Journal: Cellular and molecular life sciences : CMLS

Article Title: Loss of fragile WWOX gene leads to senescence escape and genome instability.

doi: 10.1007/s00018-023-04950-1

Figure Lengend Snippet: Fig. 6 ROS inhibition results in senescence induction in late- passage Wwox−/− MEFs. a ROS levels in Wwox+/+, Wwox+/− and Wwox−/− MEFs at early- and late-passages were determined by DHE staining and flow cyto- metric analysis. b PCR amplifi- cation of mononucleotide repeat markers Bat26, Bat30, Bat37, Bat64 and Bat67 was performed using the genomic DNA sam- ples isolated from NAC-treated late-passage Wwox+/+ and Wwox−/− MEFs, and the length of PCR products was analyzed by capillary electrophoresis for detecting microsatellite insta- bility. c, d SA-β-gal staining was performed in late-passage Wwox+/+, Wwox+/−, Wwox−/−, and 1 mM NAC-treated Wwox−/− MEFs. The representa- tive images obtained from at least three independent experi- ments are shown in d. Scale bars = 100 µm. The percentages of SA-β-gal-positive senescent cells are shown in c. *P ≤ 0.05; ***P ≤ 0.005; Two-tailed t test. e Cellular morphology of Wwox+/+, Wwox+/−, Wwox−/−, and 1 mM NAC-treated Wwox−/− MEFs at early- and late-passages was examined. Scale bars = 100 µm. f DNA methylation of p16Ink4a pro- moter at specific CpG islands was analyzed. N.S. not sig- nificant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005; Two-tailed t test. g p16Ink4a, p21Cip1/Waf1, p53 and WWOX protein expression was examined in late-passage Wwox+/−, Wwox−/−, and 1 mM NAC-treated Wwox−/− MEFs. β-actin was used as an internal control in western blotting

Article Snippet: Human p21Cip1/Waf1 and p53 expression vectors were obtained from Addgene (#20814 and #11770, respectively; Watertown, MA, USA).

Techniques: Inhibition, Staining, Isolation, Electrophoresis, Two Tailed Test, DNA Methylation Assay, Expressing, 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 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: Oncotarget

Article Title: Anti-cancer effect of novel PAK1 inhibitor via induction of PUMA-mediated cell death and p21-mediated cell cycle arrest

doi: 10.18632/oncotarget.15783

Figure Lengend Snippet: (A) IPP-14 induces p21 expression in regardless of cell lines and serum condition. HCT116, A549 (human lung cancer cells), H1299 cell lines were treated with IPP-14 (2.5 μM) for 8 hr in serum-present or absent condition and western blot was performed using the indicated antibodies. Actin was used as loading control. (B) p21 induction is accomplished at post-translational level. Actinomycin D (Act. D; 1 μg/ml, Transcription inhibitor), Cyclohexamide (CHX; 100 μg/ml, Translational elongation inhibitor) were treated to block p21 induction by IPP-14. HCT116 cells were pre-treated Act. D or CHX for 2 hr before incubating with IPP-14 (1 μM). (C) Exogenous p21 is up-regulated by IPP-14. But p21-T145D mutant is induced marginally. HCT116 cells were transfected with p21 wild or mutant form (T145A, T145D), followed by treating IPP-14 (1 μM). Western blot was performed by using indicated antibodies. p21/Actin ratio was measured by using Image J software. (D) IPP-14 induces p21 expression but FRAX486 (known as selective PAK1 inhibitor) does not affect p21 upregulation. HCT116 cells were treated with IPP-14 or FRAX486 (5 μM). (E) p21 level is increased by IPP-14 but not by FRAX486. HCT116 cells were treated indicating chemicals for time-dependent manner and expression level was measured by western blot. p21/Actin ratio was measured by using Image J software. (F) Induction of cell death by IPP-14 and derivatives (IPP-115, 120, and 159) are not fully dependent on p21 induction. Moreover, FRAX486 does affect cell viability in regardless of p21 status. HCT116 and HCT116 p21-deficient cells were treated with indicating doses of chemicals for 48 hr, then cell viability was measured by MTT assay. * P < 0.005 (Student's t -test) (G) IPP-14 derivatives shows similar effect on p21 upregulation as IPP-14. HCT116 cells were incubated with IPP-14 derivatives (1 μM) or FRAX486 (5 μM) for 8 hr. (H) IPP-14 induces G2/M arrest in regardless of p21 status. Cells were treated with IPP-14 (1 μM) for 12 hr, followed fixing by PFA, and finally cell cycle was analyzed by FACS. (I) Inhibition of Cyclin B1 expression by IPP-14 in p21 deficient cells. Differentially from HCT116, where IPP-14 induced p21, HCT116 p21−/− cells showed the reduction of cyclin B1 in response to IPP-14 (1 μM). Western blot was performed by indicated antibodies. Actin was used as loading control.

Article Snippet: FLAG-p21, T145A, T145D expression vectors were purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Western Blot, Blocking Assay, Mutagenesis, Transfection, Software, MTT Assay, Incubation, Inhibition

Journal: Oncotarget

Article Title: Anti-cancer effect of novel PAK1 inhibitor via induction of PUMA-mediated cell death and p21-mediated cell cycle arrest

doi: 10.18632/oncotarget.15783

Figure Lengend Snippet: (A) IPP-14 does not show the effect of cell viability in isogenic PUMA/BAX deficient cell lines and normal fibroblast cells. After treatment of IPP for 48 hr, cell viability was measured by MTT assay. ## mean different group by ANOVA test ( P < 0.001). (B) p21 induction by IPP-14 is not detected in normal fibroblast cells and HaCaT cells (Non-cancer cells). HCT116, Normal fibroblast, and HaCaT cells were treated IPP-14 (1 μM) for 8 hr and western blot was performed using the indicated antibodies. (C) IPP-14 increases p21 expression in HCT116 cells clearly, but not in PUMA/BAX deficient cells. HCT116 and PUMA or BAX deficient cell lines were treated with IPP-14 (1 μM) for 8 hr in serum-present (CM) or absent (SF) condition. p21/Actin ratio was measured by using Image J software. (D) Bcl-2 suppresses the IPP-14-induced p21. Moreover, Bcl-2 also suppresses p21 basal level. HCT116 cells were transfected with Bcl-2 (HA) for 24 hr, then IPP-14 (1 μM) was treated in serum contain or free condition. (E) Exogenous p21 expression is suppressed by Bcl-2 overexpression. HCT116 cells were co-transfected with Bcl-2 and p21 WT or MT (T145A and T145D) for 24 hr, then western blot was performed. (F) IPP-14 inhibits the interaction between Bcl-2 and p21. HEK293 cells, co-transfected with Bcl-2 and p21 for 24 hr, were incubated with IPP-14 (1 μM), and lysed for IP assay with anti-HA antibody. Actin was used as loading control and negative control. (G) PUMA is required for IPP-14-mediated inhibition of Bcl-2 and p21 binding. Under the same condition of above, IP assay was performed in HCT116, HCT PUMA deficient cells by anti-HA antibody. (H) Bcl-2 expression is low in normal fibroblast and HaCaT cells. HCT116, normal fibroblast, and HaCaT cells were exposed to IPP-14 (1 μM) for 8 hr.

Article Snippet: FLAG-p21, T145A, T145D expression vectors were purchased from Addgene (Cambridge, MA, USA).

Techniques: MTT Assay, Western Blot, Expressing, Software, Transfection, Over Expression, Incubation, Negative Control, Inhibition, Binding Assay

Journal: Mediators of Inflammation

Article Title: Identification of a Novel lincRNA-p21-miR-181b-PTEN Signaling Cascade in Liver Fibrosis

doi: 10.1155/2016/9856538

Figure Lengend Snippet: lincRNA-p21 is downregulated in TGF- β 1-treated HSCs and human liver fibrosis. (a) The downregulation of lincRNA-p21 expression was dose-dependently induced by TGF- β 1. LX-2 cells were treated with TGF- β 1 (0, 5, 10, and 15 ng/mL) for 24 h. (b) The downregulation of lincRNA-p21 expression was time-dependently induced by TGF- β 1. LX-2 cells were treated with TGF- β 1 (5 ng/mL) for 0, 24, 48, and 72 h. (c) Expression of lincRNA-p21 in liver tissues of healthy controls ( n = 15) and cirrhotic patients ( n = 15). Each value is the mean ± SD of three experiments. ∗ P < 0.05 compared with the control.

Article Snippet: Adenoviral vectors expressing a control scrambled sequence (Ad-Ctrl) and adenoviral vectors expressing lincRNA-p21 (Ad-lincRNA-p21) were purchased from GenePharma biotechnology (Shanghai, China).

Techniques: Expressing, Control

Journal: Mediators of Inflammation

Article Title: Identification of a Novel lincRNA-p21-miR-181b-PTEN Signaling Cascade in Liver Fibrosis

doi: 10.1155/2016/9856538

Figure Lengend Snippet: Effects of lincRNA-p21 overexpression on cell proliferation, α -SMA, type I collagen, and PTEN in LX-2 cells. Cells were transduced with Ad-lincRNA-p21 for 48 h and treated with PTEN siRNA for additional 48 h. (a) lincRNA-p21 was detected in cells transduced with Ad-lincRNA-p21. Overexpression of lincRNA-p21 suppressed cell proliferation (b), α -SMA mRNA (c), α -SMA protein (d), Col1A1 mRNA (e), and type I collagen (f), which were almost blocked down by PTEN siRNA. Cell proliferation was assessed by CCK-8 assay. PTEN mRNA (g) and protein (h) expressions were upregulated by Ad-lincRNA-p21. GAPDH was used as internal control. Each value is the mean ± SD of three experiments. ∗ P < 0.05 compared with the control and # P < 0.05 compared with Ad-lincRNA-p21 group.

Article Snippet: Adenoviral vectors expressing a control scrambled sequence (Ad-Ctrl) and adenoviral vectors expressing lincRNA-p21 (Ad-lincRNA-p21) were purchased from GenePharma biotechnology (Shanghai, China).

Techniques: Over Expression, Transduction, CCK-8 Assay, Control

Journal: Mediators of Inflammation

Article Title: Identification of a Novel lincRNA-p21-miR-181b-PTEN Signaling Cascade in Liver Fibrosis

doi: 10.1155/2016/9856538

Figure Lengend Snippet: miR-181b is involved in the effects of lincRNA-p21 on PTEN expression and HSC activation. Cells were transduced with Ad-lincRNA-p21 for 48 h and treated with miR-181b mimics for additional 48 h. (a) Expressions of miR-21, miR-24, miR-32, miR-93, miR-153, miR-181b, miR-205, and miR-214 were detected in cells transduced with lincRNA-p21. (b) Expression of miR-181b in liver tissues of healthy controls ( n = 15) and cirrhotic patients ( n = 15). (c) Expression of miR-181b in miR-181b mimics group. (d) lincRNA-p21-induced PTEN was inhibited by miR-181b. (e) The effect of Ad-lincRNA-p21 on cell proliferation was suppressed by miR-181b. (f) The reduced mRNA expressions of α -SMA and Col1A1 by Ad-lincRNA-p21 were inhibited by miR-181b. (g) The reduced protein expressions of α -SMA and type I collagen by Ad-lincRNA-p21 were inhibited by miR-181b. GAPDH was used as internal control. Each value is the mean ± SD of three experiments. ∗ P < 0.05 compared with the control and # P < 0.05 compared with Ad-lincRNA-p21 group.

Article Snippet: Adenoviral vectors expressing a control scrambled sequence (Ad-Ctrl) and adenoviral vectors expressing lincRNA-p21 (Ad-lincRNA-p21) were purchased from GenePharma biotechnology (Shanghai, China).

Techniques: Expressing, Activation Assay, Transduction, Control

Journal: Mediators of Inflammation

Article Title: Identification of a Novel lincRNA-p21-miR-181b-PTEN Signaling Cascade in Liver Fibrosis

doi: 10.1155/2016/9856538

Figure Lengend Snippet: The effect of lincRNA-p21 on PTEN expression is through competitively binding miR-181b. (a) Correlation between lincRNA-p21 level and miR-181b expression in liver tissue samples from cirrhotic patients ( n = 15) was subjected to Pearson correlation analysis. (b) Schematic diagram of the miR-181b binding site in the lincRNA-p21 based on RNA22 software. (c) Relative luciferase activities of luciferase reporters bearing wild-type or mutant lincRNA-p21 were analyzed 48 h following transfection with the indicated miR-181b mimics or miR-NC in LX-2 cells. Each value is the mean ± SD of three experiments. ∗ P < 0.05.

Article Snippet: Adenoviral vectors expressing a control scrambled sequence (Ad-Ctrl) and adenoviral vectors expressing lincRNA-p21 (Ad-lincRNA-p21) were purchased from GenePharma biotechnology (Shanghai, China).

Techniques: Expressing, Binding Assay, Software, Luciferase, Mutagenesis, Transfection

Journal: Oncogene

Article Title: 15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE 2 /PPARγ-mediated activation of p21 WAF1/Cip1

doi: 10.1038/onc.2013.69

Figure Lengend Snippet: A . Tumorigenecity assay. Huh7 cells (1 × 10 8 cells in 0.2 ml of PBS) were injected subcutaneously at armpit in SCID mice. The mice were sacrificed 4 weeks after inoculation and the tumors were recovered. The wet weight of each tumor and tumor appearance time (days) were determined for each mouse. ( Left panel ) Photograph of xenograft tumors recovered from four groups of SCID mice inoculated with Huh7 cell lines (GFP control, 15-PGDH, RNAi control, 15-PGDH RNAi). ( Mid panel ) Xenograft tumor weights (grams). The data represent mean±SEM from eight SCID mice in each group. ( Right panel ) Xenograft tumors onset time (days). The data represent mean±SEM (n=8 for each group). B . Immunohistochemical analysis of xenograft tumor tissues. ( Left panel ) Hematoxylin-eosin (H&E) stain and PCNA immunostain were performed in formalin-fixed, paraffin-embedded xenograft tumor tissues recovered from SCID mice (original magnification ×100). ( Right panel ) Semi-quantification of PCNA positive cells (the data represent mean±SEM, n=8). C . Western blotting for PCNA and p21 in xenograft tumors (8 samples each group). β-actin was used as the internal control.

Article Snippet: To select cells with 15-PGDH knockdown plus p21 overexpression, the Huh7 cells stably transfected with the 15-PGDH RNAi vector (pGFP-V-RS-15PGDH) were subsequently transfected with the p21 expression vector (pcDNA3/WAF1/Cip1/p21, obtained from Addgene).

Techniques: Injection, Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Western Blot

Journal: Oncogene

Article Title: 15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE 2 /PPARγ-mediated activation of p21 WAF1/Cip1

doi: 10.1038/onc.2013.69

Figure Lengend Snippet: Hepa1-6 cells (1×10 8 cells in 0.2 ml of PBS) were inoculated subcutaneously at armpit in C57BL/6J mice. When the tumor nodules become palpable, the pAd control virus or pAd-15-PGDH (10 10 pfu) was injected directly to the tumor nodules (the injection was performed every three days, from the 10 th day to the 28 th day). After the last injection, the mice were observed for additional 3 days before sacrifice to recover tumor nodules. The wet weight of each tumor was recorded. The tumor diameters were measured by a caliper in two dimensions. The tumor volume was calculated by using the formula V=L/2*w 2 . A . Tumor size at different days. The data represent mean±SEM (n=6; *p < 0.05; **p < 0.01). B . ( Left panels ) Photographs of C57BL/6J mice inoculated with Hepa1-6 cells (prior to sacrifice) and the recovered xenograft tumors. ( Right panel ) The average tumor weight. The data represent mean±SEM (n= 6). C . Western blotting analysis for 15-PGDH in pAd-15-PGDH and pAd treated Hepa1-6 tumor tissues. β-actin was used as the internal control. D . H&E stain and PCNA immunostain in pAd-15-PGDH and pAd treated Hepa1-6 tumors. E . Western blotting for PCNA and p21 in pAd-15-PGDH and pAd treated Hepa1-6 tumors (6 samples for each group). β-actin was used as the internal control. F . ( Left panel ) Western blotting to detect nuclear p21 in pAd-15-PGDH and pAd treated Hepa1-6 tumors (6 samples for each group). Histone was used as the internal control. ( Right panel ) Western blotting to detect cytoplasmic p21 in pAd-15-PGDH and pAd treated Hepa1-6 tumors (6 samples for each group). β-actin was used as the internal control.

Article Snippet: To select cells with 15-PGDH knockdown plus p21 overexpression, the Huh7 cells stably transfected with the 15-PGDH RNAi vector (pGFP-V-RS-15PGDH) were subsequently transfected with the p21 expression vector (pcDNA3/WAF1/Cip1/p21, obtained from Addgene).

Techniques: Injection, Western Blot, Staining

Journal: Oncogene

Article Title: 15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE 2 /PPARγ-mediated activation of p21 WAF1/Cip1

doi: 10.1038/onc.2013.69

Figure Lengend Snippet: A . The effect of 15-PGDH and 15-keto-PGE 2 on p21 promoter activity. a p21 promoter luciferase reporter activity in Huh7 cells with altered expression of 15-PGDH (**p < 0.01). b p21 promoter luciferase reporter activity in Huh7 stable cell lines with co-transfection of PGR2. c p21 promoter luciferase reporter activity in Huh7 stable cell lines treated with 10µM GW9662. d 15-keto-PGE 2 (10µM) enhanced p21 promoter luciferase reporter activity in wild type Huh7 cells; the effect was abolished by PGR2 overexpression or by GW9662 treatment (10µM). B . PPARγ CHIP-PCR assay. a PPARγ association with p21 promoter in Huh7 stable cells with or without PGR2 overexpression. b 15-keto-PGE 2 (10µM) enhanced PPARγ binding to the p21 promoter in wild type Huh7 cells and the effect was abolished by PGR2 overexpression or GW9662 treatment (10µM). IgG CHIP was used as the negative control. p21 promoter PCR product was used as the input. C . PPARγ CHIP-real-time PCR assay. a PPARγ association with p21 promoter in Huh7 stable cells with or without PGR2 overexpression. b 15-keto-PGE 2 (10µM) enhanced PPARγ binding to the p21 promoter in wild type Huh7 cells and the effect was abolished by GW9662 treatment (10µM). IgG CHIP was used as the negative control. D . Effect of the PPARγ agonist ciglitazone (10µM) in wild type Huh7 cells. a PPARγ CHIP-real-time PCR assay. b PPRE luciferase reporter activity assay. E . Western blotting for p21 and phosphorylated p21 in Huh7 cells with indicated transfections.

Article Snippet: To select cells with 15-PGDH knockdown plus p21 overexpression, the Huh7 cells stably transfected with the 15-PGDH RNAi vector (pGFP-V-RS-15PGDH) were subsequently transfected with the p21 expression vector (pcDNA3/WAF1/Cip1/p21, obtained from Addgene).

Techniques: Activity Assay, Luciferase, Expressing, Stable Transfection, Cotransfection, Over Expression, Binding Assay, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal: Oncogene

Article Title: 15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE 2 /PPARγ-mediated activation of p21 WAF1/Cip1

doi: 10.1038/onc.2013.69

Figure Lengend Snippet: A . Western blotting for p21 in the nuclear fraction (nP21) and p21 in the cytoplasmic fraction (cP21) from Huh7 stable cell lines with altered 15-PGDH expression. Histone and β-actin were used as the internal control, respectively. B . Co-immunoprecipitation and western blotting analysis using indicated antibodies in Huh7 stable cell lines with altered 15-PGDH expression. IgG IP was used as the negative control. PCNA western blotting was used as input control. C . Co-immunoprecipitation and western blotting analysis using indicated antibodies in Huh7 stable cell lines with altered 15-PGDH expression. IgG IP was used as the negative control. rIP denotes repeat co-immunoprecipitation. D . Co-immunoprecipitation and western blotting analysis using indicated antibodies in Huh7 stable cell lines with altered 15-PGDH expression. IgG IP was used as the negative control. rIP denotes repeat co-immunoprecipitation. E . Co-immunoprecipitation and western blotting analysis using indicated antibodies in Huh7 stable cell lines with altered 15-PGDH expression. IgG IP was used as the negative control.

Article Snippet: To select cells with 15-PGDH knockdown plus p21 overexpression, the Huh7 cells stably transfected with the 15-PGDH RNAi vector (pGFP-V-RS-15PGDH) were subsequently transfected with the p21 expression vector (pcDNA3/WAF1/Cip1/p21, obtained from Addgene).

Techniques: Western Blot, Stable Transfection, Expressing, Immunoprecipitation, Negative Control

Journal: Oncogene

Article Title: 15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE 2 /PPARγ-mediated activation of p21 WAF1/Cip1

doi: 10.1038/onc.2013.69

Figure Lengend Snippet: A . Western blotting analysis using indicated antibodies in HepG2 cells transfected with GFP control vector and 15-PGDH expression vector with or without co-transfection of the p53 shRNA vector. The level of p21 in the nuclear fraction (nP21) and cytoplasmic fraction (cP21) were examined. β-actin and histone were used as the internal controls. B . p21 promoter luciferase activity assay in HepG2 cells transfected with the 15-PGDH expression vector or the control vector with or without co-transfection of the p53 shRNA vector. The data are presented as mean±SEM (**p < 0.01). C . Co-immunoprecipitation and western blotting analysis in HepG2 cells transfected with the control vector or the 15-PGDH expression vector with or without cotransfection of the p53 shRNA vector. IgG IP was used as the negative control. D . PPARγ CHIP assay in HepG2 cells transfected with the control vector or the 15-PGDH expression vector with or without cotransfection of the p53 shRNA vector (Addgene). ( Upper panel ) CHIP-regular PCR assay. p21 promoter PCR product was used as input. ( Lower panel ) CHIP-real time PCR assay. IgG CHIP was used as the negative control. The data are presented as mean±SEM (**p < 0.01 compared with the corresponding control).

Article Snippet: To select cells with 15-PGDH knockdown plus p21 overexpression, the Huh7 cells stably transfected with the 15-PGDH RNAi vector (pGFP-V-RS-15PGDH) were subsequently transfected with the p21 expression vector (pcDNA3/WAF1/Cip1/p21, obtained from Addgene).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Cotransfection, shRNA, Luciferase, Activity Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: 15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE 2 /PPARγ-mediated activation of p21 WAF1/Cip1

doi: 10.1038/onc.2013.69

Figure Lengend Snippet: A . Huh7 cells transfected with 15-PGDH overexpression vector with or without co-transfection of p21 RNAi. a Western blotting for 15-PGDH and p21 (β-actin was used as loading control). b WST cell proliferation assay. Each sample was assayed in triplicate for 6 consecutive days. Data are means±SEM from three independent experiments (**p < 0.01). c Soft-agar colony formation assay. The results are representative of three independent experiments (**p < 0.01). B . Huh7 stable cell transfected with 15-PGDH RNAi vector with or without co-transfection of p21 expression vector. a Western blotting for 15-PGDH and p21 (β-actin was used as loading control). b WST cell proliferation assay. Each sample was assayed in triplicate for 6 days consecutively. Data are mean±SEM from three independent experiments (**p < 0.01). c Soft-agar colony formation assay. The results are representative of three independent experiments (**p < 0.01).

Article Snippet: To select cells with 15-PGDH knockdown plus p21 overexpression, the Huh7 cells stably transfected with the 15-PGDH RNAi vector (pGFP-V-RS-15PGDH) were subsequently transfected with the p21 expression vector (pcDNA3/WAF1/Cip1/p21, obtained from Addgene).

Techniques: Transfection, Over Expression, Plasmid Preparation, Cotransfection, Western Blot, Proliferation Assay, Soft Agar Assay, Stable Transfection, Expressing