Figure 3. The mechanism of action of emetine on CAF.

A. Fluorescent microscopic analysis of lymphoma cells and CAF treated with emetine. Lymphoma cells (#2) co-cultured with CAF were assessed after treatment with 2 μM emetine for 48 h. Live and dead cells stained with Calcein-AM (green) and PI (red), respectively, are shown. B. ATP production in lymphoma cells co-cultured with emetine-pre-treated CAF. The scheme of the experiment is shown at left.ATP levels were assessed in lysates from lymphoma cells (#2) (3 × 10⁴ cells per well) co-cultured with non-treated (Control) CAF or with CAF that were treated with emetine (3 × 10³ cells per well, 0.5 μM, for 48 h) prior to initiation of the co-culture. Each point represents the mean value taken from three independent experiments. Error bars indicate SEM. C. Assessment of the death of lymphoma cells co-cultured with non-treated or emetine-pre-treated CAF. The scheme of the experiment is shown at left in (B). The death of lymphoma cells (#1 and #2) was assessed 48 h after co-culture with non-treated or emetine-pre-treated CAF. Each point represents the mean value taken from two independent experiments. Error bars indicate SEM. Asterisks indicate the P value as follows; * P < 0.05; ** P < 0.01. D. Relative gene expression of the glucose transporter GLUT1. RNA was extracted 48 h after co-culture of lymphoma cells (#2) with non-treated or emetine-pre-treated CAF, and relative mRNA expression of GLUT1 was then assessed using quantitative RT-PCR. Each point represents the mean value taken from two independent experiments. Error bars indicate SEM. Asterisks indicate the P value as follows; ** P < 0.01. E. Intracellular GSH concentration of lymphoma cells (#2) co-cultured with non-treated (control) or emetine-pre-treated CAF, or cultured as a monoculture (No CAF). Each point represents the mean value taken from three independent experiments. Error bars indicate SEM. Asterisks indicate the P value as follows; * P < 0.05. F. Escape from the anti-tumor effect of emetine by the addition of GSH into the culture medium. Cell death of lymphoma cells (#1, left and #2, right) co-cultured with CAF in the presence of various concentrations of emetine supplemented with (squares, dotted-line) or without (circles, solid line) 2 mM GSH for 48 h is shown. Each point represents the mean value taken from three independent experiments. Error bars indicate SEM.