Journal: The EMBO Journal

Article Title: Nuclear rupture in confined cell migration triggers nuclear actin polymerization to limit chromatin leakage

doi: 10.1038/s44318-025-00566-2

Figure Lengend Snippet: ( A ) Image sequences of representative HT1080-nAC-GFP cells migrating through 3 µm microchannels, after being treated with either 0.01% DMSO or 100 µM of the Arp-2/3 inhibitor CK-666. Actin is visualized via nAC-GFP. Scale bar, 5 µm. ( B ) Percentage of HT1080-nAC-GFP displaying nuclear F-actin structures after treatment with either 0.01% DMSO or 100 µM CK-666. Data shown as mean ± s.d. Two independent experiments per condition. n ≥ 8 cells per independent experiment. ( C ) Representative immunoblots for the silencing efficiency of DIAPH1 (siDIAPH1), DIAPH2 (siDIAPH2), DIAPH3 (siDIAPH3), DIAPH1 and DIAPH3 (siDIAPH1/siDIAPH3), and INF2 (siINF2). Tubulin serves as a loading control. ( D ) Image sequences of HT1080-nAC-GFP cells that were treated with 0.01% DMSO or ROCK inhibitor 10 µM Y27132 before and during NE rupture. Actin is visualized via nAC-GFP. Scale bar, 5 μm. ( E ) Percentage of cells displaying nuclear F-actin upon NE rupture after being treated with either DMSO or Y27132 . Data shown as mean ± s.d. Three independent experiments per condition with n ≥ 6 cells per experiment. Statistical analysis was performed with an unpaired parametric t -test. ( F ) Image sequences of a representative HT1080-icGAS-mCherry cell that expresses FMNL2-GFP migrating through a microchannel. Before NE rupture; corresponds to the time before a NE rupture event had occurred. NE rupture; corresponds to the first frame in which a NE rupture event is detected via accumulation of icGAS-mCherry. After exit; corresponds to the time point at which the nucleus has exited from the narrow part of the microchannel. Scale bar, 5 µm. ( G ) Quantification of nuclear FMNL2-GFP intensity before NE rupture, during NE rupture and after exit. Data shown as individual values from n = 5 cells. Statistical analysis was performed by a paired parametric t -test. ( H ) Fold change of nuclear FMNL2-GFP and nuclear GFP-Diaph3 mean fluorescence intensities upon NE rupture. Data shown as individual values from n = 5 cells. Statistical analysis was performed by an unpaired parametric t -test. Exact P value: 3.0 × 10 − ⁷. **** P < 0.0001 and ns not significant. .

Article Snippet: DIAPH3 , Proteintech , 14342-1-AP.

Techniques: Western Blot, Control, Fluorescence

Journal: The EMBO Journal

Article Title: Nuclear rupture in confined cell migration triggers nuclear actin polymerization to limit chromatin leakage

doi: 10.1038/s44318-025-00566-2

Figure Lengend Snippet: ( A ) Top, schematic representation of a cell migrating through a 3 µm microchannel. Bottom, representative image sequences of HT1080 cells stably expressing nAC-GFP migrating through 3 µm microchannels. HT1080 cells are treated with control siRNA (siCtrl) or siRNA targeted against DIAPH1 (siDIAPH1), DIAPH2 (siDIAPH2), DIAPH3 (siDIAPH3) or DIAPH1 and DIAPH3 combined (siDIAPH1/siDIAPH3). Actin is visualized via nAC-GFP. Scale bar, 5 µm. ( B ) Percentage of HT1080 cells stably expressing nAC-GFP displaying nuclear F-actin formation after treatment with either siCtrl, siDIAPH1, siDIAPH2, siDIAPH3, siDIAPH1/siDIAPH3, or siINF2. Data shown as mean ± s.d. Three independent experiments per condition and two independent experiments for siINF2, n ≥ 9 cells per experiment. Statistical analysis was performed by one-way analysis of variance (ANOVA). Exact P values from left to right: P = 0.00296, P = 0.0006, P = 0.00003. ( C ) Representative image sequences of an HT1080 cell transiently expressing GFP-Diaph3 (green) and icGAS-mCherry (red) migrating through a microchannel. Scale bar, 4 µm. ( D ) Quantification of nuclear GFP-Diaph3 intensity before NE rupture, during NE rupture and after Diaph3 export. Data shown as individual values from n = 5 cells. Statistical analysis was performed by paired parametric t -test; Exact P value: 0.00054. ns P > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. .

Article Snippet: DIAPH3 , Proteintech , 14342-1-AP.

Techniques: Stable Transfection, Expressing, Control

Journal: The EMBO Journal

Article Title: Nuclear rupture in confined cell migration triggers nuclear actin polymerization to limit chromatin leakage

doi: 10.1038/s44318-025-00566-2

Figure Lengend Snippet: ( A ) Schematic representation of a cell migrating through a 3 µm microchannel. Nuclear deformation results in an NE rupture event as observed by perinuclear accumulation of icGAS, followed by either exit of the nucleus from the microchannel or nuclear fragmentation and halt of migration. ( B ) Representative image sequences of HT1080 cells stably expressing H2B-mCherry and icGAS-GFP migrating through 3 µm microchannels after treatment with either control siRNA (siCtrl) or siRNA against DIAPH1 and DIAPH3 (siDIAPH1/siDIAPH3), undergoing nuclear fragmentation. The white arrowhead indicates nuclear fragmentation. Scale bar, 10 µm. ( C ) Percentage of HT1080 cells stably expressing histone H2B (H2B-mCherry) and icGAS-GFP treated with siCtrl or siDIAPH1/siDIAPH3 displaying nuclear fragmentation upon NE rupture. Data shown as mean ± s.d. Four independent experiments per condition, n ≥ 27 cells per experiment. Statistical analysis was performed with unpaired parametric t -test. Exact P value: 0.0005. ( D ) Image sequences of representative HT1080-H2B-mCherry/icGAS-GFP/NLS-BFP or NLS-BFP-Actin-R62D (blue) migrating through 3 µm microchannels, undergoing nuclear fragmentation. The white arrowhead indicates nuclear fragmentation. Scale bar, 10 μm. ( E ) Percentage of HT1080 cells stably expressing H2B-mCherry/icGAS-GFP/NLS-BFP or NLS-BFP-Actin-R62D displaying nuclear fragmentation upon NE rupture. Data shown as mean ± s.d. and are displayed in a scatter dot bar plot. Three independent experiments per condition with n ≥ 38 cells per experiment. Statistical analysis was performed with an unpaired parametric t -test. Exact P value: 0.0008. ( F ) Representative image sequences of HT1080 cells expressing H2B-mCherry and icGAS-GFP migrating through 3 µm microchannels undergoing NE rupture, as observed by perinuclear accumulation of icGAS-GFP after being treated with either siCtrl or siDIAPH1/siDIAPH3. Scale bar, 10 µm. ( G ) Percentage of cells stably expressing H2B-mCherry/icGAS-GFP displaying NE rupture after being treated with either siCtrl or siDIAPH1/siDIAPH3. Data shown as mean ± s.d. Four independent experiments per condition with n = 20 cells per experiment. Statistical analysis was performed with the Mann–Whitney test. Exact P value: 0.6571. ( H ) Representative image sequences of HT1080-H2B-mCherry/icGAS-GFP expressing NLS-BFP or NLS-BFP-Actin-R62D migrating through 3 µm microchannels undergoing NE rupture, as observed by accumulation of icGAS-GFP. Scale bar, 10 µm. ( I ) Percentage of HT1080-H2B-mCherry/icGAS-GFP cells expressing NLS-BFP or NLS-BFP-Actin-R62D that undergo NE rupture as scored by perinuclear accumulation of icGAS. Data shown as mean ± s.d. Three independent experiments per condition with n = 20 cells per individual experiment. Statistical analysis was performed with the Mann–Whitney test. Exact P value: P > 0.9999. *** P < 0.001 and ns not significant. .

Article Snippet: DIAPH3 , Proteintech , 14342-1-AP.

Techniques: Migration, Stable Transfection, Expressing, Control, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Nuclear rupture in confined cell migration triggers nuclear actin polymerization to limit chromatin leakage

doi: 10.1038/s44318-025-00566-2

Figure Lengend Snippet: ( A ) Representative immunoblot of phosphorylated Chk1 (pChk1) (stripped and reprobed for total-Chk1) for the efficiency of ATR inhibitors ETP-46464 and VE-821. Loading control Lamin-A/C. ( B ) Percentage of HT1080 cells undergoing NE rupture after being treated with siCtrl or siATR. Data shown as mean ± s.d. Four independent experiments per condition with n ≥ 8 cells per experiment. Statistical analysis was performed with the Mann–Whitney test. ns not significant. ( C ) Representative images of HT1080-nAC-GFP (white) that transiently express NLS-BFP-Diaph3-wt in the presence or absence of LY2603618, with bottom right images showing nuclear expression of this construct (magenta); Scale bar, 4 µm. ( D ) Percentage of HT1080-nAC-GFP cells transiently expressing NLS-Diaph3-wt that display nuclear F-actin formation after being treated with either 0.01% DMSO or 1 µM LY2603618 for 16 h. Data shown as mean ± s.d. Three independent experiments per condition with n = 30 cells per experiment. Statistical analysis between DMSO and LY2603618 treatment after expression of NLS-BFP-Diaph3-WT was performed by an unpaired parametric t -test. ( E ) HT1080-WT cells were transfected with myc-NLS-Diaph-WT, and cells were treated with Hydroxyurea (HU) in the presence or absence of ETP-46464. Immunoprecipitation was carried out by myc-agarose beads. The amount of phosphorylated serine residues was determined via western blot and detected with a general anti-phosphoserine antibody (pSer) (stripped and reprobed for myc). pChk1 (stripped and reprobed for total-Chk1 and Tubulin) was used as a marker for ATR activation and to monitor the activity of ETP-46464. ( F ) Quantification of the phosphorylation of serine residues of myc-NLS-BFP-Diaph3-WT upon HU treatment in the presence or absence of ETP-46464. Fold change of the pSer/myc ratio was calculated. Data shown as mean ± s.d. Three independent experiments per condition. Statistical analysis was performed with a paired parametric t -test. Exact P value: 0.01948. ( G ) HT1080-WT cells were transfected with myc-NLS-Diaph-S1072A, and cells were treated with HU in the presence or absence of ETP-46464. Immunoprecipitation was carried out by myc-agarose beads. The amount of phosphorylated serine residues was determined via western blot and detected with a general anti-phosphoserine antibody (pSer) (stripped and reprobed for myc). pChk1 (stripped and reprobed for total-Chk1 and Tubulin) was used as a marker for ATR activation and to monitor the activity of ETP-46464. ( H ) Quantification of the phosphorylation of serine residues of myc-NLS-BFP-Diaph3-S0172A upon HU treatment in the presence or absence of ETP-46464. Fold change of the pSer/myc ratio was calculated. Data shown as mean ± s.d. Three independent experiments per condition. Statistical analysis was performed with a paired parametric t -test. * P < 0.05 and ns was not significant. .

Article Snippet: DIAPH3 , Proteintech , 14342-1-AP.

Techniques: Western Blot, Control, MANN-WHITNEY, Expressing, Construct, Transfection, Immunoprecipitation, Marker, Activation Assay, Activity Assay, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Nuclear rupture in confined cell migration triggers nuclear actin polymerization to limit chromatin leakage

doi: 10.1038/s44318-025-00566-2

Figure Lengend Snippet: ( A ) MS/MS spectrum identifying the tryptic peptide Q1069-K1081 of Diaph3 carrying a phosphorylation (PH) at serine S1072. Fragments matched to masses of b and y ions are marked in the scheme and by red labels in the spectrum. Yellow labels denote matches for fragment or precursor (M) ions after neutral loss of the phosphate group. Neutral loss of H 2 O or NH is indicated by subscripts of ° or *. ( B ) Schematic representation of DIAPH3 functional domains and the identified consensus sequence, a S-Q motif (red) adjacent to DAD (Diaphanous-autoregulatory domain). The conserved sequence of 12 aminoacids displays the serine residue where ATR can phosphorylate human DIAPH3 or the mouse orthologue mDia2 (red). ( C ) Representative images of HT1080-nAC-GFP (white) that transiently express either NLS-BFP, NLS-BFP-Diaph3-wt, NLS-BFP-Diaph3-S1072D or NLS-BFP-Diaph3-S1072A with bottom right images showing nuclear expression of these constructs (magenta); Scale bar, 5 µm. ( D ) Percentage of HT1080 cells stably expressing nAC-GFP displaying nuclear F-actin formation after co-expressing NLS-BFP, NLS-BFP-mDia2-wt, NLS-BFP-Diaph3-S1072D, or NLS-BFP-Diaph3-S1072A. Data shown as mean ± s.d. Three independent experiments per condition with n ≥ 100 cells per experiment. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. Exact P values for NLS-BFP vs NLS-BFP-Diaph3-wt, NLS-BFP-Diaph3-S1072D, and NLS-BFP-Diaph3-S1072A, respectively: P = 5.0 × 10⁻⁹. P = 3.0 × 10⁻⁹. P = 9.0 × 10⁻⁸. Exact P values for NLS-BFP-Diaph3-WT vs NLS-BFP-Diaph3-S1072D and NLS-BFP-Diaph3-S1072A are respectively: P = 0.0371. P = 6.0 × 10⁻⁶. Exact P values for NLS-BFP-Diaph3-S1072D vs NLS-BFP-Diaph3-S1072A: P = 1.0 × 10⁻⁶. ( E ) Representative images of HT1080-nAC-GFP (white) that transiently express either NLS-BFP-Diaph3-wt or NLS-BFP-Diaph3-S1072D in the presence or absence of ETP-46464, with bottom right images showing nuclear expression of these constructs (magenta); Scale bar, 5 µm. ( F ) Percentage of HT1080-nAC-GFP cells transiently expressing either NLS-Diaph3-wt or NLS-Diaph3-S1072D that display nuclear F-actin formation after being treated with either 0.01% DMSO or 3 µM ETP-46464 for 3 h. Data shown as mean ± s.d. Three independent experiments per condition with n = 50 cells per experiment. Statistical analysis between DMSO and ETP-46464 treatment after expression of NLS-BFP-Diaph3-WT was performed by an unpaired parametric t -test. Exact P value: 3.0 × 10⁻⁷. Statistical analysis between DMSO and ETP-46464 treatment after expression of NLS-BFP-Diaph3-S1072D was performed by the Mann–Whitney test. Exact P value: 0.3000. ns: P > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. .

Article Snippet: DIAPH3 , Proteintech , 14342-1-AP.

Techniques: Tandem Mass Spectroscopy, Phospho-proteomics, Functional Assay, Sequencing, Residue, Expressing, Construct, Stable Transfection, Comparison, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Nuclear rupture in confined cell migration triggers nuclear actin polymerization to limit chromatin leakage

doi: 10.1038/s44318-025-00566-2

Figure Lengend Snippet: ( A ) Nuclear stiffness of cells transiently expressing NLS-mScarlet, NLS-mScarlet-Diaph3-wt, NLS-mScarlet-Diaph3-S1072A and NLS-mScarlet-Diaph3-S1072D was measured with AFM. Data shown as mean ± s.d. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. Exact P value for NLS-mScarlet vs NLS-mScarlet-Diaph3-S1072D: P = 0.0045. Other P values from left to right: P = 0.00014. P = 0.0003. P = 0.0092. ( B ) Nuclear stiffness after ATR inhibitor ETP-46464 was added (3 µM, 3 h) in the same dish. Data shown as mean ± s.d. Statistical analysis was performed by the Kruskal–Wallis test. Exact P value: P = 0.0286. ( C ) Quantification of the change in Young’s modulus (YM) after ATR inhibition. Nuclear stiffness of cells expressing NLS-mScarlet-Diaph3-S1072D and NLS-mScarlet-Diaph3-S1072A is unaffected by ETP-46464 treatment. Nuclei of cells expressing NLS-mScarlet-Diaph3-wt show a significant reduction in YM in comparison to NLS-Diaph3-S1072D and NLS-Diaph3-S1072A expressing cells. Data shown as mean ± s.d. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. Exact P values: P = 0.0068. * P < 0.05, ** P < 0.01. *** P < 0.001. .

Article Snippet: DIAPH3 , Proteintech , 14342-1-AP.

Techniques: Expressing, Comparison, Inhibition