Journal: bioRxiv

Article Title: Chronic lysosome damage boosts interferon responses to Palbociclib through a mitochondrial signalling axis

doi: 10.1101/2025.03.13.642980

Figure Lengend Snippet: A - C) A549 NT (non-targeted) or Δ ATG5 cells were treated with DMSO or 10 μM Palbociclib (Pb) for 48 h then analysed by TEM. Representative images shown in A) , dotted lines delineate zoom area showing swollen lysosomes contacting mitochondria (arrowheads delimit a contact site, defined as continuous membrane opposition of < 30 nm). B) The proportional cross-sectional area of lysosomes within the cytoplasm. C) The mean length of lysosome-mitochondria (LM) contact sites per unit area of cytoplasm (n = 20 images, ± S.D., * = p < 0.05, **= p < 0.01, *** = p < 0.001, 2-way ANOVA, Holm-Šídák comparisons). Scale bars, 0.5 μm. D - E) A549 Δ ATG5 cells expressing YFP-GAL8 were treated with 10 mM Pb for 24 h, then stained for TOM20 (mitochondrion) and imaged by Super Resolution Optical Photon Reassignment Microscopy (SoRA). D) Representative raw images in upper panels, dotted lines (1-3) delineate the zoom areas in the row below and the cognate mitochondrial surface rendered images in the bottom row. E) Relative frequency distribution of distances between YFP-GAL8 foci and rendered mitochondria (Actual), compared with in silico shuffling of these foci within the cytoplasm (Random) (n = 3, 400 measurements from 5 cells, Kolmogorov-Smirnov test, p < 0.0001, representative of 3 independent experiments). See also Supp. Movie 1 . Scale bars, 5 μm (merge), 1 μm (zooms). F) A549 Δ ATG5 cells expressing YFP-GAL8 were treated with 10 mM Pb for 6 h, then MitoTracker-Red added 30 mins prior to super-resolution radial fluctuation (SRRF) processing-facilitated time-lapse microscopy. White arrows: YFP-GAL8 positive membranes associated with the mitochondrial network. See also: Suppl. Movies 2 and 3 . Scale bars, 2 mm. Representative of 4 experiments.

Article Snippet: The following antibodies were used: anti-DNA (EMD Milipore, #CBL186), anti-FASTKD2 (Proteintech, #17464-1-AP), anti-LAMP2 (Abcam, #ab25631), anti-TFAM (CST, #8076), anti-TOM20 (CST, #42406) (used when co-staining with mouse antibodies), anti-TOM20 (Santa Cruz, #sc-17764) (used when co-staining with rabbit antibodies and YFP-GAL8) and anti-DNA (Merck-Millipore, #CBL186).

Techniques: Membrane, Expressing, Staining, Microscopy, In Silico, Time-lapse Microscopy

Journal: bioRxiv

Article Title: Chronic lysosome damage boosts interferon responses to Palbociclib through a mitochondrial signalling axis

doi: 10.1101/2025.03.13.642980

Figure Lengend Snippet: A-F) A549 NT (non-targeting wild-type) or Δ ATG5 cells were treated with DMSO or 10 μM Palbociclib (Pb) for 48 h. Cells were stained for mitochondria (TOM20) and mitochondrial nucleoids using A,C) anti-TFAM; B,D) anti-DNA; E,F) anti-FASTKD2 antibodies. A,B) Representative SoRA and E) confocal microscopy images. C-D,F) Wide-field fluorescence microscopy was used to quantify the aggregated nucleoids (TFAM foci with > 0.7 μm 2 cross-sectional area or prominent DNA/FASTKD2 foci) (n =3, ± S.E.M., ns = non-significant, > 250 cells/condition, * = p < 0.05, **= p < 0.01, **** = p < 0.0001, 2-way ANOVA, Holm-Šídák comparisons DMSO vs. Pb). Dotted lines delineate zoom areas. G-H) A549 NT or Δ ATG5 cells were treated with DMSO or 700 μM LLOMe for 24 h. Cells were analysed for aggregated mitochondrial nucleoids as in A-F , quantifications presented here (n =3, ± S.E.M, > 220 cells/condition, ns = non-significant, * = p < 0.05, ** = p < 0.01, **** = p < 0.0001, 2-way ANOVA, Holm-Šídák comparisons DMSO vs. LLOMe). I-J) MCF7 cells were treated with DMSO, 700 μM LLOMe or 10 μM Pb, for 24 h, then analysed for aggregated TFAM-positive nucleoids as in A and C. I) Representative widefield fluorescence images. J) Quantifications (n = 3, ± S.E.M, > 150 cells/condition, * = p < 0.05, t-tests vs. DMSO). Scale bars, 10 μm.

Article Snippet: The following antibodies were used: anti-DNA (EMD Milipore, #CBL186), anti-FASTKD2 (Proteintech, #17464-1-AP), anti-LAMP2 (Abcam, #ab25631), anti-TFAM (CST, #8076), anti-TOM20 (CST, #42406) (used when co-staining with mouse antibodies), anti-TOM20 (Santa Cruz, #sc-17764) (used when co-staining with rabbit antibodies and YFP-GAL8) and anti-DNA (Merck-Millipore, #CBL186).

Techniques: Staining, Confocal Microscopy, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Chronic lysosome damage boosts interferon responses to Palbociclib through a mitochondrial signalling axis

doi: 10.1101/2025.03.13.642980

Figure Lengend Snippet: A) A549 Rescue (Δ ATG5 + GFP- ATG5 ) and Δ ATG5 cells were treated with DMSO (-) or 10 μM Palbociclib, Pb (+), for 48 h. Pure cytosolic fraction (Cytosol) was separated from remaining cellular material including mitochondria (Insoluble) using digitonin extraction and immunoblotted to confirm purity (mitochondrial markers: TOM20; Cytochrome C; ATP5A, ER marker: Calnexin) B-C) Nucleic acids ( B , DNA; C , RNA) were isolated from the cytosolic fraction and qPCR performed for mitochondrial genes ND1 , ND6 , CYTB and COII , normalised to ACTB from whole cell DNA (n >= 4 after outliers removed by Grubb’s test, ± S.E.M., normalisation to Rescue + DMSO, ** = p < 0.01, *** = p < 0.001, ratio paired t-tests).

Article Snippet: The following antibodies were used: anti-DNA (EMD Milipore, #CBL186), anti-FASTKD2 (Proteintech, #17464-1-AP), anti-LAMP2 (Abcam, #ab25631), anti-TFAM (CST, #8076), anti-TOM20 (CST, #42406) (used when co-staining with mouse antibodies), anti-TOM20 (Santa Cruz, #sc-17764) (used when co-staining with rabbit antibodies and YFP-GAL8) and anti-DNA (Merck-Millipore, #CBL186).

Techniques: Extraction, Marker, Isolation

Journal: bioRxiv

Article Title: Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

doi: 10.1101/2025.05.01.651794

Figure Lengend Snippet: ( a, b) Age-related decrease in MITOL expression. Western blot analysis of MITOL expression in the heart at each indicated month of age. Signal intensities were normalized to Tubulin and presented as values relative to the 1-month group. (c) Schematic representation of the compound evaluation strategy. Various herbal extracts and previously reported mitochondria-activating bioactive compounds were individually administered to human dermal fibroblasts. Candidate compounds identified based on MITOL mRNA upregulation by qRT-PCR were subsequently administered to mice to assess MITOL expression in multiple organs. (d) Relative MITOL mRNA levels in human dermal fibroblasts after 24-hour treatment with each compound, quantified by qRT-PCR. mRNA levels were normalized to GAPDH and expressed as a percentage relative to the control group (without test substances). Data represent the average of three independent experiments (n = 3). (e, f) Increased MITOL expression in mouse tissues following herbal extract administration. Mice were administered each herbal extract ( Phellodendron amurense , Coptis japonica , or Paeonia suffruticosa ) intraperitoneally, and 9 days later, MITOL expression levels in various organs were assessed by Western blot. f shows the graph of MITOL expression levels in the heart, normalized to tubulin (n = 5– 6 per group). Paeonia suffruticosa extract was included based on its prior effect on MITOL expression in hair follicle keratinocytes (data not shown); however, it did not increase MITOL expression in the heart, liver, or kidney in this experiment. (g, h) Upregulation of MITOL and mitochondrial proteins in C2C12 myoblasts following berberrubine treatment. C2C12 cells were treated with berberine (10 μM) or quinoid-type berberrubine (10 μM) for 48 hours, and protein expression levels were analyzed by Western blot ( g ). h shows the quantification of Western blot signals for MITOL, Tom20, Mfn2, HSP60, and ATP5a, normalized to Vinculin and expressed as values relative to the None group (n = 4 per group). The None group indicates cells cultured without any additives, including solvents. Statistical significance was determined using two-way ANOVA followed by Bonferroni post hoc test (f) or one-way ANOVA followed by Tukey’s HSD test (h) . * p < 0.05; ** p < 0.01; *** p < 0.001; ** p < 0.0001; ns, not significant.

Article Snippet: The following antibodies were used: anti-MITOL (LS-C164034, LSBio); anti-α-Tubulin (T-9026, Sigma-Aldrich); anti-Mfn2 (sc-100560, Santa Cruz Biotechnology); anti-HSP60 (sc-136291, Santa Cruz Biotechnology); anti-ATP5a (ab14748, Abcam); anti-Tom20 (11802-1-AP, Proteintech); and anti-Vinculin (V9131, Sigma-Aldrich).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Cell Culture

Journal: bioRxiv

Article Title: Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

doi: 10.1101/2025.05.01.651794

Figure Lengend Snippet: (a) C2C12 cells were treated with Q-BRB or AcA-BRB (5 μM, 10 μM), and the protein levels of mitochondrial-localized proteins (outer membrane: MITOL, Tom20, Mfn2; matrix: HSP60; inner membrane: ATP5a) were detected by Western blotting. Signal intensities were normalized to Vinculin and expressed as values relative to the 0 μM group (n = 4 per group). The 0 μM group indicates cells cultured without any additives, including solvents. (b) mRNA levels of MITOL and Tom20 were measured by qRT-PCR and normalized to GAPDH and expressed as fold change relative to the 0 μM group (n = 3 per group). (c) Mitochondrial content was quantified by real-time PCR based on the ratio of mtDNA (16S rRNA or ND1) to nuclear DNA (Hexokinase 2; HK2) and expressed as fold change relative to the 0 μM group (n = 3 per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s HSD test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The following antibodies were used: anti-MITOL (LS-C164034, LSBio); anti-α-Tubulin (T-9026, Sigma-Aldrich); anti-Mfn2 (sc-100560, Santa Cruz Biotechnology); anti-HSP60 (sc-136291, Santa Cruz Biotechnology); anti-ATP5a (ab14748, Abcam); anti-Tom20 (11802-1-AP, Proteintech); and anti-Vinculin (V9131, Sigma-Aldrich).

Techniques: Membrane, Western Blot, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

doi: 10.1101/2025.05.01.651794

Figure Lengend Snippet: (a) C2C12 cells were treated with Q-BRB or AcA-BRB (10 μM) for 48 h, followed by immunostaining with an anti-Tom20 antibody to visualize mitochondrial morphology (green: anti-Tom20; blue: Hoechst). Representative images are shown in the upper panels. Enlarged views of the boxed regions are displayed in the lower panels. Scale bars: 20 μm (upper), 10 μm (lower). The None group indicates cells cultured without any additives, including solvents. (b) Mitochondrial length was quantified in 30 randomly selected cells per group using MiNA (Mitochondrial Network Analysis). Mitochondria were classified into four categories based on length: Short (≤1.855 μm), Middle (1.855–3.599 μm), Long (3.599–8.103 μm), and Very long (≥8.103 μm), and presented as the proportion of mitochondria in each category within each group. Representative images for each category are shown on the right. Scale bar: 10 μm. (c, e) C2C12 cells were treated with Q-BRB or AcA-BRB (30 μM) for 24 h, and mitochondrial oxygen consumption rate (OCR) was measured (n = 3 per group). (d, f) Basal respiration, ATP synthesis-linked respiration, and maximal respiration were calculated from the OCR data obtained in (c, e) after sequential administration of oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Statistical significance was determined by comparing each treatment group (Q-BRB or AcA-BRB) with the corresponding solvent control group (DMSO or H 2 O) using a two-sided unpaired Student’s t -test. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were used: anti-MITOL (LS-C164034, LSBio); anti-α-Tubulin (T-9026, Sigma-Aldrich); anti-Mfn2 (sc-100560, Santa Cruz Biotechnology); anti-HSP60 (sc-136291, Santa Cruz Biotechnology); anti-ATP5a (ab14748, Abcam); anti-Tom20 (11802-1-AP, Proteintech); and anti-Vinculin (V9131, Sigma-Aldrich).

Techniques: Immunostaining, Cell Culture, Solvent, Control

Journal: International Journal of Biological Sciences

Article Title: Pyroptotic Macrophage-Derived Microvesicles Accelerate Formation of Neutrophil Extracellular Traps via GSDMD-N-expressing Mitochondrial Transfer during Sepsis

doi: 10.7150/ijbs.87646

Figure Lengend Snippet: Pyroptotic Macrophage-derived MVs Altered Mitochondrial Homeostasis in Neutrophils. Human peripheral neutrophils were isolated and cultured with PBS, control macrophage-derived MVs, or pyroptotic macrophage-derived MVs. (A, B and C) ΔΨ of human peripheral neutrophils was assessed using JC-1staining. JC-1 monomers (green) and aggregates (red) were assessed using fluorescence microscopy (A) and flow cytometry (B and C). Scale bar, 20 µm. (D and E) MitoSOX of human peripheral neutrophils was assessed using fluorescence microscopy (D) and flow cytometry (E). Scale bar, 50 µm. (F and G) Mitochondrial mass in human peripheral neutrophils was detected using flow cytometry (F) and fluorescence microscopy (G). Scale bar, 20 µm. (H) Representative images showing neutrophil staining of DNA (DAPI, blue), TOM20 (green), and 8-OHG (red) in human peripheral neutrophils following exposure to MVs for 4 h. Results are represented as mean ± SEM.

Article Snippet: Cells were blocked with 1% BSA for 30 min and stained with rabbit anti-histone H3 antibody (Abcam; 1:500); mouse anti-myeloperoxidase antibody (Abcam 1:500); rabbit anti-GSDMD (Abcam; 1:500); rabbit anti-TOM20 antibody (Abcam 1:500); mouse anti-8-OHdG antibody (Novus; 1:200).

Techniques: Derivative Assay, Isolation, Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Staining

Journal: Antioxidants

Article Title: Thunbergia laurifolia Leaf Extract Inhibits Glutamate-Induced Neurotoxicity and Cell Death through Mitophagy Signaling

doi: 10.3390/antiox10111678

Figure Lengend Snippet: TLE inhibits glutamate-induced excessive mitophagy. HT-22 cells (passage 14,16,17) were pretreated with 50 µg/mL of TLE or 100 nM selenium followed by 5 mM glutamate for 18 h. ( a ) The protein expression level of LC3B (autophagy) and TOM20 (mitochondria) were analyzed by Western blot, and β-actin served as the loading control. Relative protein levels of ( b ) LC3B and ( c ) TOM20 were quantified by densitometry and the mean data from at least three independent experiments were normalized to the results. Cont, untreated control; Starv, starvation. The data represent the means ± SEM. * p value < 0.05, ** p value < 0.01, *** p value < 0.005 compared with untreated control # p value < 0.05, ## p value < 0.01 compared with only the glutamate-treated group.

Article Snippet: Alexa Fluor 488, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mouse monoclonal anti-β-actin antibody, mouse monoclonal anti-rabbit IgG, HRP-linked antibody, rabbit polyclonal anti-LC3B antibody, rabbit monoclonal anti-TOM20 (D8T4N) antibody and tetramethylrhodamine ethyl ester (TMRE) were purchased from Cell Signaling Technology (Denvers, MA, USA).

Techniques: Expressing, Western Blot, Control