Supplementary Figures S1-S21 from PIN1 Prolyl Isomerase Promotes Initiation and Progression of Bladder Cancer through the SREBP2-Mediated Cholesterol Biosynthesis Pathway

Supplementary Figure S1. Luminal urothelial cells show higher PIN1 expression levels. Supplementary Figure S2. PIN1 overexpression in normal immortalized SV-HUC-1 and RT4 huBLCA cells promotes cell proliferation, cell motility, invasion, migration and urothelium clearance. Supplementary Figure S3. PIN1 positively regulates T24 and 5637 cell proliferation and spheroid formation in vitro. Supplementary Figure S4. PIN1 positively regulates T24 and 5637 cell invasion, migration, cell motility and urothelium clearance in vitro. Supplementary Figure S5. Tumor masses derived from PIN1-KO T24, and 5637 cells show declining urothelial cell layers, and PIN1 promotes T24 cell tumor growth in vivo. Supplementary Figure S6. PIN1 promotes 5637 cell orthotopic tumor growth in vivo. Supplementary Figure S7. Treatment with the PIN1 specific inhibitor, sulfopin, lowers T24 cell proliferation and negatively regulates 5637 and T24 cell spheroid formation, cell motility, cell invasion and migration in vitro. Supplementary Figure S8. Sulfopin administration reduces urothelial cell layers in tumor masses derived from T24 and 5637 control cells and suppresses T24 cell tumor growth in vivo. Supplementary Figure S9. MB49 cells instilled through the urethra metastasize to distant organs, lung, and kidney. Supplementary Figure S10. Reducing Pin1 expression in organoids derived from primary mouse urothelial cells shows lower cell proliferation. Supplementary Figure S11. PIN1 positively affects cell cholesterol levels and regulates cholesterol metabolism–related gene signatures. Supplementary Figure S12. SREBP2, a crucial transcriptional factor, associates with PIN1 and promotes huBLCA cell proliferation. Supplementary Figure S13. Ablation of Pin1 in mouse MB49 bladder cancer cells reduces the cholesterol level and expression levels of SREBP2 and its downstream targets, HMGCS1 and MVD. Supplementary Figure S14. PIN1 enhances the DNA-binding activity of the SREBP2 transcription factor. Supplementary Figure S15. TNFα promotes JNK phosphorylation and activation, and the interaction between PIN1 and SREBP2. Supplementary Figure S16. JNK-mediated phosphorylation of Ser455 in SREBP2 is required for the interaction between PIN1 and SREBP2 in T24 cells. Supplementary Figure S17. PIN1 inhibition in SREBP2 S455A mutant re-expressing huBLCA cells slightly reduced the SREBP2 expression level compared to that of SREBP2 S455A mutant re-expressing cells. Supplementary Figure S18. The sulfopin PIN1 inhibitor treatment in SREBP2 S455A mutant re-expressing cells slightly reduced the cholesterol biosynthesis, bladder cancer cell proliferation and migration in vitro. Supplementary Figure S19. PIN1 knockout in SREBP2 S455A mutant re-expressing cells slightly reduced the cholesterol biosynthesis, bladder cancer cell proliferation and migration in vitro. Supplementary Figure S20. PIN1 knockout in SREBP2 S455A mutant re-expressing cells slightly reduced the 5637-cell tumor growth in vivo. Supplementary Fig. S21. The cholesterol biosynthesis pathway is affected by the combination therapy of sulfopin plus simvastatin.