Figure 7. Reduced O-GalNAc Glycosylation in Cells Lacking GALNT3 that Is Restored by Re-expression of GALNT3.

(A) Total protein O-GalNAc glycosylation is reduced with the loss of GALNT3 in TS^WTGsh1 and TS^WTGsh2 cells. IP with VVL agarose and blotting with biotinylated VVL are shown. Western blots of TS^WT and TS^KI4 cells expressing control shRNA (C) or TS^WT cells expressing two independent Galnt3 shRNAs (TS^WTGsh1 and TS^WTGsh2) are representative of three independent experiments.

(B) Re-expression of GALNT3 in TS^KI4 cells (TS^KI4Gab1 and TS^KI4Gab2) increases protein O-GalNAc glycosylation. Experiments were performed as in (A). Western blots of TS^WT and TS^KI4 cells expressing a control lentiviral vector (C) or or TS^KI4 cells infected with a lentiviral vector expressing human Galnt3 (TS^KI4Gab1 and TS^KI4Gab2) are representative of three independent experiments.

(C) Knockdown of HDAC6 in TS^KI4 cells (TS^KI4H6sh) increases total protein O-GalNAc glycosylation similar to TS^WT cells. Experiments were performed as in (A). Western blots of TS^WT cells and TS^KI4 cells expressing control shRNA (C) or TS^KI4 cells expressing HDAC6 shRNA (TS^KI4H6sh) are representative of two independent experiments.

(D) Absence of cell surface O-GalNAc glycosylation in TS^KI4 and TS^WTGsh cells with reduced GALNT3. VVL immunofluorescence staining in non-permeabilized TS^WT and TS^KI4 cells infected with a control virus, TS^WT cells infected with Galnt3 shRNA (TS^WTGsh), and TS^KI4 cells re-expressing GALNT3 (TS^KI4Gab) are shown. Images are representative of three independent experiments.

(E) O-GalNAc glycosylation of the trophoectoderm (TE) of intact wild-type E3.5 hatched blastocyst is shown by confocal microscopy using biotinylated VVL. Image is representative of seven blastocysts. DAPI (blue) and VVL (green).

(F) Reduced GALNT3 expression in TS^KI4 cells induces retention of O-GalNAc glycosylated proteins in the Golgi. DAPI (blue), VVL (red), and Giantin (Golgi) (green) staining of Triton-permeabilized cells. Arrowheads show co-localization between VVL and Giantin in the Golgi. Images are representative of three independent experiments.

(G and H) O-GalNAc glycosylation of E-cadherin is reduced in cells lacking GALNT3. IP with VVL agarose and blotting with anti-E-cadherin antibody are shown for TS cells (G) and HMECs (H). Western blots are representative of three independent experiments.

(I) Knockdown of HDAC6 increases E-cadherin O-GalNAc glycosylation in TS^KI4H6sh cells. Experiments were performed as in (G and H). Western blots are representative of two independent experiments.

IP, immunoprecipitation; MW, molecular weight in kDa; white bar represents 50 μm.

See also Figures S6 and S7.