anti phospho wee1 ser642 rabbit  (Cell Signaling Technology Inc)

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

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a MTS assay shows decreased growth rate of the mutant. Cells were assayed at 0, 24, 48, and 72 h of growth. Values corrected for background absorbance were normalized to 0 h of growth. Error bars denote SEM. The mean of three independent experiments, including triplicates per experiment, was plotted. Statistical significance was determined using an unpaired two-tailed t test: p = 0.08 at 24 hrs, (ns = not significant), **** p = 0.00006 at 48 hrs, ** p = 0.008 at 72 hrs. b Bar graph depicts Caspase 3/7 activity. Data corrected for background absorbance plotted as fold change over WT-control. Error bars denote SEM of three independent experiments with two or three replicates assayed per experiment. p = 0.94 (ns) (unpaired two-tailed t test). c Western blot represents p53 and phospho-S317-Chk1 levels in asynchronous (Async) WT and mutant HCT116 cells. Vinculin probed as a loading control. The data represent three independent experiments. d Representative immunofluorescent images of asynchronous cells stained with DAPI (nuclear marker) and anti-phospho-S10-Histone H3 antibody (mitosis). e Percent of mitotic cells (red) in the images shown in ( d ). were quantitated using multiple image panels with an average of 350 cells per panel, and plot shows 2 panels assayed per experiment from three independent experiments. Error bars denote SEM and significance determined by an unpaired two-tailed t test: *** p = 0.0008. f , g Western blot analysis of asynchronous cells probed with indicated antibodies with Tubulin and Vinculin as loading controls. h , i Plots represent phospho-Y15-CDK1 levels, and WEE1 levels normalized to the loading control. The data are representative of three experiments. Error bars denote SEM. Statistical significance determined by unpaired two-tailed t test: ** p = 0.0018 ( h ) and * p = 0.0208 ( i ). All molecular weight markers in the figure denoted in kDa.

Article Snippet: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4 °C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), CDK1 (1:1000; Santa Cruz Biotechnology, sc-54), WEE1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-245).

Techniques: MTS Assay, Mutagenesis, Two Tailed Test, Activity Assay, Control, Western Blot, Staining, Marker, Molecular Weight

Journal: Nature Communications

Article Title: Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit

doi: 10.1038/s41467-025-66794-6

Figure Lengend Snippet: a Western blot analysis of in vitro kinase assay of recombinant RPA (150 nM or 250 nM) incubated with DNA-PK for 5 min. Blots probed with indicated antibodies. Data represent three to four independent experiments. b Quantitation of blots shown in ( a ). and phospho-S4/8 RPA32 levels were normalized to RPA70 control and expressed as fold over WT-RPA. * p = 0.0185 and ** p = 0.0036 (unpaired two-tailed t test). Error bars denote SEM of four independent experiments. c Western blot analysis of in vitro kinase assay of recombinant RPA (250 nM) incubated with DNA-PK for 5 min. d Quantitation of blots shown in c. and phospho-S4/8 RPA32 levels were normalized to RPA70 control and expressed as fold over WT-RPA. * p = 0.0337 and *** p = 0.0001 (unpaired two-tailed t test). Error bars denote SEM of three (T191D) or four independent experiments. e Western blot analysis of in vitro kinase assay of recombinant RPA (250 nM) incubated with DNA-PK for 5 min in the presence and absence of (dT) 97 ssDNA (50 nM). Blot is representative of three independent experiments. f Model depicts the positive feedback loop between RPA and the cell cycle-specific kinases that phosphorylate RPA. It remains unclear if the activity of kinases is regulated by RPA through a direct effect on their catalytic activity or through modulation of mediators such as WEE1. The OB-domains A, B, C, D, E are depicted in gray. The two protein interaction domains (F and wh) are colored purple and green. The disordered N-terminus of RPA32 is shown as a black line. g Model illustrates the release of the N-terminus of RPA32 (black line) along with the F and Wh domains upon cell cycle-specific priming phosphorylation at T191, S23, and S29 sites by CDK1 kinase. The phosphocode primes RPA32 for efficient downstream hyperphosphorylation by kinases such as DNA-PK in response to DNA damage. Thus, the structural re-organization induced by cell cycle-specific priming phosphorylation of both RPA70 and RPA32 works synergistically and is crucial for hyperphosphorylation of RPA32 in response to DNA damage. All molecular weight markers in the figure denoted in kDa.

Article Snippet: Blots were blocked in 5% non-fat dry milk dissolved in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) at RT for 1 h. The following antibodies were diluted in TBS-T buffer and incubated overnight at 4 °C, except β-Actin which was incubated at room temperature for 30 min: β-Tubulin (1:2000; Cell Signaling, 2128), Vinculin (1:2000; Cell Signaling, 13901), β-Actin (1:1000; Cell Signaling, 3700), RPA70 (1:2000; Cell Signaling, 2198), p53 (1:1000; Santa Cruz Biotechnology, sc-126), RPA32 (1:5000; Cell Signaling, 35869), CDK1 (1:1000; Santa Cruz Biotechnology, sc-54), WEE1 kinase (1:1000; Cell Signaling, 13084), Cyclin B1 (1:1000; Santa Cruz Biotechnology, sc-245).

Techniques: Western Blot, In Vitro, Kinase Assay, Recombinant, Incubation, Quantitation Assay, Control, Two Tailed Test, Activity Assay, Phospho-proteomics, Molecular Weight