Journal: Communications Biology

Article Title: Identification of responsible sequences which mutations cause maternal H19 -ICR hypermethylation with Beckwith–Wiedemann syndrome-like overgrowth

doi: 10.1038/s42003-024-07323-x

Figure Lengend Snippet: ChIP-qPCR analysis of CTS1-4 in H19 -ICR mSOC3/+ ( n = 6) and WT ( n = 4), H19 -ICR mSOC4/+ ( n = 7) and WT (n = 6), and H19 -ICR ΔC3,4/+ ( n = 7) and WT ( n = 5) embryos from 2 litters at 10.5 dpc, as performed using CTCF ( a ) and RAD21 ( b ) antibodies. White and red bars indicate the WT and mutant, respectively. Error bars indicate standard deviation. P -values are indicated (unpaired two-tailed t -test). ns; not significant.

Article Snippet: Chromatin was immunoprecipitated using the following antibodies: rabbit monoclonal anti-CTCF (#3418; Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal anti-Rad21 (ab992; Abcam, Cambridge, UK).

Techniques: Mutagenesis, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Differential Regulation of Large-scale Chromosome Conformations in Osteoblasts and Osteosarcoma

doi: 10.1101/2024.11.01.621571

Figure Lengend Snippet: (A) A schematic of chromatin architectural proteins, CCCTC binding factor (CTCF, green) and cohesin. RAD21 (grey) is the kleisin subunit of cohesin critical for ring formation. (B) Western blotting shows that CTCF and RAD21 protein expression levels were significantly reduced 24 hours post siRNA transfection in osteosarcoma cells (siCTCF and siRAD21 respectively). (C) Western blotting shows RAD21 knockdown does not result in changes of H3K27me3 levels in OS cells. (D) (left to right) Images of C19q in osteosarcoma cells in the indicated conditions (37°C, 5% CO 2 and humidity). Images were taken 24 hours post-transfection for knockdown cells. (E) The area calculation of C19q in osteosarcoma cells upon siRNA transfection and negative control (siControl, N C19q =10 for each condition) (F) Percentages of cells with collapsed (green) and extended (orange) C19q conformations in osteosarcoma cells (N C19q =110 for wild type, N C19q =110 for negative control, N C19q = 110 for CTCF knockdown, N C19q = 110 for siRAD21, and N C19q = 110 for double knockdown) (G) Volcano plots of osteosarcoma cells with and without RAD21 knockdown (24 hours post-knockdown). Downregulated transcripts (log2FoldChange ≧ –1, p<0.05) are colored in blue and upregulated transcripts (log2FoldChange ≦ 1, p<0.05) are colored in pink. Top 5 genes are labeled. (H) Genome wide gene set enrichment analysis (GSEA) of RNA seq with (siRAD21) and without (siControl) RAD21 knockdown (24 hours post-knockdown, ontology, p<0.05). (I) Genome wide gene set enrichment analysis (GSEA) of RNA seq in osteoblasts and osteosarcoma cells (ontology, p<0.05). The significance tests in the were calculated by using Welch’s T-test. The significance test in was calculated by using Fisher’s exact test. *, p<0.05; **p<0.01, ***p<0.001; n.s., non-significant.

Article Snippet: Rabbit polyclonal antibodies against RAD21 (Cat#27071-1-AP), GAPDH (Cat#10494-1-AP) and alpha-Tubulin (Cat#11224-1-AP) were obtained from Proteintech.

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Knockdown, Negative Control, Labeling, Genome Wide, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Differential Regulation of Large-scale Chromosome Conformations in Osteoblasts and Osteosarcoma

doi: 10.1101/2024.11.01.621571

Figure Lengend Snippet: (A) Western blotting shows that protein levels of CTCF and RAD21 (a cohesin subunit) are higher in osteosarcoma cells. (B) Western blotting of core histones. (C) Western blotting on the extraction of histone by acid extraction. While H3K9me2/3 and H3K4me2 levels are similar, a gain of H3K27ac and a loss of H3K27me3 were found in osteosarcoma. One million cells were used in each sample. All experiments were repeated at least three times.

Article Snippet: Rabbit polyclonal antibodies against RAD21 (Cat#27071-1-AP), GAPDH (Cat#10494-1-AP) and alpha-Tubulin (Cat#11224-1-AP) were obtained from Proteintech.

Techniques: Western Blot, Extraction

Journal: bioRxiv

Article Title: Differential Regulation of Large-scale Chromosome Conformations in Osteoblasts and Osteosarcoma

doi: 10.1101/2024.11.01.621571

Figure Lengend Snippet: (A) ChIP seq data showing H3K27me3 and H3K27ac occupancy along chr 19 in OB and OS cells. (B) Schematic diagram illustrating the action of EPZ005687, which prevents deposition of H3K27me3 by blocking the SET domain of EZH2 in PRC2 complexes. (C) Western blotting shows that H3K27me3 expression levels were significantly reduced within 8 days post EPZ005687 treatment in osteoblast cells. (D) Images of untreated osteoblast cells with extended C19q conformations and EPZ005687 treated osteoblast cells with collapsed C19q conformations. (E) Percentages of cells with collapsed (green) and extended (orange) C19q conformations in osteoblast cells under indicated conditions (N C19q =50 for each condition). The significance was calculated using Fisher’s exact test. * p <0.05, ** p <0.01, ***, p<0.001. (F) Western blotting shows that reduction of H3K27me3 in OB upon EPZ005687 treatment does not change the levels of Rad21 and CTCF. However, a mild reduction of H3K9me2/3 was observed, suggesting collapsed C19q was not due to the increase of CTCF, cohesin, or H3K9me2/3.

Article Snippet: Rabbit polyclonal antibodies against RAD21 (Cat#27071-1-AP), GAPDH (Cat#10494-1-AP) and alpha-Tubulin (Cat#11224-1-AP) were obtained from Proteintech.

Techniques: ChIP-sequencing, Blocking Assay, Western Blot, Expressing

Journal: Nature Genetics

Article Title: Cohesin prevents cross-domain gene coactivation

doi: 10.1038/s41588-024-01852-1

Figure Lengend Snippet: a , Schematic diagram showing that the correlation of two genomic features A and B at the single-cell level may vary drastically even when their average levels remain the same in the cell population. b , The workflow for Smart-SCRB sequencing of control and RAD21-depleted mouse ES cells; Ctrl, control; RT, Reverse Transcription. c , Acute cohesin depletion results in only subtle changes in gene expression at the population level. Each circle represents a detected gene. Average gene expression was quantified by calculating the logarithmic value of averaged sequencing counts of ~400 individual cells. The red line marks the diagonal y = x . d , ACDs were identified based on ATAC peak densities by Gaussian peak fitting and thresholding for each chromosome. In total, 776 ACDs were identified across the genome. Red triangles indicate ATAC peaks. e , Statistics of Δ C RNA ( i , j ) per ACD pair after cohesin depletion in chromosomes 1–19 and X. Each circle indicates the value of the differential coexpression coefficient for one ACD pair. The red line indicates the median value, and the dotted line indicates the zero-change line. Only active ACDs (those ACDs with detectable gene expression by Smart-SCRB) were included for analysis. The statistics were derived from 76,943 intrachromosomal ACD pairs with quantifiable values over 20 different chromosomes. f , Heat maps show elevated differential coexpression coefficients per active ACD pair in Chr 2 after cohesin depletion.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-RAD21 (D213; Cell Signaling, 4321, 1:1,000 dilution), rabbit polyclonal anti-MED1 (CRSP1/TRAP220; Bethyl Laboratories, A300-793A, 1:1,000 dilution), rabbit polyclonal anti-MED6 (Abcam, ab220110, 1:1,000 dilution), rabbit monoclonal anti-BRD4 (E8V7I; Cell Signaling, 54615, 1:1,000 dilution), rabbit polyclonal anti-TBP (Cell Signaling, 8515, 1:1,000 dilution), rabbit polyclonal anti-OCT4 (Abcam, ab19857, 1:2,000 dilution), anti-histone H2A.Z (EPR18090; Abcam, ab188314, 1:2,000 dilution) and rabbit monoclonal anti-α-tubulin (11H10; Cell Signaling, 2125, 1:2,000 dilution).

Techniques: Sequencing, Control, Reverse Transcription, Expressing, Derivative Assay

Journal: Nature Genetics

Article Title: Cohesin prevents cross-domain gene coactivation

doi: 10.1038/s41588-024-01852-1

Figure Lengend Snippet: a , A schematic workflow for scATAC-seq (10x Genomics) of control and RAD21-depleted mouse ES cells. b , Acute cohesin loss results in no significant alteration of normalized average counts of ATAC peaks per ACD at the cell population level. Each circle represents one ACD, and empty ACDs without ATAC peaks were omitted from analysis. Normalized average counts of ATAC peaks per ACD were calculated by dividing the accumulated ATAC peak count per ACD by M Average (the average ATAC peak counts across all ACDs and cells). The red line marks the diagonal y = x . c , Acute cohesin loss increases chromatin coaccessibility per ACD pair globally. Coaccessibility Spearman correlation coefficients per ACD pair ( S ATAC ( i , j )) under control (black) and cohesin-depleted (red) conditions were plotted as a function of the genomic distance after a five-point smoothing. Only the data from ACD pairs within the same chromosome were used to generate the plot. Data are presented as mean values ± s.e., and shadow regions indicate s.e.m. d , Heat map showing that acute cohesin loss selectively increases intrachromosome chromatin coaccessibility per ACD pair across 20 chromosomes in the mouse genome. After filtering out cells with low read counts and batch normalization, around 3,000 cells were analyzed for each condition. e , The heat map shows elevated differential coaccessibility per ACD pair within each chromosome. Differential coaccessibility Spearman correlation coefficients per ACD pair were calculated by subtracting the coaccessibility Spearman correlation coefficient value under control conditions ( d , top left) from that under cohesin-depleted conditions ( d , top right). f , Heat map showing elevated differential coaccessibility per ACD pair in Chr 2. g , Dot plots of differential coaccessibility Spearman correlation coefficients (Δ S ATAC ( i , j )) before and after cohesin depletion for chromosomes 1–19 and X. Every circle indicates the value of the differential coaccessibility Spearman correlation coefficient per ACD pair. The red line indicates the median value, and the dotted line indicates the zero-change line. The statistics were derived from 15,308 intrachromosomal ACD pairs with quantifiable values over 20 different chromosomes.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-RAD21 (D213; Cell Signaling, 4321, 1:1,000 dilution), rabbit polyclonal anti-MED1 (CRSP1/TRAP220; Bethyl Laboratories, A300-793A, 1:1,000 dilution), rabbit polyclonal anti-MED6 (Abcam, ab220110, 1:1,000 dilution), rabbit monoclonal anti-BRD4 (E8V7I; Cell Signaling, 54615, 1:1,000 dilution), rabbit polyclonal anti-TBP (Cell Signaling, 8515, 1:1,000 dilution), rabbit polyclonal anti-OCT4 (Abcam, ab19857, 1:2,000 dilution), anti-histone H2A.Z (EPR18090; Abcam, ab188314, 1:2,000 dilution) and rabbit monoclonal anti-α-tubulin (11H10; Cell Signaling, 2125, 1:2,000 dilution).

Techniques: Control, Derivative Assay

Journal: Nature Genetics

Article Title: Cohesin prevents cross-domain gene coactivation

doi: 10.1038/s41588-024-01852-1

Figure Lengend Snippet: a-b . PONDR score charts indicate predicted ordered and disordered regions within MED1 ( a ) and MED6 ( b ). c-d . PCR genotyping results showing bi-allelic fusion of HaloTag to MED1 c; N-terminus) and MED6 ( d ; C-terminus). Genomic DNA from wild-type mouse ES cells was used as the control. e-f . Western blots showing HaloTag-MED1 ( e ) and MED6-HaloTag ( f ) protein levels before and after RAD21 depletion by auxin-induced degron system. α-tubulin protein was blotted and used as a loading control. g-h . Western blots show the efficacy of RAD21 degron system in parental cell lines and established MED1 ( g ) and MED6 ( h ) knockin cell lines before and after auxin treatment for 6 hours. The normalized RAD21 or RAD21-mAID-eGFP protein level to the loading α-tubulin level for each condition was quantified and shown below each lane. i-j . Fluorescence images showing RAD21-mAID-eGFP (Green) and HaloTag-MED1 ( i ; Red) or MED6-HaloTag ( j ; Red) levels without or with the auxin treatment (6 hrs). DNA was counter-stained with DAPI (Blue). Scale bar, 5 μm. For experiments from c to j , the measurement was repeated three times independently with similar results. k . Propidium iodide (PI) staining and flow cytometry analysis of DNA contents (CD4-FITC) from parental RAD21-mAID-eGFP cell line, and established MED1 and MED6 knockin cell lines before and after acute cohesin loss.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-RAD21 (D213; Cell Signaling, 4321, 1:1,000 dilution), rabbit polyclonal anti-MED1 (CRSP1/TRAP220; Bethyl Laboratories, A300-793A, 1:1,000 dilution), rabbit polyclonal anti-MED6 (Abcam, ab220110, 1:1,000 dilution), rabbit monoclonal anti-BRD4 (E8V7I; Cell Signaling, 54615, 1:1,000 dilution), rabbit polyclonal anti-TBP (Cell Signaling, 8515, 1:1,000 dilution), rabbit polyclonal anti-OCT4 (Abcam, ab19857, 1:2,000 dilution), anti-histone H2A.Z (EPR18090; Abcam, ab188314, 1:2,000 dilution) and rabbit monoclonal anti-α-tubulin (11H10; Cell Signaling, 2125, 1:2,000 dilution).

Techniques: Control, Western Blot, Knock-In, Fluorescence, Staining, Flow Cytometry

Journal: Nature Genetics

Article Title: Cohesin prevents cross-domain gene coactivation

doi: 10.1038/s41588-024-01852-1

Figure Lengend Snippet: a , Diagram showing biallelic integration of HaloTag and green fluorescent protein (GFP)–mini-AID (mAID) into endogenous Med6 and Rad21 gene loci (for auxin-induced protein degradation). b , Three-dimensional isosurface reconstruction of MED6 hubs (color coded by 3D volumes). Inlets are MED6 fluorescence images; scale bars, 2 μm. c , d , Violin plots show the size distribution of MED6 ( c ) and MED1 ( d ) hubs. Black lines are median values, and dotted lines are 25% and 75% quantiles. A non-parametric two-sided Wilcoxon test was used for statistical testing. e , MED6 hub sizes are inversely correlated with residual RAD21 protein levels in single cells. An F -test indicates that the slope is significantly non-zero with P < 0.0001; AU, arbitrary units. f , Timelapse imaging of MED6 hubs during cohesin depletion; scale bar, 2 μm. g , Quantification of MED6 hub sizes (red) and RAD21 residual levels (gray) in f . For MED6 hub size analysis, the statistics were derived from the top 50 hubs, and data are presented as mean values ± s.e. h , Three-dimensional isosurface rendering of overlaps between MED6 hubs (color coded by 3D volumes) and transcription bursting sites for 208 genes in Chr 2 (magenta); scale bars, 2 μm. i , Representative 3D volume overlaps between MED6 hubs and Chr 2 intron clusters; scale bar, 1 μm. j , Violin plots show the statistics of 3D volume overlaps between MED6 hubs and transcription bursting sites for 208 genes in Chr 2. Black lines are the median values, and dotted lines are 25% and 75% quantiles. The measurements were obtained from 20 cells, and a non-parametric two-sided Wilcoxon test was used for statistical testing. k , Three-dimensional isosurface reconstruction of MED6 hubs and intron-FISH signals showing a representative case that co-bursting gene loci are connected by MED6 hubs after cohesin loss; scale bar, 500 nm; smRNA-FISH, single-molecule RNA-FISH. l , Bar plots for the fraction of co-bursting loci connected by MED6 hubs for paired genes within (shadowed) and across ACDs. The fraction was calculated by dividing the number of co-bursting loci that share a common MED6 hub by the total number of co-bursting loci. The measurement was repeated three times, and two-sided Student’s t -tests were used for statistical testing. Data are presented as mean values ± s.d.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-RAD21 (D213; Cell Signaling, 4321, 1:1,000 dilution), rabbit polyclonal anti-MED1 (CRSP1/TRAP220; Bethyl Laboratories, A300-793A, 1:1,000 dilution), rabbit polyclonal anti-MED6 (Abcam, ab220110, 1:1,000 dilution), rabbit monoclonal anti-BRD4 (E8V7I; Cell Signaling, 54615, 1:1,000 dilution), rabbit polyclonal anti-TBP (Cell Signaling, 8515, 1:1,000 dilution), rabbit polyclonal anti-OCT4 (Abcam, ab19857, 1:2,000 dilution), anti-histone H2A.Z (EPR18090; Abcam, ab188314, 1:2,000 dilution) and rabbit monoclonal anti-α-tubulin (11H10; Cell Signaling, 2125, 1:2,000 dilution).

Techniques: Fluorescence, Imaging, Derivative Assay

Journal: Nature Genetics

Article Title: Cohesin prevents cross-domain gene coactivation

doi: 10.1038/s41588-024-01852-1

Figure Lengend Snippet: a . Western blots indicate HaloTag fusion protein levels for either stably expressed (OCT4, TBP1 and H2A.Z) or endogenously labelled (BRD4) transcriptional regulators before and after RAD21 depletion. α-tubulin was used as the loading control. The measurement was repeated three times independently with similar results. b . A diagram shows the labeling of HaloTag fusion proteins with JF549 dye. c . A schematic diagram illustrates the procedures for single-molecule imaging, localization, and tracking.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-RAD21 (D213; Cell Signaling, 4321, 1:1,000 dilution), rabbit polyclonal anti-MED1 (CRSP1/TRAP220; Bethyl Laboratories, A300-793A, 1:1,000 dilution), rabbit polyclonal anti-MED6 (Abcam, ab220110, 1:1,000 dilution), rabbit monoclonal anti-BRD4 (E8V7I; Cell Signaling, 54615, 1:1,000 dilution), rabbit polyclonal anti-TBP (Cell Signaling, 8515, 1:1,000 dilution), rabbit polyclonal anti-OCT4 (Abcam, ab19857, 1:2,000 dilution), anti-histone H2A.Z (EPR18090; Abcam, ab188314, 1:2,000 dilution) and rabbit monoclonal anti-α-tubulin (11H10; Cell Signaling, 2125, 1:2,000 dilution).

Techniques: Western Blot, Stable Transfection, Control, Labeling, Imaging

Journal: Nature Genetics

Article Title: Cohesin prevents cross-domain gene coactivation

doi: 10.1038/s41588-024-01852-1

Figure Lengend Snippet: a , Transcription factors search for targets in a local interaction hub by a bound-state-dominant mode. Local exploration is proposed to be guided by transient interactions with other co-regulators (proteins or non-coding RNAs) in the hub; TF, transcription factor. b , The diagram illustrates the quantification of the RoC and the calculation of its differential cumulative probability Δ C (Δ C = Δ C Ctrl – Δ C RAD21– ) over analyzed tracks. For this analysis, both loosely and tightly bound fractions were obtained by using a slower frame rate (20 Hz). c , Differential cumulative probability (Δ C ) of the RoC for histone subunits (H2B and H2A.Z) and a broad range of transcriptional regulators (MED1, MED6, BRD4, OCT4 and TBP). Histone subunits H2B and H2A.Z were analyzed as controls. The inset shows curves illustrating the cumulative probability of the RoC for H2B before (black) and after (red) cohesin loss. d , Cohesin loss leads to disruption of chromatin loops and spatial mixing of ACDs and Mediator hubs. As a result, the average distance between distant active genes (red and gray dots) in cis decreases, accompanied by their colocalization into a shared transcriptional hub and an elevated chance for their co-regulation. e , Schematic diagrams illustrating that an increase in cofluctuation of genes A (red) and B (gray; top) would alter gene coexpression correlation in single cells (bottom) without affecting their average levels (blue dots) at the cell population level. Gray dotted lines reflect sampling points of single-cell genomics for the correlation analysis in the bottom plots.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-RAD21 (D213; Cell Signaling, 4321, 1:1,000 dilution), rabbit polyclonal anti-MED1 (CRSP1/TRAP220; Bethyl Laboratories, A300-793A, 1:1,000 dilution), rabbit polyclonal anti-MED6 (Abcam, ab220110, 1:1,000 dilution), rabbit monoclonal anti-BRD4 (E8V7I; Cell Signaling, 54615, 1:1,000 dilution), rabbit polyclonal anti-TBP (Cell Signaling, 8515, 1:1,000 dilution), rabbit polyclonal anti-OCT4 (Abcam, ab19857, 1:2,000 dilution), anti-histone H2A.Z (EPR18090; Abcam, ab188314, 1:2,000 dilution) and rabbit monoclonal anti-α-tubulin (11H10; Cell Signaling, 2125, 1:2,000 dilution).

Techniques: Disruption, Sampling

Journal: Life Science Alliance

Article Title: BiFCo: visualizing cohesin assembly/disassembly cycle in living cells

doi: 10.26508/lsa.202301945

Figure Lengend Snippet: (A) Whole YFP fusions to Rad21 and Psm1, respectively, and the BiFCo strain, were mounted onto the same coverslip but spatially separated. This is achieved by sticking the cells to separate and confined soybean lectin drops. Time-lapse microscopy (traveling over the three strains at each time point) was performed under an identical illumination/capturing setup to compare fluorescent signals in cells getting into mitosis. Scale bar: 2 μm. Note that each strain’s brightness and contrast levels presented in this figure are adjusted separately to distinguish nuclear foci in all strains. Real signal comparison with identical brightness and contrast levels can be seen in section B, , and . (B) Average fluorescence intensity and SD (arbitrary units) are plotted. 10 nuclei for each strain were analyzed in the same cell cycle stage. The equivalent background area at each time-point for each cell was subtracted from the actual signal within the nucleus. It should be noted that the BiFCo system plotting appears to be flat because of the large increase in the scale of the arbitrary fluorescence units when whole individual fusion proteins are plotted along.

Article Snippet: After transferring to a nitrocellulose membrane, Rad21 was detected using a rabbit polyclonal anti-Rad21 antibody (1:2,000, A313; Antibodies.com ) and a secondary anti-rabbit IgG-peroxidase antibody (1:5,000, A8275; Sigma-Aldrich).

Techniques: Time-lapse Microscopy, Fluorescence

Journal: Life Science Alliance

Article Title: BiFCo: visualizing cohesin assembly/disassembly cycle in living cells

doi: 10.26508/lsa.202301945

Figure Lengend Snippet: (A) The BiFCo tagging was expressed into the temperature-sensitive cut9-665 mutant background. The APC/C function is abolished at high temperatures, so Rad21 is not cleaved. We cultured these cells until the early log phase at a permissive temperature of 25°C and shifted them to 36°C for 4 h before mounting them for time-lapse microscopy, keeping the sample at a high temperature under the microscope. Contrary to wild-type mitosis, the assembled complex’s YFP signal does not decay in cells that go through anaphase. The time-lapse interval is indicated at the bottom: 9 min (Scale bar: 3 μm). (B) BiFCo complex and Htt1-mRFP (red chromatin marker) were expressed into mis4 + and mis4-242 ts backgrounds. Exponentially growing cultures at 25°C and shifted to 36°C for 4 h were processed for live cell microscopy. Maximal projections from 21 Z-stacks every 0.2 μm are shown for both strains and temperatures under identical settings. Scale bar: 7 μm. Fluorescence from the assembled complex is fully dependent on the Mis4 function.

Article Snippet: After transferring to a nitrocellulose membrane, Rad21 was detected using a rabbit polyclonal anti-Rad21 antibody (1:2,000, A313; Antibodies.com ) and a secondary anti-rabbit IgG-peroxidase antibody (1:5,000, A8275; Sigma-Aldrich).

Techniques: Mutagenesis, Cell Culture, Time-lapse Microscopy, Microscopy, Marker, Fluorescence

Journal: Life Science Alliance

Article Title: BiFCo: visualizing cohesin assembly/disassembly cycle in living cells

doi: 10.26508/lsa.202301945

Figure Lengend Snippet: (A) BiFCo maximal projections (21 slices) from either asynchronous cells, nitrogen-starved cells for 4 h, cdc10-129 -arrested cells at 36°C for 4 h, hydroxyurea-treated cells (10 mM, 4 h) or cdc25-22 -arrested cells at 36°C for 4 h. Scale bar: 7 μm. The inset shows a representative nucleus enlargement in each case. Scale bar: 1 μm. Images are taken and adjusted identically for brightness and contrast. (B) Box and whisker distributions for signal quantification of 50 random nuclei in each condition. We used ordinary one-way ANOVA comparison against asynchronous control distribution to test statistical significance (GraphPad Prism 9.0.2 software. Dunnett’s multiple comparisons test). Confidence interval P < 0.05. ns, non-significant. Asterisks indicate P < 0.0001. (C) Total Rad21 levels at each cell cycle stage. Total protein extracts were prepared from each cell cycle phase arrest. We used rabbit polyclonal anti-Rad21 antibody (A313; Antibodies.com ) to assess total Rad21 protein by Western blot and monoclonal anti-PSTAIRE antibody (P7962; Sigma-Aldrich) for loading control. Densitometry analysis was performed with Image Lab 6.0.1 software. The graph on the right shows protein levels standardized to respective loading controls and relative to the asynchronous culture.

Article Snippet: After transferring to a nitrocellulose membrane, Rad21 was detected using a rabbit polyclonal anti-Rad21 antibody (1:2,000, A313; Antibodies.com ) and a secondary anti-rabbit IgG-peroxidase antibody (1:5,000, A8275; Sigma-Aldrich).

Techniques: Whisker Assay, Software, Western Blot