Journal: International journal of molecular sciences

Article Title: Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe.

doi: 10.3390/ijms241914646

Figure Lengend Snippet: Figure 4. Bortezomib (BTZ) changed mRNA and Protein Expression Profile of Cell-Cycle Regulatory Proteins in T-ALL. T-ALL cell lines were cultured and grown to attain the highest percentage of viability. The cells were then treated for 24 h with different concentrations of BTZ, followed by RNA extraction for real-time quantitative PCR analysis and protein extraction for Western blot analysis. (A): Shows the mRNA levels of cell-cycle regulatory genes in T-ALL cell lines. (B): Corresponds to Western blot expression profile of cell-cycle regulatory proteins. The result shows that BTZ treatment induced downregulation of WEE1 in all T-ALL cell lines we tested. The experiments were performed independently three times (n = 3), and the data were presented as the mean ± standard error of mean (SEM). Significant differences are denoted as ***: p < 0.001, **: p < 0.01, *: p < 0.05.

Article Snippet: For generating WEE1 overexpressing cell lines, WEE1 (NM_003390) Human Tagged ORF Clone (RC209760), purchased from OriGENE (Rockville, MD 20850, USA), was transfected to T-ALL cell lines using Lipofectamine 3000 Transfection Reagent (ThermoFisher) according to the instruction of package insert.

Techniques: Expressing, Cell Culture, RNA Extraction, Real-time Polymerase Chain Reaction, Protein Extraction, Western Blot

Journal: International journal of molecular sciences

Article Title: Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe.

doi: 10.3390/ijms241914646

Figure Lengend Snippet: Figure 6. Bortezomib (BTZ) Efficiency Decreases Against WEE1 Overexpressing T-ALL Cells. WEE1 overexpressing cell lines were cultured and then treated with BTZ along with wild-type cell lines for 24 and 72-h. The figure shows a decrease in viability of cells upon treatment with BTZ for 24 and 72 h. The degree of reduction in cell viability was higher in wild-type (red) compared to WEE1-expressing cells (black). Furthermore, the IC50 values for BTZ were calculated and presented in each figure; the result shows reduced IC50 values for wild-type cells compared to WEE1 overexpressing cells both in 24 and 72 h treatment. The experiments were performed independently three times (n = 3), and the data were presented as mean ± standard error of mean (SEM).

Article Snippet: For generating WEE1 overexpressing cell lines, WEE1 (NM_003390) Human Tagged ORF Clone (RC209760), purchased from OriGENE (Rockville, MD 20850, USA), was transfected to T-ALL cell lines using Lipofectamine 3000 Transfection Reagent (ThermoFisher) according to the instruction of package insert.

Techniques: Cell Culture, Expressing

Journal: International journal of molecular sciences

Article Title: Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe.

doi: 10.3390/ijms241914646

Figure Lengend Snippet: Figure 7. Combined Bortezomib (BTZ) and WEE1 inhibitor (AZD) induce apoptosis in T-ALL Cells. T-ALL cells, including wild-type, WEE1 overexpressing cell lines, and primary patient’s sample (Patient CSH T-ALL), were treated for 24 h and 72 h with BTZ (4 nM) and AZD (160 nM) alone and in

Article Snippet: For generating WEE1 overexpressing cell lines, WEE1 (NM_003390) Human Tagged ORF Clone (RC209760), purchased from OriGENE (Rockville, MD 20850, USA), was transfected to T-ALL cell lines using Lipofectamine 3000 Transfection Reagent (ThermoFisher) according to the instruction of package insert.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: Cells commit to forward mitotic progression in prometaphase. (A) Chemical Cdk inhibition in mitosis induces cyclin B1 and securin degradation that requires APC/C-Cdc20. HeLa cells were transfected with 50 nM Cdc20 siRNA for 24 h or with 50 nM Cdh1 siRNA for 48 h. Mitotic cells were collected in nocodazole, treated with 10 μM Flavopiridol for 30, 60, and 90 min, then lysed and processed for Western blotting. Depletion of Cdc20, but not Cdh1, inhibits degradation of cyclin B and securin. (B) Summary of the live imaging data from Xenopus S3 cells treated with Cdk inhibitor, Flavopiridol, and Wee1/Myt1 inhibitor, PD 0166285 at different stages of the mitotic progression. Flavopiridol was washed out 1 h after addition. (C) A prophase Xenopus S3 cell expressing alpha tubulin-GFP was treated with Cdk inhibitor, Flavopiridol, and Wee1/Myt1 inhibitor, PD 0166285. After treatment with Cdk inhibitor, mitotic progression stopped, the chromosomes decondensed, and the cell returned to an interphase morphology. Flavopiridol was washed out at 1 h, and the cell re-entered mitosis, indicating that Cdk1-activating cyclins were preserved. The cell then progressed normally trough mitosis. (D) An early prometaphase Xenopus S3 cell expressing alpha tubulin-GFP was treated with Flavopiridol and PD 0166285. After treatment at time 0, the cell underwent cytokinesis without chromosome segregation, the chromosomes decondensed, the nuclear envelope reformed, and an interphase array of microtubules appeared. Flavopiridol was washed out at 1 h. However, the cell did not re-enter mitosis, indicating that it had advanced to a G1-like state. The complete time-lapse sequences for (A) and (B) are shown in Supplemental Videos 1 and 2. Bar, 10 μm.

Article Snippet: Cdh1, pT14Cdk1, and Nucleolin antibodies were from Abcam (Cambridge, MA); cyclin B1 antibody was from BD Biosciences (San Jose, CA); Cdc20(p55) antibody was a gift from Jasminder Weinstein (Amgen, Thousand Oaks, CA), securin-1 antibody was from Zymed; pY15Cdk1, pS10 histone H3, Wee1, anti-Myt1Cdc25C, and Cdk1 antibodies were from Cell Signaling.

Techniques: Inhibition, Transfection, Western Blot, Imaging, Expressing

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: Mitotic progression in cells synchronized at S/G2 and treated with Wee1/Myt1 and Cdc25 inhibitors. (A, B) HeLa cells stably expressing fluorescent histone H2B fused to GFP were synchronized by the double thymidine block at the S/G2 border and treated with the Wee1/Myt1 inhibitor, PD0166285, alone (A) or in combination with Cdc25 inhibitor, NSC663284 (B). While the Wee1/Myt1 inhibitor alone rapidly triggers mitosis in the majority of cells, the combination of the Wee1/Myt1 and Cdc25 inhibitors results in slow mitotic entry followed by mitotic collapse. The complete time-lapse sequence is shown in Supplemental Videos 7 and 8. Bar, 10 μm. (C) Synchronized HeLa cells were treated with the Wee1/Myt1 inhibitor, PD0166285, alone or in combination with Cdc25 inhibitor, NSC663284, for 90 min. Cells were then fixed and processed by immunofluorescence for alpha-tubulin and phosphorylated-histone H3 on S10 (mitotic marker). Labeling shows disorganized mitotic spindle, and in some cells, reduced mitotic marker.

Article Snippet: Cdh1, pT14Cdk1, and Nucleolin antibodies were from Abcam (Cambridge, MA); cyclin B1 antibody was from BD Biosciences (San Jose, CA); Cdc20(p55) antibody was a gift from Jasminder Weinstein (Amgen, Thousand Oaks, CA), securin-1 antibody was from Zymed; pY15Cdk1, pS10 histone H3, Wee1, anti-Myt1Cdc25C, and Cdk1 antibodies were from Cell Signaling.

Techniques: Stable Transfection, Expressing, Blocking Assay, Sequencing, Immunofluorescence, Marker, Labeling

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: Inhibition of Wee1/Myt1 and Cdc25 in synchronized cells causes mitotic collapse. (A) HeLa cells were synchronized at the S/G2 border after double thymidine block and then treated with the Wee1/Myt1 inhibitor, PD0166285, Cdc25 inhibitor, NSC663284, and the combination of the two drugs. Nocodazole was added to the medium to prevent mitotic exit. Cells were then collected at indicated time points, fixed and stained with antibody to phospho-histone H3 (mitotic marker) conjugated with Alexa Fluor 647, and processed by flow cytometry. In cells treated with vehicle only (DMSO, blue line), the mitotic index progressively increased, with more than half the cells being in mitosis by the end of the experiment. Cdc25 inhibitor, NSC663284, blocked mitotic entry (brown line). Wee1 inhibitor, PD0166285 (green line), caused rapid mitotic entry during the first hour after its addition. In cells treated with both PD0166285 and NSC663284 (orange line), the mitotic index first increased then fell. (B) HeLa cells were treated as in (A), lysed and analyzed by SDS–PAGE. In cells not treated with inhibitors (blue lanes), phosphorylations on histone H3 and nucleolin appeared by 8 h after second thymidine release and increased for the duration of the experiment. Phosphorylation of Cdk1 on inhibitory T14 and Y15 decreased over time, indicating the activation of the Cdk1/cyclin B complex. As cells were entering mitosis, a portion of Wee1, Myt1 Cdc25C, Cdc27, and MastL acquired an electrophoretic mobility shift. Cyclin B1 levels were increasing, and cyclin A2 levels dropped slightly as cells accumulated in mitosis. Inhibition of Wee1 and Myt1 kinases with PD0166285 (green lanes) resulted in rapid phosphorylation of Nucleolin and histone H3 that peaked 2 h after the drug addition and remained steadily high for the duration of the experiment. Cdk1 was rapidly dephosphorylated on inhibitory T14 and Y15. Wee1, Myt1, Cdc25, and Cdc27 rapidly shifted up. By 1 h after drug addition, Cyclin A2 was largely degraded and cyclin B1 was stable. Inhibition of Wee1 and Myt1 together with Cdc25 by addition of both PD0166285 and NSC 663284 (orange lanes) triggered the a weak phosphorylation on Nucleolin and histone H3 that peaked at 1–2 h and disappeared at 3–4 h after addition of the two drugs. Reduced mitotic phosphorylation shifts of Wee1, Myt1, Cdc25, and Cdc27 indicated that these proteins were not fully phosphorylated. Note that cyclin B and most of the cyclin A were not degraded in these cells. Panels on the right show quantifications of indicated Western blots. All values were adjusted for loading and normalized to the 4-h time point of DMSO-treated cells.

Article Snippet: Cdh1, pT14Cdk1, and Nucleolin antibodies were from Abcam (Cambridge, MA); cyclin B1 antibody was from BD Biosciences (San Jose, CA); Cdc20(p55) antibody was a gift from Jasminder Weinstein (Amgen, Thousand Oaks, CA), securin-1 antibody was from Zymed; pY15Cdk1, pS10 histone H3, Wee1, anti-Myt1Cdc25C, and Cdk1 antibodies were from Cell Signaling.

Techniques: Inhibition, Blocking Assay, Staining, Marker, Flow Cytometry, SDS Page, Phospho-proteomics, Activation Assay, Electrophoretic Mobility Shift Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: Deposphorylation of mitotic substrates in “collapsed” cells is a result of incomplete inhibition of Cdk-opposing phosphatases. (A) Cdk1/cyclin B1 activity does not drop in mitotic collapse cells. HeLa cells were synchronized at the S/G2 border and treated with the Wee1/Myt1 inhibitor, PD0166285, Cdc25 inhibitor, NSC663284, and the combination of the two in the presence of nocodazole. Cells were then collected at indicated time points and lysed. An aliquot of the lysate was analyzed by Western blotting for Nucleolin phosphorylation. β-Actin served as a loading control. Cyclin B1/Cdk1 complex was immunoprecipitated from the rest of the lysate and subjected to an in vitro kinase assay using histone H1 as a substrate. The kinase reaction mixture was resolved by SDS–PAGE, and the gel was exposed to phosphor-screen, which was then scanned with phosphor-imager. For a control, samples derived from the 4-h time point of DMSO-treated cells were treated with Cdk inhibitor (lane labeled “+Flavopiridol”), or processed omitting cyclin B1 antibody from immunoprecipitation (lane labeled “mock”). The gel was subsequently stained with Coomassie blue for loading. Panel on the right shows quantifications of histone H1 phosphorylation normalized to the 4 h time point of DMSO-treated cells. An average of three independent assays is shown. Error bars denote SD. (B) Simultaneous inhibition of Wee1/Myt1 and Cdc25 in cells already in mitosis does not cause mitotic substrate dephosphorylation. Mitotic HeLa cells were collected in nocodazole and then treated with Wee1/Myt1 and Cdc25 inhibitors for the indicated time, lysed, and analyzed by Western blotting. Mitotic substrates nucleolin and histone H3 remained phosphorylated throughout the experiment. (C) The phosphatase inhibitor, okadaic acid, prevents dephosphorylation of mitotic substrates in cells treated with a combination of Wee1/Myt1 and Cdc25 inhibitors. HeLa cells were synchronized at the S/G2 border after double thymidine block and treated with the Wee1/Myt1 inhibitor, PD0166285, and Cdc25 inhibitor, NSC663284, for the indicated time in the presence or absence of okadaic acid. Addition of the okadaic acid resulted in robust and sustained phosphorylation of mitotic substrates.

Article Snippet: Cdh1, pT14Cdk1, and Nucleolin antibodies were from Abcam (Cambridge, MA); cyclin B1 antibody was from BD Biosciences (San Jose, CA); Cdc20(p55) antibody was a gift from Jasminder Weinstein (Amgen, Thousand Oaks, CA), securin-1 antibody was from Zymed; pY15Cdk1, pS10 histone H3, Wee1, anti-Myt1Cdc25C, and Cdk1 antibodies were from Cell Signaling.

Techniques: Inhibition, Activity Assay, Western Blot, Phospho-proteomics, Control, Immunoprecipitation, In Vitro, Kinase Assay, SDS Page, Derivative Assay, Labeling, Staining, De-Phosphorylation Assay, Blocking Assay

Journal: Molecular Biology of the Cell

Article Title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

doi: 10.1091/mbc.E10-07-0599

Figure Lengend Snippet: (A) Cdk substrate phosphorylation regulatory network. The phosphorylation of mitotic substrates (enzymes and structural proteins) by Cdk1/cyclin B complex underlies mitotic entry. Cdk1/cyclin B is antagonized by phosphatases PP1 and PP2A that dephosphorylate mitotic substrates. Wee1 kinase and Cdc25 phosphatases regulate Cdk1 activity: Wee1 inhibits Cdk1 (green inhibitory line) and Cdc25 activates it (blue arrow). Wee1 and Cdc25 are themselves Cdk substrates. Cdk1 phosphorylates and inhibits Wee1, preventing Wee1 from inactivating Cdk1. Also, Cdk1 phosphorylates and activates its activator Cdc25. Active Cdk also inhibits antagonists PP1 and PP2A by at least two known mechanisms. First, Cdk1 can inhibit PP1 directly by phosphorylating T320 residue on a catalytic subunit of the phosphatase (black inhibitory line). Second, Cdk1 phosphorylates and activates the Greatwall/MastL kinase, which inhibits PP2A and possibly PP1 by yet unidentified mechanisms (red inhibitory line). Therefore as Cdk activation is fueled by positive feedback, it also promotes the inactivation of its antagonists, ensuring the stability of substrate phosphorylation. (B) Failure to activate Cdk rapidly results in mitotic collapse after nuclear envelope breakdown. The feedback-mediated activation of the Cdk1/Cyclin B complex may be required to prevent the dilution of the kinase activity throughout the cytoplasm when the nuclear envelope becomes permeable. Cdk1 activity appears to spike around the time of the nuclear envelope disassembly, when the activated Cdk/cyclin B complex spreads through the cytoplasm. In the absence of the positive feedback, active Cdk1 would be diluted in the cytoplasm when the nuclear envelope becomes permeable. In the absence of positive feedback mechanisms, the concentration of the active kinase per unit of cytosol may fall below the level that is needed to efficiently counteract Cdk-opposing phosphatases, which leads to the mitotic collapse.

Article Snippet: Cdh1, pT14Cdk1, and Nucleolin antibodies were from Abcam (Cambridge, MA); cyclin B1 antibody was from BD Biosciences (San Jose, CA); Cdc20(p55) antibody was a gift from Jasminder Weinstein (Amgen, Thousand Oaks, CA), securin-1 antibody was from Zymed; pY15Cdk1, pS10 histone H3, Wee1, anti-Myt1Cdc25C, and Cdk1 antibodies were from Cell Signaling.

Techniques: Phospho-proteomics, Activity Assay, Residue, Activation Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe

doi: 10.3390/ijms241914646

Figure Lengend Snippet: Bortezomib (BTZ) changed mRNA and Protein Expression Profile of Cell-Cycle Regulatory Proteins in T-ALL. T-ALL cell lines were cultured and grown to attain the highest percentage of viability. The cells were then treated for 24 h with different concentrations of BTZ, followed by RNA extraction for real-time quantitative PCR analysis and protein extraction for Western blot analysis. ( A ): Shows the mRNA levels of cell-cycle regulatory genes in T-ALL cell lines. ( B ): Corresponds to Western blot expression profile of cell-cycle regulatory proteins. The result shows that BTZ treatment induced downregulation of WEE1 in all T-ALL cell lines we tested. The experiments were performed independently three times ( n = 3), and the data were presented as the mean ± standard error of mean (SEM). Significant differences are denoted as ***: p < 0.001, **: p < 0.01, *: p < 0.05.

Article Snippet: For generating WEE1 overexpressing cell lines, WEE1 (NM_003390) Human Tagged ORF Clone (RC209760), purchased from OriGENE (Rockville, MD 20850, USA), was transfected to T-ALL cell lines using Lipofectamine 3000 Transfection Reagent (ThermoFisher) according to the instruction of package insert.

Techniques: Expressing, Cell Culture, RNA Extraction, Real-time Polymerase Chain Reaction, Protein Extraction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe

doi: 10.3390/ijms241914646

Figure Lengend Snippet: Bortezomib (BTZ) Efficiency Decreases Against WEE1 Overexpressing T-ALL Cells. WEE1 overexpressing cell lines were cultured and then treated with BTZ along with wild-type cell lines for 24 and 72-h. The figure shows a decrease in viability of cells upon treatment with BTZ for 24 and 72 h. The degree of reduction in cell viability was higher in wild-type (red) compared to WEE1-expressing cells (black). Furthermore, the IC50 values for BTZ were calculated and presented in each figure; the result shows reduced IC50 values for wild-type cells compared to WEE1 overexpressing cells both in 24 and 72 h treatment. The experiments were performed independently three times ( n = 3), and the data were presented as mean ± standard error of mean (SEM).

Article Snippet: For generating WEE1 overexpressing cell lines, WEE1 (NM_003390) Human Tagged ORF Clone (RC209760), purchased from OriGENE (Rockville, MD 20850, USA), was transfected to T-ALL cell lines using Lipofectamine 3000 Transfection Reagent (ThermoFisher) according to the instruction of package insert.

Techniques: Cell Culture, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe

doi: 10.3390/ijms241914646

Figure Lengend Snippet: Combined Bortezomib (BTZ) and WEE1 inhibitor (AZD) induce apoptosis in T-ALL Cells. T-ALL cells, including wild-type, WEE1 overexpressing cell lines, and primary patient’s sample (Patient CSH T-ALL), were treated for 24 h and 72 h with BTZ (4 nM) and AZD (160 nM) alone and in combination, followed by staining with Annexin V-FITC/PI for flow cytometric analysis of apoptosis. ( A ): Representative flow cytometry plots after 24 h treatment, indicating dramatic increase in apoptotic plus pre-apoptotic cells after treatment. ( B ): Representative flow cytometry plots after 72 h treatment, indicating dramatic increase in apoptotic plus pre-apoptotic cells after treatment. ( C ): Histograms representing the percentage of pre-apoptotic and apoptotic cells in control, BTZ, AZD, and combined treated T-ALL cells. The percentages were statistically compared, and significant differences are denoted as ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05. The experiments were performed independently three times ( n = 3), and the data were presented as the mean ± standard error of mean (SEM). FITC: Fluorescein Isothiocyanate. PI: Propidium Iodide.

Article Snippet: For generating WEE1 overexpressing cell lines, WEE1 (NM_003390) Human Tagged ORF Clone (RC209760), purchased from OriGENE (Rockville, MD 20850, USA), was transfected to T-ALL cell lines using Lipofectamine 3000 Transfection Reagent (ThermoFisher) according to the instruction of package insert.

Techniques: Staining, Flow Cytometry, Control

Journal: International Journal of Molecular Sciences

Article Title: Bortezomib Is Effective in the Treatment of T Lymphoblastic Leukaemia by Inducing DNA Damage, WEE1 Downregulation, and Mitotic Catastrophe

doi: 10.3390/ijms241914646

Figure Lengend Snippet: Forward and Reverse Primers Used in RT-qPCR.

Article Snippet: For generating WEE1 overexpressing cell lines, WEE1 (NM_003390) Human Tagged ORF Clone (RC209760), purchased from OriGENE (Rockville, MD 20850, USA), was transfected to T-ALL cell lines using Lipofectamine 3000 Transfection Reagent (ThermoFisher) according to the instruction of package insert.

Techniques: Sequencing

Journal: Cells

Article Title: Role of Alternative Splicing and Polyadenylation in Regulation of Spleen Development

doi: 10.3390/cells15060496

Figure Lengend Snippet: Confirmation of the regulatory roles of transcription factors E2F1 and MYBL2 in WEE1 gene expression. ( A ) Schematic structure of the WEE1 promoter and the putative binding sites of E2F1 and MYBL2. Binding sites and the position are given in the schematic structure, while sequences for each motif in the wild-type and mutant-type (Mut) promoters are given below. Sequences shown with strikethrough were deleted, and those highlighted in yellow were mutated in the Mut reporter constructs. ( B ) Log2Fold-change in E2F1, MYBL2 and WEE1 in 90- compared to 30-day-old pigs as revealed with RNA-seq. ( C ) Characterization of the promoter activity of WEE1 gene with dual-luciferase reporter analysis. WT, wild-type reporter gene. ( D ) Deletion of each putative binding site significantly decreased promoter activity. ( E ) Expression efficiency of plasmids overexpressing MYBL2 or E2F1 as revealed with Western blotting. ( F ) MYBL2 or E2F1 regulated reporter gene expression via the putative binding sites. ( G ) Overexpression of MYBL2 or E2F1 upregulated the mRNA level of WEE1 in PK-15 cells. ( H ) Overexpression of MYBL2 or E2F1 upregulated the protein level of WEE1 in PK-15 cells. ( I ) Direct binding of MYBL2 or E2F1 to the motifs as revealed by EMSA. EV, empty vector. * p < 0.05, ** p < 0.01, compared with WT reporters in Panels ( D , F ).

Article Snippet: The transferred PVDF membranes underwent protein blocking and were incubated with primary antibodies against WEE1 (1:5000 dilution; Catalog Number: 29474-1-AP; Proteintech, Wuhan, China) at 4 °C overnight, followed by incubation with secondary antibodies (1:20,000 dilution; LI-COR) at 37 °C for 1 h. Finally, the PVDF membranes were detected using a LI-COR near-infrared fluorescence imaging system (LI-COR, Lincoln, NE, USA).

Techniques: Gene Expression, Binding Assay, Mutagenesis, Construct, RNA Sequencing, Activity Assay, Luciferase, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Transcriptomic Screening Identifies ATR Pathway Inhibitor AZ20 as a Promising Candidate to Overcome SN-38 Resistance in TP53 -Mutant DIPG. ( A ) KEGG analysis of 190313 cells treated with SN-38 (10 nM, 72 h) revealed upregulation of cell cycle and DNA replication pathways (* P < .05). (B) HALLMARK pathway analysis identified enrichment of E2F target genes (* P < .05). (C) Correlation analysis showed strong associations between E2F (E2F1, E2F2, E2F7, E2F8) and DNA damage repair genes (ATR, CHK1, PARP1, etc.) (* P < .05). (D) Western blot analysis showed significant upregulation of ATR, CHK1, PARP1, and WEE1 protein expression in TP53 -mutant DIPG cell lines (190313, 190326, 150728) treated with 10 nM SN-38 for 72 h, compared to untreated controls. (E-G) PARP1 inhibitor Olaparib had minimal effect on TP53-mutant DIPG (IC50 > 10 μM) and showed no synergy with SN-38 (NS). (H) The CHK1 inhibitor (SCH900776) exhibited potent cytotoxic effects on TP53 -mutant DIPG cells (190326), with an IC50 of ∼100 nM and minimal toxicity to PPCs. (I-J) Co-treatment with SCH900776 (100 nM) and SN-38 (10 nM) demonstrated significant synergy, reducing cell viability (* P < .05). (K) Screening of 23 ATR pathway inhibitors identified AZ20 as the most potent (>70% viability reduction at 1 μM). Heatmap includes TP53-KD and PPM1D-KD isogenic lines. (L) AZ20 exhibited strong activity in TP53-mutant DIPG (IC50 ∼200 nM) and limited toxicity to PPCs (IC50 > 1 μM). Viability assessed by CellTiter-Glo (mean ± SD, n = 3).

Article Snippet: P53 (DO-1, Cat# 18032S), WIP1 (E2X1I, Cat# 94886S), and GAPDH (D4C6R, Cat# 97166S) antibodies were obtained from Cell Signaling Technology (CST); BCL2 (Cat# 68103-1-Ig), BAX (Cat# 60267-1-Ig), Vinculin (Cat# 66305-1-Ig), and PARP1 (Cat# 66520-1-Ig) antibodies were purchased from ProteinTech; CHK1 (Cat# BM3968), WEE1 (Cat# A01319-2), and ATR (Cat# A00262-3) antibodies were sourced from Boster.

Techniques: Mutagenesis, Western Blot, Expressing, Activity Assay

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Synergistic anti-tumor effects of AZ20 and SN-38 in TP53 -mutant DIPG cells through inhibition of ATR pathway signaling and induction of apoptosis. (A) Twenty-one ATR pathway inhibitors were screened in combination with SN-38 (1 μM each) in TP53-mutant DIPG cells. Viability was measured by CellTiter-Glo ( n = 3) analyzed by a two-tailed unpaired t -test. (B-D) Synergy analysis using the BLISS model confirmed a robust synergistic interaction between SN-38 and AZ20 in 190326 cells (D). In contrast, this synergistic effect was not observed in TP53 wild-type DIPG cells (150714, DIPG17) (B and C). (E-G) Cell viability was measured after 24, 48, and 72 h of treatment with DMSO, SN-38 (10 nM), AZ20 (10 nM), or both in 190326, 150714, and DIPG17 cells. Combination significantly reduced viability in 190326 (**** P < .0001). (H) Western blot analysis of protein expression in 190326 cells following 72 h of treatment with DMSO (vehicle control), SN-38 (10 nM), AZ20 (10 nM), or their combination. SN-38 monotherapy activated ATR and its downstream targets, CHK1 and WEE1, while combination treatment with SN-38 and AZ20 suppressed ATR activation and downregulated CHK1 and WEE1 expression. The combination treatment also induced apoptosis, as evidenced by increased levels of cleaved PARP1. (I) Chou-Talalay-based combination index (CI) heatmap for SN-38 and AZ20 in TP53-mutant DIPG cell line 190326. Combination index values were calculated from a 72-h viability assay using fixed-ratio matrix combinations of SN-38 and AZ20. CI < 1 indicates synergy, CI = 1 indicates additivity, and CI > 1 indicates antagonism. (J) 190326 cells transfected with siATR and treated with SN-38 or AZ20 showed reduced viability in SN-38 + siATR and AZ20 + SN-38 + siATR groups (**** P < .0001).

Article Snippet: P53 (DO-1, Cat# 18032S), WIP1 (E2X1I, Cat# 94886S), and GAPDH (D4C6R, Cat# 97166S) antibodies were obtained from Cell Signaling Technology (CST); BCL2 (Cat# 68103-1-Ig), BAX (Cat# 60267-1-Ig), Vinculin (Cat# 66305-1-Ig), and PARP1 (Cat# 66520-1-Ig) antibodies were purchased from ProteinTech; CHK1 (Cat# BM3968), WEE1 (Cat# A01319-2), and ATR (Cat# A00262-3) antibodies were sourced from Boster.

Techniques: Mutagenesis, Inhibition, Two Tailed Test, Western Blot, Expressing, Control, Activation Assay, Viability Assay, Transfection