Figure 1
Expression of LAT1 in B16-F10 melanoma cells and the effect of LAT1 suppression on cell proliferation and tumor growth. (A) Quantification of LAT1, LAT2, LAT3, and LAT4 mRNA levels by RT-qPCR. Data were normalized to the expression of GAPDH mRNA and shown as relative expression levels compared to LAT1. PCR analyses were performed in triplicate (n = 3) and shown as mean ± SEM. (B) Protein expression of LAT1 and 4F2hc in B16-F10 cells. LAT1 and 4F2hc were detected by western blot using an anti-LAT1 antibody (left panel) and anti-4F2hc antibody (right panel), respectively, under reducing (DTT( +)) and non-reducing (DTT(-)) conditions. Arrows indicate the bands corresponding to LAT1-4F2hc heterodimer at 150 kDa. The bands of LAT1 (37 kDa) and 4F2hc (70 kDa) monomers are indicated by black and white arrowheads, respectively. Original, uncropped electrophoretic blots are presented in Supplementary Fig. S5. (C) Concentration-inhibition curve of nanvuranlat. B16-F10 cells were treated with nanvuranlat (1.5, 3, 7, 15, and 30 μM) for 48 h. The concentration-inhibition curve shows the percentage of inhibition of B16-F10 cell growth versus the concentration of nanvuranlat (n = 4, data are mean ± SEM). The GI50 value was calculated by nonlinear regression analysis: GI50 = 23.8 ± 3.6 μM. (D) Suppression of cell proliferation in B16-F10 LAT1-knockdown cells. Cell proliferation of B16-F10 LAT1-knockdown cells (shLAT1#1, #2, and #3) and control cells (B16-F10 cells transfected with control shRNA) was determined by the absorbance at 450 nm in the CCK-8 assay and shown at 24, 48, and 72 h after cell seeding. (E) Inhibitory effect of nanvuranlat on tumor growth compared with placebo control. On Day 0, B16-F10 cells were transplanted subcutaneously into the footpad of the hind leg of the mice. Nanvuranlat administration (i.v., 25 mg/kg, daily) started on Day 7 and finished on Day 20. Tumor size was measured from Day 7 to Day 21. (F) Suppression of B16-F10 tumor growth by LAT1 knockdown. Statistical significance was determined using two-way ANOVA with Sidak's post-test (n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Data are mean ± SEM.
