mouse ifn beta  (R&D Systems)

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Induction of mtRNA leakage by IFN-α-100 and ICs treatment. BMDMs (2 × 10 6 ) were stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h, and the levels of cytosolic mtRNA were determined by measuring both the heavy chain and the light chain of several mtRNA genes as indicated by RT-qPCR ( A and C ) and confocal microscopy ( B and D ). The heavy and light chains of some mtRNA genes in the supernatant were determined in BMDMs treated with IFN-α (100 U/ml) or 10 µg/ml ICs for different periods of time ( E and F ). The levels of cytosolic mtRNA in BMDMs treated with anti-chicken egg albumin Abs, albumin (ovalbumin) or a combination of anti-albumin Abs and albumin (ICs) were determined ( G ). Each data point represents one mouse, and the values are fold changes relative to the mean value of the unstimulated controls determined by RT-qPCR ( A , C , E , F , and G ). Representative results from at least 3 independent experiments are shown ( B and D ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( A , C , E , and F ) and two-way ANOVA with the Holm-Šídák’s multiple comparisons test to compare differences among different treatments ( G ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ND, NADH dehydrogenase; CYTB, cytochrome b

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Quantitative RT-PCR, Confocal Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: IFN-α-100 and ICs treatment selectively induces mitochondrial pore opening. BMDMs (2 × 10 6 ) were stimulated with IFN-α (100 U/ml) ( A ) or 10 µg/ml ICs ( B ) for 24 h, and mPTP opening was measured by calcein AM staining, and the results were analyzed by flow cytometry as described in the Materials and Methods. Images of calcein AM staining were analyzed by confocal microscopy ( C ). The protein levels of ANT1 and ANT2 were determined ( D ). The oligomerization of VDAC1 ( E and F ) and BAX ( G and H ) with or without EGS, a cross-linking reagent used to stabilize oligomers in electrophoresis, was evaluated, and the results of the statistical analysis are presented individually ( E to H ). As a positive control for BAX oligomerization, the treatment with LPS (200 ng/ml) for 4 h and then ATP (4 mM) for 1 h ( G ) or TNF-α (20 ng/ml) plus cycloheximide (CHX; 1 µg/ml) for 3 h ( H ) was given. BMDMs (2 × 10 6 ) were pre-treated with BAI-1 (2 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. As positive controls, BMDMs were pre-treated with BAI-1 (2 µM) for 2 h and then stimulated with staurosporine (STS; 2 µM) or TNF-α (20 ng/ml) plus cycloheximide (CHX; 30 µg/ml) for 3 h. MtRNA release was measured by RT-qPCR ( I ). Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A and B ). For Western blotting, the ratio of protein levels was calculated by comparing the intensity of a protein of interest to that of a housekeeping protein or monomer of protein as indicated ( D to H ). The samples were derived from the same experiment, and both the gels and the blots were processed in parallel. In ( I ), the values are fold changes relative to the mean value of the controls determined in RT-qPCR. Statistical analysis was performed with an unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ANT, adenine nucleotide translocase; Tri, trimer; Di, dimer; PDTC, pyrrolidine dithiocarbamate ammonium

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Staining, Flow Cytometry, Confocal Microscopy, Electrophoresis, Positive Control, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Inhibition of mitophagy does not block IFN-α-100 and ICs-induced mtRNA release. BMDMs (2 × 10 6 ) were stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h, and the intensity of mitophagy was determined by flow cytometry as described in the Materials and Methods section, and the statistics are presented ( A and B ). C The results of the confocal microscopic imaging studies. D and E show the inhibitory effects of Mdivi-1 (50 µM), a mitophagy inhibitor, on IFN-α-100 and ICs-induced mitophagy by flow cytometry. Mdivi-1 did not affect cytosolic mtRNA release, as determined by measuring ND1 and Co-2 mRNA expression in the cytosol ( F and G ). Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A , B , D , and E ). The values are fold changes relative to the mean value of the controls in RT‒qPCR ( F and G ). Representative results from at least 3 independent experiments are shown ( C ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( A and B ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( D to G ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The arrows show the colocalization of mitophagy dye and lysosomal marker

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Inhibition, Blocking Assay, Flow Cytometry, Imaging, Expressing, Marker

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Inhibition of mPTP opening and VDAC1 oligomerization attenuates IFN-α-100 and ICs-induced mtRNA release and downstream signaling. BMDMs (2 × 10 6 ) were pretreated with cyclosporin A (5 µM) or VBIT-12 (80 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. The intensity of mPTP opening ( A and D ), mtRNA release ( B , E , H , and K ), mRNA levels of several inflammatory genes ( C , F , I , and L ), and VDAC1 oligomerization ( G and J ) were measured as described in Materials and Methods. Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A and D ). The values are fold changes relative to the mean value of the controls as determined by RT-qPCR ( B , C , E , F , H , I , K , and L ). Representative results from at least 3 independent experiments are shown ( G and J ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( C , F , I , and L ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( A , B , D , E , H , and K ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. RIG1, retinoic acid-inducible gene I; ISG15, interferon-stimulated gene 15; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; OAS, 2′-5′-oligoadenylate synthetase; Tri, trimer; Di, dimer

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Inhibition, Flow Cytometry, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Blockade of mitochondrial calcium overload inhibits IFN-α-100 and ICs-induced mtRNA release. BMDMs (2 × 10 6 ) were pretreated with RuR (30 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. The mitochondrial Ca 2+ concentration ([Ca 2+ ]m) was measured by Rhod-2 AM staining and analyzed by flow cytometry ( A and B ). ( C ) shows the results of confocal microscopy. mPTP opening was determined with calcein AM staining and analyzed by flow cytometry ( D and E ). The levels of cytosolic mtRNA, ND1 and Co-2, ( F and H ) or mRNA of inflammatory signaling molecules ( G and I ) were measured by RT-qPCR. Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A , B , D , and E ). The values are fold changes relative to the mean value of the controls according to the results from RT-qPCR ( F , G , H , and I ). Representative results from at least 3 independent experiments are shown ( C ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( G and I ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( A , B , D , E , F , and H ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. RIG1, retinoic acid-inducible gene I; ISG15, interferon-stimulated gene 15; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; OAS, 2′-5′-oligoadenylate synthetase, TNF-α, tumor necrosis factor-α

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Concentration Assay, Staining, Flow Cytometry, Confocal Microscopy, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Endoplasmic reticulum calcium release regulates IFN-α-100 and ICs-induced mtRNA release. BMDMs (2 × 10 ) were pretreated with XeC (5 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. The signal and mitochondrial Ca 2+ concentrations ([Ca 2+ ]m) ( A , B , and C ), mPTP opening ( D and E ), cytosolic mtRNA levels ( F and H ), and mRNA levels of inflammatory signaling molecules ( G and I ) were measured as described in Fig. . Each data point represents one mouse, and the values for the flow cytometry results are presented as the geomeans (MFIs) ( A , B , D , and E ). The values are fold changes relative to the mean value of the controls ( F , G , H , and I ). Representative results from at least 3 independent experiments are shown ( C ). Statistical analysis was performed with unpaired Student’s t test to compare the means between two groups ( G and I ). Two-way ANOVA with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments ( A , B , D , E , F , and H ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. RIG1, retinoic acid-inducible gene I; ISG15, interferon-stimulated gene 15; IL, interleukin; MDA5, melanoma differentiation-associated protein 5; OAS, 2′-5′-oligoadenylate synthetase, TNF-α, tumor necrosis factor-α

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: Effects of IFN-α-100 or ICs treatment on mitochondrial membrane potential, apoptosis and cell viability. BMDMs (2 × 10 6 ) were treated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. To measure changes in mitochondrial membrane potential, JC-1 green/red staining was performed, after which the cells were analyzed by flow cytometry ( A ). Several approaches to measure cell viability, apoptosis and cell death as described in Materials and Methods were performed. These include the measurements of SYTOX staining ( B ), LDH release ( C ), CCK-8 assays ( D ), annexin-V staining ( E ), and ATP levels ( F ). BMDMs (2 × 10 6 ) were pre-treated with Z-VAD-FMK (20 µM) for 2 h and then stimulated with IFN-α (100 U/ml) or 10 µg/ml ICs for 24 h. As controls, BMDMs were pre-treated with Z-VAD-FMK (20 µM) for 2 h and then stimulated with TNF-α (20 ng/ml) plus cycloheximide (CHX; 30 µg/ml) for 3 h. The cleavaged products from PARP and caspase 3 were detected by Western blotting ( G ). Cytosolic mtRNA release was measured by RT-qPCR ( H ). Each data point represents one mouse, and the values are fold changes relative to the mean value of the controls ( A and F ). For measuring SYTOX staining and annexin V staining, the values are the percentages of cells with SYTOX Green-positive nuclei (pseudocolored red) or annexin V-positive relative to the total cell population ( B and E ). The values of LDH release are the percentage relative to the mean value of the positive controls ( C ). The values of the CCK-8 and ATP level are the readouts from the absorbance microplate reader and luminescence reader ( D and F ). Representative results from at least 3 independent experiments are shown ( B and G ). One-way ANOVA ( B , D and F ) or two-way ANOVA ( A , C , E and H ) with Holm-Šídák’s multiple comparisons was used to compare differences among different treatments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Membrane, Staining, Flow Cytometry, CCK-8 Assay, Western Blot, Quantitative RT-PCR, Microplate Reader Absorbance Measurement

Journal: Cell Communication and Signaling : CCS

Article Title: Interferon-alpha selectively signals mitochondrial pore opening to allow mitochondrial RNA release in systemic lupus erythematosus: pathophysiological implications

doi: 10.1186/s12964-026-02910-3

Figure Lengend Snippet: The sequential events involved in the regulation of mtRNA release into the cytosol by IFN-α-100 and IC treatment. Stimulation of IFN-α-100 and ICs resulted in the release of calcium from the endoplasmic reticulum that entered the mitochondria, induced mitochondrial calcium overload, and triggered the opening of mitochondrial pores, including the oligomerization of voltage dependent anion channel (VDAC) and mPTP. The activation of adenine nucleotide translocase (ANT) also participated in the opening of mitochondrial pores in the inner mitochondrial membrane. Although ANT is not an essential component of the mPTP, ANT can regulate mPTP opening. The opening of the mPTP in the inner mitochondrial membrane and the oligomerization of VDAC1 resulted in the release of mtRNA into the cytosol, which activated its sensors RIG-I and MDA5 as well as several downstream inflammatory signaling pathways to induce innate immunity

Article Snippet: Recombinant mouse IFN-α (12100–1) was purchased from PBL Assay Science (Piscataway, NJ, USA).

Techniques: Activation Assay, Membrane, Protein-Protein interactions

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Reverse transcriptase PCR gel for CFTR mRNA (predicted size of 125 bp; arrow) in Cftr +/+ and Cftr −/− mouse CD4 + T cells. Lane 1 is a 100 bp ladder. ( B ) qPCR analysis of Cftr expression in Cftr +/+ and Cftr −/− mouse CD4 + T cells at 0 and 24 hours following TCR ligation with anti-CD3 and anti-CD28 mAbs ( n = 4 per genotype and time point). ( C ) Immunostaining of CFTR (green), plasma membrane lectin (red), and nuclei (blue) in 24-hour–cultured mouse CD4 + cells (3 different biologic replicates per genotype, Cftr +/+ and Cftr −/− ). Scale bar: 10 μm. ( D ) Immunoprecipitated CFTR protein in immortalized human CD4 + T cells (Jurkat) using UNC-450 anti-CFTR monoclonal antibody (mAb) for pulldown and UNC-596 anti-CFTR mAb for detection compared with immunoprecipitation isotype control. ( E – H ) qPCR analysis of Cftr expression in Cftr +/+ and Cftr −/− cultured mouse CD4 + T cells at 0, 6, 18, 48, 72, and 78 hours in Th2 ( E ), Th1 ( F ), Th17 ( G ), and Tregs ( H ) ( n = 3 per genotype per time point). Dotted lines represent TCR ligation with anti-CD3 (1 μg/mL) and anti-CD28 (0.5 μg/mL) mAbs and (a) anti–IFN-γ (10 μg/mL) and mouse IL-4 (10 ng/mL) for Th2 cells, (b) anti–IL-4 (10 μg/mL) and mouse IL-12 (10 ng/mL) for Th1 cells, and (c) human TGF-β (0.5 ng/mL), mouse IL-23 (10 ng/mL), mouse IL-6 (40 ng/mL), mouse IL-1b (10 ng/mL), anti–IL-4 (10 μg/mL), and anti–IFN-γ (10 μg/mL) for Th17 cells. Tregs were polarized and restimulated with anti-CD3 (1 μg/mL), human IL-2 (100 IU/mL), and recombinant human TGF-β (1ng/mL). Data are shown as mean ± SD. Statistical analysis were done in B and E – H by 1-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Reverse Transcription, Expressing, Ligation, Immunostaining, Clinical Proteomics, Membrane, Cell Culture, Immunoprecipitation, Control, Recombinant

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Schematic diagram showing isolation and polarization of naive CD4 + T cells to Th2 cells for whole transcriptome analysis. ( B ) PCA plot of gene expression data for 3 biological replicates used for bulk RNA-Seq of Cftr +/+ (black dots) and Cftr −/− (red dots) Th2 cells. ( C ) Volcano plot depicting DESeq2 analysis of differentially expressed genes in Cftr +/+ and Cftr −/− Th2 cells. Red dots represent genes expressed at higher levels in Cftr −/− Th2 cells, while black dots represent genes with higher expression levels in Cftr +/+ Th2 cells. The y axis denotes −log 10 FDR values while the x axis shows log 2 fold change values. Select genes indicated. ( D ) Advanced bubble plot showing KEGG pathways enriched in Cftr −/− Th2 cells. The y axis denotes enrichment score, and −log 10 FDR values are shown on the x axis. The size of the bubble represents the number of genes enriched in each pathway. Select pathways indicated. ( E – H ) Heatmaps showing the differential gene expression profile of core Th2 associated genes including surface markers ( E ), cytokines ( F ), transcription factors ( G ), and CD4 + pan markers ( H ) in Cftr −/− versus Cftr +/+ Th2 cells. Normalized log 2 gene expression determined by RNA-Seq shown to the right of each heatmap with statistically significant DEGs denoted (*) in Cftr +/+ (gray bars) and Cftr −/− (red bars) Th2 cells. RNA-Seq data were generated in biological triplicates from 3 mice and analyzed via DESeq2. KEGG was used for pathway analysis.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Isolation, Gene Expression, RNA Sequencing, Expressing, Generated

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Schematic diagram showing isolation and polarization conditions of naive CD4 + T cells to Th2 cells for IL-4 studies. ( B ) Representative flow cytometry histogram showing the median fluorescence intensity (MFI) of IL-4Rα at 72 hours for Cftr +/+ and Cftr −/− Th2 cells. ( C ) The quantified MFI of IL-4Rα in Cftr +/+ and Cftr −/− Th2 cells at 72 hours ( n = 5 mice per genotype). ( D ) Representative flow cytometry histogram showing the MFI of GATA3 at 72 hours for Cftr +/+ and Cftr −/− Th2 cells. ( E ) The quantified MFI of GATA3 in Cftr +/+ and Cftr −/− Th2 cells at 72 hours ( n = 5 mice per genotype). ( F ) Representative CD4 + populations showing the MFI of GATA3 at 72 hours in the presence of increasing doses of polarizing IL-4 (0–40 ng/mL) for Cftr +/+ and Cftr −/− Th2 cells. ( G and H ) GATA3 MFI and secreted IL-13 from cellular supernatant in cultured Cftr +/+ (black) and Cftr −/− (red) Th2 cells in the presence of increasing doses of IL-4 (0–40 ng/mL). ( C and E ) Data are shown as mean ± SD. Statistical analysis in C and E were performed using unpaired Student’s t test and, in G and H , by 4-parameter logistic regression algorithm (sigmoidal curve fit) to fit. For G and H , data are shown as mean values with the accompanying curve fit (solid line), the 95% CI displayed as a band as well as mean data points for each genotype and concentration. ** P < 0.01 and **** P < 0.0001.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Isolation, Flow Cytometry, Fluorescence, Cell Culture, Concentration Assay

Journal: JCI Insight

Article Title: CFTR negatively reprograms Th2 cell responses, and CFTR potentiation restrains allergic airway inflammation

doi: 10.1172/jci.insight.191098

Figure Lengend Snippet: ( A ) Schematic diagram detailing the isolation and Th2 polarization of naive human CD4 + T cells used for flow cytometry and cytokine analysis. ( B ) Viability of ivacaftor (IVA) or DMSO (control) cultured human Th2 cells at 7 days. ( C ) Representative flow cytometry histogram showing the median fluorescence intensity (MFI) of GATA3 at 7 days for DMSO- and IVA-treated Th2 cells. ( D ) The quantified MFI of GATA3 in DMSO- and IVA-treated Th2 cells at 72 hours ( n = 6 paired human samples). ( E ) Representative flow cytometry histogram showing the median fluorescence intensity (MFI) of IL-13 at 7 days for DMSO- and IVA-treated Th2 cells. ( F ) The quantified MFI of IL-13 in DMSO- and IVA-treated Th2 cells at 72 hours ( n = 6 paired human samples). ( G ) IL-13 by ELISA in cellular supernatant from cultured DMSO- and IVA-treated Th2 cells. Statistical analysis in B , D , F , and G was performed using paired Student’s t test. * P < 0.05.

Article Snippet: The media were supplemented with ImmunoCult Human Th2 Differentiation Supplement (containing recombinant human IL-4 and mouse anti–human IFN-γ, StemCell Technologies) and 100 IU/mL IL-2 (NIH).

Techniques: Isolation, Flow Cytometry, Control, Cell Culture, Fluorescence, Enzyme-linked Immunosorbent Assay