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Figure Lengend Snippet: ( a ) Type I interferon (IFN) pathway activation in THP1-Dual™ cells after cGAMP and LPS challenges ( n = 4 independent experiments). THP1-Dual™ cells were pre-treated for 24 h with cGAMP (0.5–1.25–2.5–5–10–20 μg/mL), and afterward stimulated for 1 h with LPS (0.5 μg/mL). Luminescence values were normalized on the untreated condition. ( b ) Type I IFN production in bone marrow-derived dendritic cells (BMDCs) after cGAMP and LPS challenges ( n = 3 independent experiments). BMDCs were pre-treated for 24 h with cGAMP (0.025–0.5–1 μg/mL) and afterward stimulated for 16 h with LPS (1 μg/mL). Secreted type I IFN was evaluated by B16-Blue IFN-α/β™ reporter cells assay. Results are reported as mean ± SEM. Statistically significant p -values are shown (Two-way ANOVA: **** p < 0.0001).

Article Snippet: THP1-Dual™ cells (InvivoGen) are derived from the human THP1 monocyte cell line by stable integration of secreted inducible reporter construct to monitor type I IFN pathway activation.

Techniques: Activation Assay, Derivative Assay

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Figure Lengend Snippet: Evaluation of CDN potency in THP-1 (human) and J774 (mouse) reporter cells; a) CDNs were added at 1 µg/mL to 1×105 THP-1 Dual or 5×104 J774-dual reporter cells for 20 hours and IF3 activity was measured by luciferase assay. b) CDNs were added at the indicated concentrations in the 0.5 – 50 µg/mL dose range to 1×105 THP-1 Dual reporter cells for 20 hours and IF3 activity was measured by luciferase assay.

Article Snippet: The compounds were tested at 1µg/mL in human THP-1-Dual™ and mouse J774-Dual™ cells (Invivogen), featuring stable integration of two inducible reporter constructs enabling simultaneous study of the two main signaling pathways activated by STING, the NF-kB pathway, and the interferon regulatory factor 3 (IRF3) pathway.

Techniques: Activity Assay, Luciferase

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Article Snippet: Human: THP1-Dual™ KI-hSTING-R232 , InvivoGen , Cat#thpd-r232.

Techniques: Western Blot, Flow Cytometry, Recombinant, Transfection, Isolation, Membrane, Staining, Sequencing, CRISPR, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Stress-responsive Mycobacterium tuberculosis subpopulations manipulate macrophage polarization and can be targeted to limit inflammation

doi: 10.64898/2026.03.13.711673

Figure Lengend Snippet: GroEL2 mRuby phenotypic variation predicts different macrophage responses (A–D) Snapshot images of THP-1 cells infected with GroEL2 mRuby -GFP cyt reporter at 24 and 48 hpi (n = 3). GroEL2 mRuby -Dim (A, C) and -Bright (B, D) intracellular bacilli are shown (dashed boxes). Maximum intensity projections of four images from a z-stack of 8 µm; bright-field (blue), GFP cyt (green), and GroEL2 mRuby (magenta) channels are merged. Scale bars, 10 µM. (E) GroEL2 mRuby normalized to GFP cyt mean fluorescence of exponential-phase (EP) bacilli and intracellular infectious foci at 24 and 48 hpi. Lines represent mean ± SD (n = 2, with cell number shown at the bottom of the graphs). Significance by nested one-way ANOVA (P = 0.034) followed by Tukey multiple comparisons test (adjusted P values). (F) Time course of GroEL2 mRuby /GFP cyt ratio for single infectious foci, classified as Dim (mean ratio < 1, n = 31) or Bright (mean ratio > 1, n = 20). Dashed line indicates the threshold ratio. Data are from three independent experiments. (G) Pearson’s correlation between intracellular growth rate and GroEL2 mRuby /GFP cyt ratio of individual intracellular infectious foci over the infected cell lifetime (n = 64 from two independent experiments). The dotted line indicates the threshold ratio. Simple linear regression fit (solid line, R 2 = 0.36, P < 0.0001) and 95% confidence bands (dashed lines). (H) Flow cytometry analysis of infected THP-1 macrophages at 24 hpi, sorted into GroEL2 mRuby -Dim (gray) and -Bright (pink) subpopulations based on intracellular fluorescence. Representative overlays are normalized to 10,000 events (n = 7 independent experiments). (I) Bacterial load in GroEL2 mRuby -Dim and -Bright THP-1 subpopulations at 24 hpi. Lines represent mean ± SD (n = 7). Significance by two-tailed unpaired t -test (95% confidence level). (J) Immunoluminometric quantification of cytokines at 48 hpi in GroEL2 mRuby -Dim (gray) and -Bright (pink) THP-1 subpopulations sorted at 24 hpi as in (n = 7). Significance by multiple paired t -tests with Holm-Sidak correction (adjusted P values). (K) Quantification of oxidative burst in THP-1 in subpopulations sorted at 48 hpi based on intracellular GroEL2 mRuby levels. Lines represent mean ± SD (n = 4 independent experiments). Significance by two-tailed unpaired t -test (95% confidence level). (L) Representative snapshot images of THP-1 cells infected with GroEL2 mRuby -GFP cyt reporter at 48 hpi, stained with annexin V (A5) and DRAQ7 (D7) (n = 3 independent experiments). Maximum intensity projections of four images from a z-stack of 6 µm; bright-field (blue), A5 (cyan), GFP cyt (green), GroEL2 mRuby and D7 (magenta) channels are merged. Scale bars, 10 µm. (M) GroEL2 mRuby /GFP cyt ratio for single intracellular infectious foci at 48 hpi in THP-1 macrophages, classified according to A5 and D7 negative (–) or positive (+) staining. Lines represent mean ± SD (n = 3, with cell number shown at the bottom of the graphs). Significance by two-way ANOVA (P = 0.009), followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (individual P values).

Article Snippet: THP-1 DualTM and THP-1 DualTM MyD88 knock-out cells (InvivoGen) were grown in RPMI 1640 (Gibco) supplemented with 10% FBS as above, 25 mM HEPES, 100 μg/mL Pen-Strep, 100 μg/mL Normocin, 10 μg/mL Blasticidin, and 100 μg/mL Zeocin.

Techniques: Infection, Fluorescence, Flow Cytometry, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Stress-responsive Mycobacterium tuberculosis subpopulations manipulate macrophage polarization and can be targeted to limit inflammation

doi: 10.64898/2026.03.13.711673

Figure Lengend Snippet: GroEL2 H triggers NF-κB and IRF signaling and can be targeted to decrease inflammation (A) Immunoblotting of M. tuberculosis extracellular (EC) and intracellular (IC) fractions using anti-HSP65 mouse monoclonal IgG 2a . Control (CT) and rv2224c -silenced (si) strains in the absence (–) or presence (+) of ATC inducer (200 ng/mL). Loaded sample amounts in percentage, high (H) and low (L) molecular-weight GroEL2 bands, and Ag85 as a control are indicated. (B and C) Activation of NF-κB (B) and IRF (C) in WT (circles) and KO MyD88 (squares) dual-reporter THP-1 cells 24 hpi. Cells were infected with either M. tuberculosis CT (filled symbols) or rv2224c si (open symbols) at MOI 2 for 2 hours, 4 days after induction of silencing. Absence (–) or presence (+) of inducer and TLR4 inhibitor (760 nM) is indicated. NF-κB_SEAP (B) and ISRE_Lucia (C) signals are normalized to uninfected cells. Significance by one-way ANOVA (P < 0.0001), followed by Šidák’s multiple comparisons test (adjusted P values). Lines represent mean ± SEM (n = 3). (D–F) Activation of NF-κB (D) and IRF (E), and TNF-α production (F) in WT THP-1 dual-reporter cells infected with M. tuberculosis H37Rv (MOI 5 or 10) for 3 hours. Cells were left untreated (circles) or treated with mr38548 (MIC: 0.88 µg/mL; inverted triangles) or RIF (MIC: 0.076 µg/mL; triangles) at different concentrations relative to the MIC (x) for 24 hours. Values are normalized to uninfected and untreated cells. Significance by non-parametric Friedman test: P = 0.016 (D) and P < 0.0001 (E and F), followed by Dunn’s multiple comparisons test (adjusted P values). Lines represent mean ± SEM (n ≥ 6). (G–I) Activation of NF-κB (G) and IRF (H), and TNF-α production (I) in WT THP1 dual-reporter cells infected with M. tuberculosis H37Rv (MOI 5 or 10; circles) or stimulated with LPS (100 ng/mL; diamonds) for 5 hours. Cells were left untreated or exposed to different concentrations of monoclonal anti-HSP65 IgG 2a . Values are normalized to uninfected and untreated cells. Significance by mixed-model one-way ANOVA: P = 0.0014 (G), P = 0.0002 (H), and P = 0.0274 (I), followed by Šidák’s multiple comparisons test (adjusted P values). Lines represent mean ± SEM (n ≥ 3).

Article Snippet: THP-1 DualTM and THP-1 DualTM MyD88 knock-out cells (InvivoGen) were grown in RPMI 1640 (Gibco) supplemented with 10% FBS as above, 25 mM HEPES, 100 μg/mL Pen-Strep, 100 μg/mL Normocin, 10 μg/mL Blasticidin, and 100 μg/mL Zeocin.

Techniques: Western Blot, Control, Molecular Weight, Activation Assay, Infection