Journal: bioRxiv

Article Title: Phosphorylated ubiquitin is a secondary messenger and an epigenetic mark mediating mitochondria to nucleus signaling

doi: 10.64898/2026.04.24.719390

Figure Lengend Snippet: A - Human fibroblasts were treated with mitochondrial toxins (CCCP, 50µM; valinomycin, 1µM; oligomycin 10µM + antimycin, 4µM) or DMSO for 0, 4, 8 and 24 hrs. Top: Representative immunofluorescence images from 24 hr timepoint showing distribution of pS65Ub (green). Bottom: Quantification shows mean +/- SEM pS65Ub intensity in cytoplasm, nucleus and nucleus:cytoplasm relative to 0 hrs timepoint (separated with vertical dashed line) from 1 experiment with 3 technical repeats. The horizontal line shows the 0hr-normalized baseline. B - Fibroblasts were treated with valinomycin (1µM, 24 hrs) before pS65Ub (green) was visualized by immunofluorescence using multiple specific antibodies. Nuclei (blue) are annotated with green asterisks. C - Confocal Z-stack max projections showing human induced dopaminergic neurons treated with oligomycin + antimycin (OA, 1µM, 24 hrs) and stained for pS65Ub (green), the dopaminergic neuron marker tyrosine hydroxylase (TH; magneta), MAP2 (white) and Hoechst (blue). Dashed boxes indicate areas magnified in pS65Ub channel inset and dashed circles delineate nuclei. D - Wild type (WT) and PINK1 knockout (KO) HeLa cells were treated with CCCP (20µM, 4 hrs) as indicated, then subcellular fractionations were analysed by Western blot. E - HeLa were treated with CCCP (20µM, 4 hrs) or OA (1µM, 24 hrs) before immunoblot analysis of whole-cell lysates using two pS65Ub antibodies. F - HeLa, HEK293T, NCI-H226, murine melanoma, murine PDAC or human skin fibroblast cells were treated with OA, then pS65Ub levels were assessed by Western blot. G - NGN2-induced iPSC-derived neurons were treated with OA (1µM, 6 or 24 hrs) before subcellular fractionation and Western blot analysis. H - HeLa were treated with CCCP (20µM, 4 hrs), rotenone (Rot - 20µM, 20 hrs), paraquat (P3/P6 - 3/6mM, 20 hrs) or CCCP+i (PINK1 inhibitor/PRT062607, 2.5µM, 4 hrs). Quantifications show mean +/-SEM from three independent repeats, *** p<0.0001 (CCCP+i treatment performed twice only so excluded from quantification). I - HeLa cells were treated with 10µM Gamitrinib-TPP (GTPP) for 0, 3 or 20hrs before analysis of whole-cell lysates by Western blot. Red asterisks mark the pS65Ub-histone band. Scale bars 10µm (50µM for panel D). Dashed lines in panels E and F indicate where identical samples were run on separate blots.

Article Snippet: Control primary fibroblasts (#106-05A) were from Cell Applications, Inc., PINK1 (Q456X/Q456X, #sc1028) and Parkin (Ex4-7 del / c.203_204 del AG, #sc1064) mutant fibroblasts are available from the NINDS stem cell repository.

Techniques: Immunofluorescence, Staining, Marker, Knock-Out, Western Blot, Derivative Assay, Fractionation

Journal: bioRxiv

Article Title: Phosphorylated ubiquitin is a secondary messenger and an epigenetic mark mediating mitochondria to nucleus signaling

doi: 10.64898/2026.04.24.719390

Figure Lengend Snippet: A - Schematic depiction of subcellular fraction experiment performed in . Detergent buffers used for fractionation are given in italics. CIB and MIB buffers are from Cell Fractionation Kit (Cell Signaling Technologies, #9038), while HNTE was made in house. B - Cells were treated with OA (1µM, 18 hrs), MG132 (20µM, 4 hrs) or DMSO before subcellular fractionation and Western blotting. Protein subcellular localizations are annotated (IMM = inner mitochondrial membrane, OMM = outer mitochondrial membrane) and arrows indicate both full length and cleaved PINK1 bands. For the pS65Ub blot, identical samples were analyzed on a separate gel (separated by dashed lines). C - PINK1-HA was transiently expressed in HeLa cells before treatment with OA, MG132 or DMSO as before. The distributions of pS65Ub (green), HA (red) and DNA (blue) were assessed by immunofluorescence. Magnified regions of interest are indicated by dashed boxes. Red dashed lines indicate regions captured by intensity profile (performed in the red/HA channel), graphed below. D - WT or PINK1 KO HeLa were transiently transfected with empty vector (EV), PINK1-FKBP (P) or FIS1-FRB (F) in combinations indicated before 18 hrs treatment with OA (1μM) or rapalog (500nM). A dashed line reveals where identical samples were analyzed on a separate gel. E - Schematic depiction of experiments performed in Extended Data Figures 4F, G. F - WT or PINK1 KO HeLa cells were transiently transfected with EV, PINK1 WT, PINK1-NES or PINK1-NLS before treatment with CCCP and Western blot analysis. Two plasmid amounts were used for transfection to achieve high and low relative PINK1 expression, captured by high and low exposures (HiExp/LoExp respectively). G - PINK1-myc tagged with NES/NLS signals were transiently expressed in HeLa cells before treatment with CCCP (20µM, 4 hrs) or MG132 (20µM, 4 hrs). Cells were fixed and stained for myc (green), pS65Ub (blue, grayscale in the far right column) or mitochondrial marker HSP60 (red). Nuclear exclusion (NES) and sequestration (NLS) is observed after MG132 treatment. H - Quantification of nuclear vs cytoplasmic pS65Ub signal density (arbitrary units/µm 2 ) in healthy control, PRKN (left) and PINK1 (right) mutant iPSC-derived dopaminergic neurons after treatment with CCCP (10µM, 6 hrs). The horizontal line shows the signal on DMSO-treatment, considered background due to negligible PINK1 activation/pS65Ub levels. Mean +/- SEM from three independent cell lines per genotype. I - iPSC-derived dopaminergic neurons from PD patients with mutations in PINK1 were treated with CCCP (10µM, 6 hrs). Dashed boxes annotate the area magnified in the inset and dashed circles show nuclear borders. Inset zoom in of pS65Ub channel. See for Control and PRKN mutant conditions. J - Skin fibroblasts from PINK1 / PRKN mutant PD donors and healthy controls were treated with valinomycin (1µM, 8 hours) before Western blot analysis. Maximal Parkin activity (revealed by MFN2 ubiquitination, see band shift) and PINK1 activity (substrate pS65Ubiquitination) is only detected in WT cells treated with valinomycin. K - Fibroblasts were treated with valinomycin (1µM, 0-24 hrs) before fixation and immunofluorescence analysis of pS65Ub (green, grayscale in the left hand column), TOM20 (orange) and DNA (blue). Nuclear:cytoplasmic pS65Ub signal intensity from three experiments was quantified, with mean +/-SEM, 2-way ANOVA shown. Red asterisks identify the pS65Ub-histone band *p<0.05, **p<0.01, ***p<0.001. Scale bars 10µm.

Article Snippet: Control primary fibroblasts (#106-05A) were from Cell Applications, Inc., PINK1 (Q456X/Q456X, #sc1028) and Parkin (Ex4-7 del / c.203_204 del AG, #sc1064) mutant fibroblasts are available from the NINDS stem cell repository.

Techniques: Fractionation, Cell Fractionation, Western Blot, Membrane, Immunofluorescence, Transfection, Plasmid Preparation, Expressing, Staining, Marker, Control, Mutagenesis, Derivative Assay, Activation Assay, Activity Assay, Ubiquitin Proteomics, Electrophoretic Mobility Shift Assay

Journal: Cell Death & Disease

Article Title: Pharmacological activation of p53 induces dose-dependent changes in endothelial cell fate during angiogenic sprouting

doi: 10.1038/s41419-025-08292-7

Figure Lengend Snippet: A Concentration-dependent growth inhibition of human umbilical vein endothelial cells (HUVEC) by three p53-activators (MDM2 inhibitors navtemadlin and nutlin-3a; MDM2/MDMX inhibitor sulanemadlin), but not by non-specific control peptide. Cell growth was measured by live-cell imaging as the percent confluence normalized to untreated wells following 72 h treatment. Data points show averaged value from one experiment ( n = 3 experiments) and are fitted with a best fit model for concentration-growth response. B Growth inhibition of human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) by navtemadlin, as measured by live-cell imaging. Data points show averaged value from one experiment ( n = 3 experiments) and are fitted with a best fit model for concentration-growth response. C Growth inhibition of HUVEC recovers within 48 h following an initial 24 h treatment using ≤ 0.1 μM navtemadlin. Cell growth was measured by live-cell imaging and quantified over 72 h as cell counts per well and normalized to counts at time 0. Data points show mean ± SD ( n = 3 independent experiments). * P adj < 0.05, ** P adj < 0.01 using 1-way, repeated measures ANOVA with adjustment using Dunnett’s correction. D Morphological abnormalities are visible in phase-contrast images of venous ECs (HUVEC) and capillary ECs (HDMEC), but not in those of fibroblasts (NHDF) after 72 h of navtemadlin treatment, but not after 24 h. Scale bar = 200 μm. E Increased expression of proteins involved in p53 signaling (MDM2, clone IF2, 0.5 μg/mL; p53, clone DO-1, 0.4 μg/mL), cell cycle arrest (p21, clone 12D1, 0.24 μg/mL), and apoptosis (PUMA, clone D30C10, 0.96 μg/mL) in HUVEC following 24 h navtemadlin treatment, as determined by western blot analysis. Total protein controls correspond to distinct membranes (L1 or L2). Images are cropped from full-length blots of one biological experiment (see ‘Full Length Western Blots’) and are representative of at least two experiments. Further details regarding antibodies used are shown in SI Table . F Increased expression of p53 (clone DO-1, 12 μg/mL), cell cycle arrest (p21, clone 12D1, 1.22 μg/mL), apoptosis (PUMA, clone D30C10, 4.8 μg/mL), dead cells (Sytox green, 100 nM), and senescence (β-galactosidase), and reduced expression of active cell cycle (Ki67, clone SP6, 0.12 μg/mL), following 24 h treatment of HUVEC using navtemadlin as visualized by immunofluorescence, live-cell fluorescence imaging, and colorimetric staining. Scale bar = 50 μm. Further details regarding antibodies and concentrations used are shown in SI Table . G –K Quantification of fluorescence and colorimetric levels of protein markers, showing increased expression of p53, cell cycle arrest (p21), apoptosis (PUMA), cell death (Sytox green), and senescence, and reduced activity in cell cycle (Ki67). Data points indicate value from one experiment ( n = 3 experiments). ** P adj < 0.01, *** P adj < 0.001 using one-way ANOVA with adjustment using Dunnett’s correction. Horizontal black line indicates the mean value.

Article Snippet: For in vitro assays, we used commercially available primary cultures of three cell lines: human umbilical vein endothelial cells (HUVEC) from pooled donors (cat # C-12203, Promocell), human dermal microvascular endothelial cells (HDMEC) from adult donors (cat # C-12212, Promocell), and normal human dermal fibroblasts (NHDF) from adult donors (cat # 106-05 A, Cell Applications Inc).

Techniques: Concentration Assay, Inhibition, Control, Live Cell Imaging, Expressing, Western Blot, Immunofluorescence, Fluorescence, Imaging, Staining, Activity Assay