Absence of the Integrin α3 Subunit Induces an Activated Phenotype in Human Keratinocytes Chiara Pazzagli, Yinghong He, Hauke Busch, Philipp Esser, Dimitra.

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Absence of the Integrin α3 Subunit Induces an Activated Phenotype in Human Keratinocytes Chiara Pazzagli, Yinghong He, Hauke Busch, Philipp Esser, Dimitra Kiritsi, Yannick Gache, Leena Bruckner-Tuderman, Melanie Boerries, Cristina Has Journal of Investigative Dermatology Volume 137, Issue 6, Pages 1387-1391 (June 2017) DOI: 10.1016/j.jid.2017.01.018 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Effects of the constitutional absence of integrin α3 on keratinocyte spreading and adhesion. (a) F-actin and vinculin in control (Co) and ILNEB (A3−) immortalized keratinocytes on uncoated, laminin 332- or fibronectin-coated coverslips; nuclei are stained in blue. Scale bar: 20 μm. (b) Cell index of primary normal human keratinocytes (NHK), primary ILNEB keratinocytes (PatientA3−), Co and A3− keratinocytes was measured during 120 minutes (2 hours). (c) Quantification of adhesion on uncoated, laminin 332- and fibronectin-coated wells; ∗∗∗P < 0.0001. (d) Immunoblot analysis of P-FAK(Tyr397), P-ERK1/2(Thr183/Tyr185), and P-Akt(Ser473) in lysates of suspended cells (CS), and 15 or 30 minutes after seeding on laminin 332- or fibronectin-coated dishes. Total FAK, ERK, Akt, and GAPDH proteins are loading controls. (e) Quantification of the de-adhesion assay (∗∗P < 0.01; ∗∗∗P < 0.001). Akt, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ERK, extracellular signal-regulated kinases; FAK, focal adhesion kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ILNEB, interstitial lung disease, congenital nephrotic syndrome and junctional epidermolysis bullosa. Journal of Investigative Dermatology 2017 137, 1387-1391DOI: (10.1016/j.jid.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Integrin landscape in integrin α3-deficient keratinocytes. (a) Flow cytometry for the integrin α3, α5, and αv subunits. (b) Cells treated with integrin blocking antibodies or untreated were seeded on fibronectin; absorbance was measured at 540 nm (mean ± SD; ∗∗∗P < 0.0001). (c) Immunoblots with A3− cell lysates, with/without integrin α5 blocking antibodies, and antibodies to the indicated proteins, P-ERK1/2(Thr183/Tyr185), P-Akt(Ser473), Akt, and GAPDH. (d) Immunofluorescence staining of normal and ILNEB skin sections with integrin α5 and αv antibodies. Scale: 100 μm; line represents basement membrane. (e) Directionality and velocity of single-cell migration were quantified during time-lapse recording with/without blocking antibodies (mean and s.e.m.; ∗∗P < 0.001; ∗∗∗P < 0.0001). (f) Outlines of Co and A3− keratinocytes without (−) or with α integrin blocking antibodies, obtained by drawing the cell contours in merged time-lapse pictures. Akt, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ERK, extracellular signal-regulated kinases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ILNEB, interstitial lung disease, congenital nephrotic syndrome and junctional epidermolysis bullosa; SD, standard deviation; s.e.m., standard error of the mean. Journal of Investigative Dermatology 2017 137, 1387-1391DOI: (10.1016/j.jid.2017.01.018) Copyright © 2017 The Authors Terms and Conditions