Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: NaBu and TSA induce p21 mRNA expression in HT-29 cells in an immediate-early fashion. (A) Dose response for p21 induction by NaBu. HT-29 cells were treated with various concentration of NaBu, 0–20 mM for 24 hr, and p21 mRNA expression was examined by Northern blot analyses. (B) Time course for induction of p21 by NaBu. Cells were treated with 5 mM NaBu for varying lengths of time, from 0 to 24 hr, and p21 induction was examined by Northern analysis. (C) p21 induction by NaBu is not blocked by protein synthesis inhibition. HT-29 cells were treated concomitantly with 5 mM NaBu and the protein synthesis inhibitor, cycloheximide (CHX, 10 μg/ml), for 48 hr. (D) Time course for p21 induction by TSA. Cells were treated with 0.3 μM TSA for varying lengths of time. (E) p21 induction by TSA is not blocked by protein synthesis inhibition. Cells were treated concomitantly with 0.3 μM TSA and 10 μg/ml CHX for 6 hr. Representative Northern blots (n = 4) are depicted in each figure. The actin control is shown in the lower panel. Twenty micrograms of total RNA was loaded in each lane, with equal loading verified by ethidium bromide staining.

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Expressing, Concentration Assay, Northern Blot, Inhibition, Control, Staining

Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: Overexpression of histone deacetylase (HDAC1) blocks induction of the p21 promoter by NaBu and TSA. (A) NaBu and TSA induce p21 promoter activity. HT-29 cells were transiently transfected with CAT reporter plasmids under the control of various amounts of the mouse p21 promoter (0–4.7 kb upstream from the transcriptional start site). Transfectants were treated without or with 5 mM NaBu or 0.3 μM TSA, and total cellular protein was examined for CAT expression. Results are shown for the 0-kb (pCAT), 1.4-kb (p211.4CAT), and 4.7-kb (p214.7CAT) p21 promoter constructs and are expressed as fold CAT induction compared with the untreated negative control, arbitrarily taken as 1. (B) Cotransfection of HDAC1 blocks p21 promoter induction by NaBu and TSA. Transfections were performed with the p214.7 CAT −/+ HDAC1, and cells were treated without or with 5 mM NaBu or 0.3 μM TSA. The results are expressed as fold CAT induction compared with the untreated negative control (arbitrarily taken as 1). All results are shown as mean ± SEM (n ≥ 4, in all cases).

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Over Expression, Histone Deacetylase Assay, Activity Assay, Transfection, Control, Expressing, Construct, Negative Control, Cotransfection

Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: NaBu, TSA, and p21 overexpression inhibit growth of HT-29 cells. (A) NaBu and TSA inhibit growth of HT-29 cells. Cells were plated at equal density in six-well plates. At 80% confluence, cells were treated without or with 5 mM NaBu or 0.3 μM TSA for 24 hr. 3H-thymidine (1 μCi/ml) was added for the last 6 hr of treatment, and incorporation was measured by scintillation counting. Treatment results are expressed as percent change compared with untreated negative control, arbitrarily taken as 1. (B) p21 overexpression inhibits growth of HT-29 cells. Cells were stably transfected with an expression plasmid containing the human p21 gene, and overexpression of the gene in pooled stable transfectants compared with the parental and NEO controls (n = 5 each) was determined by Northern analyses (see Inset). 3H-thymidine incorporation was measured in exponentially growing cells under basal conditions, and results are expressed as percent change compared with parental control, arbitrarily taken as 100%, and are shown as mean ± SEM (n ≥ 4, in all cases).

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Over Expression, Negative Control, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Northern Blot, Control

Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: p21 is required for NaBu-induced growth inhibition of colon cancer cells. (A) p21 mRNA is induced early in HCT116 +/+ cells. Cells were treated at 80% confluence with 1 mM NaBu or 0.15 μM TSA (higher concentrations of either agent led to cell death), and p21 mRNA expression was examined by Northern analysis. (B) p21 deletion prevents growth inhibition by butyrate and TSA. HCT116 +/+ and −/− cells were treated with 1 mM NaBu or 0.15 μM TSA for 24 hr, and 1 μCi/ml 3H-thymidine was added for the last 6 hr of treatment. Incorporation was measured by scintillation counting. (C) HCT116 −/− cells are growth-inhibited by serum starvation and postconfluent growth. HCT116 +/+ and −/− cells were serum-starved for 24 hr or grown to 2 weeks postconfluence, and 3H-thymidine incorporation was measured. Cells grown in standard McCoy’s 5A medium with 10% fetal bovine serum and that were preconfluent were used as controls. Results are expressed as percent change compared with control groups (arbitrarily taken as 1) and are shown as mean ± SEM (n ≥ 4, in all cases).

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Inhibition, Expressing, Northern Blot, Control

Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 expression is associated with senescence. A Human fibroblasts were exposed to 20 Gy ionizing irradiation (IR). On day 1, 5 and 7, NLRP1 and senescence protein expression were analyzed by immunoblotting. B Representative images of Ki67 and NLRP1 immunofluorescence. Scale bar = 50 μm. C , D IL-1β, IL18, IL-6 and IL-8 were quantified by ELISA. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 differences between time points after irradiation and day 0. E , F Human fibroblasts were treated with Valboropro (Vbpro) to induce NLRP1 expression. After 24 h, NLRP1 and IL-6 protein expression were analyzed by immunoblotting and cytokines were analyzed by ELISA. Human fibroblasts were irradiated ( G ) or stimulated with palbociclib (Palbo) ( H ) to induce two different senescence models. Then, cells were transfected with a non-targeting control siRNA (Control) or with siRNAs against NLRP1 (siNLRP1). Expression of NLRP1 and senescence-associated proteins p16, p21, p53 and IL-6 were assessed by immunoblotting and IL6, IL-8 or IL-18 were quantified by ELISA. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 irradiated vs. control. aaa P < 0.001, IR + siRNA vs. IR cells (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: Expressing, Irradiation, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 contributes to cellular senescence in vivo. A Nlrp1 protein expression in liver from WT mice at 1 month after IR. B Serum levels of IL-18 after IR. C Effect of IR on the bodyweight of WT and Nlrp1 knockout (KO) mice. D Protein expression in liver from IR and non-IR WT and Nlrp1 KO mice of senescent markers (IL-6, p16 and p21). Densitometry in Supplementary Fig. 6. E Serum levels of IL-6 in serum from IR and non-IR WT and Nlrp1 KO mice. F Heat map depicting expression of 44 mouse cytokines in serum at 5 weeks after IR of WT and Nlrp1 KO mice. n = 6 mice per group. G IL-6 releases from healthy fibroblasts was assessed after 24 and 48 h of incubation with media containing serum from IR and non-IR WT and Nlrp1 KO mice. H Representative liver section stainings of hematoxylin and eosin (H&E). I Representative liver section immunostainings of NLRP1 and p16. All data are presented as means ± SEM, n = 6–8 mice per group; * P < 0.05, ** P < 0.005, *** P < 0.001 irradiated vs. control. aa P < 0.005 IR WT vs. IR KO mice; bb P < 0.005 IR KO vs. IR KO mice (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: In Vivo, Expressing, Knock-Out, Incubation, Irradiation, Control

Journal: Inflammation Research

Article Title: NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype

doi: 10.1007/s00011-024-01892-7

Figure Lengend Snippet: NLRP1 senses DNA damage dependent of cGAS activation. A Protein expression levels of NLRP1 and cGAS after 24 h exposition to gDNA from non-irradiated and irradiated cells. B IL-6 and IL-18 release to the medium after the same experimental condition. Levels were determined by ELISA assay. C , D Protein expression levels of NLRP1 and cGAS and IL-6 and IL-18 release after 24 h exposition to a non-irradiated or irradiated synthetic double-stranded DNA sequence, poly(dA-dT), Cytokine levels were determined by ELISA assay. Cytokine levels were determined by ELISA assay. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, *** P < 0.001 gDNA vs. control cells. a P < 0.05, aa P < 0.005, aaa P < 0.001, IR gDNA vs. control cells. E Human fibroblasts were irradiated to induces senescence. Then, cells were transfected with a non-targeting control siRNA (Control) or with siRNAs against cGAS (sicGAS). Expression of NLRP1, NLRP3, IL-1β, cGAS and senescent protein p16, p21 was assessed by immunoblotting. F IL-18 release of non-irradiated, irradiated and irradiated and transfected with siRNAs against cGAS. Cytokine levels were determined by ELISA assay. All data are presented as means ± SEM, n = 4 independent experiments; ** P < 0.005, IR vs. control cells. aa P < 0.005, IR vs. sicGAS (color figure online)

Article Snippet: Monoclonal antibodies specific for NLRP1 (NBP1-54899), NLRP3 (NBP2-12446) and p21/CIP1/CDKN1A (NBP2-29463) were purchased from Novus Biologicals (Colorado, USA).

Techniques: Activation Assay, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Transfection, Western Blot

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: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor Receptor Pathway Analysis Identifies Amphiregulin as a Key Factor for Cisplatin Resistance of Human Breast Cancer Cells

doi: 10.1074/jbc.m706287200

Figure Lengend Snippet: FIGURE 3. Analysis of AKT kinase, downstream signaling reveals inactiva- tion of the p53 pathway in MCF-7 CisR cells. A, quantification of AKT kinase activity. To measure AKT kinase activity, a solid phase ELISA, which utilizes a specific synthetic peptide as a substrate and a polyclonal antibody that rec- ognizes the phosphorylated form of the substrate, was used. (n 3, ***, p 0.001). B, detection of p53 protein by immunoblotting (IB) using a polyclonal affinity-purified goat Ab specific for p53. MCF-7 cells (lane 1) and MCF-7 CisR cells (lane 2). Lane M, molecular weight marker. p53 is indicated by an arrow. C, quantification of p53 by a sandwich ELISA that measures human total p53 in cell lysates (n 3, ***, p 0.001). D, p21 expression indicates p53 pathway activity. Detection of p21 in whole cell lysates of MCF-7 (lane 1) and MCF-7 CisR cells (lane 2) by immunoblotting using a polyclonal affinity-purified goat Ab specific for p21. Lane M, molecular weight marker. p21 indicated by an arrow. E, to quantify the levels of p21 protein a sandwich ELISA that measures p21 in cell lysates was used. (n 3, ***, p 0.001). F, to quantify BCL-2 expression an ELISA that detects human BCL-2 in cell lysates was used (n 3, ***, p 0.001).

Article Snippet: For immunoblotting we used a polyclonal affinity-purified goat Ab specific for p53 at a concentration of 1 g/ml (AF1355, R & D Systems, Wiesbaden, Germany) and a polyclonal affinity-purified goat Ab specific for p21 at a concentration of 1 g/ml (AF1047, R & D Systems, Wiesbaden, Germany).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Affinity Purification, Molecular Weight, Marker, Sandwich ELISA, Expressing

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The total p21 protein expression (pg/mL) in normal cell line-HUVEC and in tumor cell line-PE/CA-PJ49 cells treated 24 h with CisPt and/or RSV, CRM. The experiments were performed in triplicates. Results are expressed as mean values of three determinations ± standard deviation (SD). (** p < 0.005, *** p < 0.0005; **** p < 0.00005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Standard Deviation

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effects of CisPt, RSV, CRM treatment applied alone or in combination for 24 h on the level of p21 protein expression (n-fold p21protein expression) in normal cell line-Huvec and in tumor cell line-PE/CA-PJ49. The n-fold p21expression was calculated using the formula: n-fold p21expression = p21 pg/mL treatment/p21 pg/mL control.

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Expressing, Control

Journal: Nutrients

Article Title: The Effect of Resveratrol or Curcumin on Head and Neck Cancer Cells Sensitivity to the Cytotoxic Effects of Cisplatin

doi: 10.3390/nu12092596

Figure Lengend Snippet: The effect of treatment with CisPt, RSV, CRM applied independently or in combination on P21 gene expression in tumor cells PE/CA-PJ49 compared to normal cells HUVEC. Each sample was performed in duplicate. The samples were analyzed using the formula 2-ΔΔCt = gene expression. (* p < 0.05, ** p < 0.005; *** p < 0.0005).

Article Snippet: DuoSetR IC ELISA-Human Total p21/CIP1/CDKN1A(: DYC1047-2) was purchased from R&D Systems Inc. and contains the basic components required for the development of sandwich ELISAs to measure human p21 protein also known as CIP1 and CDKN1A in cell lysates.

Techniques: Gene Expression