Journal: Molecular Biology of the Cell
Article Title: Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells
Figure Lengend Snippet: Effect of C -mannosylation on Rspo1 function. (A) Effect of C -mannosylation on Rspo1 secretion using CHO-K1 and Lec15.2 cells. CHO-K1 and Lec15.2 cells were transiently transfected with pCI-neo-Rspo1/N137Q-MH vector for 6 h and then cultured in serum-free medium with 50 μg/ml soluble heparin for 18 h. The protein samples were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (CHO-K1) was defined as 1.0. (B, C) Establishment of mutant form of Rspo1 (W 153 and W 156 replaced by alanine residues; W153A/W156A: 2WA)–overexpressing HT1080 cell line, HT1080-Rspo1/2WA-MH. HT1080-neo (neo), HT1080-Rspo1-MH (wt), and HT1080-Rspo1/2WA-MH (2WA) cells were lysed, and aliquots of the cell lysates were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin (B). Total RNA was isolated from each cell line, and semiquantitative (left) and quantitative (right) RT-PCR was performed (C). Equal amounts of exogenous Rspo1 in these cells were confirmed. ns, not significant. (D, E) Effect of C -mannosylation on intracellular trafficking. Cells were cultured with 50 μg/ml soluble heparin, stained with Hoechst 33258 (blue), anti–c-myc (green), and anti-KDEL (red; D) or anti-GRASP65 (red; E), and examined by fluorescence microscopy. Areas of overlapping stains are represented in yellow in the superimposed images. Bars, 10 μm. (F) Effect of C -mannosylation on Rspo1 secretion using a mutant Rspo1-overexpressing cell line. Cells were cultured with 50 μg/ml soluble heparin, and cell lysates and conditioned media were electrophoresed and immunoblotted with anti–c-myc and anti–α-tubulin. Signal intensities of Rspo1 were quantified and normalized to α-tubulin expression using ImageJ software. The Rspo1/α-tubulin ratio (wt) was defined as 1.0. (G) Effect of C -mannosylation on the kinetics of Rspo1 secretion. Cells were cultured with 50 μg/ml soluble heparin, and conditioned media were collected at the indicated times, electrophoresed, and immunoblotted with anti–c-myc (top). Protein bands were quantified by using ImageJ software (bottom). The secreted amount of wild-type Rspo1 at 24 h was defined as 100%. (H) Effect of C -mannosylation on Rspo1-mediated enhancement of Wnt signaling. Recombinant Rspo1 and Rspo1/2WA were purified from each cell line, and the amounts of proteins were equalized by Western blot (inset). 293T cells were transfected with TOPFlash or FOPFlash in the presence of 10% Wnt3a-conditioned medium and treated with equal amounts of purified Rspo1. After 24 h, luciferase activities were measured and normalized to Renilla luciferase. Non– C -mannosylated Rspo1 slightly enhanced Wnt signaling activity. Data shown are means ± SD. * p
Article Snippet: After 24 h, the cells were transiently transfected with 400 ng of canonical Wnt signaling reporter Super 8×TopFlash (Addgene plasmid 12456) or mutant reporter Super 8×FopFlash (Addgene plasmid 12457; ) and 20 ng of phRL-TK vector (Promega) in the presence of 10% (vol/vol) Wnt3a-conditioned medium ( ) from L-Wnt3a cells (ATCC CRL-2647) as described ( ).
Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Software, Mutagenesis, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining, Fluorescence, Microscopy, Recombinant, Purification, Western Blot, Luciferase, Activity Assay