Figure 6.

Positive Feedback Loop Makes a Bi-stable Switch

(A) The core regulatory network of the SRF-mediated double loop, comprising KLF4/5, MAL-SRF complex, focal adhesion proteins, and G-actin.

(B) Bifurcation diagram of the KLF4/5 expression level as a function of the cell-matrix adhesion, for a MAL expression level approximately corresponding to that of the AST cells. Solid black lines represent stable cell states, with the SRF-repressed state in the upper branch and the SRF-activated state in the lower branch. For cell-matrix adhesion between the two limiting points LP1 and LP2, the cell can be in either of the two stable states. With changes of cell-matrix adhesion, the cell can switch between the two states reversibly. The region of negative substrate adhesion (gray bar) is unreachable. Blue line indicates the transition of AST cells.

(C) AIT and AST cell adhesion curves on Matrigel-coated substrates with gradient of coating concentrations (0.1–40 μg/cm²) (mean ± SD, n = 4 independent biological replicates, ^∗p < 0.05, ^∗∗p < 0.01).

(D) Relative expression of KLF4, KLF5, and NANOG in AIT and AST cells on Matrigel-coated substrates with three different coating concentrations (0.1, 1, and 10 μg/cm²) (mean ± SD, n = 3 independent experiments, ^∗p < 0.05, NS, not significant).

(E) Bifurcation diagrams of KLF4/5 expression level as a function of cell-matrix adhesion. The three diagrams correspond to the three values of MAL expression level indicated on the right. For a MAL expression level approximately that of an AIT cell (1.59 unit), the cell cannot switch to the upper branch by just changing the cell-matrix adhesion. With reduced total MAL expression, the upper branch can become reachable. Below a critical value (0.72 unit) of MAL expression, there is no bi-stability and the properties of the cell change smoothly. Green line indicates the irreversible transition of AIT cells. Violet line indicates the reversible transition of the small interfering RNA (siRNA)-treated AIT cells.

(F) The relative change of the position of the two limiting points LP1 and LP2, when each of the 20 parameters in the model is increased or decreased by 15%.

(G) Western blot result indicated the interference efficiency of MAL protein in AIT and AST cells.

(H) siRNAs were transfected with lipofection. Suspended AIT cells were treated with siRNA for 2 hr, and then seeded on the GNF substrate. These cells formed a dome-like morphology after MAL siRNA transfection. Samples with only addition of liposome were used as a control.

(I) Relative expression of KLF4, KLF5, and NANOG in AIT cells on low-adhesion GNF substrate with or without siRNA treatment (mean ± SD, n = 3 independent biological replicates, ^∗p < 0.05, ^∗∗p < 0.01). To avoid the interference of ROCK inhibitor on cell adhesion, data in this figure were obtained more than 48 hr after withdrawal of the ROCK inhibitor from cell cultures.

See also Figure S7.