Fig. 1: Hepatocyte TM4SF5 has differential effects on filopodia-like processes depending on available nutrients.
a Enrichment plot by the Kyoto Encyclopedia of Genes and Genomes pathway of actin cytoskeleton organization depending on ectopic TM4SF5 expression in SNU449 hepatocytes. b TM4SF5 immunoblots of representative clones of TM4SF5-KO or control (non-KO) hepatocyte variants via a CRISPR–Cas9 approach from parental Huh7 and Hep3B cells with endogenous TM4SF5. The subscripts indicate different Huh7KO cell variants. The band intensity ratio values were measured by ImageJ and normalized to those of the loading control. c, d TM4SF5WT-HA cDNA transfected into Huh7-KO1#2 cells for 24 h were replated on collagen I (10 μg/ml) without (SFM) or with 10% FBS or SFM + ALB (3.6 mg/ml) with basal GLU before phalloidin staining (c) or onto collagen I in basal GLU-containing SFM with or without ALB (d). In c, representative images with pseudo-colors per HA tag-stained cell are presented (n ≥ 20, each data point is a value from one individual cell). In d, the automatic quantification of filopodia-like processes (or protrusions) per cell visualized by rhodamine–phalloidin staining was performed using the Fiji plugin ADAPT (v1.193). Each dot indicates the protrusion numbers of a cell. e–g Huh7-KO1#2 (e and f) or Hep3B-KO1#6 (g) cells reconstituted with the indicated cDNA were replated to different ECM or poly-l-lysine (10 μg/ml) for 16 h in the GLU + SFM condition before fluorescence staining of the HA tag: rcn (e) and quantified for filopodia-like protrusions (f and g). h–j Huh7KO cell variants on collagen I were treated with DMSO, control compound or TSAHC as above (h), with replenishment of GLU alone or GLU + ALB to SFM (i) or of none or GLU alone (j), before imaging and protrusion quantification. P values were calculated via one-way ANOVA or unpaired Student’s t-tests, and P < 0.05 was considered statistically significant. k Huh7-KO1#2 cells transduced with lentivirus for different cDNAs were manipulated as in c, before immunoblots. Data represent three independent experiments. See also Supplementary Figs. 1, 2 and 4.
