Journal: Oncotarget

Article Title: Inhibition of RAC1 GTPase sensitizes pancreatic cancer cells to γ-irradiation.

doi: 10.18632/oncotarget.2500

Figure Lengend Snippet: Figure 5: Rac1 inhibition abolishes IR-induced activation of both ATM and ATR signaling pathways. CD18/HPAF cells were treated with/without 10 Gy IR in the presence of NSC23766 at the indicated doses and incubated for 1 h at 37oC. (A) To assess ATR and ATM kinase activities, ATR and ATM were immunoprecipitated from the cell lysates using anti-ATR (N-19) and anti-ATM (2C1) antibodies respectively and assayed for relative kinase activity using recombinant p53 protein as substrate. (B) To measure Chk1 and Chk2 activity, Chk1 and Chk2 were immunoprecipitated from the cell lysates using anti-Chk1 (G-4) and anti-Chk2 (B-4) antibodies respectively and assayed for relative kinase activity using recombinant Cdc25C protein as substrate. As controls, protein levels of ATR, ATM, Chk1 and Chk2 in the immunoprecipitates (IP-WB) as well as in the cell lysates (WB) were assessed by immunoblotting. (C) Cdc2 was immunoprecipiated from the cell lysates using anti-Cdc2 (17) antibody and analyzed for Cdc2-Y15 phosphorylation and Cdc2 protein by immunoblotting.

Article Snippet: Recombinant p53 protein for ATM and ATR kinase assays was a glutathione S-transferase (GST) fusion protein containing full-length human p53 (Addgene, Cambridge, MA).

Techniques: Inhibition, Activation Assay, Protein-Protein interactions, Incubation, Immunoprecipitation, Activity Assay, Recombinant, Western Blot, Phospho-proteomics

Journal: Nature Communications

Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

doi: 10.1038/s41467-021-26901-9

Figure Lengend Snippet: A OWCM-155 NEPC organoid cells (5000/well) were plated with or without stable H19 knockdown (Lv-shH19 or Lv-Scr). Line graphs demonstrate the quantification of organoid growth. B Trp53 flox/flox /Rb1 flox/flox mouse organoid cells (5000/well/condition) were plated. Bar plot represents organoid area quantification ( n = 10; biological replicates) C Representative fluorescence images of organoids in Fig. 4B. Scale bar: 1000 μm. D Time course of OWCM-155 control (shScr) and H19 KD (shH19) tumor xenograft growth in n = 5 NSG mice. E Mean tumor weight of the tumor xenografts ( n = 4) harvested at 11-week post-injection. F Relative mRNA expression of H19 and NE markers in OWCM-155 shH19 vs. OWCM-155 shScr xenografts. Each bar represents pooled data from tumors from three different mice per group. G Organoid invasion assay. Transwell assay using FluoroBlock inserts with 50,000 cells/well of Trp53 flox/flox / Rb1 flox/flox organoids transduced with EV-GFP, Cre-GFP, and Cre-GFP + shH19 ( n = 3). Quantification by fluorescence intensity of the migrated cells on the bottom of the transwell, five days post-plating. EV-GFP was used as a control. H Growth response (left) of LNCaP cells with WT and P53/RB1 knockdown (shP53/RB1) with and without H19 knockdown (shH19 vs. Scr) treated with ENZA (5 µM) for 5 days. DMSO was used for the control treatment. WB (right) of these cells shows the RB and P53 knockout. I WB of LNCaP shP53/Rb1 cells with and without two different shH19 (shH19-C, shH19-D) treated with ENZA (2, 5 µM). DMSO was used as a control. J , Growth response of LNCaP cells overexpressing H19 vs. empty vector (EV) treated with ENZA (2, 5 µM) for 5 days. DMSO was a control treatment at 0 μM. K Western blot of LNCaP cells used in ( J ) treated with ENZA (2, 5 µM; 72 h). In H , I , and K , WB images were quantified using ImageJ (Methods) with Actin as a control. Data are mean ± SD ( D , G , H ), or mean ± SEM ( A , B , E , F , J ); n = 3 ( F – H , J ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test ( A , B , D – G , J ) or Tukey’s multiple comparisons test ( h ).

Article Snippet: For H19 knockdown in mouse organoids, plasmid GIPZ Mouse H19 shRNA purchased from Dharmacon (RMM4431), and for Cre recombinase expression, the plasmid FUGW-Cre (Addgene) was kindly provided by Dr. Owen Witte (UCLA, Los Angeles, CA). shOCT4 (LL-hOCT4i-1) (Addgene plasmid # 12198; http://n2t.net/addgene:12198 ; RRID:Addgene_12198) and shNANOG (LL–hNANOGi) were a gift of George Daley (Addgene plasmid # 12196; http://n2t.net/addgene:12196 ; RRID:Addgene_12196) . shSOX2 (pLKO.1 Sox2 HM) was a gift from Matthew Meyerson (Addgene plasmid # 26353; http://n2t.net/addgene:26353 ; RRID:Addgene_26353) . shP53 (pLKO-p53-shRNA-941) (Addgene plasmid # 25637; http://n2t.net/addgene:25637 ; RRID:Addgene_25637) and shRB1 (pLKO-RB1-shRNA19) (Addgene plasmid# 25640; http://n2t.net/addgene:25640 ; RRID:Addgene_25640) , were a gift from Todd Waldman.

Techniques: Knockdown, Fluorescence, Control, Injection, Expressing, Invasion Assay, Transwell Assay, Transduction, Knock-Out, Plasmid Preparation, Western Blot, Two Tailed Test

Journal: PLoS ONE

Article Title: Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

doi: 10.1371/journal.pone.0056912

Figure Lengend Snippet: (A) Serum from vector, mp53, and hp53 vaccinated mice showed different antibody responses to p53 protein on the Western blot. (B) ELISA demonstrates p53-specific Ab's in mice vaccinated with various DNA vaccines. The result shows the sequential dilution 1∶100, 1∶500, 1∶2500, and 1∶12500, detected by mean absorbance (OD450 nm) ± S.E. N = 3 per group (*, P<0.05).

Article Snippet: Human p53 DNA (pCMV-hp53) was purchased from Addgene (Cambridge, MA).

Techniques: Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Vaccines

Journal: PLoS ONE

Article Title: Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

doi: 10.1371/journal.pone.0056912

Figure Lengend Snippet: Splenocytes from vaccinated mice were cultured with p53 epitope 232 under IL-2 stimulation for one week (A) (B) Mice vaccinated with hp53 DNA vaccine generate 345±58.6 (mean ± S.E) IFN-γ/CD8+ cells per 30 0 ,000 splenocytes, comparing to mp53 vaccination group was 128±14.4. N = 3 for each group (*, P<0.05 **, P<0.01).

Article Snippet: Human p53 DNA (pCMV-hp53) was purchased from Addgene (Cambridge, MA).

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

doi: 10.1371/journal.pone.0056912

Figure Lengend Snippet: (A) Therapeutic schedule. (B) Mice (n = 5) were inoculated s.c. with 2 ×10 5 MC38 cells. Mice were immunized using either vector only, mp53, or hp53 DNA vaccine administered via injection with electroporation, starting on day 5 at 5 day intervals. Tumors were measured with digital calipers and tumor volumes were calculated. Data are expressed as volume ± S.E. N = 5 in each group (**, P<0.01).

Article Snippet: Human p53 DNA (pCMV-hp53) was purchased from Addgene (Cambridge, MA).

Techniques: Plasmid Preparation, Injection, Electroporation

Journal: PLoS ONE

Article Title: Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

doi: 10.1371/journal.pone.0056912

Figure Lengend Snippet: (A) Therapeutic schedule. (B) Mice (n = 5) were immunized by hp53 DNA vaccine administered via injection with electroporation and challenged with 2 ×10 5 MC38 cells to determine the effect of lymphocyte subsets on the potency of the hp53 DNA vaccine. CD4, CD8, and NK1.1 depletions were initiated one week before tumor challenge and lasted 30 days after tumor challenge, with treatments at one day intervals. (C) Tumor volume was measured weekly with digital calipers. Data are expressed as volume ± S.E. N = 5 in each group . N = 5 in each group (*, P<0.05 **, P<0.01).

Article Snippet: Human p53 DNA (pCMV-hp53) was purchased from Addgene (Cambridge, MA).

Techniques: Injection, Electroporation

Journal: PLoS ONE

Article Title: Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

doi: 10.1371/journal.pone.0056912

Figure Lengend Snippet: (A, B) C57/BL6 mice (5 per group) were vaccinated with DNA encoding mP53 or hP53. Splenocytes from vaccinated mice were isolated and stimulated in vitro with mP53-specifc CTL epitope (KYMCNSSCM) and IL-2. Splenocytes were then incubated with RMA-GFP-Luc or MC38-GFP-Luc cell line (5×10 4 cells/well) in different E/T ratios. The luciferase signal of live tumor cells was detected by IVIS imaging system. (C, D) Control group was set as 100% survival rate of tumor cells. Luciferase activity correlates with survival of tumor cells (*P<0.01).

Article Snippet: Human p53 DNA (pCMV-hp53) was purchased from Addgene (Cambridge, MA).

Techniques: Isolation, In Vitro, Incubation, Luciferase, Imaging, Control, Activity Assay

Journal: Stem Cells and Development

Article Title: P53 Promotes Retinoid Acid-induced Smooth Muscle Cell Differentiation by Targeting Myocardin

doi: 10.1089/scd.2017.0244

Figure Lengend Snippet: Elevated p53 expression following cell apoptosis is induced during mESC/SMC differentiation. (A) Outline of RA-induced SMC differentiation from mESCs in adherent monolayer culture. (B) Immunofluorescence staining on day 6 of RA-induced SMC differentiation. SM α-actin-positive staining indicated induced cells (upper left). Nuclei were counterstained with Hochest33258 (upper right) and a large number of apoptotic bodies were found mainly in SM α-actin-negative cells, which were magnified in the white rectangle area (lower right). Scale bar = 100 μm. (C) Electrophoretogram of DNA Ladder assay for mESCs treated by RA or DMSO for 3 days. (D) The protein levels of p53 during RA-induced SMC differentiation were detected by western blotting (left) and relative protein levels were further quantified and presented with bar plot (right, ***P < 0.05). β-tubulin was used as the loading control. (E) The relative expression levels of p53 and its targeted genes, p21, Mdm2, and Bax, were determined by qRT-PCR. mESC, mouse embryonic stem cell; RA, retinoid acid; SMC, smooth muscle cell. Color images available online at www.liebertpub.com/scd

Article Snippet: The pcDNA3 p53 WT (p53) and pcDNA3 p53 S15A (p53-Mut) plasmids were gifts from David Meek (Addgene plasmids #69003 and #69004) [ 20 ].

Techniques: Expressing, Immunofluorescence, Staining, DNA Laddering, Western Blot, Control, Quantitative RT-PCR

Journal: Stem Cells and Development

Article Title: P53 Promotes Retinoid Acid-induced Smooth Muscle Cell Differentiation by Targeting Myocardin

doi: 10.1089/scd.2017.0244

Figure Lengend Snippet: Suppression of p53 by shRNA attenuates SMC differentiation from ESCs. (A) Knocking down of p53 mRNA reduced its protein expression. (B) Representative immunofluorescence staining images from mESCCtrl and mESCsh-p53 differentiation into SMC by 10 μM RA treatment for 9 days. (C) qPCR and (D) western blotting assay demonstrated that downregulation of p53 attenuated SMC differentiation by decreasing the expression of SMC markers: Myocardin, SM α-actin, and SMMHC. β-tubulin was used as the loading control and the quantitative analysis of relative protein levels from western blotting was demonstrated with bar plot (the right panel of D, ***P < 0.05). (E) FACS assay revealed that the proportion of SM α-actin-positive cells was significantly reduced from 90.7% to 71.1% by shRNA-mediated p53 knockdown in RA-induced mESC/SMC differentiation (left), and the statistical result presented with bar plot (right, ***P < 0.05). FACS, fluorescence-activated cell sorting; SMMHC, smooth muscle myosin heavy chain. Color images available online at www.liebertpub.com/scd

Article Snippet: The pcDNA3 p53 WT (p53) and pcDNA3 p53 S15A (p53-Mut) plasmids were gifts from David Meek (Addgene plasmids #69003 and #69004) [ 20 ].

Techniques: shRNA, Expressing, Immunofluorescence, Staining, Western Blot, Control, Knockdown, Fluorescence, FACS

Journal: Stem Cells and Development

Article Title: P53 Promotes Retinoid Acid-induced Smooth Muscle Cell Differentiation by Targeting Myocardin

doi: 10.1089/scd.2017.0244

Figure Lengend Snippet: P53 activates Myocardin transcription by binding to the promoter directly. (A) Bioinformatic analysis revealed two predicted binding sites of p53 on Myocardin promoter. (B) ChIP-qPCR demonstrated that p53 binds to the Myocardin promoter at P1 during RA-induced ESC/SMC differentiation. (C) The effects of p53 and p53-Mut acting on Myocardin promoter were further measured by luciferase reporter assay. ***P < 0.05. (D) qRT-PCR revealed that the upregulation of Myocardin level was synchronized with the increasing of p53 expression. ***P < 0.05.

Article Snippet: The pcDNA3 p53 WT (p53) and pcDNA3 p53 S15A (p53-Mut) plasmids were gifts from David Meek (Addgene plasmids #69003 and #69004) [ 20 ].

Techniques: Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Quantitative RT-PCR, Expressing

Journal: Stem Cells and Development

Article Title: P53 Promotes Retinoid Acid-induced Smooth Muscle Cell Differentiation by Targeting Myocardin

doi: 10.1089/scd.2017.0244

Figure Lengend Snippet: Increased p53 expression promotes A404 differentiation into SMC. (A) After an 8-h doxorubicin treatment, the increased expression of p53 was measured by western blotting. The expressions of SMC markers in RA-induced A404/SMC differentiation were detected by (B) qPCR and (C) western blotting. The right panel (C) shows quantitative analysis of SMC-specific markers from western blotting. ***P < 0.05. (D) A schematic diagram exhibits RA-p53-Myocardin-mediated differentiation of mESCs into SMCs.

Article Snippet: The pcDNA3 p53 WT (p53) and pcDNA3 p53 S15A (p53-Mut) plasmids were gifts from David Meek (Addgene plasmids #69003 and #69004) [ 20 ].

Techniques: Expressing, Western Blot

Journal: Stem Cells and Development

Article Title: P53 Promotes Retinoid Acid-induced Smooth Muscle Cell Differentiation by Targeting Myocardin

doi: 10.1089/scd.2017.0244

Figure Lengend Snippet: SMC-selective p53 transgenic overexpression inhibits injury-induced neointimal formation in vivo. (A) A schematic diagram of the SMMHC-p53 transgenic expression cassette. The p53 cDNA was cloned into the expression vector, which carries SMMHC promoter and the BGH polyadenylate DNA fragments. (B) P53 was overexpressed in SMC tissues of transgenic mice. (C) Representative photomicrographs from elastica-van Gieson-stained sections. Images showed the neointimal development of mouse arteries with 14 days after wire-mediated injury in wild-type or SMMHC-p53 mice (left). Relative intima/medial ratios were calculated by measuring area of each vascular layer and lumen (n = 6, ***P < 0.05) (right). Color images available online at www.liebertpub.com/scd

Article Snippet: The pcDNA3 p53 WT (p53) and pcDNA3 p53 S15A (p53-Mut) plasmids were gifts from David Meek (Addgene plasmids #69003 and #69004) [ 20 ].

Techniques: Transgenic Assay, Over Expression, In Vivo, Expressing, Clone Assay, Plasmid Preparation, Staining