Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet:

Article Snippet: Escherichia coli BL21 (DE3) , New England Biolabs , Cat#C2527I.

Techniques: Virus, Recombinant, Cell Recovery, Modification, Gentle, Flow Cytometry, Reverse Transcription, Bicinchoninic Acid Protein Assay, RNA Extraction, Western Blot, Cloning, Mutagenesis, Plasmid Preparation, Software, Cell Culture, Microscopy, Nucleic Acid Electrophoresis

Journal:

Article Title: Saccharomyces cerevisiae DNA Polymerase ? and Polymerase ? Interact Physically and Functionally, Suggesting a Role for Polymerase ? in Sister Chromatid Cohesion

doi: 10.1128/MCB.23.8.2733-2748.2003

Figure Lengend Snippet: (A) Pol2 coimmunoprecipitates with Trf4. Coexpression of FLAG-Pol2 and His-Trf4 and immunoprecipitation protocols are described in Materials and Methods. Western blots of crude extract (labeled “I” for input protein) from insect cells expressing various combinations of Trf proteins and FLAG-Pol2 were probed with antibody against Trf4 or anti-FLAG Pol2 as indicated. Lane 1, no recombinant protein; lane 2, His-Trf4; lane 3, His-Trf4 plus FLAG-Pol2. These extracts were incubated with anti-FLAG beads. After washing of the beads, proteins that bound from extracts (labeled “B” for bound protein) were eluted by boiling. The proteins were analyzed on Western blots probed with antibody against Trf4 or anti-FLAG Pol2 as indicated on the right. Lane 4, no recombinant protein; lane 5, His-Trf4; lane 6, His-Trf4 plus FLAG-Pol2. (B) Pol2 coimmunoprecipitates with Trf5. Coexpression of FLAG-Pol2 and Trf5-His is described in Materials and Methods. Western blots of crude extract from insect cells are represented in the same order as those in Fig. ​Fig.2A.2A. (C) Recombinant Trf4 prepared in E. coli stimulates Pol ɛ holoenzyme. Trf4 was purified exactly as described previously (70). The oligo(dT)12-18 primer extension assay is described in Materials and Methods. Reaction mixtures contained 680 ng of Trf4, the amount required to observe Trf4 DNA polymerase activity (lanes 2 and 3), and/or 0.15 U of Pol ɛ, as indicated, are shown. The high level of Trf4 is saturating for stimulatory activity (see panel D). Lane 1, no protein; lane 2, Trf4-His with 0.1 mM dTTP; lane 3, Trf4-His with 1 mM dTTP; lane 4, Pol ɛ with 0.1 mM dTTP; lane 5, Pol ɛ with 1 mM dTTP; lane 6, Pol ɛ plus Trf4-His with 0.1 mM dTTP; and lane 7, Pol ɛ plus Trf4-His with 1 mM dTTP. (D) Titration of stimulatory activity of scTrf4 made in bacteria. The indicated amounts of scTrf4 were assayed for stimulation of [3H]dTMP incorporation by 0.15 U of Pol ɛ on an oligo(dT)-poly(dA) substrate as described in Materials and Methods. (E) scTrf4-His expressed in insect cell cochromatographs with Pol ɛ-stimulatory activity. scTrf4-His was expressed in insect cells. Silver staining of Trf4-His after purification through Ni2+-nitrilotriacetic acid and Mono Q columns, as described in Materials and Methods, and gel electrophoresis is shown at the top. Numbers refer to MonoQ fraction numbers. The same fractions are assayed for stimulation of primer extension by pol ɛ. Each fraction from the Mono Q column was dialyzed, and 2 μl of each fraction was used in a 20-μl reaction. Fraction 13 contained 17 ng of Trf4 protein (13 nM); but 2.7 nM Trf4 gave equivalent stimulation (not shown). The first lane shows no primer extension, the second lane shows activity of 0.15 U of Pol ɛ (0.5 nM) alone, and the subsequent lanes are the Mono Q fractions of the Trf4 purification assayed with 0.15 U of Pol ɛ (0.5 nm). The fraction numbers are identified above. (F) scTrf4-His and Pol ɛ-stimulatory activity copurify. [3H]dTMP incorporation assay: the same fractions from the Mono Q column were assayed for [3H]dTMP incorporation on an oligo(dT)-poly(dA) substrate as described in Materials and Methods in the presence of 0.15 U of Pol ɛ (0.5 nm). (G) Trf4 from the MonoQ column is highly purified. Coomassie-stained gel of Trf4 from fraction 14 of the MonoQ column. (H) scTrf4-His does not efficiently stimulate Pol2-140 lacking the C-terminal 1,000 amino acids. Three levels (0.075, 0.15, or 0.3 U) of either Pol ɛ (solid dots) or truncated Pol2 protein (open dots) were assayed with saturating amounts (40 ng) of Trf4-His purified from insect cells. Similar results were obtained with scTrf4 prepared in E. coli.

Article Snippet: As controls that the stimulation was not due to contaminating E. coli DNA polymerase I (the most abundant DNA polymerase in the bacterial extracts) in the scTrf4 preparation, we showed that DNA polymerase I of E. coli (Gibco BRL) did not stimulate Pol ɛ and antibody to E. coli Pol I (gift of S. Linn, U.C.

Techniques: Immunoprecipitation, Western Blot, Labeling, Expressing, Recombinant, Incubation, Purification, Primer Extension Assay, Activity Assay, Titration, Silver Staining, Nucleic Acid Electrophoresis, Staining

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 1. PWP1 Regulates Tissue Growth and Proliferation (A) dPWP1 RNAi in the posterior compartment of the developing wing (En-Gal4) leads to reduced compartment size. (B) Quantification of the ratio of posterior (P) (n = 10) and anterior (A) (n = 10) wing areas in (A). (C) Cell density ratio between posterior (P) (n = 10) and anterior (A) (n = 10) compartments in (A). (D) Pupation kinetics of control (n = 5) and dpwp1nclb1/2 (n = 5) larvae. dAEL, days after egg laying. (E) Representative images of control and dpwp1nclb1/2 pupae. (F) Quantification of pupal volumes of control (n = 4) and dpwp1nclb1/2 (n = 4) pupae. (G) Proliferation in Ctrl (Lac dsRNA) (n = 3) and dPWP1-specific dsRNA (n = 3) treated S2 cells. (H) Proliferation of HeLa cells after transfection with non-targeting (Ctrl) (n = 3) or PWP1-specific (n = 3) siRNAs. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S1.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Control, Transfection

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 2. PWP1 Regulates Pol I-Mediated Ribosomal Gene Expression (A) dpwp1nclb1/2 mutants display short and thin bristles. (B) Pupation kinetics of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted larvae. dAEL, days after egg laying. (C) Representative images of control and dPWP1 fat-body (Cg-Gal4)-depleted pupae. (D) Quantification of volumes of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted pupae. (E) Representative immunofluorescent images of endogenous dPWP1 localization in comparison with fibrillarin in fat bodies of early third instar larvae. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, and 28S rRNA (RNA polymerase I targets) expression in control larvae (n = 3) and dpwp1 mutants (n = 3). cdk7 was used as a reference gene. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S2.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Gene Expression, Control, Comparison, Quantitative RT-PCR, Expressing

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 4. PWP1 Functionally Cooperates with MYBBP1A and Nucleolin (A) Summary of dPWP1 interacting ribosome biogenesis regulators in S2 cells. (B) Summary of PWP1 interacting ribosome biogenesis regulators in HEK293 cells. (C) Co-purification of HA-tagged dPWP1 with dMYBBP1A upon pull-down of V5-tagged dMYBBP1A from S2 cells. Tubulin serves as a loading control. (D) Representative immunofluorescent images of PWP1 and Nucleolin localization in U2OS cells. Scale bar, 5 mm. (E) Representative immunofluorescent images of Nucleolin localization in U2OS cells followed by PWP1 depletion. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, 28S, and 5S rRNA expression in control larvae (n = 3) and dmybbp1a mutants (n = 3). cdk7 was used as a reference gene. (G) Representative images of control (w-) and mybbp1a mutant larvae at 96 hr after egg laying. (H) Representative immunofluorescent images of dPWP1 localization in fat bodies of early third instar larvae. Depletion of dMYBBP1A from the fat body (Fb-GAL4) leads to the dissociation of nucleolar dPWP1. Scale bar, 5 mm. (I) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (H). A total of 15 nuclei from 3 independent fat bodies were quantified. (J and K) Gel electrophoresis analysis of total RNA (lower panel, visualized by Midori green or ethidium bromide staining) and newly transcribed RNA (upper panel, visualized by streptavidin-HRP detection) prepared from U2OS cells transfected with indicated siRNAs followed by 30 min of 4sU labeling. RNA from non-transfected U2OS cells cultured in the absence of 4sU (No 4sU) was used as a control for streptavidin-HRP signal specificity. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S4.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Control, Quantitative RT-PCR, Expressing, Mutagenesis, Nucleic Acid Electrophoresis, Staining, Transfection, Labeling, Cell Culture

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 5. mTORC1-Dependent Phosphorylation Regulates Nucle- olar PWP1 (A) Representative immunofluorescent images of endogenous dPWP1 locali- zation in fat bodies of early third instar fed and starved (6 hr) larvae. Scale bar, 5 mm. (B) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (A). A total of 15 nuclei from 3 independent fat bodies were quantified. (C) Representative immunofluorescent images of endogenous dPWP1 locali- zation in fat bodies of early third instar starved (4 hr) and re-fed (6 hr) larvae. Scale bar, 5 mm. (D) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (C). A total of 15 nuclei from 3 independent fat bodies were quantified. (E) Representative immunofluorescent images of dPWP1 localization in fat bodies dissected from early third instar larvae fed without or with rapamycin (14 hr). Scale bar, 5 mm. (F) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (E). A total of 15 nuclei from 3 independent fat bodies were quantified. (G) Immunoblot of S2 cell lysates expressing a V5-tagged form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin alone (10 min) or in combination with rapamycin (2 hr). Phospho-dPWP1 species (anti-V5) are indicated by arrowheads. (H) Immunoblot of lysates of S2 cell expressing a V5-tagged form of dPWP1 together with RNAi against LacZ (ctrl), dS6K, or dRaptor resolved on Phos-tag SDS-PAGE. Cells were treated without or with insulin (10 min). (I) Quantification of phospho-dPWP1 species of (H). (J) Immunoblot of S2 cell lysates expressing a V5-tagged wild-type or S384 alanine mutated form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin (10 min). (K) Quantification of phospho-dPWP1 species of (J). (L) Immunofluorescent analysis of fat body expressing the wild-type and S384A form of dPWP1 dissected from early third instar larvae. Scale bar, 5 mm. (M) Quantification of the nucleolus/nucleoplasm localization ratio in (L). A total of 15 nuclei from 3 independent fat bodies were quantified. **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S5.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Phospho-proteomics, Western Blot, Expressing, SDS Page

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-d, Validation of mitotic exit in spindle-less cells by comparing H3-S10 dephosphorylation kinetics during unperturbed anaphase and spindle-less mitotic exit. a , Example images of wild-type HeLa cells during unperturbed anaphase fixed after time-lapse imaging, in metaphase, 6 min (maximally clustered), and 10 min after anaphase onset. Upper panel: chromatin labelled with H2B-mCherry, lower panel: H3-pS10 immunofluorescence. Single Z-slice shown. b , Quantification of H3-pS10 mean fluorescence and chromatin area in unperturbed mitosis, as shown in ( a ). Cell numbers: n = 9 (metaphase), n = 14 (3–6 min after anaphase onset), n = 8 (≥8 min after anaphase onset). Normalization to average value of metaphase time point. c , Example images of wild-type HeLa cells during spindle-less mitosis fixed after time-lapse imaging, in prometaphase (no flavopiridol), 10 min (maximally clustered), and 20 min after flavopiridol addition. Imaging as in ( a ). d , Quantification of H3-pS10 mean fluorescence and chromatin area in spindle-less mitosis, as shown in ( c ), demonstrates that histone 3-serine 10 was efficiently dephosphorylated in flavopiridol-induced mitotic exit and chromosomes cluster to a degree comparable to that of normal late anaphase. Cell numbers: n = 11 (nocodazole arrested pro-metaphase), n = 12 (10 min after flavopiridol addition), n = 13 (20 min after flavopiridol addition). Normalization to average value of prometaphase time point. e , Time-lapse microscopy of HeLa cell expressing IBB-EGFP and H2B-mCherry incubated in nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. f, Quantification of IBB-EGFP mean fluorescence within the chromosomal region, normalized to pre-flavopiridol time points, during spindle-less mitotic exit as in ( e ). n = 20 cells. g , Time-lapse microscopy of a HeLa cell expressing H2B-mCherry, progressing through reversine-induced mitotic exit in the absence of spindle. Yellow line indicates convex hull around chromosomes, single Z-slice shown. Time is relative to onset of clustering. h , Quantification of chromosome convex hull area of 11 cells as in ( g ). Individual cell curves were aligned based on half-maximum value of convex hull area. Normalization to average of first 4 time points. i , Live HeLa cell undergoing mitosis upon RNAi-mediated depletion of the spindle checkpoint protein Mad2 in the absence of a spindle. The cell line stably expresses H2B-mCherry and membrane marker AcGFP-Lap2β. Time relative to nuclear envelope breakdown (NEBD), single Z-slice shown. Representative example of 14 cells shown. Bars indicate mean in ( b, d ), lines and shaded areas indicate mean ± SD in ( f, h ). Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Biomarker Discovery, De-Phosphorylation Assay, Imaging, Immunofluorescence, Fluorescence, Time-lapse Microscopy, Expressing, Incubation, Stable Transfection, Membrane, Marker

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Chromosome organization during spindle-less mitotic exit. 3D-video of HeLa cell expressing H2B-mCherry imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Yellow line indicates convex hull around chromosomes. b , Quantification of convex hull area of 16 cells as in ( a ), and inter-chromosomal area. c, HeLa cell expressing GEMs and H2B-mCherry, during spindle-less mitotic exit as in ( a ). Dashed lines indicate chromosomal area; single Z-slices shown. d, GEM particle count within chromosomal area as in ( c ), normalized to pre-flavopiridol time points (green). Chromosome convex hull area, normalized to pre-flavopiridol time points (magenta). n = 35 cells. Lines and shaded areas represent mean ± SD, dashed vertical lines indicate flavopiridol addition. Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Expressing

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, b, Effect of actin depolymerization on chromosome clustering. a , Live HeLa cells stably expressing actin-EGFP and H2B-mCherry were imaged in absence (control) or presence of latrunculin B. Nocodazole was present in both conditions. b , Quantification of chromosome convex hull area during a flavopiridol induced mitotic exit in the presence of nocodazole, in the absence (control) and presence of latrunculin B as shown in ( a ), normalized to average area pre-flavopiridol addition. Cell numbers: n = 13 (control), n = 22 (latrunculin B). c , Timing of chromosome clustering relative to nuclear envelopment. Live mitotic HeLa cell expressing H2B-mCherry and the inner nuclear envelope protein AcGFP-LAP2β were imaged in presence of nocodazole; flavopiridol was added at t = 0 min to induce mitotic exit. d, Quantification of chromosome area by convex hull (yellow line in H2B channel in ( c )) and quantification of AcGFP-LAP2β accumulation at the surface of the chromatin region, within a rim of 1.6 μm width (yellow ROI in Lap2β channel in ( c )). n = 21 cells. e-h , Kinetochore tracking in cells progressing through spindle-less mitotic exit. e , Live mitotic HeLa cell stably expressing H2B-mCherry and CENP-A-EGFP imaged in the presence of nocodazole before and after flavopiridol addition, time-lapse = 10 s; Z-projection. f , 240 s long kinetochore trajectories starting 240 s before and at 400 after flavopiridol addition. g , Representative mean squared displacement (MSD) analyses of kinetochore tracks (CENP-A–EGFP) of nocodazole-treated mitotic HeLa cells before and after flavopiridol addition. h , Diffusion coefficients derived from mean square displacement (MSD) analyses of trajectories as in ( f ). Significance was tested by a two-tailed ratio paired t-test (****P = 2.9 × 10 −11 ). n = 13 cells. Bars and lines indicate mean, shaded areas indicate ± SD, dashed vertical lines refer to flavopiridol addition. Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Stable Transfection, Expressing, Control, Diffusion-based Assay, Derivative Assay, Two Tailed Test

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: Live cell microscopy of wild-type HeLa cells stably expressing H2B-mCherry synchronized to metaphase by MG132 treatment. a , Upper panel: negative control treated with dimethyl sulfoxide (DMSO) solvent, lower panel: mitotic exit induced through acute flavopiridol addition at t = 0 min. Chromosome arms extend out of the metaphase plate before flavopiridol addition, but densely cluster on the metaphase plate 8 min after flavopiridol addition. b , Quantification of chromosome convex hull area of 24 control and 25 flavopiridol treated cells, as in ( a ). c , Upper panel: acute mitotic spindle depolymerization by addition of nocodazole at t = 2 min leads to chromosome movement out of the metaphase plate; lower panel: mitotic exit was induced by flavopiridol (0 min), briefly before nocodazole-mediated spindle disassembly (2 min), preventing movement of chromosomes out of the metaphase plate. d , Chromosome convex hull area measurements of 27 nocodazole and 23 flavopiridol and nocodazole treated cells, as shown in ( c ). Values normalized to average of all frames prior to first drug treatment. Showing single Z-slices, lines and shaded areas indicate mean ± SD, dashed vertical lines indicate drug addition, scale bars 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Microscopy, Stable Transfection, Expressing, Negative Control, Solvent, Control

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-c, Effect of BAF depletion on chromosome clustering. a , Live mitotic HeLa cells stably expressing H2B-mCherry and GEMs were imaged 72 h after siRNA transfection, in the presence of nocodazole; flavopiridol was added at t = 0 min to induce mitotic exit. White dashed lines indicate chromosome areas, single Z-slice shown. b , Quantification of chromosome convex hull area, normalized to pre-flavopiridol addition. c , GEM particle count within chromosomal area normalized to pre-flavopiridol addition. n = 23 cells (siBAF), n = 23 cells (siControl). d , Immunoblot analysis of BAF and actin 72 h after siRNA transfection, showing one of two biological replicates. For gel source data, see . e , Localization of BAF-EGFP in live mitotic HeLa cell imaged in the presence of nocodazole; flavopiridol was added at t = 0 s to induce mitotic exit. f , Quantification of chromosome convex hull area and BAF-EGFP accumulation at the surface of the chromatin region as in ( e ), normalized to pre-flavopiridol. n = 21 cells. g , Localization of Ki-67, EGFP tagged on endogenous loci in live HeLa cell progressing from metaphase to anaphase (anaphase onset = 0 min), DNA was stained with SiR-Hoechst, Z-projection. h, Quantification of chromosome convex hull area and EGFP-Ki-67 mean fluorescence on chromosomes, normalized to pre-anaphase, for 41 cells as in ( g ). i , Localization of Ki-67 during spindle-less mitotic exit. Time-lapse microscopy of mitotic HeLa cell as in ( g ), in the presence of nocodazole; flavopiridol was added (t=0 min) to induce mitotic exit. Z-projection. j , Quantification of chromosome convex hull area and EGFP-Ki-67 mean fluorescence on chromosomes of 27 cells as in ( i ). Shaded areas indicate ± SD, scale bars 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Staining, Fluorescence, Time-lapse Microscopy

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a-d , Molecular organization of Ki-67 on the surface of mitotic chromosomes before and after flavopiridol addition. a , Ki-67 was tagged on the N-terminus by mCherry, on the C-terminus by EGFP and expressed in HeLa cells. Sister chromatid pairs oriented perpendicular to the imaging plane were imaged in live mitotic cells, lines indicate measurement regions for ( b ). b , Relative positions of mCherry and EGFP along an axis perpendicular to the chromosome surface. Fluorescence densities (dots) were measured along line profiles as in ( a ) and a sum of two Gaussian functions (lines) was separately fitted to the EGFP and mCherry channels, respectively, to determine peak positions (dashed lines). c , The radial displacement of EGFP relative to mCherry peaks was determined based on line profile measurements as in ( a, b ) for mCherry-Ki-67-EGFP (R-Ki-67-G: n = 34 chromosomes pre-flavopiridol, n = 46 chromosomes post-flavopiridol) and a construct where the fluorophores were linked in reverse order, EGFP-Ki-67-mCherry (G-Ki-67-R: n = 40 chromosomes pre-flavopiridol, n = 39 chromosomes post-flavopiridol). Bars represent mean, significance was tested by a two-tailed unpaired t-test (****P = 2.1 ×10 −8 , ****P = 2.3 × 10 −6 ). d, Model of Ki-67 organization on chromosome surfaces during early mitosis and during mitotic exit. e, Ki-67 KO cell expressing low levels of H2B-mNeonGreen. Representative example of 14 cells. f , Spindle-less mitotic exit in wild-type cells and in Ki-67 knockout (KO) cells overexpressing H2B-mNeonGreen to high levels to suppress the Ki-67 knockout individualization failure phenotype. Live cells were imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Z-projection. g , Normalized chromosome convex hull area quantification of 24 wild-type and 22 Ki-67 KO cells as in ( f ). Lines and shaded areas represent mean ± SD. Scale bars 1 μm ( a ), all others 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Imaging, Fluorescence, Construct, Two Tailed Test, Expressing, Knock-Out

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Time-lapse microscopy of clustering-deficient HeLa Ki-67 KO cell stably expressing H2B-mCherry to high levels during a flavopiridol-induced mitotic exit in the presence of nocodazole. Transient expression of EGFP-Ki-67 to levels at least matching Ki-67 endogenous levels (lower panel). Single Z-slice shown. b , Chromosome convex hull area quantification, normalized to pre-flavopiridol time points, of 29 Ki-67 KO and 28 Ki-67 KO + EGFP-Ki-67 cells as in ( a ). c , Immunoblot analysis of H2B from cell lysates, isolated from wildtype cells (1) or cells transiently expressing H2B-mNeonGreen (2,3). Sample 3 was lysed after 48 h while sample 2 was additionally FACS sorted for the 10% brightest cells which we estimated to represent the population of cells that suppress the Ki-67 knockout individualization failure phenotype. For gel source data, see . n = 2 biological repeats. d, e , Immunofluorescence of acetylated H3 of Ki-67 KO cells arrested in nocodazole with or without trichostatin A. d , Representative examples. Single Z-slices shown. e , Quantification of acetylated H3 mean fluorescence intensity in Ki-67 KO cells arrested in nocodazole with and without trichostatin A. Bars represent mean, significance was tested with a two-tailed Kolmogorov-Smirnov test. (****P = 2.22×10 −16 ). n = 115 cells (Ki-67 KO), n = 111 cells (Ki-67 KO + TSA). f , Time-lapse microscopy of HeLa wild-type or clustering-deficient Ki-67 KO cells with or without transient re-expression of Ki-67 during a flavopiridol-induced mitotic exit in the presence of nocodazole. Cells were treated with trichostatin A 2 h before imaging to rescue the Ki-67 knockout individualization failure phenotype in Ki-67 KO cells. Representative examples stained with SiR-Hoechst, single Z-slices shown. g , Chromosome convex hull area, normalized to pre flavopiridol time points, quantification of 21 Ki-67 KO, 31 wild-type and 32 Ki-67 KO + EGFP-Ki-67 cells as in ( f ). Lines and shaded areas indicate mean ± SD ( b, g ), scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Stable Transfection, Expressing, Western Blot, Isolation, Knock-Out, Immunofluorescence, Fluorescence, Two Tailed Test, Imaging, Staining

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a , Schematic of genotyping strategy to endogenously mutate Ki-67’s PP1 binding motif RVSF to RASA. A newly generated SacII restriction site generated by CRISPR/Cas9 nickase as depicted was used to detect correctly mutated alleles. b , SacII restriction fragments were detected by gel electrophoresis following the assay depicted in ( a ), showing successful recombination of all three Ki-67 alleles present in HeLa cells for clone 43 and 96. Showing one example of 2 biological replicates. c-d , Spindle-less mitotic exit in wild-type cells and in homozygous Ki-67 RASA mutant cells. Live cells were imaged in the presence of nocodazole; flavopiridol was added (t = 0 min) to induce mitotic exit. Quantification of chromosome convex hull area, normalized to pre-flavopiridol time points ( c ) and representative examples stained with SiR-Hoechst ( d ). Z projection. Lines and shaded areas indicate mean ± SD. n = 22 cells (wild-type), n = 23 cells (RASA) . e , Molecular organization of the Ki-67 RASA mutant on the surface of mitotic chromosomes before and after flavopiridol addition to taxol treated cells. Ki-67(RASA) was tagged by mCherry and EGFP on either protein end, respectively, and expressed in HeLa cells bearing the endogenous RASA mutation in all three copies of Ki-67. Bars represent mean, significance was tested by a two-tailed unpaired t-test (****P = 8.3 ×10 −8 , ***P = 0.00049). Chromosome numbers: n = 32 (R-Ki-67-G, Pre), n = 28 (R-Ki-67-G, Post), n = 37 (G-Ki-67-R, Pre), n = 34 (G-Ki-67-R, Post). f, g , Immunofluorescence of H3-pS10 during spindle-less mitotic exit in wild-type and Ki-67 KO cells. f , Representative examples of wild-type and Ki-67 KO cells before, 10 and 20 min after flavopiridol addition. Single Z-slice is shown. g , Quantification of H3-pS10 mean fluorescence intensity before (Wild-type n = 38 cells, Ki-67 KO n = 23 cells), 5 (Wild-type n = 61 cells, Ki-67 KO n = 61 cells), 10 (Wild-type n = 72 cells, Ki-67 KO n = 65 cells) and 20 min (Wild-type n = 73 cells, Ki-67 KO n = 55 cells) after mitotic exit induction with flavopiridol in wild-type and Ki-67 KO cells. Values normalized to average of wild-type 5 min time point. Showing combined data of two independent biological replicates. Bars represent mean, significance tested with a two-tailed Mann Whitney test (P = 0.72 for pre-flavopiridol time point, P = 0.96 for 5 min time point, P = 0.71 for 10 min time point and P = 0.26 for 20 min time point). Scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Binding Assay, Generated, CRISPR, Nucleic Acid Electrophoresis, Mutagenesis, Staining, Two Tailed Test, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, Time-lapse microscopy of clustering-deficient HeLa Ki-67 KO cell transiently expressing H2B-mCherry to high levels, and stably expressing IBB-EGFP, during a flavopiridol-induced mitotic exit in the presence of nocodazole. b , Quantification of IBB-EGFP mean fluorescence in chromosomal area (green) and quantification of chromosome convex hull area (magenta) in clustering-deficient cells as in ( a ), normalized to pre-flavopiridol. n = 15 cells. c, d , H2B-mCherry expression levels and chromosome area measurements for cells shown in – . , Quantification of H2B-mCherry total fluorescence for cells analyzed in (all data points normalized to mean of wild-type dataset). d , Chromosome convex hull area quantification (normalized to pre-flavopiridol area) for cells quantified in . n = 46 cells (wild-type), n = 29 cells (Ki-67 KO). e , Clustering-proficient HeLa cell imaged as in , but treated with leptomycin B. f , Quantification of total L10 fluorescence in nuclear regions as shown in ( e ), normalized to average of pre-flavopiridol addition. n = 22. g, h , Quantification of H2B-mCherry total fluorescence and chromosome convex hull area for cells analyzed in and in ( f ). Normalizations as in ( c, d ), cell numbers: n = 23 wild-type, n = 8 Ki-67 KO, n = 22 leptomycin B. Individual Ki-67 KO curves shown in . Significance tested with two-sided unpaired t-test (P = 0.28 in GEM cell lines wild-type vs Ki-67 KO, P = 0.12 in L10 cell lines wild-type vs LMB treated and P = 0.13 in L10 cell lines wild-type vs Ki-67 KO). Lines and shaded areas indicate mean ± SD, dashed vertical lines indicate flavopiridol addition. Scale bars 10 μm, showing single Z-slices.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Expressing, Stable Transfection, Fluorescence

Journal: Nature

Article Title: Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

doi: 10.1038/s41586-020-2672-3

Figure Lengend Snippet: a, b, Time-lapse microscopy of spindle-less mitotic exit in clustering-proficient wild-type HeLa cell ( a ) and clustering-deficient Ki-67 KO HeLa cell ( b ). Cells stably expressed GEMs and transiently expressed H2B-mCherry; images show time point 1 min before and 20 min after flavopiridol-induced mitotic exit in presence of nocodazole. c, Quantification of GEM localization in 46 clustering-proficient wild-type and 29 clustering-deficient Ki-67 KO cells as in ( a, b ). Particle numbers were quantified in chromosomal area as in , and normalized to average of particle number pre flavopiridol. t = 0 min refers to addition of flavopiridol. Analyzed cells expressed H2B-mCherry at similar levels in both genetic backgrounds . d, e, Time-lapse microscopy of clustering-proficient wild-type cells and clustering-deficient Ki-67 KO cells ( e ) stably expressing ribosomal protein L10-EGFP and transiently expressing H2B-mCherry, before and after mitotic exit ind-ction in presence of nocodazole. f, Quantification of L10-EGFP total fluorescence within chromosomal area for conditions shown in ( d, e ). t = 0 min refers to flavopiridol addition, data normalized to average values of pre-flavopiridol addition. Analyzed cells expressed H2B-mCherry at similar levels in both conditions . n = 23 cells (wild-type), n = 8 cells (Ki-67 KO). Individual Ki-67 KO curves shown in . g, Model of nuclear assembly during mitotic exit. Lines and shaded areas represent mean ± SD, dashed vertical lines indicate flavopiridol addition. Single Z-slices shown; scale bars, 10 μm.

Article Snippet: Mitotic exit was induced by addition of flavopiridol (Tocris Bioscience) to a final concentration of 20 μM or reversine (Sigma Aldrich) to a final concentration of 1 μM.

Techniques: Time-lapse Microscopy, Stable Transfection, Expressing, Fluorescence

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 1. Replication Fork Stalling Induced by Co-transcriptional R-Loops Is Followed by Replication Restart via the SLX4-MUS81-RAD52- POLD3 Axis (A) Co-localization of PCNA and elongating RNA polymerase II (RNAPII pS2) in S phase nuclei of U2OS cells after 1 h of treatment with camptothecin (CPT; 100 nM) or pyridostatin (PDS; 10 mM), as determined by proximity ligation assay (PLA) and EdU-pulse labeling. Representative images (left panel) and quanti- fication of the percentage (right panel) of EdU+ and EdU cells with R5 PLA foci per nucleus are shown. EdU (10 mM) was added 10 min before CPT/PDS treatment. Where indicated, cordycepin (CORD; 50 mM) or DRB (100 mM) were added 2 h before CPT/PDS treatment. Data represent the means ± SDs, n = 3. Scale bar, 10 mm. (B) Effect of RNase H1 (RNH1) overexpression and transcription inhibition on replication fork slowing induced by CPT (100 nM) or PDS (10 mM) in U2OS T-REx/ RNH1-GFP cells. Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of values of the IdU:CldU tract length ratio obtained for indicated conditions (n R 200, whiskers: 10th–90th percentiles). RNH1 expression was induced with doxycycline (Dox). ns, not significant; ***p < 0.001; ****p < 0.0001 (Mann-Whitney test). (C) PDS and CPT induce sister fork asymmetry in a manner dependent on R-loop formation. Top panel: representative images of symmetric and asymmetric replication tracts of sister forks identified on DNA fibers in (B). Bottom panel: boxplot of the values of the sister fork IdU tract length ratio measured for the indicated conditions (n R 100, whiskers: 10th–90th percentiles). ns, not significant; ***p < 0.001; ****p < 0.0001 (Mann-Whitney test). (D) Effect of transcription inhibition and RNH1 overexpression on the frequency of reversed replication forks in U2OS T-REx/RNH1-GFP cells treated with CPT (100 nM) or PDS (10 mM) for 1 h. RNH1 expression was induced by the addition of Dox at 24 h before treatment. CORD (50 mM) was added 2 h before treatment. Data represent the means ± SDs, n = 3. p values: paired t test. (E) Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. (F) Effect of the depletion of MUS81, EME1, SLX4, RAD52, and POLD3, respectively, on the rescue of CPT-induced replication fork slowing by PARP inhibition (PARPi) with 10 mM olaparib. Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of values of the IdU:CldU tract length ratio obtained for indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ***p < 0.001; ****p < 0.0001 (Mann-Whitney test).

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Proximity Ligation Assay, Labeling, Over Expression, Inhibition, Expressing, MANN-WHITNEY, Western Blot, Transfection

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 3. Restart of R-Loop-Stalled Forks Requires Fork Cleavage by MUS81 Endonuclease (A) Genomic DNA breakage stimulated by PARPi in CPT- and PDS-treated U2OS T-REx/RNH1-GFP depends on DNA replication, transcription, and R-loop formation. Cells were treated with CPT (1 mM) or PDS (20 mM) for 5 h. Olaparib (PARPi; 10 mM), CORD (50 mM), and APH (5 mM), respectively, were added 2 h before CPT/PDS treatment. DNA breakage was monitored by pulsed-field gel electrophoresis (top panels) and quantified using ImageJ software (bottom panels). Data were normalized and represent the means ± SDs, n = 3. ****p < 0.0001 (unpaired t test). DSB, double-strand break. (B) Effect of the depletion of the indicated proteins on the level of DNA breakage in U2OS cells treated for 5 h with 1 mM CPT (black bars) or 20 mM PDS (gray bars) in the presence of PARPi. Genomic DNA was analyzed as in (A). Data represent the means ± SDs, n = 4. (C) The PARPi-mediated rescue of CPT- and PDS-induced replication fork slowing in U2OS cells requires MUS81 endonuclease activity. Top panel: experimental workflow of DNA fiber assays with U2OS cells stably transfected with WT MUS81 or MUS81(D338A/D339A) (MUS81Mut) cDNA constructs. Endogenous MUS81 was depleted with siRNA targeting MUS81 30 UTR (siMUS81UTR). Bottom panel: boxplot of the values of the IdU:CldU tract length ratio obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann-Whitney test). (D) Western blot analysis of the extract of cells in (C). (E) Effect of CPT (100 nM) and PDS (10 mM) on replication fork progression in RA3331/E6E7/hTERT fibroblasts, complemented with either SLX4 WT or SLX4 DSAP cDNAs, before and after PARPi. Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of the values of the IdU:CldU tract length ratio obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann-Whitney test). (F) Representative immunofluorescence images (top panel) and quantification (bottom panel) of MUS81 foci (green) colocalizing with FANCD2 foci (red) in U2OS cell nuclei (DAPI, blue) before and after treatment with CPT (100 nM) or PDS (10 mM) for 1 h. Data represent the means ± SDs, n = 3. Scale bar, 10 mm. (G) Effects of RNase H1 (RNH1) overexpression and transcription inhibition on the formation of MUS81 foci in U2OS T-REx/RNH1-GFP cells treated with CPT (100 nM) or PDS (10 mM) for 1 h. RNH1-GFP expression was induced 24 h before CPT/PDS treatment. CORD (50 mM) was added 2 h before the addition of CPT or PDS. Horizontal lines represent the means ± SEMs (n R 300). ns, not significant; ****p < 0.0001 (Mann-Whitney test). (H) Effects of SLX4 and EME1 depletions on the formation of FANCD2+ MUS81 foci in U2OS cells treated with CPT for 1 h. Data represent the means ± SDs, n = 3. See also Figure S4.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Nucleic Acid Electrophoresis, Software, Activity Assay, Stable Transfection, Transfection, Construct, MANN-WHITNEY, Western Blot, Over Expression, Inhibition, Expressing

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 5. Restart of R-Loop-Stalled Forks Depends on the Catalytic Activity of the LIG4/XRCC4 Complex (A) Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. (B) Effect of the depletion of the indicated proteins on replication fork progression in U2OS cells upon treatment with 100 nM CPT, and on the rescue of CPT-induced replication fork slowing by PARPi (10 mM olaparib). Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of the values of the IdU:CldU tract length ratio obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann- Whitney test). (C) Effect of depletion of the indicated proteins on replication restart following the exposure of U2OS cells to CPT (100 nM) or PDS (10 mM). Top panel: experimental workflow of DNA fiber assays. Bottom panel: quantification of replication fork stalling events performed as in Figure 1G. Data represent the means ± SDs, n = 3. (D) LIG4/XRCC4 is required for mitotic DNA synthesis (MiDAS) in U2OS cells. MiDAS assay was performed as depicted in Figure S6B. The data points represent the number of EdU incorporation events per metaphase spread (n R 150). Horizontal lines represent the means ± SEMs. ns, not significant; ****p < 0.0001 (Mann- Whitney test). (E) Effect of CPT and PDS on replication fork progression in human fibroblasts expressing WT (LIG4WT, 1BR) or catalytically inactive (LIG4MUT, 411 BR) forms of LIG4, with or without PARPi. DNA fiber assays were performed as in Figure 3E. (F) The levels of spontaneous (DMSO) and CPT-induced DNA breakage in U2OS cells depleted for the indicated proteins. Cells were treated with 1 mM CPT for 5 h. Genomic DNA was analyzed as in Figure 3A. Data represent the means ± SDs, n = 4. (G) Effect of the depletion of LIG4 and RAD52 on the sensitivity of WT and MUS81 knockout (KO) HeLa Kyoto cells to PARPi, as determined by clonogenic assay. Data represent the means ± SDs, n = 3. Also see Figure S6.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Activity Assay, Western Blot, Transfection, MANN-WHITNEY, DNA Synthesis, Expressing, Knock-Out, Clonogenic Assay

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 6. Restart of R-Loop-Stalled Forks Requires Reactivation of Transcription (A) Effect of DRB (100 mM) and CORD (50 mM) on replication restart following the treatment of U2OS cells with 100 nM CPT or 10 mM PDS. Top panel: experimental workflow of DNA fiber assays. Bottom panel: quantification of replication fork stalling events performed as in Figure 1G. Data represent the means ± SDs, n = 3. (B) Depletion of MUS81, LIG4, or ELL impairs the resumption of transcription following the exposure of U2OS cells to CPT. Nascent RNA strand production was quantified using 5-ethynyl uridine (EU; 1 mM) labeling during a 30-min treatment of cells with 100 nM CPT (or DMSO, control) and during a subsequent 30-min chase with CPT-free medium. The data represent the mean intensity of the EU signal in the nucleus. Horizontal lines represent median (n > 1,800). ****p < 0.0001 (Mann-Whitney test). (C) Effect of ELL depletion on replication restart following the treatment of U2OS cells with CPT (100 nM) or PDS (10 mM). Replication restart was quantified as in (A). (D) Effect of ELL depletion on replication fork progression in U2OS cells upon treatment with 100 nM CPT or 10 mM PDS, and on the rescue of CPT- or PDS- induced replication fork slowing by PARPi (10 mM olaparib). Top panel: experimental workflow of DNA fiber assay. Bottom panel: boxplot of the values of the IdU:CldU tract length obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann- Whitney test). (E) Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. (F) The levels of spontaneous (DMSO) and CPT-induced DNA breakage in U2OS cells depleted for the indicated proteins. Cells were treated with 1 mM CPT for 5 h. Genomic DNA was analyzed as in Figure 3A. Data represent the means ± SDs, n = 3. (G) Effect of ELL depletion on the sensitivity of WT and MUS81 KO HeLa Kyoto cells to PARPi as determined by clonogenic assay. Data represent the means ± SDs, n = 3. See also Figure S7.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Labeling, Control, MANN-WHITNEY, Western Blot, Transfection, Clonogenic Assay

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 7. Model for the Resolution of R-Loop-Mediated TRCs The blockage of replication fork progression by an oncoming transcription complex results from the buildup of positive supercoiling within the intervening DNA region and the formation of an R-loop. Replication fork stalling leads to the assembly of RAD51 filament at the fork junction, which promotes fork reversal in conjunction with the DNA translocase ZRANB3. RECQ1 DNA helicase counteracts replication fork reversal to promote replication restart. In this pathway, RECQ5 DNA helicase disrupts the RAD51 filament on the stalled fork to facilitate fork cleavage by MUS81/EME1 endonuclease. This relieves the topological barrier in the DNA template, allowing ELL-mediated tran- scription restart. After re-annealing of the parental strands by RAD52 and sealing the nick in the parental duplex by the LIG4/XRCC4 complex, the re- activated transcription complex bypasses the replication-stalling site. This is followed by the POLD3-mediated restart of semiconservative DNA replication. It is assumed that after fork stalling, the replicative helicase CMG traverses the fork junction onto dsDNA via its ssDNA gate (not shown). After fork religation, CMG translocates back onto ssDNA to nucleate a functional replisome.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Functional Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 1. Alteration of multidrug resistance protein 2 (MRP2) and P- glycoprotein (P-gp)/MDR1 protein and mRNA levels in HepG2 in re- sponse to interleukin-1 (IL-1) or tumor necrosis factor- (TNF) administration. (A) For Western blot analysis, 200 g of protein was loaded, and blots were incubated with an antibody, M2III-6 for MRP2 and C219 for P-gp/MDR1. (B,C) Northern blot analysis. (B) HepG2 cells were treated with the indicated dose of IL-1 (left) or TNF (right) for 24 hours. (C) HepG2 cells were treated with 20 ng/mL IL-1 (left) or TNF (right) for the indicated time. Ten micrograms total RNA was loaded and hybridized with MRP2 cDNA. The results were representative of three experiments.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Western Blot, Incubation, Northern Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 2. Extracellular signal-regulated kinase (ERK) pathways were involved in interleukin-1 (IL-1)-induced MRP2 downregulation. 20 M PD98059 (MEK1/2 inhibitor), 25 M SB203580 (p38 inhibitor), or 20 M SP600125 (JNK inhibitor) were pretreated for 30 minutes before IL-1 stimulation (20 ng/mL) in HepG2 cells. After 24 hours, total RNA were harvested and hybridized with multidrug resistance protein 2 (MRP2) cDNA. Relative MRP2 mRNA expression levels are shown, com- pared with the expression in untreated cells. This result was representa- tive of three experiments.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 3. Rates of MRP2 mRNA decay. 5 g/mL actinomycin D was incubated for 30 minutes with HepG2 cells, and interleukin-1 (IL-1; 20 ng/mL) was administrated for the indicated time. Total RNA was harvested, and 10 g of RNA was loaded on a 1% formaldehyde- agarose gel and transferred to a membrane. This result was representa- tive of three experiments.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Incubation, Agarose Gel Electrophoresis, Membrane

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 4. Maps of the MRP2 promoter luciferase constructs and the transcriptional activity of multidrug resistance protein 2 (MRP2) promot- er-luciferase fusion plasmids transfected into HepG2 cells. Various lengths of human MRP2 gene promoter plasmids containing the lucif- erase gene in the upstream region were constructed. Putative transcrip- tion factor binding sites on the MRP2 promoter are also indicated. Luciferase activities were measured after 24 hours and were correlated for differences in transfection efficiency by Renilla luciferase activity, then normalized to the activity of the p-491 MRP2-Luci construct transfected into cells. Data are shown as the mean SD (error bars) of three independent experiments. *P .01. ISRE, interferon stimulatory re- sponse element; USF, upstream stimulatory factor; HNF-1, hepatic nu- clear factor 1.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Luciferase, Construct, Activity Assay, Transfection, Binding Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 5. Transcriptional activity of p-491 MRP2-Luci and 4 p-491 MRP2 mutant-Luci (interferon stimulatory response element [ISRE], C/EBP, hepatic nuclear factor 1 [HNF1], and upstream stimulatory factor [USF]) transfected into HepG2 cells with or without 20 ng/mL of interleukin-1 (IL-1) for 24 hours. Various mutant forms of human MRP2 gene promoter plasmids containing the luciferase gene in the upstream region were constructed. Luciferase activities were measured after 24 hours and were corrected for differences in transfection effi- ciency by Renilla luciferase activity, then normalized to the activity of the p-491 MRP2-Luci construct transfected into cells. This result was repre- sentative of three experiments. *P .01. MRP2, multidrug resistance protein 2.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Activity Assay, Mutagenesis, Transfection, Luciferase, Construct

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 6. Effects of PD98059 on interleukin-1 (IL-1)-induced MRP2 promoter suppression. p-491MRP2-Luci and p-491MRP2(ISREmt)-Luci were transfected into HepG2 cells. Six hours later, 20 M PD98059 was administrated for 30 minutes and IL-1 was treated (20 ng/mL; 24 hours). This result was representative of three experiments. *P .05. MRP2, multidrug resistance protein 2; ISRE, interferon stimulatory re- sponse element.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Transfection

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 7. Binding of IRF3 to MRP2 promoter interferon stimulatory response elements (ISREs). Electrophoretic mobility shift assay (EMSA) was performed using radiolabeled probes, including interferon stimulatory response element (ISRE; 171/159, 5-GCAGCAGAAGCGAAACTGCAC-3). For the competition assay, the unlabeled ISRE mutant probe (179/153: 5-GCAGCAcgtGCGcgtCTGCA-3) was used as a competitor. (A) Effects of interleukin-1 (IL-1) on nuclear protein-ISRE binding. Cells were incubated with 20 ng/mL IL-1 for 0 to 8 hours. The nuclear extracts were prepared, and the extracts (4 g of protein) that were incubated with 32P-labeled oligonucleotide were resolved by gel electrophoresis. (B) Effects of PD98059 on IL-1-induced MRP2 downregulation. Before IL-1 stimulation, 20 M PD98059 was administrated for 30 minutes. (C) Super shift and competition assay using nuclear extract with or without IL-1 for 8 hours. Anti-IRF1-4, 7-9, Sp1, and RXR antibodies were added to the nuclear extracts. A 50-fold excess of the unlabeled oligonucleotide was added for the competition. (D) EMSA using nuclear extract from normal human hepatocytes (hNHeps). A bracket (]) indicates the DNA protein complex. These results were representative of three experiments. IRF, interferon regulatory factor; RXR, retinoid X receptor; MRP2, multidrug resistance protein 2.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Competitive Binding Assay, Mutagenesis, Incubation, Labeling, Nucleic Acid Electrophoresis

Journal: Hepatology (Baltimore, Md.)

Article Title: Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells.

doi: 10.1002/hep.20216

Figure Lengend Snippet: Fig. 9. Alteration of MRP2 promoter-luciferase activity by expression of rIRF3 in HepG2 cells. Cells were transiently transfected with each construct, pRL-TK, and Flag or Flag-IRF3 for 24 hours and assayed for luciferase activity. Luciferase activities were corrected for differences in transfection efficiency by Renilla luciferase activity and were normalized to the activity of the pGL3 basic vector transfected into cells. Each dose of Flag or Flag-IRF3 was transiently transfected. This result was repre- sentative of three experiments. *P .01. IRF3, interferon regulatory factor 3.

Article Snippet: The antibodies used in these experiments are as follows: MRP2 (M2III-6; Alexis, San Diego, CA), P-gp (C219; Centacor, Malvern, PA), IRF3 (FL425; Santa Cruz Biotech, Santa Cruz, CA), high mobility group protein-I (T-16; Santa Cruz Biotech), and glucose-6-phosphate dehydrogenase (A9521; Sigma-Aldrich, Saint Louis, MO).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, Construct, Plasmid Preparation

Journal: Kidney international

Article Title: Ubiquitin-dependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury.

doi: 10.1038/sj.ki.5000261

Figure Lengend Snippet: Figure 5 | Immunoblot analysis and effects of Smurf2 immunodepletion on ubiquitination activity against SnoN and Ski. (a) Western blot analysis demonstrated that Smurf2 was weakly expressed in sham-operated kidneys. In UUO, the increases of Smurf2 were noted in almost inverse proportion to the levels of SnoN. (b) Renal extracts from obstructed or sham-operated kidneys (input) were pre-incubated with anti-Smurf2 antibody and protein G–Sepharose. After centrifugation, the remnant amounts of Smurf2 in the supernatants were checked by immunoblotting using anti-Smurf2 antibody (aSmurf2), and then subjected to the following assay. As a control, we used renal extracts that had been pre-incubated with rabbit immunoglobulin G (aIgG). (c, left panel) In obstructed kidneys, significant smeared bands were observed when HA-tagged SnoN was incubated with the control extracts that had not been immunodepleted of Smurf2 (; lanes 3 and 4); however, the bands were much weaker when HA-tagged SnoN was incubated with Smurf2-immunodepleted extracts ( þ ; lanes 5 and 6). In sham-operated kidneys, no significant bands were detected (lanes 1 and 2). (Right panel) We also performed a similar ubiquitination assay for Ski. No notable differences in the intensity of the bands were observed between the control (; lanes 9 and 10) and the Smurf2-immunodepleted extracts ( þ ; lanes 11 and 12) in obstructed kidneys.

Article Snippet: Equal amounts of proteins (40 mg) were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously.29–31 The primary antibodies were goat anti-human SnoN (Santa Cruz Biotechnology), rabbit anti-human c-Ski (Santa Cruz Biotechnology), rabbit antihuman Smurf2 (sc-25511; Santa Cruz Biotechnology), and mouse monoclonal anti-b-actin (Sigma, St Louis, MO, USA). b-Actin was used as an internal control.

Techniques: Western Blot, Immunodepletion, Ubiquitin Proteomics, Activity Assay, Incubation, Centrifugation, Control