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. 2018 Jun 28;11(1):142–156. doi: 10.1016/j.stemcr.2018.06.003

Figure 5.

Figure 5

A Regulatory Circuitry in AST and AIT Cells

(A) Immunofluorescence images of F-actin and G-actin in AIT and AST cells on the GNF substrate. Confocal microscopy images show the basal surface.

(B) Quantification of F-actin and G-actin levels 2 hr after seeding. Total integrated fluorescence of phalloidin (anti-F-actin) and DNaseI (anti-G-actin) was normalized to the fluorescence of AIT cells (mean ± SD, n = 3 independent biological replicates, ∗p < 0.05, ∗∗p < 0.01).

(C) MAL localization in AIT and AST at the colony and single-cell levels. White arrows indicate dividing cells with a disassembled nuclear membrane and MAL distributed in the whole cell bodies.

(D) The intensity correlation quotients (ICQ) indicating the colocalization of MAL and the nucleus in AIT and AST cells (mean ± SD, n = 3 independent biological replicates, ∗p < 0.05).

(E) Relative expression of focal adhesion genes with 2-fold or larger changes in AIT and AST cells. These genes were selected from 84 genes involved in cellular adhesion (mean ± SD, n = 3 independent experiments, ∗p < 0.05, ∗∗p < 0.01).

(F) Western blot of SRF targeted focal adhesion proteins: zyxin, vinculin, and talin in AIT and AST cells.

(G) Relative expression of miRNA143 and miRNA145 in AIT and AST cells (mean ± SD, n = 3 independent experiments, ∗p < 0.05).

(H) The relative expression of KLF4 and KLF5 in AIT and AST cells under self-renewal conditions at 48 hr after transfection of miRNA-mimic-miR143 and 145 (mean ± SD, n = 3 independent biological replicates, ∗p < 0.05, ∗∗p < 0.01).

(I) Phase contrast images of AIT and AST cells at 72 hr after miR143/145-mimic transfection. The cells undergoing differentiation could be observed (red arrows), and the AST cells formed heterogeneous colonies with both flat and domed morphology. Scale bars, 250 μm.

(J) Western blot analysis of KLF4, KLF5, and NANOG expression in AIT and AST cells on GNF substrates.

(K) SRF-based double-loop-coupling cell morphology, adhesion property, and the expression of KLF4/5 and NANOG. 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 S6.