thp1  (ATCC)

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: The Journal of Experimental Medicine

Article Title: Tet2 deficiency–induced expansion of monocyte-derived macrophages promotes liver fibrosis

doi: 10.1084/jem.20251114

Figure Lengend Snippet: Tet2 deficiency enhances Ccl2 and Ccl8 mRNA stability by modifying 5hmC-dependent RNA – protein interactions. (A and B) Ccl2 (A) and Ccl8 (B) mRNA decay in Tet2 +/+ and Tet2 −/− MDMs ( n = 6 for each group). (C) Tet2 -mediated oxidation of Ccl2 and Ccl8 mRNA 5mC disrupts its binding with Ybx1, Elavl1, and Zfp36. Pull-down assay was performed by incubating C, 5mC, and 5hmC oligos of Ccl2 and Ccl8 mRNA with cell lysate from THP1-derived pMDMs ( n = 3 for each group). (D) Effect of Tet2 deficiency on the binding enrichment of Ybx1, Elavl1, and Zfp36 at 3′UTR of Ccl2 and Ccl8 mRNA. Tet2-binding sites were mapped in the mRNA of Ccl2 and Ccl8 by qPCR of Ybx1, Elavl1, and Zfp36 RIP product in THP1-derived pMDMs ( n = 3 for each group). (E and F) Effect of enzymatic inactivation of Tet2 via catalytic domain mutation on stabilization of Ccl2 (F) and Ccl8 (G) transcripts ( n = 4 for each group). Data are the accumulative results from at least two independent experiments (A, B, D, E, and F) or are representative of at least two independent experiments with similar results (C and D). All data are shown as mean ± SD and were analyzed by two-tailed, unpaired Student’s t test (A, B, and D–F). ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Source data are available for this figure: .

Article Snippet: THP1 cell line was purchased from ATCC and was cultured in high-glucose DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (P/S, 100 U/ml; Hycone).

Techniques: Binding Assay, Pull Down Assay, Derivative Assay, Mutagenesis, Two Tailed Test