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Fig. 4 | BMC Genomics

Fig. 4

From: The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins

Fig. 4

Relative expression levels of candidate genes linked to inferred TF activities. a-c Histograms representing the mean fold change of expression (+/− s.d.), estimated from normalised TPM (transcripts per million) counts, of the indicated genes in 3D culture normalised to their expression levels in Control 2D condition of growth. Results were obtained from three independent biological replicates (except Matrigel 1.5 mg/ml which had two replicates). Three top gene candidates of the statistically significant transcription factors are shown along with the expression level of the TF themselves for comparison. d Histograms represent the relative mean (+/− s.d.) expression levels of hepatocyte marker ALB, growth marker Ki-67 and mature miR-122-5p in cells grown on Collagen and Matrigel scaffolds. ALB, Ki-67 levels were estimated from normalised TPM counts. miR-122-5p levels were estimated by qPCR relative to U6snRNA levels. Histograms represent the enrichment in the quantities of the indicated genes in Collagen and Matrigl culture conditions normalised to their levels in the Control 2D growth condition. e-f Scheme of Plasmids used for transfection of Huh-7 cells and the basis of calculation of fold repression (e). Histograms represent relative repression levels of a miR-122 reporter with multiple bulged miR-122 binding sites in Huh-7 cells. Cells were co-transfected with Renilla / RL-3xBulge-miR 122 along with Firefly luciferase expression plasmid, split and re-seeded onto plates coated with Collagen (CG 0.5 mg/ml) or Matrigel (MG 1.5 mg/ml) or Control for a duration of 72 h prior to lysis and luminescence measurements. Experiments were performed with two biological replicates (with two technical replicates each) in separate batches for Control / MG and Control / CG. For comparison, the fold repression level was normalised to unit value in the Control condition (f). g Representative microscopic images of P bodies in Huh-7 cells grown on Control or Matrigel (1.5 mg/ml) reveals loss of discrete P bodies on Matrigel. For ectopic expression, a GFP tagged DCP1a expression construct was transfected into Huh-7 cells and subsequently the cells were split into Control or Matrigel coated chamber slides. Prior to imaging, Hoechst was added to stain the nucleus. A DIC image was also captured for visualisation of cell boundaries. A normal field of view image (top panel) and a digitally zoomed region (lower panel) of the field in the indicated conditions are shown. The P values of the unpaired non parametric t tests comparing the expression of the designated genes in the Matrigel and Collagen growth conditions, with respect to the Control, is denoted (when significant). The P values comparing the luciferase reporter repression levels, computed by a paired t test, is also denoted. * represents a P value of ≤0.05, ** represents a P value ≤0.01, *** represents a P value of ≤0.001. P values > 0.05 were considered non-significant

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