Journal: STAR Protocols

Article Title: Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells

doi: 10.1016/j.xpro.2025.104304

Figure Lengend Snippet: Validation of IRG1 KO by Immunoblot Analysis WT and IRG1 KO THP-1 cells were differentiated with PMA (100 nM, 72 hr) and subsequently simulated with vehicle control or LPS (200 ng/mL) and IFNγ (200 ng/mL) for 6 and 24 hr. The abundance of IRG1 is demonstrated by immunoblot analysis with β-Actin serving as a loading control.

Article Snippet: THP-1 Cells , ATCC , Cat#TIB-202.

Techniques: Biomarker Discovery, Western Blot, Control

Journal: STAR Protocols

Article Title: Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells

doi: 10.1016/j.xpro.2025.104304

Figure Lengend Snippet: Endogenously produced itaconate is required to produce IFNβ protein WT and IRG1 KO THP-1 cells were differentiated with PMA (100 nM, 72 hr) and subsequently simulated with vehicle control or LPS (200 ng/mL) and IFNγ (200 ng/mL) for 6 and 24 hr ( n = 3 biological replicates for each timepoint). Data are represented as mean ± SD.

Article Snippet: THP-1 Cells , ATCC , Cat#TIB-202.

Techniques: Produced, Control

Journal: STAR Protocols

Article Title: Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells

doi: 10.1016/j.xpro.2025.104304

Figure Lengend Snippet: Representative chromatograms for itaconate, citraconate, mesaconate, and the 13 C 5 -itaconic acid internal standard (A) neat standards for itaconate and its isomers citraconate and mesaconate. Shown are the 129.000/85.100 m/z MRM channel for itaconate, citraconate, and mesaconate (blue; 50 ng/mL each) and the 133.926/89.100 m/z MRM channel for 13 C 5 -itaconic acid (pink; 100 ng/mL). Expected retention times for itaconate/ 13 C 5 -itaconate, citraconate, and mesaconate are 2.83 min, 2.17 min, and 2.73 min, respectively. (B) cell lysate from a wild type THP-1 sample stimulated for 24 hours with LPS and IFNγ (200 ng/mL each). (C) supernatant from a wild type THP-1 culture stimulated for 24 hours with LPS and IFNγ (200 ng/mL each).

Article Snippet: THP-1 Cells , ATCC , Cat#TIB-202.

Techniques:

Journal: STAR Protocols

Article Title: Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells

doi: 10.1016/j.xpro.2025.104304

Figure Lengend Snippet: Itaconate levels quantified by LC-MS (A) Itaconate in culture supernatant and (B) cell lysate from PMA-differentiated WT and IRG1 KO THP-1 cultures stimulated with LPS and IFNγ (200 ng/mL each) ( n = 8 biological replicates for each timepoint). Data are represented as mean ± SD.

Article Snippet: THP-1 Cells , ATCC , Cat#TIB-202.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) Flowchart of the CXCL-10 experiment. ( B ) Compounds 2 , 3 , and 15 repressed ISG signaling in CXCL-10 production. THP-1 cells were pre-treated with individual compounds 48 h prior to 100 ng/mL TNFa stimulation for 48 h. CXCL-10 levels were measured. ( C ) Flowchart of the ISRE experiment. ( D ) Compounds 2 , 3 , and 15 repressed ISG signaling in IRF3 production. THP-1-Dual cells were pre-treated with individual compounds 24 h prior to 100 ng/mL TNFa stimulation for 48 h. The inhibition of type I IFN response was monitored via ISRE reporter activation. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (raw data and analysis). Figure 2—source data 1. Raw data and analysis results to generate the graphs shown in .

Article Snippet: THP-1-Dual cells (thpd-nifs, 1 × 10 6 /mL) treated with 100 ng/mL TNF (Biolegend, 575204) for 48 h, or cGAMP (InvivoGen, tlrl-nacga23-02) for 24 h to induce a cGAS-STING-dependent interferon response.

Techniques: Inhibition, Activation Assay

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) Chemical structures of hit compounds and related analogs. ( B ) TFAM modulators were profiled in the ISRE assay and display dose-dependent suppression of ISG signaling. THP-1-Dual cells were pre-treated with individual compounds 24 h prior to 100 ng/mL TNFa stimulation for 48 h. ISRE reporter activation was measured. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (raw data and analysis). Figure 3—source data 1. Raw data and analysis results to generate the graphs shown in .

Article Snippet: THP-1-Dual cells (thpd-nifs, 1 × 10 6 /mL) treated with 100 ng/mL TNF (Biolegend, 575204) for 48 h, or cGAMP (InvivoGen, tlrl-nacga23-02) for 24 h to induce a cGAS-STING-dependent interferon response.

Techniques: Activation Assay

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) ISRE reporter activation of THP-1 dual cells stimulated with 100 ng/mL TNFa for 48 h. Compounds were added to the cells 5 min before the assay detection. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (raw data and analysis).

Article Snippet: THP-1-Dual cells (thpd-nifs, 1 × 10 6 /mL) treated with 100 ng/mL TNF (Biolegend, 575204) for 48 h, or cGAMP (InvivoGen, tlrl-nacga23-02) for 24 h to induce a cGAS-STING-dependent interferon response.

Techniques: Activation Assay

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) Compounds 2 , 3 , and 15 do not repress cGAMP induced ISG signaling. THP-1-Dual cells were pre-treated with individual compounds 48 h prior to 10 ug/mL cGAMP stimulation for 24 h. ISRE reporter activation was measured. ( B ) Compounds 2 , 3 , and 11 impart a dose-dependent increase in TFAM protein levels. Immunoblot analysis of TFAM from T47D cells treated with indicated compounds for 5 days. ( C ) Compounds exhibit minimal impact on TFAM mRNA levels. ( D ) Downregulation of TFAM attenuates the function of compounds 2 and 3 in repression of ISG signaling. THP-1-Dual cells were treated with individual compounds 24 h after siRNA transfection. After incubation for 24 h, THP-1-Dual cells were stimulated with 100 ng/mL TNFa for another 48 h. ISRE reporter activation was measured and normalized to protein concentration. ( E ) Compound 3 inhibits mtDNA cytosolic leakage. THP-1 cells were pre-treated with individual compounds 72 h prior to 100 ng/mL TNFa stimulation for 48 h. Cytosolic mtDNA was extracted and quantified using a qPCR assay. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (original uncropped blots) and (annotated uncropped blots), and (raw data and analysis). Figure 4—source data 1. Original uncropped western blot images for . Figure 4—source data 2. Annotated uncropped western blot images for , with treatment conditions and protein identities indicated. Figure 4—source data 3. Raw data and analysis results to generate the graphs shown in .

Article Snippet: THP-1-Dual cells (thpd-nifs, 1 × 10 6 /mL) treated with 100 ng/mL TNF (Biolegend, 575204) for 48 h, or cGAMP (InvivoGen, tlrl-nacga23-02) for 24 h to induce a cGAS-STING-dependent interferon response.

Techniques: Activation Assay, Western Blot, Transfection, Incubation, Protein Concentration

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) CXCL10 protein levels from THP-1 cells stimulated with increasing concentrations of TNFa for 24 h and 48 h. ( B ) Pre-treatment of cells with H151 for 48 h inhibited CXCL10 secretion in a dose-dependent fashion. THP-1 cells were pre-treated with H151 for 48 h prior to TNFa (100 ng/mL) stimulation for 48 h followed by measurement of CXCL-10. ( C ) Pre-treatment of cells with VBIT-4 for 48 h inhibited CXCL10 secretion in a dose-dependent fashion. THP-1 cells were pre-treated with VBIT-4 for 48 h prior to TNFa (100 ng/mL) stimulation for 48 h followed by measurement of CXCL-10. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (raw data and analysis).

Article Snippet: THP-1 (TIB-202) and T-47D (HTB-133) cell lines were obtained from ATCC.

Techniques:

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) Flowchart of the CXCL-10 experiment. ( B ) Compounds 2 , 3 , and 15 repressed ISG signaling in CXCL-10 production. THP-1 cells were pre-treated with individual compounds 48 h prior to 100 ng/mL TNFa stimulation for 48 h. CXCL-10 levels were measured. ( C ) Flowchart of the ISRE experiment. ( D ) Compounds 2 , 3 , and 15 repressed ISG signaling in IRF3 production. THP-1-Dual cells were pre-treated with individual compounds 24 h prior to 100 ng/mL TNFa stimulation for 48 h. The inhibition of type I IFN response was monitored via ISRE reporter activation. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (raw data and analysis). Figure 2—source data 1. Raw data and analysis results to generate the graphs shown in .

Article Snippet: THP-1 (TIB-202) and T-47D (HTB-133) cell lines were obtained from ATCC.

Techniques: Inhibition, Activation Assay

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) Chemical structures of hit compounds and related analogs. ( B ) TFAM modulators were profiled in the ISRE assay and display dose-dependent suppression of ISG signaling. THP-1-Dual cells were pre-treated with individual compounds 24 h prior to 100 ng/mL TNFa stimulation for 48 h. ISRE reporter activation was measured. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (raw data and analysis). Figure 3—source data 1. Raw data and analysis results to generate the graphs shown in .

Article Snippet: THP-1 (TIB-202) and T-47D (HTB-133) cell lines were obtained from ATCC.

Techniques: Activation Assay

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) ISRE reporter activation of THP-1 dual cells stimulated with 100 ng/mL TNFa for 48 h. Compounds were added to the cells 5 min before the assay detection. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (raw data and analysis).

Article Snippet: THP-1 (TIB-202) and T-47D (HTB-133) cell lines were obtained from ATCC.

Techniques: Activation Assay

Journal: eLife

Article Title: Suppression of interferon signaling via small-molecule modulation of TFAM

doi: 10.7554/eLife.108742

Figure Lengend Snippet: ( A ) Compounds 2 , 3 , and 15 do not repress cGAMP induced ISG signaling. THP-1-Dual cells were pre-treated with individual compounds 48 h prior to 10 ug/mL cGAMP stimulation for 24 h. ISRE reporter activation was measured. ( B ) Compounds 2 , 3 , and 11 impart a dose-dependent increase in TFAM protein levels. Immunoblot analysis of TFAM from T47D cells treated with indicated compounds for 5 days. ( C ) Compounds exhibit minimal impact on TFAM mRNA levels. ( D ) Downregulation of TFAM attenuates the function of compounds 2 and 3 in repression of ISG signaling. THP-1-Dual cells were treated with individual compounds 24 h after siRNA transfection. After incubation for 24 h, THP-1-Dual cells were stimulated with 100 ng/mL TNFa for another 48 h. ISRE reporter activation was measured and normalized to protein concentration. ( E ) Compound 3 inhibits mtDNA cytosolic leakage. THP-1 cells were pre-treated with individual compounds 72 h prior to 100 ng/mL TNFa stimulation for 48 h. Cytosolic mtDNA was extracted and quantified using a qPCR assay. Figures are representatives of at least two independent experiments. Graph shows one representative experiment of two independent experiments. Error bars represent ± SD from n=3 biological replicates. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Source data for this figure are available in (original uncropped blots) and (annotated uncropped blots), and (raw data and analysis). Figure 4—source data 1. Original uncropped western blot images for . Figure 4—source data 2. Annotated uncropped western blot images for , with treatment conditions and protein identities indicated. Figure 4—source data 3. Raw data and analysis results to generate the graphs shown in .

Article Snippet: THP-1 (TIB-202) and T-47D (HTB-133) cell lines were obtained from ATCC.

Techniques: Activation Assay, Western Blot, Transfection, Incubation, Protein Concentration

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Targeting HMGB1 signalling improves therapeutic outcomes in NSCLC. (A) Correlation analysis between immune infiltration scores and HMGB1 expression in 491 LUAD and 500 LUSC patients from the TCGA database. (B) Correlation between HMGB1 expression and the distribution of various immune cell subsets in LUAD and LUSC patients. (C, D) THP‐1–derived M0 macrophages were treated with PBS, HMGB1 (10 or 100 ng) or exosomes derived from vector or HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). M1 macrophage markers (CD86, CD80, iNOS) and M2 markers (CD206, IL‐10, Arg1) were quantified by PCR. (E) Lewis tumour‐bearing mice were treated with PBS, HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse, twice per week), anti‐PD‐1 antibody (RMP1‐14, 200 μg per mouse, twice per week) or combination therapy ( n = 5 per group). Tumour volumes and apoptosis levels in tumour tissues (day 25) were assessed. (F) PC9 cells were treated with PBS or exosomes from HMGB1 OE cells (cell‐to‐exosome ratio = 1:10), followed by Osimertinib (50 nM, 48 h), and apoptosis was measured. (G) A549 and PC9 cells were similarly treated with PBS or HMGB1 OE‐derived exosomes, followed by Cisplatin (5 μM, 48 h), and apoptosis was analysed. (H) A549 and PC9 cells were similarly treated with paclitaxel (10 μM, 48 h) under the same conditions, and cell apoptosis was determined. (I) A549‐bearing mice were treated with HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse), followed by PBS, paclitaxel (PTX, 10 mg/kg, twice per week), STAT3 inhibitor (5 mg/kg, twice per week) or combination therapy. (J) Schematic diagram illustrating the proposed mechanism: HMGB1 upregulates TLR4, thereby activating the NF‐κB–IL‐6 axis and stimulating JAK2/STAT3 signalling to promote tumour progression. Concurrently, HMGB1 facilitates M2 macrophage polarisation.

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Derivative Assay, Plasmid Preparation