Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Colocalization between SINV RNA FISH and Broccoli. The images (objective lens 20×) show infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS 6 h after infection. After adding DFHBI-1T, RNA FISH E1 + E2 (red) colocalizes well with Broccoli-DFHBI-1T (green) signal for both TEUTR4Br and TEds10Br. The scatter plot (X – red, Y – green) shows strong colocalization (R colocalization = 0.9049). Scale bar = 100 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Infection

Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Rare discrepancy between RNA FISH and Broccoli signals. The images (objective lens 63×) show TEds10Br-infected BHK cells (MOI = 5), fixed with 3.7% formaldehyde in PBS at 6 h after infection. Although rare, some cells showed a discrepancy between FISH and Broccoli signals (white arrow), resulting in a separated cluster in the scatter plot (X – red, Y – green). Scale bar = 20 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Infection

Journal: Scientific Reports

Article Title: Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

doi: 10.1038/s41598-020-61573-3

Figure Lengend Snippet: Live cell imaging of infected cells in mouse brain slices incubated with DFHBI-1T. ( A ) Channel mode and ( B ) lambda mode imaging of fresh TEds10Br-infected brain or ( C ) TE-infected brain. Broccoli-DFHBI-1T signal (green) in cells of the corpus callosum. ( D ) RNA E1 FISH imaging (red) of infected neurons in formalin-fixed brain tissue counterstained with DAPI. ( E ) Image of the 250 μm slice (McIlwain tissue chopper) of TEds10Br-infected mouse brain (3 days after infection) in a 35-mm glass-bottom dish in imaging medium containing DFHBI-1T with area of imaging for A, B indicated in the red square. Scale bar = 50 μm.

Article Snippet: Cells in each well were then incubated with two custom Stellaris RNA FISH probe sets each containing 48 short Quasar 570-labeled non-overlapping oligonucleotides against SINV E1 and E2 genes (LGC Biosearch Technologies), 25 nM each, in hybridization buffer (working buffer plus 10% dextran) at 37 °C overnight.

Techniques: Live Cell Imaging, Infection, Incubation, Imaging

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) IFNAR1 blockade does not enhance Nrf2 upregulation in TNF-stimulated B6.Sst1S macrophages. B6 and B6.Sst1S BMDMs were treated with 10 ng/m TNF, with or without IFNAR1-blocking antibodies or isotype control (Isotype C Ab) at 5 μg/mL concentration for 4, 8, and 12 hr. Nrf2 protein levels were quantified by western blot. Average densitometric values from two separate experiments were included above the blot. ( B ) IFNAR1 blockade does not increase Ftl expression in TNF-stimulated B6.Sst1S macrophages. B6.Sst1S BMDMs were treated with 10 ng/mL TNF, with or without IFNAR1-blocking antibodies (5 μg/mL) or Isotype C Ab (5 μg/mL), for 8 and 12 hr. Ftl protein levels were quantified by western blot. Average densitometric values from two independent experiments were included above the blot. ( C ) IFNAR1 blockade does not increase mRNA levels of Fth , Ftl , and Gpx1 . B6 and B6.Sst1S BMDMs were treated with 10 ng/mL TNF, with or without IFNAR1-blocking antibodies or Isotype C Ab, for 12 hr. Blocking antibodies (5 μg/mL) or isotype C antibodies (5 μg/mL) were added 2 hr after TNF stimulation. Fold induction was calculated using B6 untreated control as average one-fold by utilizing the ΔΔCt method with β-actin as the internal control. ( D and E ) IFNAR1 blockade reduces Rsad2 mRNA levels ( E ) but does not affect Ifnb1 mRNA levels ( D ) B6 and B6.Sst1S BMDMs were treated with 10 ng/mL TNF, with or without IFNAR1-blocking antibodies or Isotype C Ab for 16 hr. Blocking antibodies (5 μg/mL) or isotype C antibodies (5 μg/mL) were added 2 hr after TNF stimulation. Fold induction was calculated using B6 untreated control as average onefold by utilizing the ΔΔCt method with β-actin as the internal control. ( F ) Lipid peroxidation inhibition prevents the superinduction of Ifnb1 mRNA. B6.Sst1S BMDMs were treated with 10 ng/mL TNF, and the lipid peroxidation inhibitor (Fer-1) was added 2 hr post-TNF stimulation. Ifnb1 mRNA levels were measured using qRT-PCR after 16 hr of TNF treatment. Fold induction was calculated using untreated control as average onefold by utilizing the ΔΔCt method with 18 S as the internal control. ( G ) Lipid peroxidation inhibition reverses the superinduction of Ifnb1 mRNA. B6.Sst1S BMDMs were stimulated with 10 ng/mL TNF for 18 hr, then the LPO inhibitor (Fer-1) was added for the remaining 12 hr. Ifnb1 mRNA levels were measured using qRT-PCR. Fold induction was calculated using untreated control as average onefold by utilizing the ΔΔCt method with 18 S as the internal control. ( H and I ) IFNAR1 blockade reduces 4-HNE adduct accumulation in B6.Sst1S BMDMs treated with TNF (10 ng/mL) for 48 hr. Blocking antibodies (5 μg/mL) or Isotype C Ab (5 μg/mL) were added 2 hr post-TNF stimulation. The data are presented as means ± standard deviation (SD) from three to five samples per experiment, representative of three independent experiments. The statistical significance was performed by two-way ANOVA using Tukey’s multiple comparison test (Panels C-E), Ordinary one-way ANOVA using Bonferroni’s multiple comparison test (Panels F-G and I). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Blocking Assay, Control, Concentration Assay, Western Blot, Expressing, Inhibition, Quantitative RT-PCR, Standard Deviation, Comparison

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) The lack of Myc mRNA downregulation after prolonged TNF stimulation in B6.Sst1S macrophages. BMDMs from B6 and B6.Sst1S were treated with 10 ng/mL TNF for 6, 12, and 24 hr. Expression of Myc was quantified by the ΔΔCt method using qRT-PCR and expressed as a fold induction compared to the untreated B6 BMDMs. β-actin was used as the internal control. ( B ) Myc protein levels expressed by B6 and B6.Sst1S BMDMs during the course of stimulation with TNF(10 ng/mL) for 6 and 12 hr. (western blot). Average densitometric values from two independent experiments were included above the blot. ( C ) Myc inhibition restored the levels of Fth and Ftl proteins in TNF-stimulated B6.Sst1S macrophages to the B6 levels. B6 and B6.Sst1S BMDMs were treated with 10 ng/mL TNF alone or in combination with Myc inhibitor, 10058-F4 (10 μM) for 24 hr. 10058-F4 was added 2 hr post TNF stimulation. Protein levels of Fth and Ftl were observed using western blot. Average densitometric values from two independent experiments were included above the blot. ( D ) Myc inhibition decreased the labile iron pool in TNF-stimulated B6.Sst1S macrophages. B6.Sst1S BMDMs were treated with 10 ng/mL TNF or left untreated for 48 hr. The 10058-F4 inhibitor was added 2 hr post TNF stimulation. The labile iron pool (LIP) was measured using the Calcein AM method and represented as fold change as compared to untreated control. DFO was used as a negative control, and FeSO4 was used as a positive control. ( E and F ) Myc inhibition reduced lipid peroxidation in TNF-stimulated B6.Sst1S BMDMs. Cells were treated with 10 ng/mL TNF in the presence or absence of 10058-F4 for 48 hr. The inhibitor was added 2 hr post TNF stimulation. The MDA production was measured using commercial MDA assay ( E ) The lipid peroxidation was measured by fluorometric method using C11-Bodipy 581/591 ( F ). ( G ) B6.Sst1S BMDMs were treated as above in E. The accumulation of lipid peroxidation product, 4-HNE after 48 hr was detected by confocal microscopy using 4-HNE-specific antibody. The 4-HNE adducts accumulation was quantified using ImageJ and plotted as fold accumulation compared to untreated group. ( H ) The BMDMs from B6.Sst1S were treated with 10 ng/mL TNF alone or in combination with Myc inhibitor, 10058-F4 (10 μM) for 24 hr. 10058-F4 was added 2 hr post TNF stimulation. Expression of Ifnb1 , Rsad2, Trib3, and Chac1 was quantified by the ΔΔCt method using qRT-PCR and expressed as a fold induction compared to the untreated group. 18 S was used as the internal control. ( I and J ) B6 ( I ) and B6.Sst1S ( J ) BMDMs were treated with TNF (10 ng/ml) for 6, 12, and 24 hr in the presence or absence of JNK inhibitor D-JNK1 (2 μM). The cells were harvested and the protein levels of c-Myc and p-cJun were determined by western blotting. JNK inhibitor D-JNK1 was added 2 hr post TNF stimulation. Average densitometric values from two independent experiments were included above the blot. The data are presented as means ± standard deviation (SD) from three to five samples per experiment, representative of three independent experiments. The statistical significance was performed by two-way ANOVA using Šídák’s multiple comparison test (Panel A) and ordinary one-way ANOVA using Šídák’s multiple comparison test (Panels D-F and H). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Figure 5—source data 1. PDF file containing original western blots for indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Inhibition, Negative Control, Positive Control, Multiple Displacement Amplification, Confocal Microscopy, Standard Deviation, Comparison

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) Myc inhibitor 10058-F4 (10 μΜ) prevents the 4-HNE adducts accumulation in TNF-stimulated B6.Sst1S BMDMs. B6.Sst1S BMDMs were treated with 10 ng/mL TNF for 48 hr. The inhibitor was added 2 hr post TNF stimulation. The accumulation of lipid peroxidation product, 4-HNE, was detected by confocal microscopy using 4-HNE-specific antibody. Scale bar = 20 μm. ( B ) Myc inhibition reduced lipid peroxidation in TNF-stimulated B6.Sst1S BMDMs. Cells were treated with 10 ng/mL TNF in the presence or absence of 10058-F4 for 48 hr. The inhibitor was added 2 hr post TNF stimulation. The accumulation of lipid peroxidation product, 4-HNE, was detected by confocal microscopy using 4-HNE-specific antibody. The 4-HNE adducts accumulation was quantified using ImageJ and plotted as fold accumulation compared to untreated group. ( C ) Media change induces Myc upregulation and similar downregulation in B6 and B6.Sst1S BMDMs. Myc protein levels were monitored using western blot. ( D ) Selection of non-toxic concentration of CSF1R inhibitors. BMDMs from B6.Sst1S were either left untreated or treated with 10 ng/mL TNF. Post 2 hr of TNF stimulation, the inhibitors of CSF1R were added at different concentrations. PLX3397 (30, 10, and 3 nM), BLZ945 (100, 30, and 10 nM), and GW2580 (30, 10, and 3 nM) for 46 hr. Percent of cell number was determined by automated microscopy. ( E and F ) CSF1R inhibitors do not prevent the IFN-I pathway hyperactivity in TNF-stimulated B6.Sst1S macrophages. BMDMs from B6.Sst1S were treated with 10 ng/mL TNF alone or in combination with CSF1R inhibitors, PLX3397 (3 nM), BLZ945 (10 nM), and GW2580 (10 nM) for 20 hr. CSF1R inhibitors were added 2 hr post TNF stimulation. Expression of Ifnb1 and Rsad2 were quantified by the ΔΔCt method using qRT-PCR and expressed as a fold induction compared to the untreated group. 18 S was used as the internal control. The data are presented as means ± standard deviation (SD) from three to five samples per experiment, representative of three independent experiments. The statistical significance was performed by ordinary one-way ANOVA using Šídák’s multiple comparison test (Panels B, E and F ). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Figure 5—figure supplement 1—source data 1. PDF file containing original western blots for indicating the relevant bands and treatments. Figure 5—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Confocal Microscopy, Inhibition, Western Blot, Selection, Concentration Assay, Microscopy, Expressing, Quantitative RT-PCR, Control, Standard Deviation, Comparison

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) Representative 3D confocal images of paucibacillary (n=16) and multibacillary (n=16) pulmonary TB lesions of B6.Sst1S, Ifnb1 -YFP reporter mice stained with anti-4-HNE antibody (yellow). Cells expressing YFP are green, Mtb reporter Mtb (smyc':: mCherry) is red. Arrows indicate Mtb reporter strain expressing mCherry. The mice were infected for 20 weeks. ( B ) Representative fluorescent multiplexed immunohistochemistry (fmIHC) images of pauci-bacillary and multi-bacillary PTB lesion in B6.Sst1S mice at high magnification (×600). 4-HNE adducts (magenta), CD11b (green), and DAPI (gray). White areas showing 4-HNE adducts and CD11b co-localization. The mice were infected for 20 weeks. ( C ) Heatmap of interferon-inducible genes differentially expressed in Iba1 + cells within multibacillary vs paucibacillary lesions (fold change 1.5 and above). Pooled gene list of IFN type I and II regulated genes was assembled using public databases (as described in Materials and methods). The mice were infected for 14 weeks. ( D ) Representative fmIHC images of IFN-I producing (YFP positive) myeloid cells in pauci-bacillary (n=8) and multi-bacillary (n=8) lesion of B6.Sst1S, Ifnb1 -YFP reporter mice. The different markers are shown as Iba1 (red), iNOS (teal), and YFP (green) at ×400 magnification. The mice were infected for 20 weeks. ( E ) Representative fmIHC images of IFN-I producing (YFP positive) cells accumulating stress markers in pauci-bacillary (n=6) and multi-bacillary (n=9) lesion of B6.Sst1S, Ifnb1 -YFP reporter mice. The different markers are shown as phospho-c-Jun (peach), Chac-1 (yellow), and YFP (green) at ×400 original magnification. The mice were infected for 20 weeks.

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Staining, Expressing, Infection, Immunohistochemistry

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) 3D images of uninvolved lung and pulmonary TB lesions in Mtb-infected B6.Sst1S, Ifnb1 -YFP mice presented in at low and high magnification. 4-HNE + staining is yellow, and the reporter Mtb (smyc':: mCherry) is red. The mice were infected for 20 weeks. Scale bar = 10 μm. Arrows indicate Mtb reporter strain expressing mCherry. Lower panels are magnified areas of insets shown in top panels. ( B ) Representative fluorescent single-channel images of pauci- and multibacillary PTB lesions in B6.Sst1S mice and merged images corresponding to . 4-HNE adducts (magenta), CD11b (green), and DAPI (gray).

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Infection, Staining, Expressing

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) Confocal images of BMDMs isolated from B6.Sst1S, Ifnb1 -YFP mice stimulated with TNF (10 ng/ml) in vitro for 24 hr or left untreated. The endogenous YFP signal is shown in green. Staining with anti-YFP antibody was used to demonstrate YFP expression in activated BMDMs, as a control of specificity. Nuclei in blue (DAPI). Scale bar = 50 μm. ( B ) Confirmation of YFP signal from B6.Sst1S, Ifnb1 -YFP reporter mice using Ifnb1 mRNA in situ hybridization. 3D confocal images of fluorescent in situ hybridization with Ifnb1 mRNA probes (red) showed overlap with YFP + cells. Insets at the lower right corner are an enlarged image of the inset at the top right corner, which is showing the same cells expressing both signals. The mice were infected for 20 weeks. Scale bar = 10 μm.

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Isolation, In Vitro, Staining, Expressing, Control, In Situ Hybridization, Infection

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: ( A ) Quantification of IFN-I producing (YFP positive) myeloid cells in pauci-bacillary (n=8) and multi-bacillary (n=8) lesions of B6.Sst1S, Ifnb1 -YFP reporter mice. The quantification was performed by manually counting total and individual group of markers at different lesions at ×400 magnification and calculated the percentage cell number as compared to total DAPI. The average total cell number (DAPI) per field was 140. ( B and C ) fmIHC images and quantification of total macrophages (Iba1 positive) co-expressing stress markers in paucibacillary (n=15) and multibacillary (n=15) PTB lesions. The quantification was performed using Halo automated analysis as percent lesion area. The different markers are shown as p-c-Jun (peach), Chac-1 (yellow), and Iba1 (red) at ×200 magnification. The mice were infected for 20 weeks. ( D ) Quantification of IFN-I producing (YFP positive) cells accumulating stress markers in pauci-bacillary (n=6) and multi-bacillary (n=9) lesion of B6.Sst1S, Ifnb1 -YFP reporter mice using Halo automated analysis. ( E and F ) Quantification and representative fmIHC images of activated (iNOS+) macrophages co-expressing stress markers in paucibacillary (n=14) and multibacillary (n=14) PTB lesions. The quantification was performed using Halo automated analysis, and the signals are presented as percent lesion area. The different markers are shown as phospho-c-Jun (peach), Chac-1 (yellow), and iNOS (teal) at ×400 magnification. The mice were infected for 20 weeks. The data represent the means ± SD and the statistical significance was performed by two-tailed unpaired t-test (Panel A, C–E ). Significant differences are indicated with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Expressing, Infection, Two Tailed Test

Journal: eLife

Article Title: Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility

doi: 10.7554/eLife.106814

Figure Lengend Snippet: Common for B6 and B6.Sst1S: 1. TNF activates c-Myc expression. 2. TNF induces moderate Ifnb1 expression. 3. TNF stimulation upregulates Nrf2. B6-specific: 4. Ifnβ induces the sst1-encoded SP110 and SP140 nuclear proteins that co-activate Nrf2 and suppress c-Myc. 5. Nrf2 activates antioxidant defense (AOD) that inhibits lipid peroxidation (LPO). B6.Sst1S-specific: 6. Sp110 and Sp140 are not expressed. 7. Myc is upregulated, inhibits ferritin expression, and coactivates Ifnb1 transcription. 8. Deficient AOD activation coupled with increased labile iron pool promotes accumulation of LPO products. 9. LPO further co-stimulates Ifnb1 superinduction. 10. IFN-I hyperactivity activates ISR and induces Chac1 that further inhibits the AOD and increases LPO. 11. Alternative mechanisms of IFN-mediated dysregulation of AOD defense and iron homeostasis.

Article Snippet: Commercial assay or kit , HCR RNA-FISH probe set targeting Ifnb1 mRNA (custom design) , Molecular Instruments , N/A , .

Techniques: Expressing, Activation Assay