384 low-binding multi-well microscopy plates  (Greiner Bio)

Journal: The EMBO Journal

Article Title: Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence

doi: 10.1038/s44318-024-00212-3

Figure Lengend Snippet: ( A – D ) Representative images of electron microscopy and statistical analysis of the electron-dense areas of the heterochromatin regions at the nuclear periphery in the liver ( A , B ) and kidney ( C , D ) tissues from WT and Irtks KO mice. Parts of the upper panel were enlarged and are shown in the lower panel. Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. n = 10 cells analyzed for each condition ( B , *** p = 8.3836 × 10 −8 ; D , *** p = 1.17 × 10 −8 ). Scale bar, 1 µm. ( E ) Ectopically expressed IRTKS can rescue heterochromatin at the nuclear periphery of Irtks KO MEFs. Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. Scale bar, 1 µm. ( F ) Quantification of the electron-dense heterochromatin regions in MEFs. n = 10 cells analyzed for each condition. *** p = 7.34 × 10 −13 (IRTKS +/+ vs IRTKS −/− ) and 3.36 × 10 −5 (IRTKS −/− vs IRTKS −/− -Flag-IRTKS). ( G , H ) Immunofluorescence images ( G ) of MEFs show that IRTKS (green) co-localizes with HP1α (red) and H3K9me3 (purple). Nuclei were counterstained with DAPI (blue). ( H ) Line scans of the images of a cell co-stained for IRTKS, HP1α, H3K9me3, and DAPI at the position depicted by the white arrow. Scale bar, 5 µm. ( I – L ) Representative confocal images of HP1α foci (red) and nuclei (DAPI, blue) in the livers ( I ) and kidneys ( K ) of WT and Irtks KO mice. Quantification of lines scanned across HP1α foci and nuclei at the position depicted by the white arrow ( J , L ). Scale bar, 5 µm. Data are presented as the mean ± SD. Figure 1F was tested by one-way ANOVA followed by Tukey’s post hoc test. The remaining plots were tested by two-tailed Student’s t test. .

Article Snippet: For in vitro experiments such as FRAP and time-lapse imaging experiments, phase separation was recorded on 384 low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: Electron Microscopy, Immunofluorescence, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence

doi: 10.1038/s44318-024-00212-3

Figure Lengend Snippet: ( A , B ) Quantification of electron-dense regions (EDRs) around the nucleolus of the livers ( A , *** p = 2.72 × 10 −10 ) and kidneys ( B , *** p = 2.73 × 10 −9 ) from WT and Irtks KO mice. n = 10 cells analyzed for each condition. ( C – E ) Representative images ( C ) and quantification of EDRs at the nuclear periphery ( D , *** p = 3.09 × 10 −9 ) and around the nucleolus ( E , *** p = 5.49 × 10 −11 ) of the stomach tissues of WT and Irtks KO mice. Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. n = 10 cells analyzed for each condition. Scale bar, 1 µm. ( F ) Quantification of EDRs around the nucleolus in MEF cells. n = 10 cells analyzed for each condition. *** p = 1.22 × 10 −13 , * p = 0.0134. ( G , H ) Electron microscopy images ( G ) and quantification of EDRs at the nuclear periphery ( H , *** p = 2.14 × 10 −8 ) and around the nucleolus ( I ), *** p = 1.19 × 10 −10 ) in WT and Irtks-KO SK-Hep-1 cells. Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. n = 10 cells analyzed for each condition. Scale bar, 1 µm. ( J – O ) Electron microscopy images and quantification of the electron-dense heterochromatin regions at the nuclear periphery and around the nucleolus in MEFs ( J – L , respectively) and SK-Hep-1 cells ( M – O , respectively) that were transfected with empty vector or Flag-IRTKS construct. Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. n = 10 cells analyzed for each condition ( K , *** p = 1.39 × 10 −9 ; L , * p = 0.0370; N , *** p = 5.68 × 10 −6 ; O , *** p = 6.90 × 10 −8 ). Scale bar, 1 µm. ( P , Q ) Representative confocal images ( P ) and line scan analysis ( Q ) of HP1α foci (red) and nuclei (DAPI, blue) in the stomach tissues of WT and Irtks KO mice. Quantification of lines scanned across HP1α foci and nuclei at the position depicted by the white arrow. Scale bar, 5 µm. ( R , S ) Western blotting analyses of HP1α expression in the livers ( R ) and kidneys ( S ) of WT and Irtks-KO mice. GAPDH was used as the loading control. ( T ) Representative confocal images and line scan analysis (right) on H3K9me3 (green) and nuclei (DAPI, blue) in the livers of WT and Irtks KO mice. Quantification of lines scanned across H3K9me3 foci and nuclei at the position depicted by the white arrow. Scale bar, 5 µm. ( U ) Representative confocal microscopy and line scan analysis of SK-Hep-1 cells showing the location of EGFP-HP1α foci. Nuclei were labeled with Hoechst 33342 (blue). Quantification of lines scanned across HP1α foci and nuclei at the position depicted by the white arrow. Scale bar, 5 µm. ( V ) Live-cell images and fluorescence recovery curves of FRAP experiments of EGFP-HP1α in SK-Hep-1 cells. Red arrow indicates the bleached point, which is boxed and amplified in the images on the right. n = 8 biological replicates for the FRAP curve construction. *** p = 3.10 × 10 −9 . Scale bar, 5 µm. ( W ) Representative confocal microscopy and line scan analysis of MEF cells showing the location of EGFP-HP1α foci. Nuclei were labeled with Hoechst 33342 (blue). Quantification of lines scanned across HP1α foci and nuclei at the position depicted by the white arrow. Scale bar, 5 µm. ( X ) Live-cell images and fluorescence recovery curves of FRAP experiments of EGFP-HP1α in MEF cells. Red arrow indicates the bleached point, which is boxed and amplified in the images on the right. n = 8 biological replicates for the FRAP curve construction. *** p = 1.03 × 10 −6 . Scale bar, 5 µm. Data are presented as the mean ± SD. Figure EV1V and y were tested by two-way ANOVA. The remaining plots were tested by two-tailed unpaired Student’s t test. .

Article Snippet: For in vitro experiments such as FRAP and time-lapse imaging experiments, phase separation was recorded on 384 low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: Electron Microscopy, Transfection, Plasmid Preparation, Construct, Western Blot, Expressing, Control, Confocal Microscopy, Labeling, Fluorescence, Amplification, Two Tailed Test

Journal: The EMBO Journal

Article Title: Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence

doi: 10.1038/s44318-024-00212-3

Figure Lengend Snippet: ( A ) Western blotting analysis of several important epigenetic factors associated with heterochromatin formation in liver tissues from WT and Irtks KO mice. GAPDH was used as the loading control. ( B ) Western blotting analysis of HP1α and IRTKS expression in SK-Hep-1 cells treated with CRISPR/Cas9 single-guide RNA (sgRNA) lentivirus (sgHP1α), to knock out HP1α, coupled with the Flag-IRTKS construct. GAPDH was used as the loading control. ( C ) Western blotting analysis of HP1α and IRTKS expression in these SK-Hep-1 cells treated with CRISPR/Cas9 single-guide RNA (sgRNA) lentivirus (sgIRTKS) to knock out IRTKS, and then coupled with the Flag-HP1α construct. GAPDH was used as the loading control. ( D ) Electron microscopy images and quantification (bottom) of the electron-dense heterochromatin regions in SK-Hep-1 cells that were genetically engineered with CRISPR/Cas9 single-guide RNA (sgRNA) lentivirus (sgIRTKS) to knock out IRTKS, and then transfected with the Flag-HP1α construct. Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. n = 10 cells analyzed for each condition. *** p = 7.52 × 10 −11 (sgControl vs sgIRTKS) and 4 × 10 −5 (sgIRTKS vs sgIRTKS-Flag-HP1α). Scale bar, 1 µm. ( E , F ) IRTKS and HP1α reciprocally interact directly in a GST pull-down assay. Equivalent amounts of His-HP1α were incubated with either GST (negative control) or GST-IRTKS. After GST pulldown, HP1α was detected by western blotting. The mirror experiment was performed using His-IRTKS and GST-HP1α. ( G , H ) The reciprocal interaction between IRTKS and HP1α was detected by co-immunoprecipitation (co-IP) with anti-IRTKS and anti-HP1α antibodies in MEFs ( G ) and HEK293T cells ( H ). The immunoglobulin G (IgG) group was the negative control. ( I ) Schematic summaries of the interactions between diverse IRTKS and HP1α truncations. ( J ) Direct interaction between full-length or truncated GST-HP1α proteins and His-IRTKS revealed by GST pulldown assay. Full-length and truncated GST-HP1α proteins were visualized by Coomassie blue staining. ( K ) GST pulldown assays were performed with recombinant His-HP1α and full-length or truncated GST-IRTKS. The pulldown samples were analyzed by western blotting. ( L ) A 3D structural model of the IRTKS-HP1α complex was constructed using the Z-DOCK server. The 3D structures of IRTKS and HP1α were predicted by AlphaFold algorithms (upper panel). The interaction between IRTKS and HP1α is depicted by the yellow and blue colors, respectively. Details of the key residues of HP1α that interact with IRTKS are also shown in the lower panel. Data are presented as the mean ± SD. Figure EV2D was tested by one-way ANOVA followed by Tukey’s post hoc test. .

Article Snippet: For in vitro experiments such as FRAP and time-lapse imaging experiments, phase separation was recorded on 384 low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: Western Blot, Control, Expressing, CRISPR, Knock-Out, Construct, Electron Microscopy, Transfection, Pull Down Assay, Incubation, Negative Control, Immunoprecipitation, Co-Immunoprecipitation Assay, GST Pulldown Assay, Staining, Recombinant

Journal: The EMBO Journal

Article Title: Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence

doi: 10.1038/s44318-024-00212-3

Figure Lengend Snippet: ( A ) Electron microscopy images and quantification of the electron-dense heterochromatin regions in SK-Hep-1 cells treated with CRISPR/Cas9 single-guide RNA (sgRNA) lentivirus (sgHP1α) to knock out HP1α coupled with the Flag-IRTKS construct. Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. n = 8 cells analyzed for each condition. *** p = 3.39 × 10 −11 , ns = 0.967612. Scale bar, 1 µm. ( B ) HP1α was abundantly SUMOylated by SUMO-1 when co-expressed with IRTKS in HEK293T cells, as detected by immunoprecipitation assay. HEK293T cells transfected with HA-IRTKS, Flag-HP1α, and GFP-SUMO1 were immunoprecipitated with an anti-Flag antibody for the SUMOylation assay, followed by western blotting with the indicated antibodies. ( C ) In vitro SUMOylation assay showing that IRTKS visibly enhanced HP1α SUMOylation in the presence of SUMO E1, E2 and SUMO-1 proteins. ( D ) Droplet formation assays showing that SUMO-mCherry-HP1α forms liquid-like droplets. mCherry-HP1α or SUMO-mCherry-HP1α was added to the droplet formation buffer to 40 µM. Scale bars, 10 μm. n = 8 fields for each group were quantified. *** p = 2.39 × 10 −7 . ( E ) Representative images and quantification of droplet formation at various protein concentrations ( n = 6 fields for each group were quantified). *** p = 1.34 × 10 −8 (10 μM vs 20 μM), 2.58 × 10 −13 (10 μM vs 40 μM), and 1.96 × 10 −9 (20 μM vs 40 μM). SUMO-mCherry-HP1α was added to droplet formation buffer to final concentrations as indicated. Scale bar, 10 μm. ( F ) Time-lapse fluorescence images showing that the droplets of SUMO-mCherry-HP1α rapidly fused. Scale bar, 2 μm. ( G ) Representative images and the fluorescence recovery curve of the SUMO-mCherry-HP1α FRAP experiments. Scale bar, 10 μm. n = 8 biological replicates for the FRAP curve construction. Data are presented as the mean ± SD. Figure 2D was tested by two-tailed Student’s t test. The remaining plots were tested by one-way ANOVA followed by Tukey’s post hoc test. .

Article Snippet: For in vitro experiments such as FRAP and time-lapse imaging experiments, phase separation was recorded on 384 low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: Electron Microscopy, CRISPR, Knock-Out, Construct, Immunoprecipitation, Transfection, Western Blot, In Vitro, Fluorescence, Two Tailed Test

Journal: The EMBO Journal

Article Title: Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence

doi: 10.1038/s44318-024-00212-3

Figure Lengend Snippet: ( A ) Representative images of droplet formation at different concentrations of IRTKS and HP1α protein. Concentrations of IRTKS and HP1α are indicated at the bottom and left of the images, respectively. Scale bars, 5 μm. ( B ) Representative images of droplet formation at various concentrations of EGFP-IRTKS and SUMO-mCherry-HP1α protein. Concentrations of EGFP-IRTKS and SUMO-mCherry-HP1α are indicated at the bottom and left of the images, respectively. Scale bars, 10 μm. ( C , D ) In vitro phase separation assay of EGFP-IRTKS protein at various concentrations mixed with nucleosomal DNA. A total of 6 nM nucleosomal DNA for the droplet assay was stained using DAPI ( n = 8 fields for each group were quantified). *** p = 3.3 × 10 −6 (5 μM vs 10 μM), 8.34 × 10 −17 (5 μM vs 20 μM), and 3.42 × 10 −14 (10 μM vs 20 μM). Scale bar, 5 µm. ( E , F ) Droplet formation of various concentrations of EGFP-IRTKS protein mixed with reconstituted native 12× nucleosomal arrays (NA). A total of 330 nM reconstituted native 12× NA for the droplet assay was stained using DAPI ( n = 8 fields for each group were quantified). * p = 0.0162 (5 μM vs 10 μM), *** p = 1.22 × 10 −15 (5 μM vs 20 μM), and 1.71 × 10 −14 (10 μM vs 20 μM). Scale bar, 5 µm. ( G ) Liquid‒liquid phase separation assay with nucleosomal DNA to examine the ability of IRTKS to form condensates with HP1α and nucleosomal DNA stained using DAPI. Scale bar, 5 µm. ( H – J ) A phase diagram of IRTKS and HP1α mixed with Cy5-labeled DNA oligos ( H ), nucleosomal DNA ( I ), and reconstituted H3K9me3 12× NA ( J ). n = 8 fields for each group were quantified. ( K , L ) Representative images ( K ) and line scan analysis ( L ) of EGFP or EGFP-IRTKS with different truncations (full-length, I-BAR, IDR and IDR-mutant) in NIH3T3 cells. Nuclei were labeled with Hoechst 33342. Quantification of lines scanned across EGFP or EGFP-IRTKS with different truncations and nuclei at the position depicted by the white arrow. Scale bar, 2 µm. ( M , N ) Live-cell images ( M ) and line scan analysis ( N ) of EGFP or EGFP-IRTKS-expressing SK-Hep-1 cells. Nuclei were labeled with Hoechst 33342. Quantification of lines scanned across EGFP or EGFP-IRTKS and nuclei at the position depicted by the white arrow. Scale bar, 5 µm. ( O , P ) Electron microscopy images ( O ) and quantification of the electron-dense heterochromatin regions ( P ) in MEF cells overexpressed EGFP or EGFP-IRTKS with different truncations (full-length, I-BAR, IDR, and IDR-mutant). Red arrows indicate the electron-dense heterochromatin regions. Nu, nucleolus. n = 8 cells analyzed for each condition. Scale bar, 1 µm. ( Q ) Enrichment of H3K9me3 and HP1α within the regions of repetitive sequences (LINE1, IAP, and SINE) in the MEFs of WT and Irtks KO mice as measured by ChIP-qPCR. n = 3 technical replicates in independent experiments. The p values of Fig. EV5P and Q were provided in Source data. Data are presented as the mean ± SD or mean ± SEM and the p value of one-way ANOVA followed by Tukey’s post hoc test. .

Article Snippet: For in vitro experiments such as FRAP and time-lapse imaging experiments, phase separation was recorded on 384 low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: In Vitro, Staining, Labeling, Mutagenesis, Expressing, Electron Microscopy, ChIP-qPCR

Journal: Fundamental Research

Article Title: CEBIT screening for inhibitors of the interaction between SARS-CoV-2 spike and ACE2

doi: 10.1016/j.fmre.2022.01.034

Figure Lengend Snippet: Validation of CEBIT-based system for detecting RBD-ACE2 interaction. (a) Schematic diagram showing the strategy of SmF-based phase separation system. The condensates formed by SmF-mCherry-SIM and SmF-GFP-SUMO3 serve as the “reactor” for PPI recruitment (top left). With the addition of (SUMO3) 5 -SA and RBD-Strep-tag in turn (top right and bottom right), ACE2-His was recruited into the condensates via its interaction with RBD-Strep-tag. ACE2-His is fluorescently tagged to monitor its recruitment (bottom right). Disruption of the RBD-ACE2 interaction by an inhibitor or competitor evicts ACE2 from the condensates (bottom left). (b) Microscopy analysis of the recruitment of ACE2 labeled by Alexa 647 into the condensates formed by SMF-mCherry-SIM and SmF-GFP-SUMO3. (c) Relative fluorescence intensity ratio of ACE2 (Alexa 647) versus condensates (GFP).

Article Snippet: All the phase separation experiments were performed on 384-low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: Biomarker Discovery, Strep-tag, Disruption, Microscopy, Labeling, Fluorescence

Journal: Fundamental Research

Article Title: CEBIT screening for inhibitors of the interaction between SARS-CoV-2 spike and ACE2

doi: 10.1016/j.fmre.2022.01.034

Figure Lengend Snippet: Optimization of a multivalent recruitment system driven by polySUMO-polySIM. (a) Schematic diagram showing the strategy for the phase separation system mediated by polySUMO-polySIM. The scaffolds used here to mediate the formation of condensates are (SUMO3) 5 -SA and SIM 11 . The colocalization of the client protein ACE2-His with the condensates was observed in the presence of the bridging protein RBD-Strep-tag. (b) Microscopy analysis of the recruitment of ACE2 labeled by Alexa 647 into the condensates formed by (SUMO3) 5 -SA and SIM 11 (Alexa 488). (c) Relative fluorescence intensity ratio of ACE2 (Alexa 647) versus condensates (Alexa 488). (d-e) Competitive binding assay between RBD-His and RBD-Strep-tag for ACE2 protein.

Article Snippet: All the phase separation experiments were performed on 384-low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: Strep-tag, Microscopy, Labeling, Fluorescence, Competitive Binding Assay

Journal: Fundamental Research

Article Title: CEBIT screening for inhibitors of the interaction between SARS-CoV-2 spike and ACE2

doi: 10.1016/j.fmre.2022.01.034

Figure Lengend Snippet: Optimization of a multivalent recruitment system driven by HP1α. (a) Schematic diagram showing the strategy for the HP1α-driven phase separation system. Partitioning of ACE2-His into the HP1α-PDZ-driven condensates is mediated by its interaction with RBD- KKETPV. (b) Microscopy analysis of the recruitment of ACE2 labeled by Alexa 546 into the condensates of HP1α-PDZ (Alexa 647), and the competitive binding assay by RBD-His protein. (c) Analysis of the relative fluorescence intensity ratio of ACE2 (Alexa 546) versus condensates (Alexa 647).

Article Snippet: All the phase separation experiments were performed on 384-low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: Microscopy, Labeling, Competitive Binding Assay, Fluorescence

Journal: Fundamental Research

Article Title: CEBIT screening for inhibitors of the interaction between SARS-CoV-2 spike and ACE2

doi: 10.1016/j.fmre.2022.01.034

Figure Lengend Snippet: High throughput screening for inhibitors of the RBD-ACE2 interaction. (a) Microscopy analysis of the inhibition activity of positive compounds. (b) Quantitative analysis of the relative ratio of fluorescence intensities of ACE2 (Alexa 546) and HP1α-PDZ (Alexa 647). (c) Schematic draws of the two-dimensional structures of the positive compounds.

Article Snippet: All the phase separation experiments were performed on 384-low-binding multi-well 0.17 mm microscopy plates (Cellvis).

Techniques: High Throughput Screening Assay, Microscopy, Inhibition, Activity Assay, Fluorescence