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Fig. 3 | Molecular Medicine

Fig. 3

From: COX-2 Specific Inhibitor, NS-398, Increases Macrophage Migration Inhibitory Factor Expression and Induces Neuroendocrine Differentiation in C4-2b Prostate Cancer Cells

Fig. 3

NS-398 effects on MIF and cox-2 expression in LNCaP parental and C4-2b cells. Cells were cultured as in Fig. 1. At 24 h intervals culture medium was collected and cells harvested by gentle scraping. Half of the collected cells were used to isolate total protein, the remaining cells were used to isolate total RNA. (A) Secreted MIF levels: ELISA determined MIF in culture medium. Open, LNCaP control; hatched left, LNCaP NS-398; hatched right, C4-2b control; cross-hatched, C4-2b NS-398. Data are means of three separated experiments performed in triplicate. Error bars = SE of means. (B) MIF mRNA concentrations: MIF mRNA was determined by relative Q-RT-PCR. MIF specific primers and 18S rRNA primers: competimers (3:7) were simultaneously added to PCR reaction tubes and used to co-amplify MIF and 18S rRNA. PCR products were separated by agarose gel electrophoresis and areas as well as relative intensity were determined using AlphaImager. The area intensity of the MIF PCR product band (254 bp) was divided by area intensity of the 18S rRNA PCR product band (488 bp). Gel is representative of three independent experiments performed in triplicate. Lanes 1–2, C4-2b; lanes 3–4, LNCaP; lanes 1–3, DMSO controls; lanes 2–4, NS-398 treatment (10 µM). Gel is representative of two independent experiments performed in triplicate. (C) Intracellular COX-2 levels: ELISA determined cell lysate COX-2. Closed circle, LNCaP control; open circle, LNCaP NS-398; closed triangle, C4-2b control; open triangle, C4-2b NS-398. Data are means of three separate experiments performed in triplicate. Error bars = SE of means. (D) COX-2 mRNA levels: COX-2 mRNA was determined by relative Q-RT-PCR. COX-2 specific primers and 18S rRNA primers:competimers (3:7) were simultaneously added to PCR reaction tubes and used to co-amplify COX-2 and 18S rRNA. PCR products were separated by agarose gel electrophoresis and areas and relative intensity determined using AlphaImager. The area intensity of the COX-2 PCR product band (696 bp) was divided by area intensity of the 18S rRNA PCR product band (488 bp). Lanes 1–2, LNCaP; lanes 3–4, C4-2b; lanes 1–3, DMSO controls; lanes 2–4, NS-398 treatment (10 µM). Gel is representative of three independent experiments performed in triplicate.

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