Figure 1. 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 classify the region of measurement (lower panels). Scale bar: 30 µm. (B) Schematic overview of the screening process. Images from the RNAi screen in Chia et al. (2012) were quantified for HPL intensities. Non-targeting (NT) siRNA and Syntaxin 5 (STX5) siRNA were used as negative and positive controls, resepectively. (C) Fold-change of HPL intensities normalised to NT siRNA treatment (green dots) and STX5 (orange dots). Primary hits were selected based on a threshold of a nine-fold increase (red dashed line) and the final validated genes are labelled in red (Hit genes). (D) Images from the screen of HPL staining in HeLa cells depleted of ERK8. MannII-GFP labels the Golgi apparatus. Scale bar: 30 μm. (E) HPL staining in cells knockdown of ERK8 with a control siRNA or GalNAc-T1 and -T2 siRNA. Scale bar: 30 μm.

DOI: http://dx.doi.org/10.7554/eLife.01828.003

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) application (Chia et al., 2012) and MetaXpress transfluor HT (Molecular Devices). (C) Quantification of the Tn levels of ERK8 depletion with pooled and deconvoluted siRNAs (dERK8-1 to 4). (D) Hit validation using deconvoluted siRNA pools. HPL intensities were quantified in HeLa cells treated with each of the four individual duplex siRNAs from the pool for the 19 primary hits. A gene was validated if at least two unique siRNAs reproduced at least a 4.5-fold increase in HPL intensities. (E) Comparison of HPL and Vicia Villosa Lectin (VVL) staining intensities in ERK8 and MAP4K2 depletion. (F) Quantification of Tn levels upon co-knockdown (coKD) of each of the 12 validated Tn regulators with GalNAc-T1 and -T2. (G) SDS-PAGE analysis of ERK8 expression levels in single and co-knockdown with GalNAc-T1 and -T2. Values on graphs indicate mean ± SEM. **p<0.0001, *p<0.05 by two-tailed unpaired t test, relative to the non-targeting (NT) siRNA-treated cells. NS, not significant.

DOI: http://dx.doi.org/10.7554/eLife.01828.004