Figure 3. Knockown of GALNT3 in TS^WT Cells Induces EMT.

(A) Transcripts were measured using qPCR in TS^WT and TS^KI4 cells expressing control shRNA (C) or TS^WT cells expressing two independent Galnt3 shRNAs (TS^WTGsh1 and TS^WTGsh2).

(B) Western blots of whole-cell lysates are representative of three independent experiments. N.S., Non-Specific.

(C) GALNT3 knockdown in TS^WT cells induces a mesenchymal morphology. Representative phase microscopy images from three independent experiments are shown. Black bar represents 100 μm.

(D) GALNT3 knockdown in TS^WT cells reduces epithelial marker expression in TS^WTGsh1 and TS^WTGsh2 cells.

(E and F) Western blots of E-cadherin (E) and Claudin-6 (F) are representative of three independent experiments.

(G and H) Increased transcripts of mesenchymal markers (G) and EMT-inducing transcription factors (H) with reduced Galnt3 expression are shown.

(I) Impaired barrier formation in TS^WTGsh1 and TS^WTGsh2 cells with reduced GALNT3 expression. Diffusion of fluorescent dye across a confluent monolayer of cells is expressed as a fold-change in fluorescence relative to TS^WT cells. Data shown are the mean ± SEM of three independent experiments.

(J) Loss of GALNT3 increases invasiveness in TS^WTGsh1 and TS^WTGsh2 cells through growth-factor-reduced Matrigel-coated transwells. Data show cells per 103 field and are the mean ± range of two independent experiments performed in triplicate.

(A, D, G, and H) qPCR data normalized to Actb are expressed as a fold-change relative to TS^WT cells and are the mean ± SEM of three independent experiments. *p < 0.05; ***p < 0.001; Student’s t test.

See also Figure S2.