ( N = 3 each). c Protein levels and quantification of p-ZAKα, ZAKα, p-p38, p38, NLRP3, and GSDMD (full-length, FL; N-terminal fragment, NT) in eNAs-transfected HaCaT cells with or without UVB exposure (55 mJ/cm²) ( N = 3 each). d Protein levels and quantification of p-p38, p38, NLRP3, GSDMD-FL, and GSDMD-NT in UVB-exposed eNAs-transfected HaCaT cells pre-treated with DMSO or p38 inhibitor SB203580 ( N = 3 each). e Protein levels and quantification of GSDMD-FL and GSDMD-NT in UVB-exposed eNAs-transfected HaCaT cells pre-treated with DMSO or NLRP3 inhibitor MCC950 for 2 h ( N = 3 each). f MIF secretion in supernatants of eNAs-transfected HaCaT cells quantified by ELISA under basal conditions (CTR), following ZAKα activation by anisomycin (ANS), and post-UVB exposure with DMSO, SB203580 (p38 inhibitor), MCC950 (NLRP3 inhibitor), disulfiram (DSF, GSDMD pore inhibitor), GW4869 (exosome inhibitor), or brefeldin A (Golgi inhibitor) treatment ( N = 4 each). g LDH release in supernatants from the same experimental conditions as in f (N = 4 each). h Protein levels and quantification of p-p38, p38, NLRP3, and GSDMD in eNAs-transfected HaCaT cells treated with conditioned media from untreated (CTR-CM) or UVB-exposed HaCaT cells (UVB-CM), with or without ISO-1 ( N = 3 each). i Protein levels and quantification of CD74, p-p38, p38, NLRP3, and GSDMD in eNAs-transfected HaCaT cells transfected with a control siRNA (si-CTR) or two CD74-targeting siRNAs (si-CD74 #1 and #2) followed by PBS or MIF treatment ( N = 3 each). Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To investigate MIF release mechanisms, cells were exposed to UVB in the presence of 40 μM disulfiram (DSF, a GSDMD pore inhibitor, MCE), 10 μM GW4869 (an exosome inhibitor, APExBIO), or 5 μg/mL brefeldin A (a Golgi-mediated secretion inhibitor, APExBIO) [ – ].

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Activation Assay, Control

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: THP-1 cells ( A , C , D , E ) and hMDMs ( B ) were infected with H37Rv Mtb at an MOI of 5. A Mean percentages of AK release relative to the positive control. Each colored dot represents an independent experiment (N = 4). B Mean percentages of LDH release relative to the positive control. Each dot represents an independent experiment (N = 6), with colors indicating distinct human monocyte donors (N = 3). C Representative western blot images showing STAT1 expression and phosphorylation following Mtb infection and IFNAR neutralization. Data are representative of four independent experiments (N = 4). D Quantification of band intensities shown in panel C. p-STAT1α intensity was normalized to β-actin from the same lane. E Mean percentages of LDH release relative to the positive control. Each colored dot represents an independent experiment (N = 4). Welch’s t-tests were performed for panels A , B , and D ; p -values are shown on the graphs. Two-way ANOVA was performed for panel E , with p -values presented accordingly.

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Infection, Positive Control, Western Blot, Expressing, Phospho-proteomics, Neutralization

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: A, B Percentages of early apoptotic cells among total cells assessed by fluorescence microscopy. THP-1 cells or hMDMs were infected with dsRed-expressing H37Rv Mtb at MOI:5 for 1–3 d, stained with YO-PRO-1 (YO) and Draq7, and imaged at 1, 2, or 3 d post-infection. More than 200 cells were analyzed per time point in each replicate (N = 3). Multiple paired t-tests were performed to compare infected groups with uninfected groups. and p -values are shown in the graphs. Each colored data point represents an independent experiment ( A , THP-1, N = 4) or a distinct donor ( B , hMDM, N = 3). C–H THP-1 cells were infected with w.t. H37Rv Mtb at MOI:5 for 1, 2, and 3 d or left uninfected. C–F Representative western blot images showing the cleavage of CASP3 ( C , D ) and GSDME ( E , F ) in THP-1 cells ( C , E ) or hMDMs ( D , F ), respectively. FL, full-length target protein; CL, cleaved target protein. STS, Staurosporine (1 μM, 6 h); RAP, Raptinal (10 μM, 6 h). G, H Bar graphs showing the quantification of normalized band intensities corresponding to THP-1 cleaved CASP3 ( G ), and GSDME ( H ). Each value in the infected groups was normalized to its uninfected counterpart from the same experiment. Band intensities were quantified using ImageLab software. One-sample t-tests were performed to compare the normalized values to 1, and p -values are shown in the graphs. Each colored data point represents an independent experiment (N = 4).

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Fluorescence, Microscopy, Infection, Expressing, Staining, Western Blot, Software

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: THP-1 cells ( A , B , E , and F ) or hMDMs ( B , D , F , and H ) were infected with w.t. H37Rv Mtb at MOI:5 for 1-3 d or left uninfected. A, C Representative western blot images showing GSDMD cleavage in THP-1 cells ( A ) and hMDMs ( C ). E, G Representative western blot images showing MLKL phosphorylation in THP-1 cells ( E ) and hMDMs ( G ). FL, full-length target protein; CL, cleaved target protein. L/N, LPS (100 ng/mL, 4.5 h) plus nigericin (20 μM, 30 min) treatment, as a positive control. B, D, F, and H Quantification of band intensities corresponding to cleaved GSDMD ( B and D ) or phosphorylated MLKL ( F and H ), as indicated next to each Western blot. Band intensities were quantified using ImageLab software, normalized to the corresponding β-actin bands, and then expressed relative to the uninfected counterparts from the same experiment. Bar graphs display the normalized band intensities of the infected groups. One-sample t-tests were used to compare normalized values to 1, and p -values are shown in the graphs. Each colored data point represents an independent experiment (N = 4 or 3) for THP-1 cells ( B and F ) or an individual human donor (N = 3) for hMDMs ( D and H ).

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Infection, Western Blot, Phospho-proteomics, Positive Control, Software

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: A Confocal time-lapse images of THP-1 cells infected with dsRed H37Rv Mtb (blue, MOI: 2) and stained with Syt G (orange) for 20 h in the presence or absence of 20 μM disulfiram (DIS). UT, untreated. White arrowheads: extracellular DNA webs. B, C Quantification of DNA release events ( B ) and Syt G⁺ cells ( C ). D, E Percentages of LDH release relative to the lysis control. Validation of disulfiram efficacy ( E ): THP-1 cells were treated with 20 μM disulfiram prior to LPS and nigericin (L/N) stimulation, treated with L/N in the absence of DIS, or left untreated. SC, solvent control. F Confocal images of Mtb-infected CASP4/5 −/− and WT cells. THP-1 cells were a kind gift from Dr. Claire Bryant. G, H Quantification of DNA release events ( G ) and Syt G⁺ cells ( H ). I, J LDH, and IL-1β release profiles in CASP4/5 −/− cells. Data represent N = 3 independent experiments. p -values determined by Welch’s t -test ( B , C , E , F , H ) or two-way ANOVA ( D , I , J ). Scale bars: 50 μm.

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Infection, Staining, Lysis, Control, Biomarker Discovery, Solvent

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: A, C THP-1 cells or hMDMs were infected with H37Rv Mtb at MOI:5 for 1-3 d or left uninfected. Representative western blot images show the GPX4 levels in THP-1 cells ( A ) (N = 4) or hMDMs ( C ) (N = 6). B, D Band intensities of GPX4 were quantified using ImageLab software, normalized to the corresponding β-actin bands, and then expressed relative to the uninfected counterparts from the same experiment. Bar graphs display the normalized band intensities of the infected groups. One-sample t-tests were used to compare normalized values to 1, and p -values are shown in the graphs. One-sample t-tests were performed to compare normalized values to 1, and p -values are shown in the graphs. Each colored data point represents an independent experiment (N = 4) for THP-1 cells ( B ) or an individual human donor (N = 3) for hMDMs ( D ). E Representative confocal microscopic images showing the level of lipid peroxidation in THP-1 cells (N = 3). Cells were infected with dsRed H37Rv Mtb (yellow) at MOI:5 for 4 or 24 h, respectively, treated with cumene hydroperoxide (CuPx) for 2 h, or left uninfected and untreated. Staining was performed with Liperfluo (cyan) and Draq7 (magenta). Scale bars: 50 µm. F–I Quantification of normalized Liperfluo fluorescence intensities ( F and H ), or the percentage of Draq7-positive cells relative to the total cells in each field ( G and I ). Each dot represents the mean measurement from an independent experiment (N = 3), with 6 fields imaged per condition. Regions of interest (ROIs) were defined using Cellpose 2.0, and Raw Integrated Densities (RawIntDen) were calculated using FIJI. Nested One-way ANOVA ( F and H ) or One-way ANOVA with Brown-Forsythe correction ( G and I ) was performed; p -values are presented in decimal format. J LDH assay validating the efficacy of Ferrostatin-1. THP-1 cells were treated with solvent control (SV), RSL-3, or a combination of RSL-3 and Ferrostatin-1 (Fer-1) at the indicated concentrations. Background-corrected absorbance values for each group are shown. K LDH assay measuring necrosis in infected and uninfected THP-1 cells in the presence or absence of Fer-1. THP-1 cells were infected with w.t. H37Rv Mtb at MOI:5 for 1–3 d with or without Fer-1 (10 μM) (N = 4). The percentage of LDH release was calculated by normalizing background-corrected absorbance values to the lysis control.

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Infection, Western Blot, Software, Staining, Fluorescence, Lactate Dehydrogenase Assay, Solvent, Control, Lysis

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: A Representative time-lapse live-cell imaging of THP-1 cells showing changes in Lysoview (LV, blue) fluorescence intensity over time. Cells were stained with LV and Sytox Green (Syt G, green), then treated with LLoMe or DPBS approximately 25 minutes after imaging began. White arrowheads indicate tracked cells. B THP-1 cells were infected with dsred H37Rv Mtb at MOI:2 then stained with LV and Syt G. Representative time-lapse live-cell imaging of THP-1 cells show changes in LV fluorescence intensity over time. 0 min is defined as the time when a cell first becomes Syt G + . Negative time points indicate the duration (up to 120 min) before time point 0, during which LV intensities were quantified. The line graphs next to the confocal microscopic images display the logarithm (log) (base 2) of fold change (FC) over time ( A and B ). C, D corresponding to A or B , the line graphs show the log2 FC changing over time. In each independent experiment, 2 scenes were imaged for each condition, and 4 for the untreated (UT) (uninfected) condition. A total of 3 independent experiments were performed (N = 3). The UT dataset is the same as the UI from the infection experiments. 39, 48, and 71 cells were tracked and included in the statistical analysis for the DPBS, LLoMe-treated, and UT groups. For D , 4 biological replicates were conducted for the Mtb and bystander (Bys) conditions (N = 4), and 3 for the UI condition (N = 3). 35, 41, and 71 cells were tracked and analyzed for the Mtb, Bys, and UI groups. In C and D , each line represents mean; error bars indicate SD. Modified Chi-squared analyses were performed. p -values are shown in graphs.

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Live Cell Imaging, Fluorescence, Staining, Imaging, Infection, Modification

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: A , THP-1 cell images extracted from the same time-lapse video shown in and time-lapse confocal microscopic images of Mtb-infected hMDMs. THP-1 cells or hMDMs were infected with dsRed H37Rv Mtb (blue) at MOI:2 and then stained with Syt G (orange). The white arrowheads indicate extracellular, web-like DNA structures, while asterisks label cells that do not exhibit DNA release. Scale bars represent 50 μm. B, C Quantification of DNA release or plasma membrane rupture (PMR) ratios in uninfected and Mtb-infected THP-1 cells. DNA release events and total cell numbers in frame 1 of each video were quantified using FIJI software (the same for E and F ). Each colored data point represents the average of 4 fields (N = 4 for Mtb; N = 3 for UI) and includes over 200 cells. Statistical analysis was performed using unpaired Welch’s t-tests, and p -values are shown in the graphs. D Quantification of the time duration from PMR to DNA release in Mtb-infected THP-1 cells. The number of frames between the two events was manually counted and converted to time (in minutes). The black dot line indicates the median, and the white dashed lines represent the 25th and 75th percentiles. Each dot represents a single trackable cell. The mean (μ) and standard deviation (σ) are indicated; the same notation is used hereinafter. Each color represents an independent experiment (N = 4). E, F Quantification of DNA release or PMR ratios in Mtb-infected or uninfected hMDMs. Each data point represents the average of 4 fields from an independent experiment (N = 6), with more than 200 cells analyzed per condition. Each color corresponds to a distinct human donor (N = 3). μ and σ are shown only for the infected groups. N.A., Not applicable.

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Infection, Staining, Clinical Proteomics, Membrane, Software, Standard Deviation

Journal: bioRxiv

Article Title: Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis -infected human macrophages

doi: 10.64898/2026.01.30.702894

Figure Lengend Snippet: A THP-1 cells or human neutrophils (hNeu) (from 1 donor) were infected with dsred H37Rv Mtb (red) at MOI:2 for 20 hrs and fixed and stained with DAPI (blue) to visualize DNA. Alexa Fluor 647-conjugated antibodies (yellow) were applied to detect myeloperoxidase (MPO) or citrullinated Histone 3 (CitH3). White arrowheads indicate extracellular web-like DNA structures. Scale bars represent 50 μm. B, C Bar graphs showing the mean fluorescence intensity (MFI) ratios for DAPI and antibody staining on extracellular DNA. Intracellular fluorescence signals were manually excluded, and the MFI of DAPI or antibody staining on extracellular web-like DNA was quantified using FIJI. Paired t-tests were performed; p -values are reported in each graph. Each data point represents the average of 3-4 fields from an independent experiment, with each color denoting an independent experiment (N = 3). D Airyscan confocal image showing extracellular DNA released by infected THP-1 cells interacting with extracellular Mtb (N = 1). The left image is a 0.53 μm optical slice with merged signals from brightfield, DAPI (blue), and dsRed Mtb (red). 3D volume rendering (on the right) was performed using ZEISS Zen 3.6, showing the X-Y view. Each square in the background grid represents a 10 μm × 10 μm area. E LDH release assay showing the percentage of cell death in each group, calculated by normalizing absorbance values to the lysis control. THP-1 cells were infected with w.t. H37Rv Mtb at MOI:5 for 4 hours, washed with DPBS twice, and replenished with fresh medium. DNase I was added to half of the UI and infected groups at 100 U/mL, while the remaining groups were left untreated. Supernatants were collected at 2 and 3 d post-infection for LDH release analysis ( E ) and CFU enumeration ( F ) (N = 4 for both panels). F CFU analysis reflecting the number of viable extracellular Mtb bacilli. Supernatants from infected groups were harvested and plated on 7H11 agar plates. Samples were diluted 1:10 and 1:100 prior to plating, and only plates containing >20 colonies were used for quantification. Plates were monitored daily to ensure optimal colony size for counting, and CFUs were enumerated at 21 days post-plating.

Article Snippet: To induce GSDMD cleavage and pyroptosis, THP-1 cells were treated with LPS (InvivoGen, CA, USA; tlrl-eblps) at 100 ng/mL for 4 h, treatment with 20 μM nigericin (InvivoGen, tlrl-nig) for 30 min. To inhibit GSDMD activation, THP-1 cells were treated with disulfiram (MedChemExpress, HY-B0240) at 20 μM.

Techniques: Infection, Staining, Fluorescence, Lactate Dehydrogenase Assay, Lysis, Control