Journal: Carcinogenesis

Article Title: Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells.

doi: 10.1093/carcin/bgr192

Figure Lengend Snippet: Fig. 1. WA inhibits cervical cancer cell proliferation. (A) Structure of WA. (B) Data represent percent viability measured by MTTassay in the CaSki, HeLa, SiHa and C33a cervical cancer cell lines after treatment with WA at the 0-2 lM dose. (C) Cervical cancer cell lines were treated with indicated dose of WA for 24 h, protein lyastes were prepared and probed with HPV16/18 E6, p53 and p21 antibodies for western blot analysis. (D) Dose- and time-dependent effect of WA on CaSki cells. (E) Effect of WA on cell proliferation of primary epidermal keratinocytes (HEKn) at 24 h, CaSki cells included for comparison.

Article Snippet: Plasmid pZMZ70 (kind gift from Prof Zheng, Zhi-Ming, NIH) contains the E6 and E7 coding regions, fused in frame to the C-terminus of GFP and GFP-p53 (Addgene, Cambridge, MA) were used for transient transfection in C33a (HPV-negative cervical cancer cell line) and H1299 (p53 null lung cancer cell line) cells, respectively.

Techniques: Western Blot, Comparison

Journal: Carcinogenesis

Article Title: Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells.

doi: 10.1093/carcin/bgr192

Figure Lengend Snippet: Fig. 3. WA modulates HPVoncogenes and restores p53. (A) CaSki cells were treated with vehicle or WA for 12 and 24 h. HPV16 E6 (left) and E7 (right) mRNA levels were measured by qPCR. Data represent mean ± SD of fold downregulation of mRNA levels relative to the control. P , 0.01, P , 0.001. (B) CaSki cells were treated for 6 h with or without WA (0.5 lM) and RNA synthesis blocked with actinomycin D (5 lg/ml). Cells were harvested at indicated times and transcript levels of HPV16 E6 (left); E7 (center) and p53 (right) were determined by qPCR. Data represent mean ± SD of three experiments. (C), Transient transfection of a C33a (HPV negative) cells with pZMZ70 (GFP-HPV16 E6E7) (top row) and H1299 (p53 null) cells (bottom row) with GFP-p53 reporter is shown. Fluorescent microscope images show 60 and 90% transfection efficiency in C33a and H1299 cells, respectively. Cells were treated with vehicle or WA (0.5 lM) for 24 h and cell lysates were analyzed by western blotting using anti-GFP antibody. (D) Confocal photomicrographs (20) of representative CaSki cells showing nuclear localization p53 protein. Cells were treated with vehicle (first row) and WA (0.5 lM) (second row) and isotype-matched negative control (third row) are shown. 4#,6-Diamidino-2-phenylindole was used to visualize DNA (first lane) and Phalloidin 488 (green) or 594 (red) used to visualize actin filaments (second lane). The scale represents a length of 40 lm. Western blot analysis of p53 protein from cytoplasmic (left) and nuclear (right) fractions of WA-treated CaSki cells at indicated doses. (E) Western blot analysis of indicated proteins after 12 (left) and 24 h (right) of WA treatment at indicated doses. (F) CaSki cells

Article Snippet: Plasmid pZMZ70 (kind gift from Prof Zheng, Zhi-Ming, NIH) contains the E6 and E7 coding regions, fused in frame to the C-terminus of GFP and GFP-p53 (Addgene, Cambridge, MA) were used for transient transfection in C33a (HPV-negative cervical cancer cell line) and H1299 (p53 null lung cancer cell line) cells, respectively.

Techniques: Control, Transfection, Microscopy, Western Blot, Negative Control

Journal: Carcinogenesis

Article Title: Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells.

doi: 10.1093/carcin/bgr192

Figure Lengend Snippet: Fig. 5. WA modulates STAT3 and apoptosis-related proteins. (A) CaSki cells were treated with vehicle or WA at indicated doses for 12 h (left) and 24 h (right). Levels of indicated proteins were analyzed by western blot analysis. Cytosolic fractions were analyzed for cytochrome c levels (B) CaSki cells were transfected with control siRNA, si STAT3 or si p53, followed by treatment with vehicle or WA (0.5 lM) for 24 h. Cell lysates were used for western blot analysis with STAT3, p53 and b-actin antibodies. Representative blots are shown.

Article Snippet: Plasmid pZMZ70 (kind gift from Prof Zheng, Zhi-Ming, NIH) contains the E6 and E7 coding regions, fused in frame to the C-terminus of GFP and GFP-p53 (Addgene, Cambridge, MA) were used for transient transfection in C33a (HPV-negative cervical cancer cell line) and H1299 (p53 null lung cancer cell line) cells, respectively.

Techniques: Western Blot, Transfection, Control

Journal: Gene

Article Title: An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

doi: 10.1016/j.gene.2012.01.011

Figure Lengend Snippet: Live-cell imaging of p53-GFP-expressing Saos-2 cells. Saos-2 cells were transfected as described previously with pJCPAC-Mam1-p53-GFP. Three days post-transfection, Saos-2 cells were photographed and UV and white light images were merged. (A). Exogenous p53-GFP localizes predominantly to the nuclei of Saos-2 cells. (B). Membrane blebbing, a hallmark event in apoptosis occurs in a subset of p53-GFP-expressing Saos-2 cells.

Article Snippet: A p53-GFP cassette (Gift of Jon Jarvik, SpectraGenetics) was subcloned into the unique NotI site of pJCPAC-Mam1 and Saos-2 cells were also transfected with this p53-GFP fusion protein-expressing construct using PEI as previously described.

Techniques: Live Cell Imaging, Expressing, Transfection

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A, B ) p53 −/− kidneys exhibit 50% fewer and less complex LTA-positive proximal tubules. Metanephroi were harvested at E11.5, cultured on trans-well filters for 72 h and stained for cytokeratin and LTA. LTA counts were averaged from kidneys collected from at least 4 embryos. Mutant kidneys exhibit significantly fewer LTA+ structures than wild-type kidneys (p<0.005). C) E17.5 kidneys were harvested from embryos from p53 +/− crosses, formalin-fixed and sectioned for immunostaining (Methods). Sections were stained with WT1 and cytokeratin antibodies. P53 −/− kidneys show paucity of WT1 stained nephrons. D ) Kidneys from mice with conditional p53 deletion from Six2+ cap mesenchyme (CM p53−/− ). Hypoplasia persists post-natally, shown in P0 kidneys, top panels at 4x. Bottom panels (x10) show fewer Lhx1-positive nascent nephrons (red). At P0, 6/8 (75%) examined CM p53−/− kidneys were hypoplastic compared to wild-type littermate kidneys (n = 8).

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Cell Culture, Staining, Mutagenesis, Immunostaining

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) p53 expression overlaps that of Pax2 in the developing kidney. Immunofluorescence staining was done on E15.5 kidney sections to visualize Pax2 and p53 protein expression. p53 co- localizes with Pax2 in the cap mesenchyme (CM) and in the ureteric tip (UB). B ) In situ hybridization. Decreased expression of Pax2 mRNA at E12.5. C ) QPCR. Pax2 mRNA is significantly lower in germ-line p53 −/− kidneys. QPCR was done on RNA from individual E15.5 kidney pairs from wild-type and p53 −/− embryos (n = 4). D ) Pax2 protein in E15.5 kidneys, detected by IF staining. Both wild-type and mutant kidney sections were processed for IF staining simultaneously and images captured at identical exposure setting. After normalizing intensity to that in wild-type, images were converted to a heat-map to demonstrate differential Pax2 staining between wild-type and mutant kidneys. High intensity staining corresponds to red/yellow areas and lower intensity corresponds to green/blue. Top panels at x4 and bottom panels at x20. E ) Quantitation of staining intensity was done using Slidebook software and is shown graphically. UB, ureteric bud/tips; CM, cap mesenchyme; Nn, nascent nephrons including pretubular aggregates and renal vesicles.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Expressing, Immunofluorescence, Staining, In Situ Hybridization, Mutagenesis, Quantitation Assay, Software

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) Reduced Pax2 staining in the CM of E12.5 CM p53−/− kidneys in comparison to wild-type CM, indicative of lower Pax2 protein levels. B ) Pax2 mRNA is ∼40% lower in CM p53−/− kidneys. QPCR was done on RNA from E15.5 littermate kidneys from wild-type (n = 5 pairs) and CM p53−/− (n = 5 pairs).

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Staining, Comparison

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: ) Genomic view of p53 occupancy at the Pax2 gene locus identified by ChIP-Seq and visualized using the Integrated Genome Browser (IGB). Orange bars denote p53 enriched regions after background (Input) subtraction. Green bars show MACS peaks which represent points of highest density of sequenced fragments within the orange interval. Peak 1 and 2 are in the proximal promoter and in intron2-exon3, respectively. Two additional peaks were identified far distal to the Pax2 gene at ∼−14 kb (peak 3) and ∼−83 kb (peak 4) from the transcription start site. Yellow box shows region validated for p53-enrichment by ChIP-PCR in Fig. 5. B ) High sequence conservation in mammals of regions encompassing p53-enriched areas, including the non-genic distal regions 3 and 4, visualized in the UCSC genome browser ( http://genome.ucsc.edu ). C ) Red bars show location of p53 binding motifs in and around p53-occupied regions 1 and 2 in the Pax2 promoter/gene. Although p53 binding sites are broadly scattered across the entire region including the intervening region between regions 1 and 2 (see ), p53 occupancy occurs at specific regions.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: ChIP-sequencing, Sequencing, Binding Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: ) (a) ChIP-Seq track showing p53 occupancy at Pax2 proximal promoter (Region 1). (b) That this enrichment is not an artifact of amplification and sequencing is shown by lack of enrichment in the Input sample. Presence of any spurious false peaks in the Input sample eliminates that region from an interval, reflecting high stringency set for the MACS program. (c) Grey bar shows region of p53 enrichment designated as an interval. The dotted green arrow denotes RefSeq TSS, solid green arrow shows TSS described by . (d) p53 motifs (blue bars) identified by Genomatix and manually. (e) Position of amplicons to validate ChIP-Seq data and show differential p53 occupancy between mK3 and mK4 cells by ChIP-PCR. (f) Co-ordinates on mus chromosome 19. B ) Chromatin was immunoprecipitated from both cell lines with anti-p53 antibody and amplified by PCR. PCR band intensities were quantified using the Alphaimager software as described in , and band intensity of immunoprecipitated fragments was normalized to Input band intensity for each cell line. Normalized values for each amplicon were plotted as mK4/mK3 ratios. Values greater than 1.0 indicate fragment enrichment in mK4 relative to mK3, as a result of increased p53 binding and immunoprecipitation as seen for amplicons P3 and P7 (blue line). Values are a mean of three independent ChIP experiments.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: ChIP-sequencing, Amplification, Sequencing, Immunoprecipitation, Software, Binding Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) p53 −/− H1299 cells were co-transfected with a p53 expression plasmid (pCMV-p53) and a 4.1-Pax2-CAT reporter construct. Dose-dependent increase in reporter activity was observed with wild-type p53 (WT-p53). Reporter activity was maintained at baseline when reporter plasmid was co-transfected with a non-DNA binding mutant p53 (Mt-p53, pCMVp53-E258K). This mutant acts as a dominant-negative and inhibits Pax2 promoter activation by wild-type p53. B ) mK4 cells were transiently co-transfected with a p53 expression plasmid (pCMV-p53) and a 4.1-Pax2-Luc reporter construct. Absolute values of luciferase activity are plotted as Relative Light Units (RLU). Luciferase activity was normalized to protein concentration. C ) p53 knock-down in mK4 cells using 4 different p53 shRNA plasmids showed corresponding decrease in Pax2 mRNA, relative to mRNA levels from GFP- cells (dotted red line). mK4 cells were transfected with p53-shRNA-GFP plasmid, and GFP+ cells were sorted 48 h post-transfection, RNA harvested and used for QPCR. Gene expression was normalized to β-actin or GAPDH expression. D ) Schematic of deletion mutant Pax2 promoter constructs, shown with respect to p53 occupancy determined by ChIP-Seq. Orange bar denotes p53 occupancy in Pax2 promoter, corresponding to region 1 in Fig. 2A. Green solid arrow shows TSS used in this study, dotted arrow denotes possible alternate TSS in RefSeq. Purple bars indicate location of p53 binding sites identified in silico and tested for p53 binding by EMSA, numbered 1–14. Mutagenized sites shown in red. E ) Full-length or truncated Pax2-promoter-CAT reporter plasmids were transfected into UB cells either without or with pCMV-p53 (50 ng). CAT activity was normalized to protein concentration. Fold-induction by p53 of CAT activity over baseline (red-line) is plotted. F ) Fold-change in reporter activity by p53 after site-directed mutagenesis of p53-binding site 7 (Mut 7–1 and 7–2) and 8 (Mut 8–1 and 8–2) individually or together (Mut 7/8). Two clones were tested in transient transfection assays per site mutated.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Activity Assay, Binding Assay, Mutagenesis, Dominant Negative Mutation, Activation Assay, Luciferase, Protein Concentration, Knockdown, shRNA, Gene Expression, ChIP-sequencing, In Silico, Clone Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: Percent conservation in mammals of p53 binding sites in the Pax2 promoter.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) E13.5 kidneys from Pax2 LacZ/+; p53 +/− crosses were stained with cytokeratin and UB tips counted. B ) UB tip number was used as a phenotypic readout and measure of renal hypoplasia in Pax2 LacZ/+; p53 −/− animals compared to wild type, or Pax2 LacZ/+ ;p53 +/+ . Reduction of either Pax2 gene dosage or elimination of p53 gene reduced UB tip number by ∼12–15%. Complete p53 deficiency with Pax2 haploinsufficiency resulted in ∼55% decrease in tip number. For each genotype, N = 3−5 kidney pairs from at least 3 independent litters. All animals were harvested at ∼E13.0, cultured overnight and fixed for whole mount cytokeratin staining to enable counting of UB tips.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Staining, Cell Culture

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A) Model for autonomous and interactive gene regulation by p53 and Pax2. Genes regulated independently by each transcription factor. P53 positively regulates Pax2 expression, and the two may co-operatively regulate genes shown in box below dotted arrow. Expression of these genes is altered in p53 −/− E15.5 kidneys and these genes contain p53-binding motifs that are occupied by p53 in vivo as determined by ChIP-Seq. Genomatix search revealed Pax2 binding motifs in these genes. B) Model for p53-Pax2 cross-talk in the developing kidney. p53 enhances Pax2 expression in mesenchyme cells and promotes their transition to epithelia. Thus, p53 serves to promote differentiation of progenitor cells to nascent nephrons, and in its absence Pax2 down-regulation is a possible mechanism of nephron deficit observed in these kidneys.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Expressing, Binding Assay, In Vivo, ChIP-sequencing