Journal: Clinical Epigenetics

Article Title: DNA hypermethylation of sirtuin 1 ( SIRT1 ) caused by betel quid chewing—a possible predictive biomarker for malignant transformation

doi: 10.1186/s13148-019-0806-y

Figure Lengend Snippet: Flow chart of cell culture. Human gingival epithelium progenitors (HGEPs), cells were treated with arecoline at a concentration of 50 μg/mL. The culture media was replaced every 3 days, alternating media with and without arecoline for 30 days. Untreated samples were used as controls. DDW, double-distilled water; ARE, arecoline

Article Snippet: Human gingival epithelial progenitors (HGEPs) and primary keratinocytes derived from healthy gingival epithelium were purchased from CELLnTEC Advanced Cell Systems (Basel, Switzerland) and cultured in CnT-Prime epithelial culture medium (CELLnTEC Advanced Cell Systems) at 37 °C in a humidified atmosphere of 95% air and 5% CO . HGEPs were spread onto 100 mm tissue culture plates at a density of 4.0 × 10 4 cells/mL.

Techniques: Cell Culture, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) and PH Domain and Leucine-rich Repeat Phosphatase Cross-talk (PHLPP) in Cancer Cells and in Transforming Growth Factor β-Activated Stem Cells *

doi: 10.1074/jbc.M113.537241

Figure Lengend Snippet: Epithelial-mesenchymal transition of prostate stem cells activates cross-talk between PHLPP and PTEN. A , WPE stem cells were treated with TGFβ-1 (4 ng/ml) for up to 8 days as indicated. Representative images by light microscopy (original magnification ×10) are shown. B , WPE cells were treated with TGFβ-1 for 24 h and cell proliferation was estimated by MTT assay. C , WPE cells were treated with TGFβ-1 for 8 days and lysates were analyzed for Slug, Snail, β-catenin, MMP9, and MMP2. Cdk2 was used as loading control. D , WPE cells were treated with TGFβ-1 for 8 days and the invasiveness was analyzed by invasion assay. Representative images are shown as well as a graph presenting data from three independent experiments. Results are presented as mean ± S.D. *, significantly different from controls ( p < 0.05). E and F, TGFβ-1-treated (4 ng/ml, for 8 days) WPE cells were transfected with PTEN, PHLPP1, or PHLPP2 and samples were analyzed for proteins indicated. Cdk2 was used as loading control. B , D , E, and F, results from three independent experiments are presented as mean ± S.D. *, significantly different from controls ( p < 0.05).

Article Snippet: The human prostate carcinoma cell lines DU145, 22RV1, LNCaP, PC3, prostate stem cell line WPE, immortalized prostate luminal epithelial (nontumorigenic) RWPE-1, and human breast adenocancinoma MCF7 cells were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Light Microscopy, MTT Assay, Control, Invasion Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) and PH Domain and Leucine-rich Repeat Phosphatase Cross-talk (PHLPP) in Cancer Cells and in Transforming Growth Factor β-Activated Stem Cells *

doi: 10.1074/jbc.M113.537241

Figure Lengend Snippet: Phosphatase cross-talk is dependent on P2X4 receptor. A , HEK293 cells stably overexpressing empty vector were transfected with PTEN, PHLPP2, or PHLPP1 for 40 h. Cell lysates were analyzed by Western blotting, employing antibodies for PHLPP1, PHLPP2, and PTEN. B , HEK293 cells expressing P2X7 or P2X4 were transfected with PTEN, PHLPP1, or PHLPP2 for 40 h. Cell lysates were analyzed for PHLPP1, PHLPP2, and PTEN. C, Western blot analysis of the levels of P2X7, P2X4, PTEN, PHLPP1, and PHLPP2 in HEK293 stably overexpressing P2X7 or P2X4 cells. D, PC3 cells were transfected with PTEN and siRNA P2X7 or siRNA P2X4 for 40 h. Cell lysates were analyzed for P2X4, P2X7, PHLPP2, and PTEN. E , the levels of PHLPP1, PHLPP2, P2X4, P2X7, and PTEN in RWPE-1, WPE, and TGFβ-1 (4 ng/ml for 8 days) treated WPE cells were analyzed. F , TGFβ-1-treated (4 ng/ml for 8 days) WPE cells were transfected with PTEN, PHLPP1, or PHLPP2 and siRNA control or siRNA P2X4 for 40 h. Cell lysates were analyzed for P2X4, PHLPP1, PHLPP2, and PTEN. A–F , Cdk2 was used as loading control. Data from three independent experiments are presented as mean ± S.D. *, significantly different from controls ( p < 0.05).

Article Snippet: The human prostate carcinoma cell lines DU145, 22RV1, LNCaP, PC3, prostate stem cell line WPE, immortalized prostate luminal epithelial (nontumorigenic) RWPE-1, and human breast adenocancinoma MCF7 cells were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Stable Transfection, Plasmid Preparation, Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) and PH Domain and Leucine-rich Repeat Phosphatase Cross-talk (PHLPP) in Cancer Cells and in Transforming Growth Factor β-Activated Stem Cells *

doi: 10.1074/jbc.M113.537241

Figure Lengend Snippet: P2X4 is essential for TGFβ-1-induced invasiveness and EMT. A and B , PC3 cells were treated with TGFβ-1 (0,1 ng/ml) and TNP-ATP (30 μ m ) for 48 h. A, invasiveness was analyzed and representative images (original magnification ×10) are shown as well as data from three independent experiments. Results are presented as mean ± S.D. *, significantly different from controls ( p < 0.05); #, significantly different from TGFβ-1-treated cells (*, p < 0.05). B , lysates were analyzed for MMP9. Cdk2 was used as loading control. C , PC3 cells were transfected for siRNA P2X4 for 40 h and thereafter treated with TGFβ-1 (0.1 ng/ml) for 48 h. The samples were analyzed for MMP9 and P2X4. Cdk2 was used as loading control. D , 22RV1 cells were transfected with PHLPP2 for 40 h. During transfection cells were treated with BAPTA-AM (2 μ m ). Data from three different experiments are presented. Results are presented as mean ± S.D. *, significantly different from empty vector-transfected cells (*, p < 0.05); #, significantly different from PHLPP2-transfected cells ( p < 0.05). E , WPE cells were treated with TGFβ-1 (4 ng/ml) and TNP-ATP (30 μ m ) for up to 8 days. Representative images by light microscopy (original magnification ×10) are shown. F , WPE cells were treated with TGFβ-1 (4 ng/ml) and TNP-ATP (30 μ m ) for 8 days as indicated. The lysates were analyzed for MMP9, MMP2, and β-catenin. Cdk2 was used as loading control. Data from three independent experiments are presented as mean ± S.D. *, significantly different from controls ( p < 0.05). G , WPE cells were treated with TGFβ-1 (4 ng/ml) and TNP-ATP (30 μ m ) for 8 days. Invasiveness was analyzed and representative images (original magnification ×10) are shown as well as data from three different experiments. Results are presented as mean ± S.D. *, significantly different from controls ( p < 0.05); #, significantly different from TGFβ-1-treated cells ( p < 0.05). H, levels of PTEN, PHLPP1, and PHLPP2 in liver preneoplastic tissue and control tissues from rats treated with diethyl nitrosamine (300 μmol/kg body weight) weekly for 11 weeks. Samples were analyzed for PTEN, PHLPP1, and PHLPP2. Cdk2 were used as a loading control. Densitometric analysis of data from three different rats. Results are presented as mean ± S.D. I , negative cross-talk between PTEN and PHLPP in cancer cells or activated stem cells. A scheme depicting factors implicated in the down-regulation of PHLPP in response to PTEN transfection and a similar scenario involving miR214 is envisioned for PTEN down-regulation in response to PHLPP transfection.

Article Snippet: The human prostate carcinoma cell lines DU145, 22RV1, LNCaP, PC3, prostate stem cell line WPE, immortalized prostate luminal epithelial (nontumorigenic) RWPE-1, and human breast adenocancinoma MCF7 cells were purchased from American Type Culture Collection (Manassas, VA).

Techniques: Control, Transfection, Plasmid Preparation, Light Microscopy

Journal: Biomaterials

Article Title: Development of switchable polymers to address the dilemma of stability and cargo release in polycationic nucleic acid carriers

doi: 10.1016/j.biomaterials.2017.02.036

Figure Lengend Snippet: (A) In vitro transfection of HeLa cells with different formulations. (B) GFP expression mediated by different polyplexes at N/P ratio of 5 analyzed by flow cytometry. (C). Representative images of GFP expression mediated by various polyplexes in HeLa cells. Scale bar: 50 μm. Data are shown as mean ± SD (n = 3; student's t-test, *p < 0.05, **p < 0.01).

Article Snippet: 2.8.1 Gene transfection in HeLa, Z310 cells and primary neural progenitor cells HeLa cells (ATCC CCL-2TM) were plated in a 24-well plate (20,000 cells/well) in minimum essential medium (MEM) with 10% fetal bovine serum (FBS) and antibiotics/antimyotics (AbAm) (100 IU of penicillin, 100 ug/mL of streptomycin, and 0.25 ug/mL of amphotericin B).

Techniques: In Vitro, Transfection, Expressing, Flow Cytometry

Journal: Biomaterials

Article Title: Development of switchable polymers to address the dilemma of stability and cargo release in polycationic nucleic acid carriers

doi: 10.1016/j.biomaterials.2017.02.036

Figure Lengend Snippet: (A). In vitro cellular uptake of YOYO-1 labeled DNA delivered by IP and SP. Data are shown as mean ± SD (n=3). (B). Confocal images of HeLa cells treated with polyplexes containing YOYO-1 labeled DNA for 4 h and stained with LysoTracker Red. DAPI (4′,6-diamidino-2-phenylindole, blue) was used to stain cell nuclei. Scale bar: 10 μm. (C). Colocalization ratio of YOYO-1-DNA with LysoTracker Red stained endosomes. Results were presented as the mean of 15 individual cells. Data are shown as mean ± SD (n=15; student's t test, *p<0.001). (D). Unpacking study of IP and SP polyplexes in different conditions. Data are shown as mean ± SD (n=3; student's t test, *p<0.001).

Article Snippet: 2.8.1 Gene transfection in HeLa, Z310 cells and primary neural progenitor cells HeLa cells (ATCC CCL-2TM) were plated in a 24-well plate (20,000 cells/well) in minimum essential medium (MEM) with 10% fetal bovine serum (FBS) and antibiotics/antimyotics (AbAm) (100 IU of penicillin, 100 ug/mL of streptomycin, and 0.25 ug/mL of amphotericin B).

Techniques: In Vitro, Labeling, Staining