Morphology and cell counts of HepG2/C3A cells cultured in media containing 10%FBS
HEPG2/C3A cells grown in media supplemented with 10% foetal bovine serum (FBS) characteristically display a regular polygonal morphology and grow in monolayer colonies (A) after 72 hours in culture, (B) after 120 hours in culture (confluent). (C) Bar chart demonstrating the cell cycle stages calculated using the Watson pragmatic algorithm. Values are mean ± SD (n=2).
Individual cell cycle histograms
Individual histograms of the cell cycle analysis that were presented as overlays in figure 2. Gating, calculations and images presented in this figure were carried out using FlowJo 10.5.3.
Cell cycle analysis of serum starved HepG2/C3A cells
Cell cycle analysis of HEPG2/C3A hepatocytes after 48 hours and 72 hours serum starvation demonstrate that cell cycle arrest gradually increases over time. Data represented in the graph are mean percentages of G1, S and G2/M cell cycle stages at 48 hours and 72 hours of serum starvation. Values are mean ± SD (n=2).
TUNEL assay of serum starved HepG2/C3A cells
TUNEL assay of HEPG2/C3A cells demonstrates apoptosis at 4% and 16.6% after (A) 48 and (B) 72 hours (image from fig 3 a of main text) of serum starvation respectively.
Individual dot plots of TUNEL assay
Individual dot plots of the TUNEL assay that were presented as overlays in figure 3.
Analysis of HepG2/C3A cells cultured in media containing 10% FBS
HepG2/C3A cells cultured in media containing 10% FBS for 72 hours were analysed by (A) TUNEL assay using a flow cytometer for apoptosis (0.8 ± 0.2 %) (n=2). (B) fluorescence microscopy demonstrates 1) DAPI (nuclear), 2) calcein AM (cytoplasmic) and 3) ethidium bromide (nuclear) staining. Scale bar=100 µm. A necrotic index of 1.7 ± 0.8% was calculated as the percentage of necrotic cells (ethidium bromide) from the total cell count (DAPI) (n=2).
Treatment of HepG2/C3A cells cultured in media containing 10% FBS with human serum albumin
Mean cell counts of HepG2/C3A cells cultured for 72 hours in media containing 10% FBS (5830000 ± 40000) and media containing 10% FBS treated with 10mg/ml albumin (6470000 ± 1083097). Values are mean ± SD (n=3).