Figure 4: GALNT14 promotes self-renewal of BCCs by suppressing BMP signalling.

(a) The role of GALNT14 in overcoming inhibitory effects of BMPs on self-renewal. Top: The number of spheres formed by the indicated 231-LM2 cells in the presence of PBS or BMP4 (30 ng ml⁻¹). Bottom: Representative images of spheres. Images of single sphere are shown in the inset. shG14 indicates shGALNT14. Scale bar, 500 μm. n=6. (b) Similar experiment as in a except that cells were treated with DMSO or BMPR kinase inhibitor DMH1 (5 μM). Scale bar, 500 μm. n=6. (c) Similar experiment as in a,b except that control (shCntr) and GALNT14-silenced (shG14) CN34-LM1 cells were used. BMP4 (10 ng ml⁻¹). n=6 (d,e) The indicated 231-LM2 cells (d) and CN34-LM1 cells (e) were treated with PBS or BMP4 (30 ng ml⁻¹ for 231-LM2, 10 ng ml⁻¹ for CN34-LM1), followed by immunoblotting analyses with antibodies against phospho-SMAD1/5/8, total SMAD1, and SMAD5. (f) O-GalNAcylation of BMPRs. HEK 293T cells were transfected with BMPR1A (ALK3), ActR-IIB and GALNT14 expression vectors as indicated and subjected to pull-down with Vicia villosa lectin (VVA)-agarose, followed by the western blot analyses with the indicated antibodies. P values were calculated using two-tailed unpaired Student’s t-test. **P<0.001; ***P<0.0001. Data in a–c are mean±s.e.m. from three independent experiments and western blots in d–f are representatives of two to three independent experiments.