Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: PP4R3β S840 phosphorylation is required for 53BP1 recruitment to damaged chromatin. a MS/MS spectrum of the PP4R3β phospho-peptide harboring S840. Phosphorylated residue is highlighted in green. b Co-immunoprecipitation of 53BP1 or PP4C with PP4R3β S840 phospho-variants. HeLa cells transiently transfected with the indicated FLAG-53BP1 and Myc-PP4R3β constructs were left in an asynchronous state (Asyn) or synchronized to prometaphase (M) by treatment with nocodazole (Supplementary Fig. ). Whole cell extracts were immunoprecipitated (IP) with indicated agarose beads and analyzed by immunoblot using indicated antibodies. EV: empty vector; S/D: S840D; S/F: S840F; S/A: S840A. c Immunoblots showing the kinetics of 53BP1 phosphorylation throughout mitosis in cells expressing PP4R3β S840 phospho-variants . HeLa cells were transfected with the indicated siRNAs and complemented with the indicated siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs. Transfected cells were released from prometaphase arrest and harvested at indicated time points for immunoblot analysis using indicated antibodies. Phospho-histone H3 at Ser10 (H3pS10) indicates mitotic state. d Irradiation-induced 53BP1 foci formation in cells expressing PP4R3β S840 phospho-variants. Mitotic HeLa cells were complemented with Myc-PP4R3β S840 phospho-variant constructs as described in c , collected from prometaphase arrest and seeded on poly-D-lysine-coated coverslips. Four hours after release, cells were irradiated with 5 Gy. Two hours post-irradiation, cells were fixed and analyzed by immunofluorescence with indicated antibodies. γ-H2AX staining marks sites of DNA damage. e Quantifications of 53BP1 foci in d , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. f Radiosensitivity of cells expressing PP4R3β S840 phospho-variants. HeLa cells were complemented with siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs as described in c . Viability was evaluated by clonogenic survival. Immunoblots confirm siRNA efficiency and expression of siRNA-resistant constructs. Data are expressed as mean ± s.d; n = 3. g Response of BRCA1-null UWB1.289 cells expressing PP4R3β S840 phospho-variants to indicated concentration of clinical-grade PARP inhibitor Olaparib. Cells were complemented as described in c . Viability was assessed by CellTiter-Glo colorimetric assay. Immunoblots confirm siRNA efficiency and expression of siRNA-resistant constructs. Data are expressed as mean ± s.d; n = 3

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Phospho-proteomics, Tandem Mass Spectroscopy, Residue, Immunoprecipitation, Transfection, Construct, Western Blot, Plasmid Preparation, Expressing, Variant Assay, Irradiation, Immunofluorescence, Staining, MANN-WHITNEY, Concentration Assay, Colorimetric Assay

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: CDK5 is active in mitosis. a Phosphorylation of 53BP1 T1609/S1618 at distinct stages of mitosis and in G1. RPE1 cells were fixed at 0, 15, 30, 60, 90, and 120 min after release from G2/M arrest and stained with phospho-specific antibody against T1609/S1618 (pT1609/pS1618). Cells at indicated stages of mitosis were selected based on chromatin morphology, as indicated by DAPI staining. b Fluorescence intensity of phosphorylated 53BP1 over area of interest was quantified. Staining intensities from number of events per stage (indicated in the parenthesis) were expressed as mean ± s.d. A.U: arbitrary unit. c Phosphorylation of PP4R3β S840 at distinct stages of mitosis and in G1. RPE1 cells were synchronized and fixed at time points after release as described in a and stained with a phospho-specific antibody against S840 (pS840). d Fluorescence intensity of phosphorylated PP4R3β over area of interest was quantified as described in b . e Distribution of HeLa cells across distinct stages of mitosis at indicated time points after release from prometaphase arrest. HeLa cells were arrested at G2/M by RO-3306 and released into nocodazole to enrich for cells in prometaphase for 1 h. Prometaphase-arrested cells were collected by shake-off and released to progress through mitosis into G1 and fixed at indicated time points. Cells corresponding to stages of mitosis were tallied based on chromatin morphology, as indicated by DAPI staining. f Kinase activity levels of CDK5 in mitosis and G1. HeLa cells were synchronized and released as described in e . CDK5, immunoprecipitated from cells collected at indicated time points after release, was incubated with substrate peptide and [ 32 P]-ATP. Radioactivity of labeled substrate peptide was measured in a liquid scintillation counter. Data are expressed as mean ± s.d; n = 3. Representative immunoblots show indicated proteins present in the cell lysate for kinase assay. g Immunoblots showing the kinetics of 53BP1 T1609/S1618 phosphorylation and PP4R3β S840 phosphorylation throughout mitosis. HeLa cells were collected at indicated time points after release from synchronization as described in e and were analyzed with indicated antibodies

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Phospho-proteomics, Staining, Fluorescence, Activity Assay, Immunoprecipitation, Incubation, Radioactivity, Labeling, Western Blot, Kinase Assay

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: PP4R3β is a bona fide substrate of CDK5. a Reconstituted CDK5/p25 complex was incubated with purified wild-type or S840A PP4R3β. 32 P-ATP signal indicates CDK5/p25-mediated phosphorylation. Ponceau-stained blots show input proteins. b HeLa cells stably expressing FLAG-HA empty vector (EV) or FLAG-HA PP4R3β were arrested in prometaphase, lysed, and immunoprecipitated for FLAG-HA PP4R3β. Elution from FLAG immunoprecipitation was analyzed by immunoblot with indicated antibodies. c RPE1 cells released from RO-3306-induced G2/M arrest were immediately treated with DMSO or 10 µM of CDK5 inhibitor compound 20-223. One hour after release from RO-3306, prometaphase cells were collected by shake-off and seeded on poly-D-lysine-coated coverslips. Three hours after release, cells were irradiated with 5 Gy IR. Two hours post-irradiation, cells were fixed and stained with indicated antibodies to detect irradiation-induced 53BP1 foci. γ-H2AX staining marks sites of DNA damage. d Quantifications of 53BP1 foci in c , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. e Effect of CDK5 inhibition on radiosensitivity. Hela cells were treated with DMSO or 250 nM of 20-223 for 48 h, followed by irradiation at the indicated doses. Cell viability was evaluated by clonogenic survival. Data are expressed as mean ± s.d; n = 3. Immunoblots indicate abrogation of PP4R3β S840 phosphorylation from treatment with 20-223. f Response of BRCA1-null UWB1.289 cells treated with DMSO or 250 nM of 20-223 to indicated concentration of clinical-grade PARP inhibitor Olaparib. Cell viability was assessed by CellTiter-Glo assay. Data are expressed as mean ± s.d; n = 3. Immunoblots indicate abrogation of PP4R3β S840 phosphorylation from treatment with 20-223. g Melanoma A375 cells stably expressing CDK5 analog-sensitive variant (A375-AS) were arrested in prometaphase. Mitotic cells were released into media containing either DMSO or 10 µM of non-hydrolyzable ATP analog, 1NMPP1, to specifically inhibit CDK5. Cells were harvested at indicated time points for immunoblot analysis with indicated antibodies. h Quantification of immunoblot intensity in g , from duplicate repeat experiments, by ImageJ

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Incubation, Purification, Phospho-proteomics, Staining, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Irradiation, MANN-WHITNEY, Inhibition, Concentration Assay, Glo Assay, Variant Assay

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: CDK5 regulates dephosphorylation and the recruitment of 53BP1 to DNA damage. a Endogenous 53BP1 was immunoprecipitated from DMSO-treated or 1NMPP1-treated A375-AS cells and collected at indicated time points after release from arrest at prometaphase. Levels of co-immunoprecipitated endogenous PP4R3β were analyzed by immunoblot. b Endogenous 53BP1 was immunoprecipitated from DMSO-treated or 1NMPP1-treated A375-AS cells, collected at indicated time points, after release from nocodazole-induced arrest at prometaphase. Levels of 53BP1 T1609/s1618 phosphorylation from immunoprecipitated endogenous 53BP1 were analyzed by immunoblot. c Mitotic A375-AS cells, collected by shake-off from arrest at prometaphase, were immediately treated with DMSO or 10 µM of 1NMPP1 and seeded on poly-D-lysine-coated coverslips. 90 min after release, treated cells were irradiated with 5 Gy. 240 min after release, cells were fixed and stained with indicated antibodies to detect irradiation-induced 53BP1 foci. γ-H2AX was stained to mark sites of DNA damage. d Quantifications of 53BP1 foci in c , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. e A375-AS cells were exposed to 0.2 µM Aphidicolin and DMSO or 10 µM 1NMPP1 for 12 h. Cells were fixed and stained for 53BP1 and Cyclin-A, a negative marker for G1 cells. f Quantification of percentage of cells containing 53BP1-containing nuclear bodies in e out of total cells, indicated in parenthesis, pooled from triplicate repeat experiments. P -values, Mann–Whitney U Test

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Irradiation, Staining, MANN-WHITNEY, Marker

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: A CDK5-PP4 signaling axis drives 53BP1 recruitment to damaged chromatin. a A375-AS cells were depleted of endogenous PP4R3β by siRNA knockdown and transfected with indicated siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs. Transfected cells were synchronized to prometaphase. Mitotic cells collected by shake-off were immediately treated with DMSO or 10 µM of 1NMPP1 and seeded on poly-D-lysine–coated coverslips. Ninety minutes after release, treated cells were irradiated with 5 Gy. 240 min after release, cells were fixed and stained with indicated antibodies to detect irradiation-induced 53BP1 foci. γ-H2AX was stained to mark sites of DNA damage. b Quantifications of 53BP1 foci in a , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. c A375-AS cells were depleted of endogenous PP4R3β by siRNA knockdown and transfected with indicated siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs. The transfected cells were exposed to 0.2 µM Aphidicolin and DMSO or 10 µM 1NMPP1 for 12 h. Cells were fixed and stained for 53BP1 and Cyclin-A, a negative marker for G1 cells. d Quantification of percent cells containing 53BP1-containing nuclear bodies in c out of total cells (indicated in parenthesis) pooled from triplicate repeat experiments. P -values, Mann–Whitney U Test. e Model depicting CDK5 as a regulator of 53BP1 recruitment to damaged chromatin

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Knockdown, Transfection, Variant Assay, Construct, Irradiation, Staining, MANN-WHITNEY, Marker

Journal: bioRxiv

Article Title: Ubiquitin-dependent degradation of p21 Waf1/Cip1 is mediated by AMBRA1 to limit DNA replication stress

doi: 10.1101/2025.07.28.666126

Figure Lengend Snippet: a, ( Left panel ) Stacked barplot of UW228 in control condition constitutively downregulated for Rb gene expression ( shRb ) (n = 400 in 3 biological independent replicates). ( Right panel ) Table summarizing Rb status across the cell lines used in this study. b , Barplot of γH2AX geomean intensity analysed by flow cytometry in DAOY-FUCCI (CA), UW228-FUCCI (CA) and ONS76-FUCCI (CA) in control or AMBRA1-depleted condition (n=10000 events in three independent technical replicates). c , ( Left panel ) QIBC analysis of γH2AX total intensity along cell cycle of control ( shCTRL ) or shRb ONS76 cells in scramble or AMBRA1-depleted condition stained for EdU and Hoechst (2000 cells are displayed per condition). ( Right panel ), IB for the indicated proteins of whole-cell extracts from of control ( shCTRL ) or shRb ONS76. d , IB for the indicated proteins of whole-cell extracts from of control ( shCTRL ) or shRb ONS76 cells in scramble or AMBRA1-depleted condition, untreated (CTRL) or treated with 200 nM AZD7762 for 24 hrs (n=3). h/e: high exposure time; l/e: low exposure time. e , ( Left panel ) Representative images from DAOY cells engineered to constitutively express a CDK4/6 (red) and CDK2 (green) activity reporter. Barplot of CDK2 activity ( Top Right panel ) and CDK4/6 corrected activity ( Bottom Right panel ). Cells were depleted for scramble or AMBRA1 (siRNA) and FBS-deprived (FBS 2%) or not for 24 hrs and then analysed by high-content imaging (n=4). Scale bar 100 µm. g , Representative images of DAOY-FUCCI (CA) cells treated for 24 hrs with 1µM palbociclib and 500 nM abemaciclib and analysed by high-content imaging. Scale Bar: 100 µm. h , Barplot for G1 phase cells % analysed in g (n=3). i , Barplot for CDK4/6 and CDK2 (green) activity reporter. Cells were depleted for scramble or AMBRA1 (siRNA) and then treated or not with 500 nM abemaciclib for 24 hrs and then analysed by high-content imaging (n=3). j , ( Top panel ) IB for the indicated proteins of whole-cell extracts in ONS76 cells silenced or not for the indicated genes. ( Bottom panel ) Barplot of corresponding quantification of normalized phosphorylated levels over total (n=3). Unless otherwise stated data are presented as mean value ±SEM and n refers to biological independent samples. Data were analysed using One-way ANOVA ( b , f ).

Article Snippet: Primary antibodies used for immunoblot (IB), immunoprecipitation (IP) immunohistochemistry (IHC), Proximity-Ligation Assay (PLA) and immunofluorescence (IF) were: α-TUBULIN (GeneTex GTX628802 - IB 1:10000), β-actin (Sigma A2066 - IB 1:5000), Myc (Santa Cruz sc-40 – IB 1:1000, IP 500 ng), HSP90 (Santa Cruz sc13119 IB -1:10000), AMBRA1 (Millipore ABC131 - IB 1:1000), AMBRA1 (G-6) (Santa Cruz sc-398204 - IB 1:1000), AMBRA1 (Novus 26190002 - 1:100), cyclin D1 (Cell Signaling 2978 - IB 1:1000), cyclin D1 (Abcam 16663 - IF 1:300 / IP 1:100), cyclin D2 (Cell Signaling - 3741 IB 1:1000), cyclin D2 (Santa Cruz sc-452 - IF 1:50), cyclin D3 (Thermo Fisher Scientific - MA5-12717 WB 1:1000), cyclin E2 (Cell Signaling 4132 - IB 1:1000), p21 Waf1/Cip (Cell Signaling 2947 – IB 1:1000 / IF 1:100 / IHC 1:100, IP 250 ng), p27 Kip (Cell Signaling 3686 – IB 1:1000, IP 250 ng), p27 Kip (Santa Cruz sc-528 – 1:100), Ki67 (Abcam 15580 IF 1:200), HA (Y11) (Santa Cruz sc-805 - 1-500), CUL1-NEDD8 (Cell Signaling 4995 - IB 1:1000), PARP / cleaved-PARP (Abcam ab32138 - IB 1:1000), pRb 780 (Cell Signaling 8307 - IB 1:1000) pRb 807/811 (Cell Signaling 9308 - IB 1:1000), Cdt2 (Abcam ab72264 IB - 1:500), FEN1 (Novus NB100-150 PLA - 1:100), Rb (Cell Signaling 9313 IB - 1:1000), CDK4 (Cell Signaling 12790 – IB 1:1000), CDK4 (Santa Cruz sc-23896 – PLA 1:50), CDK2 (Cell Signaling 2546 – IB 1:1000), CDK2 (Santa Cruz sc-6248 – PLA 1:50), DDB1 (Novus NBP1-33061 – IB 1:1000), DDB1 (BD Biosciences 612488 – PLA 1:100), PCNA (Santa Cruz sc-56 – IB 1:1000), PCNA (Abcam ab18197 PLA – 1:100) Chk1 (Santa Cruz sc-8408 - IB 1:1000), Chk1(Ser345) (Cell Signaling 2341 - IB 1:1000), H2AX p-S139 (Merck Millipore 05-636 - IF 1:500), H2AX p-S139 (Abcam ab22551 - IB 1:500), CDK2 pT160 (Cell Signaling 2561 - IB 1:500), ATM (Abcam ab2618 – IB 1:1000), ATM pS1981 (Abcam ab81292 – IB 1:5000), p53 (Santa Cruz sc-126 – IB 1:500), UBIQUITIN (Santa Cruz sc-8017 - IB 1:500), SKP2 (Santa Cruz sc-7164 – IB 1:1000).

Techniques: Control, Gene Expression, Flow Cytometry, Staining, Activity Assay, Imaging