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Cell Signaling Technology Inc 9282s
9282s, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 6276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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9282s - by Bioz Stars, 2026-06

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p53 differentially regulates tamoxifen-induced proliferation of immortalized endometrial epithelial cells depending on its mutational status. (A, B) Immortalized human endometrial epithelial cells were stably transduced with lentiviral vectors encoding wild-type (WT) p53, mutant p53 (R248Q), or empty vector control. Western blotting was performed to confirm p53 expression levels using anti-p53 antibodies, with GAPDH as the loading control. Densitometric quantification of protein bands was conducted using ImageJ software. (C) Cells were treated with 4-hydroxytamoxifen (4-OHT, 1 μM) or vehicle (ethanol) for 72 hours, and cell viability was measured using the CCK-8 assay. Absorbance was recorded at 450 nm using a microplate reader. (D, E) For colony formation assays, 400 cells per well were seeded in six-well plates and cultured for 14 days in the presence or absence of 4-OHT (1 μM). Colonies were fixed with methanol, stained with 0.1% crystal violet, and counted using ImageJ software. All experiments were independently repeated at least three times (n = 3 biological replicates per group). P < 0.01; NS, not significant.

Journal: Frontiers in Oncology

Article Title: Tamoxifen differentially modulates endometrial hyperplasia via wild-type and mutant p53 regulation of the ALKBH5-REG1A axis

doi: 10.3389/fonc.2026.1784356

Figure Lengend Snippet: p53 differentially regulates tamoxifen-induced proliferation of immortalized endometrial epithelial cells depending on its mutational status. (A, B) Immortalized human endometrial epithelial cells were stably transduced with lentiviral vectors encoding wild-type (WT) p53, mutant p53 (R248Q), or empty vector control. Western blotting was performed to confirm p53 expression levels using anti-p53 antibodies, with GAPDH as the loading control. Densitometric quantification of protein bands was conducted using ImageJ software. (C) Cells were treated with 4-hydroxytamoxifen (4-OHT, 1 μM) or vehicle (ethanol) for 72 hours, and cell viability was measured using the CCK-8 assay. Absorbance was recorded at 450 nm using a microplate reader. (D, E) For colony formation assays, 400 cells per well were seeded in six-well plates and cultured for 14 days in the presence or absence of 4-OHT (1 μM). Colonies were fixed with methanol, stained with 0.1% crystal violet, and counted using ImageJ software. All experiments were independently repeated at least three times (n = 3 biological replicates per group). P < 0.01; NS, not significant.

Article Snippet: For immunoprecipitation, chromatin equivalent to approximately 1 × 10 6 cells (corresponding to ~500 μg of DNA–protein complex) was incubated overnight at 4 °C with 5 μg of anti-p53 antibody (Cell Signaling Technology, #9282) or normal rabbit IgG (Cell Signaling Technology, #2729) as a negative control.

Techniques: Stable Transfection, Transduction, Mutagenesis, Plasmid Preparation, Control, Western Blot, Expressing, Software, CCK-8 Assay, Cell Culture, Staining

P53 mutational status determines REG1A expression in response to tamoxifen. (A, B) Immortalized human endometrial epithelial cells were stably transduced with lentiviral vectors expressing wild-type (WT) p53, mutant p53 (R248Q), or empty vector control. Cells were treated with 4-hydroxytamoxifen (4-OHT, 1 μM) for 48 h, and REG1A protein and mRNA levels were analyzed by Western blotting and qRT-PCR. (C, D) To confirm p53 dependence, cells were transfected with siRNA targeting p53 (si-p53) prior to 4-OHT treatment. Silencing of p53 abolished the differential regulation of REG1A at both the protein (C) and mRNA (D) levels induced by WT or mutant p53. P < 0.05, P < 0.01.

Journal: Frontiers in Oncology

Article Title: Tamoxifen differentially modulates endometrial hyperplasia via wild-type and mutant p53 regulation of the ALKBH5-REG1A axis

doi: 10.3389/fonc.2026.1784356

Figure Lengend Snippet: P53 mutational status determines REG1A expression in response to tamoxifen. (A, B) Immortalized human endometrial epithelial cells were stably transduced with lentiviral vectors expressing wild-type (WT) p53, mutant p53 (R248Q), or empty vector control. Cells were treated with 4-hydroxytamoxifen (4-OHT, 1 μM) for 48 h, and REG1A protein and mRNA levels were analyzed by Western blotting and qRT-PCR. (C, D) To confirm p53 dependence, cells were transfected with siRNA targeting p53 (si-p53) prior to 4-OHT treatment. Silencing of p53 abolished the differential regulation of REG1A at both the protein (C) and mRNA (D) levels induced by WT or mutant p53. P < 0.05, P < 0.01.

Techniques: Expressing, Stable Transfection, Transduction, Mutagenesis, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Transfection

Tamoxifen induces p53 binding to the ALKBH5 promoter and regulates ALKBH5 transcription in a p53-allele–dependent manner. (A, B) Immortalized human endometrial epithelial cells stably expressing wild-type (WT) p53 or the R248Q mutant were treated with 4-hydroxytamoxifen (4-OHT, 1 μM) for 48 h. ALKBH5 mRNA (A) and protein (B) levels were analyzed by qRT-PCR and Western blotting, respectively. (C) Schematic representation of the ALKBH5 promoter showing predicted p53-binding sites identified by bioinformatic analysis. (D) Chromatin immunoprecipitation (ChIP) assays using anti-p53 antibodies were performed to assess 4-OHT–induced recruitment of WT or mutant p53 to the ALKBH5 promoter. (E, F) Luciferase reporter assays were performed using ALKBH5 promoter–driven luciferase constructs containing either the wild-type promoter or promoters with mutations in the predicted p53-binding sites. Cells expressing wild-type (WT) p53 or the R248Q mutant were treated with 4-hydroxytamoxifen (4-OHT) for 24 h. Firefly luciferase activity was normalized to Renilla luciferase activity. (G) Cells expressing WT or mutant p53 were transfected with control siRNA (si-NC) or p53-targeting siRNA (si-p53) prior to 4-OHT treatment. ALKBH5 mRNA levels were quantified by qRT-PCR to assess p53 dependency at the transcriptional level. Data are presented as mean ± SD from at least three independent experiments. P < 0.05, P < 0.01.

Journal: Frontiers in Oncology

Article Title: Tamoxifen differentially modulates endometrial hyperplasia via wild-type and mutant p53 regulation of the ALKBH5-REG1A axis

doi: 10.3389/fonc.2026.1784356

Figure Lengend Snippet: Tamoxifen induces p53 binding to the ALKBH5 promoter and regulates ALKBH5 transcription in a p53-allele–dependent manner. (A, B) Immortalized human endometrial epithelial cells stably expressing wild-type (WT) p53 or the R248Q mutant were treated with 4-hydroxytamoxifen (4-OHT, 1 μM) for 48 h. ALKBH5 mRNA (A) and protein (B) levels were analyzed by qRT-PCR and Western blotting, respectively. (C) Schematic representation of the ALKBH5 promoter showing predicted p53-binding sites identified by bioinformatic analysis. (D) Chromatin immunoprecipitation (ChIP) assays using anti-p53 antibodies were performed to assess 4-OHT–induced recruitment of WT or mutant p53 to the ALKBH5 promoter. (E, F) Luciferase reporter assays were performed using ALKBH5 promoter–driven luciferase constructs containing either the wild-type promoter or promoters with mutations in the predicted p53-binding sites. Cells expressing wild-type (WT) p53 or the R248Q mutant were treated with 4-hydroxytamoxifen (4-OHT) for 24 h. Firefly luciferase activity was normalized to Renilla luciferase activity. (G) Cells expressing WT or mutant p53 were transfected with control siRNA (si-NC) or p53-targeting siRNA (si-p53) prior to 4-OHT treatment. ALKBH5 mRNA levels were quantified by qRT-PCR to assess p53 dependency at the transcriptional level. Data are presented as mean ± SD from at least three independent experiments. P < 0.05, P < 0.01.

Techniques: Binding Assay, Stable Transfection, Expressing, Mutagenesis, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Luciferase, Construct, Activity Assay, Transfection, Control

Tamoxifen regulates endometrial epithelial cell proliferation through a p53-dependent ALKBH5–REG1A axis. (A, B) Western blot and qRT-PCR analyses of ALKBH5, p53, and REG1A expression in immortalized human endometrial epithelial cells expressing wild-type (WT) or mutant p53 (R248Q). Cells were further subjected to ALKBH5 knockdown (WT p53 + ALKBH5 shRNA) or ALKBH5 overexpression (R248Q p53 + ALKBH5 OE), as indicated, and treated with vehicle or 4-hydroxytamoxifen (4-OHT, 1 μM) for 48 h. (C, D) Cell proliferation was assessed by CCK-8 assays, and long-term clonogenic capacity was evaluated by colony formation assays under the indicated genetic manipulations and 4-OHT treatment. ALKBH5 and REG1A knockdown or overexpression was achieved using lentiviral shRNA or overexpression constructs. Data are presented as mean ± SD from three independent experiments. P < 0.05, P < 0.01, P < 0.001.

Journal: Frontiers in Oncology

Article Title: Tamoxifen differentially modulates endometrial hyperplasia via wild-type and mutant p53 regulation of the ALKBH5-REG1A axis

doi: 10.3389/fonc.2026.1784356

Figure Lengend Snippet: Tamoxifen regulates endometrial epithelial cell proliferation through a p53-dependent ALKBH5–REG1A axis. (A, B) Western blot and qRT-PCR analyses of ALKBH5, p53, and REG1A expression in immortalized human endometrial epithelial cells expressing wild-type (WT) or mutant p53 (R248Q). Cells were further subjected to ALKBH5 knockdown (WT p53 + ALKBH5 shRNA) or ALKBH5 overexpression (R248Q p53 + ALKBH5 OE), as indicated, and treated with vehicle or 4-hydroxytamoxifen (4-OHT, 1 μM) for 48 h. (C, D) Cell proliferation was assessed by CCK-8 assays, and long-term clonogenic capacity was evaluated by colony formation assays under the indicated genetic manipulations and 4-OHT treatment. ALKBH5 and REG1A knockdown or overexpression was achieved using lentiviral shRNA or overexpression constructs. Data are presented as mean ± SD from three independent experiments. P < 0.05, P < 0.01, P < 0.001.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Mutagenesis, Knockdown, shRNA, Over Expression, CCK-8 Assay, Construct

Schematic illustration of the p53-dependent mechanism governing endometrial responses to tamoxifen. (Left) In the presence of wild-type (WT) p53, tamoxifen stimulation promotes the recruitment of WT p53 to the ALKBH5 promoter, thereby transcriptionally activating ALKBH5. Upregulation of the m 6 A demethylase ALKBH5 facilitates the removal of m 6 A modifications from REG1A mRNA, preventing YTHDF2-mediated mRNA decay. Stabilization of REG1A leads to increased protein abundance, which functions as a negative feedback regulator to constrain endometrial cell proliferation. (Right) In contrast, mutant p53 (R248Q) fails to transactivate ALKBH5 in response to tamoxifen. The resultant reduction in ALKBH5 expression causes increased m 6 A methylation of REG1A mRNA, enhancing its recognition and degradation by YTHDF2. Loss of REG1A expression abolishes this proliferative restraint, ultimately driving tamoxifen-induced endometrial hyperplasia.

Journal: Frontiers in Oncology

Article Title: Tamoxifen differentially modulates endometrial hyperplasia via wild-type and mutant p53 regulation of the ALKBH5-REG1A axis

doi: 10.3389/fonc.2026.1784356

Figure Lengend Snippet: Schematic illustration of the p53-dependent mechanism governing endometrial responses to tamoxifen. (Left) In the presence of wild-type (WT) p53, tamoxifen stimulation promotes the recruitment of WT p53 to the ALKBH5 promoter, thereby transcriptionally activating ALKBH5. Upregulation of the m 6 A demethylase ALKBH5 facilitates the removal of m 6 A modifications from REG1A mRNA, preventing YTHDF2-mediated mRNA decay. Stabilization of REG1A leads to increased protein abundance, which functions as a negative feedback regulator to constrain endometrial cell proliferation. (Right) In contrast, mutant p53 (R248Q) fails to transactivate ALKBH5 in response to tamoxifen. The resultant reduction in ALKBH5 expression causes increased m 6 A methylation of REG1A mRNA, enhancing its recognition and degradation by YTHDF2. Loss of REG1A expression abolishes this proliferative restraint, ultimately driving tamoxifen-induced endometrial hyperplasia.

Techniques: Quantitative Proteomics, Mutagenesis, Expressing, Methylation

(A) Schematic of full genome CRISPR screens. After four weeks of growth with no selection pressure, gRNAs selectively lost in WT but not p53 -/- and USP28 -/- cells were used to identify genes whose functions are guarded or masked by EMS. (B) GO Cellular Component Enrichment Analysis on the 79 genes that were considered essential in the WT but neutral or near neutral in p53 -/- and USP28 -/- backgrounds. The RZZ complex was revealed as a top hit, with two of three components, KNTC1 (ROD) and ZWILCH, identified. (C) Growth curve analyses showed that following induction of shRNA-KNTC1, WT (black) cells were fully arrested, while p53 -/- (red), USP28 -/- (green), and 53BP1 -/- (blue) cells continued to grow, albeit at slightly slower rates compared to controls (the respective shRNA clonal line without Dox induction). Data are mean ± SD, N = 3, initial cell count = 100,000, and doxycycline (Dox) at 1 μg/mL. (D) BrdU labeling assays revealed that KNTC1 knockdown has a widespread, detrimental growth impact on WT cells (left) but not p53 -/- , USP28-/-, and 53BP1 -/- cells (center), compared to the non-targeting shRNA control (shRNA-NTC, right). Note that KNTC1 knockdown does have a non-population-level growth impact on EMS-knockout cells ( p53 -/- , USP28 -/- , or 53BP1 -/- ), consistent with (C). Cells were fixed at the end of indicated days, with BrdU (10 μM) added 24 hours prior to fixation. Data are mean ± SD, n > 300, N = 3, statistical significance calculated via T-test with Welch’s correction. (E) Quantification of nuclear p21 intensity at indicated days following knockdown of KNTC1 in WT (left), USP28 -/- (right), or 53BP1 -/- (right) cells. At Day 10 after KNTC1 knockdown, ∼70% of WT cells showed a nuclear p21 intensity that is 3 standard deviations above the mean in control cells, which are defined as high outliers (above dashed blue lines). In contrast, only 10-11% of USP28 -/- or 53BP1 -/- cells were high outliers by Day 10, consistent with the observation that KNTC1 loss has a mild, non-population-level growth impact on EMS-knockout cells. Data are mean signal intensity, n > 240. Black lines indicating median, dashed blue line marking 3 standard deviations above the control mean, and statistical significance calculated via T-test with Welch’s correction.

Journal: bioRxiv

Article Title: Fidelity-Ensuring Consistency of Mitosis is Safeguarded by the 53BP1-USP28-p53 Pathway

doi: 10.64898/2026.03.16.712228

Figure Lengend Snippet: (A) Schematic of full genome CRISPR screens. After four weeks of growth with no selection pressure, gRNAs selectively lost in WT but not p53 -/- and USP28 -/- cells were used to identify genes whose functions are guarded or masked by EMS. (B) GO Cellular Component Enrichment Analysis on the 79 genes that were considered essential in the WT but neutral or near neutral in p53 -/- and USP28 -/- backgrounds. The RZZ complex was revealed as a top hit, with two of three components, KNTC1 (ROD) and ZWILCH, identified. (C) Growth curve analyses showed that following induction of shRNA-KNTC1, WT (black) cells were fully arrested, while p53 -/- (red), USP28 -/- (green), and 53BP1 -/- (blue) cells continued to grow, albeit at slightly slower rates compared to controls (the respective shRNA clonal line without Dox induction). Data are mean ± SD, N = 3, initial cell count = 100,000, and doxycycline (Dox) at 1 μg/mL. (D) BrdU labeling assays revealed that KNTC1 knockdown has a widespread, detrimental growth impact on WT cells (left) but not p53 -/- , USP28-/-, and 53BP1 -/- cells (center), compared to the non-targeting shRNA control (shRNA-NTC, right). Note that KNTC1 knockdown does have a non-population-level growth impact on EMS-knockout cells ( p53 -/- , USP28 -/- , or 53BP1 -/- ), consistent with (C). Cells were fixed at the end of indicated days, with BrdU (10 μM) added 24 hours prior to fixation. Data are mean ± SD, n > 300, N = 3, statistical significance calculated via T-test with Welch’s correction. (E) Quantification of nuclear p21 intensity at indicated days following knockdown of KNTC1 in WT (left), USP28 -/- (right), or 53BP1 -/- (right) cells. At Day 10 after KNTC1 knockdown, ∼70% of WT cells showed a nuclear p21 intensity that is 3 standard deviations above the mean in control cells, which are defined as high outliers (above dashed blue lines). In contrast, only 10-11% of USP28 -/- or 53BP1 -/- cells were high outliers by Day 10, consistent with the observation that KNTC1 loss has a mild, non-population-level growth impact on EMS-knockout cells. Data are mean signal intensity, n > 240. Black lines indicating median, dashed blue line marking 3 standard deviations above the control mean, and statistical significance calculated via T-test with Welch’s correction.

Article Snippet: The following antibodies were used in this study, with the company, catalog number, species/immunotype, and working concentration used noted in parenthesis: anti-BRDU (BioRad, MCA6144, rat, 1:500), anti-BUBR1 (Abcam, ab6437, mouse IgG 1, 1:400, note – discontinued), anti-Centrin (Millipore-Sigma, 04-1624, mouse IgG 2a, 1:1000), anti-CREST (Immunovision hct-0100, human, 1:50,000), anti-KNTC1 (Santa-Cruz, sc-81853, mouse IgG 2a, 1:50), anti-p21 (Abcam, ab7960, rabbit, 1:200), anti-p53 (Cell Signaling Technology, 9282S, rabbit, 1:400), anti-USP28 (Invitrogen, PA5-52346, rabbit, 1:200), anti-53BP1 (Millipore-Sigma, MAB3802, mouse IgG 1, 1:400), and anti-γH2AX (Millipore-Sigma, 05-636, mouse IgG 1, 1:200).

Techniques: CRISPR, Selection, shRNA, Cell Characterization, Labeling, Knockdown, Control, Knock-Out

(A) Analyses of clonal isolations for cells undergoing CRISPR-mediated KNTC1 targeting in p53 -/- , USP28 -/- , 53BP1 -/- , and WT backgrounds. The table starts with the total number of clones examined, among which how many were visibly arrested. Also tabulated are numbers of clones checked for KNTC1 staining at early mitotic kinetochores, of which how many exhibited normal or disrupted (reduced or absent) signals. Indicated numbers of clones with disrupted signals were further selected for sequencing, determining if they are heterozygous or homozygous KNTC1 knockouts. (B) Representative images of normal, reduced, and absent KNTC1 signals in p53 -/- ; KNTC1 +/+ (WT), p53 -/- ; KNTC1 +/- (heterozygous knockout), and p53 -/- ; KNTC1 -/- (homozygous knockout), respectively. (C) Quantification of mitotic durations prior to slippage in the presence of nocodazole. KNTC1 +/+ cells in both p53 -/- and WT backgrounds could arrest in nocodazole-treated mitosis for a mean duration of 11.5 and 10.7 hours, respectively, before slippage. In contrast, both KNTC1 +/- (heterozygous) and KNTC1 -/- (homozygous) cells in the p53 -/- background exited mitosis after a short arrest of 1.8 hours, indicating a severe disruption of SAC. The singular KNTC1 +/- clone (Singleton-Het) survived in the WT background, however, could stay in mitotic arrest for a mean of 8.3 hours, indicating a mildly compromised SAC. n > 200, nocodazole added at 50 ng/mL. (D) Quantification of normal mitotic duration in the absence of nocodazole perturbation. KNTC1 +/- and KNTC1 -/- cells obtained from the p53 -/- background showed a small increase in average mitotic duration from 23 to 34 and 36 minutes, respectively, suggesting the presence of sub-optimal kinetochore function. The singular heterozygous KNTC1 clone survived in the WT background in our screen showed no significant increase in mitotic duration, remaining at 24 minutes, suggesting the presence of functional kinetochores or SAC. n > 200, statistical significance calculated via T-test with Welch’s correction.

Journal: bioRxiv

Article Title: Fidelity-Ensuring Consistency of Mitosis is Safeguarded by the 53BP1-USP28-p53 Pathway

doi: 10.64898/2026.03.16.712228

Figure Lengend Snippet: (A) Analyses of clonal isolations for cells undergoing CRISPR-mediated KNTC1 targeting in p53 -/- , USP28 -/- , 53BP1 -/- , and WT backgrounds. The table starts with the total number of clones examined, among which how many were visibly arrested. Also tabulated are numbers of clones checked for KNTC1 staining at early mitotic kinetochores, of which how many exhibited normal or disrupted (reduced or absent) signals. Indicated numbers of clones with disrupted signals were further selected for sequencing, determining if they are heterozygous or homozygous KNTC1 knockouts. (B) Representative images of normal, reduced, and absent KNTC1 signals in p53 -/- ; KNTC1 +/+ (WT), p53 -/- ; KNTC1 +/- (heterozygous knockout), and p53 -/- ; KNTC1 -/- (homozygous knockout), respectively. (C) Quantification of mitotic durations prior to slippage in the presence of nocodazole. KNTC1 +/+ cells in both p53 -/- and WT backgrounds could arrest in nocodazole-treated mitosis for a mean duration of 11.5 and 10.7 hours, respectively, before slippage. In contrast, both KNTC1 +/- (heterozygous) and KNTC1 -/- (homozygous) cells in the p53 -/- background exited mitosis after a short arrest of 1.8 hours, indicating a severe disruption of SAC. The singular KNTC1 +/- clone (Singleton-Het) survived in the WT background, however, could stay in mitotic arrest for a mean of 8.3 hours, indicating a mildly compromised SAC. n > 200, nocodazole added at 50 ng/mL. (D) Quantification of normal mitotic duration in the absence of nocodazole perturbation. KNTC1 +/- and KNTC1 -/- cells obtained from the p53 -/- background showed a small increase in average mitotic duration from 23 to 34 and 36 minutes, respectively, suggesting the presence of sub-optimal kinetochore function. The singular heterozygous KNTC1 clone survived in the WT background in our screen showed no significant increase in mitotic duration, remaining at 24 minutes, suggesting the presence of functional kinetochores or SAC. n > 200, statistical significance calculated via T-test with Welch’s correction.

Techniques: CRISPR, Clone Assay, Staining, Sequencing, Knock-Out, Disruption, Functional Assay

(A & B) Cells arrested at G2 by RO3306 (9.5 μM) were released into mitosis under the indicated conditions for the indicated amounts of time before fixation for PLA analysis of complex formation. In control cells no increase in the total area ( A ) or number ( B ) of PLA foci was seen up to 40 minutes after RO3306 washout (first panel). In the presence of nocodazole (50 ng/mL), an increase in both the area and number of foci was seen between 40 and 80 minutes after washout (second panel) but not before. Following inducible KNTC1 knockdown (Day 3 under doxycycline), an increase in both the area and number of PLA foci was detectable between 10 and 20 minutes after washout, with an additional significant increase between 20 and 30 minutes (third panel). PLA reactions were carried out using primary antibodies against endogenous 53BP1 and p53, n > 75, area quantified as the number of pixels above the background threshold, and statistical significances calculated via T-tests with Welch’s correction. Similar PLA results were seen with antibodies against 53BP1 and USP28 (data not shown; see methods). (C) Mitotic durations of WT cells treated with low-dose ProTAME (2 μM) resembled those of KNTC1-knockdown cells (left) but did not induce fast formation of 53BP1-USP28-p53 complexes in early mitosis (middle and right). The mean duration in both conditions is ∼36 minutes with no significant difference in distributions (calculated by T-test with Welch’s correction). Cells arrested at G2 with RO3306 (9.5 μM) were released into mitosis in the presence of ProTAME (2 μM) for the indicated amounts of time, fixed, and examined by PLA with primary antibodies against endogenous 53BP1 and p53. No increase in total area (middle) or number (right) of PLA foci was seen up to 40 minutes after RO3306 washout. n > 75, statistical significances calculated via T-tests with Welch’s correction.

Journal: bioRxiv

Article Title: Fidelity-Ensuring Consistency of Mitosis is Safeguarded by the 53BP1-USP28-p53 Pathway

doi: 10.64898/2026.03.16.712228

Figure Lengend Snippet: (A & B) Cells arrested at G2 by RO3306 (9.5 μM) were released into mitosis under the indicated conditions for the indicated amounts of time before fixation for PLA analysis of complex formation. In control cells no increase in the total area ( A ) or number ( B ) of PLA foci was seen up to 40 minutes after RO3306 washout (first panel). In the presence of nocodazole (50 ng/mL), an increase in both the area and number of foci was seen between 40 and 80 minutes after washout (second panel) but not before. Following inducible KNTC1 knockdown (Day 3 under doxycycline), an increase in both the area and number of PLA foci was detectable between 10 and 20 minutes after washout, with an additional significant increase between 20 and 30 minutes (third panel). PLA reactions were carried out using primary antibodies against endogenous 53BP1 and p53, n > 75, area quantified as the number of pixels above the background threshold, and statistical significances calculated via T-tests with Welch’s correction. Similar PLA results were seen with antibodies against 53BP1 and USP28 (data not shown; see methods). (C) Mitotic durations of WT cells treated with low-dose ProTAME (2 μM) resembled those of KNTC1-knockdown cells (left) but did not induce fast formation of 53BP1-USP28-p53 complexes in early mitosis (middle and right). The mean duration in both conditions is ∼36 minutes with no significant difference in distributions (calculated by T-test with Welch’s correction). Cells arrested at G2 with RO3306 (9.5 μM) were released into mitosis in the presence of ProTAME (2 μM) for the indicated amounts of time, fixed, and examined by PLA with primary antibodies against endogenous 53BP1 and p53. No increase in total area (middle) or number (right) of PLA foci was seen up to 40 minutes after RO3306 washout. n > 75, statistical significances calculated via T-tests with Welch’s correction.

Techniques: Control, Knockdown

Representative images of PLA foci using primary antibodies against endogenous p53 and 53BP1 to visualize 53BP1-USP28-p53 complexes in mitosis. Cells were arrested at G2 by RO3306 and released into mitosis under indicated conditions for indicated amounts of time before being fixed and examined by PLA. Cells were released into mitosis without additional drugs (control), in the presence of nocodazole (NOC, 50 ng/mL) or ProTAME (2 μM), or following KNTC1-knockdown (doxycycline 1 μg/mL, Day 3) as indicated.

Journal: bioRxiv

Article Title: Fidelity-Ensuring Consistency of Mitosis is Safeguarded by the 53BP1-USP28-p53 Pathway

doi: 10.64898/2026.03.16.712228

Figure Lengend Snippet: Representative images of PLA foci using primary antibodies against endogenous p53 and 53BP1 to visualize 53BP1-USP28-p53 complexes in mitosis. Cells were arrested at G2 by RO3306 and released into mitosis under indicated conditions for indicated amounts of time before being fixed and examined by PLA. Cells were released into mitosis without additional drugs (control), in the presence of nocodazole (NOC, 50 ng/mL) or ProTAME (2 μM), or following KNTC1-knockdown (doxycycline 1 μg/mL, Day 3) as indicated.

Techniques: Control, Knockdown

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