Journal: Journal of Translational Autoimmunity

Article Title: Exploring the immunomodulatory effects of environmental contaminants on autoimmune patients: An in vitro approach

doi: 10.1016/j.jtauto.2025.100341

Figure Lengend Snippet: Modulation of intracellular signaling pathways in HD, RA, and SLE cells exposed to environmental contaminants. Heatmap showing the relative expression and phosphorylation levels of key signaling proteins involved in immune regulation, inflammation, and cell survival (STAT1, STAT3, p38, AKT, and NFκB) and their phosphorylated forms in HD, RA, and SLE after exposure to PM, silica, and TCDD. Data are represented as z-score normalized values. Asterisks indicate statistically significant differences compared with the unstimulated control within each group (p < 0.05). AKT: protein kinase B; HD: healthy donors; NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells; P: phosphorylated form; p38: p38 mitogen-activated protein kinase; PM: particulate matter; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; STAT: signal transducer and activator of transcription; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Article Snippet: Membranes were stripped and re-probed for total AKT, NFκB p65, p38 MAPK, STAT1, STAT3, and β-actin (Cell Signaling Technology, Danvers, MA, USA; Santa Cruz Biotechnology, Dallas, TX, USA) as a loading control.

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: (A) Growth curves of wild-type (WT) and mNeonGreen-expressing B. ovis in rich medium over 72 hours post-inoculation. (B) Percentage of THP-1 macrophages infected with mNeonGreen B. ovis after 3 hours of infection at multiplicities of infection (MOIs) of 100 or 1,000, as assessed by flow cytometry. (C) Percentage of THP-1 macrophages infected with mNeonGreen B. ovis after 2 or 3 hours of infection at MOI 1,000. (D) Representative flow cytometry plots gated on live, single THP-1 macrophages. Cells were stained with Zombie NIR viability dye and infected with mNeonGreen B. ovis (MOI 1,000). The left panel shows a control population of heat-treated, uninfected macrophages. Percentages of live/dead (Zombie NIR) and infected (mNeonGreen-positive) cells are indicated. (E) Representative flow cytometry plots of THP-1 macrophages infected with mNeonGreen B. abortus (MOI 1,000), showing a distinct population of mNeonGreen-positive infected cells. Data in panels B and C represent means ± standard deviation (SD) from three biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test (***, P < 0.001; ****, P < 0.0001).

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Expressing, Infection, Flow Cytometry, Staining, Control, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: (A) Representative flow cytometry plots showing mNeonGreen-positive THP-1 macrophages at 3 hours post-infection at increasing MOI. Plots are gated on live, single cells (gate shown as green box on flow plots) and the percentage of mNeonGreen-positive (infected) cells is indicated in each plot. (B) Representative flow cytometry plots showing mNeonGreen-positive macrophages at 2 and 3 hours post-infection with B. ovis at MOI 1,000. All plots are gated on live, single cells.

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Flow Cytometry, Infection

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: (A) Schematic of the genome-wide loss-of-function screen. A CRISPR-Cas9 pooled sgRNA library was introduced into Cas9-expressing THP-1 macrophage-like cells, followed by infection with mNeonGreen-expressing Brucella for 3 hours. Infected (mNeonGreen + ) and uninfected (mNeonGreen − ) populations were separated by FACS, and the abundance of sgRNAs in each population was quantified by sequencing. (B) Plot of gene enrichment P values from the B. abortus CRISPR-Cas9 screen, calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. P -value threshold of 10 -2 (1/p of 10 2 ) is drawn as a dotted line. The small GTPases RAB14 and RAB5C are highlighted in red. (C) Validation of RAB14 as a host factor. THP-1 macrophages expressing a RAB14 -targeting sgRNA were infected with mNeonGreen B. abortus or B. ovis at MOI 1,000. The percentage of infected cells was quantified by flow cytometry and normalized to non-targeting control (NTC). Data represent means ± SD from three biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test (**, P < 0.01). Editing efficiency for both sgRNAs > 60% (D) Latex bead uptake assay in NTC and RAB14 -deficient macrophages. Cells were incubated with yellow-green fluorescent beads (bead:cell ratio = 10) for 3 hours. Representative flow cytometry plots show comparable bead uptake between genotypes, indicating that phagocytic capacity is preserved in RAB14 -deficient cells.

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Genome Wide, CRISPR, Expressing, Infection, Sequencing, Biomarker Discovery, Flow Cytometry, Control, Comparison, Incubation

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: THP-1 macrophages expressing non-targeting control (sgNTC) or the indicated sgRNAs were infected for 3 h with mNeonGreen-expressing B. abortus or B. ovis at MOI 1,000. Live, single cells were gated (gate shown as green box on flow plots), and the percentage of mNeonGreen-positive (infected) cells is indicated in each plot. (A) Representative plots for sgNTC cells and two independent RAB14-targeting sgRNA lines infected with B. abortus (top row) or B. ovis (bottom row). (B) sgNTC and CSK-deficient (sgCSK) cells, shown uninfected (left panels) or infected with B. abortus (top row) or B. ovis (bottom row). (C) sgNTC and TMEM30A-deficient (sgTMEM30A) cells, with uninfected sgNTC controls (left panels) and infections with B. abortus (top row) or B. ovis (bottom row). (D) Latex bead uptake in sgNTC, CSK-deficient, and TMEM30A-deficient macrophages. Cells were incubated with yellow-green fluorescent beads (bead:cell ratio 10:1) for 3 h. Representative plots show similar proportions of bead-positive cells across genotypes, indicating that phagocytic capacity is preserved in the mutant THP-1 lines.

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Expressing, Control, Infection, Incubation, Mutagenesis

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: (A, D) Statistical enrichment plots (1/ P ; dotted line) calculated using the α-RRA algorithm in MAGeCK. CSK (A) and TMEM30A (D) met the criteria for B. abortus host factors (≥1 log 2 fold change, ≥2 independent sgRNAs, P < 0.01). (B, E) Validation of CSK (B) and TMEM30A (E) as host factors. THP-1 cells expressing sgRNAs targeting either gene were infected with m NeonGreen B. ovis or B. abortus at MOI 1,000 for 3 hours. The percentage of infected macrophages was quantified by flow cytometry and normalized to the non-targeting control (NTC). Data represent mean ± SD from three biological replicates. Significance in panels B and E was assessed by an unpaired t -test (***, P < 0.001). (C, F) Phagocytosis assays using yellow-green fluorescent latex beads (bead:cell ratio = 10) in THP-1 cells expressing NTC or gene-targeting sgRNAs. The percentage of bead-positive cells was quantified by flow cytometry and normalized to NTC. Editing efficiency - CSK: 61%; TMEM30A: 71%.

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Biomarker Discovery, Expressing, Infection, Flow Cytometry, Control

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: (A) Plot showing gene enrichment P values for the B. abortus CRISPR-Cas9 screen, ranked using the α-RRA algorithm in MAGeCK ( P -value threshold of 10 -2 (1/p of 10 2 ) is drawn as a dotted line). TORC1-related genes LAMTOR2 and AKT1 are highlighted. (B-C) Validation of AKT1 and LAMTOR2 as host factors. THP-1 cells expressing sgRNAs targeting either gene were infected with m NeonGreen B. abortus (B) or B. ovis (C) at MOI 1,000 for 3 hours. The percentage of infected macrophages was quantified by flow cytometry and normalized to the non-targeting control (NTC). Data represent mean ± SD from three biological replicates. Significance in panels B and C was assessed using one-way ANOVA with Dunnett’s multiple comparison test (****, P < 0.0001; ***, P < 0.001; **, P < 0.01). Editing efficiency - AKT1: 63%; LAMTOR2: 87%. (D) Phagocytosis assay in THP-1 macrophages expressing non-targeting control (NTC), AKT1 , or LAMTOR2 sgRNAs. Cells were incubated with yellow-green fluorescent latex beads (bead:cell ratio = 10) for 3 hours, and bead-positive cells were quantified by flow cytometry. Data are normalized to the mean uptake in NTC cells. (E) THP-1 viability following treatment with increasing concentrations of AKT inhibitor X for 48 hours, assessed by XTT assay and normalized to untreated controls. (F) Axenic growth of B. abortus in broth culture in the presence of AKT inhibitor X. Cultures were incubated for 48 hours, and OD 600 values were normalized to untreated samples. (G) THP-1 macrophages pretreated with 10 µM AKT inhibitor X showed reduced B. abortus infection, as measured by flow cytometry. Data represent mean ± SD from three biological replicates; unpaired t -test ( P < 0.01).

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: CRISPR, Biomarker Discovery, Expressing, Infection, Flow Cytometry, Control, Comparison, Phagocytosis Assay, Incubation, XTT Assay

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: THP-1 macrophages expressing non-targeting control (sgNTC) or sgRNAs targeting AKT1 or LAMTOR2 were infected with mNeonGreen-expressing B. abortus or B. ovis (MOI = 1,000) and analyzed 3 h post-infection by flow cytometry. (A, B) Representative flow cytometry plots showing the percentage of infected (mNeonGreen-positive) cells within the live, single-cell gate for each genotype after infection with B. abortus (A) or B. ovis (B). (C) Latex bead uptake in sgNTC, AKT1-deficient, and LAMTOR2-deficient macrophages. Cells were incubated with yellow-green fluorescent beads (bead:cell ratio 10:1) for 3 h. Representative plots show similar proportions of bead-positive cells across genotypes, indicating that phagocytic capacity is preserved in the mutant THP-1 lines. (D) Representative viability and infection profiles of THP-1 macrophages 3 h after infection with mNeonGreen B. abortus under the indicated conditions. Cells were gated (horizontal and vertical lines on the flow plot) on live, single events and stained with Zombie NIR viability dye. The left panel shows uninfected, heat-treated macrophages used to define live/dead gates. The middle and right panels show macrophages that were either untreated or pre-treated with 10 micromolar AKT inhibitor X prior to infection. Quadrants indicate the percentages of live/dead (Zombie NIR) and infected (mNeonGreen-positive) cells.

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Expressing, Control, Infection, Flow Cytometry, Single Cell, Incubation, Mutagenesis, Staining

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: THP-1 macrophages expressing sgRNAs targeting AKT1, LAMTOR2, CSK , or RAB14 , along with a non-targeting control (NTC), were infected with mEmerald-expressing M. abscessus at MOI 5 for 4 hours. The percentage of infected cells was quantified by flow cytometry and normalized to NTC. Data represent means ± SD from three biological replicates. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test (****, P < 0.0001).

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Expressing, Control, Infection, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

doi: 10.64898/2026.05.18.725962

Figure Lengend Snippet: (A) Flow cytometry plots showing THP-1 macrophages expressing non-targeting control (sgNTC) or sgRNAs targeting AKT1, LAMTOR2, or CSK, infected with mNeonEmerald -expressing Mycobacterium abscessus at MOI = 5 for 4 hours. The percentage of mEmerald-positive (infected) cells is indicated for each genotype within the live, single-cell gate (gate shown as green box in the flow plots). (B) Representative flow cytometry plots for THP-1 cells expressing sgNTC or sgRNAs targeting RAB14, infected with M. abscessus under the same conditions. Unlike other validated Brucella host factors, disruption of RAB14 did not reduce M. abscessus infection, indicating a Brucella -specific requirement for this gene.

Article Snippet: We infected mutant and non-targeting control THP-1 cells with a previously optimized fluorescent Mab strain (ATCC 19977) that constitutively expresses mEmerald GFP ( ) and quantified infection at 4 h post-infection.

Techniques: Flow Cytometry, Expressing, Control, Infection, Single Cell, Disruption

Journal: bioRxiv

Article Title: Identification of non-covalent inhibitors for the atypical peroxiredoxin PRDX5 as a therapeutic strategy in malignant pleural mesothelioma

doi: 10.64898/2026.05.13.724787

Figure Lengend Snippet: A. IST-MES-1, IST-MES-2, MSTO-211H and MeT-5A cells were incubated for 5 days in presence of exogenous Peroxynitrite (left panel) or H 2 O 2 (right panel) at different concentrations. Cell viability was measured using CellTiter assay (luminescence). B. Digital representation of data obtained for PRDX3 and PRDX5 detection (capillary western blot) in cell line panel (left panel). Bar plots showing data from one individual run (2 replicates per condition, SD value, right panel).

Article Snippet: MSTO-211H and the normal mesothelial control cell line MeT-5A were sourced from ATCC and Addexbio, respectively.

Techniques: Incubation, Western Blot