Figure 4. PC3-DR cells acquire glutamine addiction.

A. Immunoblot analysis of c-Myc in PC3 and PC3-DR cells treated with or without 10 nM docetaxel for 48 h in serum-free medium. Actin immunoblot was used for normalization. B. Evaluation of PC3 and PC3-DR cells growth in medium containing either glucose (25 mM), pyruvate (1 mM) or glutamine (2 mM) was analyzed by crystal violet assay for 48 h. Results are representative of three experiments. *p<0.005 PC3-DR vs PC3; ≠p<0.05 PC3-DR vs PC3. C. Boyden invasion assay of PC3 and PC3-DR cells treated or not with 10 μM glutaminase inhibitor (compound 968). Bar graph represents the mean of invaded cells in six field for sample. Number of invaded cells was expressed as fold change respect to PC3 cells without metformin. Results are representative of three independent experiments. *p<0.005 treated vs untreated. D. Evaluation of [¹⁴C]-glutamine uptake of PC3 and PC3-DR cells treated or not with 10 nM docetaxel for 48 h and normalized on protein content. Results are representative of three experiments. *p<0.005 vs PC3. E. Respiration of [¹⁴C]-glutamine in PC3 and PC3-DR cells treated as in D) was evaluated as [¹⁴C]-CO₂ release and normalized on total protein content. Results are shown as fold change relative to PC3 cells. Results are representative of four experiments. *p<0.005 vs PC3. F. Immunoblot analysis of c-Myc in PC3-DR cells silenced with control siRNA or c-Myc siRNA after 48 h from transfection. Actin immunoblot was used for normalization. G. PC3-DR cells were transfected with control siRNA or c-Myc siRNA. After 24 h from transfection cells were treated or not with 20 nM docetaxel for 48 h. Cells viability was then quantified by crystal violet assay. Bar graph represents the percentage of dead cells following docetaxel treatment with respect to untreated cells. Results are representative of three experiments. *p<0.005 c-myc sil PC3-DR vs control PC3-DR.