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: 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