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. 2019 Mar 4;13(3):2913–2926. doi: 10.1021/acsnano.8b06998

Figure 2.

Figure 2

Nanoneedles inhibit focal adhesion formation and generation of intracellular tension. (A) Confocal maximum projection images 6 h postseeding. On flat substrates, dense vinculin staining is observed in stable focal adhesion (FA) complexes. Strong phosphorylated paxillin (pPax) and phosphorylated myosin light chain (pMLC) signal on flat substrates indicate FA maturation and active actomyosin contractile machinery, respectively. Cells on nN display diffuse vinculin staining and severely reduced pPax and pMLC signal. Scale bars: vinculin = 25 μm; pPax and pMLC = 50 μm. (B) Significant reduction in vinculin signal reveals reduced FA density on nN (box plots, minimum/maximum; N = 3). (C) qPCR indicates that culture on nN yields downregulation in gene expression for multiple FA components (focal adhesion kinase (FAK), paxillin (PAX), vinculin (VCL), and zyxin (ZYX); qPCR, N = 3, mean ± SD). (D) Western blot shows downregulation of vinculin protein expression on nN (HUVEC: N = 2, hMSC: N = 3, mean ± SD). (E) Quantification of pMLC signal intensity via image analysis confirms a significant reduction for both cell types cultured on nN, as compared to their respective controls (box plots, minimum/maximum, N ≥ 4); *p < 0.05, **p < 0.01, ***p < 0.001 between groups as indicated by the lines.