Volume 71, Issue 1, Pages 8-12 (January 2017) Single-cell Sequencing Reveals Variants in ARID1A, GPRC5A and MLL2 Driving Self- renewal of Human Bladder Cancer Stem Cells  Zhao Yang, Chong Li, Zusen Fan, Hongjie Liu, Xiaolong Zhang, Zhiming Cai, Liqin Xu, Jian Luo, Yi Huang, Luyun He, Chunxiao Liu, Song Wu  European Urology  Volume 71, Issue 1, Pages 8-12 (January 2017) DOI: 10.1016/j.eururo.2016.06.025 Copyright © 2016 Terms and Conditions

Fig. 1 Single-cell sequencing and phylogenetic analysis of bladder cancer cells. (A) The neighbor joining phylogenetic tree is constructed by Euclidean distance using filtered polymorphic SNP/SNV sites during BCSCs (orange), BCNSCs (yellow), BESCs (blue) and BENSCs (green). (B) Key genes with somatic mutation. The somatic mutation spectra of single cells is present on the left. The heat map in central shows the distribution of mutations across all 31 single-cells. Unreported key genes are asterisked. (C) Hills representing mutated genes are randomly distributed on the plate, and the heights of hills suggest the relative frequency each somatic mutation in single cells. (D) The key genes selected for functional investigation. The horizontal axis contained genes ranked by alphabet. The vertical axis showed the somatic mutant frequency of genes. The circle size (diameter) indicated the observed number of cells with each gene mutations. European Urology 2017 71, 8-12DOI: (10.1016/j.eururo.2016.06.025) Copyright © 2016 Terms and Conditions

Fig. 2 ARID1A, GPRC5A and MLL2 mutations promote the self-renewal and tumor-initiating of bladder cancer non-stem cell. (A) Data points indicate average number of spheres formed by bladder cancer non-stem cells isolated from primary bladder cancer tissues with distinct mutations in serum-free conditions. Each of the 15 mutations in Fig. 1D was tested separately (first column), or in combination with MLL2 mutation (second column) or in combination with MLL2 and ARID1A alterations (third column). Other mutations were also tested in combination with MLL2, ARID1A, and GPRC5A alterations (fourth column). (B) BCNSCs Mut formed spheres efficiently in contrast to BCNSCs WT. Spheres were counted in six separate fields after 14 days and shown as means±SD. Scale bar=100μm. This assay was repeated four times. (C) The expression levels of stemness-related genes were elevated in BCNSCs Mut. The mRNA expression levels of GLI1, STAT3 and CD44 were increased in BCNSCs Mut. (D) BCNSCs Mut formed bigger tumors than that of BCNSCs WT, n=5. (E) The percentage of tumor-free immunodeficient mice four months after subcutaneous injection of different dilutions of BCNSCs WT or BCNSCs Mut. (n=6 grafted tumors per dilution). (F) Serial tumor formation assays of BCNSCs WT and BCNSCs Mut. Tumor volumes were measured and calculated at the indicated time points, n=3. Data are representative of three independent experiments and shown as means±SD. *P<0.05. European Urology 2017 71, 8-12DOI: (10.1016/j.eururo.2016.06.025) Copyright © 2016 Terms and Conditions