Journal: PLoS ONE

Article Title: p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21 Waf1/CIP1

doi: 10.1371/journal.pone.0008369

Figure Lengend Snippet: HFKs were induced to differentiate in CaCl 2 medium and harvested at the indicated time points (t = 0, 6, 16, 48 hrs). ( A ) Western blot analysis of levels of p300 in control and p300 depleted HFKs using scrambled shRNA (scr) two shRNA molecules (shp300A and shp300B) directed against p300. ( B ) Cells stably expressing p300 targeting shRNAs were induced to differentiate and harvested at the indicated time points. Western blot analysis indicates that knocking down p300 dramatically reduces the p300 mediated acetylation and expression of p53. There is also a decrease in p21 Waf1/CIP1 and K1 levels in p300 depleted HFKs. The results shown are representative of three independent experiments.

Article Snippet: All these constructs were sub-cloned into the XbaI site of pGL-3 basic (Promega), except the 0-luc that was inserted into a blunt-ended Xho1 site of pGL3-basic. p21 Waf1/Cip1 -mut1 luciferase report vector construct was kindly provided by Professor Wei-Guo Zhu . pMT5-p21 Waf1/CIP1 -Flag over-expression vector was purchased from Addgene.

Techniques: Western Blot, shRNA, Stable Transfection, Expressing

Journal: PLoS ONE

Article Title: p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21 Waf1/CIP1

doi: 10.1371/journal.pone.0008369

Figure Lengend Snippet: Stably expressing p300 targeted shRNAs cells were transfected with p21 Waf1/CIP1 promoter luciferase construct. Transfected cells were either kept in low calcium (0.5 mM) or high calcium (1.5 mM) for the indicated times prior to the termination of the experiment (72 hours after transfection). Doxorubicin (0.1 µg/ml) was used as a positive control in this experiment. (A) Depletion of p300 down-regulates p21 Waf1/CIP1 promoter activity after 16 hours of calcium treatment (Mean +/− SE, three independent biological replicates; asterisk (*) p<0.01 relative to relevant control, Student's t test). (B) Real time quantification confirms that depletion of p300 inhibits the transcription of p21 Waf1/CIP1 during differentiation (Mean +/− SE, two independent biological replicated; asterisk (*) p<0.05 relative to relevant control, Student's t test). (C) ChIP assay for p53 and p300 binding to two p53 response elements located in the p21 Waf1/CIP1 promoter in HFKs induced to differentiate by addition of calcium. PCR analysis of DNA precipitated by a p300 or p53 antibodies indicates that p300 only interacts with both p53 response elements within the p21 Waf1/CIP1 promoter after 6 hours calcium treatment. Left panel: p53 binding to the p53 respond elements (RE1 and RE2) increases over a 16 hour periods. Right panel: Depletion of p53 results absence of p53 and p300 at both p53 response sites. Mouse (IgGM) and Rabbit IgG (IgGR) are included as negative control. (D) Western blot of the proteins from the same extracts as in (C).

Article Snippet: All these constructs were sub-cloned into the XbaI site of pGL-3 basic (Promega), except the 0-luc that was inserted into a blunt-ended Xho1 site of pGL3-basic. p21 Waf1/Cip1 -mut1 luciferase report vector construct was kindly provided by Professor Wei-Guo Zhu . pMT5-p21 Waf1/CIP1 -Flag over-expression vector was purchased from Addgene.

Techniques: Stable Transfection, Expressing, Transfection, Luciferase, Construct, Positive Control, Activity Assay, Binding Assay, Negative Control, Western Blot

Journal: PLoS ONE

Article Title: p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21 Waf1/CIP1

doi: 10.1371/journal.pone.0008369

Figure Lengend Snippet: HFKs were transiently tranfected with p21 Waf1/Cip1 targeting siRNA molecules. ( A ) Immunoblotting of the whole cell lysates with p21 Waf'1/CIP1 antibody shows that both siRNA molecules, sip21 Waf'1/CIP1 A and sip21 Waf'1/CIP1 B, were able to knockdown p21 Waf'1/CIP1 protein levels efficiently (∼90%) ( B ) Western blot shows p21 Waf1/CIP1 depletion with sip21 Waf1/CIP1 A and a decreased in K1 levels after 48 hours of differentiation. ( C ) Transfected cells were seeded onto coverslips, incubated with 1.5 mM calcium for 48 hours, and were pulsed with BrdU prior to fixation. BrdU uptake indicates a 90% increased proliferation of p21 Waf1/CIP1 knockdown cells prior to the addition differentiation (t = 0). After 48 hours calcium treatment, around 40% of the p21 Waf1/CIP1 knock-down cells remained BrdU positive. ( D ) H&E staining shows an increase in thickness of epithelia in p21 Waf1/CIP1 depleted cells (upper panel). Immunohistochemisty staining of organotypic raft cultures indicates that the differentiation marker K1 is reduced in p21 Waf1/CIP1 knockdown rafts with low levels of K1 (middle panel). Rafts were pulsed with BrdU for 16 hours prior to harvest and BrdU positive cells counted (lower panel). BrdU positive cells were observed in most parabasal cells. ( E ) Graph represents BrdU uptake expressed as percentage of scrambled control (Mean +/− SE, two independent experiments). (F) Stably expressing p300 targeted shRNA cells were transiently transfected with either 1 µg pMT5-p21 Waf1/CIP1 -Flag vector or pCMV empty vector (negative control) for 24 hours. Transfected cells were then incubated with calcium for 48 hours. Western blot analysis indicates that exogenous expression of p21 Waf1/CIP1 rescued the expression of K1 in p300 depleted cells.

Article Snippet: All these constructs were sub-cloned into the XbaI site of pGL-3 basic (Promega), except the 0-luc that was inserted into a blunt-ended Xho1 site of pGL3-basic. p21 Waf1/Cip1 -mut1 luciferase report vector construct was kindly provided by Professor Wei-Guo Zhu . pMT5-p21 Waf1/CIP1 -Flag over-expression vector was purchased from Addgene.

Techniques: Western Blot, Transfection, Incubation, Staining, Marker, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Negative Control

Journal: Oncology Reports

Article Title: let-7 and miR-17 promote self-renewal and drive gefitinib resistance in non-small cell lung cancer

doi: 10.3892/or.2019.7197

Figure Lengend Snippet: miR-17 affects gefitinib resistance and self-renewal by targeting CDKN1A. (A) Luciferase assays of the miR-17 targeting effects on CDKN1A. Mutations were generated in the CDKN1A 3′-UTR sequence in the complementary site for the seed region of miR-17. (B) mRNA levels of CDKN1A were decreased in PC9/GR cells compared with PC9 cells. (C) Inhibition of miR-17 increased CDKN1A expression in PC9/GR cells and overexpression of let-7 decreased CDKN1A expression in PC9 cells. (D) mRNA levels of CDKN1A decreased after PC9 cells were treated with CDKN1A siRNA and increased after PC9/GR cells were treated with CDKN1A plasmids. (E) Overexpression of CDKN1A decreased gefitinib resistance in PC9/GR cells and inhibition of CDKN1A increased gefitinib resistance in PC9 cells. (F) Overexpression of CDKN1A decreased self-renewal in PC9/GR cells and inhibition of CDKN1A increased self-renewal in PC9 cells. (G) Overexpression of CDKN1A or inhibition of miR-17 increased the G1 phase and decreased the G2/M phase in PC9/GR cells and inhibition of CDKN1A or overexpression of let-7 decreased the G1 phase and increased the G2/M phase in PC9 cells. (H) Inhibition of CDKN1A or overexpression of let-7 decreased the G1 phase and increased the G2/M phase in HCC827 cells. (n=3, *P<0.05). GR, gefitinib resistant.

Article Snippet: The wild-type (WT) and mutant-type (MUT) 3′untranslated region (3′-UTR) of MYC and CDKN1A were synthesized chemically and inserted into the psiCHECK™-2 vector (Promega Corp., Madison, WI, USA) to obtain psiCHECK™-2-MYC-3′-UTR (WT or MUT plasmids) and psiCHECK™-2-CDKN1A-3′-UTR (WT or MUT plasmids).

Techniques: Luciferase, Generated, Sequencing, Inhibition, Expressing, Over Expression

Journal: The Journal of biological chemistry

Article Title: Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter.

doi: 10.1074/jbc.M306694200

Figure Lengend Snippet: FIG. 3. Che-1 increases p21 expression. A, lysates from HCT116 and LoVo cells transiently transfected with the indicated amounts of Myc-Che-1 were subjected to Western blot analysis with anti-Myc and anti--tubulin antibodies to verify protein expression. B, HCT116 and LoVo cells were transiently transfected with 1 g of the p21 promoter luciferase reporter and the indicated amounts of Myc-Che-1. The total amount of transfected DNA was normalized with pCMV empty vector (). Data are presented as the mean S.D. from three independent experiments performed in duplicate. C, RNA from HCT116 cells transiently transfected as described in panel A was isolated. Equal amounts of RNA (RNA input) were analyzed by RT-PCR (25 cycles) for expression of the p21 gene. The RT control lanes represent RT-PCR in the absence of reverse transcription. D, lysates from HCT116 cells transiently transfected with empty vector (Control) or Myc-Che-1, respectively, were subjected to Western blot analysis with anti-p21, anti-Myc, and anti--tubulin antibodies.

Article Snippet: Mouse monoclonal antibodies anti-Myc 9e10 (Invitrogen), -tubulin and human p21 (sc-8035 and sc-6246; Santa Cruz Biotechnology), and bromodeoxyuridine (BrdUrd) (Roche Applied Science) were also used.

Techniques: Expressing, Transfection, Western Blot, Luciferase, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription

Journal: The Journal of biological chemistry

Article Title: Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter.

doi: 10.1074/jbc.M306694200

Figure Lengend Snippet: FIG. 4. p21, but not p53, is required for Che-1-mediated growth inhibition. HCT116 p21/ (A) and HCT116 p53/ (B) cells were transiently transfected with empty pCMV expression vector (Control) or Myc-Che-1 and a vector carrying puromycin resistance gene (10:1 ratio) and selected for 7 days in purumycin. Parallel plates were stained with methylene blue and scored. In the lower panels, the expression levels of Myc-Che-1 and -tubulin were assayed by Western blotting. C, HCT116 and HCT116 p53/ cells were transiently transfected with 1 g of the p21 promoter luciferase reporter and the indicated amounts of Myc-Che-1. The total amount of transfected DNA was normalized with pCMV empty vector (). Data are presented as means S.D. from three independent experiments performed in duplicate.

Article Snippet: Mouse monoclonal antibodies anti-Myc 9e10 (Invitrogen), -tubulin and human p21 (sc-8035 and sc-6246; Santa Cruz Biotechnology), and bromodeoxyuridine (BrdUrd) (Roche Applied Science) were also used.

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Control, Staining, Western Blot, Luciferase

Journal: The Journal of biological chemistry

Article Title: Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter.

doi: 10.1074/jbc.M306694200

Figure Lengend Snippet: FIG. 6. Che-1 displaces the HDAC1 associated to Sp1 binding sites of the p21 promoter in vivo. A, DNA sequence of the 5-end region of the p21 promoter; nucleotides are numbered from the transcription initiation site. Consensus sites for Sp1 binding are indicated. Arrows indicate starting points of primers used for ChiP analysis. B, lysates from HCT116 cells transiently transfected with empty vector (Control) or Myc-Che-1, respectively, were subjected to Western blot analysis with anti-Myc and anti--tubulin antibodies to verify protein expression. C and D, lysates from HCT116 cells transfected as described in panel B were subjected to ChiP using specific polyclonal antibodies, anti-HDAC1, anti-Che-1, anti-Sp1, or anti-acetylated histone H3. C, increasing amounts of input samples (0.25, 0.5, 1 l) were used as template in PCR amplifications performed using primers specific for the different regions of the p21 promoter that include the Sp1 binding sites. D, immunoprecipitates from each sample were analyzed by PCR, and a sample representing linear amplification (0.5–1 l) of the total input chromatin (Input) was included in the PCRs as control. Additional control included a precipitation performed with no specific IgGs.

Article Snippet: Mouse monoclonal antibodies anti-Myc 9e10 (Invitrogen), -tubulin and human p21 (sc-8035 and sc-6246; Santa Cruz Biotechnology), and bromodeoxyuridine (BrdUrd) (Roche Applied Science) were also used.

Techniques: Binding Assay, In Vivo, Sequencing, Transfection, Plasmid Preparation, Control, Western Blot, Expressing, Amplification

Journal: The Journal of biological chemistry

Article Title: Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter.

doi: 10.1074/jbc.M306694200

Figure Lengend Snippet: FIG. 7. Che-1 silencing inhibits p21 transcription and increases cell proliferation. A, silencing of Che-1 in HCT116 cells. Western blots of cells transfected with GFP siRNA (Control) or Che-1 siRNA, probed with anti-Che-1 antibody. The blot was stripped and reprobed with anti--tubulin antibody. B, double fluorescence analysis with anti Che-1-specific antibody and Hoechst of HCT116 cells transfected with GFP siRNA (Control) or Che-1 siRNA. C, HCT116 cells were transiently transfected with 1 g of the p21 promoter luciferase reporter and GFP siRNA (Control) or Che-1 siRNA. Data are presented as the mean S.D. from three independent experiments performed in duplicate. D, cell proliferation analyses of HCT116 cells transiently transfected with GFP siRNA (Control) or Che-1 siRNA. Data are presented as means S.D. from three independent experiments performed in duplicate.

Article Snippet: Mouse monoclonal antibodies anti-Myc 9e10 (Invitrogen), -tubulin and human p21 (sc-8035 and sc-6246; Santa Cruz Biotechnology), and bromodeoxyuridine (BrdUrd) (Roche Applied Science) were also used.

Techniques: Western Blot, Transfection, Control, Fluorescence, Luciferase

Journal: Cancer Research

Article Title: Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo

doi: 10.1158/0008-5472.can-10-0801

Figure Lengend Snippet: Figure 1. Serial 3T3 cultivation of primary MEFs of different genotypes. A and B, MEFs derived from wild-type (WT), p16-KO, p21-KO, and DKO mice were cultivated according to the 3T3 protocol. The graphs represent the accumulated population doublings at every passage (A, in 20% oxygen; B, in 3% oxygen). The means ± SD of triplicate experiments are shown. C, protein levels of the cell cycle inhibitors p16INK4a, p21Waf1/Cip1, p15INK4b, and p19Arf. Note that passage 20 of the wild-type and p16-KO MEFs in 20% oxygen culture could not be analyzed because a very long period of time was required to generate an immortalized culture. To compare the levels of proteins across the gels, the same sample (immortalized MEFs) was loaded on every gel (lanes 1, 10, 19, and 30). β-Actin was used as a loading control. Representative data of two independent experiments are shown. Uncropped images are shown in Supplementary Data.

Article Snippet: The primary antibodies used were 4a (11104 IBL), p21Waf1/Cip1 (sc-6246, Santa Cruz Biology, Inc.), p15INK4b (sc-613, Santa Cruz Biotechnology), C12, Cell Signaling), p19Arf [ab80 (Abcam) or sc-32748 a Cruz Biotechnology)], H-Ras (sc-29, Santa Cruz hnology), pRb (554136, BD Pharmingen), β-actin , Sigma-Aldrich), and α-tublin (DM-1A, Sigma-Aldrich). dary antibodies were detected by enhanced chemilumice (Amersham). virus infection and focus formation assay oviral gene transfer was done by transient transfection LinxE ecotropic packaging cells with a pBabe-puro and the vector containing the human H-rasV12 oncoas previously described (6).

Techniques: Derivative Assay, Control

Journal: Cancer Research

Article Title: Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo

doi: 10.1158/0008-5472.can-10-0801

Figure Lengend Snippet: Figure 2. Induction of oncogene-induced cellular senescence by retrovirally transduced oncogenic Ras in wild-type, p16-KO, p21-KO, and DKO MEFs. MEFs of the indicated genotypes were infected with control retrovirus or retrovirus encoding oncogenic Ras, and a series of experiments were performed a week after infection. A, representative photographs of cells; B, percentage of senescence-associated β-galactosidase (SA-β-gal) positive MEFs; C, relative ROS levels. Columns, mean of three independent experiments; bars, SD. D, protein levels of p15INK4b, p19Arf, p53, Rb, and H-Ras in MEFs infected with retrovirus encoding oncogenic Ras (H-RasV12) or control empty vector (Vector). β-Actin was used as a loading control. Representative data of two independent experiments are shown. Uncropped images are shown in Supplementary Data.

Article Snippet: The primary antibodies used were 4a (11104 IBL), p21Waf1/Cip1 (sc-6246, Santa Cruz Biology, Inc.), p15INK4b (sc-613, Santa Cruz Biotechnology), C12, Cell Signaling), p19Arf [ab80 (Abcam) or sc-32748 a Cruz Biotechnology)], H-Ras (sc-29, Santa Cruz hnology), pRb (554136, BD Pharmingen), β-actin , Sigma-Aldrich), and α-tublin (DM-1A, Sigma-Aldrich). dary antibodies were detected by enhanced chemilumice (Amersham). virus infection and focus formation assay oviral gene transfer was done by transient transfection LinxE ecotropic packaging cells with a pBabe-puro and the vector containing the human H-rasV12 oncoas previously described (6).

Techniques: Infection, Control, Plasmid Preparation

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