Fig. 3: TM4SF5 WT promoted PIP3-mediated macropinocytosis depending on nutrient replenishment.
a SNU761 hepatocytes stably transfected with pmCherry-TM4SF5WT were live imaged in normal culture media at 37 °C and 5% CO2. b TM4SF5-negative SNU449Cp or TM4SF5-positive SNU449Tp cells in normal culture conditions were processed for scanning electron microscopy. The yellow circles were enlarged for membrane ruffling (bottom). c SNU449 cells exogenously transfected with TM4SF5WT conjugated with APEX2 were processed for transmission electron microscopy33. Note that membrane edges showed ruffling with TM4SF5-APEX2-positive dark dots. d Huh7KO cell variants replenished with GLU alone or ALB + GLU to SFM for 16 h were processed for immunoblotting. The band intensities measured by ImageJ software were normalized to those of the loading control. e A schematic of possible molecular linkage from TM4SF5 to PIP3 for ruffling and macropinocytosis. f, g Huh7-KO1#2 cells transduced with control, TM4SF5WT or TM4SF5A132V lentivirus were replated on collagen I and cells were then imaged to observe nondifferential TM4SF5 expression (f) or transiently transfected with pEGFP-AKT-PH for 24 h and manipulated as above for serum starvation and nutrient replenishment, before live imaging (15 s intervals for 20 min) around cellular edges (g). Representative images were collected and are presented. h, i Huh7KO cell variants were manipulated as in g with the indicated nutrients: after live imaging for specific durations, chambers on the microscope stage were replenished with ALB (3.6 mg/ml, h) or GLU (25 mM, i), and live imaging continued. Data represent three independent experiments. See also Supplementary Movies 1–4.
