Journal: Oncotarget

Article Title: Barrier-to-Autointegration Factor (BAF) involvement in prelamin A-related chromatin organization changes

doi: 10.18632/oncotarget.6697

Figure Lengend Snippet: ( A ) Western blotting evaluation of total lysates from HEK293 cells subjected to BAF depletion by siRNA treatment. Cells treated with scramble or BANF1 siRNA (si BANF1 ) were untransfected (−) or transfected with LA-WT or LA-C661M constructs. FLAG, LAP2α, HP1α and BAF proteins bands are shown. Actin: protein loading control. Western blotting densitometric analysis is reported. ( B ) Immunofluorescence evaluation of BAF in HEK293 cells treated with scramble or BANF1 siRNA. BAF (green) and DNA staining (DAPI) are shown. Bar 10 mm. ( C ) Immunofluorescence detection of LA-WT, LA-C661M, HP1α and LAP2α in cells treated with scramble siRNA or si BANF1 . FLAG (red), HP1α and LAPα (green). DNA was costained by DAPI and shown in merge. Arrowheads: nuclear lamina rarefaction staining of FLAG tagged proteins. Empty arrows: absence of the HP1α nuclear periphery staining increase. Arrow indicates the defective LAP2α nuclear periphery recruitment. Higher magnification of nuclear periphery distribution of HP1α and LAP2α staining, merged with DAPI, is shown. Bar 10 μm.

Article Snippet: BAF depletion by siRNA treatment (Santa Cruz biotechnology SC-44804) was performed according to the manufacturer's protocol.

Techniques: Western Blot, Transfection, Construct, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Emerin oligomerization and nucleoskeletal coupling at the nuclear envelope regulate nuclear mechanics against stress

doi: 10.1101/2021.02.12.429834

Figure Lengend Snippet: a) Diffusion coefficients (± s.e.m.) and population percentages of wild-type emerin (untreated, n =71004 trajectories in 14 nuclei) after depletion of lamin A/C (lamin A/C KD, n =60569 trajectories in 11 nuclei), depletion of nuclear actin (IPO9 KD, n =74501 trajectories in 17 nuclei) or replacement of endogenous BAF with BAF L58R mutant (BAF KD + BAF L68R, n =62714 trajectories in 8 nuclei) (F-test, ns: non-significant, **: p <0.01). b) Schematic of emerin fused to complementary split-GFP fragments and co-expressed to track the mobility of emerin oligomers by single molecule imaging of complemented GFP. c) Map of individual trajectories for complemented emerin-GFP-emerin species at the nuclear envelope (left) and examples of oligomer domains (squares) where trajectories often overlap (left). Scales: 2 µm (left) and 50 nm (right). d) Comparison of diffusion coefficients (± s.e.m.) and population percentages for individual wild-type emerin assessed by sptPALM and complemented emerin-GFP-emerin species (n=4833 trajectories in 13 nuclei) assessed by CALM (F-test, ns: non-significant). e) Diffusion map of wild-type PA-TagRFP-emerin at the nuclear envelope where emerin oligomers form slow mobility domains (blue, O) surrounded by larger areas where emerin monomers diffuse faster (red, M). Scale: 500 nm.

Article Snippet: BAF L58R was expressed from an EGFP-BAF L58R lentiviral plasmid (Addgene #101776) and lentiviral particles were produced by the UCLA Vector Core.

Techniques: Diffusion-based Assay, Mutagenesis, Imaging, Comparison

Journal: bioRxiv

Article Title: Emerin oligomerization and nucleoskeletal coupling at the nuclear envelope regulate nuclear mechanics against stress

doi: 10.1101/2021.02.12.429834

Figure Lengend Snippet: a) Super-resolved 3D-dSTORM positions (top half) and non-super-resolved imaging (bottom half) of wild-type SNAP-emerin at the bottom nuclear envelope of an EMD -/y HDF. Scale: 5 µm. b) Neighborhood densities (± s.d.) and cluster analyses of wild-type emerin at the nuclear membrane of untreated HDF (n=189,331 localizations in 10 nuclei) and HDF after lamin A/C knock down (n=178,206 localizations in 6 nuclei). Neighborhood densities at various length scales are compared to MonteCarlo simulations of complete spatial randomness and fitted to measure molecular densities above random and length scale of significant clustering (green). c) Relative nuclear envelope molecular densities above random (± s.e.m.) for wild-type emerin oligomers (O) and monomers (M) in untreated EMD -/y HDF treated with control siRNA (n= 180,546 localizations in 5 nuclei), depleted for lamin A/C, depleted for nuclear actin after IPO9 knock down (n=225,394 localizations in 9 nuclei), with endogenous BAF replaced by BAF L58R (n=90,241 localizations in 6 nuclei) or depleted for SUN1 (n=258,300 localizations in 6 nuclei). Values in parenthesis represent the typical length scale (± s.e.m.) of each domain in nanometers. d) Local cluster map of wild-type emerin at the nuclear envelope of an untreated HDF, for a search radius of 25 nm ( L (r 25 )). e) Local cluster map of wild-type emerin after lamin A/C knock down. f) Local cluster map of wild-type emerin after destabilization of the LINC complex by SUN1 knock down. Cluster values in maps are assigned to all localized emerin, and values L (r 25 )=25 represent areas where emerin is randomly distributed while L (r 25 )=70 values represent areas with emerin local densities (70/25) = ∼8-fold higher than expected for a random distribution. M: monomer areas, O: oligomer nanodomains. Scales for d, e and f: 250 nm.

Article Snippet: BAF L58R was expressed from an EGFP-BAF L58R lentiviral plasmid (Addgene #101776) and lentiviral particles were produced by the UCLA Vector Core.

Techniques: Imaging, Membrane, Knockdown, Control