Journal: Oncogenesis

Article Title: FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization

doi: 10.1038/s41389-018-0033-y

Figure Lengend Snippet: a COV434-Mock and COV434-FGF1 cells were treated or not with 25 µg/mL etoposide for 1, 2, 3 or 16 h. Total protein extracts were analyzed for p53 and Ser15-phosphorylated p53 by western blotting. b COV434 Mock and COV434-FGF1 cells were treated or not with etoposide for 1, 2, 3 or 16 h. Total proteins were analyzed for PUMA, Bax and p21 protein levels by western blotting. c Average apoptosis rates ± SEM for 2 experiments done in triplicate measured by flow cytometry of non-transfected COV434 (NT), Mock and FGF1 cells treated with etoposide for 16 h. Cells were pretreated or not for 90 min with the p53 transcriptional inhibitor pifithrin-alpha (PFT-α, 30 µM). d Western blot analysis of p21 and Bax protein levels in COV434 Mock and FGF1 cells pretreated with PFT-α (30 µM for 90 min), followed by an etoposide (25 µg/mL) treatment for 6 or 16 h. Two-tailed t -tests results are shown by * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001

Article Snippet: At 30% confluence, siRNA transfection was done using 80 pmol of human p21 siRNA, human p53 siRNA or siRNA-A control (Santa Cruz, Dallas, TX, USA) and 4 μl lipofectamine RNAimax (Invitrogen).

Techniques: Western Blot, Flow Cytometry, Transfection, Two Tailed Test

Journal: Oncogenesis

Article Title: FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization

doi: 10.1038/s41389-018-0033-y

Figure Lengend Snippet: a COV434 Mock and COV434 FGF1 cells were transfected or not with scramble (scr) or p21 siRNA. Upper panel: average apoptosis rates ± SEM for 2 experiments done in duplicate were measured by flow cytometry in cells treated with etoposide for 6 h, or not treated (Ctl). Lower panel: Western blot analysis for p21 protein levels in COV434 Mock and COV434 FGF1 cells transfected with scr siRNA or p21 siRNA. b Western blot analysis of total proteins for p21 and for procaspase-9, cleaved caspase-9, and cleaved caspase-3, and PARP levels. COV434 Mock and FGF1 cells transfected with scr or p21 siRNA were treated or not with etoposide for 16 h. One experiment representative of 3 independent experiments is shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Article Snippet: At 30% confluence, siRNA transfection was done using 80 pmol of human p21 siRNA, human p53 siRNA or siRNA-A control (Santa Cruz, Dallas, TX, USA) and 4 μl lipofectamine RNAimax (Invitrogen).

Techniques: Transfection, Flow Cytometry, Western Blot

Journal: Oncogenesis

Article Title: FGF1 induces resistance to chemotherapy in ovarian granulosa tumor cells through regulation of p53 mitochondrial localization

doi: 10.1038/s41389-018-0033-y

Figure Lengend Snippet: a COV434 cells were transfected or not (NT) with scramble (scr) or p53 siRNA. Upper panel: histogram represents average apoptosis rates ± SEM for 2 experiments done in duplicate measured by flow cytometry in cells treated with etoposide for 17 h, or not treated (Ctl). Lower panel: Western blot analysis for p53 protein levels in COV434 cells transfected with scr siRNA or p53 siRNA. b COV434 cells were transfected with an empty vector (Mock), a vector encoding p53 wild-type (p53 WT ) and a vector encoding p53 fused to the mitochondrial transmembrane domain of BCL-X L (p53 CTB ). Cells were treated with of G418 for one week and then stained with ethidium bromide and visualized using Chemidoc (Biorad)

Article Snippet: At 30% confluence, siRNA transfection was done using 80 pmol of human p21 siRNA, human p53 siRNA or siRNA-A control (Santa Cruz, Dallas, TX, USA) and 4 μl lipofectamine RNAimax (Invitrogen).

Techniques: Transfection, Flow Cytometry, Western Blot, Plasmid Preparation, Staining

Journal: International Journal of Biological Sciences

Article Title: BET inhibition induces synthetic lethality in PTEN deficient colorectal cancers via dual action on p21 CIP1/WAF1

doi: 10.7150/ijbs.91867

Figure Lengend Snippet: BETi reduced p21 phosphorylation at Thr145 and promoted p21 nucleus importing by repressing AKT in PTEN-deficient cells. A, B Western blot illustrating AKT, p-AKT (ser473), p21, p-p21(thr145) levels in HCT116 ( A ) and DLD1 ( B ) PTEN isogenic cell pairs via treatment with OTX-015 for 24 h. C Immunofluorescence analysis of p21 localization inHCT116 PTEN isogenic cells treated with or without BETi. D Endogenous p21 in HCT116 PTEN-deficient cells were repressed by siRNA and the cells were transfected with Thr145 mutant p21 (p21-T145D) and wild type p21 (p21-WT) respectively. The transfected cells were treated with OTX-015 or DMSO for 24 h. The percentages of cell confluence were shown. E Cell images represented the cell confluence taken by Incucyte Zoom.

Article Snippet: pCDH-puro-MYC plasmid was a gift from Jialiang Wang (Addgene plasmid #46970) , and Flag p21 WT and T145D plasmids were gifts from Mien-Chie Hung (Addgene, plasmid # 16240 and 16242) .

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence, Transfection, Mutagenesis

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High p21 expression correlates with poor survival in breast cancer patients . A-C , The relationship of higher p21 expression and breast cancer outcome was assessed by Kaplan-Meier survival analysis. The survival rates were analyzed using three different and separate splits of the patients, based on p21 expression levels (A, median; B, upper quartile; C, lower quartile). For each split, the patients were then divided into high and low groups with respect to p21 expression. Overall survival (left panels) and distant metastasis-free survival (right panels) correlate with p21 gene expression. Number of breast cancer patients at risk with higher expression (red) and lower expression (black) of p21 at the indicated time points.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Gene Expression

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Silencing p21 prevents breast tumor local invasion in vivo and cancer cell migration and invasion . A , Total lysates from parental and shRNA p21 SCP2 cells were analyzed by immunoblotting for the protein levels of p21 and β-tubulin. B , Parental and shRNA p21 SCP2 cells were injected into the mammary glands of four- to six-week-old female Balb/c nude mice. The size of mammary tumor was measured from two sets of mice (eight per group; error bars indicate SEM). C , Representative photographs show hematoxylin and eosin staining of the mammary gland (tumor and fat pad) of 12- to 15-month-old mice. D and E , Transwell cell migration (left panel) and GFR-Matrigel invasion assay (right panel) of parental and shRNA p21 SCP2 cells were performed. Graphs show total migrated and invaded cell number counted by Image J (error bars indicate SEM; n = 3 independent experiments). Student's unpaired t -test was used to compare parental vs. shRNA p21 SCP2 groups. Differences were considered significant at * P < 0.05.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: In Vivo, Migration, shRNA, Western Blot, Injection, Staining, Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: TGFβ induces p21 expression in migratory and invasive human breast cancer cells . A , Real-time PCR was performed to measure the mRNA level of p21 gene (error bars indicate SD; n = 3 independent experiments) for the indicated cell lines. B , Cells were treated with or without 5 ng/ml TGFβ for the indicated times. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. C , Total cell lysates were analyzed for c-myc, p15 and β-tubulin protein levels by Western blotting. D , SCP25 cells were pretreated with 10 µM TGFβ type I receptor (TβRI) inhibitor (SB431542) or vehicle (DMSO) for 30 minutes and then stimulated with TGFβ. Total cell lysates were analyzed for p21 and β-tubulin protein levels by Western blotting. E , MDA cells were transfected with 40 nM Scrambled (Scr), Smad2 or Smad3 siRNAs in response to TGFβ. Total cell lysates were analyzed for Smad2/3, p21 and β-tubulin protein levels by Western blotting.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell migration . A , Representative images of phase contrast and wound mask of indicated cell lines stimulated with TGFβ in scratch/wound healing assay. The initial wound mask (black) and wound closure (grey) were measured using the Essen Instruments Scratch Wound Module. B , The time course of cell migration for the indicated cell lines was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with Scr or p21 siRNAs and then stimulated with or without TGFβ for 24 hrs. Total cell lysates were analyzed for p21 and β-tubulin by Western blotting. D , Representative images of phase contrast (top panels) and wound mask (bottom panels) of transfected SCP2 cells with the indicated siRNAs in scratch/wound healing assay. E , The time course of transfected SCP2 cell migration was quantified using the relative wound density metrics at two-hour time intervals (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Migration, Wound Healing Assay, Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 expression is required for TGFβ-mediated cell invasion . A , SCP2 cells were transfected with the indicated siRNAs and cultured in the presence or absence of TGFβ. GFR-Matrigel coated Transwell invasion assay was performed and images of the invading cells were photographed. B , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments). C , SCP2 cells were transfected with a Scr or p21 siRNA as well as a flag-tagged p21 cDNA in the presence or the absence of TGFβ. p21 protein levels were then analyzed by Western blotting. D , Cell invasion was assessed using the Transwell Invasion assay. The number of invaded cells was counted by Image J (error bars indicate SD; n = 3 independent experiments). E , Cell invasion of transfected SUM159 was assessed using the Transwell Invasion assay. F , Total cell number was counted by Image J and fold induction was quantified (error bars indicate SD; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Cell Culture, Transwell Invasion Assay, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 interacts with Smad3 and modulates TGFβ-induced transcriptional activity . A , HEK293 and SCP2 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21. Transfected cells were stimulated TGFβ for 8 hrs. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and p21 antibodies. B , Transfected MDA cells were immunoblotting by phospho-Smad3 (p-Smad3), Smad2/3 and β-tubulin antibodies in response to TGFβ for 30 minutes. C , SCP2 cells were co-transfected with either mock, Scr siRNA, p21 siRNA or flag-tagged p21 construct (p21 cDNA) and SBE promoter construct (CAGA12-luc). Transfected cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicate SEM; n = 3 independent experiments). D and E , SUM159 and SCP2 cells were treated with or without TGFβ for the indicated times. The mRNA levels of indicated genes were then analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Western Blot, Construct, Luciferase, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21 regulates TGFβ-induced downstream genes involved in cell invasion . A , SUM159 cells were transfected with Scr or p21 siRNA as well as p21 cDNA. Cells were then treated with or without TGFβ and the mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). B , SUM159 cells transfected with Scr siRNA and p21 siRNA were treated with TGFβ for the indicated times. The mRNA level of TGFBI gene was measured by real-time PCR (error bars indicate SEM; n = 3 independent experiments). C , SCP2 cells were transfected with the indicated siRNAs. Cell invasion was assessed using the Transwell Invasion assay. D , Total cell number was counted by Image J and number of invaded cells was quantified (error bars indicate SEM; n = 3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Transwell Invasion Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: p21/p/CAF regulates TGFβ transcriptional activity and Smad3 occupancy on SBE . A , SCP2 and SUM159 cells were treated with TGFβ. Cell lysates were analyzed by co-immunoprecipitation using specific antibodies, as indicated. B , HEK293 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21 with or without p/CAF siRNA. Transfected cells were stimulated with TGFβ for eight hours. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and flag antibodies. C and D , SUM159 cells were transfected with Scr or p/CAF siRNAs as well as a flag-tagged p21 cDNA, treated with or without TGFβ. The mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). E , SCP2 cells transfected with Scr and p/CAF siRNA were stimulated with or without TGFβ. Cell invasion was quantified by relative TGFβ fold induction (error bars indicate SEM; n = 3 independent experiments). F , Transfected SCP2 cells were subjected to immunoblotting p/CAF and β-tubulin. G , HEK293 cells were co-transfected with myc-Smad3, myc-Smad2 and p/CAF. Immunoprecipitated Smad2/3 using an anti-myc antibody was subjected to Western blotting. H , DNA precipitation (DNA IP) was performed using biotinylated control and 4× CAGA SBE oligonucleotides, following by streptavidin precipitation. Western blotting of Smad3 and p/CAF is shown. I , SCP2 cells were transfected with p21 or p/CAF siRNAs. Samples were subjected to DNA IP and immunoblotting of Smad3. J , Transfected SCP2 cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicated SEM; n =3 independent experiments).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Activity Assay, Immunoprecipitation, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Control, Luciferase

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: High expression of p/CAF/p21/pSmad3 is associated with lymph node positivity . A , Representative immunohistochemistry images of pSmad3, p21 and p/CAF in breast cancer tissue microarray samples. B , Overall pSmad3, p21 and p/CAF immunohistochemistry staining intensity between lymph node negative (LN-) and positive (LN+) tissues. C , Percentage of case distribution according to immunoreactivity of pSmad3, p21 and p/CAF in tumor cells, the score for negative (0), low (1 to 2) and high (3 to 4).

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining

Journal: Breast Cancer Research : BCR

Article Title: A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

doi: 10.1186/bcr3322

Figure Lengend Snippet: Model of pro-invasive function for the cell cycle regulator p21 in human breast cancer . The role of p21 as both a direct transcriptional target of TGFβ and a co-stimulatory factor of p/CAF/Smad3 in regulation of pro-invasive genes in triple negative breast cancer cells.

Article Snippet: Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941) [ ] and Flag-tagged human p21 cDNAs (Addgene plasmid 16240) [ ] using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol.

Techniques: