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: VBIT-4 (Selleck Chemicals, s3544); H-151 (InvivoGen, INHH151); TNF-α (Biolegend, 575204); cGAMP (InvivoGen, tlrl-nacga23-02); TFAM siRNAs (Thermo Fisher Scientific, s14000 and s14001).
Techniques: Activation Assay, Western Blot, Transfection, Incubation, Protein Concentration