Fig 2: ITPR3 knockdown suppresses the stemness properties of bladder cancer cells. a, b The cancer stemness of 5637 and 253 J cells transfected with shITPR3 or shCon was evaluated by a tumorsphere assay. The number and diameter were also quantified and plotted. The scale bar represents 100 µm. c A colony formation assay was conducted in 5637 and 253 J cells transfected with shITPR3 or shCon, and the colony number was counted and plotted. Quantification analysis was shown below. d CSC-related markers, such as CD44, OCT4, SOX2, P63 and KLF4, were examined in 5637 and 253 J cells transfected with ITPR3 shRNA or shCon. ß-actin was used as a loading control. **p < 0.05, ***p < 0.001

Fig 3: Cancer stemness markers in cells treated with chemotherapeutic agents in vivo, and downregulation of lncRNA-LET enhances CSC-like properties. (A) In vivo GEM chemotherapy simulates clinical regimen with multiple GEM treatment cycles (dashed boxes) and gap periods. Tumor sizes of T24 xenografts were measured for GEM treatment and vehicle control group (n = 6 per group). (B) Sphere formation assay of primary cells derived from T24 xenografts of control group (Veh) and GEM (n=3 per group). (C) Representative H&E and IHC data showing the expression levels of CSC markers (CK5 and CK14) in T24 xenografts of control and GEM groups (n = 6 per group). (D) Western blotting of CSC markers (ALDH1A1, CD44, KLF4, and HMGA2) in T24 xenografts of control and GEM groups. (E) The levels of lncRNA-LET in T24 and 5637 cells treated with GEM or vehicle. (F) Knockdown efficiency of lncRNA-LET in T24 cells and forced expression of lncRNA-LET in J82 cells. (G-H) ALDHhigh population (G) and CSC markers (H) were determined by flow cytometer and Western blotting in T24 with knockdown and J82 cells with overexpression of lncRNA-LET (n=3 per group). (I) Knockdown efficiency of lncRNA-LET in T24 cells infected with shRNA targeting lncRNA-LET virus (shLET), compared to control (shCTL). (J) Tumor volume of shCTL and shLET T24 cells with limiting dilution. Each dot represents an individual mouse (n=6 per group). Data are shown as mean ± SD and represent three independent experiments with similar results. * P < 0.05; ** P < 0.01, *** P < 0.001 (Student's unpaired two-tailed t-test). (K) CSC frequency was calculated using extreme limiting dilution analysis (ELDA). There was a significant difference in CSC frequency between two groups, with a P value of 0.025.

Fig 4: BMP4 was selected as a key downstream target of KLF4 via RNA sequencing. (A) Heatmap for the visualization of a clustering analysis of differentially expressed genes (DEGs). (B) Venn diagram of the overlap number of DEGs between comparisons. (C) DEGs, differentially expressed genes. (C) Volcano plots showing the upregulated (red) and downregulated (blue) DEGs for each comparison. BMP4 was clearly marked. (D) KEGG pathway network analysis of differentially expressed genes.

Fig 5: Effects of microRNA-92a (miR-92a) on KLF4 expression in vascular smooth muscle cells (VSMCs). A and B, The protein level of KLF4 were measured by Western blot analyses in HASMCs. Results of statistical analyses are shown below. C and D, Immunofluorescent staining for KLF4 (green) in Gba (C) or A7r5 (D) cells treated with 10 ng/ml PDGF-BB after transfected. Nuclei demonstrated by DAPI (blue). Scale bars: 100 µm (C) and 200 µm (D). nc, negative control. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01