Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 1 | Phylogenetic analysis of complete genome (A) and VP24 amino acid sequences (B) of nine filoviruses. Sequences were obtained from Genbank: Bombali (BOMV; Accession number MF319185), Bundibugyo (BDBV; KC545394), Ebola (EBOV; KM233113), Reston (RESTV; KY798006), Sudan (SUDV; KC545389), Taï Forest (TAFV; KU182910), Lloviu (LLOV; NC016144.1), Marburg (MARV; NC001608.3), and Mengla virus (MLAV; KX371887) sequences were aligned with MUSCLE. Table (C) presents the amino acid sequence identities between filovirus VP24s. Phylogenetic analysis was carried out using maximum likelihood method based on the general time reversible (GTR) model for nucleotide sequences and general reverse transcriptase model for amino acid sequences with correction for gamma distribution to reconstruct the phylogenetic trees in MEGA 7 software platform. Evolutionary invariable sites were allowed in the GTR-model. The substitution models were chosen based on the model test in MEGA 7. One thousand bootstrap pseudoreplicates were used to estimate the reliability of the analysis.

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Virus, Sequencing, Reverse Transcription, Software

Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 2 | Subcellular location of nine filovirus VP24 proteins. Huh7 cells were transfected for 24 hours with different filovirus VP24 expression plasmids (2.5µg/well in 6-well plates). For imaging purposes cells were fixed and permeabilized followed by labeling with anti-HA antibody and fluorescent secondary antibodies. (A) Representative images of filovirus VP24 expressing Huh7 cells. BDBV* and MARV* refer to codon optimized expression constructs of the respective genes due to the low or undetectable expression of the original gene sequences in Western blotting. (B) Quantification of subcellular localizations of different filovirus VP24s. The quantitation was based on 3 experimental repeats in each of which 100-200 cells per VP24 were analyzed, scale bar 10µm, error bars are standard error of means. (C) Representative image of RESTV VP24 expressing Huh7 cell stained with anti-HA antibody (for VP24) and MitoTracker® stain is shown. (D) Western blot analysis of indicated VP24 proteins and controls in HEK293 cells. The cells were transfected with 1200 ng in 12-well plate for 24 h, protein samples were collected and 20% of total cellular proteins were separated on 10% SDS-PAGE, transferred onto PVDF membranes and detected with anti-HA and anti-GAPDH antibodies followed by Alexa-anti-mouse-568.

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Transfection, Expressing, Imaging, Labeling, Construct, Western Blot, Quantitation Assay, Staining, SDS Page

Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 3 | Binding of filovirus VP24 proteins to human importin a5. (A) HEK293 cells were transiently transfected in a 6-well format with BOMV, RESTV, SUDV, TAFV, EBOV or LLOV VP24 expression plasmids (4 mg/well). At 24 h post-transfection, soluble cell extracts were prepared, and proteins in cell extracts were allowed to bind to empty beads or bead-immobilized GST, GST-importin a5, and influenza A virus GST-nucleoprotein (GST-IAV-NP) as another nonspecific binding control. Bound proteins were separated on 12% SDS-PAGE, and the presence of VP24 proteins were analyzed by immunoblotting with anti-HA antibodies. Control lanes include ca. 5% of the amount of input cell extracts for different VP24 proteins (input 1:20). (B) [35S]Met/Cys-labeled and in vitro-translated BDBV, MARV and MLAV VP24 proteins were allowed to bind to beads alone and immobilized GST, GST-importin a5 and GST-IAV-NP. Bound proteins were separated on 12% SDS-PAGE, and the presence of VP24 proteins were analyzed by autoradiography. Control lanes show in vitro-translated BDBV, MARV and MLAV VP24 proteins, and each lane represents 1:20 of the amount of reticulate lysate that was used in each binding experiment. (C) Coomassie Blue-stained gel is shown to visualize the amount of Glutathione Sepharose-bound GST, GST-Importin a5, and GST-IAV-NP. (D) Quantitation of relative VP24 binding efficacy to GST-importin a5. VP24 band intensities in immunoblots were scanned and the percentage of GST-importin a5 bound VP24 of the input is shown. The percentage was obtained by dividing the intensity of bound VP24 signal with the input signal multiplied by 20 (loading included 1:20 of the amount of VP24 in binding experiments).

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Binding Assay, Transfection, Expressing, Virus, Control, SDS Page, Western Blot, Labeling, In Vitro, Autoradiography, Staining, Quantitation Assay

Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 5 | Comparative ability of MDA5 and RIG-I to stimulate IFN-b and IFN-l1 promoter activation. (A). HEK293 cells were transfected in a 96-well format with expression plasmids for MDA5, wtRIG-I and DRIG-I (from left to right 1, 3, 10, 30, 100 ng/well) and reporter plasmids IFN-b or IFN-l1-promoter-luciferase (30 ng/ well) and RSV-Renilla (50 ng/well). wtRIG-I expression plasmid transfected cells were stimulated with polyI:C (10 mg/ml) at 4 h post-transfection. After 24 h incubation luciferase activities were measured. (B) Comparative presentation of luciferase activity obtained with 30ng/well of expression plasmids. Data obtained from panel (A) A representative experiment of 3 is shown. (C) HEK293 cells were transfected with increasing amounts of MDA5 and DRIG-I expression plasmids in a 12-well format (10, 30, 100, 300, 900 ng/well) for 24 h. Cells were collected and 20% of cell extracts were separated on 10% SDS-PAGE, transferred to PVDF membranes and stained with rabbit anti-MDA5 and anti-RIG-I antibodies followed by goat anti-rabbit IRDye 800CW.

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Activation Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Incubation, Activity Assay, SDS Page, Staining

Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 6 | Inhibitory effects of filovirus VP24 proteins on RIG-I induced IFN-l1 and IFN-b gene expression. HEK293 cells were transfected with increasing amounts (3, 10 or 30 ng/well) of filovirus VP24 expression plasmids (*codon optimized) together with expression plasmid DRIG-I and reporter plasmids IFN-l1 (A) or IFN-b (B) promoter-luciferase (30 ng/well) and RSV-Renilla (50 ng/well). Cells were harvested after overnight incubation and measured for the luciferase activity. Controls include DRIG-I, promoter-reporter plasmids and HCV NS3/4 plasmids (3, 10 or 30 ng/well). Luciferase values were normalized with RSV-Renilla expression control. The bars represent the mean values of three independent experiments with three replicates (n=9). Error bars indicate standard errors. Statistical differences were calculated by ordinary one-way ANOVA Dunnett’s multiple comparisons test with a single pooled variance. Each column value was compared to the positive control. P values, *p = < 0.05, **p = < 0.005, ***p = < 0.0005, ****p = < 0.0001.

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Gene Expression, Transfection, Expressing, Plasmid Preparation, Luciferase, Incubation, Activity Assay, Control, Positive Control

Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 7 | Inhibition of MDA5 and RIG-I induced IFN-b and IFN-l1 gene expression by wild type and NLS-mutated EBOV VP24. (A) HEK293 cells were transfected with increasing amounts (3, 10, 30 ng/well in 6-well plates) of EBOV VP24 and VP40 and Zika virus NS3 expression constructs. Positive control refers to MDA5 induced promoter activity in the absence of viral proteins. (B) Experimental setting as in panel (A), but promoter activation is induced by DRIG-I. (C, D) HEK293 cells were transfected with increasing amounts of EBOV VP24 or NLS-mutated VP24 (VP24mut) expression plasmids (3, 10 or 30ng/well) together with an expression plasmid DRIG-I and reporter plasmids IFN-b/IFN-l1 -promoter-luciferase (30 ng/well) and RSV-Renilla. VP24 (wt+mut) refers to combinations of wt and NLS-mutated VP24 expression plasmids (3+27 ng, 10+20 ng and 30+0 wt-NLS mutant plasmid ratios). Cells were harvested at 24 h after incubation and luciferase values were normalized with RSV-Renilla control. The experiment was repeated three times and values represent the mean values. Error bars indicate standard errors of the mean. Positive controls refer to MDA5 or DRIG-I induced promoter activity without VP24 expression plasmids.

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Inhibition, Gene Expression, Transfection, Virus, Expressing, Construct, Positive Control, Activity Assay, Activation Assay, Plasmid Preparation, Luciferase, Mutagenesis, Incubation, Control

Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 8 | The effect of RESTV, SUDV, EBOV and LLOV VP24 on RIG-I and MDA5 activated IRF3 phosphorylation. HEK293 cells were transfected with increasing amounts of expression plasmids for VP24s (200–2000 ng/well in 6-well plates) together with (A) DRIG-I or (B) MDA5 and IRF3 expression constructs (30 ng/well). After overnight incubation cells were harvested, separated on SDS-PAGE, transferred to nitrocellulose membranes and stained with specific antibodies against different proteins as indicated in the figure. GAPDH detection with anti-GAPDH antibody was used as a loading control. P-IRF3 refers to activated, phosphorylated form of IRF3. The experiment was repeated twice with similar results. A representative experiment is shown.

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Phospho-proteomics, Transfection, Expressing, Construct, Incubation, SDS Page, Staining, Control

Journal: Frontiers in immunology

Article Title: Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

doi: 10.3389/fimmu.2021.694105

Figure Lengend Snippet: FIGURE 9 | The effect of EBOV VP24 on nuclear import of IRF3. Huh7 cells we transfected with EBOV VP24 expression plasmids for 24 h (1200ng of plasmid per 12-well), followed by stimulation of the cells with 10µg of polyI:C for 18 h. (A) Fixed and permeabilized cells were stained with anti-HA and anti-IRF3 antibodies and fluorescent secondary antibodies. (B) Nuclear localization of IRF3 was quantified. Bars represent the average of IRF3 nuclear localization in each sample. From each variable 100 to 110 cells were analyzed and statistical analysis was performed using Chi-square test. ns, non-significant.

Article Snippet: Mitotracker staining of TAFV VP24 gene transfected cells was performed according to manufacturer’s instructions (MitoTracker® Red CMXRos, Cell Signaling Technology) followed by detection of VP24 protein as described above.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining

Journal: Frontiers in medicine

Article Title: Ketone Bodies Improve Human CD8 + Cytotoxic T-Cell Immune Response During COVID-19 Infection.

doi: 10.3389/fmed.2022.923502

Figure Lengend Snippet: FIGURE 2 | Beta-hydroxybutyrate shifts human T-cell metabolism toward oxidative phosphorylation enabling higher mROS production. Human peripheral blood mononuclear cells (PBMC) were cultivated for 5 days in RPMI containing 80 mg/dl glucose (NC) and supplemented with 10 mM D/L-beta-hydroxybutyrate (BHB). T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD8+ T cells were isolated via magnetic cell separation. (A–D) Oxygen consumption rate [OCR] (A), basal (B), maximum (C) and spare respiratory capacity (D) were measured using a Seahorse HS mini Analyzer, n = 5 individual patient samples, each performed in 2–3 technical replicates. (E) Mitochondrial mass determined via MitoTracker green, indicated by MFI FITC in human CD8+ T cells, n = 12 individual patient samples. Histogram depicting exemplary change of MitoTracker green. (F) Quantification of mitochondrial superoxide production using MitoSOX, displayed as MFI PE in human CD8+ T cells, n = 11 individual patient samples. Histogram depicting exemplary change of MitoSOX fluorescence. *p < 0.05, **p < 0.01.

Article Snippet: MitoTracker Green FM (#9074, Cell Signaling Technology, Danvers, MA, United States) was used for flow cytometric determination of mitochondrial mass (200 nM MitoTracker in the dark, 37◦C, 15 min).

Techniques: Phospho-proteomics, Cell Stimulation, Isolation, Magnetic Cell Separation

Journal: Nature Communications

Article Title: DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos

doi: 10.1038/s41467-022-31497-9

Figure Lengend Snippet: a Ca 2+ microdomains in 2-cell embryos show a similar distribution to that of DDX1. Arrows point to a subset of Ca 2+ microdomains. b Ca 2+ microdomains (Fluo-4 AM, green) do not co-compartmentalize with MitoTracker Deep Red (magenta). c Ca 2+ microdomains (Fluo-4 AM, green) co-compartmentalizes with DDX1 (magenta) in 2-cell embryos. d Ca 2+ microdomains (Fluo-4 AM, green) do not co-compartmentalize with ER-Tracker Red (magenta). e Statistical analysis of ( b ) Fluo-4 AM/Mitotracker, ( c ) Fluo-4 AM/DDX1 and ( d ) Fluo-4 AM/ER-Tracker, with 2-cell embryos obtained from 3, 4 and 2 pairs, respectively, of wild-type natural matings. Pearson’s correlation coefficients were calculated by ImageJ and plotted with Prism. Statistical analysis of single plane confocal images of each embryo (middle sections) was performed with one-way ANOVA and Tukey multiple comparison test. ****indicates p < 0.0001. Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. Scale bars = 10 µm.

Article Snippet: For JC-1, MitoSOX, ER-Tracker, MitoTracker Deep Red and BioTracker ATP-red live cell staining, embryos were incubated for 30 min in equilibrated M16 medium containing 10 μg/ml JC-1 dye (Thermo Fisher), 2 μM MitoSOX (Thermo Fisher), 200 nM ER-Tracker Red (Thermo Fisher), 100 nM MitoTracker Deep Red (Cell Signaling Technology), or 15 min in 10 μM BioTracker ATP-red (Millipore).

Techniques: Comparison, Standard Deviation

Journal: Nature Communications

Article Title: DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos

doi: 10.1038/s41467-022-31497-9

Figure Lengend Snippet: Two-cell embryos were collected from natural matings. a Embryos were treated with 100 µM EGTA-AM or equivalent DMSO for 3 h. Ca 2+ microdomains (green) show similar but weaker patterns in EGTA-AM treated embryos compared to DMSO control. Scale bars = 10 µm. b Statistical analysis of ( a ) with data from 3 pairs of wild-type natural matings. Ca 2+ intensities were plotted with Prism. Statistical analysis was performed with two-sided Students’ t -test. ****indicates p < 0.0001 (with the exact p = 7.91E-10). Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. c EGTA-AM treated embryos show reduced size of DDX1 aggregates compared to DMSO control (DDX1, magenta; DAPI, blue). Maximum intensity projections of the Z-stack images are shown. Scale bars = 10 µm. d Size distribution analysis of ( c ) with data from 4 pairs of wild-type natural matings. Data were plotted with Prism. Statistical analysis was performed with two-sided multiple t -test using the Holm-Sidak method, with alpha = 0.05. *indicates p < 0.05. The exact p values are 0.024 for volumes <0.5 µm 3 , 0.023 for volumes <1 µm 3 , 0.046 for volumes <1.5 µm 3 , 0.026 for volumes <2.5 µm 3 , 0.021 for volumes <3 µm 3 and 0.021 for volumes >5 µm 3 . Each circle represents the mean value, and the error bars represent standard deviation. Solid circles represent DMSO-treated controls; empty circles represent EGTA-treated embryos. e Co-staining of Ca 2+ (Fluo-4 AM; green) and mitochondria (MitoTracker, magenta) with or without saponin treatment. Arrows and arrowheads point to Fluo-4 AM and MitoTracker co-compartmentalization at the inner and subplasmalemmal cytoplasm, respectively. Scale bars = 10 µm. Images were obtained with a Zeiss LSM710 confocal microscope. f TEM shows that DDX1 remains in MARVs after treatment with the membrane permeabilizing glycoside, saponin. Arrows point to DDX1-containing vesicles. A MARV aggregate of DDX1 vesicles is outlined by the dots. Four embryos from 2 wild-type crosses were independently collected with similar results obtained for all embryos. Scale bar = 0.5 µm. g Statistical analysis of ( e ) with data collected from 2-cell embryos obtained from three pairs of wild-type natural matings (single plane mid-sections for each embryo). Pearson’s correlation coefficients were calculated by ImageJ and plotted with Prism. Statistical analysis was performed with two-sided Students’ t -test. ****indicates p < 0.0001 (with the exact p = 3.39E-14). Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation.

Article Snippet: For JC-1, MitoSOX, ER-Tracker, MitoTracker Deep Red and BioTracker ATP-red live cell staining, embryos were incubated for 30 min in equilibrated M16 medium containing 10 μg/ml JC-1 dye (Thermo Fisher), 2 μM MitoSOX (Thermo Fisher), 200 nM ER-Tracker Red (Thermo Fisher), 100 nM MitoTracker Deep Red (Cell Signaling Technology), or 15 min in 10 μM BioTracker ATP-red (Millipore).

Techniques: Control, Standard Deviation, Staining, Microscopy, Membrane

Journal: Nature Communications

Article Title: DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos

doi: 10.1038/s41467-022-31497-9

Figure Lengend Snippet: One-cell embryos from Ddx1 heterozygote crosses were cultured in M16 medium for 72 h. One-cell embryos from Ddx1 wild-type crosses were cultured for up to 96 h. Images were captured with a Zeiss LSM710 confocal microscope. a Fluo-4 AM staining (green) of stalled 2-cell embryos and wild-type 2-cell, 4-cell, 16-cell and blastocyst embryos. Three independent wild-type crosses were used for each stage with the exception of blastocysts where 2 crosses were used. Similar results were obtained for all embryos at the same stage. Arrows point to Fluo-4 AM staining in embryos. b JC-1 staining of stalled and wild-type 2-cell embryos. Stalled embryos have a stronger J-aggregate (magenta)/monomer (green) signal ratio compared to wild-type embryos indicating that the mitochondrial membrane potential is higher in stalled embryos. Single plane images are shown to better illustrate high potential mitochondria distribution. c Statistical analysis of mitochondrial membrane potential in wild-type ( n = 36) and stalled ( n = 13) embryos. Mean intensity values were plotted with Prism. Statistical analysis was performed with two-sided Students’ t -test. ****indicates p < 0.0001 (with the exact p = 2.24E-05). Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. d Mitochondrial (top) and nuclear (bottom) fragmentation detected in stalled embryos. Arrows point to fragmented nuclei. Maximum intensity projections of the Z-stack images are shown for DAPI (MitoTracker, magenta; DAPI, blue). e The reduced size of MitoTracker Deep Red aggregates suggests fragmentation of mitochondria. Data were plotted with Prism. Statistical analysis was performed with two-sided multiple t -test using the Holm-Sidak method, with alpha = 0.05. ****indicates p < 0.0001 (both p values were less than 1E-06). Each circle represents the mean value, and the error bars represent standard deviation. Solid circles represent wild-type controls; empty circles represent stalled embryos. f The number of nuclei (or nuclei fragments) per cell was quantified and plotted with Prism. Statistical analysis was performed with two-sided Students’ t -test. ****indicates p < 0.0001 (with the exact p = 1.92E-06). Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. g Mitochondrial ROS in wild-type and stalled 2-cell embryos was detected using the MitoSOX dye (green). Maximum intensity projections of the Z-stack images at each stage are shown. h Data obtained from ( g ) were plotted with Prism. Statistical analysis was performed with two-sided Students’ t -test. ****indicates p < 0.0001 (with the exact p = 1.59E-08). Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. Scale bars = 10 µm. Z-stack images of each embryo was used for the statistical analysis in ( c ), ( f ) and ( h ). Single plane images of each embryo (middle sections) were used for the statistical analysis in ( e ). WT = wild-type.

Article Snippet: For JC-1, MitoSOX, ER-Tracker, MitoTracker Deep Red and BioTracker ATP-red live cell staining, embryos were incubated for 30 min in equilibrated M16 medium containing 10 μg/ml JC-1 dye (Thermo Fisher), 2 μM MitoSOX (Thermo Fisher), 200 nM ER-Tracker Red (Thermo Fisher), 100 nM MitoTracker Deep Red (Cell Signaling Technology), or 15 min in 10 μM BioTracker ATP-red (Millipore).

Techniques: Cell Culture, Microscopy, Staining, Membrane, Standard Deviation

Journal: Nature Communications

Article Title: DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos

doi: 10.1038/s41467-022-31497-9

Figure Lengend Snippet: One-cell embryos from Ddx1 heterozygote crosses were cultured for 72 h. One-cell embryos from Ddx1 wild-type crosses cultured to the 2-cell stage were used for comparison. a BioTracker ATP-red staining (green) of stalled 2-cell embryos show higher ATP levels compared to wild-type 2-cell embryos. Maximum intensity projections of the Z-stack images at each stage are shown. b Statistical analysis of ( a ) using Z-stack images for each embryo. Mean intensity values were plotted with Prism. Statistical analysis was performed with two-sided Students’ t -test. ***indicates p < 0.001 (with the exact p = 1.34E-04). Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. c Triple staining of nuclei (Hoechst 33342) (blue), mtROS (MitoSOX) (green) and mitochondria (MitoTracker) (magenta) in stalled versus wild-type two-cell embryos. d Stalled embryos have higher mtROS and more fragmented nuclei and mitochondria based on statistical analysis of each channel in ( c ) plotted individually. Statistical analysis was performed with either two-sided Students’ t -test or two-sided multiple t -test using the Holm-Sidak method, with alpha = 0.05. ***indicates p < 0.001. **indicates p < 0.01. Exact p value for left panel is 0.0002, center panel is 0.0045, and right panel is 0.00045. For the box plots, center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. For the symbol plot, each circle represents the mean value, and the error bars represent standard deviation. Solid circles represent wild-type controls; empty circles represent stalled embryos. e Statistical analysis of fatality index constructed with weighted average index construction using the z -scores of nuclei (or nuclei fragments) number/cell, MitoSOX intensity and percentage of mitochondria with size <0.2 µm 2 , and plotted with Prism. Statistical analysis was performed with two-sided Students’ t -test. ****indicates p < 0.0001 (with the exact p = 2.66E-05). Center line, median; box limits, 25th and 75th percentiles; whiskers, minimum to maximum. Error bars represent standard deviation. Scale bars = 10 µm. Numbers of embryos analysed are indicated on the right hand side. WT = wild-type. Z-stack images were used for statistical analysis of nuclei and mtROS whereas single plane images of each embryo (middle sections) were used for statistical analysis of mitochondria.

Article Snippet: For JC-1, MitoSOX, ER-Tracker, MitoTracker Deep Red and BioTracker ATP-red live cell staining, embryos were incubated for 30 min in equilibrated M16 medium containing 10 μg/ml JC-1 dye (Thermo Fisher), 2 μM MitoSOX (Thermo Fisher), 200 nM ER-Tracker Red (Thermo Fisher), 100 nM MitoTracker Deep Red (Cell Signaling Technology), or 15 min in 10 μM BioTracker ATP-red (Millipore).

Techniques: Cell Culture, Comparison, Staining, Standard Deviation, Construct

Journal: Journal of Nanobiotechnology

Article Title: Lacc1-engineered extracellular vesicles reprogram mitochondrial metabolism to alleviate inflammation and cartilage degeneration in TMJ osteoarthritis

doi: 10.1186/s12951-025-03355-5

Figure Lengend Snippet: The application of Lacc1-engineered EVs in TMJOA chondrocytes. ( A ) The construction of Lacc1-engineered EVs. ( B ) Lacc1-engineered EVs alleviated TMJOA inflammation via modulating mitochondrial metabolism in chondrocytes. (By Figdraw.)

Article Snippet: For mitochondrial superoxide detection, the chondrocytes were incubated with 100 nM MitoTracker Green FM (MCE, USA) and 5 µM MitoSox Red (Invitrogen, USA) for 15 min at 37℃.

Techniques:

Journal: Journal of Nanobiotechnology

Article Title: Lacc1-engineered extracellular vesicles reprogram mitochondrial metabolism to alleviate inflammation and cartilage degeneration in TMJ osteoarthritis

doi: 10.1186/s12951-025-03355-5

Figure Lengend Snippet: OE-EVs attenuate IL-1β-induced inflammation in chondrocytes. ( A ) TEM and NTA characterization of NC-EVs and OE-EVs. ( B ) PKH26-labeled EVs internalized by chondrocytes. ( C ) Western blot analysis of exosomal markers and Lacc1 in NC-EVs and OE-EVs. ( D ) qRT-PCR of inflammatory and cartilage matrix markers. ( E & G ) Immunofluorescence staining of Aggrecan and MMP13. ( F ) IL-1β levels measured by ELISA. ( H ) Western blot of inflammation- and ECM-related markers. ( n = 3, * p < 0.05, ** p < 0.01, and *** p < 0.001)

Article Snippet: For mitochondrial superoxide detection, the chondrocytes were incubated with 100 nM MitoTracker Green FM (MCE, USA) and 5 µM MitoSox Red (Invitrogen, USA) for 15 min at 37℃.

Techniques: Labeling, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Nanobiotechnology

Article Title: Lacc1-engineered extracellular vesicles reprogram mitochondrial metabolism to alleviate inflammation and cartilage degeneration in TMJ osteoarthritis

doi: 10.1186/s12951-025-03355-5

Figure Lengend Snippet: OE-EVs reprogram metabolism in chondrocytes. ( A & B ) qRT-PCR and Western blot analysis of glycolytic enzyme expression. ( C & D ) ATP production and lactate levels in chondrocytes. ( E ) JC-1 staining of mitochondrial membrane potential (red: JC-1 aggregates, green: JC-1 monomers). ( F ) Mitochondrial ROS analysis using MitoSOX and Mitotracker staining. ( G ) TEM images of mitochondria in different groups. ( n = 3, * p < 0.05, ** p < 0.01, and *** p < 0.001)

Article Snippet: For mitochondrial superoxide detection, the chondrocytes were incubated with 100 nM MitoTracker Green FM (MCE, USA) and 5 µM MitoSox Red (Invitrogen, USA) for 15 min at 37℃.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, Membrane

Journal: Journal of Nanobiotechnology

Article Title: Lacc1-engineered extracellular vesicles reprogram mitochondrial metabolism to alleviate inflammation and cartilage degeneration in TMJ osteoarthritis

doi: 10.1186/s12951-025-03355-5

Figure Lengend Snippet: Transcriptomic and signaling pathway analysis of IL-1β- and OE-EV-treated chondrocytes. ( A ) Heatmap of differentially expressed genes. ( B & C ) KEGG and GO enrichment analysis. ( D ) GSEA analysis of key pathways. ( E & F ) Western blot and semi-quantification of JAK-STAT signaling. ( n = 3, * p < 0.05 and ** p < 0.01)

Article Snippet: For mitochondrial superoxide detection, the chondrocytes were incubated with 100 nM MitoTracker Green FM (MCE, USA) and 5 µM MitoSox Red (Invitrogen, USA) for 15 min at 37℃.

Techniques: Western Blot

Journal: Science Advances

Article Title: CD38 reduces mitochondrial fitness and cytotoxic T cell response against viral infection in lupus patients by suppressing mitophagy

doi: 10.1126/sciadv.abo4271

Figure Lengend Snippet: ( A ) Oxygen consumption rate (OCR) of CD38 hi CD8 + and CD38 lo CD8 + T cells sorted from healthy donor at baseline and after addition of oligomycin, carbonyl cyanide p -trifluoromethoxyphenylhydrazone, and rotenone/antimycin A. ( B ) Basal and maximal OCR of CD38 hi CD8 + and CD38 lo CD8 + T cells sorted from healthy donor in (A). ( C ) Representative flow cytometry plot of MitoTracker Green and MitoTracker Deep Red staining in CD38 hi CD8 + and CD38 lo CD8 + T cells from the peripheral blood of SLE patients. Percentage of depolarized mitochondria in CD38 hi CD8 + and CD38 lo CD8 + T cells. ( D and E ) Representative flow cytometry plot of MitoTracker Green (D) and MitoSOX (E) staining and the mean fluorescence intensity (MFI) in CD38 hi CD8 + and CD38 lo CD8 + T cells from lupus patient peripheral blood. ( F to I ) Representative electron microscopy of CD38 hi with reduced number of cristae (F), no cristae (G), and CD38 lo CD8 + T cells (H) sorted from lupus patient peripheral blood. Percentage of mitochondria observed with partial or complete loss of cristae in CD38 hi CD8 + and CD38 lo CD8 + T cells (I). Data are means ± SD; statistical analysis by two-tailed t test (B and F), paired t test (C to E), and chi-square test (I). ** P < 0.01, *** P < 0.001.

Article Snippet: For MitoTracker and immunofluorescence staining, control OFPSpark Vector (Sino Biological, CV025) and CD38 overexpression vector with OFPSpark (Sino Biological, HG10818-ACR) were used because of the consideration of fluorescence colors.

Techniques: Flow Cytometry, Staining, Fluorescence, Electron Microscopy, Two Tailed Test