Fig. 1
AST factors reprogram anchorage dependency. A Schematic of the transcriptomic analysis of 141 adhesion cells and 39 suspension cell lines using RNA expression data from the ENCODE and The Human Protein Atlas databases to identify anchorage-dependent differentially expressed gene profile. B Correlation plot of gene expression in adhesion cells versus suspension cells. Correlations were calculated using the Pearson method. C Volcano plot showing differentially expressed genes selected based on |fold changes|> 2, |differences|> 1, and p-values < 0.05. D Selection of 20 hematopoietic transcriptional regulators as candidate AST factors. E Heatmap of the 20 candidate AST factors in 90 adhesion and 23 suspension cell lines. F Schematic overview of the experimental design for generating HEK293A-20AST cells by lentiviral expression of AST factors. G, Representative cell images of HEK293A-mock or 20AST cell morphology in culture plate and media. H Immunoblotting analysis of the expression of V5-tagged AST factors in HEK293A-20AST cells. I Representative fluorescence images of cell viability in cell culture media of HEK293A-mock and 20AST cells stained using LIVE/DEAD cell imaging assay. Green, Live; Red, Dead. Scale bar, 100 μm. J Measurements of cell proliferation rate between adherent mock and suspended 20 AST cells. K Images of time-lapse microscopy showing spontaneous cell detachment of HEK293A-4ASTTetR cells upon doxycycline treatment. L Immunoblotting analysis of the expression of V5-tagged AST factors in HEK293A-20ASTTetR cells upon doxycycline treatment M-P Schematic for narrowing down from 20 to 10 (M), and then from 10 to 4 (N) AST candidates via multiple trials of AST induction. Representative cell images of HEK293A-10AST (N) or 4AST (P) cells in culture plates and media. Q Measurement of AST efficiency by subtracting individual factors from 4 AST factors. R Volcano plot showing mRNA expression of transcription factors. x-axis, contrast in expression between suspension and adhesion cells; y-axis, p-value. 20 candidate AST factors (orange) were statistically upregulated in suspension cells versus adhesion cells. Upregulation, fold changes > 2, p-values < 0.05. S Consecutive reprogramming of anchorage dependency in HEK293A-ASTTetR cells by the addition and removal of doxycycline. Scale bars, 50 μm