thp 1 dual cells  (InvivoGen)

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: Small Science

Article Title: Triplet RNA Lipid Nanoparticles for Locoregional Cancer Immunotherapy

doi: 10.1002/smsc.202500506

Figure Lengend Snippet: Nanoparticulate pIpC induces IRF responses in vitro and significantly reduces tumor growth resulting in increased survival after IT injection. A) THP‐1 dual monocytic cells were simulated with either soluble pIpC (Sol. pIpC) or pIpC incorporated into a lipid nanoparticle (pIpC‐LNP) at a range of concentrations for 24 h. Following the simulation, supernatant was removed and assayed for IRF reporter using Quanti‐Luc reagent. Data plotted represents the relative light unit (RLU) readout from n = 3 repeat studies. Data was analyzed with a 2‐way ANOVA followed by Sidak's multi comparison test. LNPs were formulated with either negative siRNA (siNEG) or pIpC conjugated to fluorescein (pIpC‐FL); DiR dye was incorporated at a final concentration of 1% molar ratio. B16F10 cells were incubated with LNP (1 μg mL −1 ) for 24 h before being stained with Zombie Aqua viability dye; LNP uptake was assessed using flow cytometry. B) The uptake of LNP in viable cells. C) Confocal images of B16‐F10 cells incubated with DiI‐labeled pIpC‐FL‐LNP for 2, 4, 8, and 24 h. The cell nuclei were stained using Hoechst 33342 (Blue); the endo/lysosomes were stained using LysoTracker Red (red); pIpC‐FL and LNP were tracked using lipophilic dye DiI (magenta). Individual confocal channels, brightfield, and merged images were presented. Column Merged 1 combined the blue‐red‐green channel and column Merge 2 combined the blue‐red‐green‐magenta channel. Scale bar represents 10 μm. C57BL/6 ( n = 8) were implanted with 1 × 10 6 B16F10 melanoma cells on day 0, and at days 7 and 9, mice were treated intratumorally with Sol. pIpC or pIpC LNP (15 μg per injection). Tumor growth was monitored, and mice were culled at their humane endpoint (tumor diameter 15 mm). D) The mean ± SEM of tumor volume is presented in tumor growth curve. E) Statistical analysis was carried out using a Student's T test; mouse survival is presented in Kaplan–Meier plot, followed by Mantel–Cox test; ns, nonsignificant, * p < 0.05, *** p < 0.005.

Article Snippet: THP‐1 dual cells were purchased from InvivoGen and cultured in RPMI 1640 medium supplemented with 10% heat‐inactivated FBS and selective antibiotics Zeocin (100 μg mL −1 ) and Blasticidin (10 μg mL −1 ).

Techniques: In Vitro, Injection, Comparison, Concentration Assay, Incubation, Staining, Flow Cytometry, Labeling