Fig 2: Effect of ME3 silencing on lactate production: (A) qPCR determination of ME3 mRNA level after 48 h of ME3 silencing (siME3) compared to control (NC1). (B) Representative western blot of ME3 after 48 h of ME3 silencing in PC3 cells (right panel); densitometric analysis of ME3, GAPDH was used as a loading control. Uncropped Western Blots of Figure 7B are available in Figure S14. (C) Lactate concentration analysis by colorimetric assay in PC3 cells after ME3 silencing collected at 48 h after transfection. Results are plotted as fold change vs. mean NC1. Individual values (n = 3) with mean ± SEM are shown. Non-parametric paired two-tailed Student’s t-test determined statistical significance. * p = 0.05 MALAT1gapmer vs. LacZgapmer.

Fig 3: Validation of array data in PCa cells by qRT-PCR. (A) Differential expression of genes as emerging from transcriptomic analysis before/after MALAT1 depletion in C27IM, DU145, and PC3 cells. Transcript levels of ME3, PDK3, PDK1, and PCho-related genes (choline kinase A (CHKA), Phospholipase C Gamma 1 (PLGC1), sphingomyelin phosphodiesterase 1 (SMPD1), and sphingomyelin synthase 1 (SGMS1)) are expressed as fold change vs. LacZgapmer (mean ± SEM are showed). (B,C) mRNA expression level of those genes emerging as differentially modulated were validated in different PCa cells (PC3, DU145, C27IM, and LNCaP) transfected with specific gapmer for MALAT1 or LacZ. Results are plotted as fold induction vs. LacZgapmer (placed to 1 and depicted as a black line). Individual values (n = 3–8) with mean ± SEM are showed. Non-parametric paired two-tailed Student’s t-test determined statistical significance. * p = 0.05 MALAT1gapmer vs. LacZgapmer. * p < 0.05 vs. gapmerLacZ.

Fig 4: Validation of PCa cells array data set. (A–C) Representative western blot and densitometry analysis for specific metabolic enzymes assessed in 3 different PCa cells (PC3, DU145, and C27IM) not transfected or transfected with specific gapmers for MALAT1 or LacZ: ME3 ((A), black arrow indicates specific signal), PDK3 (B), ?-PDHa1, and total PDHa1 (C). ß-actin or fibrillarin were used as a loading control. ME3 and PDK3 protein levels were normalized versus ß-actin and fibrillarin, respectively; ?-PDHa1 versus total PDHa1. Results are plotted as fold change vs. LacZgapmer taken as reference value 1 (straight black line). Individual values (n = 4 to 8) with mean ± SEM are shown. Non-parametric paired two-tailed Student’s t-test determined statistical significance. * p = 0.05 MALAT1gapmer vs. LacZgapmer. Uncropped Western Blots of Figure 5 are available in Figures S11–S13.