ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration
- Institute of Molecular and Cell Biology, Singapore
- National University of Singapore, Singapore
Figures
Figure 1 with 1 supplement
RNAi screening reveals 12 negative regulators of Tn expression.
(A) Helix pomatia lectin (HPL) staining was analysed using the ‘Transfluor HT’ module of MetaXpress software (Molecular Devices). A mask was generated for both HPL and nuclei (Hoechst) staining to …
Figure 1—figure supplement 1
Helix Pomatia Lectin (HPL) stains reliably and specifically for Tn antigen.
(A) Comparison of the HPL intensities between the two screen replicates. (B) Comparison of HPL intensities generated from different analysis algorithms from the HCSU (high content screening unit) …
Figure 2 with 1 supplement
Tn regulators control Tn expression through GalNAc-T subcellular localisation.
(A) Peanut Agglutinin (PNA) lectin staining in ERK8-depleted HeLa cells. Scale bar: 30 μm. (B) PNA lectin staining quantification after depletion of the 12 Tn regulators, using COSMC knockout HeLa …
Figure 2—figure supplement 1
Tn regulators control GalNAc-T1 and -T2 localisation.
(A) Peanut Agglutinin (PNA) lectin staining quantification after depletion of the 12 Tn regulators with a single siRNA from the pool used in the screen and in COSMC knockout cells. (B) Pearson's …
Figure 3 with 1 supplement
ERK8 regulates ER-localised O-glycosylation initiation.
(A) Protein replacement by expression of siRNA-resistant wild-type ERK8 or the kinase inactive mutant in ERK8-depleted HeLa cells. Cells were stained for Helix pomatia lectin (HPL) and ERK8. Scale …
Figure 3—figure supplement 1
ERK8 regulates Tn expression through GalNAc-T relocation and not related to transcriptional or translational events.
(A) Tn staining with Helix Pomatia Lectin (HPL) and quantification of cells expressing mirRNA (Invitrogen) for ERK8. Scale bar: 10 μm. (B) SDS-PAGE analysis of ERK8 expression levels in cells …
Figure 4 with 1 supplement
ERK8 is dynamically localised at the Golgi.
(A) High magnification of HeLa MannII-GFP expressing cells stained for endogenous ERK8 following cytosol extraction. (B) Cytosol-depleted cells treated with platelet-derived growth factor (PDGF; 50 …
Figure 4—figure supplement 1
ERK8 is dynamically localised when SRC is increasingly activated at the Golgi.
(A) MannII-GFP- and Src-8A7F-mcherry-expressing cells were treated with imidazole for the indicated times, cytosol-depleted and stained for ERK8. Scale bar: 10 μm. (B) Pearson's correlation …
Figure 5 with 1 supplement
ERK8 regulates COPI-dependent GalNAc-T traffic.
(A) Co-knockdown of ERK8 with Arf1 or GTP exchange factor, GBF1, and staining with Helix pomatia lectin (HPL). NT, non-targeting. Scale bar: 30 μm. (B) Quantification of Tn levels upon ERK8 …
Figure 5—figure supplement 1
ERK8 regulated GalNAc-T traffic depends on the activity of COPI regulators.
(A) Tn expression levels, as determined by Helix Pomatia Lectin (HPL) staining in cells expressing dominant-negative mutant Arf1 (Q71L) compared to wild-type Arf1. GRASP55 labels the Golgi …
Figure 6 with 1 supplement
ERK8 regulates cell migration through ER O-glycosylation.
(A) Phase contrast images and (B) actin and tubulin staining of non-targeting (NT) siRNA-treated and ERK8-depleted cells. Scale bars: 100 μm in (A) and 10 μm in (B). (C) Migration assay using …
Figure 6—figure supplement 1
ERK8 inhibits cell motility by controlling Tn expression on cell-surface O-glycoproteins.
(A) Cell-surface staining using a specific Tn antibody (green) and Helix Pomatia Lectin (HPL) (red) on non-permeabilised ERK8-depleted cells. Scale bar: 10 μm. (B) Scratch wound assay of cellular …
Figure 7 with 1 supplement
ERK8 is downregulated in human breast and lung carcinoma.
(A) Quantification of ERK8 staining in human breast biopsies. Each point represents the staining of one tissue core normalised to the average staining of the normal tissue cores. (B) Quantification …
Figure 7—figure supplement 1
ERK8 levels are frequently reduced in human lung carcinoma.
(A) H&E staining of 40 breast biopsies on BRF404 slides purchased from US Biomax, Inc. Image is from Biomax website (http://www.biomax.us/). (B) ERK8 and (C) Vicia Villosa Lectin (VVL) were …
