Journal: Nature Communications

Article Title: Loss of phosphatase CTDNEP1 potentiates aggressive medulloblastoma by triggering MYC amplification and genomic instability

doi: 10.1038/s41467-023-36400-8

Figure Lengend Snippet: a CTDNEP1-interating proteins identified by mass spectrographic analysis in 293 T cells which was expressing HA-tag CTDNEP1. b Mass spectroscopy analysis of phosphorylated proteins in Ctdnep1 -cKO NPCs (cKO) at DIC 10 compared with Ctrl NPCs. Two-tailed unpaired Student’s t test. c Pathway analysis of the most differentially upregulated phospho-proteins in Ctdnep1 -cKO NPCs compared with wild-type NPCs. Fisher exact test. d Upper, representative phosphorylated proteins involved in cell-cycle progression that are enriched in Ctdnep1 -cKO NPCs compared to control NPCs. Lower panel; the phosphorylation intensity of mitosis and chromosome segregation proteins in the NPCs detected by mass spectrometry. Data represent means, n = 2 independent experiments. e Venn diagram of CTDNEP1 binding proteins and phospho-proteins enriched in Ctdnep1 -cKO NPCs compared to wild-type NPCs. f GO analysis of candidate CTDNEP1 interacting phospho-proteins in Ctdnep1 -cKO NPCs. Fisher exact test. g Representative immunoblots from 3 independent experiments for p-TOP2A, p-CDK1, p-SRPK1 and p-CHEK1 in Ctdnep1- cKO and wild-type NPCs at late-stages. h Representative immunoblots from 3 independent experiments for the indicated phospho-proteins in D425 cells transfected with control siRNA or siCTDNEP1 after treatment with nocodazole for 14 h and sampled at indicated time points in fresh medium. NOW; nocodazole washout. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: Nestin (Mouse, Abcam; Cat#ab22035, 1:500), Ki67 (Rabbit, Thermo Fisher; Cat#MA5-14520, 1:1000), BrdU (Mouse, BD Bioscience; Cat#347580, Abcam; Cat#ab6326, 1:500), Cleaved Caspase 3 (Rabbit, Cell Signaling; Cat#9661, 1:500), c-Myc (Rabbit, Cell Signaling; Cat#5605 S, 1:1000), γH2A.X (Rabbit, Cell Signaling; Cat# 9718 S, 1:1000), p53 (Rabbit, Cell Signaling; Cat# 2524 S, 1:1000), p-S15 p53 (Rabbit, Cell Signaling; Cat#9284, 1:1000), GAPDH (Mouse, Thermo Sci; Cat# 39–8600, 1:5000), phosphor-Ser/Thr-Pro MPM2 (Mouse, Millipore Sigma; Cat#05-368, 1:1000), p-S62 c-Myc (Rabbit, Abcam; Cat#ab51156, 1:1000), p-S1525 TOP2A (Rabbit, Cedarlanelabs; Cat#E-AB-21933, 1:1000), TOP2A (Rabbit, Proteintech; Cat#20233-1-AP, 1:1000), p-S317 Chk1 (Rabbit, Cell Signaling; Cat#12302, 1:1000), Chk1 (Rabbit, Proteintech; Cat#25887-1-AP, 1:1000), Cdc2 (Rabbit, Cell Signaling; Cat#9116 T, 1:1000), p-T14 Cdc2 (Rabbit, Cell Signaling; Cat#2543 S, 1:1000), SRPK1 (Rabbit, BD biosciences; Cat#611072, 1:1000), HA-Tag (Mouse, Cell Signaling; Cat#2367, 1:1000), DYKDDDDK-Tag (Mouse, Thermo Fisher, Cat# MA1-91878, 1:1000), Phosphor-Ser/Thr (Rabbit, Abcam; Cat#ab117253, 1:1000) and GFAP (Goat, Santa Cruz; Cat#sc-6170, 1:500).

Techniques: Expressing, Mass Spectrometry, Two Tailed Test, Binding Assay, Western Blot, Transfection

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) In vitro CDK1 activity (top2 plots) was analyzed in EOC cells with or without ODF2L knockdown and then treated with DMSO or AZD1775 (treatment: DMSO/200 nM AZD1775, 4 hours). ( B ) Immunoblots of the T14 and Y15 phosphorylation status of CDK1 in the indicated EOC cells (treatment: DMSO/200 nM AZD1775, 24 hours). ( C and D ) In vitro CDK1 activity (top 2 plots) was analyzed in CDK1 T14A–expressing ( C ), CDK1 Y15F–expressing ( D ), and CDK1 WT–expressing ( C and D ) EOC cells with endogenous CDK1 removed (treatment: DMSO or 200 nM AZD1775 for 4 hours). ( E and F ) Representative images and quantification of colony formation ( E ) and apoptotic cell death ( F ) of EOC cells with or without ODF2L knockdown. Cells were treated with sublethal doses of AZD1775 (A2780, 200 nM; SKOV3, 200 nM) together with or without 5 μM Ro-3306 for 72 hours. Data are the mean ± SD from 3 technical replicates of each sample and are representative of 3 ( A ), 2 ( B ), and 3 ( C – F ) independent biological experiments. Blots shown were run in parallel, contemporaneously using the same cell lysate harvested from 1 representative experiment for each cell line in A – D . *** P < 0.001 and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vitro, Activity Assay, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) Analysis of correlations between CDK1 expression levels and those of WEE1, PKMYT1, and ODF2L using TCGA OV data. ( B ) Analysis of the endogenous interaction between ODF2L and PKMYT1 by reverse coimmunoprecipitation followed by immunoblotting. ( C and D ) Analysis of the interaction between CDK1 and PKMYT1 in ODF2L-knockdown EOC cells ( C ) and the interaction between CDK1 and ODF2L in PKMYT1 knockdown EOC cells ( D ) by immunoprecipitation of endogenous CDK1 followed by immunoblotting. ( E ) BRET assay for the binding of PKMYT1 and CDK1 (plasmid design strategy is shown on left) measured in the indicated cells transfected with Halo-tagged CDK1 and Nluc-tagged PKMYT1 in the absence or presence of ODF2L. ( F ) Analysis of the binding of CDK1 and PKMYT1 to the C-terminally, middle-range, and N-terminally truncated ODF2L by coimmunoprecipitation. The indicated cells were cotransfected with Myc-PKMYT1, CDK1-V5, or Flag-tagged truncated ODF2L domains. ( G and H ) Analysis of cell viability upon AZD1775 treatment with or without ODF2L knockdown upon overexpression of PKMYT1 ( G ) or knockdown of PKMYT1 ( H ). AZD1775 (A2780, 200 nM; SKOV3, 200 nM). Data are the mean ± SD from 3 technical replicates in E , G , and H . Data are representative of 2 ( B ), 2 ( C ), 2 ( D ), 3 ( E ), 2 ( F ), 2 ( G ), and 2 ( H ) independent biological experiments. Blots shown were run in parallel, contemporaneously using the same cell lysate harvested from 1 representative experiment for each cell line in H . ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Bioluminescence Resonance Energy Transfer, Binding Assay, Plasmid Preparation, Transfection, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A and B ) In vitro CDK1 activity, total ODF2L expression levels ( A ) and cell viability ( B ) were analyzed in AZD1775-treated EOC cells derived from the primary tumor tissue of 57 ovarian cancer patients. Patient 1 was used as a control for different batches and labeled blue. ( C and D ) Analysis of correlations between ODF2L expression levels and CDK1 activity ( C ) or cell viability ( D ) in AZD1775-treated primary EOC cells. ODF2L expression levels and CDK1 activity were measured by quantification of the blots in A using ImageJ software. ( E ) Effect of ODF2L expression levels on in vivo tumor growth of PDXs treated with AZD1775. Tumor tissue from patients 1, 5, and 10; patients 3 and 21; and patients 33 and 36 were chosen for the xenograft on the basis of the differential expression levels of ODF2L in the primary cells confirmed by Western blotting. Mice were evenly grouped when the volume of their tumors reached approximately 100 mm 3 25 days after xenografting and were treated with vehicle or AZD1775 (40 mg/kg, orally, once per day). ID, identification. Data are representative of 2 ( A ) and 3 ( B ) independent biological experiments and represent the mean ± SD of 3 technical replicates of each sample ( B ). Error bars in E represent the SEM for tumor volume ( n = 6). ** P < 0.01 and *** P < 0.0001, by 2-tailed Pearson’s correlation coefficient ( C and D ), 2-way ANOVA for tumor volume ( E ), and 1-way ANOVA for tumor weight ( F ).

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vitro, Activity Assay, Expressing, Derivative Assay, Labeling, Software, In Vivo, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) Schematic illustration of targeted LNP against ovarian ID8 cells using the ASSET platform (upper panel) and experimental design (lower panel). ( B and C ) Effect of combination treatment with siODF2L-LNP and AZD1775 on ID8 tumor growth. In vivo bioluminescence image ( B ) and average photonic flux ( C ) at week 7. LNPs (0.75 mg/kg, twice a week); AZD1775 (30 mg/kg, orally, once per day). n = 10. ( D ) Survival curves of ID8-bearing mice in the indicated groups. n = 10. ( E ) Quantification of peritoneal ID8 tumor nodule numbers in mice at the endpoint. ( F and G ) In vitro CDK1 activity and total ODF2L expression levels were analyzed by Western blotting ( F ), and the level of the DNA damage marker γH2AX was analyzed ( G ) by flow cytometry in the 2 or 3 ID8 tumors harvested from mice in each group at the endpoint. Data represent the mean ± SD; n = 10 ( B – E ). Data are the mean ± SD; n = 2 or n = 3 ( G ). Data are representative of 2 ( F ) and 3 ( G ) independent biological experiments. ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vivo, In Vitro, Activity Assay, Expressing, Western Blot, Marker, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) In vitro CDK1 activity (top2 plots) was analyzed in EOC cells with or without ODF2L knockdown and then treated with DMSO or AZD1775 (treatment: DMSO/200 nM AZD1775, 4 hours). ( B ) Immunoblots of the T14 and Y15 phosphorylation status of CDK1 in the indicated EOC cells (treatment: DMSO/200 nM AZD1775, 24 hours). ( C and D ) In vitro CDK1 activity (top 2 plots) was analyzed in CDK1 T14A–expressing ( C ), CDK1 Y15F–expressing ( D ), and CDK1 WT–expressing ( C and D ) EOC cells with endogenous CDK1 removed (treatment: DMSO or 200 nM AZD1775 for 4 hours). ( E and F ) Representative images and quantification of colony formation ( E ) and apoptotic cell death ( F ) of EOC cells with or without ODF2L knockdown. Cells were treated with sublethal doses of AZD1775 (A2780, 200 nM; SKOV3, 200 nM) together with or without 5 μM Ro-3306 for 72 hours. Data are the mean ± SD from 3 technical replicates of each sample and are representative of 3 ( A ), 2 ( B ), and 3 ( C – F ) independent biological experiments. Blots shown were run in parallel, contemporaneously using the same cell lysate harvested from 1 representative experiment for each cell line in A – D . *** P < 0.001 and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vitro, Activity Assay, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) Analysis of correlations between CDK1 expression levels and those of WEE1, PKMYT1, and ODF2L using TCGA OV data. ( B ) Analysis of the endogenous interaction between ODF2L and PKMYT1 by reverse coimmunoprecipitation followed by immunoblotting. ( C and D ) Analysis of the interaction between CDK1 and PKMYT1 in ODF2L-knockdown EOC cells ( C ) and the interaction between CDK1 and ODF2L in PKMYT1 knockdown EOC cells ( D ) by immunoprecipitation of endogenous CDK1 followed by immunoblotting. ( E ) BRET assay for the binding of PKMYT1 and CDK1 (plasmid design strategy is shown on left) measured in the indicated cells transfected with Halo-tagged CDK1 and Nluc-tagged PKMYT1 in the absence or presence of ODF2L. ( F ) Analysis of the binding of CDK1 and PKMYT1 to the C-terminally, middle-range, and N-terminally truncated ODF2L by coimmunoprecipitation. The indicated cells were cotransfected with Myc-PKMYT1, CDK1-V5, or Flag-tagged truncated ODF2L domains. ( G and H ) Analysis of cell viability upon AZD1775 treatment with or without ODF2L knockdown upon overexpression of PKMYT1 ( G ) or knockdown of PKMYT1 ( H ). AZD1775 (A2780, 200 nM; SKOV3, 200 nM). Data are the mean ± SD from 3 technical replicates in E , G , and H . Data are representative of 2 ( B ), 2 ( C ), 2 ( D ), 3 ( E ), 2 ( F ), 2 ( G ), and 2 ( H ) independent biological experiments. Blots shown were run in parallel, contemporaneously using the same cell lysate harvested from 1 representative experiment for each cell line in H . ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Bioluminescence Resonance Energy Transfer, Binding Assay, Plasmid Preparation, Transfection, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A and B ) In vitro CDK1 activity, total ODF2L expression levels ( A ) and cell viability ( B ) were analyzed in AZD1775-treated EOC cells derived from the primary tumor tissue of 57 ovarian cancer patients. Patient 1 was used as a control for different batches and labeled blue. ( C and D ) Analysis of correlations between ODF2L expression levels and CDK1 activity ( C ) or cell viability ( D ) in AZD1775-treated primary EOC cells. ODF2L expression levels and CDK1 activity were measured by quantification of the blots in A using ImageJ software. ( E ) Effect of ODF2L expression levels on in vivo tumor growth of PDXs treated with AZD1775. Tumor tissue from patients 1, 5, and 10; patients 3 and 21; and patients 33 and 36 were chosen for the xenograft on the basis of the differential expression levels of ODF2L in the primary cells confirmed by Western blotting. Mice were evenly grouped when the volume of their tumors reached approximately 100 mm 3 25 days after xenografting and were treated with vehicle or AZD1775 (40 mg/kg, orally, once per day). ID, identification. Data are representative of 2 ( A ) and 3 ( B ) independent biological experiments and represent the mean ± SD of 3 technical replicates of each sample ( B ). Error bars in E represent the SEM for tumor volume ( n = 6). ** P < 0.01 and *** P < 0.0001, by 2-tailed Pearson’s correlation coefficient ( C and D ), 2-way ANOVA for tumor volume ( E ), and 1-way ANOVA for tumor weight ( F ).

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vitro, Activity Assay, Expressing, Derivative Assay, Labeling, Software, In Vivo, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: ODF2L acts as a synthetic lethal partner with WEE1 inhibition in epithelial ovarian cancer models

doi: 10.1172/JCI161544

Figure Lengend Snippet: ( A ) Schematic illustration of targeted LNP against ovarian ID8 cells using the ASSET platform (upper panel) and experimental design (lower panel). ( B and C ) Effect of combination treatment with siODF2L-LNP and AZD1775 on ID8 tumor growth. In vivo bioluminescence image ( B ) and average photonic flux ( C ) at week 7. LNPs (0.75 mg/kg, twice a week); AZD1775 (30 mg/kg, orally, once per day). n = 10. ( D ) Survival curves of ID8-bearing mice in the indicated groups. n = 10. ( E ) Quantification of peritoneal ID8 tumor nodule numbers in mice at the endpoint. ( F and G ) In vitro CDK1 activity and total ODF2L expression levels were analyzed by Western blotting ( F ), and the level of the DNA damage marker γH2AX was analyzed ( G ) by flow cytometry in the 2 or 3 ID8 tumors harvested from mice in each group at the endpoint. Data represent the mean ± SD; n = 10 ( B – E ). Data are the mean ± SD; n = 2 or n = 3 ( G ). Data are representative of 2 ( F ) and 3 ( G ) independent biological experiments. ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 1-way ANOVA.

Article Snippet: In the immunoblots using the whole-cell lysate, antibodies against p-ATM (Ser1981) (catalog 5883); ATM (catalog 2873); p-CHK2 (Thr68) (catalog 2197), CHK2 (catalog 6334); p-RPA32/RPA2 S8 (catalog 83745); RPA32/RPA2 (catalog 35869); p-CDK1 (Tyr15) (catalog 4539); p-CDK1 T14 (catalog 2543); CDK1 (catalog 77055); p–histone γH2AX (Ser139) (catalog 9718); and PKMYT1 (catalog 4282) were all obtained from Cell Signaling Technology.

Techniques: In Vivo, In Vitro, Activity Assay, Expressing, Western Blot, Marker, Flow Cytometry