Fig. 2 ITGA5 knockdown attenuates hPSC activation, differentiation, and functions. ITGA5 knockdown attenuates hPSC activation, differentiation, and functions.

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Fig. 2 ITGA5 knockdown attenuates hPSC activation, differentiation, and functions. ITGA5 knockdown attenuates hPSC activation, differentiation, and functions. Immunofluorescence staining showing ITGA5 (A) and α-SMA (B) expression in hPSCs after ITGA5 knockdown (sh-ITGA5) compared to NC (sh-NC) hPSCs with or without TGF-β activation. (C) Differential gene expression of adhesion molecules and ECM using human ECM RT2 Profiler PCR Array in sh-ITGA5 with or without TGF-β activation. Left graph: sh-ITGA5 data are relative to sh-NC (set at 1.0) showing top 20 down-regulated genes after ITGA5 knockdown. Right graph: sh-NC + TGF-β data are relative to sh-NC hPSCs, while sh-ITGA5 + TGF-β data are relative to sh-ITGA5 hPSCs, showing the most induced genes after TGF-β activation, which are not induced after ITGA5 knockdown. (D) Cell adhesion assay shows that ITGA5 knockdown in hPSCs (sh-ITGA5) reduced cell attachment to the coated FN at t = 30 min compared to control hPSCs (sh-NC). Scale bar, 100 μm. (E) Spheroid formation assay shows that sh-ITGA5 hPSCs do not form compact spheroid due to lowered cell-to-cell attachment. (F) BrdU enzyme-linked immunosorbent assay (ELISA) shows that sh-ITGA5 hPSCs had a reduced cell proliferation compared to sh-NC assessed for 3 days. (G) Representative microscopic images of wound closure assay and quantitative analyses at t = 15 hours shows that sh-ITGA5 hPSC had a reduced migration ability. (H) Representative images from 3D collagen gel contractility assay show that TGF-β–induced contractility was inhibited in sh-ITGA5 hPSCs after 96 hours. (I) Western blot analysis (bands and quantitative analysis) shows that sh-ITGA5 hPSCs had a reduced TGF-β–induced pSmad2 and pFAK signaling in comparison to sh-NC hPSCs. The pSmad2/Smad2 ratio was analyzed at t = 30 min, while the pFAK-Y397/FAK ratio was analyzed at t = 48 hours. Densitometry analyses were performed using ImageJ software. Data represent means ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar, 200 μm. n.d., not determined. Praneeth R. Kuninty et al. Sci Adv 2019;5:eaax2770 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).