Fig. 5

Expression analysis of LDHB in MCF7Ecadvar and MCF7pcDNA3 cells. a Western immunoblotting of the 35 kDa LDHB form in total protein extracts of MCF7pcDNA3 and MCF7Ecadvar cells. The immunodetection of β-tubulin (55 kDa) was used as loaded control. b Immunofluorescence analysis of LDHB (× 200 magnification, scale bar 50 μm) coupled to nuclear staining with dye Hoechst 33342 in MCF7pcDNA3 and MCF7Ecadvar cells. Negative controls were included for both cell lines. Quantification of the LDHB specific signal was also done and indicated in the bar graph. c, d Determination of c glucose and d lactate concentrations (mg/dL) in the 72-h-conditioned media of MCF7Ecadvar and MCF7pcDNA3 culture cells. Measurements were done with a Cobas 8000 equipment. e Cellular viability analysis of MCF7pcDNA3 and MCF7Ecadvar culture cells after 24 h treatment with 0, 0.5, or 5 mM of the 2DG glycolytic inhibitor. f Quantitative analysis of LDHB by real-time PCR in MCF7Ecadvar siScr and MCF7Ecadvar siLDHB cells. The relative expression was calculated as described in the “Materials and Methods” section, using GAPDH as the endogenous gene and the MCF7Ecadvar siScr cell line as reference. g Western immunoblotting of the 35 kDa LDHB form in total protein extracts of MCF7Ecadvar siScr and MCF7Ecadvar siLDHB cells. The immunodetection of β-tubulin (55 kDa) was used as loaded control. Quantification of the LDHB specific signal was also done and indicated in the bar graph. h Cellular viability analysis of MCF7Ecadvar siScr and MCF7Ecadvar siLDHB culture cells after 72 h treatment with 100 pmoL/mL of a LDHB specific siRNA. *p < 0.05, ***p < 0.001