High expression of KRT6B in CD44+ bladder cancer stem cells (in vitro) Initially, the cell suspension was prepared by digesting the 10 bladder cancer samples. The CD44v6+EMA- bladder cancer stem cells were isolated by magnetic bead cell sorting. The results of transcriptome sequencing compared the expression profiles of the bladder cancer genes in the CD44v6+EMA- BCSCs, control subjects, and the KRT6B expression levels in BCSCs were significantly higher. To explore the expression of KRT6B in bladder cancer cell lines, the CD44+ cells were isolated by magnetic bead cell sorting from 5637 cell line. RT-PCR assay was used for detection of CD44 expression after magnetic sorting (P < 0.05; Figure 1). RT-PCR results showed that CD44+ cells expressed higher level of CSCs-related genes (Sox2, Oct4, and Nanog; P < 0.05; Figure 2). In the next step, colony formation assays indicated a higher proliferation and colony formation ability of CD44+ cells (P < 0.05; Figure 3A). Sphere formation assays showed that CD44+ BCSCs could form larger and more spheres than CD44- bladder cancer cells (P < 0.05; Figure 3B). It is demonstrated that CD44+ cells possessed enhanced self-renewal ability. Wound healing assays showed that the migration capacity of CD44+ cells was higher compared with the control group (P < 0.05; Figure 3C). A series of experiments showed that the CD44+ cells possessed the characteristic of BCSC populations, including self-renewal and spheroid formation capacity. Subsequently, the expression of KRT6B in CD44+ BCSCs and CD44- bladder cancer cells was analyzed. RT-PCR indicated that KRT6B was enriched in CD44+ cells of 5637 human bladder cancer cell line (P < 0.05; Figure 4). These results indicated that the KRT6B expression trend remained consistent between RNA sequencing and RT-PCR."

"

"

"

Silencing KRT6B in CD44+ BCSCs inhibited spheroid formation ability (in vitro) We explored the role of KRT6B in the CD44+ bladder cancer stem-like properties by silencing KRT6B in CD44+ BCSCs. KRT6B was knockdown by siRNA in CD44+ BCSCs and the downregulation of KRT6B was confirmed by RT-PCR (P < 0.05; Figure 5). Colony formation assays showed that KRT6B knockdown in CD44+ BCSCs inhibited proliferation and colony formation of CD44+ BCSCs (P < 0.05; Figure 6A). Sphere formation assay indicated that silence of KRT6B expression decreased the spheroid formation and self-renewal ability in CD44+ BCSCs (P < 0.05; Figure 6B). Wound healing assay displayed that ability of migration in KRT6B siRNA cells was decreased compared with the control group (P < 0.05; Figure 6C). This suggested that the downregulated KRT6B inhibited cell self-renewal and spheroid formation ability in CD44+ bladder cancer stem cells. To further explore the effect on a cellular level, we assessed the effect of KRT6B down-regulation in CD44- cells. Next, we aimed to initially explore the potential mechanism by which KRT6B maintains stemness in BCSCs. The RT-PCR analysis demonstrated that the silence of KRT6B in CD44+ cells increased the expression of NOTCH1 and HES1 in mRNA level (P < 0.05; Figure 7). Collectively, we speculated that KRT6B maintained the CD44+ BCSCs stemness which might be related to the Notch signal pathway."

"

"

Expression of KRT6B in immunohistochemistry and immunocytochemistry (in vitro) Immunohistochemistry was performed in 24 bladder cancer tissues to analyze the expression of CD44v6 and KRT6B. Immunohistochemical analysis showed that the cells with KRT6B expression located in the basal and intermediate layer of normal bladder uroepithelium. During uroepithelium carcinoma differentiation, immunohistochemical analysis indicated that basal cells expressed KRT6B and KRT6B+ cells distributed in nest-pattern in bladder cancer (Figure 8A). Immunocytochemistry analysis showed that KRT6B was mainly located in the cytoplasm and expressed in different subsets of the bladder cancer cells (Figure 9B). It is proposed that CD44v6+ cells located in the basal layer of bladder cancer. Immunohistochemical analysis confirmed co-expression of CD44v6 and KRT6B defined basal layer cells in bladder cancer and expanded from basal towards differentiated layer. Furthermore, it was observed that KRT6B expression was correlated with CD44v6 expression in bladder cancer specimens (n=24, P=0.006, Bilateral, Fisher’s exact test; Table 3). In summary, these results showed that the expression of KRT6B was correlated with CD44v6 and KRT6B+ cells, which might be related to CD44+ BCSCs."

"

"

Expression and prognosis of KRT6B in normal bladder tissue and bladder cancer (in vitro) To further explore the effect of KRT6B expression on the clinical outcome of bladder cancer patients, we used the Lee database from ONCOMINE aimed at mining bladder gene information. We analyzed the expression of KRT6B at the protein level by GraphPad Prism 7.0 statistical software. The result displayed that the expression of KRT6B in bladder cancer tissues was significantly increased compared with normal bladder tissues (P=0.022; Figure 10A). Subsequently, we evaluated the possible association between KRT6B expression and the prognostic role of KRT6B in human bladder cancer tissues. The result showed that bladder cancer patients with high KRT6B expression had a shorter overall survival rate than that of patients with low KRT6B expression (median survival=66.3 months vs. 98 months, n=164, P=0.044; Figure 10B). Therefore, these results indicate that the high expression of KRT6B is related to the poor prognosis of bladder cancer patients. "