Journal: The EMBO Journal

Article Title: Plakoglobin transmits tension across VE-cadherin for vascular leak formation and leukocyte diapedesis

doi: 10.1038/s44318-026-00732-0

Figure Lengend Snippet: ( A , B ) HUVEC were treated with histamine (100 µM, Sigma-Aldrich) or thrombin (1 U/ml, CalBiochem) for 10 min, or left untreated with equivalent volumes of vehicle (media) as controls. Cells were subsequently fixed, permeabilized and stained for plakoglobin, β-catenin, and VE-cadherin, all visualized in grayscale to ensure unbiased comparative analysis. Higher magnification images (63X) were acquired using a Zeiss LSM 980 confocal microscope equipped with an Airyscan detector ( A ), while lower magnification images (40X) were captured on a Zeiss LSM 880 confocal microscope ( B ). Scale bars, 25 µm.

Article Snippet: Zeiss LSM 980 Airyscan acquisitions were processed with Airyscan Joint Deconvolution processing module in ZEN blue software (Zeiss) to improve signal-to-noise and spatial resolution; LSM 880 images were used without further deconvolution.

Techniques: Staining, Microscopy

Journal: bioRxiv

Article Title: Alpha-synuclein regulates nucleolar DNA double-strand break repair in melanoma

doi: 10.1101/2024.01.13.575526

Figure Lengend Snippet: A, B) SK-Mel28 cells were seeded on PLL-coated coverslips and then fixed and stained for alpha-synuclein (Syn1), nucleolar markers (nucleophosmin, treacle, and nucleostemin), and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with Airyscan and colocalization was analyzed in Imaris software. Error bars represent Standard Error of the Mean (SEM) with quantification from 3 biological replicates. ****p<0.0001 by T-test. C). SK-Mel28 cells were seeded on PLL-coated coverslips and then fixed and stained for alpha-synuclein (Syn1), fibrillar center marker (RPA194), dense fibrillar component marker (fibrillarin), granular component marker (nucleophosmin), and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with Airyscan oversampling and Joint Deconvolution and Channel Alignment post-processing. 3D renderings were produced using Imaris software. D) SK-Mel28 control and KO cell pellets were fixed with 0.1M sodium cacodylate buffer (pH 7.2) containing 0.05% glutaraldehyde, 4% paraformaldehyde, and 0.1% picric acid for 2 hours at RT. Thin sections were cut on an ultramicrotome (EM UC7) using a diamond knife. Sections were permeabilized and stained for alpha-synuclein (MJFR1, 1:75) (12nm colloidal gold particles). The stained sections were observed by a JEOL 1400 transmission electron microscope. Red labelling denotes nuclear membrane. Blue labelling denotes the outline of the nucleolus. FC=Fibrillar Center. Arrows point to representative MJFR1 staining. Quantification of 3 biological replicates. ****p<0.0001 by T-test. Error bars denote SEM.

Article Snippet: These images underwent Airyscan Joint Deconvolution and Channel Alignment post-processing steps, prior to Imaris 3D analysis.

Techniques: Staining, Microscopy, Software, Marker, Produced, Control, Transmission Assay, Membrane

Journal: bioRxiv

Article Title: Alpha-synuclein regulates nucleolar DNA double-strand break repair in melanoma

doi: 10.1101/2024.01.13.575526

Figure Lengend Snippet: A, C) SK-Mel28 cells were seeded on PLL-coated coverslips and then fixed and stained for alpha-synuclein (Syn1), DSB marker (γH2AX), nucleolar mask (nucleostemin), and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with airyscan and data was analyzed using FIJI (intensity) or Imaris (colocalization). Quantification represents quantification from 3 biological replicates. * p<0.05, *** p<0.001, **** p<0.0001 by T-test or ANOVA. Error bars denote SEM for all graphs. N=nucleus, Nu=nucleolus. Same γH2AX quantification of control cells between A and C. B) SK-Mel28 control and KO cells were seeded on PDL-coated coverslips and then fixed in 4% paraformaldehyde. Proximity Ligation Assay (Duolink) was completed using antibodies against Syn1 and γH2AX. Cells were imaged on the Zeiss 980 confocal microscope and number of foci per nucleus was measured using CellProfiler while masking for the nucleus using DAPI. Each figure shows representative images and quantification from 3 biological replicates. ****p<0.0001 by T-test. Error bars denote SEM. D) SK-Mel28 cells (control/KO/KI) were lysed and a nuclear fractionation was performed. Nuclear protein was run out on SDS-PAGE and probed for γH2AX and total protein. Western blots were imaged on Licor CLx imager. *** p<0.001 by ANOVA. Error bars denote SEM. Quantification from 4 biological replicates. E, F) SK-Mel28 cells were seeded on PLL-coated coverslips and treated with DMSO, KU-60019 (10µM), VE-822 (0.1µM), or NU-7441 (1µM) for 24 hours. Cells were fixed and stained for Syn1, γH2AX, nucleostemin and DAPI. Mean intensity of γH2AX signal within DAPI and nucleostemin masks analyzed using FIJI. ** p<0.01, *** p<0.001, **** p<0.0001 by ANOVA. Error bars denote SEM. Quantification from 3 biological replicates.

Article Snippet: These images underwent Airyscan Joint Deconvolution and Channel Alignment post-processing steps, prior to Imaris 3D analysis.

Techniques: Staining, Marker, Microscopy, Control, Proximity Ligation Assay, Fractionation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Alpha-synuclein regulates nucleolar DNA double-strand break repair in melanoma

doi: 10.1101/2024.01.13.575526

Figure Lengend Snippet: A) SK-Mel28 cells were seeded on PLL-coated coverslips and then treated with WT I-PpoI mRNA for 6 hours or 100ng/ml Actinomycin D for 1 hour prior to fixation. Cells were stained for Syn1, nucleolar caps marker (UBF), nucleostemin, and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with Airyscan oversampling and Joint Deconvolution and Channel Alignment post-processing. 3D renderings and distance from cap analysis were produced using Imaris software. Quantification from 5 biological replicates. Error bars denote SEM. Statistical significance was calculated via nonlinear regression (99% confidence interval). B) SK-Mel28 cells (control/KO/KI) were seeded on PDL-coated 8-well Ibidi plates. Cells were transfected with 800ng GFP-Treacle using Lipofectamine 3000. Twenty-four hours post-transfection, cells were treated with WT I-PpoI mRNA. Four hours after treatment, live-cell imaging was performed using the Zeiss Celldiscoverer 7 and imaged for 15 hours. Quantification from 5 biological replicates. *p<0.05, ****p<0.0001 by two-way ANOVA or Mantel-Cox test.

Article Snippet: These images underwent Airyscan Joint Deconvolution and Channel Alignment post-processing steps, prior to Imaris 3D analysis.

Techniques: Staining, Marker, Microscopy, Produced, Software, Control, Transfection, Live Cell Imaging