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: Nutrients

Article Title: ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis

doi: 10.3390/nu14142786

Figure Lengend Snippet: Hyperglycaemia-induced senescence promotes barrier disruption in endothelial cells. ( A ) Experimental design and treatment conditions. ( B , C ) Dot plot summarizing data of barrier integrity measured by FITC dextran leakage ( B ) and TEER ( C ). ( D – G ) Exemplary immunoblot of p21, p16, and p53 (( D ), loading control: GAPDH) and dot plot summarizing p21 ( E ), p16 ( F ) and p53 ( G ) densitometric quantifications of immunoblots. ( H – L ) Representative immunofluorescence images showing endothelial cell staining for p21 (( H ), top panel, red; nuclear counterstain: DAPI, blue), p16 (( H ), first middle panel, red; nuclear counterstain: DAPI, blue), p53 (( H ), second middle panel, red; nuclear counterstain: DAPI, blue) and senescence-associated beta galactosidase staining (( H ), bottom panel, blue). Dot plots summarizing data for p21 ( I ), p16 ( J ), p53 ( K ) and SAβ gal ( L ). Scale bar: 20 µm ( H ). HCAEC maintained under control (non-treated, C), high glucose (25 mM; HG) or oxidized low density lipoprotein (oxLDL 50 µg/mL, oxLDL) conditions. Each dot represents data obtained from one biological specimen; * p < 0.05, ** p < 0.01; ANOVA.

Article Snippet: The following reagents and antibodies were used in this study: p21 Waf1/Cip1 (12D1) rabbit, p16 INK4A (D7C1M) rabbit, p53 (1C12) mouse (Cell Signaling Technology, Frankfurt, Germany); anti-phospho-IRE1 (S724) rabbit monoclonal antibody (Boster, Germany); human XBP1 antibody (R & D System, Wiesbaden, Germany); anti ATF6 alpha (Rockland, Germany); alexa fluor FITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-mouse IgG and alexa fluor FITC goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany).

Techniques: Disruption, Western Blot, Control, Immunofluorescence, Staining

Journal: Nutrients

Article Title: ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis

doi: 10.3390/nu14142786

Figure Lengend Snippet: Senescence is induced within plaques of diabetic ApoE −/− mice. ( A ) Representative immunofluorescence images showing staining of brachiocephalic arteries for p21 (( A ), top panel, p21, green; nuclear counterstain: DAPI, blue), p16 (( A ), middle panel, p16, red; nuclear counterstain: DAPI, blue) and p53 (( A ), bottom panel, p16, red; nuclear counterstain: DAPI, blue). ( B – D ) Dot plots summarizing immunofluorescence data for p21 ( B ) p16 ( C ), p53 ( D ) and Scale bar: 20 µm ( A ). ApoE −/− control mice (Cont, normal chow diet, citrate instead of streptozotocin injections), DM mice (normal chow diet, streptozotocin injections) or HFD mice (fed high fat diet). Each dot represents data obtained from one mouse specimen; ** p < 0.01; ANOVA.

Article Snippet: The following reagents and antibodies were used in this study: p21 Waf1/Cip1 (12D1) rabbit, p16 INK4A (D7C1M) rabbit, p53 (1C12) mouse (Cell Signaling Technology, Frankfurt, Germany); anti-phospho-IRE1 (S724) rabbit monoclonal antibody (Boster, Germany); human XBP1 antibody (R & D System, Wiesbaden, Germany); anti ATF6 alpha (Rockland, Germany); alexa fluor FITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-mouse IgG and alexa fluor FITC goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany).

Techniques: Immunofluorescence, Staining, Control

Journal: Nutrients

Article Title: ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis

doi: 10.3390/nu14142786

Figure Lengend Snippet: aPC reduces glucose-induced senescence. ( A – C ) Representative immunoblots showing p21 and p16 expressions (( A ), loading control: GAPDH). Dot plots summarize densitometric quantifications of immunoblotting results for p21 ( B ) and p16 ( C ). ( D – F ) Representative immunofluorescence images showing endothelial cells staining for p21 (( D ), upper panel, p21, red; nuclear counterstain: DAPI, blue) and p16 (( D ), lower panel, p16, red; nuclear counterstain: DAPI, blue). Dot plots summarizing immunofluorescence data for p21 ( E ) and p16 ( F ). Scale bar: 20 µm ( D ). ( G , H ) Dot plot summarizing barrier integrity data measured by FITC dextran leakage ( G ) and TEER ( H ). HCAECs maintained under control ( C ), high glucose (25 mM; HG), or HG+aPC (25 mM glucose + 20nM of exogenous activated protein C) conditions. Each dot represents data obtained from one biological specimen. ** p < 0.01; ANOVA.

Article Snippet: The following reagents and antibodies were used in this study: p21 Waf1/Cip1 (12D1) rabbit, p16 INK4A (D7C1M) rabbit, p53 (1C12) mouse (Cell Signaling Technology, Frankfurt, Germany); anti-phospho-IRE1 (S724) rabbit monoclonal antibody (Boster, Germany); human XBP1 antibody (R & D System, Wiesbaden, Germany); anti ATF6 alpha (Rockland, Germany); alexa fluor FITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-rabbit IgG, alexa fluor TRITC goat anti-mouse IgG and alexa fluor FITC goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany).

Techniques: Western Blot, Control, Immunofluorescence, Staining

Journal: EBioMedicine

Article Title: MDM2 Antagonists Counteract Drug-Induced DNA Damage

doi: 10.1016/j.ebiom.2017.09.016

Figure Lengend Snippet: P21 deficiency sensitizes cells to MDM2i and AURKAi therapy. (a) Hs294T cells were transfected with control siRNA or p21-specific siRNA and treated with 10 μM nutlin-3a (MDM2i) ± 1 μM alisertib (AURKAi) for 3 days and pulsed with BRDU for 2 h. Representative histograms of flow cytometric analysis of BRDU, γH2AX and cleaved PARP are shown. (b) Quantitation from 3 independent experiments performed as described in (a). Indicated groups were compared using ANOVA with Bonferroni test. (c) Western blot analysis of lysates from Hs294T cells treated as described in (a). *p < 0.05, ***p < 0.001, ****p ≤ 0.0001. All experiments were repeated at least 3 times with consistent results.

Article Snippet: P21 siRNA (Cell Signaling, #6456) was mixed with in vivo JetPei (Polyplus-transfection SA, Illkirch, France) in accordance with manufacturer's recommendations and injected into the tumor.

Techniques: Transfection, Control, Quantitation Assay, Western Blot

Journal: EBioMedicine

Article Title: MDM2 Antagonists Counteract Drug-Induced DNA Damage

doi: 10.1016/j.ebiom.2017.09.016

Figure Lengend Snippet: P21 as a potential therapeutic target in melanoma. (a) Analysis of the TCGA dataset of 479 melanoma specimens using cBio portal. Tumors were sorted into high and low expression of p21, based on p21 protein expression by RPPA analysis (left panel). Disease-free survival was compared between these groups (right panel). (b) Nude mice bearing Hs294T melanoma xenograft tumors were treated QD with 30 mg/kg alisertib (AURKAi) and 150 mg/kg idasanutlin (HDM2a). Animals also received injections of control siRNA (no-target siRNA) or p21 siRNA mixed with in vivo siRNA delivery reagent JetPei directly into the tumor twice a week. Tumor area (length x width) was measured every 3–4 days. Average tumor area ± SD is shown. N = 6 in vehicle groups and n = 7 in AURKAi and HDM2a treatment groups. Mixed-effects statistical model was used to assess group differences in tumor area over days. Expression of p21 in tumors from vehicle-treated mice injected with control or p21 siRNA was evaluated in whole tumor lysates by western blot with human p21-specific antibodies (right panel).

Article Snippet: P21 siRNA (Cell Signaling, #6456) was mixed with in vivo JetPei (Polyplus-transfection SA, Illkirch, France) in accordance with manufacturer's recommendations and injected into the tumor.

Techniques: Expressing, Control, In Vivo, Injection, Western Blot

Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet: List of metal conjugated antibodies for stress response pathway analysis by CyTOF.

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques:

Journal: Oncology Letters

Article Title: Increased soluble E‑cadherin of spheroid formation supplemented with fetal bovine serum in colorectal cancer cells

doi: 10.3892/ol.2023.13793

Figure Lengend Snippet: Cell viability changes after spheroid formation of colorectal cancer cells in different environments. (A) The SNU-C5 and SNU-C5/5-FUR cells were mock-treated with DMSO or treated with indicated doses of 5-FU for 3 days. The extent of cell viability was determined by MTT assay. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. DMSO; # P<0.05 and ## P<0.01 vs. sphere/GF. (B) The SNU-C5 and SNU-C5/5-FUR cells were mock-treated with DMSO or treated with indicated doses of 5-FU for indicated days. The extent of cell viability was determined by MTT assay using the aforementioned procedures. Data are presented as the mean ± SD (n=3). (C) Expression levels of cell cycle-related proteins in monolayer and spheroid formation cultures in SNU-C5 and SNU-C5/5-FUR cells were detected by immunoblotting. Immunoblotting analysis was performed for p21, β-catenin and GSK3β, while GAPDH was used for a loading control. Band density was analyzed by AzureSpot analysis software, and results are expressed as the mean ± SD (n=3). **P<0.01 and ***P<0.001 vs. monolayer; ## P<0.01 vs. sphere/FBS. 5-FU, 5-fluorouracil; FBS, fetal bovine serum; GF, growth factor.

Article Snippet: CREB (1:1,000; cat. no. CSB-PA005947HA01HU; Cusabio Technology, LLC), fibronectin (1:2,000; cat. no. CL54951AP; Cedarlane Laboratories), p21 (1:1,000; cat. no. 60214-1; Proteintech Group, Inc.) and p90RSK (Ab348; 1:1,000; cat. no. 79-554; Prosci, Inc.) were obtained from the corresponding listed company.

Techniques: MTT Assay, Expressing, Western Blot, Control, Software

Journal: Oncology Letters

Article Title: Increased soluble E‑cadherin of spheroid formation supplemented with fetal bovine serum in colorectal cancer cells

doi: 10.3892/ol.2023.13793

Figure Lengend Snippet: Densitometric results of western blotting on SNU-C5 and SNU-C5/5-FUR cells.

Article Snippet: CREB (1:1,000; cat. no. CSB-PA005947HA01HU; Cusabio Technology, LLC), fibronectin (1:2,000; cat. no. CL54951AP; Cedarlane Laboratories), p21 (1:1,000; cat. no. 60214-1; Proteintech Group, Inc.) and p90RSK (Ab348; 1:1,000; cat. no. 79-554; Prosci, Inc.) were obtained from the corresponding listed company.

Techniques: Western Blot, Marker