GPR81 nuclear transportation is critical for cancer growth and progression in lung and other solid cancers

Figure 1 Lactate promotes GPR81 expression and GPR81 nuclear translocation in cancer cells. A: GPR81 expression level in lung cancer and control cells were measured with reverse transcriptase-PCR (RT-PCR) (left panel) and western blot (middle panel). Densitometry values summarizing western blot data are shown in the right panel. GAPDH served as a loading control; B: GPR81 expression level in lung cancer and control cells cultured under vehicle/Lactate (5 mmol/L) conditions were measured with RT-PCR (left panel) and western blot (middle panel). Densitometry values summarizing western blot data are shown in the right panel. GAPDH served as a loading control; C: Lung cancer cells self-renewal was assessed in the colony forming assay; D: Cancer stemness marker CD44 and cell proliferation marker Ki67 were quantified with RT-PCR and western blot analysis; E: Lung cancer cell invasion was assessed; F: Cell motion marker MMP2 was quantified in RT-PCR and Western blot analyses. A-F: n = 4, each of control and lung cancer cells (Control 202, 205, 272, 392; lung cancer A549, 661, 858, 645); G: Lung cancer cells were cultured with varying concentration of lactate or vehicle and the cells were collected at 4 hours and nuclear fractions isolated for Western blot analysis. Lamin served as a loading control; H: Lung cancer cells were cultured with 5 mmol/L of lactate and the cells were collected at the indicated time and nuclear fractions were isolated for Western blot analysis. G and H: n = 4 Lung cancer cell lines (A549, 645, 661, 858); I: GPR81 expression level in cancer and control cells was measured with RT-PCR (left panel) and western blot (middle panel). Densitometry values summarizing western blot data are shown in the right panel. GAPDH served as a loading control; J: GPR81 Level in cancer and control cells cultured under vehicle/Lactate (5 mmol/L) conditions were measured with RT-PCR (left panel) and the nuclear fraction was analysed with western blot (middle panel). Densitometry values summarizing western blot data are shown in the right panel. Lamin served as a loading control.