Journal: bioRxiv

Article Title: Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

doi: 10.64898/2026.03.22.713508

Figure Lengend Snippet: A . SDS-PAGE separation and immunoblotting of mitochondria isolated from WT, SLC35A4–MP KO and SLC35A4-MP KO + SLC35A4-MP FLAG U2OS cells. SLC35A4-MP FLAG expression was induced with 5 ng/ml doxycycline for 48 h. SDHA serves as a loading control. Representative of n = 3 biological replicates. B. Representative confocal images of SLC35A4-MP KO + SLC35A4-MP FLAG U2OS cells. SLC35A4-MP FLAG expression was induced with 5 ng/ml doxycycline for 48 h. Immunofluorescence was used to label mitochondria (Anti-TOM20, magenta), and determine SLC35A4-MP localisation (anti-FLAG, yellow). Scale bar indicates 5 µm. C. Electron microscopy of WT and SLC35A4-MP KO U2OS cells. Scale bars indicate 500 nm. D. The steady-state whole cell proteome of SLC35A4-MP KO vs WT. MitoCarta 3.0 proteins are highlighted in black, and those with OMM or IMM localisations are highlighted in blue and pink respectively. E. Whole cell proteome of SLC35A4-MP KO vs WT U2OS in apoptotic vs non-apoptotic conditions. The log 2 (fold change) of significantly altered ( p < 0.05) WT and KO proteins following 1 h apoptotic treatment was plotted against each other; WT (x-axis) and KO (y-axis). Mitochondrial proteins are highlighted in black, and those with OMM or IMS/IMM localisation are highlighted in blue or pink respectively.

Article Snippet: Human osteosarcoma cells (U2OS) were purchased from the ATCC, and a clonal line was established.

Techniques: SDS Page, Western Blot, Isolation, Expressing, Control, Immunofluorescence, Electron Microscopy

Journal: bioRxiv

Article Title: Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

doi: 10.64898/2026.03.22.713508

Figure Lengend Snippet: A . The steady-state SLC35A4-MP interactome. Proteins with a log 2 (SLC35A4-MP FLAG 0 h / WT 0 h) > 1, p value < 0.05, were considered significantly enriched. Proteins with MitoCarta 3.0 annotations have been coloured as indicated B. Comparison of log 2 (SLC35A4-MP FLAG / WT) enrichment of the SLC35A4-MP FLAG interactome at 0 h and 1 h apoptosis. Mitochondrial proteins are coloured in black. Proteins preferentially enriched at 0 h or 1 h post induction of apoptosis are highlighted by the blue and red regions respectively. Stable interactors are highlighted by the yellow region (refer to Fig. S4B) C-D. BN-PAGE analysis of 1% digitonin soluble complexes from isolated mitochondria, immunoblotting using antibodies against C) MIC10 (MICOS complex) and D) OPA1 respectively. NDUFA9 (CI) and SDHA (CII) serve as a loading controls. Representative of n = 3 independent experiments. For densitometric quantification of soluble MIC10 or OPA1 containing complexes, complex abundance was normalised to NDUFA9 or SDHA respectively and displayed relative to WT levels. Data represents n = 3 independent experiments. Error bars indicate mean ± SEM. * = p value < 0.05, ** = p value < 0.01 by one-way ANOVA with Tukey’s multiple comparison test. E. SDS-PAGE analysis of OPA1 isoforms in mitochondria isolated from control, SLC35A4-MP KO and SLC35A4-MP rescue U2OS cells (SLC35A4-MP FLAG) . SLC35a4-MP FLAG expression was induced with doxycycline for 48 h (concentrations as indicated). LONP1 serves as a loading control. Representative of n = 3 independent experiments. F-G. Densitometric quantification of L-OPA1 ‘a’ isoform, and S-OPA1 isoforms ‘c’ and ‘e’ in F) SLC35A4-MP KO (without doxycycline treatment), or G) SLC35A4-MP KO cells overexpressing SLC35A4-MP FLAG with increasing concentrations of doxycycline. OPA1 isoform levels were normalised to LONP1 and displayed relative to WT (without doxycycline treatment). n = 3 independent experiments. Error bars indicate mean ± SEM. F: *** = p value < 0.005 by Student’s t-test. G: ** = p value < 0.01 *** = p value < 0.005 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Human osteosarcoma cells (U2OS) were purchased from the ATCC, and a clonal line was established.

Techniques: Comparison, Isolation, Western Blot, SDS Page, Control, Expressing

Journal: bioRxiv

Article Title: Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

doi: 10.64898/2026.03.22.713508

Figure Lengend Snippet: A. Live-cell imaging of WT, SLC35A4-MP KO and SLC35A4-MP rescue U2OS cells (SLC35A4-MP FLAG) stably expressing TOM20 Halo following treatment with ABT-737 [10 µM], S63845 [2 µM] and 1 h pre-treatment with QVD-OPh [20 µM]. Representative images from n=3 independent experiments. Scale bars indicate 5 µm. B-C. Quantification of apoptosis induced mtDNA release and mitochondrial fragmentation respectively. Quantification was performed on 10-15 cells from n = 3 independent experiments. Data points indicate mean ± SEM. Dashed line indicates the start of image acquisition D. Live cell imaging of SLC35A4-MP KO U2OS cells following CCCP [20µM] treatment. Representative images from n = 3 independent experiments. Scale bars indicate 10 µm. E. Quantification of mitochondrial fragmentation. Quantification was performed on 10-15 cells from n=3 independent experiments. Dashed line indicates start of image acquisition.

Article Snippet: Human osteosarcoma cells (U2OS) were purchased from the ATCC, and a clonal line was established.

Techniques: Live Cell Imaging, Stable Transfection, Expressing

Journal: iScience

Article Title: Predicting DNA damage response using synthetic cell painting profiles and experimental analysis

doi: 10.1016/j.isci.2026.115000

Figure Lengend Snippet: Application of the model to external datasets and experimental testing (A) Principal component analysis plot for the idr-0080 and cpg-0012 datasets. (B) Classification of cpg-0012 compounds into high-DDR (9,923) and low-DDR (20,694) groups. The high-DDR group includes known DNA damage inducers. Test compounds without prior DDR annotation were selected from the high-DDR (tetrindole, KF38789, and LY2183240) and low-DDR (amoxapine, acetazolamide, and captopril) groups. Flow cytometric (C) and Western blot (D) analyses of γH2AX expression in U2OS cells following 18 h treatment with 10 μM test compounds. (E) U2OS cells were treated with high-DDR candidates at increasing doses or with 10 μM low-DDR candidates for 72 h, and cell viability was measured using the CellTiter-Glo assay. Data are shown as means ± SD from independent experiments. One-way ANOVA with LSD post hoc test was used for statistical analysis. ∗ , p < 0.05; ∗∗∗ , p < 0.001 versus vehicle.

Article Snippet: U2OS human osteosarcoma cells (ATCC, HTB-96), originally derived from a moderately differentiated sarcoma of the tibia of a 15-year-old White female osteosarcoma patient, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and authenticated by ATCC-provided STR profiling.

Techniques: Western Blot, Expressing, Glo Assay

Journal: iScience

Article Title: Predicting DNA damage response using synthetic cell painting profiles and experimental analysis

doi: 10.1016/j.isci.2026.115000

Figure Lengend Snippet:

Article Snippet: U2OS human osteosarcoma cells (ATCC, HTB-96), originally derived from a moderately differentiated sarcoma of the tibia of a 15-year-old White female osteosarcoma patient, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and authenticated by ATCC-provided STR profiling.

Techniques: Recombinant, Flow Cytometry, Software