Fig 2: Detection of LAT1 and 4F2hc by western blotting and immunofluorescence analyses in 8505C, OCUT-2, and OCUT-6 human ATC cells. (A) Expression of LAT1 and 4F2hc levels in the indicated cells detected by western blotting. (B) The densitometry value of LAT1 was normalized α-tubulin. (C) The densitometry value of 4F2hc was normalized α-tubulin. (D) DAPI (4,6-diamidino-2-phenylindole is a blue fluorescent stain specific for DNA) nuclear staining was shown in blue. Both LAT1 (green) and 4F2hc immunoreactivities (red) were detected on the plasma membrane of 8505C, OCUT-2, or OCUT-6 cells. LAT1 (green) and 4F2hc immunostaining (red), and DAPI (blue) were merged and shown in 2D, indicating the coexistence of LAT1 with 4F2hc in the plasma membrane of 8505C, OCUT-2, or OCUT-6 cells, respectively.

Fig 3: Anti-tumor effect of JPH203 in 8505C-inoculated athymic BALB/c nude mice. An equal number of 8505C cells (1 × 106 cells) were injected into the flanks of each mouse before treatment. When tumors started to develop (average tumor size reached 100 mm3), JPH203 or vehicle was administered intraperitoneally for 18 day (12.5 mg/kg/d). JPH203 treatment decreased (A) tumor growth ratio, (B) tumor size. ‡: #4 mouse was just died before euthanasia. Therefore, this mouse was excluded from following analysis. (C) Representative images of H&E-stained tumor sections (panels a and b). There was no difference between vehicle treated mice (panel a) and JPH203 treated mice (panel b). Immunohistochemical analysis showed the LAT1 and 4F2hc express expression at cell surface, and their expressions had little effect on JPH203 treatment (panel c to f). However, JPH203 treatment decreased the Ki67 immunoreactivity (panel g and h). (D) The counting data showed the number of Ki67 immunoreactive positive cells were decreased in the group of JPH203 treated mice (***p < 0.001). (E) Western blot for LAT1, 4F2hc, cyclin D1, phosphorylated S6, total S6 and α-tubulin was carried out to access mTOR signal and cell cycle regulators. (F) The band intensities of these proteins detected in (E) were quantified and compared. JPH203 treatment suppresses the expression of cyclin D1 (*p < 0.05) and the phosphorylation of S6 (*p < 0.05).

Fig 4: Representative images of immunoreactivity for LAT1, 4F2hc, and Ki67 in human ATC and AG tissues. (a) immunoreactivity 0 for LAT1 in ATC tissues; (b) immunoreactivity 1 for LAT1 in ATC tissues; (c) immunoreactivity 2 for LAT1 in ATC tissues; (d) immunoreactivity 3 for LAT1 in ATC tissues. (e) immunoreactivity 0 for 4F2hc in AG tissues; (f) immunoreactivity 1 for 4F2hc in AG tissues; (g) immunoreactivity 2 for 4F2hc in ATC tissues; (h) immunoreactivity 3 for 4F2hc in ATC tissues. (i) immunoreactivity 0 for Ki67 in AG tissues; (j) immunoreactivity 1 for Ki67 in ATC tissues; (k) immunoreactivity 2 for Ki67 in ATC tissues; (l) immunoreactivity 3 for Ki67 in ATC tissues. (m) Cause-Specific Survival (CSS) rates of ATC patients according to LAT1 expression.