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: 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: Science advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: Fig. 9. Rac1 via actomyosin regulates the G2-M checkpoint kinase Wee1 to prevent premature mitotic entry. (A and B) CD cells were G2-synchronized using RO- 3306, and lysates were collected at the indicated time points after RO-3306 washout (“G2 release”). Lysates were immunoblotted for pH3 to monitor mitotic entry. Three repeat experiments were quantified in (B). Fold change values ± SD. Arrows highlight the first pH3 peak of the respective groups indicating mitotic entry. (C and D) G2- synchronized CD cells were immunoblotted for cleaved caspase 3 (cl-Casp3) after G2 release and quantified in (D) as fold change values ± SD (n = 3). Asterisk (*) denotes between-group significance at the corresponding time point. (E and F) CD cells were G2-synchronized, and lysates were obtained immediately upon G2 washout (G2, T0) and immunoblotted in biological duplicates. p-Cdk1 Y15: phosphorylated Cdk1 tyrosine-15. Three repeat experiments were quantified in (F). Fold change values ± SD. (G and H) Asynchronous Rac1f/f (+DMSO) and Rac1−/− [+DMSO or blebbistatin (5 μM)] CD cells were treated with cycloheximide (CHX; 100 μg/ml), and lysates were obtained at the indicated time points and immunoblotted for Wee1. Three repeat experiments are quantified in (H) as fold change ± SD. Asterisk (*) denotes significance between Rac1−/− and Rac1f/f or blebbistatin-treated Rac1−/− at the corresponding time point. (I and J) Rac1f/f and Rac1−/− CD cells were G2-synchronized and treated with vehicle (DMSO) or 5 μM blebbistatin upon G2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry. Three independent repeat experiments are quantified (for Rac1f/f, only the vehicle control is shown) in (J) as fold change values ± SD. Asterisk (*) denotes significance between Rac1−/− and Rac1f/f or blebbistatin-treated Rac1−/− at the corresponding time point. *P < 0.05.

Article Snippet: Primary antibodies used are pH3 (Cell Signaling Technology, #9701), cleaved caspase 3 (Cell Signaling Technology, #9664), Wee1 (Novus Biologicals, #NBP1- 33506), actin (Cell Signaling Technology, #4967), cyclin B1 (Cell Signaling Technology, #4138), Rac1 (Millipore, #05- 389), α- tubulin (Cell Signaling Technology, #2144), Cdk1 (Cell Signaling Technology, #77055), and phospho–(Y15) Cdk1 (Cell Signaling Technology, #4539).

Techniques: Control

Journal: Science advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: Fig. 10. Wee1 inhibition phenocopies Rac1 deficiency in mitosis. (A to D) Rac1f/f and Rac1−/− CD cells were G2-synchronized using RO-3306 and treated with the Wee1- specific inhibitor MK-1775 (1 μM) upon G2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry or cleaved caspase 3 to monitor cell death. Densitometry was used to quantify fold changes ± SD of three repeat experiments in (B) and (D). Arrows in (B) highlight the first pH3 peak indicating mitotic entry. (E) F-actin (white)– and DNA (blue)–labeled Rac1f/f and Rac1−/− (+MK-1775; 1 μM) CD cell monolayers analyzed by confocal microscopy with a mitotic metaphase cell shown in the center (scale bars, 10 μm). The top row column depicts metaphase F-actin (scale bars, 5 μm) as outlined by a red continuous box in the bottom row. Images are representative of at least three experiments. (F) Circularity quantification of mitotic metaphase F-actin as shown in (E) with a minimum of 10 measurements shown per group. Bars are means ± SD. (G) Live confocal mitosis imaging of SPY650-DNA (blue)– and SPY555-actin (white)–labeled vehicle (DMSO)– or MK-1775 (1 μM)–treated Rac1f/f CD cells in vitro. The mitotic cell was manually segmented and colored in green. Scale bars, 10 μm. Sequences are representative of three repeat experiments with at least three to four mitoses analyzed per experiment. (H) Relative distribution of mitotic defects in vitro during live imaging cell division se- quences with at least 10 mitoses analyzed per group. *P < 0.05.

Article Snippet: Primary antibodies used are pH3 (Cell Signaling Technology, #9701), cleaved caspase 3 (Cell Signaling Technology, #9664), Wee1 (Novus Biologicals, #NBP1- 33506), actin (Cell Signaling Technology, #4967), cyclin B1 (Cell Signaling Technology, #4138), Rac1 (Millipore, #05- 389), α- tubulin (Cell Signaling Technology, #2144), Cdk1 (Cell Signaling Technology, #77055), and phospho–(Y15) Cdk1 (Cell Signaling Technology, #4539).

Techniques: Inhibition, Labeling, Confocal Microscopy, Imaging, In Vitro

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Differential FLNA and Wee1 protein expression in ACC, ACA, and NAG. (A) Representative immunoblots of FLNA, Wee1 and GAPDH expression in 6 patient‐derived ACC, 8 ACA, and 8 NAG. (B) The graphs show densitometric analysis of FLNA and Wee1 expression normalized to GAPDH and expressed as fold over NCI‐H295R. NCI‐H295R cell lysates were included to normalize all blots to the same control sample. Horizontal bars represent median. * p < 0.05; *** p < 0.001. Kruskal–Wallis multiple comparison test with Dunn's post‐hoc test.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Expressing, Western Blot, Derivative Assay, Control, Comparison

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Expression of FLNA and Wee1 in human ACC cell lines, and effects of FLNA silencing and overexpression on Wee1 protein levels. (A) Protein expression levels of FLNA and Wee1 in NCI‐H295R, MUC‐1, and TVBF‐7 cell lines. Representative immunoblots of FLNA and Wee1 expression normalized to GAPDH are shown. (B) NCI‐H295R, MUC‐1, and TVBF‐7 cell lines were transfected with FLNA siRNA or negative control (C‐) siRNA for 6 days. The graphs show densitometric analysis of Wee1 normalized to GAPDH (median and IQR of at least 3 independent experiments, respectively). Representative immunoblots are shown. * p < 0.05 vs C‐ siRNA. Mann–Whitney test. (C) MUC‐1 cells were transfected with FLNA siRNA or negative control (C‐) siRNA for 6 days. The graphs show densitometric analysis of phosphorylated CDK1 (Tyr15) normalized to total CDK1 (median and IQR of at least 3 independent experiments), and of cyclin B1 normalized to GAPDH (median and IQR of at least 3 independent experiments). Representative immunoblots are shown. * p < 0.05 vs C‐ siRNA. Mann–Whitney test. (D) Patient‐derived primary cultured ACC cells (ACC #1) were transfected with FLNA siRNA or negative control (C‐) siRNA for 6 days. The values above immunoblot images indicate densitometric analysis of Wee1 normalized to GAPDH. (E) MUC‐1 cells were transiently transfected with myc‐tagged FLNA plasmid for 72 h. Mock‐transfected cells were used as negative control. The graphs show densitometric analysis of Wee1 and cyclin B1 normalized to GAPDH (median and IQR of at least 3 independent experiments, respectively), and phosphorylated CDK1 (Tyr15) normalized to total CDK1 (median and IQR of at least 3 independent experiments). Representative immunoblots are shown. *** p < 0.001 vs mock. Mann–Whitney test.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Expressing, Over Expression, Western Blot, Transfection, Negative Control, MANN-WHITNEY, Derivative Assay, Cell Culture, Plasmid Preparation

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Lactacystin treatment reverted Wee1 depletion in FLNA‐transfected in MUC‐1 cell line. (A) MUC‐1 cells were transfected with FLNA siRNA or C‐ siRNA for 6 days, and with myc‐tagged FLNA plasmid for 72 h, and then RNA was extracted. The graphs show expression levels of Wee1 transcript measured by qRT‐PCR after FLNA genetic silencing and FLNA overexpression normalized to GAPDH (median and IQR of at least 3 independent experiments). Mann–Whitney test. (B) MUC‐1 cells were transiently transfected with myc‐tagged FLNA plasmid for 72 h, and then incubated with lactacystin 10 μM for 20 h. Mock‐transfected cells were used as negative controls. The graph shows densitometric analysis of Wee1 normalized to GAPDH (median and IQR of at least 3 independent experiments), expressed as fold vs mock (untreated). Representative immunoblots are shown. * p < 0.05 of untreated myc‐FLNA vs untreated mock, and of lactacystin‐treated myc‐FLNA vs untreated myc‐FLNA. Kruskal–Wallis test with Dunn's post‐hoc test. (C) MUC‐1 cells were transiently transfected with myc‐tagged FLNA plasmid for 72 h. Mock‐transfected cells were used as negative control. Representative immunoblots are shown. Due to the poor quality of p‐Wee1 (Ser123) antibody, densitometrical analysis could not be performed. Three independent experiments were carried out.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Over Expression, MANN-WHITNEY, Incubation, Western Blot, Negative Control

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Effects of Wee1 knockdown and Wee1 kinase inhibitor AZD1775 on ACC cells. (A) Proliferation assay in Wee1‐silenced MUC‐1 cells. Cells were transfected with Wee1 siRNA or negative control (C‐) siRNA for 72 h. Cells were incubated with BrdU for 2 h, and its incorporation into newly synthetized DNA was measured (median and IQR of at least 3 independent experiments). Representative immunoblots of Wee1 silencing are shown. *** p < 0.001 vs C‐ siRNA. Mann–Whitney test. (B) Detection of MUC‐1 cell apoptosis by Pacific Blue Annexin V/SYTOX AAdvanced Apoptosis kit. Unlabelled cells were used as negative control. After 72 h of transfection, the percentage of Annexin V+ and SYTOX+ cell fractions were measured. Representative flow cytometric plots are shown. Early apoptotic, late apoptotic, and necrotic cells are plotted graphically and are expressed as fold vs C‐ siRNA (median and IQR of at least 3 independent experiments). ** p < 0.01 vs C‐ siRNA. Mann–Whitney test. (C) Cell proliferation assay in MUC‐1, NCI‐H295R, and TVBF‐7 cell lines. Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. After 2 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of at least 3 independent experiments, respectively). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal condition. Friedman test with Dunn's post‐hoc test. (D) Cell viability assay in MUC‐1, NCI‐H295R, and TVBF‐7 cell lines. Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. MTT assay was used to verify cell viability in response to AZD1775 treatment (median and IQR of at least 3 independent experiments, respectively). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal condition. Friedman test with Dunn's post‐hoc test. (E) Cell proliferation assay in two different patient‐derived primary cultures of ACC (ACC #1 and ACC #2). Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. After 24 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of 5 replicates for each condition). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal absorbance value. Kruskal–Wallis test with Dunn's post‐hoc test. (F) Detection of MUC‐1 cell apoptosis by Pacific Blue Annexin V/SYTOX AAdvanced Apoptosis kit. Unlabelled cells were used as negative control. After 72 h of treatment with AZD1775, the percentage of Annexin V+ and SYTOX+ cell fractions were measured. Representative flow cytometric plots are shown. Early apoptotic, late apoptotic, and necrotic cells are plotted graphically and are expressed as fold vs basal condition (median and IQR of at least 3 independent experiments). * p < 0.05; ** p < 0.01 vs basal condition. Friedman test with Dunn's post‐hoc test.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Knockdown, Proliferation Assay, Transfection, Negative Control, Incubation, Western Blot, MANN-WHITNEY, Viability Assay, MTT Assay, Derivative Assay

Journal: Heliyon

Article Title: Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

doi: 10.1016/j.heliyon.2024.e40299

Figure Lengend Snippet: Wee1 inhibition plus gemcitabine inhibits H1975 tumor formation. (A) Schema showing the experimental timeline. (B) There were no significant differences in the body weights of xenografted mice treated with vehicle, gemcitabine, MK-1775, or combined with these two drugs. (C) Tumor volume raised in the vehicle, gemcitabine, and MK-1775 groups but not in the gemcitabine plus MK1775 group. (D) The harvested tumors are shown. (E) The tumor weights show the mean values of D, along with the standard errors and p values. Data are expressed as mean ± SEM. Scale bar: 1 cm ∗, p < 0.05. Gem: gemcitabine.

Article Snippet: Primary antibodies against Wee1 (GTX111392, 1:1000, GeneTex), phospho-Wee1 (#4910, 1:1000, Cell Signaling Technology), cdc2 (GTX108120, 1:1000, GeneTex), phospho-cdc2 (GTX128155, 1:1000, GeneTex), phospho-histone H2AX Ser139 (γ-H2AX; #05–636, 1:1000, EMD Millipore), poly (ADP-ribose) polymerase (PARP; #9532, 1:1000, Cell Signaling Technology), α-Tubulin (T9026, 1:1000, Sigma-Aldrich), along with secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#115-035-003/111-035-003, Jackson ImmunoResearch Laboratories), were used.

Techniques: Inhibition

Journal: Heliyon

Article Title: Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

doi: 10.1016/j.heliyon.2024.e40299

Figure Lengend Snippet: Wee1 inhibition plus pemetrexed inhibits H460 tumor formation. (A) Schema showing the experimental timeline. (B) There were no significant differences in the body weights of xenografted mice treated with vehicle, pemetrexed, MK-1775, or pemetrexed plus MK-1775. (C) Tumor volume raised in the vehicle and pemetrexed groups but not in the combination group. (D) The harvested tumors are shown. (E) The tumor weights show the mean values of D, along with the standard errors and p values. Data are expressed as mean ± SEM. Scale bar: 1 cm ∗, p < 0.05.

Article Snippet: Primary antibodies against Wee1 (GTX111392, 1:1000, GeneTex), phospho-Wee1 (#4910, 1:1000, Cell Signaling Technology), cdc2 (GTX108120, 1:1000, GeneTex), phospho-cdc2 (GTX128155, 1:1000, GeneTex), phospho-histone H2AX Ser139 (γ-H2AX; #05–636, 1:1000, EMD Millipore), poly (ADP-ribose) polymerase (PARP; #9532, 1:1000, Cell Signaling Technology), α-Tubulin (T9026, 1:1000, Sigma-Aldrich), along with secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#115-035-003/111-035-003, Jackson ImmunoResearch Laboratories), were used.

Techniques: Inhibition

Journal: Heliyon

Article Title: Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

doi: 10.1016/j.heliyon.2024.e40299

Figure Lengend Snippet: MK-1775 in combination with gemcitabine increased the cytotoxic activity in NSCLC. The survival rate of gemcitabine alone or plus wee1 inhibitor in p53 wt NSCLC A549 (A) and H460 (B). The p53 mut NSCLC HCC827 (C) and H1975 (D) cells treated with the same drug therapy for 72 h. The IC 50 is displayed in bar graph (E) or detailed data in the table (F). The p values (mean ± SEM) presented are from three independent experiments. ∗ p < 0.05 by Student's t-tests.

Article Snippet: Primary antibodies against Wee1 (GTX111392, 1:1000, GeneTex), phospho-Wee1 (#4910, 1:1000, Cell Signaling Technology), cdc2 (GTX108120, 1:1000, GeneTex), phospho-cdc2 (GTX128155, 1:1000, GeneTex), phospho-histone H2AX Ser139 (γ-H2AX; #05–636, 1:1000, EMD Millipore), poly (ADP-ribose) polymerase (PARP; #9532, 1:1000, Cell Signaling Technology), α-Tubulin (T9026, 1:1000, Sigma-Aldrich), along with secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#115-035-003/111-035-003, Jackson ImmunoResearch Laboratories), were used.

Techniques: Activity Assay

Journal: Heliyon

Article Title: Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

doi: 10.1016/j.heliyon.2024.e40299

Figure Lengend Snippet: Wee1 inhibition plus pemetrexed improved cell death in NSCLC except for HCC827 cells. The cell survival of pemetrexed only or in combination with MK-1775 in A549 (A), H460 (B), HCC827 (C), and H1975 (D). The bar graph (E) and the table (F) presented the IC 50 and p values (mean ± SEM) after NSCLC treated with the above monotherapy or combination therapy. The p values presented are from three independent experiments. ∗ p < 0.05 by Student's t-tests.

Article Snippet: Primary antibodies against Wee1 (GTX111392, 1:1000, GeneTex), phospho-Wee1 (#4910, 1:1000, Cell Signaling Technology), cdc2 (GTX108120, 1:1000, GeneTex), phospho-cdc2 (GTX128155, 1:1000, GeneTex), phospho-histone H2AX Ser139 (γ-H2AX; #05–636, 1:1000, EMD Millipore), poly (ADP-ribose) polymerase (PARP; #9532, 1:1000, Cell Signaling Technology), α-Tubulin (T9026, 1:1000, Sigma-Aldrich), along with secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#115-035-003/111-035-003, Jackson ImmunoResearch Laboratories), were used.

Techniques: Inhibition

Journal: Heliyon

Article Title: Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

doi: 10.1016/j.heliyon.2024.e40299

Figure Lengend Snippet: MK-1775 in combination with chemotherapy induced apoptosis in NSCLC. Western blot analyzed the expression of Wee1, cdc2, γ-H2AX, and PARP after MK-1775 monotherapy or combined with gemcitabine in A549 (A), H460 (B), HCC827 (C), and H1975 (D) or four cells treated MK-1775 plus pemetrexed for 24 h in E (The original uncropped images are provided in the ).

Article Snippet: Primary antibodies against Wee1 (GTX111392, 1:1000, GeneTex), phospho-Wee1 (#4910, 1:1000, Cell Signaling Technology), cdc2 (GTX108120, 1:1000, GeneTex), phospho-cdc2 (GTX128155, 1:1000, GeneTex), phospho-histone H2AX Ser139 (γ-H2AX; #05–636, 1:1000, EMD Millipore), poly (ADP-ribose) polymerase (PARP; #9532, 1:1000, Cell Signaling Technology), α-Tubulin (T9026, 1:1000, Sigma-Aldrich), along with secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#115-035-003/111-035-003, Jackson ImmunoResearch Laboratories), were used.

Techniques: Western Blot, Expressing

Journal: Heliyon

Article Title: Synergistic effects of MK-1775 and gemcitabine on cytotoxicity in non-small cell lung cancer

doi: 10.1016/j.heliyon.2024.e40299

Figure Lengend Snippet: Wee1 inhibition by MK-1775 plus gemcitabine stimulated the G2/M phase in p53 mut HCC827. FACS evaluated the cell cycle after MK-1775 alone or plus gemcitabine in HCC827 (A), and H1975 (B) for 24 and 48 h. The mean ± SEM presented are from three independent experiments.

Article Snippet: Primary antibodies against Wee1 (GTX111392, 1:1000, GeneTex), phospho-Wee1 (#4910, 1:1000, Cell Signaling Technology), cdc2 (GTX108120, 1:1000, GeneTex), phospho-cdc2 (GTX128155, 1:1000, GeneTex), phospho-histone H2AX Ser139 (γ-H2AX; #05–636, 1:1000, EMD Millipore), poly (ADP-ribose) polymerase (PARP; #9532, 1:1000, Cell Signaling Technology), α-Tubulin (T9026, 1:1000, Sigma-Aldrich), along with secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#115-035-003/111-035-003, Jackson ImmunoResearch Laboratories), were used.

Techniques: Inhibition

Journal: Nature Communications

Article Title: Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

doi: 10.1038/s41467-024-47668-9

Figure Lengend Snippet: a Wee1 expression in whole MG tissue from nulliparous, PD6.5, PD10.5, PD15.5, PD18.5 and LD2 MGs, as detected by RT-qPCR. n = 4. b Wee1 expression in sorted luminal, basal and stromal populations from PD18.5 and LD2 MGs, as detected by RT-qPCR. Populations FACS-sorted based on CD24 and CD29 expression. c , d Representative western blot ( c ) and quantifications ( d ) of WEE1 (top), pCDK1 and CDK1 (bottom) expression in nulliparous, PD10.5, PD15.5, PD18.5, and LD2 MGs. Quantification ( d , bottom) shown as pCDK1/CDK1 ratio. e Percentage of 2C, 4C and >4C CK8 + cells from MG organoids differentiated for 3 days, as detected by FACS analysis, after DMSO treatment (Ctr) or doxorubicin (Doxo), hydroxyurea (Hu), Ro-3306, nucleosides (Nucs) or Mk-1775. f Milk protein gene expression, Csn2 , Wap , or Lalba , in MG organoids differentiated for 3 days after DMSO treatment (Ctr) or Doxo, Hu, Ro-3306, Nucs or Mk-1775. g Cartoon representing contralateral intraductal injection (IDI) of MGs performed. Created with BioRender.com. h Percentage of CK8 + cells with >4C DNA content, as detected by FACS analysis, after contralateral IDI with DMSO or MK-1775 in LD5 MGs. Colored data points and dashed lines represent paired samples. i Milk protein gene expression, Lalba, Wap, Csn2, Plin2, Xdh1 , and Btn1 , in LD5 MGs after contralateral IDI with DMSO or Mk-1775, as detected by RT-qPCR. Colored data points and dashed lines represent paired samples. j , k Representative images of milk (white) in LD5 MGs after contralateral IDI with DMSO or Mk-1775. CK8 shown in magenta. Yellow squares indicate magnified areas in ( k ), illustrating milk contained within alveoli. l Quantification of milk per alveolus after contralateral IDI with DMSO or Mk-1775, as detected by IHC. Colored data points and dashed lines represent paired samples. Data presented as mean ± SEM ( a , b , i , l ) and mean ± SD ( d , e , f , h ). Data analyzed by one-way ANOVA with Tukey’s multiple comparison test ( a , d ) and paired, two-tailed Student’s t test ( b , e , f , h , i , l) . Data representative of n = 4 biologically independent experiments except for ( b – f ), representative of n = 3. p values: *<0.05, **<0.01, ***<0.001, ****<0.0001.

Article Snippet: Primary antibodies [anti-GAPDH (SCBT, sc-365062; 1:1000), anti-Actin (SCBT, sc-47778; 1:5000), anti-HSP70 (SCBT, sc-24; 1:1000), anti- Cyclin B1 (SCBT, sc-245; 1:1000), anti-Cyclin E1 (Millipore-Sigma, SAB4503516; 1:1000) and anti-CSN2 (ABclonal, A12749; 1:1000), anti-pATR (GeneTex, GT128145; 1:500), anti-ATR (Cell Signaling, 2790 S; 1:500), anti-pATM (GeneTex, GTX132146; 1:1000), anti-ATM (GeneTex, GTX70103; 1:500), anti-pCHK1 (Cell Signaling, 23485; 1:1000), anti-CHK1 (SCBT, sc-8408; 1:1000), anti-pCdc2 (Cell Signaling, 10A11; 1:1000), anti-Cdc2 (SCBT, sc-454; 1:1000), anti-WEE1 (Abnova, H00007465-M01A; 1:1000, anti-WEE1 (Thermofisher, PA5-29303; 1:500), anti-pSTAT5 (Cell Signaling, 9351 S; 1:1000), anti-STAT5 (SCBT, sc-836; 1:1000), anti-pSTAT3 (Cell Signaling, 9145 S; 1:1000), and anti-STAT3 (Cell Signaling, 9139 S; 1:1000)] were incubated overnight at 4 °C in a rocker.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Injection, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

doi: 10.1038/s41467-024-47668-9

Figure Lengend Snippet: a Quantification of the body weight (g) of pups nursed by Wee1 +/+ or Wee1 fl/fl dams. b Representative images of pups nursed by Wee1 +/+ (top) or Wee1 fl/fl (bottom) dams. Black dashed-line squares indicate the stomach of the pups. c Milk protein gene expression, Lalba, Wap, Csn2, Plin2, Xdh1 and Btn1 , in Wee1 +/+ or Wee1 fl/fl LD2 MGs, as detected by RT-qPCR. d , e Representative images of milk (white) by IHC in LD2 MGs from Wee1 +/+ or Wee1 fl/fl mice. CK8 shown in magenta. Yellow squares indicate the magnified areas in ( e ), illustrating the milk contained within the alveoli. f Quantification of total milk per MG area in Wee1 +/+ and Wee1 fl/fl mice at LD2, as detected by IHC. g – i Quantification of number of alveoli with lumens per MG area ( g ), alveoli lumen size ( h ), and fat pad filling ( i ) in LD2 MGs from Wee1 +/+ or Wee1 fl/fl mice, as detected by IHC. For ( a ), data show the mean values of 5 pups per timepoint from each n = 3 biologically independent experiments. Data presented as mean ± SD ( a , f , g , i) and mean ± SEM ( c , h ). Data analyzed by unpaired, two-tailed Student’s t test. Data representative of n = 3 biologically independent experiments. p values: *<0.05, **<0.01.

Article Snippet: Primary antibodies [anti-GAPDH (SCBT, sc-365062; 1:1000), anti-Actin (SCBT, sc-47778; 1:5000), anti-HSP70 (SCBT, sc-24; 1:1000), anti- Cyclin B1 (SCBT, sc-245; 1:1000), anti-Cyclin E1 (Millipore-Sigma, SAB4503516; 1:1000) and anti-CSN2 (ABclonal, A12749; 1:1000), anti-pATR (GeneTex, GT128145; 1:500), anti-ATR (Cell Signaling, 2790 S; 1:500), anti-pATM (GeneTex, GTX132146; 1:1000), anti-ATM (GeneTex, GTX70103; 1:500), anti-pCHK1 (Cell Signaling, 23485; 1:1000), anti-CHK1 (SCBT, sc-8408; 1:1000), anti-pCdc2 (Cell Signaling, 10A11; 1:1000), anti-Cdc2 (SCBT, sc-454; 1:1000), anti-WEE1 (Abnova, H00007465-M01A; 1:1000, anti-WEE1 (Thermofisher, PA5-29303; 1:500), anti-pSTAT5 (Cell Signaling, 9351 S; 1:1000), anti-STAT5 (SCBT, sc-836; 1:1000), anti-pSTAT3 (Cell Signaling, 9145 S; 1:1000), and anti-STAT3 (Cell Signaling, 9139 S; 1:1000)] were incubated overnight at 4 °C in a rocker.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

doi: 10.1038/s41467-024-47668-9

Figure Lengend Snippet: a – c Representative western blot ( a ) and quantifications of STAT5 ( b ) and pSTAT5 ( c ) expression in Wee1 +/+ or Wee1 fl/fl LD2 MGs. Quantification ( c ) shown as pSTAT5/STAT5 ratio. d Percentage of CK8 + cells, as detected by FACS analysis, in Wee1 +/+ or Wee1 fl/fl LD2 MGs. e Representative dot plots showing the SSC-A and FSC-A parameters of the CK8 + population from Wee1 +/+ or Wee1 fl/fl LD2 MGs, as detected by FACS analysis. Red-to-blue color scale shows high-to-low density of data points. Black line shows the gating strategy for identifying cells with high scatter parameters. f Percentage of CK8 + cells from Wee1 +/+ or Wee1 fl/fl LD2 MGs with high scatter (HS) parameters, as detected by FACS analysis. g Percentage of CK8 + cells with 2C, 4C or >4C DNA content, as detected by FACS analysis, in Wee1 +/+ or Wee1 fl/fl LD2 MGs. Data presented as mean ± SD. Data analyzed by unpaired, two-tailed Student’s t test. Data representative of n = 3 biologically independent experiments. p values: *<0.05, **<0.01, ***<0.001.

Article Snippet: Primary antibodies [anti-GAPDH (SCBT, sc-365062; 1:1000), anti-Actin (SCBT, sc-47778; 1:5000), anti-HSP70 (SCBT, sc-24; 1:1000), anti- Cyclin B1 (SCBT, sc-245; 1:1000), anti-Cyclin E1 (Millipore-Sigma, SAB4503516; 1:1000) and anti-CSN2 (ABclonal, A12749; 1:1000), anti-pATR (GeneTex, GT128145; 1:500), anti-ATR (Cell Signaling, 2790 S; 1:500), anti-pATM (GeneTex, GTX132146; 1:1000), anti-ATM (GeneTex, GTX70103; 1:500), anti-pCHK1 (Cell Signaling, 23485; 1:1000), anti-CHK1 (SCBT, sc-8408; 1:1000), anti-pCdc2 (Cell Signaling, 10A11; 1:1000), anti-Cdc2 (SCBT, sc-454; 1:1000), anti-WEE1 (Abnova, H00007465-M01A; 1:1000, anti-WEE1 (Thermofisher, PA5-29303; 1:500), anti-pSTAT5 (Cell Signaling, 9351 S; 1:1000), anti-STAT5 (SCBT, sc-836; 1:1000), anti-pSTAT3 (Cell Signaling, 9145 S; 1:1000), and anti-STAT3 (Cell Signaling, 9139 S; 1:1000)] were incubated overnight at 4 °C in a rocker.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation

doi: 10.1038/s41467-024-47668-9

Figure Lengend Snippet: Proliferation of alveolar cells results in accumulation of DNA damage during pregnancy and activation of the ATR pathway. As a result, ATR mediates the transition from a mitotic cell cycle into an endocycle through the activation of the CDK1 inhibitor WEE1, which activates the G2/M checkpoint and regulates alveolar endoreplication during lactation. Created with BioRender.com.

Article Snippet: Primary antibodies [anti-GAPDH (SCBT, sc-365062; 1:1000), anti-Actin (SCBT, sc-47778; 1:5000), anti-HSP70 (SCBT, sc-24; 1:1000), anti- Cyclin B1 (SCBT, sc-245; 1:1000), anti-Cyclin E1 (Millipore-Sigma, SAB4503516; 1:1000) and anti-CSN2 (ABclonal, A12749; 1:1000), anti-pATR (GeneTex, GT128145; 1:500), anti-ATR (Cell Signaling, 2790 S; 1:500), anti-pATM (GeneTex, GTX132146; 1:1000), anti-ATM (GeneTex, GTX70103; 1:500), anti-pCHK1 (Cell Signaling, 23485; 1:1000), anti-CHK1 (SCBT, sc-8408; 1:1000), anti-pCdc2 (Cell Signaling, 10A11; 1:1000), anti-Cdc2 (SCBT, sc-454; 1:1000), anti-WEE1 (Abnova, H00007465-M01A; 1:1000, anti-WEE1 (Thermofisher, PA5-29303; 1:500), anti-pSTAT5 (Cell Signaling, 9351 S; 1:1000), anti-STAT5 (SCBT, sc-836; 1:1000), anti-pSTAT3 (Cell Signaling, 9145 S; 1:1000), and anti-STAT3 (Cell Signaling, 9139 S; 1:1000)] were incubated overnight at 4 °C in a rocker.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Fcp1-dependent dephosphorylation is required for M-phase-promoting factor inactivation at mitosis exit

doi: 10.1038/ncomms1886

Figure Lengend Snippet: ( a ) SAC-arrested HeLa cells were released from arrest (nocodazole wash out; NWO) and sampled at indicated incubation time points. Indicated antigens were probed on total sample blots except p-T239-Wee1 and Wee1, probed on blots of Wee1 immunoprecipitates (Ips). Cdc27 and Mad2 were also probed on blots of Cdc27 Ips. All antigens were analyzed from samples of the same cell lysates except V5-tagged USP44, analysed by probing with an anti-V5 antibody lysates from HeLa cells previously transfected with a V5-tagged USP44 expression vector (V5-USP44-Tf; Mk-Tf=mock-transfected cells). ( b ) Densitometric quantification of signals from the immunoblots shown in a , expressed as percent of the time 0 value: (open triangles) Cyclin B1, (open diamonds) p-T239-Wee1, (closed diamonds) Cdc27-bound Mad2. ( c ) Densitometric quantification of signals from the immunoblots shown in a , expressed as percent of the time 0 value: (open triangles) Cyclin B1, (closed triangles) p-T244-Cdc27, (open squares) p-T48-Cdc25C and (closed squares) p-S83-Myt1. The data shown are representative of four independent experiments.

Article Snippet: Rabbit polyclonal antibodies against phosphothreonine 239 of human Wee1 were raised using PQVNINPF(p)TPDSLL peptide as immunogen; peptide synthesis, rabbit inoculation, serum production and cross-affinity antibody purification were carried out by Polypeptide Group.

Techniques: Incubation, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Nature Communications

Article Title: Fcp1-dependent dephosphorylation is required for M-phase-promoting factor inactivation at mitosis exit

doi: 10.1038/ncomms1886

Figure Lengend Snippet: Checkpoint extracts were mock-depleted (Mk-dep.), Fcp1-depleted (Fcp1-dep.) and 3F-Fcp1-reconstituted as in . At indicated time of incubation, ( a ) p-T239-Wee1 and Wee1 were probed on Wee1 immunoprecipitates (Ips), while Cdc20 on total samples. USP44 was analysed by mixing extracts with V5-USP44, immunopurified from transfected, SAC-arrested, cells before starting incubation and probing samples for V5 epitope. ( b ) Wee1 activity was assayed by mixing 3XFlag-tagged Wee1, immunopurified from transfected, SAC-arrested, cells with mock-depleted extract aliquots taken at time 0 and after a 4-h incubation at 23 °C and with Fcp1-depleted and 3F-Fcp1-reconstituted extracts taken after a 4-h incubation at 23 °C. After further 30 min incubation, kinase activity of reisolated Wee1 was assayed in vitro using recombinant, catalytic dead, cyclin B-cdk1(KR) complex as substrate. Samples taken at 0, 5 and 10 min of kinase reaction were probed with an anti-p-Y15-cdk1 antibody. ( c ) USP44 activity was assayed by mixing V5-USP44, immunopurified from transfected, SAC-arrested, cells with mock-depleted extract aliquots taken at time 0 and after a 4-h incubation at 23 °C and with Fcp1-depleted and 3F-Fcp1-reconstituted extracts taken after a 4-h incubation at 23 °C. After further 30 min incubation, deubiquitinating activity of reisolated V5-USP44 was assayed in vitro on polyubiquitin chains (3/7 Poly-Ub) as substrate. The reactions were probed with an anti-ubiquitin antibody, the positions of the mono-, di- and poly-ubiquitin forms are indicated. ( d ) Cdc20 and Mad2 were probed on Cdc20 Ips taken from mock-depleted, Fcp1-depleted and 3F-Fcp1-reconstituted samples taken at indicated incubation times (asterisk, Ig). Mad2 was also probed in total extract samples (Total Mad2). The bar graph shows the level of (open bar) Cdc20 and (closed bar) Mad2 in Cdc20 Ips expressed as percent of time 0 sample values. ( e ) Mock-depleted extracts were incubated for 30 min at 23 °C minus (lane 1) or plus (lane 2) the cdk1 inhibitor RO 3306, along with Fcp1-depleted extract plus RO 3306 (lane 3). MPM-2 was probed from total samples, other antigens analysed as described in a . The data shown are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibodies against phosphothreonine 239 of human Wee1 were raised using PQVNINPF(p)TPDSLL peptide as immunogen; peptide synthesis, rabbit inoculation, serum production and cross-affinity antibody purification were carried out by Polypeptide Group.

Techniques: Incubation, Transfection, Activity Assay, In Vitro, Recombinant, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Fcp1-dependent dephosphorylation is required for M-phase-promoting factor inactivation at mitosis exit

doi: 10.1038/ncomms1886

Figure Lengend Snippet: ( a ) Wee1, Cdc20 and Cdc27 were immunoprecipitated (Ip) from nocodazole-arrested HeLa cell samples and from cell samples taken after 120 min incubation from nocodazole wash out (NWO). USP44 was immunopurified from previously V5-USP44-transfected HeLa cells under analogous conditions. Ips from nocodazole-arrested cells were divided into three samples and incubated for 30 min at 23 °C with buffer, as control (Contr.), or with active recombinant 3F-Fcp1 in the absence (3F-Fcp1) or in the presence of 120 mM KCl (3F-Fcp1+KCl), a salt that inhibits Fcp1 catalytic activity . After incubation, the Ips were resolved, along with those from NWO cells, on SDS–PAGE and blots probed for the indicated antigens. ( b ) Wee1 was Ip from nocodazole-arrested HeLa cells. The Ip was split into five samples and incubated (lane 1) with Fcp1 phosphatase buffer, (lane 2) buffer+active 3F-Fcp1, (lane 3) buffer, (lane 4) buffer+inactive 3F-Fcp1-CD and (lane 5) buffer+active 3F-Fcp1+okadaic acid (o.a., 100 nM, a concentration shown to inhibit both PP2A and PP1 catalytic activities in vitro . After incubation, samples were split in two, resolved on parallel SDS–PAGE and blots probed for the indicated antigens. The bar graph shows densitometric quantification of the p-T239-Wee1 signal expressed as normalized percent of the value from control sample in lane 1. The data shown are representative of at least three independent experiments. ( c ) V5 Ips were performed from mock- (Mk) and V5-USP44-transfected (V5-USP44), nocodazole-arrested, HeLa cell lysates. The V5 Ip from V5-USP44-transfected cells was split into three portions and incubated with either buffer, recombinant 3F-Fcp1 or inactive 3F-Fcp1-CD for 30 min at 23 °C. V5-USP44 was reisolated and its deubiquitinating activity assayed in vitro as previously described, the positions of the mono-, di- and poly-ubiquitin (Poly-Ub) forms are indicated. ( d ) An anti-V5 blot from samples of Mk, wild-type (WT) V5-USP44- and mutant V5-USP44-4A-transfected SAC-arrested HeLa cells. ( e ) Deubiquitinating activity of wild-type V5-USP44 and mutant V5-USP44-4A, isolated from SAC-arrested HeLa cells.

Article Snippet: Rabbit polyclonal antibodies against phosphothreonine 239 of human Wee1 were raised using PQVNINPF(p)TPDSLL peptide as immunogen; peptide synthesis, rabbit inoculation, serum production and cross-affinity antibody purification were carried out by Polypeptide Group.

Techniques: Immunoprecipitation, Incubation, Transfection, Control, Recombinant, Activity Assay, SDS Page, Concentration Assay, In Vitro, Ubiquitin Proteomics, Mutagenesis, Isolation

Journal: Nature Communications

Article Title: Fcp1-dependent dephosphorylation is required for M-phase-promoting factor inactivation at mitosis exit

doi: 10.1038/ncomms1886

Figure Lengend Snippet: ( a ) SAC-arrested HeLa cells were released from arrest (nocodazole wash out; NWO) and sampled at indicated incubation time points. Top panel: Fcp1 and Cdc27 were probed on total extract samples. Bottom panel: Fcp1 and Cdc27 were probed on blots of Cdc27 Ips. ( b ) 48 h post-transfection, control (Cont.), non-targeting, or Cdc27-targeting siRNAs-transfected HeLa cells were treated with nocodazole for 14 h. SAC-arrested cells, that detached from the substrate, were released from arrest (nocodazole wash out; NWO), sampled at indicated incubation time points and lysates were directly probed for Cdc27, whereas p-T239-Wee1 and total Wee1 were probed on blots of Wee1 Ips. ( c ) Indirect fluorescence immunostaining of Fcp1 (red), and chromatin (blue) staining, of HeLa cells at the indicated mitotic stages. Scale bars, 5 μm.

Article Snippet: Rabbit polyclonal antibodies against phosphothreonine 239 of human Wee1 were raised using PQVNINPF(p)TPDSLL peptide as immunogen; peptide synthesis, rabbit inoculation, serum production and cross-affinity antibody purification were carried out by Polypeptide Group.

Techniques: Incubation, Transfection, Control, Fluorescence, Immunostaining, Staining