Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: TREK2 channel expression in human bladder cancer cells. (A) Messenger RNA (mRNA) of ion channels related to the TREK1 (Accession No; AF129399) and TREK2 (Accession No; AF279890) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) analysis. TREK1 (355base pair [bp]) and TREK2 (291 bp) were detected. (B) Immunoblot showed presence of TREK2 channel protein in the human bladder cancer 253J cell line. TREK2 transfected CHO cells were used as a control. (C) Representative confocal microscopic analysis of TREK2 in bladder cancer cell line 253J. Cells were stained with an anti-TREK2 antibody and F-actin. Scale bar, 20 µm.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Staining

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: The physiologic properties of TREK2 at a single channel level in bladder cancer 253J cells. (A) TREK2 in CHO cells transfected with DNA encoding TREK2 and GFP and in 253J cells was measured in the excised inside-out patch configuration at the holding potential values shown on the left. The current trace was obtained in symmetrical 150 mM KCl solutions. The letters "c" and "o" represent the "closed" and "open" states of the channels, respectively. (B) The I-V relationships showed inward rectification, and each point is the mean of 4 experiments with standard error (S.E) represented by the error bars. (C~E) Current tracing showed arachidonic acid, intracellular pH, and mechanosensitivity of the native TREK2-like channel in 253J cells at -60 mV, +40 mV, and -40 mV. Negative pressure (-10 mmHg or -20 mmHg) was applied through the pipette. The panel below each of the figures shows the single channel trace on an expanded time scale.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Transfection, Transferring

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: The effect of TREK2 knockdown on the growth of 253J bladder cancer cells. (A and B) TREK2 mRNA and protein levels in 253J cell were determined after knockdown of TREK2 by siRNA through qRT-PCR and Western blot. Expression of TREK2 mRNA after transfection of TREK2 siRNA or negative control siRNA was normalized. TREK2 protein was examined by Western blot analysis after transfection of the negative control or TREK2 siRNA in 253J cells. GAPDH was used as a control. (C) The membrane potential was measured at current clamp (I=0) in a whole cell patch configuration. The 253J cells were treated with FITC-labeled negative control siRNA and FITC-labeled TREK2 siRNA for 72 hours. The data were represented as the mean±S.E. (t-test, p value <0.05). (D) The antiproliferative effect of TREK2 knockdown by siRNA in 253J cells. Cells were treated for three days with TREK2 siRNA or (-)control siRNA in 2% serum culture media. After treatment, proliferation was measured by XTT assay. Error bars represent the mean±S.E for 38 separate experiments. Asterisks indicate values that are different from the respective control (t-test, p<0.05). (E) Effect of TREK2 siRNA on 253J cell growth. Cells were captured 48 hours after transfection with TREK2 siRNA using a Nikon microscope at 10×10 magnification. Scale bar, 100 µm.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Negative Control, Labeling, XTT Assay, Microscopy

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

doi: 10.4196/kjpp.2013.17.6.511

Figure Lengend Snippet: Cell cycle arrest at G0/G1 after TREK2 siRNA transfection in bladder cancer 253J cells. (A) The bar graph shows that TREK2 siRNA treated cells resulted in an increased percentage of cells in G0/G1 and a decreased percentage of cells in the S phase of the cell cycle (t-test p<0.05) (B) Representative Western blot showing changes in the levels of associated proteins in cell cycle arrest of human bladder cancer 253J cells. Knockdown of TREK2 decreased the expression of cyclin D1, cyclin D3, cdk2, cdk4, and cdk6 and increased protein levels of p21 and p53.

Article Snippet: For the staining, the cells were incubated with rabbit polyclonal anti-hKCNK10 (TREK2) (concentration of 1:200, Alomone Labs, Jerusalem, Israel) for overnight at 4℃, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (concentration of 1:200, Santa Cruz Biotechnology, Santa Cruz, USA) for 1 h. To visualize F-actin, cells were stained with Alexa Fluor 594 conjugated phalloidin (concentration of 1:100, Life Technologies, Grand lsland, USA) for 30 min at 25℃.

Techniques: Transfection, Western Blot, Expressing