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Fig. 5 | Molecular Cancer

Fig. 5

From: ITIH5 mediates epigenetic reprogramming of breast cancer cells

Fig. 5

ITIH5 alters integrin signaling impairing single-cell polarization. a Integrin protein expression/stability in ITIH5 and mock clones. β -actin served as loading control. b Densitometric evaluation of western blot results demonstrating an integrin protein shift. Relative protein expression levels are normalized to β-actin. Mean protein level of mock clones was set to 100%, respectively. c Analysis of integrin downstream signaling. Representative western blot results illustrate activated Rac1 and RhoA GTPases in two independent ITIH5 and mock clones. Total Rac1 and RhoA served as loading control. d Densitometric evaluation of GTPases activation. Relative protein expression levels are normalized to total Rac1 and total RhoA, respectively. Mean protein level of mock clones was set to 100%. e Cell migration was analyzed by using a wound healing assay. Mean migration rate of a control cell set (n = 4, WT and mock clones) and ITIH5 MDA-MB-231 clones (n = 4) was analyzed over 4 days. Vertical lines: standard deviation (S.D.) of triplicates. Cell-free area on day 0 was set as 100% and used for standardization. Δday1: differences of cell-free areas on day 1. f Documentation of the wounded area by SEM 24 h after scratching. Left rectangle regions: separately enlarged. Scale bar = 100 μm. g Detailed comparison of wound closure after 24 h for each single-cell clone. h Visualization of F-actin architecture and focal adhesion are shown of ITIH5 and mock clones. Upper rows: Representative micrographs of ITIH5 clone #7 and ITIH5 clone #4. White arrows indicate cortical actin bundles (red) and less elongated focal adhesions (green dots). Lower row: Representative micrographs of mock clone #1. White arrows: F-actin stress fibers (red) co-localized with elongated focal-adhesion sites (green) in the cell body of single-cells. Scale bar = 10 μm. i Illustration of the ITIH5-associated impact on cell-polarization necessary for cell migration. a: ITIH5 clones showed tight clusters lacking cell polarization. b: mock cells are able to form a distinct protrusive front and a retracting rear. Scale bar = 10 μm j Real-time PCR analysis demonstrating significant upregulation of DSP, DSC2 and DSG2 in ITIH5 (n = 5) compared to mock clones (n = 4). Horizontal lines: grouped medians. Boxes: 25–75% quartiles. Vertical lines: range, peak and minimum; *p < 0.05, **p < 0.01, ***p < 0.001

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