Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a IB analysis of WCLs derived from HEK293T cells transfected with His-E4F1 and increasing doses of Flag-PA28γ. b Quantitative real-time PCR analysis of the indicated genes in HEK293T cells transfected with EV and increasing doses of Flag-PA28γ. Endo., endogenous. c HSC-3 cells stably expressing EV or Flag-PA28γ were treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of E4F1 levels relative to tubulin levels is shown. d , e HEK293T cells stably expressing EV or Flag-PA28γ were transfected with Myc-E4F1 truncations, and treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of Myc-E4F1 levels relative to tubulin levels is shown. f HEK293T cells transfected with the indicated plasmids were treated without or with 10 μM MG132 for 6 h before harvesting, and WCLs were collected for IB analysis. g HEK293T cells stably expressing shRNA against endogenous PA28γ were transfected with the indicated plasmids, and WCLs were collected for IB analysis. h Purified PA28γ, E4F1 and 20S proteasome in the absence or presence of 100 nM proteasome inhibitor epoxomicin (Epox) were incubated as indicated for 45 min, followed by IB analysis. i Purified PA28γ WT, PA28γ-T23A, E4F1, p21 (as a positive control for E4F1) and 20S proteasome were incubated as indicated for 45 min, followed by IB analysis. Data in ( b – e ) represent the mean ± SD of three biological replicates; statistical significance was assessed by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human E4F1 protein (H00001877-P01) and recombinant human p21/CDKN1A protein (NBP2-22976) were purchased from Novus Biologicals.

Techniques: Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, shRNA, Purification, Incubation, Positive Control

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Immunohistochemistry with primary anti-p21 antibody with positive reaction visible as brown staining. A – weak nuclear reaction in a grade 1 mast cell tumour (MCT); B – moderate nuclear reaction in a grade 2 MCT; C – strong nuclear reaction in a grade 3 MCT

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques: Immunohistochemistry, Staining

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Frequency of immunoreactivity for p21 protein in grade 1, 2 and 3 mast cell tumours (MCTs). * – P-value < 0.05; **_ P-value < 0.01; *** P-value < 0.001 compared to grade 1 MCTs; (−) – no reaction; (+) – weak reaction; (++) – moderate reaction; (+++) – strong reaction

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques:

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: p21 in MDA‐MB‐231 cells treated with abemaciclib and/or ABT‐263. (A, B) Cancer cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 2 days. After harvesting, the cytoplasmic and nuclear fractions were separated and subjected to immunoblotting. TBP and GAPDH were used as controls. (C) Immunoblotting was performed similarly using whole lysates. (D) MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) with zVAD (10 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. The numbers are proportions of the subsets. (Left) Data of the means ± SD of three cells are shown. * p < 0.05, ** p < 0.01. (E) MDA‐MB‐231 cells were treated similarly with zVAD (10 µM) for 24 h and subjected to immunoblotting. (F) Untreated MDA‐MB‐231 cells were cultured with Hoechst 33342 (5 µg/ml) and stained with anti‐p21 and anti‐caspase‐3 antibodies followed by an Alexa 488‐conjugated anti‐rabbit antibody and Cy5‐conjugated anti‐mouse IgG. Confocal imaging reveals nuclei (blue), p21 (green), and caspse‐3 (white). Scale, 10 µm. (G) Control and p21‐overexpressing MDA‐MB‐231 cells were examined for their p21 expression by immunoblotting. (H) Control and p21‐overexpressing MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐APC. Numbers are the proportions of the subset (Left). The means ± SD of four wells are shown. ** p < 0.01. (I) Cancer cells were treated with abemaciclib (1.5 µM) for 24 h, and the cell lysates were subjected to immunoblot to examine the expression of p21 and c‐Myc

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Western Blot, Staining, Cell Culture, Imaging, Control, Expressing

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Effect of genetic knockdown of p21 in MCF‐7 cells on their sensitivity to drugs. (A) siRNA‐transfected cancer cells were cultured for 48 h and subjected to immunoblotting. (B) MDA‐MB‐231 and MCF‐7 cells transfected with siRNA p21(I) 2 days prior were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 48 h. After staining with annexin V‐FITC and PI, flow cytometric analysis was performed. Data are the means of three wells. * p < 0.05, ** p < 0.01. (C) Representative flow cytometry results; numbers are percentages of the subsets. (D) MDA‐MB‐231 cells were cultured with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) in the presence of caspase inhibitors (10 µM) for 48 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. Data are the means of three wells. ** p < 0.01. (E) siRNA p21(I)‐transfected MDA‐MB‐231 and MCF‐7 cells were cultured with TRAIL for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. ** p < 0.01

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Knockdown, Transfection, Cell Culture, Western Blot, Staining, Flow Cytometry

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Poor prognosis of breast cancer patients with p21 high compared with those with p21 low . (A, B, C) Kaplan–Meier plotter univariate analysis of survival time in CDKN1A mRNA expression in breast cancer. Version 2021 of the database was used for analysis. Outlier array data were excluded for array quality control. Patients were split into low‐ and high‐expression groups based on the optimal cutoff. (D, E) TRGAted was used for survival analysis according to p21 protein level in patients with invasive breast carcinoma. All subtypes of TCGA‐BRCA‐L4 were used in the analysis. Patients were split into low‐ and high‐expression groups based on the optimal cutoff

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Expressing, Control

Journal: Experimental cell research

Article Title: KIF20A promotes the development of fibrosarcoma via PI3K-Akt signaling pathway.

doi: 10.1016/j.yexcr.2022.113322

Figure Lengend Snippet: Fig. 2. KIF20A downregulation inhibits cell prolifer ation, migration and invasion in vitro. (A) After Kif20a gene knockdown, cell cycle was measured by flow cytometry. (B) KIF20A, Cyclins and cyclin-dependent kinase inhibitor p21Waf1/Cip1 levels were measured by Western blotting. GAPDH was used as an internal control. (C) Cell proliferation was evaluated by CCK-8 assay at 12, 24, 36, and 48 h. (D) Cell proliferation was evaluated by cell clone formation assay. (E) Cell migration was measured via Transwell assay. (F) Cell invasion was measured via Transwell assay, and matrigel was added to the upper chambers of Trans well. Scale bar represents 10 μm. The cells used were stably-transfected HT-1080. Data are representative of 3 independent experiments. *: p < 0.05, **: p < 0.01, ***p < 0.001.

Article Snippet: The primary antibody for KIF20A was purchased from Santa Cruz (USA); GAPDH, β-Actin, Phospho-Akt Pathway Antibody Sampler Kit #9916, NF-κB Pathway Sampler Kit #9936, PI3K, p21 Waf1/Cip1 (DCS60), Cyclin E1, Cyclin A2, Caspase 3, Caspase 7, Caspase 8, Caspase 9, Bax, and Bcl-2 were purchased from Cell Signaling Technology (USA).

Techniques: Migration, In Vitro, Knockdown, Flow Cytometry, Western Blot, Control, CCK-8 Assay, Tube Formation Assay, Transwell Assay, Stable Transfection, Transfection

Journal: International Journal of Biological Sciences

Article Title: BET inhibition induces synthetic lethality in PTEN deficient colorectal cancers via dual action on p21 CIP1/WAF1

doi: 10.7150/ijbs.91867

Figure Lengend Snippet: BETi reduced p21 phosphorylation at Thr145 and promoted p21 nucleus importing by repressing AKT in PTEN-deficient cells. A, B Western blot illustrating AKT, p-AKT (ser473), p21, p-p21(thr145) levels in HCT116 ( A ) and DLD1 ( B ) PTEN isogenic cell pairs via treatment with OTX-015 for 24 h. C Immunofluorescence analysis of p21 localization inHCT116 PTEN isogenic cells treated with or without BETi. D Endogenous p21 in HCT116 PTEN-deficient cells were repressed by siRNA and the cells were transfected with Thr145 mutant p21 (p21-T145D) and wild type p21 (p21-WT) respectively. The transfected cells were treated with OTX-015 or DMSO for 24 h. The percentages of cell confluence were shown. E Cell images represented the cell confluence taken by Incucyte Zoom.

Article Snippet: pCDH-puro-MYC plasmid was a gift from Jialiang Wang (Addgene plasmid #46970) , and Flag p21 WT and T145D plasmids were gifts from Mien-Chie Hung (Addgene, plasmid # 16240 and 16242) .

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence, Transfection, Mutagenesis

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High p21 expression correlates with poor survival in breast cancer patients . A-C , The relationship of higher p21 expression and breast cancer outcome was assessed by Kaplan-Meier survival analysis. The survival rates were analyzed using three different and separate splits of the patients, based on p21 expression levels (A, median; B, upper quartile; C, lower quartile). For each split, the patients were then divided into high and low groups with respect to p21 expression. Overall survival (left panels) and distant metastasis-free survival (right panels) correlate with p21 gene expression. Number of breast cancer patients at risk with higher expression (red) and lower expression (black) of p21 at the indicated time points.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Gene Expression

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Silencing p21 prevents breast tumor local invasion in vivo and cancer cell migration and invasion . A , Total lysates from parental and shRNA p21 SCP2 cells were analyzed by immunoblotting for the protein levels of p21 and β-tubulin. B , Parental and shRNA p21 SCP2 cells were injected into the mammary glands of four- to six-week-old female Balb/c nude mice. The size of mammary tumor was measured from two sets of mice (eight per group; error bars indicate SEM). C , Representative photographs show hematoxylin and eosin staining of the mammary gland (tumor and fat pad) of 12- to 15-month-old mice. D and E , Transwell cell migration (left panel) and GFR-Matrigel invasion assay (right panel) of parental and shRNA p21 SCP2 cells were performed. Graphs show total migrated and invaded cell number counted by Image J (error bars indicate SEM; n = 3 independent experiments). Student's unpaired t -test was used to compare parental vs. shRNA p21 SCP2 groups. Differences were considered significant at * P < 0.05.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: In Vivo, Migration, shRNA, Western Blot, Injection, Staining, Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: TGFβ induces p21 expression in migratory and invasive human breast cancer cells . A , Real-time PCR was performed to measure the mRNA level of p21 gene (error bars indicate SD; n = 3 independent experiments) for the indicated cell lines. B , Cells were treated with or without 5 ng/ml TGFβ for the indicated times. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. C , Total cell lysates were analyzed for c-myc, p15 and β-tubulin protein levels by Western blotting. D , SCP25 cells were pretreated with 10 µM TGFβ type I receptor (TβRI) inhibitor (SB431542) or vehicle (DMSO) for 30 minutes and then stimulated with TGFβ. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. E , MDA cells were transfected with 40 nM Scrambled (Scr), Smad2 or Smad3 siRNAs in response to TGFβ. Total cell lysates were analyzed for Smad2/3, p21 and β-tubulin protein levels by Western blotting.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell migration . A , Representative images of phase contrast and wound mask of indicated cell lines stimulated with TGFβ in scratch/wound healing assay. The initial wound mask (black) and wound closure (grey) were measured using the Essen Instruments Scratch Wound Module. B , The time course of cell migration for the indicated cell lines was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with Scr or p21 siRNAs and then stimulated with or without TGFβ for 24 hrs. Total cell lysates were analyzed for p21 and β-tubulin by Western blotting. D , Representative images of phase contrast (top panels) and wound mask (bottom panels) of transfected SCP2 cells with the indicated siRNAs in scratch/wound healing assay. E , The time course of transfected SCP2 cell migration was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Migration, Wound Healing Assay, Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell invasion . A , SCP2 cells were transfected with the indicated siRNAs and cultured in the presence or absence of TGFβ. GFR-Matrigel coated Transwell invasion assay was performed and images of the invading cells were photographed. B , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments). C , SCP2 cells were transfected with a Scr or p21 siRNA as well as a flag-tagged p21 cDNA in the presence or the absence of TGFβ. p21 protein levels were then analyzed by Western blotting. D , Cell invasion was assessed using the Transwell Invasion assay. The number of invaded cells was counted by Image J (error bars indicate SD; n = 3 independent experiments). E , Cell invasion of transfected SUM159 was assessed using the Transwell Invasion assay. F , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Cell Culture, Transwell Invasion Assay, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 interacts with Smad3 and modulates TGFβ-induced transcriptional activity . A , HEK293 and SCP2 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21. Transfected cells were stimulated TGFβ for 8 hrs. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and p21 antibodies. B , Transfected MDA cells were immunoblotting by phospho-Smad3 (p-Smad3), Smad2/3 and β-tubulin antibodies in response to TGFβ for 30 minutes. C , SCP2 cells were co-transfected with either mock, Scr siRNA, p21 siRNA or flag-tagged p21 construct (p21 cDNA) and SBE promoter construct (CAGA12-luc). Transfected cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicate SEM; n = 3 independent experiments). D and E , SUM159 and SCP2 cells were treated with or without TGFβ for the indicated times. The mRNA levels of indicated genes were then analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Western Blot, Construct, Luciferase, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 regulates TGFβ-induced downstream genes involved in cell invasion . A , SUM159 cells were transfected with Scr or p21 siRNA as well as p21 cDNA. Cells were then treated with or without TGFβ and the mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). B , SUM159 cells transfected with Scr siRNA and p21 siRNA were treated with TGFβ for the indicated times. The mRNA level of TGFBI gene was measured by real-time PCR (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with the indicated siRNAs. Cell invasion was assessed using the Transwell Invasion assay. D , Total cell number was counted by Image J and number of invaded cells was quantified (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Transwell Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21/p/CAF regulates TGFβ transcriptional activity and Smad3 occupancy on SBE . A , SCP2 and SUM159 cells were treated with TGFβ. Cell lysates were analyzed by co-immunoprecipitation using specific antibodies, as indicated. B , HEK293 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21 with or without p/CAF siRNA. Transfected cells were stimulated with TGFβ for eight hours. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and flag antibodies. C and D , SUM159 cells were transfected with Scr or p/CAF siRNAs as well as a flag-tagged p21 cDNA, treated with or without TGFβ. The mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). E , SCP2 cells transfected with Scr and p/CAF siRNA were stimulated with or without TGFβ. Cell invasion was quantified by relative TGFβ fold induction (error bars indicate SEM; n = 3 independent experiments). F , Transfected SCP2 cells were subjected to immunoblotting p/CAF and β-tubulin. G , HEK293 cells were co-transfected with myc-Smad3, myc-Smad2 and p/CAF. Immunoprecipitated Smad2/3 using an anti-myc antibody was subjected to Western blotting. H , DNA precipitation (DNA IP) was performed using biotinylated control and 4× CAGA SBE oligonucleotides, following by streptavidin precipitation. Western blotting of Smad3 and p/CAF is shown. I , SCP2 cells were transfected with p21 or p/CAF siRNAs. Samples were subjected to DNA IP and immunoblotting of Smad3. J , Transfected SCP2 cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicated SEM; n =3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Immunoprecipitation, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Control, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High expression of p/CAF/p21/pSmad3 is associated with lymph node positivity . A , Representative immunohistochemistry images of pSmad3, p21 and p/CAF in breast cancer tissue microarray samples. B , Overall pSmad3, p21 and p/CAF immunohistochemistry staining intensity between lymph node negative (LN-) and positive (LN+) tissues. C , Percentage of case distribution according to immunoreactivity of pSmad3, p21 and p/CAF in tumor cells, the score for negative (0), low (1 to 2) and high (3 to 4).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Model of pro-invasive function for the cell cycle regulator p21 in human breast cancer . The role of p21 as both a direct transcriptional target of TGFβ and a co-stimulatory factor of p/CAF/Smad3 in regulation of pro-invasive genes in triple negative breast cancer cells.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques:

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 1 Effects of varying doxorubicin doses on cellular outcomes in HCT116 cells with different p53 and p21 statuses. A Immunofluorescence images of HCT116 WT, p53−/−, and p21−/−cells treated with 100 nM or 2 μM doxorubicin for 36 h, showing nuclear morphology (DAPI, cyan) and actin cytoskeleton (phalloidin, red). Scale bar = 20 μm. B Quantification of multinucleated (left) and apoptotic cells (right) in WT, p53−/−, and p21−/−cells treated with 100 nM or 2 µM doxorubicin (n = 30 cells per line). Data are presented as mean ± SD, **p < 0.01, ****p < 0.0001. C Flow cytometry of cell cycle distribution (sub G1, G1, S, G2/M and > 4 N) over time (0, 24, 48, and 72 h) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin, with quantification of cells with > 4 N DNA content. Histograms (left) and quantitation (right) are shown. D, E Western blot analysis of p53, p21, and cleaved caspase-3 expression in WT, p53−/−, and p21−/−cells treated with 100 nM (D) or 2 µM (E) doxorubicin. β-actin was used as a loading control.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Flow Cytometry, Quantitation Assay, Western Blot, Expressing, Control

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 3 p21 is essential for maintaining genomic stability and normal mitotic progression. A Western blot analysis of p21 in HCT116 WT and p53−/−cells transduced with non-targeting (NT) or p21 shRNAs. β-actin was used as a loading control. B, C Fluorescence microscopy of nuclear morphology in HCT116 WT (B) and p53−/−(C) cells transduced with NT or p21-targeting shRNAs, treated with vehicle or 100 nM doxorubicin for 48 h. Nuclei and actin were stained with DAPI (cyan) and phalloidin (red), respectively. Scale bar = 20 µm. Quantitation of multinucleated cells is shown on the right (n = 50 cells per group). Data are mean ± SD, ***p < 0.001, ****p < 0.0001. D, E Flow cytometry analysis of cell cycle phases (Sub G1, G1, S, G2/M and > 4 N) in WT (D) and p53−/−(E) cells transduced with NT or p21 shRNA, treated with vehicle or 100 nM doxorubicin for 48 h, including quantification of cells with > 4 N DNA content. F Western blot showing p21 levels in HCT116 p21−/−cells transfected with empty vector (EV) or p21, with β-actin as a loading control. G Fluorescence microscopy of nuclear morphology (cyan) and actin (red) in HCT116 p21−/−cells transfected with EV or p21 after treatment with vehicle or 100 nM doxorubicin for 48 h. Scale bar = 10 μm. Quantitation of multinucleated cells (%) following 24 and 48 h of treatment is shown on the right (n = 50 cells per group). Data are mean ± SD, **p < 0.01.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Western Blot, Transduction, Control, Fluorescence, Microscopy, Staining, Quantitation Assay, Flow Cytometry, shRNA, Transfection, Plasmid Preparation

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 2 Role of p21 in high-dose doxorubicin-induced apoptosis. A Flow cytometry of cell cycle in HCT116 cells transduced with NT or p21 shRNA, treated with 2 µM doxorubicin for 48 h, showing subG1 cell population percentages. B Western blot analysis of Noxa and Mcl-1 expression in WT, p53−/−, and p21−/−cells treated with 2 µM doxorubicin. β-actin was used as a loading control.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Flow Cytometry, Transduction, shRNA, Western Blot, Expressing, Control

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 4 Mitotic defects in p21-deficient cells following low-dose doxorubicin treatment. A Live-cell imaging of HCT116 p21−/−cells expressing GFP-H2B, treated with 100 nM doxorubicin for 48 h. Images were captured every 15 min over 9 h. Scale bar = 5 μm. B, C Immunofluorescence showing the localization of Aurora B (B) and MKLP1 (C) in WT, p53−/−, and p21−/−cells synchronized to early anaphase by a thymidine-RO-3306 block. Hoechst 33342 (blue), α-tubulin (red), and Aurora B or MKLP1 (green) are shown. Quantification of cells with weak Aurora B (B) and MKLP1 (C) signals in early anaphase (n = 25 cells per group, repeated 4 times). Data are mean ± SD, ****p < 0.0001. D, E Immunofluorescence showing Aurora B (D) and MKLP1 (E) localization in WT and p53−/−cells transduced with NT or p21 shRNA.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Live Cell Imaging, Expressing, Blocking Assay, Transduction, shRNA

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 5 Impaired DNA damage repair in p21-deficient cells after low-dose doxorubicin treatment. A Western blot analysis of phosphorylated ATM (p-ATM), p-Chk1, Chk1, p-Chk2, and Chk2 in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for various time points. β-actin was used as a loading control. B Fluorescence microscopy of Lamin B1 (red) and γ-H2AX foci (green) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 48 h. Scale Bar = 20 μm. C Fluorescence microscopy showing nuclei (blue) and γ-H2AX foci (green) in HCT116 WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 24 h and then released into fresh media for various time points. Scale bar = 20 μm. Quantification of nuclei with >10 γ-H2AX foci (n = 20 cells per group, repeated 4 times). Data are mean ± SD ****p < 0.0001.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Western Blot, Control, Fluorescence, Microscopy

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 6 Activation of error-prone DNA repair pathways in p21-deficient cells treated with low-dose doxorubicin. A, C Immunofluorescence showing DNA-PKcs phosphorylation (A) or Mre11 (C) (red) in WT, p53−/−, and p21−/−cells treated with vehicle or 100 nM doxorubicin for 48 h. Nuclei were stained with Hoechst 33342 (cyan). Quantification of DNA-PKCs pS2056 (A) or Mre11 (C) signal intensity is shown (n = 16 cells per group, repeated 3 times). Scale bar = 10 μm. Data are mean ± SD **p < 0.01, ***p < 0.001, ****p < 0.0001. B, D Western blot of DNA- PKcs pS2056, DNA-PKcs (B) or Mre11 (D) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for the indicated time points. β-actin was used as a loading control. E Fluorescence microscopy showing nuclear morphology (cyan) in WT, p53−/−, and p21−/−cells treated with vehicle, Mirin, or doxorubicin + Mirin for 48 h. Scale bar = 10 μm. F Quantification of multinucleated cells (%) following treatment as described in panel (E). data are mean ± SD, ****p < 0.0001 vs. vehicle, ####p < 0.0001 vs. Doxorubicin.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Activation Assay, Phospho-proteomics, Staining, Western Blot, Control, Fluorescence, Microscopy