Journal: Cell Communication and Signaling : CCS

Article Title: ZNF638 represses the transcription of HBV closed circular DNA involving HUSH complex-mediated histone modifications of epigenetic silencing

doi: 10.1186/s12964-026-02726-1

Figure Lengend Snippet: ZNF638 knockdown modulated histone modifications of HBV cccDNA microchromosomes. A Nuclear HBV cccDNA pulldown-WB procedures. After transfection with siRNAs targeting ZNF638, TASOR, and SETDB1 for 48 h, HepAD38 cells were transferred to Tet-free medium to induce HBV replication and cultured for an additional 6 days; HepG2-NTCP cells were infected with 6 × 10 5 copies/ml HBV for 6 days. T5 exonuclease was used to digest isolated extracts to remove rcDNA, ssDNA and dsDNA. Following incubation with a specific biotinylated cccDNA bait, streptavidin-coated magnetic beads were employed to precipitate the baited cccDNA and associated proteins in the complex. B Western blotting for the detection of proteins of interest alone with various histone H3 in HepG2-NTCP cells (left) and HepAD38 cells (right). Semiquantitative measures from densitometry were normalized to controls and plotted as changes in folds (n = 3). C Southern blotting for HBV cccDNA from HepG2-NTCP (left) and HepAD38 (right) cells. (Nondig.: Non-digested DNAs)

Article Snippet: The probing antibodies against the following antigens: ZNF638 (Bethyl, USA), MPP8 (Proteintech), H3K9me3 (Abcam, UK), H3K27ac (Gene Tex, San Antonio, USA), H3K4me3 (Active motif, Carlsbad, CA, USA), H3 (Cell Signaling Technology, Massachusetts USA), SETDB1 (Proteintech) and GAPDH (Proteintech) were used for overnight incubation at 4 °C following the blocking procedure.

Techniques: Knockdown, Transfection, Cell Culture, Infection, Isolation, Incubation, Magnetic Beads, Western Blot, Southern Blot

Journal: Cell Communication and Signaling : CCS

Article Title: ZNF638 represses the transcription of HBV closed circular DNA involving HUSH complex-mediated histone modifications of epigenetic silencing

doi: 10.1186/s12964-026-02726-1

Figure Lengend Snippet: ZNF638 dependent epigenetic silencing of HBV cccDNA transcription required marking of SETDB1-mediated H3K9me3. A Levels of HBV DNA, cccDNA and pgRNA in SETDB1 knockdown HepG2-NTCP cells at 6 d of 6 × 10 5 copies/ml HBV infections after SETDB1 siRNA transfection for 48 h. B Dose response of SETDB1 knockdown on increased HBV DNA levels. C Time course of HBV cccDNA transcription shown as the ratio of pgRNA to cccDNA for efficacy comparison. D-G Effect of SETDB1 siRNA transfection at 48 h on normalized HBV transcription levels at 6 d of 6 × 10⁵ copies/ml HBV infections in ZNF638 knockdown or overexpressing HepG2-NTCP cells. H-I ChIP-qPCR assays on cccDNAs for H3K9me3 enrichment at the ZNF638 binding regions using cccDNAs isolated from sgZNF638 or ZNF638oe HepG2-NTCP cells at 6 d post HBV infection

Article Snippet: The probing antibodies against the following antigens: ZNF638 (Bethyl, USA), MPP8 (Proteintech), H3K9me3 (Abcam, UK), H3K27ac (Gene Tex, San Antonio, USA), H3K4me3 (Active motif, Carlsbad, CA, USA), H3 (Cell Signaling Technology, Massachusetts USA), SETDB1 (Proteintech) and GAPDH (Proteintech) were used for overnight incubation at 4 °C following the blocking procedure.

Techniques: Knockdown, Transfection, Comparison, ChIP-qPCR, Binding Assay, Isolation, Infection

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. Schematic representation of the cell lines exhibiting different SETDB1 expression levels. A549 Ctrl cells exhibit endogenous overexpression of SETDB1. SETDB1 KO cells show complete loss of SETDB1. In the inducible shRNA-mediated knockdown model (SETDB1 KD), cells are either untreated (0) or treated with doxycycline for 3 or 7 days (3d, 7d), resulting in a progressive reduction of SETDB1 levels. B. SETDB1 mRNA levels measured by RT-qPCR and normalized to PPIA . Values are shown relative to A549 cells (Ctrl). Bars represent the mean of N=3 biological replicates +/− SD error bars., with individual data points indicated. Statistical significance was assessed using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. C. Principal Component Analysis (PCA) based on gene expression profiles for A549 cells (Ctrl), SETDB1 KO, SETDB1 KD 0 (no doxycycline), and SETDB1 KD 7d-dox. D. Venn diagram of differentially expressed genes (DEGs). Comparison of Control vs SETDB1 KO (light grey) and SETDB1 KD 0 vs 7d-dox (dark grey). DEGs were selected based on a specified log2 fold change (log2FC) threshold: log2FC > +1 or <-1. E. Volcano plots showing -log10(p-value) (-Log10 P) versus log2 fold change for gene expression comparisons: Control vs SETDB1 KO and SETDB1 KD 0 vs 7d-dox. F. Heatmap of log2 fold changes for genes associated with cell migration, comparing Ctrl vs SETDB1 KO and SETDB1 KD 0 vs 7d-dox. Migration-related genes were selected from Gene Ontology database ( GO:0016477 ). G. Representative immunofluorescence images of Ctrl, SETDB1 KO, SETDB1 KD 7 days-dox cells stained with anti-CDH1 (E-cadherin, green). Nuclei are counterstained with DAPI (blue). Merged images show overlay of CDH1 and DAPI signals. Scale bar: 50 µm. H. Left: Representative images from Transwell migration assays for Control, SETDB1 KO, and SETDB1 KD (0, 3, 7 days-dox). Migrated cells are stained in violet; images acquired at 40× magnification. Scale bar: 500 µm. Right: Quantification of migrated cells by absorbance measurement at 590 nm. Data represents N=4 replicates; red lines indicate the sample mean +/−SD error bars. Statistical analysis performed via unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Expressing, Over Expression, shRNA, Knockdown, Quantitative RT-PCR, Gene Expression, Comparison, Control, Migration, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. Western blot analysis of SETDB1 protein levels in A549 cells (Ctrl), SETDB1 KO, shCTR (0, 3d, 7d), and SETDB1 KD (0, 3d, 7d dox-treated) cells. H3 is used as a loading control. B. Quantification of SETDB1 protein level normalized to H3 used for A549 cells (Ctrl), SETDB1 KO, shCTR (0, 3d, 7d), and SETDB1 KD (0, 3d, 7d dox-treated) cells. Bars represent the mean of N=4 biological replicates +/− SD error bars, with individual values shown. Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. C. Volcano plot showing -log10(p-value) (-Log10 P) versus log2 fold change for gene expression comparison between shCTR-0d and shCTR-7d dox-treated cells, indicating minimal transcriptional effects of doxycycline or the shRNA system alone. D. mRNA levels of SETDB1, ATF7IP, and PCDHB6 measured by RT-qPCR and normalized to PPIA. Expression levels are relative to A549 cells (Ctrl), Bars represent the mean of N=3 biological replicates +/− SD error bars, with individual values shown. Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. E. Gene Set Enrichment Analysis (GSEA) plots for MYC targets and WNT/β-catenin signaling pathways comparing Control vs SETDB1 KO and SETDB1 KD 0 vs 7d dox-treated conditions. Normalized Enrichment Scores (NES) are reported. F. NES values for the Hallmark “Apical Junction” gene set in Ctrl vs SETDB1 KO (light grey) and SETDB1 KD 0 vs 7d dox-treated (dark grey) comparisons. G. Quantification of E-cadherin signal (marker of cell–cell adhesion) from immunofluorescence images (see ) of Control, SETDB1 KO and SETDB1 KD 7d dox-treated cells. Modal grey value per field is measured, >30 fields per condition are analyzed, N= 3 biological replicates, in the graph is indicated the mean +/− SD error bars; Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. H. Representative immunofluorescence images of Ctrl, SETDB1 KO and SETDB1 KD 7d dox-treated cells stained with anti-Paxillin (green). Nuclei are counterstained with DAPI (blue). Merged images show colocalization. Scale bar: 50 µm. I. Quantification of Paxillin signal (marker of focal adhesions) from images in panel S1H. Dots stand for the mean grey value of the field / number of nuclei x field, 15 fields are analyzed from N= 3 biological replicates, in the graph is indicated the mean +/− SD error bars; Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. J. Left: Representative wound healing images at 0, 24, and 48 hours after wound for SETDB1 KD 0 and 7d dox-treated cells. Lines on pictures indicate wound edges over time Right: Quantification of wound gap size (%) over time for Ctrl, SETDB1 KO, and SETDB1 KD (0, 3d, 7d dox-treated). Wound size is normalized at time 0 (100%). Error bars stand for −/+ SD and statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. K. Cell proliferation curves showing the number of cells (×10□) over time (0, 3, 5, 7 days) for Ctrl, SETDB1 KO, shCTR (0 and 7d dox-treated), and SETDB1 KD (0 and 7d dox-treated). Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Western Blot, Control, Gene Expression, Comparison, shRNA, Quantitative RT-PCR, Expressing, Protein-Protein interactions, Marker, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. Quantification of the normalized to the Ctrl level of H3K9me3 in the nucleus in different cell lines normalized to Ctrl. Bars represent the mean of N=3 biological replicates +/− SD error bars, with individual values shown. Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. B. Representative immunofluorescence images taken with the automated Operetta system used for the quantification for shCTR control cells and dox-treated cells. Cells stained with anti-H3K9me3 antibody, represented in orange. DNA was labelled with DAPI in blue. Scale bar: 50 µm. C. Quantification of H3K9me3 signal at nuclear periphery measured with the automated microscope (Operetta) corresponding to the phenotype observed in panel B. Each point represents the ratio between the intensity of the H3K9me3 signal at the nuclear periphery (defined by DAPI) and the total nuclear H3K9me3 signal intensity. 500 cells were randomly picked over more than 10 000 cells per condition. The red dot indicates the mean of N=3 biological replicates +/− SD error bars. Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. D. MA plot depicting lamina-associated domains (LADs) ≥100 kb identified in Ctrl and SETDB1 KO cells. Each dot represents an individual LAD. Red and blue dots indicate LADs significantly gained or lost in SETDB1 KO relative to Ctrl cells (log ₂ fold change > 1.5; false Discovery Rate, FDR < 0.05). E. Boxplot comparing the comparable genomic span of gained and lost LADs upon SETDB1 KO. Both categories span approximately 22 Mb. F. Graphical representation of the chromosomal distribution and positioning of gained (red) and lost (blue) Lamina-Associated Domains (LADs) in SETDB1 KO compared to Ctrl cells. The magnitude of change is indicated by a color gradient reflecting the log ₂ fold change (log2FC). G. Integrated Genome Viewer (IGV) tracks displaying DamID-seq and ChIP-seq data from Ctrl and SETDB1 KO cells. Tracks represent Dam-Lamin B1 (DamID) and H3K9me3 (ChIP_H3K9me3) normalized signals, respectively. LADs are highlighted in red, while inter-LAD regions are shown in blue. The figure illustrates a loss of LADs on chromosome 19 (Chr19) in SETDB1 KO cells relative to Control, accompanied by a corresponding decrease in H3K9me3 signal. H3K9me3 ChIP-seq data were obtained from reference.

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Immunofluorescence, Control, Staining, Microscopy, ChIP-sequencing

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. Representative immunofluorescence images of A549 cells (Ctrl), SETDB1 KO, SETDB1 KD 0, 3d and 7d dox-treated cells stained with anti-H3K9me3 antibody. DNA was labelled with DAPI. In the merge the DAPI is represented in blue and the H3K9me3 in red. Scale bar: 5 µm. B. Violin plot showing the quantification of H3K9me3 signal at the nuclear periphery measured with the automated microscope (Operetta) corresponding to the phenotype observed in panel A. Each point represents the ratio between the intensity of the signal at the nuclear periphery (defined by DAPI) and the total nuclear H3K9me3 signal intensity. 1000 cells were randomly picked over more than 10 000 cells per condition. The red dot indicates the mean of N=3 biological replicates +/− SD error bars. Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. C. Scatter plots showing the correlation between normalized read counts (log ₂ [Dam-LB1/Dam]) from DamID-seq experiments performed in Ctrl and SETDB1 KO cells, using a bin size of 10 kb. The Spearman correlation coefficient (R) is indicated on the plot. The dashed line represents the fitted correlation line. Outliers exceeding ±4 standard deviations from the correlation line are highlighted in red and denote genomic regions specifically enriched in either Ctrl or SETDB1 KO cells. D. Integrated Genome Viewer (IGV) tracks displaying DamID-seq and ChIP-seq data from Ctrl and SETDB1 KO cells. Tracks represent Dam- Lamin B1 (DamID) and H3K9me3 (ChIP_H3K9me3) normalized signals, respectively. Genomic regions corresponding to lamina-associated domains (LADs) are highlighted in red, while inter-LAD regions are shown in blue. Although LADs are broadly conserved across the depicted region of chromosome 2 (Chr2), the figure reveals a pronounced increase in H3K9me3 signal at LADs in SETDB1 KO cells compared to control cells. H3K9me3 ChIP-seq data were obtained from reference [18]. E. Boxplot comparing the size of LADs, measured in base pairs, between Ctrl and SETDB1 KO cells. Statistical analysis indicates no significant difference (ns) between the two conditions.

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Immunofluorescence, Staining, Microscopy, ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. mRNA levels of SUV39H1 measured by RT-qPCR and normalized to PPIA. Expression levels are relative to A549 cells (Ctrl). siRNA against SUV39H is indicated as “+” and siCTR as “-”. Bars represent the mean of N=3 biological replicates +/− SD error bars, with individual values shown. Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. B. Western blot analysis of SETDB1 and SUV39H1 protein levels in Ctrl and SETDB1 KO +/−siRNA against SUV39H cell lines. Lamin A/C and H3 were used as a loading control. C. Representative immunofluorescence images of Ctrl and SETDB1 KO +/− siRNA against SUV39H. Cells were stained with anti-H3K9me3 antibody. The nuclei were labelled with DAPI. The merged images show DAPI in blue and H3K9me3 in red. Scale bar: 5µm. D. Violin plot showing the quantification of H3K9me3 signal at nuclear periphery measured with the automated microscope (Operetta) corresponding to the phenotype observed in panel C. Each point represents the ratio between the intensity of the signal at the nuclear periphery (defined by DAPI) and the total nuclear H3K9me3 signal intensity. 100 cells were randomly picked over more than 10 000 cells per condition. The red dot indicates the mean of N=3 biological replicates +/− SD error bars. Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. E. Bar plot showing the quantification of the total level of H3K9me3 in the nucleus in different cell lines normalized to Ctrl measured with the automated microscope (Operetta) corresponding to the phenotype observe d in panel C. Bars represent the normalized to the Ctrl mean of N=3 biological replicates +/− SD error bars, with individual values shown. Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. F. Bar plot showing the quantification of the total level of H3K9me3 in the nucleus in different cell lines normalized to Ctrl measured with the automated microscope (Operetta) corresponding to the phenotype observe d in panel . Bars represent the normalized to the Ctrl mean of N=3 biological replicates +/− SD error bars, with individual values shown. Statistical significance was determined using an unpaired t-test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. G. Structure of SETDB1 showing the position of the two lysins (K1186 and K1194) that were modified with alanine (A) to disrupt the SETDB1-SUV39H1 interaction. H. Solubility test for SUV39H1 in Ctrl and SETDB1 KO cells using increasing NaCl concentration (500 to 700 mM). G9A, EZH2 and HP1α are used as control to show that there is no change in solubility for these proteins between Ctrl and SETDB1 KO. On the right the total nuclei extract shows that the level of SUV39H1 is similar in Ctrl and SETDB1 KO cells.

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Immunofluorescence, Staining, Microscopy, Modification, Solubility, Concentration Assay

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. Representative immunofluorescence images of Ctrl, SETDB1 KO, Ctrl + SUV39H1 WT, Ctrl + SUV39H1 catalytic mutant. Cells were stained with anti-H3K9me3 antibody and positive cells expressing exogenous SUV39H1 WT or catalytic mutant constructs were stained with anti-MYC antibody. The nuclei were labelled with DAPI. The merge images show the DAPI in blue, MYC in green and H3K9me3 in red. Scale bar: 5µm. B. Violin plot showing the quantification of H3K9me3 signal at the nuclear periphery measured with the automated microscope (Operetta) corresponding to the phenotype observed in panel A. Cells analyzed are considered positive based on the value of the total intensity of the MYC signal inside the nucleus. Each point represents the ratio between the intensity of the signal at the nuclear periphery (defined by DAPI) and the total nuclear H3K9me3 signal intensity. 1000 cells were randomly picked over more than 10 000 cells per condition. The red dot indicates the mean of N=3 biological replicates +/− SD error bars. Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. C. Representative immunofluorescence images of SETDB1 KO + empty vector, + SETDB1 WT, + SETDB1 catalytic mutant or SUV39H1 interaction mutants. Cells were stained with anti-H3K9me3 and anti-FLAG antibodies. Flag is used to identify the positive cells. DNA was labelled with DAPI. The merged images show DAPI in blue, H3K9me3 in red and the FLAG in green. Scale bar: 5 µm. D. Violin plot showing the quantification of H3K9me3 signal at nuclear periphery measured with the automated microscope (Operetta) corresponding to the phenotype observed in panel C. Cells analyzed are considered positive based on the value of the total intensity of the FLAG signal within the nucleus. Each point represents the ratio between the intensity of the signal at the nuclear periphery (defined by DAPI) and the total nuclear H3K9me3 signal intensity. 100 cells were randomly picked over more than 3000 cells per condition. The red dot indicates the mean of N=3 biological replicates +/− SD error bars. Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. E. Solubility test for SUV39H1 in SETDB1 KD 0 days-dox (top) and SETDB1 KD 7 days-dox (bottom) cells using increasing NaCl concentration (400 to 1000 mM). SETDB1 is controlled to show the efficiency of the dox treatment. EZH2 is used as control to show that there is no change in solubility between 0 and 7 days-dox SETDB1 KD. On the left the total nuclei extract shows that the level of SUV39H1 in 0 and 7 days-dox SETDB1 KD cells.

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Immunofluorescence, Mutagenesis, Staining, Expressing, Construct, Microscopy, Plasmid Preparation, Solubility, Concentration Assay, Control

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. Schematic representation of the microfluidic assay. Cells are pushed under controlled pressure drop through constrictions of square cross-section (6×6 μm 2 ) and the entry of the cell was observed using brightfield microscopy at high frame rate. B. Typical sequential images of a cell passing through a microfluidic constriction at 4 time points (msec). C. Dot plot representation of the cellular long-time viscosity h 2 , in kPa.s, of Ctrl cells and SETDB1 KO cells with siCTR and siSUV39H. The dot plots of the non-Gaussian distributions are displayed in log scale; each dot corresponds to one cell; medians and 95% CI are indicated. Data represent N = 3 biological replicates, each with more than 50 cells (n > 50). Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. D. Schematic representation of the optical tweezers nuclear indentation experiment. After internalization of 2 µm-diameter green fluorescent beads by the cells, a bead located close to the nucleus is trapped by the optical tweezers. The piezo stage is moved in order to push the bead against the nuclear envelope and indent the nucleus. E. Sequential images of a nucleus before and after the piezo stage movement and the indentation for Ctrl, SETDB1 KO, SETDB1 KD 7d cells. The nuclei were stained in blue with Hoechst; the bead is shown in green. F. Boxplot showing the quantification of the nuclear rigidity K (pN/μm) in Ctrl and SETDB1 KO cells treated with siCTR, siSUV39H, siLaminA/C and in SETDB1 KD 0, 3d and 7d dox treated cells. The line that divides the box represents the median of the data. The ends of the box indicate the upper (Q3) and lower (Q1) quartiles. The difference between Quartiles 1 and 3 is known as the interquartile range (IQR), which measures the spread of the middle 50% of the data. The lines extending from the box show the range of values within Q3 + 1.5 × IQR to Q1 - 1.5 × IQR, representing the highest and lowest values, excluding outliers. Dots beyond the whiskers indicate potential outliers in the dataset. The mean +/− SD is shown in red. Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. G. Working model for the regulation of H3K9me3 distribution and nuclear mechanics by SETDB1 and SUV39H1. The balance between SETDB1 and SUV39H1 determines the localization of H3K9me3 at LADs. Upon SETDB1 loss of function (LOF), SUV39H1 activity drives the redistribution and enrichment of H3K9me3 at the nuclear periphery. Increased H3K9me3 at LADs enhances cellular and nuclear biophysical properties: it induces an increase of cellular viscosity and nuclear stiffness, reducing nuclear deformability and impairing efficient cell invasion.

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Microscopy, Viscosity, Staining, Activity Assay

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet: A. Dot plot representation of the entry time Te (in seconds, s) of Ctrl and SETDB1 KO cells treated with siCTR and siSUV39H. The dot plots of the non-Gaussian distributions are displayed on a logarithmic scale. Each dot represents a single cell; medians and 95% CI are shown. Data represent N = 3 biological replicates, each with more than 100 cells (n > 100). Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. B. Dot plot representation of the cellular projected diameter D (as a proxy of the cell size, in μm) in Ctrl and SETDB1 KO cells treated with siCTR and siSUV39H. Each dot represents a single cell; mean +/−SD is shown in red. Data represent N = 3 biological replicates, each with more than 100 cells (n > 100). Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. C. Distributions of entry time Te (in seconds, s) for Ctrl (top) and SETDB1 KO (bottom) cells treated with siCTR and siSUV39H sorted by cell diameter D in three groups. Each dot represents a single cell; medians and 95% CI are shown. Data represent N = 3 biological replicates. Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. D. Dot plot representation of the short-time viscosity h 1 , in Pa.s, of Ctrl cells and SETDB1 KO cells treated with siCTR and siSUV39H. The dot plots of the non-Gaussian distributions are displayed on a logarithmic scale. Each dot represents a single cell; medians and 95% CI are shown. Data represent N = 3 biological replicates, each with more than 50 cells (n > 50). Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. E. Dot plot representation of the elastic modulus E in kPa of Ctrl cells and SETDB1 KO cells treated with siCTR and siSUV39H. The dot plots of the non-Gaussian distributions are displayed on a logarithmic scale. Each dot represents a single cell; medians and 95% CI are shown. Data represent N = 3 biological replicates, each with more than 50 cells (n > 50). Statistical significance was determined using the Wilcoxon test; ns indicates p > 0,05, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. F. Circularity calculated with the Fiji program from confocal images taken during optical tweezers indentation experiments in Ctrl cells. Percentage of circular nuclei (circularity > 0.7) and non-circular nuclei (circularity<0.7) in Ctrl, SETDB1 KO, SETDB1 KD 0, 3d, 7d dox treated cells. G. Scatter plot showing correlation between nucleus rigidity and nucleus circularity in Ctrl and SETDB1 KO cells (left), and in the SETDB1 KD 0, 3d, 7d doxycycline-treated cells (right).

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: Viscosity

Journal: bioRxiv

Article Title: Epigenetic control of nuclear mechanics and cellular migration via histone H3 lysine 9 methylation at Lamina-Associated Domains

doi: 10.1101/2025.11.28.690983

Figure Lengend Snippet:

Article Snippet: Stable shSETDB1-A549 cell lines were established by transducing cells with in-house–produced lentiviral particles carrying the constructs listed in , cloned into pLKO-Tet-On vectors (modified from Addgene#83481). shRNA sequences targeting SETDB1 and a scrambled shRNA used as a negative control were designed using the siRNA Wizard tool ( https://www.invivogen.com/sirnawizard/ ). shRNA expression was induced with doxycycline (1 μg/mL).

Techniques: shRNA