Fig. 6: TM4SF5-mediated macropinocytosis involves cytosolic NCOA3 stabilization.
a Liver tissues from patients with HCC (n = 7) were processed for H&E staining or immunohistochemistry. b Analysis of co-expression between TM4SF5 and ALB in the LIHC (HCC) set from the TCGA database. c A Venn diagram showing protein binding to TM4SF5 (ref. 21) or ALB (https://thebiogrid.org/106715/summary/homo-sapiens/alb.html)49. d Huh7-KO1#2 cells stably transfected with TM4SF5WT-HA cDNA were transfected with siRNA against control (NS), ITGA2 or NCOA3 sequences for 24 h and then replenished with GLU (25 mM) alone or ALB + GLU (3.6 mg/ml and 25 mM, respectively) to SFM for 15 h before analysis of the number of protrusions per cell. Each data point depicts a value from one individual cell. e Huh7KO-TM4SF5WT-HA cells were replenished with ALB + GLU to SFM for 15 h and processed for immunofluorescence. f Huh7KO cell variants were replenished with GLU alone or GLU + ALB to SFM for 15 h, before immunofluorescence for NCOA3 with DAPI co-staining. g–l Huh7KO cell variants were transfected with indicated siRNA for control sequence (siNS) or siRNA against specific sequences (Table 1) for 24 h and then replenished with nutrients as indicated for 15 h before analysis of FITC-ALB spots (green) per (red fluorescent) (g–i), cellular tracking for 17 h (j), immunofluorescence (k) or immunoblotting (l). Alternatively, cells were treated with DMSO or the PTEN inhibitor bpV (Phen) during nutrient replenishment for 15 h before analysis on FITC-ALB spots per cell. P values were calculated via one-way ANOVA or unpaired Student’s t-tests, and P < 0.05 was considered statistically significant. Data represent three independent experiments. See also Supplementary Figs. 7 and 8.
