Fig 1: Expression of protein for CTL1 and CTL2 in KYSE-180 cells. (A) Detection of CTL1 and CTL2 proteins by western blot analysis. MM, pre-stained protein molecular weight marker. (B) Subcellular distribution of CTL1 and CTL2 proteins by immunocytochemistry. (C) Fluorescence from antibodies directed to CTL1 (green), CTL2 (green), Na+/K+-ATPase (red) and COX IV (red) was imaged with a confocal microscope. Nuclei are stained with DAPI (blue). Merged images are shown in Merge, and yellow represents co-localization.
Fig 2: CTL1 and CTL2 mRNA and protein expression in Fa2N-4 cells. (A) Relative mRNA expression is presented as a ratio of target mRNA to GAPDH mRNA (n=3). Each column represents mean ± standard deviation. BLQ indicates values below the limit of quantification. (B) Fa2N-4 whole cell and mitochondrial lysates were used. With a whole cell protein lysate, choline transporter-like protein 1 (CTL1), CTL2 and ß-actin proteins were detected, whereas CTL1, CTL2, and COX–IV proteins were detected within mitochondrial lysate. (C) Localization of CTL1 and CTL2 proteins in Fa2N-4 cells were analyzed using the cell membrane and mitochondrial markers, NaK-ATPase and COX IV, respectively. The presence of CTL1 is indicated by a green signal (a), and a merged image of CTL1 and NaK-ATPase (b) is presented in panel (c). The presence of CTL2 is represented a green signal (d), and merged images of CTL2 and COX IV (e) are shown in panel (f). 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei are shown as blue signals in each panel. Images in (a-c) have a magnification, ×120 whereas (d and e) have a magnification, ×60. CTL, choline transporter-like protein; COX–IV, cytochrome c oxidase; BLQ, below the limit of quantification.
Fig 3: Effect of flutamide and bicalutamide on CTL1 protein levels in LNCaP cells. Cells were incubated with 100 µM flutamide (F) and bicalutamide (B) for 24 h. Western blot analysis (A) and densitometry (B) show CTL1 levels in total cell lysates from drug-treated LNCaP cells. The bar graph represents the densitometric measurement of CTL1 protein normalized to GAPDH. The experiment was performed in triplicate. The densitometric data are presented as the means ± SD. **p<0.01 compared with control (C).
Fig 4: PKC stimulation of choline uptake and CTL1 protein in Fa2N-4 cells. (A) Fa2N-4 cells were pre-incubated with various concentrations of PMA or 4a-PMA (negative control) for 10 min and the uptake of 10 µM [methyl-3H]choline measured for 20 min. Compared with the negative control, PMA treatment at increasing concentrations (1–200 nM) increased [methyl-3H]choline uptake. A PMA concentration of 100 nM was optimal and led to the highest [methyl-3H]choline uptake. Each point represents mean ± SD (n=3). **P<0.01 compared with 4a-PMA. (B) Fa2N-4 cells were pre-incubated with 100 nM PMA or 4a-PMA for various times, and the uptake of 10 µM [methyl-3H] choline was measured for 20 min for the acute phase, and 24 h for the chronic phase. [Methyl-3H]choline uptake was highest at 5 min after PMA treatment. Each point represents mean ± SD (n=3). **P<0.01 compared with the 4a-PMA control. (C) Western blot analysis was performed following stimulation of Fa2N-4 cells with 4a-PMA and PMA for 25 min. The protein components of cell membranes were extracted by a Trident Membrane Protein Extraction Kit and then used to detect CTL1 expression levels by western blot analysis. (D) Results were standardized using NaK-ATPase protein band levels. PMA stimulation resulted in a higher CTL1/NaK-ATPase protein ratio compared with the 4a-PMA stimulation group. Each point represents mean ± SD (n=3). *P<0.05 compared with the 4a-PMA stimulation group. PKC, protein kinase C; CTL, choline transporter-like protein; PMA, phorbol 12-myristate 13-acetate; SD, standard deviation.
Fig 5: Expression of mRNA for choline transporters in HEEpiC and KYSE cells. Real-time PCR analysis of the expression of mRNAs for CHT1, CTL1–5, and OCT1–3. Relative mRNA expression is expressed as a ratio of the target mRNA to GAPDH mRNA. Experiments were performed in triplicate. Each column represents the mean ± SD (n=3). BLQ means below the limit of quantification.
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