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

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma

doi: 10.3390/ijms22041716

Figure Lengend Snippet: Effect of tumor necrosis factor-alpha (TNF-α) and MLN4924 on matrix metalloproteinase 9 (MMP9) expression and esophageal squamous cell carcinoma (ESCC) cell migration. ( A ) After overnight serum starvation, KYSE150 cells were pretreated or not with 1 μM MLN4924 for 30 min and then stimulated with TNF-α at the indicated concentration for 24 h. Activity of MMP9 and MMP2 was analyzed by gelatin zymography in the conditioned media. The protein levels of membrane type I-matrix metalloproteinase (MT1-MMP), cyclin dependent kinase inhibitor 1A (CDKN1A/p21), c-Jun, nuclear factor kappa B (NFκB) and SP1 were determined by Western blotting. Fold change calculated as the ratio of relative levels of proteins normalized to β-actin (ACTB) between the cells treated with TNF-α in combination with MLN4924 and the cells treated with TNF-α alone is shown in . Values shown are means ± SEM: whiskers: min–max. ( B ) MMP9 messenger RNA (mRNA) expression was analyzed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in both KYSE150 and KYSE70 cells treated for 24 h with TNF-α (30 ng/mL) and MLN4924 (1 µM) alone or in combination. Results are presented as fold change of MMP9 gene in the treated cells relative to the untreated controls normalized to the expression of the reference gene encoding glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). ( C ) Representative images from wound healing assay showing the changes in KYSE70 cell migration under MLN4924 and TNF-α applied separately and in combination after 24 h and 48 h treatment in relation to the untreated controls. The cells were seeded in silicone inserts and allowed to attach and form a confluent monolayer. After the inserts were removed, the cells were treated with TNF-α (30 ng/mL) and MLN4924 (1 µM) separately or in combination and incubated for the next 48 h. Images of cell-free artificial wounds were taken at 0, 24 and 48 h. ( D ) Quantitative analysis of wound closure. Summary bar graphs illustrating the ratio of wound closure after 24 and 48 h quantified for KYSE150 and KYSE70 cells. Values shown are means ± SEM. * p < 0.001, ** p < 0.05 vs. control. Ctrl—untreated controls.

Article Snippet: Primers for the CDKN1A gene were purchased from Sino Biological, Beijing, China (HG11108-ACG).

Techniques: Expressing, Migration, Concentration Assay, Activity Assay, Zymography, Membrane, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Wound Healing Assay, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma

doi: 10.3390/ijms22041716

Figure Lengend Snippet: Effect of tumor necrosis factor-alpha (TNF-α) and MLN4924 on the signaling pathways mediating matrix metalloproteinase 9 (MMP9) gene expression in esophageal squamous cell carcinoma (ESCC) cells. ( A ) Proteome profiling of the nuclear factor kappa B (NFκB) pathway by antibody array analyses in the KYSE150 cells treated with MLN4924 and TNF-α. Protein lysates from the untreated controls and cells treated with MLN4924 or TNF-α alone or in combination were analyzed using a human NFκB array (R&D). ( B ) Bar graphs showing the phosphorylation ratio (upper) and the protein level ratio (bottom) calculated after the semi-quantitative analysis of selected proteins. Results are presented as means ± SEM from duplicates. * p < 0.001, ** p < 0.05 vs. controls. The complete array is shown in . ( C ) Western blot analysis showing time-dependent activation of inhibitor of nuclear factor kappa B-alpha (IκB-α), NFκB/p65 and c-Jun in the KYSE150 cells treated with TNF-α (30 ng/mL). ( D ) MLN4924-dependent changes in the activation of IκB-α, NFκB/p65 and c-Jun as well as increasing levels of cyclin dependent kinase inhibitor 1A (CDKN1a/p21) protein in the KYSE150 cells within 24 h. ( E ) A dose-dependent effect of MLN4924 on activation of NFκB/p65 and c-Jun signaling pathways in the KYSE150 cells treated with TNF-α (30 g/mL) for 24 h. The effect of the KYSE70 cells treatment with different concentrations (0.25, 0.5, 1.0, 2.5 and 5.0 µM) of MLN4924 for 24 and 48 h is shown in .

Article Snippet: Primers for the CDKN1A gene were purchased from Sino Biological, Beijing, China (HG11108-ACG).

Techniques: Expressing, Ab Array, Western Blot, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma

doi: 10.3390/ijms22041716

Figure Lengend Snippet: Western blot analysis of the nuclear factor kappa B (NFκB) complex and the chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) assay of the matrix metalloproteinase 9 ( MMP9 ) gene promoter in KYSE150 cells. Nuclear protein-enriched extracts from esophageal squamous cell carcinoma (ESCC) cells were used for co-immunoprecipitation (co-IP) with a mouse anti-NFκB/p65 antibody ( A ) or with a rabbit anti-c-Jun antibody ( B ) and probed for inhibitor of nuclear factor kappa B-alpha (IκB-α)/phospho-IκB-α, SP1 and cyclin dependent kinase inhibitor 1A (CDKN1A/p21) as well as phosphorylated and unphosphorylated forms of NFκB/p65 and c-Jun. Immunoglobulin G (IgG) mixture (1:1:1) of each protein extract and normal mouse IgG or normal rabbit IgG, respectively, used as negative controls; Ctrl—protein extract from untreated cells; MLN4924 and tumor necrosis factor-alpha (TNF-α)—extracts from cells treated with MLN4924 (1 µM) for 24 h and with TNF-α (30 ng/mL) for 1 h, respectively. ( C ) ChIP-qPCR assay on the MMP9 gene promoter in KYSE150 cells. Sheared chromatin was immunoprecipitated with antibodies against NFκB/p65 and with an anti-acetyl histone H3.3 antibody, used as a positive control. Normal rabbit IgG served as a negative antibody control. Box plots showing qPCR data normalized with the fold enrichment method. Values shown are means ± SEM: whiskers: min–max; * p < 0.001, ** p < 0.01 vs. controls. Agarose gels showing DNA fragments amplified with MMP9 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers.

Article Snippet: Primers for the CDKN1A gene were purchased from Sino Biological, Beijing, China (HG11108-ACG).

Techniques: Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Co-Immunoprecipitation Assay, Positive Control, Control, Amplification

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: 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

Journal: Oncotarget

Article Title: miR-320a functions as a suppressor for gliomas by targeting SND1 and β-catenin, and predicts the prognosis of patients

doi: 10.18632/oncotarget.14975

Figure Lengend Snippet: A . Growth curves from U87MG and U251 cells transfected with Scr or miR-320a and cotransfected with miR-320a plus plasmid expressing β-catenin (miR-320a+CTNNB1) or SND1 (miR-320a+SND1) assessed by CCK8 assay. B and C . EdU-positive rate (B) and representative images (C) in the indicated cells assessed by EdU assay. D and E . Representative images (left) and percentage of each phase cells (right) in the indicated cells assessed by FCM. F and G . Western blot analyses of β-catenin, cyclin D1, SND1 and p21 WAF1 (left), and comparisons among groups of their expressions (right) in the cells as indicated. Their relative expression levels were normalized against β-actin. All the experiments were performed at least in triplicate and the data are presented as the mean ± SD. * P <0.05, ** P <0.01, ***/ ▲▲▲ P <0.001. Compared with Scr group* and with miR-320a group ▲ in FCM data.

Article Snippet: Rabbit anti-human β-catenin, MMP7, cyclin D1, Smad2 and phosphorylated Smad2 (CST), mouse anti-human SND1 (Abcam), Smad4 (R&D Systems, Minneapolis, MN, USA), MMP2, p21 WAF1 and β-actin (Boster Biological Technology, Wuhan, China) were used as primary antibodies.

Techniques: Transfection, Plasmid Preparation, Expressing, CCK-8 Assay, EdU Assay, Western Blot

Journal: Oncotarget

Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells

doi: 10.18632/oncotarget.25415

Figure Lengend Snippet: (A) siSNCG-treated T47D cells showed reduced RNA (left panel) and protein (middle and right panels) expression compared to siScramble (siScr)-treated T47D cells. Relative expression of SNCG mRNA (left panel) was assessed by qRT-PCR analysis performed in triplicate (normalized against RPLP0). Representative immunoblot analysis (middle panel) was performed on whole cell lysate for SNCG expression using anti β-actin antibody as a loading control. Bar graph (right panel) shows quantitative analysis of scanning densitometric values of SNCG protein as ratio to β-actin protein. Data represent mean values ± standard error of the mean of two (RNA) and three (protein) independent experiments. (B) siSNCG-treated T47D cells showed increased apoptosis after radiation treatment (8 and 12 Gy) compared to siScramble (siScr)-treated T47D cells. Flow cytometry analysis was carried out to detect apoptotic and necrotic cells. Histogram shows percentage of apoptotic cells 72 hours after irradiation (left panel). Data represent mean values ± standard error of the mean of four independent experiments. Representative experiment of flow cytometry analysis (n=4) shows the percentage of Annexin-V and propidium iodide staining of T47D cells irradiated or not at a dose of 8 or 12 Gy (right panel). (C) Radiation sensitivity was determined from the number of viable cells at different times after irradiation at 4, 8, and 12 Gy using the resazurin-based cell viability assay. The upper panel shows representative growth curves of siScr- or siSNCG-treated cells. Curves from three independent experiments were used as basis for calculation of doubling time in hours (hr) (lower panel) ** = P value < 0.01; ns = not significant.

Article Snippet: For siRNA experiments, T47D and MCF7 cells were transfected with SNCG siRNA and control siScramble (siSrc) constructs (Origene, SR304497) and SUM-SNCG-GFP and SUM-CTL-GFP cells were transfected with p21 siRNA and control siScr constructs (Origene SR300740), using jetPRIME ® (Polyplus) according to the manufacturer's transfection protocol.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Irradiation, Staining, Viability Assay

Journal: Oncotarget

Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells

doi: 10.18632/oncotarget.25415

Figure Lengend Snippet: (A) siSNCG-treated MCF7 cells showed reduced RNA (left panel) and protein (middle and right panels) expression compared to siScramble (siScr)-treated cells. Relative expression of SNCG mRNA (left panel) was assessed by qRT-PCR analysis performed in triplicate (normalized against RPLP0). Representative immunoblot analysis (middle panel) was performed on whole cell lysate for SNCG expression using anti β-actin antibody as a loading control. Bar graph (right panel) shows quantitative analysis of scanning densitometric values of SNCG protein as ratio to β-actin protein. Data represent mean values ± standard error of the mean of two (RNA) and three (protein) independent experiments. (B) siSNCG-treated MCF7 cells showed decreased clonogenic potential after radiation treatments (4 and 8 Gy) compared to siScramble (siScr)-treated cells. Clonogenic cell survival assay was performed and curves show the percentage of survival after irradiation (left panel). Data represent mean values ± standard error of the mean of three independent experiments, each done in triplicate. Representative pictures (n=3) of 2-week-old colonies after fixation and crystal violet staining (right panel). (C) siSNCG-treated MCF7 cells showed increased cellular senescence after radiation treatments (4 and 8 Gy) compared to siScramble (siScr)-treated cells. Cellular senescence was evaluated by the detection of SA-β-galactosidase activity and curves show the percentage of SA-β-gal positive cells as mean values ± standard error of the mean of three independent experiments (left panel). Representative images of SA-β-gal positive cells (blue) are shown (right panel) (scale bar = 100 μm). *** = P value < 0.005 ; ** = P value < 0.01 ; ns = not significant.

Article Snippet: For siRNA experiments, T47D and MCF7 cells were transfected with SNCG siRNA and control siScramble (siSrc) constructs (Origene, SR304497) and SUM-SNCG-GFP and SUM-CTL-GFP cells were transfected with p21 siRNA and control siScr constructs (Origene SR300740), using jetPRIME ® (Polyplus) according to the manufacturer's transfection protocol.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Clonogenic Cell Survival Assay, Irradiation, Staining, Activity Assay

Journal: Oncotarget

Article Title: Synuclein gamma expression enhances radiation resistance of breast cancer cells

doi: 10.18632/oncotarget.25415

Figure Lengend Snippet: (A) Representatives pictures (n=3) of immunoblotting of phospho-p53 (Ser15), total-p53, p21, and β-actin on whole cell lysates of SUM-CTL-GFP and SUM-SNCG-GFP cells irradiated or not at a dose of 4, 8, or 12 Gy. Cells were lysed 24 hours after irradiation. (B) Bar graph shows quantitative analysis of scanning densitometric values of p21 protein as ratio to β-actin protein (left panel). Bar graph shows qRT-PCR analysis of p21 RNA (normalized against RPLP0) as fold enrichment over SUM-CTL-GFP cells (right panel). Data represent mean values ± standard error of the mean of three independent experiments. (C) sip21-treated cells showed reduced protein expression compared to siScramble (siScr)-treated cells. Representative immunoblot analysis was performed on whole cell lysate for p21 expression using anti β-actin antibody as a loading control (left panel). Bar graph shows quantitative analysis of scanning densitometric values of p21 protein as ratio to β-actin protein (right panel). Data represent mean values ± standard error of the mean of three independent experiments. (D) sip21-treated cells showed increased apoptosis after radiation treatment (8 and 12 Gy) compared to siScramble (siScr)-treated cells. Flow cytometry analysis was done to detect apoptotic and necrotic cells. Bar graph shows the percentage of apoptotic cells 96 hours after irradiation. Data represent mean values ± standard error of the mean of three independent experiments. *** = P value < 0.005, ** = P value < 0.01, * = P value < 0.05.

Article Snippet: For siRNA experiments, T47D and MCF7 cells were transfected with SNCG siRNA and control siScramble (siSrc) constructs (Origene, SR304497) and SUM-SNCG-GFP and SUM-CTL-GFP cells were transfected with p21 siRNA and control siScr constructs (Origene SR300740), using jetPRIME ® (Polyplus) according to the manufacturer's transfection protocol.

Techniques: Western Blot, Irradiation, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: Effects of circPCNX on p21 mRNA and p21 protein. (A–D) AUF1 binding to p21 mRNA (A), p21 mRNA stability (B), p21 mRNA levels (C), and p21 protein levels (D) were assessed after transfection with pcDNA3(EV) or with pcDNA-circPCNX to overexpress circPCNX. (A) AUF1 binding to circPCNX, PCNX mRNA and p21 mRNA was assessed one day after transfection; values were first normalized to the levels of a transcript (GAPDH mRNA) that encodes a housekeeping protein, and afterwards normalized to the RNA levels in the IgG IP. (B) One day after transfection, cells were treated with Actinomycin D to block de novo transcription for the times indicated, and the time required for p21 mRNA to reach 50% of its initial abundance (the half-life) in each transfection group was assessed by RT-qPCR analysis. A stable transcript (ACTB mRNA) was included as control. At one and three days after transfection of plasmids, changes in the levels of p21 mRNA (C) or p21 protein (D) were assessed by RT-qPCR and western blot analyses, respectively. (E–H) AUF1 binding to p21 mRNA (E), p21 mRNA stability (F), p21 mRNA levels (G), and p21 protein levels (H) were assessed after transfection with Ctrl siRNA or circPCNX siRNA to silence circPCNX. (E) AUF1 binding to circPCNX, to PCNX mRNA and to p21 mRNA was assessed one day after transfection; as above, values were first normalized to GAPDH mRNA, and afterwards normalized to RNAs in the IgG IP. (F) One day after transfection, cells were treated with Actinomycin D to block de novo transcription for the times indicated, and the time required for p21 mRNA to reach 50% of its initial abundance (the half-life) in each transfection group was assessed by RT-qPCR analysis. A stable transcript (ACTB mRNA) was included as control. At one and three days after siRNA transfections, changes in the levels of p21 mRNA (G) or p21 protein (H) were assessed by RT-qPCR and western blot analyses, respectively. Data in (A–C, E–G) represent the mean values ± SD from three biological replicates. Significance was established using Student's t-test. ** P ≤ 0.01 or *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Binding Assay, Transfection, Blocking Assay, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: AUF1 binding to p21 mRNA is attenuated by circPCNX, but not by mutant circPCNX unable to bind AUF1. (A) Twenty-four hours after transfecting HeLa cells with the plasmids shown, the association of p21 mRNA (or ACTB mRNA, in control reactions) with AUF1 was measured by RIP followed by RT-qPCR analysis and represented as ‘fold enrichment’. (B, C) In HeLa cells transfected with the plasmids indicated, the levels of p21 mRNA (B) were assessed 3 days after transfection by RT-qPCR analysis, and the levels of p21 protein (C) were assessed one and three days after transfection by western blot analysis, including GAPDH as loading control. (D) At the times shown following transfection of HeLa cells with the plasmids indicated, cell proliferation in each transfection group was assessed by using BrdU incorporation analysis. Data in (A, B, D) represent the mean values ± SD from four biological replicates. Significance was established using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Binding Assay, Mutagenesis, Quantitative RT-PCR, Transfection, Western Blot, BrdU Incorporation Assay

Journal: Nucleic Acids Research

Article Title: AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production

doi: 10.1093/nar/gkaa1246

Figure Lengend Snippet: Expression levels of p21 mRNA and p21 protein are attenuated by circPCNX, but not by a mutant circPCNX unable to bind AUF1. (A) Reporter plasmids were created using the backbone of the psiCHECK2 dual luciferase reporter by inserting the p21 3′UTR (gray) with either the full-length wild-type (3′wt) sequence or with a mutation (3′del) in the AUF1-binding site (red). Vectors using the pCMV6 backbone were also created to express p21 protein from mRNAs that had the intact 3′UTR (3′wt) or had a mutation in the AUF1-binding site (3′del). (B) Forty-eight hours after transfecting HeLa cells with either pcDNA3(EV) or pcDNA3-AUF1 or with the siRNAs shown, the reporter plasmids psiCHECK2-p21-3′wt or psiCHECK2-p21-3′del were transfected and 24 h after that, luciferase activities (RL/FL) in each transfection group were assessed. (C) Twenty-four hours after transfecting HeLa cells with the plasmids indicated, the levels of p21 mRNA (with 3′wt or 3′del 3′UTRs) were measured by RT-qPCR analysis and normalized to neo mRNA expressed from the same plasmids; the levels of control ACTB mRNA were quantified in the same reactions; the levels of p21 protein in each transfection group were assessed by western blot analysis. (D) Ctrl or circPCNX siRNAs were transfected along with the plasmids shown (left, p21 3′wt; right, p21 3′del), and the rates of proliferation were assessed by measuring BrdU incorporation at the times indicated. (E) Summary model. Top, when circPCNX accumulates in cells, it can sequester AUF1 away from its target mRNAs, notably p21 mRNA, in turn allowing p21 mRNA stabilization, increased p21 expression, and growth suppression. Bottom, when circPCNX levels decline, or if circPCNX is unable to bind AUF1 (mutated or truncated), then AUF1 associates with p21 mRNA, reducing p21 mRNA stability and p21 levels, and enhancing cell proliferation. Image was created using BioRender. Data in (B–D) represent the mean values ± SD from four biological replicates. Significance was established using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: A plasmid expressing the coding region of p21 mRNA was obtained from OriGene (Cat. # SC119947) and the aforementioned p21 3′UTRs were then ligated; the resulting plasmids were named pCMV6-p21-3′wt or pCMV6-p21-3′del harboring the entire p21 mRNA coding sequence and either an intact or truncated 3′UTR, respectively.

Techniques: Expressing, Mutagenesis, Luciferase, Sequencing, Binding Assay, Transfection, Quantitative RT-PCR, Western Blot, BrdU Incorporation Assay