Journal: Smart Molecules

Article Title: Endocytosis‐independent cytosolic entry of messenger RNA via fluorous bilayer zippering attenuating Toll‐like receptor signaling and enables ischemic tissue salvage

doi: 10.1002/smo2.70085

Figure Lengend Snippet: Biocompatibilities and immunogenicity of mRNA‐encapsulating polyplexes. (a) Cell viabilities of HUVECs upon 24 h incubation in presence of a variety of mRNA‐encapsulating polyplexes (mean ± s.d., n = 4, ** p < 0.01, student t test). (b) Hemolytic activities of sheep red blood cells upon 2 h incubation with varied concentrated delivery materials (*** p < 0.005; Student's t ‐test, mean ± s.d., n = 4). (c) confocal laser scanning microscopy (CLSM) measurement for assessment of overall cellular internalization of mRNA‐encapsulating polyplexes into RAW264.7 cells. (d) Interferon‐β (IFN‐β): at 4 h post incubation, the expression levels of inflammatory molecules were measured with qRT‐PCR. (e) Interleukin‐8 (IL‐8): at 4 h post incubation, the expression levels of inflammatory molecules were measured with qRT‐PCR. (** p < 0.01, *** p < 0.005; Student's t ‐test, n = 4).

Article Snippet: Culture supernatants (100 μL/well) were harvested and assayed for IFN‐α using the Mouse IFN‐α ELISA Kit (PBL Interferon Source) according to the manufacturer's instructions.

Techniques: Immunopeptidomics, Incubation, Confocal Laser Scanning Microscopy, Expressing, Quantitative RT-PCR

Journal: Smart Molecules

Article Title: Endocytosis‐independent cytosolic entry of messenger RNA via fluorous bilayer zippering attenuating Toll‐like receptor signaling and enables ischemic tissue salvage

doi: 10.1002/smo2.70085

Figure Lengend Snippet: Revascularization in hindlimbs by local dosage of mVEGF‐encapsulating polyplexes. (a) Therapeutic scheme. (b) Anatomy of the established hindlimb ischemia model. Ligations were made in the femoral artery at the proximal and distal sites. (c) Angiogenesis in mouse hindlimbs post ligation. (d) Visualization of blood flow by Laser Speckle Flowgraphy on Day 28 post‐dosage of mVEGF therapeutics (mVEGF: 10 μg). The magnified inset images captured by intravital confocal laser scanning microscopy (CLSM), revealing vasculature details by intravenous dosage of FITC‐dextran (MW: 10 kDa). (e) Estimation of blood perfusion volume based on quantification by laser speckle flowgraphy on Day 28 post‐dosage of mVEGF therapeutics (mVEGF: 10 μg). The data were represented as the mean ± standard deviations (s.d.) ( n = 5). (* p < 0.05, ** p < 0.01, student t test). (f) Quantification of the expressed VEGF protein on day 4 post dosage by ELISA.

Article Snippet: Culture supernatants (100 μL/well) were harvested and assayed for IFN‐α using the Mouse IFN‐α ELISA Kit (PBL Interferon Source) according to the manufacturer's instructions.

Techniques: Ligation, Confocal Laser Scanning Microscopy, Enzyme-linked Immunosorbent 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: 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