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. 2018 Jan 1;11(1):dmm031146. doi: 10.1242/dmm.031146

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© 2018. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — Hipk alters epithelial integrity and induces EMT. (A,A′) A control wing disc (A) and a cross-section of the central region of the disc (A′) stained for GFP, Mmp1 and Ndg. (B,B′) dpp>HA-hipk^1M+HA-hipk^3M+LacZ+GFP stained to detect GFP, Mmp1 and Ndg. (C,C′) dpp>HA-hipk^1M+HA-hipk^3M+P35+GFP stained to detect GFP, Mmp1 and Ndg. (D,D′) dpp>P35+GFP stained to detect GFP, MMP1 and Ndg. (E) Ndg is expressed in a uniform pattern along the basement membrane. (F-F‴) Gaps in Ndg expression are present in dpp>HA-hipk^3M+GFP discs (F′), and higher resolution (F″,F‴) shows that the location of HA-hipk^3M+GFP-expressing cells coincides with regions where Ndg is perturbed (arrowheads). (G) Wild-type eye disc stained for Dlg to reveal cell membranes. (H) ey>HA-hipk^1M+HA-hipk^3M eye disc stained for Dlg showing defects in Dlg stain and apparent cell extensions towards posterior margin of disc (chevrons). (I,J) Cross-sections of dpp>GFP (I) and dpp>HA-hipk^3M+GFP-expressing cells (J) in the center of the wing pouch, stained for E-cad. (K) Twi is expressed in the adepithelial myoblasts, located in the notum region of the wing disc. (L) Twi-positive mesenchymal cells are present in the wing pouch region of dpp>HA-hipk^3M+GFP discs (arrowheads), and Twi is induced in a swathe of cells along the dpp domain (asterisk). Scale bars: 50 μm. (M) Drosophila Hipk (dHipk) promotes significant proliferation of three cell lines. dHipk-transfected HEK293T cells (mean=2.097, s.e.m.=0.02803) display increased proliferation compared with empty vector transfected (mean=1.161, s.e.m.=0.01619) conditions; t(4)=28.92, ****P<0.0001. dHipk-transfected breast adenocarcinoma MCF7 cells (mean=1.098, s.e.m.=0.01217) display increased proliferation compared with empty vector transfected (mean=0.8671, s.e.m.=0.0035) conditions; t(4)=18.25, ****P<0.0001. dHipk-transfected breast adenocarcinoma MDA-MB-231 cells (mean=1.067, s.e.m.=0.0037) display increased proliferation compared with empty vector transfected (mean=0.6457, s.e.m.=0.0074) conditions; t(4)=50.83, ****P<0.0001. (N) dHipk-transfected MDA-MB-231 cells (mean=1.953, s.e.m.=0.2277) demonstrated significantly increased migration compared with empty vector transfected (mean=0.9999, s.e.m.=0.0375) conditions; t(19)=4.759, ***P=0.0001. (O) qRT-PCR was used to quantify the expression of the human E-cadherin gene (CDH1) after transfection of MDA-MB-231 cells with dHipk [t(2)=34.86, ***P=0.0008]. (P) Western blot analysis of E-cad expression in HEK293T cells after dHipk transfection.