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: Cancer Biology & Therapy

Article Title: Identification of WEE1 as a target to make AKT inhibition more effective in melanoma

doi: 10.1080/15384047.2017.1360446

Figure Lengend Snippet: Targeting AKT3 and WEE1 increased p53 while reducing FOXM1 and CDK1 signaling. (A) and (B). Knockdown of WEE1 kinase led to a dose-dependent decrease in the phosphorylation of its substrate CDK1. Dose-dependent increase in the phosphorylation of H2AX was observed. Consequently, this resulted in increased p53, p21 as well as p27 levels, which are known to be inhibitory to cell proliferation. ERK2 served as a control for equal protein loading.

Article Snippet: Primary antibodies used: p21 (sc-756), p27 (sc-528), p53 (sc-6243) and ERK2 (sc-1647) from Santa Cruz Biotechnology (Dallas, TX), pAKT (9271), AKT3 (3788), Total AKT (4685), pWEE1 (4910), WEE1 (4936), p-CDC2 (Y15) (9111), pRB (S807/811) (9308), pRB (S795) (9301), pRB (S780) (9307), (9309) and p-Histone H2AX (S139) (2577) from Cell Signaling (Danvers, MA).

Techniques:

Journal: Cancer Biology & Therapy

Article Title: Identification of WEE1 as a target to make AKT inhibition more effective in melanoma

doi: 10.1080/15384047.2017.1360446

Figure Lengend Snippet: Diagram showing the mechanism of synergism for co-targeting AKT and WEE1 signaling pathways. Inhibition of siWEE1 (1) suppresses inhibitory phosphorylation of CDK1 leading to early-G2/M progression. This leads DNA damage (2) and activates p53 signaling. p53 inhibits cell cycle progression by induction of p21, allowing DNA damage repair. If the DNA damage is not repairable, p53 induces apoptosis. However, in many cancer cells, apoptotic cascades are suppressed by oncogenic alterations. Over-activated AKT inhibits pro-apoptotic factors while inducing antiapoptotic factors (3). AKT signaling also enhances cell cycle progression by CyclinD1 mediated phosphorylation of RB (4) and inhibition of p27 (5). Furthermore, AKT phosphorylates and induces Polo-like kinase 1 (PLK1) (6), which in turn inhibits pro-apoptotic functions of p53 and its family members, p63 and p73 (7). In addition, PLK1 also induces FOXM1 activity and M-phase progression (8). Proteins that were validated by Western blotting are shown in bold.

Article Snippet: Primary antibodies used: p21 (sc-756), p27 (sc-528), p53 (sc-6243) and ERK2 (sc-1647) from Santa Cruz Biotechnology (Dallas, TX), pAKT (9271), AKT3 (3788), Total AKT (4685), pWEE1 (4910), WEE1 (4936), p-CDC2 (Y15) (9111), pRB (S807/811) (9308), pRB (S795) (9301), pRB (S780) (9307), (9309) and p-Histone H2AX (S139) (2577) from Cell Signaling (Danvers, MA).

Techniques: Inhibition, Activity Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA ELDR promotes cell cycle progression in normal oral keratinocytes through induction of a CTCF-FOXM1-AURKA signaling axis

doi: 10.1016/j.jbc.2022.101895

Figure Lengend Snippet: Overexpression of ELDR induces FOXM1-AURKA signaling. A , relative mRNA expression of AURKA, PLK1, CDC25C, CDK1, and CCNB1 in ELDR-overexpressed NOK was analyzed by qRT-PCR as compared to NOK. 18S rRNA was used as an internal control. Small bar indicates standard error (∗∗∗ p < 0.001). Experiments were repeated three times with technical triplicates. B , control or ELDR-overexpressed NOK lysates were subjected to Western blot analysis for AURKA and ( C ) phospho-CDK1 and total CDK1 (Cdc2) using specific antibodies. The membranes were reprobed with actin as an internal control. Right panel shows quantitation. Small bar indicates standard error (∗ p < 0.05). Experiments were repeated two times. D , the expression of AURKA and FOXM1 was shown ( red dots ) among a total 15,219 genes in the Volcano plot generated from the RNA seq data of NOK-ELDR versus NOK. Horizontal and vertical dashed red lines represent absolute 2-fold change and p -value 0.05 as the threshold cut-off. E , AURKA and FOXM1 pathway genes are strongly upregulated in ELDR-expressing NOK cells. Gene set enrichment analysis was performed using the “fgsea” R package with genes ranked based on their differential expression in NOK-ELDR and control NOK cells against the Pathway Interaction Database (PID). The AURORA A (AURKA) and FOXM1 pathways were among the upregulated pathways in cells expressing ELDR. F , relative mRNA expression of FOXM1 in ELDR-overexpressed NOK analyzed by qRT-PCR as compared to NOK. 18S rRNA was used as an internal control. Small bar indicates standard error (∗∗∗ p < 0.001). Experiments were repeated three times with technical triplicates. G , NOK-ELDR cells were transfected with two different siRNAs to ELDR and after 48 h mRNA expression of ELDR, FOXM1, and AURKA were analyzed by qRT-PCR. 18S rRNA was used as an internal control. Relative gene expression is shown with mean value ±standard error (∗ p < 0.05; ∗∗ p < 0.01). Experiments were repeated three times with technical triplicates. AURKA, Aurora kinase A; CCNB1, cyclin B1; CDC25C, cell division cycle 25C; CDK1, cyclin-dependent kinase 1; EGFR, epidermal growth factor receptor; ELDR, EGFR long noncoding downstream RNA; FOXM1, Forkhead box M1; NOK, normal oral keratinocytes; NOK-ELDR, NOKs stably expressing ELDR; PLK1, Polo-like kinase 1; qRT-PCR, quantitative real-time reverse transcription PCR.

Article Snippet: Cell lysates were prepared using 2× SDS sample buffer, and Western blot analysis was performed using specific antibodies to AURKA (1: 1000, Cell Signaling Technology, CST), p-CDK1/p-Cdc2 (Tyr-15) (1: 1000, CST), total CDK1/Cdc2 (1: 1000, CST), CTCF (1: 1000, CST), and FOXM1 (1: 1000, CST).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Generated, RNA Sequencing Assay, Transfection, Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: Long noncoding RNA ELDR promotes cell cycle progression in normal oral keratinocytes through induction of a CTCF-FOXM1-AURKA signaling axis

doi: 10.1016/j.jbc.2022.101895

Figure Lengend Snippet: Primer sequences

Article Snippet: Cell lysates were prepared using 2× SDS sample buffer, and Western blot analysis was performed using specific antibodies to AURKA (1: 1000, Cell Signaling Technology, CST), p-CDK1/p-Cdc2 (Tyr-15) (1: 1000, CST), total CDK1/Cdc2 (1: 1000, CST), CTCF (1: 1000, CST), and FOXM1 (1: 1000, CST).

Techniques: Sequencing