Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: PRC2 protein EED and EZH2 interact with AR. (a) Immunoprecipitation of VCaP cell lysates with the indicated mouse monoclonal anti-EED antibody (05–1,320, Millipore), rabbit polyclonal anti-EED antibody (09–774, Millipore), control IgG and anti-AR antibody was followed by immunoblot analysis. Representative graph from at least three independent experiments is shown. (b) Immunoprecipitation of 22Rv1,C4–2, LNCaP and VCaP cell lysates with anti-EZH2, anti-AR antibody and control IgG was followed by immunoblot analysis. (c) HEK293T cells transfected with Halo-AR (full length), Halo-DBD, Halo-LBD, Halo-NTD plasmids and empty vector were lysed and subjected to pull-down assay using HaloLink resin (Promega), followed by immunoblot analysis. (d) Purified EZH2 and EED were respectively mixed with AR (full length) and pulled down with anti-AR antibody and protein A beads. RING1B served as a negative control. (e) Purified EED was mixed with AR N-terminal fragment and pulled down with anti-AR antibody and protein A beads. RING1B served as a negative control.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Pull Down Assay, Purification, Negative Control

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: EZH2 and EED knockdown decreases AR and downstream targets. (a) EZH2 was depleted by shRNA in C4–2 cells. After 48 hr, cells were lysed and blotted by EZH2, EED (rabbit polyclonal anti-EED antibody, 09–774, Millipore), AR, PSA and GAPDH. (b) EED was depleted by shRNA in C4–2 cells. After 48 hr, cells were lysed and blotted by EZH2, EED (rabbit polyclonal anti-EED antibody, 09–774, Millipore), AR, PSA and GAPDH. (c) LNCaP and VCaP cells were subjected to cotransfection of PSA or TMPRSS2 firefly luciferase reporter constructs and pRL-TK (Renilla luciferase). Lentivirus packaged with two distinct shRNAs of EZH2 or EED were added 24 hr after the cotransfection to knockdown EZH2 or EED. The luciferase activity was normalized using Renilla bioluminescence.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: shRNA, Cotransfection, Luciferase, Construct, Activity Assay

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: EZH2 knockdown and astemizole treatment demonstrate similar inhibition patterns of AR signaling blockage.(a) EZH2 knockdown and astemizole-treated samples cluster together based on log expression of 1,571 (top 10%) high variation genes. (b) Heat maps for the expression level of genes down- or up-regulated by EZH2 knockdown, GSK126 and astemizole treatment. (c) The number of overlapped differential genes in each paired group is significantly larger than the number of genes overlapped by chance. (d) 426 AR-induced genes were compared and the expression is similar between EZH2 knockdown and astemizole-treated samples. (e) Comparison of PSA gene track between groups. (f) Comparison of PSA gene track between groups. (g) GSEA shows that AR target genes are significantly enriched (Q value = 0.0429) in downregulated genes due to EZH2 knockdown. (h) GSEA shows that AR target genes are significantly enriched (Q value = 0.0413) in downregulated genes due to astemizole treatment.

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Inhibition, Expressing

Journal: International journal of cancer

Article Title: Polycomb group proteins EZH2 and EED directly regulate androgen receptor in advanced prostate cancer

doi: 10.1002/ijc.32118

Figure Lengend Snippet: Astemizole has potent therapeutic effects on prostate cancer. (a) Astemizole critically thwarts cell proliferation in C4–2 and other AR-positive prostate cancer cell lines. (b) The wound healing assay indicates that astemizole compromises the migration of C4–2 cells. (c) Astemizole decreases the invasive abilities of C4–2 cells compared to vehicle treatment. Cell count was analyzed and the difference was statistically significant. (d)C4–2 cells were treated with 2.5, 5 and 7.5 μM of astemizole. Cells were lysed 48 hr after treatment and blotted with anti-LC3-A/B antibody. The ratio of LC3-A/B-II/I to GAPDH was elevated as dose increased, which indicates that astemizole induces autophagy in prostate cancer cells. (e) Castration-resistant VCaP xenograft mouse models were generated. Castrated mice bearing CPRC xenografts received vehicle or astemizole treatment (50 mg kg−1) daily (5 days per week). Caliper measurements were taken every 4 days to determine tumor volume. Mean tumor volume SEM, *p < 0.05, **p < 0.01 vs. vehicle was marked. (f) Kaplan–Meier survival plot compares progression-free survival. (g) Upper panel: Proteins were blotted and quantitated to compare the protein levels of EZH2 and AR in astemizole-treated group (n = 8) compared to vehicle-treated group (n = 12). Lower panel: The expression of EZH2 and AR was decreased in response to astemizole treatment. (h) The proportion of the cells stained with EZH2/AR/PSA in astemizole-treated group (n = 6) were significantly lower than that in vehicle-treated group (n = 6).

Article Snippet: For in vitro immunoprecipitation, AR-FL (346101–5,000 U, EMD Millipore), EZH2 (50,279, BPS Bioscience), EED (50,280, BPS Bioscience) and AR-NTD (ab82124, Abcam) were purchased from the vendor listed.

Techniques: Wound Healing Assay, Migration, Cell Counting, Generated, Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Betula alba Bark Extract and Empetrum nigrum Fruit Juice, a Natural Alternative to Niacinamide for Skin Barrier Benefits

doi: 10.3390/ijms232012507

Figure Lengend Snippet: Summary of IC50 of the two extracts BA (circle) and EN (triangle) in ( a ) KLK5 ( n = 3), ( b ) hyaluronidase ( n = 3), ( c ) DPPH ( n = 3) and ( d ) COX2 ( n = 4) inhibition assays. Plotted values are displayed as mean (±SD).

Article Snippet: Thirty microliter of Tris-HCl buffer (0.1 M, pH 8), 10 µL hematin (Sigma Aldrich, H3281), 10 µL of extract (1% final concentration of raw material) and 10 µL enzyme COX2 (BPS Bioscience, San Diego, CA, USA, 71111) 20 ng/µL were introduced into a 96-well plate and incubated 15 min at room temperature.

Techniques: Inhibition