Hepatocyte TM4SF5-mediated cytosolic NCOA3 stabilization and macropinocytosis support albumin uptake and bioenergetics for hepatocellular carcinoma progression

Cells were cultured in normal 10% FBS-containing media or replated on culture wares precoated with poly-lysine (P4832, Sigma) or different extracellular matrix (10 μg/ml collagen type I (5056, Advanced Biomatrix), fibronectin (CLS356008, Merck) or laminins (354232, Life Sciences)). Cells were incubated in serum-free Dulbecco’s modified Eagle’s medium or RPMI-1640 media (SFM with basal GLU containing or without GLU) for 3 h. Cells then did or did not undergo replenishment with GLU (10 or 25 mM for SNU449, HepG2 or Huh7 cells, respectively; G8270, Sigma), ALB (3.6 mg/ml; A8806, Sigma) or GLU + ALB for 15 h or the indicated times, via each addition into (basal) GLU-free SFM. Cells in normal culture or as treated as described above were collected for whole-cell lysates using modified RIPA lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS and 1% Triton ×100). Lysates were normalized before immunoblot analysis using the following primary antibodies: β-actin (sc-47778), pS⁴⁷³AKT1 (sc-7985-R) and AKT1 (sc-8312) from Santa Cruz; HA (3724), pS²⁴⁴⁸-mTOR (5536), mTOR (2983s), pT¹⁷²AMPK (2535s), AMPKα (5832s), pS⁶³-JUN (9281s), c-JUN (9165s), p-ERK1/2 (9102s), ERK1/2 (9101s), α-tubulin (2125), NCOA3 (5765), PTEN (9559s), pT²⁴NCOA3 (5765), ubiquitin (S43124) or acetyl-lysine (9441s) from Cell Signaling Technology; strep-HRP (2-1509-001) from IBA Lifesciences; ALB (213-MSM4-P1) from NeoBiotechnologies; or purified anti HA-11 epitope tag antibody (901501) from Biolegend. An anti-TM4SF5 antibody was generated by immunizing rabbits with a TM4SF5 C-terminal or long extracellular loop two-sequence peptide³³. In some cases, the ratio values of band intensities of certain immunoblots measured by ImageJ software were normalized to those of a loading control or the total form of the molecule was presented.