plasmid encoding lem2 mut mcherry  (Addgene inc)

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, Schematic representation of nuclear disruption phenotypes when BAF-LEM2 is depleted. b, Composition of the nuclear lamina, highlighting structured domains (SD) and intrinsically disordered regions (IDR) of its constituent proteins. SDS PAGE (left) and SEC Elution profile (right) of purified recombinant c, LEM2 d, BAF. e, SDS PAGE of purified recombinant LEM2 MUT . f. Domain chart showing protein domains of various recombinant constructs. Labels indicate domain names, and numbers represent amino acid positions. The red vertical lines indicate phosphomimetic mutations (PMIM; S,Y,T → D) introduced in LEM2 to abolish multivalent interactions. Full length LEM2 (LEM2 FL ) is shown as reference. g, Bulk co-condensation assay of LEM2 and BAF showing enrichment of BAF in LEM2 condensates. h, Normalized averaged line intensity profiles of BAF, LEM2 and DNA across nuclei (left, red line) for different cells plotted as mean ± sd (right). j, Quantification of mean pixel intensity of DNA in the nuclear rim across knockdowns (N= 368, 220, 196 for siCtrl, siLME2 and siBAF respectively) indicating that the enrichment of DNA does not change with depletion of BAF or LEM2. Vertical lines in the violins show median and the interquartile ranges. k, Confocal images showing bulk phase separation upon concentration titration of LEM2. l, Assay of condensate volume fraction to determine C sat for LEM2 (n=33,34,47,43,32 for input protein concentrations 17.58µM, 20.51µM, 23.44µM, 26.37µM, 29.29µM respectively, methods ). Bars and whiskers represent mean ± s.d respectively. Scale bar, 5 µm. Statistical test used is Kruskal-Wallis test with multiple comparisons using Dunn’s method. * p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, ns: non-significant.

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Disruption, SDS Page, Purification, Recombinant, Construct, Concentration Assay, Titration

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, DNA-BAF-LEM2 interfaces the chromatin surface with the INM. Numbers indicate amino acid positions. b, Confocal imaging reveals bulk phase separation of reconstituted DNA-BAF-LEM2 (methods). c, Confocal imaging of BAF, LEM2 and DNA in the nucleus shows enrichment at the nuclear envelope (methods). d, Top, schematic of the optical tweezers assay where a single λ-DNA molecule is held between two optically trapped beads (large red cones with spheres) via biotin–streptavidin interactions at a tension of ∼40 pN. Confocal imaging (small red cone) reveals BAF and LEM2 association to the stretched DNA. Bottom, confocal images of BAF (top), BAF and LEM2 (middle) and LEM2 (bottom) association to stretched λ-DNA. e, Average tension vs. end-to-end distance graphs obtained with the optical tweezer by gradually reducing DNA end-to-end distance from 16.7 µm to 5 µm (top inset) reveals a constant tension plateau indicative of protein-DNA co-condensation of DNA with BAF (n = 8), with BAF+LEM2 MUT (n = 7), and with BAF+LEM2 (n = 7; methods ). Solid colored lines, average tension; colored shaded regions, mean ± standard deviation; dotted lines, extrapolations of tension to zero end-to-end distance; dashed black line, worm-like-chain (WLC) model of λ-DNA elasticity (methods). f, Schematic representation (top) and representative example from e (bottom) of DNA-BAF-LEM2 co-condensates, DNA (yellow) is labelled with Sytox Orange (SO). Scale bar, 0.5 µm. g, Co-condensation energy per DNA base pair (bp) obtained by integration of the tension vs. distance curves in e . Co-condensation energy is significantly higher for DNA-BAF-LEM2 than for DNA-BAF and DNA-BAF-LEM2 MUT . h, Representative tension vs. end-to-end distance graphs (solid dots) obtained by gradually increasing DNA end-to-end distance from 5 µm to ∼16.5 µm (top inset) reveals characteristic ‘rip patterns’, with each rip unraveling extended amounts of DNA, during DNA-BAF and DNA-BAF-LEM2 co-condensate tension-induced disassembly. Dotted lines, WLC fits ( methods ) reveal the contour length of DNA inside co-condensates (see j ). i, Corresponding average tension vs. end-to-end distance graphs for DNA-BAF (n = 10), and DNA-BAF-LEM2 (n = 10; methods ) co-condensate tension-induced disassembly. Solid colored lines, average tension; colored shaded regions, mean ± standard deviation. DNA-BAF co-condensates disassemble at tensions slightly higher than those required to dissociate nucleosomes from DNA, while disassembling DNA-BAF+LEM2 co-condensates requires tensions above the critical melting tension of 65 pN. Dashed black line, WLC model of λ-DNA. j, Fraction DNA remaining co-condensed during tension-induced disassembly as a function of applied tension. Note that 66±22 % of DNA remains co-condensed with BAF+LEM2 at the critical melting tension of 65 pN. k, Co-condensate per-base pair disassembly work obtained by integration of the tension vs. distance curves in i (BAF and BAF-LEM2) and (BAF-LEM2 MUT ; n=5). l, Top, confocal image of SO, bright in directions of high DNA tension and dim in regions of low DNA tension, at 52 pN DNA tension reveals that DNA tension is higher outside co-condensates than inside. Bottom, corresponding profiles of normalized intensities showing alternate low and high distributions. m, Left, representative image (top) and kymograph (bottom) of DNA-BAF-LEM2 co-condensate disassembly under high tension reveals that the ssDNA binding protein FUS (green) exclusively associates with non-co-condensed regions, revealing that DNA inside co-condensates is protected from melting. Right, FUS partitions into LEM2 condensates in bulk, indicating that FUS is not excluded from DNA-BAF-LEM2 co-condensates. n, Sketch of the IDR-dependent mechanism of co-condensate tension buffering. DNA tension (pink arrows) is reduced within co-condensate (bottom) as compared to without (top), since inside tension redistributes through IDR-IDR and other molecular interactions. Arrow thickness indicates relative tension strength, and direction shows the path of redistribution. Color bar represents tension magnitude along the DNA. Scale bar, 5 µm in b, c, m- right and 2 µm in d, l, m- left.

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Imaging, Standard Deviation, Binding Assay

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a,b, Schematic diagrams and GC content profiles (Scale bar, 10%) of biotinylated (a) λ-DNA and (b) NPS DNA (left), with corresponding tension-extension curves (right). c, Confocal images of control experiment to test binding of the free chemical dye to DNA (tethered between beads), d, Confocal image of binding of LEM2 (labeled)+BAF to NPS DNA. e, One or more co-condensates of DNA and BAF in optical tweezer with 100nM BAF (fluorescently labelled). f, Average tension-distance profiles of DNA with different concentration of BAF while the end-to-end distance is decreased suggesting that BAF binding to DNA saturates around 100 nM. Black dashed line indicates the eWLC fit. Data is plotted as mean±sd (n=8 for 100nM, n=6 for 50nM, n=6 for 5nM). g, The corresponding quantification of plateau tension at 5 µm end-to-end distance from tension-distance profiles in f. h , Confocal images showing one or more co-condensates of DNA-BAF-LEM2 in optical tweezer, with 100nM BAF (unlabelled) and 500nM of LEM2 (fluorescently labelled). i, FRAP of BAF-LEM2 co-condensate in optical tweezer. Top, pre-bleach confocal slice and post-bleach kymograph of FRAP time course for DNA-BAF-LEM2 co-condensate (inside white ROI). Bottom, average recovery profile of normalized fluorescence intensity (n=7). j,l, Tension-distance profiles of DNA while the end-to-end distance is decreased in case of (j) BAF and (l) BAF-LEM2. Red lines indicate the steady-state values of force for each end-to-end distance. Black dashed lines represent eWLC fit to calculate the amount of co-condensed DNA ( methods ). k,m, Tension-time profiles of DNA while the end-to-end distance is decreased in case of (k) BAF and (m) BAF-LEM2. Inset shows the equilibration of force with time ( methods ). n, Quantification of total co-condensed DNA length as a function of end-to-end distances for BAF, BAF-LEM2 and BAF-LEM2 MUT (BAF : n=8; BAF-LEM2: n = 7; BAF-LEM2 MUT : n = 7).

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Control, Binding Assay, Labeling, Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, The tension-distance profiles when the BAF co-condensates are disassembled as the end-to-end distance is increased at different speeds. b, Temporal tension relaxation profile following an instantaneous increase in trap-to-trap distance from 5 µm to 10 µm (BAF-LEM2:n=11, BAF:n=9, methods ). c, FRAP of BAF-LEM2 co-condensate in bulk. Top: Confocal images showing fluorescence recovery after photobleaching (FRAP) of BAF-LEM2 condensates at indicated time points. Bottom, average normalized fluorescence recovery curves at different time points post-condensate formation (1.5 to 3.5 hours). Quantification shows mean ± s.d. of n = 5 replicates except for 2.0 hr, where n = 4. d, The timescales of recovery of fluorescence intensity calculated from a simple exponential fit ( methods ) e, Average tension-distance profiles during co-condensate disassembly across multiple experiments for BAF-LEM2 MUT (n = 5; BAF+LEM2 is replotted for reference). The dashed black line represents the eWLC fit for naked λ-phage DNA. f, Quantification of DNA wrapped inside the co-condensates as a function of applied tension (BAF+LEM2 is replotted for reference). g, Top, confocal image showing the binding pattern of SO on DNA in the presence of BAF-LEM2 co-condensates at high tension, with normalized intensity profiles of SO (yellow) and LEM2 (magenta) along the DNA (see methods). Bottom, co-localization of LEM2 and SO signal and corresponding normalised intensity profiles at low tension. Scale bar, 2 µm. h, Quantification of the Pearson correlation coefficient of LEM2 and SO signals at low tension (n=6) and high tension (n=8). i, Confocal slice showing the binding of FUS-GFP to ssDNA. j, Kymograph showing FUS-GFP signal emergence coinciding with melted ssDNA formation during DNA extension. k, Overlay of LEM2 (stains dsDNA) and FUS (stains ssDNA) signals on overstretched λ-DNA.

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, Top, DNA stretched to an initial ∼40 pN tension is subjected to a buffer exchange from protein-free (left) to protein-containing (right) at time t = 0s while trap positions are maintained. Bottom, tension increases with time post addition of 100nM BAF + 500nM LEM2 (BAF+LEM2) and 100nM BAF + 500nM LEM2 MUT (BAF+LEM2 MUT ) due to module assembly on DNA, but does not increase post addition of 100nM BAF alone. Solid colored lines, mean tension; colored shaded regions, mean ± standard deviation (BAF: n=7, BAF-LEM2: n=11, BAF-LEM2 MUT : n=12). b, Corresponding average tension increases ΔT. c, ΔT as a function of initial DNA tension and LEM2 concentration (100nM BAF, n=20 for 500nM LEM2, n=17 for 250nM LEM2, : n=19 for 50nM LEM2). Dashed lines, linear fit to the data within 95% confidence interval. d, Direct experimental characterization of BAF-LEM2 module compliance in the enthalpic regime of DNA stretching via average tension vs. DNA end-to-end distance graphs in absence (yellow shaded region, mean ± standard deviation, n=11) and in presence (magenta shaded region, mean ± standard deviation, n=11) of 100nM BAF and 500nM LEM2. Dotted lines, WLC model of λ-DNA. Linear fits (dashed lines) in the enthalpic regime ( methods ) reveal a stiffness but not a rest-length change between the two conditions (see main text). Red arrows illustrate tension increase ΔT when keeping DNA end-to-end distance fixed. e, Effective stiffness as a function of LEM2 concentration (100nM BAF) measured via constant tension experiments ( , methods ). Error bars, mean ± standard deviation; dashed line, fit to the stiffening reinforcement model. f, Sketch of the IDR-dependent mechanism of assembly-induced stiffening reinforcement. Top, DNA, represented as a series of springs (stiffness k o ), is held in place at fixed end-to-end distance. BAF-LEM2 module assembly results in the addition of springs in parallel (stiffness k i ). Saturated BAF-LEM2 binding increases the effective stiffness of the system from K 0 (DNA alone) to K 0 + K i . Bottom, Saturated BAF-LEM2 binding and compliant element assembly generates tension T BAF-LEM2 (magenta arrows) in addition to DNA tension T DNA (yellow arrows), increasing the total tension experienced by the system (pink arrows).

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Buffer Exchange, Standard Deviation, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, Asymptote (black dashed line) to the eWLC model (yellow line, for λ-DNA), indicating that the rest length is shorter than the contour length for linear fits (red dashed line, inset) in the 20-40 pN range. b, Tension-distance profiles of NPS-DNA with (magenta) and without (yellow) BAF(100nM)-LEM2(500nM) while the end-to-end distance is increased. Dotted lines indicate the eWLC fits, whereas the dashed lines are linear fit to the respective data ( methods ) within 95% CI. (naked DNA:n=3, BAF-LEM2:n=3). c, Steady state end-to-end distance vs. tension for λ-DNA under a constant-tension protocol, across different LEM2 concentrations (with 100 nM BAF). Linear fits (dashed lines) show progressively higher stiffness with increasing LEM2 concentration (500 nM: n=18; 250 nM: n=18; 50 nM: n=17). d. Ensemble average stretch x̅ − x 0 per binding site (solid line) and average occupation fraction p̅ (dotted line) as a function of applied tension on DNA (parameters used : k 0 = 1 pN/nm; k i = 2 pN/nm; c 0 exp(βε) = 100 μM, c = 500μM, SI). Two regimes (at low and high tension) are shown by grey dashed lines with the corresponding stiffnesses k 0 and k ( p̅ 0 ) indicated. The half-saturation tension is indicated as a vertical grey dashed line). e , Shortening of the end-to-end distance X̅ (0) − X̅ ( p̅ ) under constant-tension condition (from c ) plotted against applied tension for varying LEM2 concentrations (with 100nM BAF). Dashed lines represent a global fit to the statistical mechanics model (SI). f, Change in DNA compliance K (0) −1 − K ( p̅ ) −1 (from panel e ) shown as a function of LEM2 concentration (with 100nM BAF). The red dashed line represents a global fit to the statistical mechanics model (SI). The top axis shows the corresponding occupation fraction. g, Quantification of the microscope’s point spread function (PSF) from the intensity distributions of a single fluorophore. Individual points represent the maximum of pixel values along the x-y plane for each z-slice ( methods ). The solid line denotes the Gaussian fit. Inset, representative confocal image showing single Atto-647N dyes conjugated to λ-DNA. (h-i) Corresponding intensity distributions along ( h ) x and ( i ) y in the focal plane, used to determine the PSF in the x and y directions (n=5). j, The UV-Vis spectrum of labeled LEM2-DyLight 650 used to calculate the labeling efficiency ( methods ). k, Calibration of the mean background intensity as a function of input LEM2 concentration. Inset, representative confocal image showing LEM2 (tagged with DyLight-650) bound to DNA in optical tweezers. Cyan and white box indicate the ROIs used for background and DNA bound protein intensity quantifications ( methods ). l, Average number of LEM2 bound in presence of 100 nM BAF as a function of LEM2 concentration ( k,l LEM2:500 nM n=11; LEM2:250 nM n=10; LEM2:100 nM n=7; LEM2:50 nM n=9; LEM2:10 nM n=12). m, Average pixel intensity, as a measure of total number of LEM2 bound to DNA across different forces for 100 nM BAF and 500 nM LEM2 (n=10).

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Concentration Assay, Binding Assay, Labeling

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, Schematic showing the translation of individual springs to a 2D meshwork (left) representing the network of BAF-LEM2 around the chromatin surface (right). b, Cryo-electron tomography of the nuclear envelope. Top, tomographic slices from different positions showing a continuous amorphous layer beneath the inner nuclear membrane. Bottom, the corresponding segmentations. c, 3D segmented models of the nuclear surface showing integration of lamin filaments into the hydrogel layer, flanked by INM and nucleosomes. Chromatin was cropped from the top to reveal the lamin network. Scale bar, 100 nm. d, Structural organization of lamin filaments inside INM. Top, cross-sectional view of the nuclear envelope showing lamin filaments embedded in the hydrogel layer. Bottom, segmentation of lamins. Scale bar, 100 nm. e, Zoomed-in view of tomographic slices from replicate tomograms showing the hydrogel layer (red arrows). Scale bar, 50 nm. f, Histogram showing the distribution of hydrogel thickness across tomograms, fitted with a normal distribution (mean thickness = 14 nm, n = 130). g, Confocal images showing the distribution of BAF in different cellular compartments, nucleus, nuclear envelope and the cytoplasm. Scale bar, 5 µm. h, Representative images showing the 3D segmentation of BAF pools in the cytoplasm, nuclear envelope and the nucleus. Scale bar, 5 µm. i , The average copy numbers of BAF in the three cellular pools of BAF: nucleus, NE and cytoplasm (n = 31 cells, methods ).

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Tomography, Membrane

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, Cryo-electron tomography of the nuclear envelope from , here reprocessed and reanalyzed. Top, representative slices from reconstructed tomogram revealing an amorphous layer beneath the INM approximately 14 nm thick (region between two red arrows). Bottom, 3D segmentation highlighting the lamin network, chromatin, nuclear envelope, microtubules (MTs), and ribosomes. Chromatin was cropped from the top to reveal the lamin network. Scale bar, 200 nm. b, Left, representative AFM force-indentation curves of nuclei of latrunculin A-treated HCT-116 cells, for siCtrl and siBAF cells. The cantilever makes contact with the nucleus at a vertical tip position of 0 µm, and we measure the mechanical work (shaded areas under the curves) required to indent the nucleus by 2 µm. Indentation in absence of BAF requires less work than in presence. Right, representative live-cell images of an HCT-116 BAF-mNeonGreen cell not indented (Vertical tip position +10 µm) and indented (Vertical tip position −2 µm). Segmentation of the nuclear surface reveals the nuclear surface area increase, which together with the mechanical work ( c ) allows for determination of the effective area modulus shown in ( d ) (methods). Dashed white line, illustration of pyramidal cantilever tip. Scale bar, 5 µm (horizontal and vertical). c, Mechanical work for a 2 µm indentation of the nucleus of siCtrl and siBAF cells ( methods ), from three biological replicates (siCtrl: n = 159, siBAF: n = 319). d, Effective area modulus of nuclei of control and siBAF cells ( ; siCtrl: n = 159, siBAF: n = 319, methods ). e, Left, Confocal image stacks of chromatin (NucBlue) of siCtrl (Top) and siBAF (Bottom) cells. Right, 3D segmentations of the nuclear surfaces for determining nuclear volume, shape, and surface area. f, Top, Schematic of the REMM surface hydrogel (purple shade) formed below the INM (gray), comprising LEM2 (magenta), BAF (cyan), lamin filaments (green), DNA (yellow) and nucleosomes, with reinforcing properties as described (elastic properties indicated by coloured springs, tension indicated by colored arrows). Bottom, surface hydrogel free energy ( F ) is given by the sum of two integrals. The first integral, taken over the total area ( A ), includes the bending energy term with bending modulus κ and average and spontaneous curvatures H and C 0 . The second integral, over the strain-free area ( A * ), combines a bare area elastic energy term representing lamins with bare elastic area modulus λ and surface strain u , and a DNA-BAF-LEM module term with stiffness k , stretch relative to rest length x ( u ) – x 0 with and x * = x (0), and surface density of DNA-BAF-LEM module binding sites in the strain-free state 1/ l * 2 . ( SI ). g, Top, Plots of the normalized free energy ( F / F 0 ) of the REMM model ( f, see methods) as a function of nuclear asphericity reveal that the minimum for the siBAF condition with k = ξ −1 ( k 0 + k i ) with measured stiffness ratio ξ (see SI) is placed at a larger asphericity than for the siCtrl condition with k 0 = k + k i . Bottom, histograms of nuclear asphericity measured as in ( e ) for siCtrl and siBAF cells (n = 603 and n = 591, respectively, pooled across three biological replicates). Colored lines, log-normal fit. With REMM model parameters λ = 0. 07 pN/nm, A * = 1. 8 A V , κ = 0, rest length mismatch x 0 / x * = 0. 74 and energy scale F 0 = λ A * . The histograms peak at the values that minimize the corresponding REMM surface hydrogel free energy F (dashed lines).

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Tomography, Control, Binding Assay

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a-d, Quantification of protein knockdowns. Confocal images showing loss of fluorescence upon (a) BAF knockdown, (c) LEM2 knockdown and (b,d) corresponding quantification of the extent of knockdown from fluorescence loss (siCtrl vs siBAF; n = 12 each, siCtrl vs siLEM2; n = 15 each, each point represents independent fields of view). e, Western blot showing depletion of LEM2 in HCT116-Lamin A/C mNeonGreen cells. GAPDH is a loading control. f, Maximum projection confocal images of nuclei across knockdown showing morphology defects. g-h, Cross-sectional area (g) and circularity (h) of nuclei across knockdowns (siCtrl: n=973, siLEM2: n=790, siBAF: n= 720 cells, data pooled across three biological replicates). i, Representative image of 3D surfaces reconstructed on siCtrl and siBAF nuclei showing nuclear sphericity with a color gradient (colorbar labels indicate sphericity values). j, Histogram and corresponding lognormal fits of asphericity of siCtrl (0.0754±0.0050) and siLEM2 (0.0664±0.0066). siCtrl: n = 241, siLEM2: n = 163 cells. k, Histograms and corresponding lognormal fits of sphericity between siCtrl (0.9256±0.0020) and siBAF (0.8839±0.0042) l, Histograms and corresponding lognormal fits of nuclear volume between siCtrl (965±19 µm ) and siBAF (1012±26 µm ). m, Histograms and corresponding lognormal fits of the nuclear area between siCtrl (516±6 µm 2 ) and siBAF (556±9 µm 2 ). siCtrl: n = 603, siBAF: n = 591 cells, pooled across three biological replicates in k,l,m. Bars and whiskers / midline and whiskers in scatter plots represent mean±s.d. respectively. Scale bar, 5 µm. Statistical test used is Kruskal-Wallis test with multiple comparisons using Dunn’s method. * p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, ns: non-significant.

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Fluorescence, Knockdown, Western Blot, Control

Journal: bioRxiv

Article Title: A protein-DNA surface hydrogel mechanically protects the cell nucleus

doi: 10.1101/2025.05.21.655270

Figure Lengend Snippet: a, Representative confocal images showing DNA damage loci visualized by immunofluorescence of 53BP1 in interphase HCT116 LEM2-mNeonGreen cells following siRNA treatments as indicated and compression to 6 µm. The compression induced mechanical strain results in DNA damage foci in siLEM2 cells. b, Representative confocal images of live HCT116 LaminA/C-mNeonGreen cells with micronuclei (white arrows) following siRNA treatments and compression to 6 µm. The compression induced mechanical strain results in micronucleation in siLEM2 cells. c , Percentage of nuclei that form 53BP1 DNA damage foci following siRNA treatments as indicated and remain uncompressed or are compressed to 6 µm (see a ; three biological replicates, uncompressed: n = 26, 38, 34; compressed: n = 39, 41, 52 fields of view for siCtrl, siLEM2 and siBAF respectively). DNA damage increases ∼2.2 fold in siLEM2 cells with compression compared to without compression. Non-significant comparisons are not shown. d, Percentage of cells that form micronuclei following siRNA treatments as indicated and compression to 6 µm (see b , three biological replicates, siCtrl: n = 48; siLEM2: n = 54; siBAF: n = 53). e, The REMM surface hydrogel model and its effect on the nuclear periphery and nuclear homeostasis. Bars and whiskers represent mean ± s.d respectively. Scale bar, 5 µm. Statistical test used is Kruskal-Wallis test with multiple comparisons using Dunn’s method ( c,f) . One-way ANOVA with multiple comparisons. * p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, ns: non-significant.

Article Snippet: The plasmid encoding mApple-53BP1trunc was purchased from Addgene (#69531).The plasmid encoding LEM2 MUT -mCherry was prepared by Gibson assembly of human codon-optimised synthesised LEM2 MUT gene block and pMGF196 backbone coding for mCherry and puromycin.

Techniques: Immunofluorescence