Journal: Cell Communication and Signaling : CCS

Article Title: Itaconate and its derivatives ameliorate autoimmunity by suppressing Th17 cells via regulating mitophagy

doi: 10.1186/s12964-025-02621-1

Figure Lengend Snippet: Itaconate and its derivatives 4-OI and DMI inhibit Th17 cells via regulating mitophagy. A Structures of ITA and the ITA photo-affinity probe named x-ITA which was synthesized based on the structure of ITA. B Visualization of ITA-interacting proteins in Th17 cells by in-gel fluorescence, CBB was used for protein staining and quantitative analysis. C Principal component analysis of ITA-interacting proteins in Th17 cells ( n = 3). D Volcano plot of ITA-interacting proteins quantified in Th17 cells. E and F Biological analysis of ITA-interacting proteins in Th17 cells. ( G ) Biological process analysis of the ITA-interacting proteins quantification in Th17 cells. The enriched processes relatied to mitochondria are marked in red. H Transmission electron microscopy (TEM) showed mitochondria morphology in presence or absence of ITA. The scale bar is 1.0 μm in the normal TEM micrograph (left) and 500 nM in the enlarged (right). Green arrows showed normal mitochondria in Veh group, and red arrows showed the swollen mitochondria. I Flow cytometric analysis of MitoTracker Green fluorescence in the presence or absence of ITA ( n = 5). (J) Flow cytometric analysis of MitoSOX fluorescence in the presence or absence of ITA ( n = 5). (K) Flow cytometric analysis of JC-1 staining in the presence or absence of ITA ( n = 5). L Statistic analysis of flow cytometry data of Th17 cells for mitophagy with and without 48 h ITA treatment and/or CCCP ( n = 5). M Immunoblotting for mitophagy-related proteins in the presence or absence of ITA in Th17 cells. β-actin is shown as a loading control. Graphs show the relative abundance of the protein calculated as the density of the protein band divided by the density of the β-actin band. N Flow cytometric analysis of MitoTracker Green fluorescence in the presence or absence of ITA with or without PMI ( n = 5). O Flow cytometric analysis of IL17A frequency in the presence or absence of ITA with or without PMI ( n = 5). P ELISA analysis of IL17A secretion in the presence or absence of ITA with or without PMI ( n = 7). P -values were calculated by two-tailed Student’s t -tests ( I - K and M ) or one-way ANOVA with Tukey’s test ( L and N - P ). Data are representative of mean ± SD. Differences were considered statistically significant at * p ≤ 0.05. ** p ≤ 0.01, and *** p ≤ 0.001

Article Snippet: Mitochondrial mass was assessed by MitoTracker Green FM (Med Chem Express, # HY-135056).

Techniques: Synthesized, Fluorescence, Staining, Transmission Assay, Electron Microscopy, Flow Cytometry, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Erythroid differentiation displays a peak of energy consumption concomitant with glycolytic metabolism rearrangements

doi: 10.1371/journal.pone.0221472

Figure Lengend Snippet: Shown is the distribution of the mitotracker intensity at each time-point of the differentiation process measured using ImageStreamX. A t-test was applied to assess whether means were significantly different (*: p-value < 0.05). Data were obtained from three independent experiments. 11678 cells were analyzed for the self renewal condition, 12587 cells for 24h, 14147 cells for 48h and 12355 cells for 72h.

Article Snippet: Cell were incubated 20 min in culture media completed with a staining solution composed of MitoTracker Green FM (100 nM) (BD Horizon), TMRE (ultra pure) mitochondria dye (Enzo) (1 nM) and Hoescht 33342 (In vitrogen) (2.5 μ g/ml).

Techniques:

Journal: PLoS ONE

Article Title: Erythroid differentiation displays a peak of energy consumption concomitant with glycolytic metabolism rearrangements

doi: 10.1371/journal.pone.0221472

Figure Lengend Snippet: Cells morphology and size were observed through the bright field channel using ImageStreamX. Dead cells were identified using FVS 660 dye (red). MMP was assessed using TMRE dye (yellow), mitochondrial content was evaluated using green mitotracker intensity (green), and the nucleus was stained using Hoechst (purple). Merge corresponds to the superposition of bright field, TMRE, mitotracker and Hoechst images. Cell granularity was assessed using SSC channel (pink). A: The FCCP uncoupler was used as a negative control of MMP staining. NT: No treatment. B: Shown is an example of FVS 660 staining to discriminate between living and dead cells. C: Boxplots of normalized TMRE distributions. Outliers are not shown. A Wilcoxon test was applied to assess whether means were significantly different (*: p-value < 0.05). Data were obtained from three independent experiments. The overall numbers of cells analyzed were 11678 for the self-renewal condition, 12587 cells for 24h, 14147 cells for 48h and 12355 cells for 72h.

Article Snippet: Cell were incubated 20 min in culture media completed with a staining solution composed of MitoTracker Green FM (100 nM) (BD Horizon), TMRE (ultra pure) mitochondria dye (Enzo) (1 nM) and Hoescht 33342 (In vitrogen) (2.5 μ g/ml).

Techniques: Staining, Negative Control

Journal: PLoS ONE

Article Title: Erythroid differentiation displays a peak of energy consumption concomitant with glycolytic metabolism rearrangements

doi: 10.1371/journal.pone.0221472

Figure Lengend Snippet: T2EC were incubated with Galloflavin (30 μ M) and FX11 (50 μ M) for 24h. A: Lactate concentration was measured in T2EC media following incubation with Galloflavin or FX11 for 24h. Each value represents mean +/- S.D. of six independent experiments for the Galloflavin and five independent experiments for FX11. A t-test was applied to assess whether distributions were significantly different (p-value inf. to 0.05). B: MMP was assessed on living cells (FVS 660 dye) using TMRE dye, normalized by dividing TMRE intensity by mitotracker intensity. FCCP uncoupler was used as a negative control for MMP staining. A wilcoxon-test was applied to assess whether distributions were significantly different (p-value inf. to 0.05). Data were obtained from three independant experiments. The overall numbers of cells analyzed for Galloflavin treatment were respectively 11306 and 11994 cells for the DMSO and Galloflavin conditions. The overall numbers of cells analyzed for FX11 treatment were respectively 10904 and 12858 cells for the DMSO and FX11 conditions. C: Two respiration parameters were assessed in self-renewing T2EC and T2EC treated for 24h with Galloflavin (30 μ M) or FX11 (50 μ M). Left panel: Routine respiration corresponds to oxygen consumption without any addition. Right panel: Cytochrome C oxydase activity was assessed by subtracting respiration following ascorbate addition from maximal TMPD-related respiration. Bars represent means +/- S.E.M. from five independent experiments. A paired t-test was applied to assess whether means were significantly different (*: p-value inf. to 0.05; n.s.: non significant).

Article Snippet: Cell were incubated 20 min in culture media completed with a staining solution composed of MitoTracker Green FM (100 nM) (BD Horizon), TMRE (ultra pure) mitochondria dye (Enzo) (1 nM) and Hoescht 33342 (In vitrogen) (2.5 μ g/ml).

Techniques: Incubation, Concentration Assay, Negative Control, Staining, Activity Assay

Journal: International Journal of Cancer

Article Title: Chronic acidosis rewires cancer cell metabolism through PPARα signaling

doi: 10.1002/ijc.34404

Figure Lengend Snippet: Acid‐adapted cells exhibit reduced glucose‐dependent acid extrusion but maintain glycolytic capacity. (A) Overview of the expression level of genes involved in glycolysis and TCA‐cycle in MDA‐AA cells compared with MDA‐ctrl. Color indicates average log2 fold change across three replicates per condition. The asterisk indicates statistical significance ( P < .05, AA vs ctrl.). (B, C) Representative Western blots (B) and quantifications (C) of HK2, PKM2, LDHA and MCT4. DCTN1 or H3 was used as loading control as indicated. For PKM2 and LDHA, the same loading ctrl is used, as these two proteins were derived from the same blot. In (C) the dotted line indicates ctrl. value. A paired (MCT4) or unpaired (HK2, PKM2 and LDHA) two‐tailed t ‐test was performed for each individual cell line, AA vs ctrl. ( p values indicated on top; n = 3, 3 (HK2); 3, 5 (PKM2); 5, 6 (LDHA); 8, 11 (MCT4) for Panc‐1 and MDA, respectively). (D, F) Extracellular acidification rate (ECAR, mpH/min) as a function of time in Panc‐1 (D) and MDA (F). At the indicated time points, glucose (Glu, 10 μM), oligomycin (1 μM) and 2‐deoxy‐glucose (2‐DG, 50 mM) was added to each well followed by three sets of three measurements over a time period of ~18 min. In each biological replicate, at least 15 technical replicates were measured. n = 3. (E, G) Calculated percent increase in ECAR between glucose (average of data points 4‐6) and oligomycin (average of data points 7‐9) conditions in Panc‐1 (E) and MDA (G). A paired two‐tailed t ‐test was performed for each individual cell line, AA vs ctrl. ( p values are indicated on top; n = 3). (H) Average oxygen consumption rate (OCR, pmol/min) as a function of average ECAR (mpH/min) of Panc‐1, MDA, MCF‐7 and HCT ctrl. and AA cells. Error bars represent S.E.M values. n = 3 for Panc‐1 and MDA and 4 for MCF‐7 and HCT. (I) Mitochondrial content in MDA‐AA compared with MDA‐ctrl. cells. Mitochondria were quantified by MitoTracker Green and normalized to cell number in each experiment using Hoechst staining. In each experiment, three technical replicates were performed. A paired two‐tailed t ‐test was performed for each individual cell line, AA vs ctrl. ( p value is indicated on top; n = 8). (J) Representative IF images of mitochondria visualized with MitoTracker Deep Red (magenta) in Panc‐1 and MDA (n = 3). Image overlays and adjustments of the intensities were performed using ImageJ software. Scale bar: 10 and 5 μm for full and zoomed image, respectively. Abbreviations for Figure : ACO1/2, aconitase 1/2; ALDOA/C, fructose‐biphosphate aldolase A/C; CS, citrate synthase; ENO1/2/3, enolase 1/2/3; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GPI, glucose‐6‐phosphate isomerase; HK1/2, hexokinase 1/2; IDH1/2, isocitrate dehydrogenase 1/2; IDH3A, isocitrate dehydrogenase 3 catalytic subunit alpha; IDH3B, isocitrate dehydrogenase 3 non‐catalytic subunit beta; IDH3G, isocitrate dehydrogenase 3 non‐catalytic subunit gamma; LDHA/B, lactate dehydrogenase A/B; MP1/2, mitochondrial pyruvate carrier 1/2; OGDH, oxoglutarate dehydrogenase; PDHA1/B, pyruvate dehydrogenase E1 subunit alpha 1/beta; PFKL/M/P, phosphofructokinase, liver/muscle/platelet; PGAM1, phosphoglycerate mutase 1; PGK1, phosphoglycerate kinase 1; PKM, pyruvate kinase M1/M2; SDHA, succinate dehydrogenase complex flavoprotein subunit A; SDHB, succinate dehydrogenase complex iron sulfur subunit B; SDHC/D, succinate dehydrogenase complex subunit C/D; SLC16A1/3, solute carrier family 16 member 1/3; SLC2A1/3, solute carrier family 2 member 1/3; SUCLA2, succinate‐CoA ligase ADP‐forming subunit beta; SUCLG1, succinate‐CoA ligase GDP/ADP‐forming subunit alpha; SUCLG2, succinate‐CoA ligase GDP‐forming subunit beta; TPI1, triosephosphate isomerase 1

Article Snippet: Cells seeded in 96‐well plates were stained for 30 min with MitoTracker Green FM (200 nM) and Hoechst, washed twice in PBS, and recorded in a GloMax Discover Microplate reader (Promega).

Techniques: Expressing, Western Blot, Control, Derivative Assay, Two Tailed Test, Staining, Software

Journal: Advanced Science

Article Title: Human Schlafen 14 Cleavage of Short Double‐Stranded RNAs Underpins its Antiviral Activity

doi: 10.1002/advs.202501727

Figure Lengend Snippet: Biochemical characterization of human SLFN14. a) Size‐exclusion chromatography (SEC, left) and analytical ultracentrifugation (AUC, right) analysis of recombinant SLFN14 (strep‐strep‐SUMO tagged) expressed in HEK293F cells; both experiments were carried out in solutions with 150 m m NaCl. Left insert, SDS‐PAGE analysis of the peak fractions. SLFN14 dimer is indicated by an arrow; the molecular mass of gel‐filtration standards is indicated by arrows. b) SLFN14 exhibits different activity on various RNA substrates. Purified SLFN14 (0.5 µ m ) was incubated with single‐stranded RNA (ssRNA), G‐quadruplex RNA (G4 RNA), double‐stranded RNA (dsRNA), hairpin RNA (hRNA), single‐stranded DNA (ssDNA), double‐stranded DNA (dsDNA), and hairpin DNA (hDNA), respectively. The reaction mixtures were incubated at 37°C for 30 min and resolved using denaturing Urea‐PAGE. c) SLFN14 cleaves all three tRNA types. SLFN14 (0.5 µ m ) was incubated with type I tRNA (tRNA‐His), type II tRNA (tRNA‐Leu and tRNA‐Ser), and tRNA‐Sec, respectively. The reaction mixtures were incubated at 37°C for 30 min and resolved using denaturing Urea‐PAGE. d) Mutation E211A abrogates RNase activity of SLFN14 on hRNA and tRNA substrates. e) SLFN14 cleaves various tRNA‐Leu isoacceptors with different efficiencies. SLFN14 exhibited higher RNase activity on tRNA‐Leu UAA than on other isoacceptors. f) SLFN14 cleaves various tRNA‐Ser isoacceptors with similar efficiencies. g) Divalent ions are required for the RNase activity of SLFN14. Various ions (concentration 1 and 4µ m ) that were included in the reactions are indicated on top of the gel; the EDTA control ensured no ions were present in the reactions. Synthetic tRNA‐Leu UAA was used as the substrate. h) Subcellular distribution of SLFN11 and SLFN14. eGFP‐fused SLFN11‐WT or SLFN14‐WT were stably expressed in HEK293T cells. The subcellular distribution of SLFN11 and SLFN14 was visualized using fluorescence microscopy. Mitochondria were labeled with Mito Tracker Deep Red FM (red fluorescence), SLFN14 or SLFN11 with eGFP (green fluorescence), and nuclei with DAPI (blue fluorescence). The white box indicates the region of interest (ROI), and white arrows highlight the plot profiles (leftmost). Colocalization images and corresponding Pearson correlation coefficients (PCC) are shown adjacent to the plot profiles. Scale bars represent 10 µm.

Article Snippet: After 24 h of transfection, cells were incubated at 37°C for 15 min in PBS containing 1 μ m of Mito Tracker Deep Red FM (Yeasen Biotech) to label mitochondria.

Techniques: Size-exclusion Chromatography, Analytical Ultracentrifugation, Recombinant, SDS Page, Filtration, Activity Assay, Purification, Incubation, Mutagenesis, Concentration Assay, Control, Stable Transfection, Fluorescence, Microscopy, Labeling