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etiology p34  (ATCC)
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ATCC etiology p34
Etiology P34, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ntda ntdc ntdb rbda t31 p34 s35 s37 s38 w90 f95 n77 f75 e1 loop phev apo hku1a apo down hku1a apo active hku1a sia  (Thermo Fisher)
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Ntda Ntdc Ntdb Rbda T31 P34 S35 S37 S38 W90 F95 N77 F75 E1 Loop Phev Apo Hku1a Apo Down Hku1a Apo Active Hku1a Sia, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti-p34-arc/arpc2  (Millipore)
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Rabbit Anti P34 Arc/Arpc2, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti-p34-arc/arpc2 07–227  (Millipore)
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The Arp2/3-vinculin hybrid complex is required for pseudopod extension in 3D (A and B) Vinculin and kindlin-2, but not FAK knockout U2OS cells have spreading defects in 3D. Cells embedded in collagen gels were stained for F-actin, imaged and quantified as described in . PEI is normalized to wild-type control. Data are shown as mean ± s.d. Kruskal-Wallis test: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001 (C) Direct Arp2/3-binding by vinculin is required for vinculin function during pseudopod extension in 3D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt), Arp3-binding mutant (Δ-Arp3) or Actin-binding mutants (Δ-IA or Δ-RE) in full-length vinculin. PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. Seven data points are outside the y axis limit, and the full graph is presented in E. Data are shown as mean ± s.d. Kruskal-Wallis test: ns = not significant, ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001. (D) Arp2/3-binding by vinculin is not essential for vinculin function during pseudopod extension in 2D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt) or indicated vinculin mutants (Arp3-binding mutant (Δ-Arp3), Actin-binding mutants (Δ-IA or Δ-RE)). PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. All vinculin constructs except the Δ-RE mutant were able to restore pseudopod formation of the vinculin KO cells comparable to that of wild-type U2OS cells. All data were collected from two to five independent experiments (N) and are shown as mean ± s.d. Each data point represents the median of the PEI of all cells within one image field of view (n) and normalized to the mean of the ev-distribution. Total number of quantified individual cells are indicated in brackets, but not used to compute any statistics. Kruskal-Wallis test: ns = not significant, ∗ p < 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. (E) Hybrid Arp2/3-vinculin complex formation is not abrogated in 2D. <t>p34-ARC-vinculin</t> PLA density was detected in cells seeded on collagen-coated glass coverslips, background subtracted (see ) and normalized to wild-type cells (wt). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗ p < 0.05, ∗∗∗∗ p ≤ 0.0001. (F) Formation of the hybrid Arp2/3-vinculin complex is sensitive to the extracellular environment. p34-ARC-vinculin PLA densities were analyzed as in (E) but from cells seeded on soft substrate (∼8 kPa). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.
Rabbit Anti P34 Arc/Arpc2 07–227, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cdk1  (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology cdk1
Gene silencing of cyclin B1 and cyclin B2. (A) Conditional gene silencing strategy for mitotic cyclin B. CRISPR-Cas9 was used to disrupt the loci of both endogenous cyclin B1 and cyclin B2. The cDNA of cyclin B1 was tagged with mAID and put inside a Sleeping Beauty transposon cassette for genome delivery to rescue the KO effects. Silent mutations were introduced into mAID cyclin B1 to confer resistance to the CRISPR-Cas9. In the presence of Dox, transcription of mAID cyclin B1 is inhibited by blocking the tetracycline-controlled transcriptional activator (tTA) from binding to the TRE in the promoter. The addition of IAA triggers the degradation of residual mAID cyclin B1 in cells expressing the F-box protein TIR1. ITR: inverted terminal repeat. (B) Gene silencing of cyclin B1 and/or cyclin B2. HeLa cells were engineered to stably express mAID cyclin B1, tTA, and TIR1. CRISPR–Cas9 was used to disrupt cyclin B1 (in mAID B1 KO B1) or both cyclin B1 and B2 (in mAID B1 KO B1B2). Single-colony–derived clones were isolated and cultured with or without DI for 8 h. Lysates were prepared and analyzed with immunoblotting. Lysates from parental HeLa and cyclin B2 KO cells ( KO B2) were included as controls. Equal loading of lysates was confirmed by immunoblotting for actin. (C) Enhanced formation of cyclin <t>B2–CDK1</t> and cyclin A–CDK1 complexes in the absence of cyclin B1. mAID B1 KO B1 cells synchronized with a double thymidine block were cultured with or without DI and harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation with an antibody against CDK1. Both total lysates and immunoprecipitates were analyzed with immunoblotting. (D) Indel analysis of cyclin B1 and cyclin B2. The endogenous cyclin B1 ( CCNB1 ) and cyclin B2 ( CCNB2 ) loci in mAID B1 KO B1B2 cells were analyzed with sequencing. Sequencing traces of control (HeLa) and the edited samples were generated for indel analysis. The targeted sequence of the gRNA (solid black line), PAM sequence (dotted red line), and edited site (dotted black line) are indicated. Discordance, calculated by ICE, is shown for the edited (green) and control (orange) traces. The alignment window indicates the region of the traces with high Phred quality scores used for alignment. The inference window indicates the altered sequences around the edited site (dotted black line). Indel and corresponding prevalence were determined using ICE, with editing efficiencies of 90% for cyclin B1 and 95% for cyclin B2. (E) Efficiency of cyclin B1 silencing in mAID B1 KO B1B2 cells. After treatment with DI for 6 h, lysates were prepared and analyzed with immunoblotting. Lysates from HeLa cells were included to serve as a reference for the expression level of endogenous cyclin B1. The signals corresponding to mAID cyclin B1 were quantified using a standard curve based on serial dilutions of mAID B1 KO B1B2 cell lysates (lanes 2–10), showing that <1% of mAID cyclin B1 remained after DI treatment. (F) DI treatment does not affect the overall cell cycle distribution. HeLa cells were treated with DI for 24 h, pulsed with BrdU for 30 min, and analyzed using bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The positions of 2N and 4N DNA content are indicated. The percentage of cells at different cell cycle stage (excluding BrdU-negative S) was quantified. Mean and SEM from three independent experiments. (G) DI treatment does not affect cell cycle progression. Parental HeLa cells were treated with DI and analyzed using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown. Source data are available for this figure: .
Cdk1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse anti cdk1  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology mouse anti cdk1
Gene silencing of cyclin B1 and cyclin B2. (A) Conditional gene silencing strategy for mitotic cyclin B. CRISPR-Cas9 was used to disrupt the loci of both endogenous cyclin B1 and cyclin B2. The cDNA of cyclin B1 was tagged with mAID and put inside a Sleeping Beauty transposon cassette for genome delivery to rescue the KO effects. Silent mutations were introduced into mAID cyclin B1 to confer resistance to the CRISPR-Cas9. In the presence of Dox, transcription of mAID cyclin B1 is inhibited by blocking the tetracycline-controlled transcriptional activator (tTA) from binding to the TRE in the promoter. The addition of IAA triggers the degradation of residual mAID cyclin B1 in cells expressing the F-box protein TIR1. ITR: inverted terminal repeat. (B) Gene silencing of cyclin B1 and/or cyclin B2. HeLa cells were engineered to stably express mAID cyclin B1, tTA, and TIR1. CRISPR–Cas9 was used to disrupt cyclin B1 (in mAID B1 KO B1) or both cyclin B1 and B2 (in mAID B1 KO B1B2). Single-colony–derived clones were isolated and cultured with or without DI for 8 h. Lysates were prepared and analyzed with immunoblotting. Lysates from parental HeLa and cyclin B2 KO cells ( KO B2) were included as controls. Equal loading of lysates was confirmed by immunoblotting for actin. (C) Enhanced formation of cyclin <t>B2–CDK1</t> and cyclin A–CDK1 complexes in the absence of cyclin B1. mAID B1 KO B1 cells synchronized with a double thymidine block were cultured with or without DI and harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation with an antibody against CDK1. Both total lysates and immunoprecipitates were analyzed with immunoblotting. (D) Indel analysis of cyclin B1 and cyclin B2. The endogenous cyclin B1 ( CCNB1 ) and cyclin B2 ( CCNB2 ) loci in mAID B1 KO B1B2 cells were analyzed with sequencing. Sequencing traces of control (HeLa) and the edited samples were generated for indel analysis. The targeted sequence of the gRNA (solid black line), PAM sequence (dotted red line), and edited site (dotted black line) are indicated. Discordance, calculated by ICE, is shown for the edited (green) and control (orange) traces. The alignment window indicates the region of the traces with high Phred quality scores used for alignment. The inference window indicates the altered sequences around the edited site (dotted black line). Indel and corresponding prevalence were determined using ICE, with editing efficiencies of 90% for cyclin B1 and 95% for cyclin B2. (E) Efficiency of cyclin B1 silencing in mAID B1 KO B1B2 cells. After treatment with DI for 6 h, lysates were prepared and analyzed with immunoblotting. Lysates from HeLa cells were included to serve as a reference for the expression level of endogenous cyclin B1. The signals corresponding to mAID cyclin B1 were quantified using a standard curve based on serial dilutions of mAID B1 KO B1B2 cell lysates (lanes 2–10), showing that <1% of mAID cyclin B1 remained after DI treatment. (F) DI treatment does not affect the overall cell cycle distribution. HeLa cells were treated with DI for 24 h, pulsed with BrdU for 30 min, and analyzed using bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The positions of 2N and 4N DNA content are indicated. The percentage of cells at different cell cycle stage (excluding BrdU-negative S) was quantified. Mean and SEM from three independent experiments. (G) DI treatment does not affect cell cycle progression. Parental HeLa cells were treated with DI and analyzed using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown. Source data are available for this figure: .
Mouse Anti Cdk1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cdc2 p34 mouse igg2a kappa monoclonal  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology cdc2 p34 mouse igg2a kappa monoclonal
Gene silencing of cyclin B1 and cyclin B2. (A) Conditional gene silencing strategy for mitotic cyclin B. CRISPR-Cas9 was used to disrupt the loci of both endogenous cyclin B1 and cyclin B2. The cDNA of cyclin B1 was tagged with mAID and put inside a Sleeping Beauty transposon cassette for genome delivery to rescue the KO effects. Silent mutations were introduced into mAID cyclin B1 to confer resistance to the CRISPR-Cas9. In the presence of Dox, transcription of mAID cyclin B1 is inhibited by blocking the tetracycline-controlled transcriptional activator (tTA) from binding to the TRE in the promoter. The addition of IAA triggers the degradation of residual mAID cyclin B1 in cells expressing the F-box protein TIR1. ITR: inverted terminal repeat. (B) Gene silencing of cyclin B1 and/or cyclin B2. HeLa cells were engineered to stably express mAID cyclin B1, tTA, and TIR1. CRISPR–Cas9 was used to disrupt cyclin B1 (in mAID B1 KO B1) or both cyclin B1 and B2 (in mAID B1 KO B1B2). Single-colony–derived clones were isolated and cultured with or without DI for 8 h. Lysates were prepared and analyzed with immunoblotting. Lysates from parental HeLa and cyclin B2 KO cells ( KO B2) were included as controls. Equal loading of lysates was confirmed by immunoblotting for actin. (C) Enhanced formation of cyclin <t>B2–CDK1</t> and cyclin A–CDK1 complexes in the absence of cyclin B1. mAID B1 KO B1 cells synchronized with a double thymidine block were cultured with or without DI and harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation with an antibody against CDK1. Both total lysates and immunoprecipitates were analyzed with immunoblotting. (D) Indel analysis of cyclin B1 and cyclin B2. The endogenous cyclin B1 ( CCNB1 ) and cyclin B2 ( CCNB2 ) loci in mAID B1 KO B1B2 cells were analyzed with sequencing. Sequencing traces of control (HeLa) and the edited samples were generated for indel analysis. The targeted sequence of the gRNA (solid black line), PAM sequence (dotted red line), and edited site (dotted black line) are indicated. Discordance, calculated by ICE, is shown for the edited (green) and control (orange) traces. The alignment window indicates the region of the traces with high Phred quality scores used for alignment. The inference window indicates the altered sequences around the edited site (dotted black line). Indel and corresponding prevalence were determined using ICE, with editing efficiencies of 90% for cyclin B1 and 95% for cyclin B2. (E) Efficiency of cyclin B1 silencing in mAID B1 KO B1B2 cells. After treatment with DI for 6 h, lysates were prepared and analyzed with immunoblotting. Lysates from HeLa cells were included to serve as a reference for the expression level of endogenous cyclin B1. The signals corresponding to mAID cyclin B1 were quantified using a standard curve based on serial dilutions of mAID B1 KO B1B2 cell lysates (lanes 2–10), showing that <1% of mAID cyclin B1 remained after DI treatment. (F) DI treatment does not affect the overall cell cycle distribution. HeLa cells were treated with DI for 24 h, pulsed with BrdU for 30 min, and analyzed using bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The positions of 2N and 4N DNA content are indicated. The percentage of cells at different cell cycle stage (excluding BrdU-negative S) was quantified. Mean and SEM from three independent experiments. (G) DI treatment does not affect cell cycle progression. Parental HeLa cells were treated with DI and analyzed using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown. Source data are available for this figure: .
Cdc2 P34 Mouse Igg2a Kappa Monoclonal, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse cdc2 p34 sc-54 antibody  (Santa Cruz Biotechnology)
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Gene silencing of cyclin B1 and cyclin B2. (A) Conditional gene silencing strategy for mitotic cyclin B. CRISPR-Cas9 was used to disrupt the loci of both endogenous cyclin B1 and cyclin B2. The cDNA of cyclin B1 was tagged with mAID and put inside a Sleeping Beauty transposon cassette for genome delivery to rescue the KO effects. Silent mutations were introduced into mAID cyclin B1 to confer resistance to the CRISPR-Cas9. In the presence of Dox, transcription of mAID cyclin B1 is inhibited by blocking the tetracycline-controlled transcriptional activator (tTA) from binding to the TRE in the promoter. The addition of IAA triggers the degradation of residual mAID cyclin B1 in cells expressing the F-box protein TIR1. ITR: inverted terminal repeat. (B) Gene silencing of cyclin B1 and/or cyclin B2. HeLa cells were engineered to stably express mAID cyclin B1, tTA, and TIR1. CRISPR–Cas9 was used to disrupt cyclin B1 (in mAID B1 KO B1) or both cyclin B1 and B2 (in mAID B1 KO B1B2). Single-colony–derived clones were isolated and cultured with or without DI for 8 h. Lysates were prepared and analyzed with immunoblotting. Lysates from parental HeLa and cyclin B2 KO cells ( KO B2) were included as controls. Equal loading of lysates was confirmed by immunoblotting for actin. (C) Enhanced formation of cyclin <t>B2–CDK1</t> and cyclin A–CDK1 complexes in the absence of cyclin B1. mAID B1 KO B1 cells synchronized with a double thymidine block were cultured with or without DI and harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation with an antibody against CDK1. Both total lysates and immunoprecipitates were analyzed with immunoblotting. (D) Indel analysis of cyclin B1 and cyclin B2. The endogenous cyclin B1 ( CCNB1 ) and cyclin B2 ( CCNB2 ) loci in mAID B1 KO B1B2 cells were analyzed with sequencing. Sequencing traces of control (HeLa) and the edited samples were generated for indel analysis. The targeted sequence of the gRNA (solid black line), PAM sequence (dotted red line), and edited site (dotted black line) are indicated. Discordance, calculated by ICE, is shown for the edited (green) and control (orange) traces. The alignment window indicates the region of the traces with high Phred quality scores used for alignment. The inference window indicates the altered sequences around the edited site (dotted black line). Indel and corresponding prevalence were determined using ICE, with editing efficiencies of 90% for cyclin B1 and 95% for cyclin B2. (E) Efficiency of cyclin B1 silencing in mAID B1 KO B1B2 cells. After treatment with DI for 6 h, lysates were prepared and analyzed with immunoblotting. Lysates from HeLa cells were included to serve as a reference for the expression level of endogenous cyclin B1. The signals corresponding to mAID cyclin B1 were quantified using a standard curve based on serial dilutions of mAID B1 KO B1B2 cell lysates (lanes 2–10), showing that <1% of mAID cyclin B1 remained after DI treatment. (F) DI treatment does not affect the overall cell cycle distribution. HeLa cells were treated with DI for 24 h, pulsed with BrdU for 30 min, and analyzed using bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The positions of 2N and 4N DNA content are indicated. The percentage of cells at different cell cycle stage (excluding BrdU-negative S) was quantified. Mean and SEM from three independent experiments. (G) DI treatment does not affect cell cycle progression. Parental HeLa cells were treated with DI and analyzed using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown. Source data are available for this figure: .
Mouse Cdc2 P34 Sc 54 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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The Arp2/3-vinculin hybrid complex is required for pseudopod extension in 3D (A and B) Vinculin and kindlin-2, but not FAK knockout U2OS cells have spreading defects in 3D. Cells embedded in collagen gels were stained for F-actin, imaged and quantified as described in . PEI is normalized to wild-type control. Data are shown as mean ± s.d. Kruskal-Wallis test: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001 (C) Direct Arp2/3-binding by vinculin is required for vinculin function during pseudopod extension in 3D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt), Arp3-binding mutant (Δ-Arp3) or Actin-binding mutants (Δ-IA or Δ-RE) in full-length vinculin. PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. Seven data points are outside the y axis limit, and the full graph is presented in E. Data are shown as mean ± s.d. Kruskal-Wallis test: ns = not significant, ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001. (D) Arp2/3-binding by vinculin is not essential for vinculin function during pseudopod extension in 2D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt) or indicated vinculin mutants (Arp3-binding mutant (Δ-Arp3), Actin-binding mutants (Δ-IA or Δ-RE)). PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. All vinculin constructs except the Δ-RE mutant were able to restore pseudopod formation of the vinculin KO cells comparable to that of wild-type U2OS cells. All data were collected from two to five independent experiments (N) and are shown as mean ± s.d. Each data point represents the median of the PEI of all cells within one image field of view (n) and normalized to the mean of the ev-distribution. Total number of quantified individual cells are indicated in brackets, but not used to compute any statistics. Kruskal-Wallis test: ns = not significant, ∗ p < 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. (E) Hybrid Arp2/3-vinculin complex formation is not abrogated in 2D. p34-ARC-vinculin PLA density was detected in cells seeded on collagen-coated glass coverslips, background subtracted (see ) and normalized to wild-type cells (wt). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗ p < 0.05, ∗∗∗∗ p ≤ 0.0001. (F) Formation of the hybrid Arp2/3-vinculin complex is sensitive to the extracellular environment. p34-ARC-vinculin PLA densities were analyzed as in (E) but from cells seeded on soft substrate (∼8 kPa). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Journal: iScience

Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D

doi: 10.1016/j.isci.2025.112623

Figure Lengend Snippet: The Arp2/3-vinculin hybrid complex is required for pseudopod extension in 3D (A and B) Vinculin and kindlin-2, but not FAK knockout U2OS cells have spreading defects in 3D. Cells embedded in collagen gels were stained for F-actin, imaged and quantified as described in . PEI is normalized to wild-type control. Data are shown as mean ± s.d. Kruskal-Wallis test: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001 (C) Direct Arp2/3-binding by vinculin is required for vinculin function during pseudopod extension in 3D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt), Arp3-binding mutant (Δ-Arp3) or Actin-binding mutants (Δ-IA or Δ-RE) in full-length vinculin. PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. Seven data points are outside the y axis limit, and the full graph is presented in E. Data are shown as mean ± s.d. Kruskal-Wallis test: ns = not significant, ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001. (D) Arp2/3-binding by vinculin is not essential for vinculin function during pseudopod extension in 2D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt) or indicated vinculin mutants (Arp3-binding mutant (Δ-Arp3), Actin-binding mutants (Δ-IA or Δ-RE)). PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. All vinculin constructs except the Δ-RE mutant were able to restore pseudopod formation of the vinculin KO cells comparable to that of wild-type U2OS cells. All data were collected from two to five independent experiments (N) and are shown as mean ± s.d. Each data point represents the median of the PEI of all cells within one image field of view (n) and normalized to the mean of the ev-distribution. Total number of quantified individual cells are indicated in brackets, but not used to compute any statistics. Kruskal-Wallis test: ns = not significant, ∗ p < 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. (E) Hybrid Arp2/3-vinculin complex formation is not abrogated in 2D. p34-ARC-vinculin PLA density was detected in cells seeded on collagen-coated glass coverslips, background subtracted (see ) and normalized to wild-type cells (wt). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗ p < 0.05, ∗∗∗∗ p ≤ 0.0001. (F) Formation of the hybrid Arp2/3-vinculin complex is sensitive to the extracellular environment. p34-ARC-vinculin PLA densities were analyzed as in (E) but from cells seeded on soft substrate (∼8 kPa). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Subsequently, the samples were stained with rabbit anti-p34-ARC/ARPC2 (Millipore; 07–227; 1:250) and mouse anti-vinculin (Sigma-Aldrich; V9264; 1:500) for 1 h at RT, followed by 1 h RT incubation with the Duolink PLA probes, 30 min ligation and 200 min amplification at 37°C in a humidified chamber.

Techniques: Knock-Out, Staining, Control, Binding Assay, Plasmid Preparation, Mutagenesis, Construct

Gene silencing of cyclin B1 and cyclin B2. (A) Conditional gene silencing strategy for mitotic cyclin B. CRISPR-Cas9 was used to disrupt the loci of both endogenous cyclin B1 and cyclin B2. The cDNA of cyclin B1 was tagged with mAID and put inside a Sleeping Beauty transposon cassette for genome delivery to rescue the KO effects. Silent mutations were introduced into mAID cyclin B1 to confer resistance to the CRISPR-Cas9. In the presence of Dox, transcription of mAID cyclin B1 is inhibited by blocking the tetracycline-controlled transcriptional activator (tTA) from binding to the TRE in the promoter. The addition of IAA triggers the degradation of residual mAID cyclin B1 in cells expressing the F-box protein TIR1. ITR: inverted terminal repeat. (B) Gene silencing of cyclin B1 and/or cyclin B2. HeLa cells were engineered to stably express mAID cyclin B1, tTA, and TIR1. CRISPR–Cas9 was used to disrupt cyclin B1 (in mAID B1 KO B1) or both cyclin B1 and B2 (in mAID B1 KO B1B2). Single-colony–derived clones were isolated and cultured with or without DI for 8 h. Lysates were prepared and analyzed with immunoblotting. Lysates from parental HeLa and cyclin B2 KO cells ( KO B2) were included as controls. Equal loading of lysates was confirmed by immunoblotting for actin. (C) Enhanced formation of cyclin B2–CDK1 and cyclin A–CDK1 complexes in the absence of cyclin B1. mAID B1 KO B1 cells synchronized with a double thymidine block were cultured with or without DI and harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation with an antibody against CDK1. Both total lysates and immunoprecipitates were analyzed with immunoblotting. (D) Indel analysis of cyclin B1 and cyclin B2. The endogenous cyclin B1 ( CCNB1 ) and cyclin B2 ( CCNB2 ) loci in mAID B1 KO B1B2 cells were analyzed with sequencing. Sequencing traces of control (HeLa) and the edited samples were generated for indel analysis. The targeted sequence of the gRNA (solid black line), PAM sequence (dotted red line), and edited site (dotted black line) are indicated. Discordance, calculated by ICE, is shown for the edited (green) and control (orange) traces. The alignment window indicates the region of the traces with high Phred quality scores used for alignment. The inference window indicates the altered sequences around the edited site (dotted black line). Indel and corresponding prevalence were determined using ICE, with editing efficiencies of 90% for cyclin B1 and 95% for cyclin B2. (E) Efficiency of cyclin B1 silencing in mAID B1 KO B1B2 cells. After treatment with DI for 6 h, lysates were prepared and analyzed with immunoblotting. Lysates from HeLa cells were included to serve as a reference for the expression level of endogenous cyclin B1. The signals corresponding to mAID cyclin B1 were quantified using a standard curve based on serial dilutions of mAID B1 KO B1B2 cell lysates (lanes 2–10), showing that <1% of mAID cyclin B1 remained after DI treatment. (F) DI treatment does not affect the overall cell cycle distribution. HeLa cells were treated with DI for 24 h, pulsed with BrdU for 30 min, and analyzed using bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The positions of 2N and 4N DNA content are indicated. The percentage of cells at different cell cycle stage (excluding BrdU-negative S) was quantified. Mean and SEM from three independent experiments. (G) DI treatment does not affect cell cycle progression. Parental HeLa cells were treated with DI and analyzed using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Gene silencing of cyclin B1 and cyclin B2. (A) Conditional gene silencing strategy for mitotic cyclin B. CRISPR-Cas9 was used to disrupt the loci of both endogenous cyclin B1 and cyclin B2. The cDNA of cyclin B1 was tagged with mAID and put inside a Sleeping Beauty transposon cassette for genome delivery to rescue the KO effects. Silent mutations were introduced into mAID cyclin B1 to confer resistance to the CRISPR-Cas9. In the presence of Dox, transcription of mAID cyclin B1 is inhibited by blocking the tetracycline-controlled transcriptional activator (tTA) from binding to the TRE in the promoter. The addition of IAA triggers the degradation of residual mAID cyclin B1 in cells expressing the F-box protein TIR1. ITR: inverted terminal repeat. (B) Gene silencing of cyclin B1 and/or cyclin B2. HeLa cells were engineered to stably express mAID cyclin B1, tTA, and TIR1. CRISPR–Cas9 was used to disrupt cyclin B1 (in mAID B1 KO B1) or both cyclin B1 and B2 (in mAID B1 KO B1B2). Single-colony–derived clones were isolated and cultured with or without DI for 8 h. Lysates were prepared and analyzed with immunoblotting. Lysates from parental HeLa and cyclin B2 KO cells ( KO B2) were included as controls. Equal loading of lysates was confirmed by immunoblotting for actin. (C) Enhanced formation of cyclin B2–CDK1 and cyclin A–CDK1 complexes in the absence of cyclin B1. mAID B1 KO B1 cells synchronized with a double thymidine block were cultured with or without DI and harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation with an antibody against CDK1. Both total lysates and immunoprecipitates were analyzed with immunoblotting. (D) Indel analysis of cyclin B1 and cyclin B2. The endogenous cyclin B1 ( CCNB1 ) and cyclin B2 ( CCNB2 ) loci in mAID B1 KO B1B2 cells were analyzed with sequencing. Sequencing traces of control (HeLa) and the edited samples were generated for indel analysis. The targeted sequence of the gRNA (solid black line), PAM sequence (dotted red line), and edited site (dotted black line) are indicated. Discordance, calculated by ICE, is shown for the edited (green) and control (orange) traces. The alignment window indicates the region of the traces with high Phred quality scores used for alignment. The inference window indicates the altered sequences around the edited site (dotted black line). Indel and corresponding prevalence were determined using ICE, with editing efficiencies of 90% for cyclin B1 and 95% for cyclin B2. (E) Efficiency of cyclin B1 silencing in mAID B1 KO B1B2 cells. After treatment with DI for 6 h, lysates were prepared and analyzed with immunoblotting. Lysates from HeLa cells were included to serve as a reference for the expression level of endogenous cyclin B1. The signals corresponding to mAID cyclin B1 were quantified using a standard curve based on serial dilutions of mAID B1 KO B1B2 cell lysates (lanes 2–10), showing that <1% of mAID cyclin B1 remained after DI treatment. (F) DI treatment does not affect the overall cell cycle distribution. HeLa cells were treated with DI for 24 h, pulsed with BrdU for 30 min, and analyzed using bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The positions of 2N and 4N DNA content are indicated. The percentage of cells at different cell cycle stage (excluding BrdU-negative S) was quantified. Mean and SEM from three independent experiments. (G) DI treatment does not affect cell cycle progression. Parental HeLa cells were treated with DI and analyzed using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown. Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: CRISPR, Blocking Assay, Binding Assay, Expressing, Stable Transfection, Derivative Assay, Clone Assay, Isolation, Cell Culture, Western Blot, Immunoprecipitation, Sequencing, Control, Generated, Flow Cytometry, Live Cell Imaging

Conditional depletion of cyclin B induces defective mitotic entry and mitotic slippage. (A) DI treatment of control HeLa cells. HeLa cells expressing histone H2B-GFP were synchronized using a double thymidine block and released into a drug-free or DI-containing medium for 6 h before time-lapse imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); and cell death (truncated bars). The plots show the cumulative percentage of cells entering mitosis over time and the elapsed time between mitotic entry and exit. (B) Defective mitosis in the absence of cyclin B does not involve SAC activation. mAID B1 KO B1B2 cells were cultured in a drug-free or DI-containing medium with or without the MPS1 inhibitor AZ3146. After 6 h, individual cells were tracked using live-cell imaging. Key: interphase (grey); mitosis (red); cell death (truncated bars); pre-NEBD mitosis (blue), and interphase after pre-NEBD slippage (green). (C) Silencing of cyclin B prevents APC/C activation. mAID B1 KO B1B2 cells expressing histone H2B-GFP were transfected with an mRFP APC/C biosensor plasmid. The cells were cultured in drug-free or DI-containing medium and analyzed using live-cell imaging. Representative images show normal mitosis and abnormal mitosis without APC/C activation. Time: h:min. Scale bar: 10 µm. (D) Loss of cyclin B1 alone does not abolish mitotic entry and exit. mAID B1 KO B1 cells were synchronized using double thymidine block and released into a drug-free or DI-containing medium. The cells were harvested at the indicated time points for immunoblotting analysis. (E) CDK1 substrate phosphorylation in the absence of cyclin B1. Samples from D were subjected to immunoblotting using an antibody against phosphorylated CDK1 substrates (pTPxK). The positions of bands affected by cyclin B depletion are as described in . Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Conditional depletion of cyclin B induces defective mitotic entry and mitotic slippage. (A) DI treatment of control HeLa cells. HeLa cells expressing histone H2B-GFP were synchronized using a double thymidine block and released into a drug-free or DI-containing medium for 6 h before time-lapse imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); and cell death (truncated bars). The plots show the cumulative percentage of cells entering mitosis over time and the elapsed time between mitotic entry and exit. (B) Defective mitosis in the absence of cyclin B does not involve SAC activation. mAID B1 KO B1B2 cells were cultured in a drug-free or DI-containing medium with or without the MPS1 inhibitor AZ3146. After 6 h, individual cells were tracked using live-cell imaging. Key: interphase (grey); mitosis (red); cell death (truncated bars); pre-NEBD mitosis (blue), and interphase after pre-NEBD slippage (green). (C) Silencing of cyclin B prevents APC/C activation. mAID B1 KO B1B2 cells expressing histone H2B-GFP were transfected with an mRFP APC/C biosensor plasmid. The cells were cultured in drug-free or DI-containing medium and analyzed using live-cell imaging. Representative images show normal mitosis and abnormal mitosis without APC/C activation. Time: h:min. Scale bar: 10 µm. (D) Loss of cyclin B1 alone does not abolish mitotic entry and exit. mAID B1 KO B1 cells were synchronized using double thymidine block and released into a drug-free or DI-containing medium. The cells were harvested at the indicated time points for immunoblotting analysis. (E) CDK1 substrate phosphorylation in the absence of cyclin B1. Samples from D were subjected to immunoblotting using an antibody against phosphorylated CDK1 substrates (pTPxK). The positions of bands affected by cyclin B depletion are as described in . Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Control, Expressing, Blocking Assay, Imaging, Activation Assay, Cell Culture, Live Cell Imaging, Transfection, Plasmid Preparation, Western Blot, Phospho-proteomics

Loss of cyclin B uncouples the normal regulation of the G 2 -M kinase network. (A) Dysregulated phosphorylation and expression of G 2 –M regulators in the absence of cyclin B. mAID B1 KO B1B2 cells synchronized using double thymidine block were released into a drug-free or DI-containing medium and harvested at different time points for immunoblotting. The positions of the three isoforms of Aurora kinases are indicated. (B) Dysregulation of CDK1 substrate phosphorylation in the absence of cyclin B. Samples prepared from A were immunoblotted with an antibody against CDK1 phosphorylation substrates (pTPxK). The positions of bands that are absent in DI-treated cells are indicated with asterisks. Bands present in both DI-treated and untreated cells but lacking cell cycle variation in DI-treated samples are indicated with circles. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Loss of cyclin B uncouples the normal regulation of the G 2 -M kinase network. (A) Dysregulated phosphorylation and expression of G 2 –M regulators in the absence of cyclin B. mAID B1 KO B1B2 cells synchronized using double thymidine block were released into a drug-free or DI-containing medium and harvested at different time points for immunoblotting. The positions of the three isoforms of Aurora kinases are indicated. (B) Dysregulation of CDK1 substrate phosphorylation in the absence of cyclin B. Samples prepared from A were immunoblotted with an antibody against CDK1 phosphorylation substrates (pTPxK). The positions of bands that are absent in DI-treated cells are indicated with asterisks. Bands present in both DI-treated and untreated cells but lacking cell cycle variation in DI-treated samples are indicated with circles. Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Phospho-proteomics, Expressing, Blocking Assay, Western Blot

Cyclin A drives residual mitotic activity in the absence of cyclin B. (A) Enhanced formation of cyclin A–CDK1 complexes without cyclin B. mAID B1 KO B1B2 cells synchronized with double thymidine block were released into a drug-free or DI-containing medium and harvested at different time points. Lysates were prepared and subjected to immunoprecipitation with a CDK1 antibody. Both total lysates and immunoprecipitates (IP) were analyzed with immunoblotting. (B) Contribution of cyclin A to CDK1 substrate phosphorylation in the absence of cyclin B. mAID B1 KO B1B2 cells transfected with control siRNA (siControl) or siRNA targeting cyclin A (siCyclin A) were synchronized using double thymidine block. The cells were released into a drug-free or DI-containing medium and harvested at different time points for immunoblotting. The positions of pTPxK bands affected by cyclin B depletion are indicated as described in . (C) Depletion of cyclin A does not affect cyclin B expression. Cell lines transfected with control siRNA or siRNA targeting cyclin A were left untreated or treated with DI for 24 h. Lysates were prepared and analyzed with immunoblotting. (D) Suppression of mitotic entry in cyclin B-deficient cells upon cyclin A knockdown. Cell lines transfected with control siRNA or siRNA targeting cyclin A were analyzed using live-cell imaging after treatment with DI. The cumulative percentage of cells entering mitosis over time is shown. Note that DI-treated mAID B1 KO B1B2 cells exhibited pre-NEBD slippage (*) instead of normal mitosis. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Cyclin A drives residual mitotic activity in the absence of cyclin B. (A) Enhanced formation of cyclin A–CDK1 complexes without cyclin B. mAID B1 KO B1B2 cells synchronized with double thymidine block were released into a drug-free or DI-containing medium and harvested at different time points. Lysates were prepared and subjected to immunoprecipitation with a CDK1 antibody. Both total lysates and immunoprecipitates (IP) were analyzed with immunoblotting. (B) Contribution of cyclin A to CDK1 substrate phosphorylation in the absence of cyclin B. mAID B1 KO B1B2 cells transfected with control siRNA (siControl) or siRNA targeting cyclin A (siCyclin A) were synchronized using double thymidine block. The cells were released into a drug-free or DI-containing medium and harvested at different time points for immunoblotting. The positions of pTPxK bands affected by cyclin B depletion are indicated as described in . (C) Depletion of cyclin A does not affect cyclin B expression. Cell lines transfected with control siRNA or siRNA targeting cyclin A were left untreated or treated with DI for 24 h. Lysates were prepared and analyzed with immunoblotting. (D) Suppression of mitotic entry in cyclin B-deficient cells upon cyclin A knockdown. Cell lines transfected with control siRNA or siRNA targeting cyclin A were analyzed using live-cell imaging after treatment with DI. The cumulative percentage of cells entering mitosis over time is shown. Note that DI-treated mAID B1 KO B1B2 cells exhibited pre-NEBD slippage (*) instead of normal mitosis. Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Activity Assay, Blocking Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Transfection, Control, Expressing, Knockdown, Live Cell Imaging

Rescue of cell cycle defects caused by cyclin B deficiency with cyclin B1 and cyclin A. (A) Expression of cyclin B1-YFP in cyclin B-deficient cells. mAID B1 KO B1B2 cells were transfected with a control plasmid or plasmids expressing YFP or cyclin B1-YFP. At 16 h after transfection, the cells were treated with DI and harvested at different time points. Lysates were prepared and analyzed with immunoblotting. (B) Ectopic expression of cyclin B1 rescues cell cycle defects induced by cyclin B deficiency. mAID B1 KO B1B2 cells transfected with plasmids expressing YFP or cyclin B1-YFP were treated with DI and harvested at the indicated time points for flow cytometry analysis. The DNA profiles of transfected YFP-positive cells are shown. (C) Overexpression of cyclin A in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing either mRFP or cyclin A-mRFP were treated with DI and harvested at specific time points. Lysates were prepared and analyzed with immunoblotting. (D) Overexpression of CDK1 and CDK2 in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing mRFP, CDK1-mRFP, or CDK2-mRFP were treated with DI and harvested at specific time points for immunoblotting analysis. (E) Ectopic expression of cyclin A promotes DNA re-replication in cyclin B-deficient cells. mAID B1 KO B1B2 cells were transfected with plasmids expressing mRFP or mRFP-tagged CDK1, CDK2, or cyclin A, followed by treatment with buffer or DI for 48 h. The cells were harvested and analyzed with flow cytometry. The DNA profiles of transfected mRFP-positive cells are shown. The asterisk indicates the population containing >4N DNA content. (F) Doubling cyclin A expression is insufficient to restore normal mitosis in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing “low” level of cyclin A and “high” level of cyclin A (see ) were treated and imaged as described in . Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. (G) Localization of cyclin A-mRFP to both nucleus and cytoplasm during interphase. mAID B1 KO B1B2 cells expressing “high” level of cyclin A were treated and imaged as described in . Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Rescue of cell cycle defects caused by cyclin B deficiency with cyclin B1 and cyclin A. (A) Expression of cyclin B1-YFP in cyclin B-deficient cells. mAID B1 KO B1B2 cells were transfected with a control plasmid or plasmids expressing YFP or cyclin B1-YFP. At 16 h after transfection, the cells were treated with DI and harvested at different time points. Lysates were prepared and analyzed with immunoblotting. (B) Ectopic expression of cyclin B1 rescues cell cycle defects induced by cyclin B deficiency. mAID B1 KO B1B2 cells transfected with plasmids expressing YFP or cyclin B1-YFP were treated with DI and harvested at the indicated time points for flow cytometry analysis. The DNA profiles of transfected YFP-positive cells are shown. (C) Overexpression of cyclin A in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing either mRFP or cyclin A-mRFP were treated with DI and harvested at specific time points. Lysates were prepared and analyzed with immunoblotting. (D) Overexpression of CDK1 and CDK2 in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing mRFP, CDK1-mRFP, or CDK2-mRFP were treated with DI and harvested at specific time points for immunoblotting analysis. (E) Ectopic expression of cyclin A promotes DNA re-replication in cyclin B-deficient cells. mAID B1 KO B1B2 cells were transfected with plasmids expressing mRFP or mRFP-tagged CDK1, CDK2, or cyclin A, followed by treatment with buffer or DI for 48 h. The cells were harvested and analyzed with flow cytometry. The DNA profiles of transfected mRFP-positive cells are shown. The asterisk indicates the population containing >4N DNA content. (F) Doubling cyclin A expression is insufficient to restore normal mitosis in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing “low” level of cyclin A and “high” level of cyclin A (see ) were treated and imaged as described in . Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. (G) Localization of cyclin A-mRFP to both nucleus and cytoplasm during interphase. mAID B1 KO B1B2 cells expressing “high” level of cyclin A were treated and imaged as described in . Representative images show mitosis in the presence and absence of DI. Time: h:min. Scale bar: 10 µm. Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Western Blot, Flow Cytometry, Over Expression

Mitotic defects caused by cyclin B deficiency can be compensated by cyclin A overexpression. (A) Ectopic expression of cyclin A in cyclin B-deficient cells. mAID B1 KO B1B2 cells were transfected with mRFP cyclin A expression plasmids. Cells with varying levels of mRFP cyclin A were sorted by flow cytometry. The cell lines were left untreated or treated with DI for the indicated time before analyzed with immunoblotting. (B) Rescue of cyclin B deficiency-induced G 2 /M arrest by cyclin A overexpression. Parental and mAID B1 KO B1B2 cells expressing low or high levels of mRFP cyclin A were treated with DI to turn off mAID cyclin B1. The cells were harvested at the indicated time points and analyzed using flow cytometry. (C) Cyclin A rescues clonogenic survival in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing high levels of mRFP cyclin A were cultured with or without DI for 2 wk. Colonies were fixed and stained. Mean ± SEM from six independent experiments. Mann–Whitney test: **P < 0.01; *P < 0.05. Note that the same data for the mAID B1 KO B1B2 control cells as in were used for comparison. (D) Cyclin B deficiency-induced pre-NEBD slippage can be overcome by cyclin A overexpression. Cells expressing histone H2B-GFP were synchronized using a double thymidine block and released into a drug-free or DI-containing medium. After 6 h, individual cells were tracked using live-cell imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); cell death (truncated bars); interphase after cytokinesis failure (purple); pre-NEBD mitosis (blue), and interphase after pre-NEBD slippage (green). The plot shows the percentage of defective mitosis (mitotic slippage and cytokinesis failure). Mean ± SEM from three independent experiments. (E) Overexpression of cyclin A overcomes mitotic entry delay in cyclin B-deficient cells. Cell lines were synchronized, released into drug-free or DI-containing medium, and analyzed with live-cell imaging as described in D. The cumulative percentage of cells entering mitosis (both normal and pre-NEBD mitosis) over time is shown. Mean ± SEM from three independent experiments. Mann–Whitney test: ****P < 0.0001; ***P < 0.001; ns P > 0.05. Note that the same graph from is included for clarity for control mAID B1 KO B1B2 cells. (F) Ectopic expression of cyclin A restores mitosis in cyclin B-deficient cells. Parental and cyclin A-overexpressing mAID B1 KO B1B2 cells were synchronized using a double thymidine block. The cells were released into a drug-free or DI-containing medium and harvested at different time points. Protein expression was analyzed with immunoblotting. The positions of pTPxK bands affected by cyclin B depletion are indicated as described in . (G) Increased cyclin A–CDK1/2 complexes in the absence of cyclin B. Parental and cyclin A-overexpressing mAID B1 KO B1B2 cells were grown in drug-free or DI-containing medium for 24 h. Lysates were prepared and subjected to immunoprecipitation using an antibody against cyclin A. Both total lysates and immunoprecipitates (IP) were analyzed with immunoblotting. (H) Increased binding of both endogenous and mRFP cyclin A to CDK1 upon the loss of cyclin B. Parental and cyclin A-overexpressing mAID B1 KO B1B2 cells were synchronized using double thymidine block as described in F. Lysates were prepared and subjected to immunoprecipitation using an antibody against CDK1. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Mitotic defects caused by cyclin B deficiency can be compensated by cyclin A overexpression. (A) Ectopic expression of cyclin A in cyclin B-deficient cells. mAID B1 KO B1B2 cells were transfected with mRFP cyclin A expression plasmids. Cells with varying levels of mRFP cyclin A were sorted by flow cytometry. The cell lines were left untreated or treated with DI for the indicated time before analyzed with immunoblotting. (B) Rescue of cyclin B deficiency-induced G 2 /M arrest by cyclin A overexpression. Parental and mAID B1 KO B1B2 cells expressing low or high levels of mRFP cyclin A were treated with DI to turn off mAID cyclin B1. The cells were harvested at the indicated time points and analyzed using flow cytometry. (C) Cyclin A rescues clonogenic survival in cyclin B-deficient cells. mAID B1 KO B1B2 cells expressing high levels of mRFP cyclin A were cultured with or without DI for 2 wk. Colonies were fixed and stained. Mean ± SEM from six independent experiments. Mann–Whitney test: **P < 0.01; *P < 0.05. Note that the same data for the mAID B1 KO B1B2 control cells as in were used for comparison. (D) Cyclin B deficiency-induced pre-NEBD slippage can be overcome by cyclin A overexpression. Cells expressing histone H2B-GFP were synchronized using a double thymidine block and released into a drug-free or DI-containing medium. After 6 h, individual cells were tracked using live-cell imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); cell death (truncated bars); interphase after cytokinesis failure (purple); pre-NEBD mitosis (blue), and interphase after pre-NEBD slippage (green). The plot shows the percentage of defective mitosis (mitotic slippage and cytokinesis failure). Mean ± SEM from three independent experiments. (E) Overexpression of cyclin A overcomes mitotic entry delay in cyclin B-deficient cells. Cell lines were synchronized, released into drug-free or DI-containing medium, and analyzed with live-cell imaging as described in D. The cumulative percentage of cells entering mitosis (both normal and pre-NEBD mitosis) over time is shown. Mean ± SEM from three independent experiments. Mann–Whitney test: ****P < 0.0001; ***P < 0.001; ns P > 0.05. Note that the same graph from is included for clarity for control mAID B1 KO B1B2 cells. (F) Ectopic expression of cyclin A restores mitosis in cyclin B-deficient cells. Parental and cyclin A-overexpressing mAID B1 KO B1B2 cells were synchronized using a double thymidine block. The cells were released into a drug-free or DI-containing medium and harvested at different time points. Protein expression was analyzed with immunoblotting. The positions of pTPxK bands affected by cyclin B depletion are indicated as described in . (G) Increased cyclin A–CDK1/2 complexes in the absence of cyclin B. Parental and cyclin A-overexpressing mAID B1 KO B1B2 cells were grown in drug-free or DI-containing medium for 24 h. Lysates were prepared and subjected to immunoprecipitation using an antibody against cyclin A. Both total lysates and immunoprecipitates (IP) were analyzed with immunoblotting. (H) Increased binding of both endogenous and mRFP cyclin A to CDK1 upon the loss of cyclin B. Parental and cyclin A-overexpressing mAID B1 KO B1B2 cells were synchronized using double thymidine block as described in F. Lysates were prepared and subjected to immunoprecipitation using an antibody against CDK1. Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Over Expression, Expressing, Transfection, Flow Cytometry, Western Blot, Cell Culture, Staining, MANN-WHITNEY, Control, Comparison, Blocking Assay, Live Cell Imaging, Immunoprecipitation, Binding Assay

Cyclin B1 overexpression overcomes cyclin A KO -mediated G 2 -M delay. (A) Conditional gene silencing of cyclin A. HeLa cells expressing AID cyclin A without endogenous cyclin A were generated ( AID A KO A). CDK2 was further disrupted in AID A KO A cells using CRISPR-Cas9. The cells were either left untreated or treated with DI for the indicated time for immunoblotting analysis. (B) Codepletion of cyclin A and CDK2 leads to extensive G 2 /M delay. HeLa or AID A KO A cells with or without CDK2 were either left untreated or treated with DI for 24 h. Cell cycle distribution was analyzed with flow cytometry. (C) Loss of cyclin A results in cell cycle delay in both S and G 2 /M. Cells were treated with DI for 24 h and pulsed with BrdU (30 min) before being analyzed with bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The percentage of cells at different cell cycle stages (excluding BrdU-negative S) was quantified (mean and SEM from three independent experiments). (D) Significant cell cycle delay in the absence of cyclin A and CDK2. Cells were preincubated with DI for 6 h to deplete cyclin A before individual cells were tracked using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown (raw data for individual cells are presented in ). ****P < 0.0001. (E) Loss of cyclin A–CDK2 delays cell cycle progression. Cells were subjected to live-cell imaging analysis as described in D. Box-and-whisker plots show the elapsed time between the end of the first mitosis to the end of the second mitosis. ****P < 0.0001; ***P < 0.001; *P < 0.05. (F) Cyclin A is an essential gene in HeLa cells. AID A KO A and AID A KO A KO CDK2 cells were cultured with or without DI. After 2 wk, the cells were fixed, stained with crystal violet, and the number of colonies was quantified. Representative images and Mean ± SEM from four independent experiments are shown. ****P < 0.0001. (G) Simultaneous cyclin A and CDK2 depletion during G 2 -M. Cells were synchronized using double thymidine block, released into drug-free or DI-containing medium, and harvested at different time points for immunoblotting analysis (upper panel). DNA content was analyzed using flow cytometry (lower panel). (H) Alleviation of cyclin A KO -induced G 2 -M delay by cyclin B1 requires CDK2. A stable cell line expressing YFP-tagged cyclin B1 (B1-YFP) was established from AID A KO A cells. CDK2 was further disrupted to obtain AID A KO A KO CDK2 cells expressing B1-YFP. Following double thymidine synchronization, cells were left untreated or treated with DI for 7 h to before analyzed using time-lapse imaging. Separate plates of cells were harvested at 3 h after the start of live-cell imaging for immunoblotting analysis to confirm protein expression. Raw data for individual cells are presented in . ****P < 0.0001; **P < 0.01. (I) Enhanced interaction between cyclin B1 and CDK2 in the absence of cyclin A. AID A KO A cells, with or without ectopically expressed cyclin B1, were synchronized in G 2 using a double thymidine block. Lysates were prepared and subjected to immunoprecipitation using antibodies against CDK1 or CDK2. The total lysates and immunoprecipitates (IP) were analyzed using immunoblotting. Note that the PSTAIRE antibody recognizes both CDK1 and CDK2. The band intensities of cyclin B1-YFP in the IP were quantified and normalized to -DI (mean ± SEM from three independent experiments). Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Cyclin B1 overexpression overcomes cyclin A KO -mediated G 2 -M delay. (A) Conditional gene silencing of cyclin A. HeLa cells expressing AID cyclin A without endogenous cyclin A were generated ( AID A KO A). CDK2 was further disrupted in AID A KO A cells using CRISPR-Cas9. The cells were either left untreated or treated with DI for the indicated time for immunoblotting analysis. (B) Codepletion of cyclin A and CDK2 leads to extensive G 2 /M delay. HeLa or AID A KO A cells with or without CDK2 were either left untreated or treated with DI for 24 h. Cell cycle distribution was analyzed with flow cytometry. (C) Loss of cyclin A results in cell cycle delay in both S and G 2 /M. Cells were treated with DI for 24 h and pulsed with BrdU (30 min) before being analyzed with bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The percentage of cells at different cell cycle stages (excluding BrdU-negative S) was quantified (mean and SEM from three independent experiments). (D) Significant cell cycle delay in the absence of cyclin A and CDK2. Cells were preincubated with DI for 6 h to deplete cyclin A before individual cells were tracked using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown (raw data for individual cells are presented in ). ****P < 0.0001. (E) Loss of cyclin A–CDK2 delays cell cycle progression. Cells were subjected to live-cell imaging analysis as described in D. Box-and-whisker plots show the elapsed time between the end of the first mitosis to the end of the second mitosis. ****P < 0.0001; ***P < 0.001; *P < 0.05. (F) Cyclin A is an essential gene in HeLa cells. AID A KO A and AID A KO A KO CDK2 cells were cultured with or without DI. After 2 wk, the cells were fixed, stained with crystal violet, and the number of colonies was quantified. Representative images and Mean ± SEM from four independent experiments are shown. ****P < 0.0001. (G) Simultaneous cyclin A and CDK2 depletion during G 2 -M. Cells were synchronized using double thymidine block, released into drug-free or DI-containing medium, and harvested at different time points for immunoblotting analysis (upper panel). DNA content was analyzed using flow cytometry (lower panel). (H) Alleviation of cyclin A KO -induced G 2 -M delay by cyclin B1 requires CDK2. A stable cell line expressing YFP-tagged cyclin B1 (B1-YFP) was established from AID A KO A cells. CDK2 was further disrupted to obtain AID A KO A KO CDK2 cells expressing B1-YFP. Following double thymidine synchronization, cells were left untreated or treated with DI for 7 h to before analyzed using time-lapse imaging. Separate plates of cells were harvested at 3 h after the start of live-cell imaging for immunoblotting analysis to confirm protein expression. Raw data for individual cells are presented in . ****P < 0.0001; **P < 0.01. (I) Enhanced interaction between cyclin B1 and CDK2 in the absence of cyclin A. AID A KO A cells, with or without ectopically expressed cyclin B1, were synchronized in G 2 using a double thymidine block. Lysates were prepared and subjected to immunoprecipitation using antibodies against CDK1 or CDK2. The total lysates and immunoprecipitates (IP) were analyzed using immunoblotting. Note that the PSTAIRE antibody recognizes both CDK1 and CDK2. The band intensities of cyclin B1-YFP in the IP were quantified and normalized to -DI (mean ± SEM from three independent experiments). Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Over Expression, Expressing, Generated, CRISPR, Western Blot, Flow Cytometry, Live Cell Imaging, Whisker Assay, Cell Culture, Staining, Blocking Assay, Stable Transfection, Imaging, Immunoprecipitation

Delayed mitotic entry in the absence of cyclin A and CDK2. (A) Mitotic entry is significantly delayed in AID A KO A and AID A KO A KO CDK2 cells. Cells were pre-incubated with DI for 6 h to deplete cyclin A, followed by live-cell imaging for 48 h ( n = 50). Key: interphase (grey); mitosis (red); and cell death (truncated bars). (B) Minimal delay in S phase progression upon cyclin A silencing. AID A KO A cells were synchronized at S phase with double thymidine block and treated with DI at the second thymidine release to deplete AID cyclin A. The cells were pulsed with BrdU for 30 min before harvested at each time points for immunoblotting analysis. The band intensity of AID cyclin A was quantified and normalized to -DI control at t = 0 h. BrdU incorporation and DNA content were examined with bivariate flow cytometry (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The percentages of S (BrdU-positive) and G 2 cells were quantified (mean ± SEM from four independent experiments). (C) Alleviation of cyclin A KO -induced G 2 -M delay by cyclin B1. AID A KO A and AID A KO A KO CDK2 cells overexpressing cyclin B1 synchronized with double thymidine block were left untreated or treated with DI for 7 h to turn off cyclin A before time-lapse imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); and cell death (truncated bars). (D) Conditional gene silencing of cyclin A in H1299 cells. HeLa and H1299 cells expressing AID cyclin A without endogenous cyclin A were left untreated or treated with DI for 24 h before immunoblotting analysis. Lysates from HeLa and H1299 cells were included as controls for the relative expression of AID cyclin A and endogenous cyclin A. (E) Cyclin A depletion induces G 2 -M delay in both HeLa and H1299 cells. AID A KO A cells were synchronized using a double thymidine block and released into a drug-free or DI-containing medium. After 6 h, mitotic entry was analyzed using live-cell imaging (left panel; time indicates the duration after thymidine release). Raw data for individual cells are presented in the right panel. Key: interphase (grey); mitosis (red); and cell death (truncated bars). (F) Presence of cyclin B1–CDK2 complexes during normal G 2 . HeLa cells were synchronized at S phase with double thymidine block. After release into fresh medium for 5 h, NOC was added to prevent mitotic exit. After 4 h, mitotic cells were removed by washing, and the attached G 2 cells were harvested for immunoprecipitation using antibodies against CDK1, CDK2, or cyclin A. Protein expression in the total lysates and immunoprecipitates (IP) was detected using immunoblotting. A negative control (no antibody was added) was included to assess the specificity of the immunoprecipitation. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Delayed mitotic entry in the absence of cyclin A and CDK2. (A) Mitotic entry is significantly delayed in AID A KO A and AID A KO A KO CDK2 cells. Cells were pre-incubated with DI for 6 h to deplete cyclin A, followed by live-cell imaging for 48 h ( n = 50). Key: interphase (grey); mitosis (red); and cell death (truncated bars). (B) Minimal delay in S phase progression upon cyclin A silencing. AID A KO A cells were synchronized at S phase with double thymidine block and treated with DI at the second thymidine release to deplete AID cyclin A. The cells were pulsed with BrdU for 30 min before harvested at each time points for immunoblotting analysis. The band intensity of AID cyclin A was quantified and normalized to -DI control at t = 0 h. BrdU incorporation and DNA content were examined with bivariate flow cytometry (red: BrdU-positive; yellow: BrdU-negative S; blue: G 1 ; green: G 2 /M). The percentages of S (BrdU-positive) and G 2 cells were quantified (mean ± SEM from four independent experiments). (C) Alleviation of cyclin A KO -induced G 2 -M delay by cyclin B1. AID A KO A and AID A KO A KO CDK2 cells overexpressing cyclin B1 synchronized with double thymidine block were left untreated or treated with DI for 7 h to turn off cyclin A before time-lapse imaging. Time indicates the duration after thymidine release. Key: interphase (grey); mitosis (red); and cell death (truncated bars). (D) Conditional gene silencing of cyclin A in H1299 cells. HeLa and H1299 cells expressing AID cyclin A without endogenous cyclin A were left untreated or treated with DI for 24 h before immunoblotting analysis. Lysates from HeLa and H1299 cells were included as controls for the relative expression of AID cyclin A and endogenous cyclin A. (E) Cyclin A depletion induces G 2 -M delay in both HeLa and H1299 cells. AID A KO A cells were synchronized using a double thymidine block and released into a drug-free or DI-containing medium. After 6 h, mitotic entry was analyzed using live-cell imaging (left panel; time indicates the duration after thymidine release). Raw data for individual cells are presented in the right panel. Key: interphase (grey); mitosis (red); and cell death (truncated bars). (F) Presence of cyclin B1–CDK2 complexes during normal G 2 . HeLa cells were synchronized at S phase with double thymidine block. After release into fresh medium for 5 h, NOC was added to prevent mitotic exit. After 4 h, mitotic cells were removed by washing, and the attached G 2 cells were harvested for immunoprecipitation using antibodies against CDK1, CDK2, or cyclin A. Protein expression in the total lysates and immunoprecipitates (IP) was detected using immunoblotting. A negative control (no antibody was added) was included to assess the specificity of the immunoprecipitation. Source data are available for this figure: .

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Incubation, Live Cell Imaging, Blocking Assay, Western Blot, Control, BrdU Incorporation Assay, Flow Cytometry, Imaging, Expressing, Immunoprecipitation, Negative Control

Relative levels of intercellular and intracellular cyclins. (A) Plasticity of mitotic cyclins in human cell lines. During G 2 , cyclin B2 is more abundant than cyclin B1 and cyclin A (represented in the diagram by the length of individual bars). The absence of cyclin B1 results in a delay but not inhibition of G 2 –M. By contrast, depletion of both cyclin B1 and B2 severely curtailed mitotic entry, leading to cell rounding followed by pre-NEBD slippage. The pre-NEBD G 2 block can be rescued by overexpressing cyclin A (red bars). Similar to cyclin B1, cyclin A is rate-limiting for G 2 -M. The G 2 -M delay due to cyclin A deficiency can be rescued by overexpression of cyclin B1. Cells were blocked in G 2 in the absence of cyclin A and cyclin B1. (B) Interplay between cyclin A and cyclin B. In the absence of cyclin A, G 2 –M is delayed with an accompanied accumulation of cyclin B1–CDK1 and cyclin B1–CDK2 complexes. This delay can be alleviated by overexpression of cyclin B1 (see A). Conversely, the absence of cyclin B1 and B2 hinders proper mitotic entry, leading to pre-NEBD slippage, accompanied by an enrichment of cyclin A-CDK1 and cyclin A-CDK2. Overexpression of cyclin A effectively overcome the pre-NEBD defects.

Journal: The Journal of Cell Biology

Article Title: Plasticity of mitotic cyclins in promoting the G 2 –M transition

doi: 10.1083/jcb.202409219

Figure Lengend Snippet: Relative levels of intercellular and intracellular cyclins. (A) Plasticity of mitotic cyclins in human cell lines. During G 2 , cyclin B2 is more abundant than cyclin B1 and cyclin A (represented in the diagram by the length of individual bars). The absence of cyclin B1 results in a delay but not inhibition of G 2 –M. By contrast, depletion of both cyclin B1 and B2 severely curtailed mitotic entry, leading to cell rounding followed by pre-NEBD slippage. The pre-NEBD G 2 block can be rescued by overexpressing cyclin A (red bars). Similar to cyclin B1, cyclin A is rate-limiting for G 2 -M. The G 2 -M delay due to cyclin A deficiency can be rescued by overexpression of cyclin B1. Cells were blocked in G 2 in the absence of cyclin A and cyclin B1. (B) Interplay between cyclin A and cyclin B. In the absence of cyclin A, G 2 –M is delayed with an accompanied accumulation of cyclin B1–CDK1 and cyclin B1–CDK2 complexes. This delay can be alleviated by overexpression of cyclin B1 (see A). Conversely, the absence of cyclin B1 and B2 hinders proper mitotic entry, leading to pre-NEBD slippage, accompanied by an enrichment of cyclin A-CDK1 and cyclin A-CDK2. Overexpression of cyclin A effectively overcome the pre-NEBD defects.

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (Cat# A5316; RRID:AB_476743; Sigma-Aldrich), phospho-AURKA T288 /AURKB T232 /AURKC T198 (2914; Cell Signaling Technology; Cat# 2914; RRID:AB_2061631), CDK1 (Cat# sc-54; RRID:AB_627224; Santa Cruz Biotechnology), CDK1 (for immunoprecipitation; a custom-made polyclonal antibody raised against the peptide CHPYFNDLDNQIKKM; Genscript), CDK2 (Cat# sc-6248; RRID:AB_627238; Santa Cruz Biotechnology), phospho-CDK1/2 Y15 (Cat# 612307; RRID:AB_399622; BD Biosciences or Cat# ab76146; RRID:AB_1310069; Abcam), cyclin A2 (AT10; a gift from Tim Hunt, Cancer Research UK); cyclin B1 (Cat# sc-245; RRID:AB_627338; Santa Cruz Biotechnology or V152; a gift from Julian Gannon, Cancer Research UK), cyclin B2 (Cat# sc-22776; RRID:AB_2072392; Santa Cruz Biotechnology), cyclin E1 (Cat# sc-247; RRID:AB_627357; Santa Cruz Biotechnology), mAID (Cat# M214-3; MBL International; RRID:AB_2890014), phospho-CDK substrates (TPXK motif) (Cat# 14371; Cell Signaling Technology; RRID:AB_2798466), EMI1 (Cat# 37-6600; Zymed Laboratories; RRID:AB_2533333), phosphoryated histone H3 S10 (Cat# sc-8656R; Santa Cruz Biotechnology; RRID:AB_653256), phospho-lamin A/C S22 (S22-p) (Cat# 2026; Cell Signaling Technology; RRID:AB_2136155), cleaved PARP1 (Cat# 552597; BD Biosciences; RRID:AB_394438), PLK1 (Cat# sc-17783; Santa Cruz Biotechnology; RRID:AB_628157), phospho-PLK1 T210 (Cat# 9062; Cell Signaling Technology; RRID:AB_11127447), PSTAIRE (a gift from Masakane Yamashita, Hokkaido University, Sappoo, Japan), PTTG1 (Cat# sc-56207; Santa Cruz Biotechnology; RRID:AB_785382), TCTP (Cat# 5128; Santa Cruz Biotechnology; RRID:AB_11220419), and phospho-TCTP S46 (Cat# 5251; Cell Signaling Technology; RRID:AB_10547143).

Techniques: Inhibition, Blocking Assay, Over Expression