Fig. 5: TM4SF5-mediated ALB uptake was linked to enhanced metastatic potential.
a Control Huh7KO, Huh7KO-TM4SF5WT or Huh7KO-TM4SF5A132V cell variants replated on collagen I with or without replenishment of ALB (3.6 mg/ml) to GLU (25 mM) + SFM for 15 h were analyzed for cell migration via tracking of individual cell movement for 17 h using a time-lapse microscope (12 cells per condition, left). The mean instantaneous speed per condition was calculated using MetaMorph software and presented (in μm/min, right). The dots indicate a mean value of the instantaneous speed calculated from the total migration distance of a cell for a measurement interval of 20 min. During the measurement period of 17 h, many instantaneous speed values (3 times per h × 17 h) were calculated for the mean value of a cell. b–f Huh7KO cell variants manipulated as in a were treated with DMSO or TSAHC (5 μM) before cellular tracking for 16 h (b), with or without EIPA treatment at different concentrations before cellular tracking for 17 h (c–f) and calculation of the mean instantaneous speed. Each trajectory line depicts a trajectory of one individual cell. P values were calculated via one-way ANOVA or unpaired Student’s t-tests, and P < 0.05 was considered statistically significant. g, h A schematic for the analysis of in vivo macropinocytosis (g): six-week-old BALB/c-nude male mice were liver-orthotopically injected with TM4SF5-null SNU449Cp or TM4SF5-overexpressing SNU449T7 cells stably transfected with luciferase (luc) constructs (106 cells/injection/mouse); luciferase signal was measured from mice fed a NCD containing 20% protein or a HGProD containing 20% GLU and 40% protein (n > 8) until day 20; on day 20, FITC-BSA (0.2 mg/mouse) was intratumorally injected, and 90 min later mice were killed for imaging of green signal in the liver; and representative images of luciferase in animals and FITC-BSA signals in liver tissue were obtained using IVIS Spectrum (h). i Liver tissues from patients with HCC (n = 7) were processed for H&E staining or immunohistochemistry. Data represent three independent experiments. See also Supplementary Fig. 6.
