| Autor: |
Wang, Bingwu, Gao, Jian, Zhao, Zhongjun, Zhong, Xuefei, Cui, Hao, Hou, Hui, Zhang, Yanping, Zheng, Junnian, Di, Jiehui, Liu, Yong |
| Rok vydání: |
2022 |
| DOI: |
10.6084/m9.figshare.21062091 |
| Popis: |
Additional file 1: Figure S1. S9 binds MDM2 in Renca cells. Addition of 100 μM S9 to Renca cells followed by western blot detection of thermal stability of MDM2. Figure S2. S9 antagonizes interaction between MDM2 and RPL11. (A) U2OS cells were treated with indicated concentration of S9 for 24 h. Co-IP was performed with homemade anti-MDM2 antibody (2A10) followed by immunoblotting with anti-MDM2, anti-p53 and anti-L11 antibodies. (B) U2OS cells were cotransfected with pcDNA3-Flag-MDM2 and pcDNA3-Myc-L11 followed by treatment of Nutlin-3 for 24 h. Co-IP was performed with anti-Flag antibody followed by immunoblotting with anti-Flag and anti-Myc antibodies. Figure S3. The anti-proliferative effect of S9 in different cancer cells. (A) MDM2 and RPL11 expression pattern were analyzed in different cancer cells using the DepMap Portal database. (B–D) SJSA-1 and U2OS cells (B), FHC and HCT116 cells (C), HT29 and HCT116 cells (D) were treated with various concentrations of S9 for 24 h. Cell Viability was measured by CCK-8 assay. The inhibition of cell proliferation was achieved by comparing the values of each group with the control. Error bars represent SDs of at least three independent measurements. Figure S4. S9 induces cell cycle arrest and apoptosis in HCT116 cells. (A) HCT116 cells were treated with described concentration of S9 for 24 h, stained with PI and analyzed by flow cytometry. The dataset is representative example of triplicate experiments. Column graph was mean ± SD of three independent experiments. (B) HCT116 cells were treated with described concentration of S9 for 24 h, PARP cleavage was detected by western blot analysis. Figure S5. S9 induced upregulation of MDM2 was p53 dependent. U2OS p53+/+ and p53−/− cells were treated with described concentration of S9 for 24 h, MDM2 and p53 protein levels were detected by western blot analysis. Figure S6. S9 has minimal effect on p53 transcript level. U2OS cells were treated with described concentration of S9 for 24 h, and the mRNA level of p53 was analyzed by RT-PCR. Fold expression changes are relative to the control and correspond to mean ± SD of three independent experiments. Figure S7. MDM2 is required for S9-induced cell cycle arrest in HCT116 cells. (A) HCT116 cells were transfected with MDM2 siRNA, 48 h after transfection, MDM2 protein levels was detected by western blot analysis. (B) HCT116 cells were transfected with MDM2 siRNA followed with described concentration of S9 treatment for 24 h, stained with PI and analyzed by flow cytometry. Figure S8. p53 is required for S9-induced apoptosis but not cell cycle arrest in HCT116 cells. HCT116 p53+/+ and p53−/− cells were treated with described concentration of S9 for 24 h, PARP and p53 protein levels were detected by western blot analysis (A); stained with PI and analyzed by flow cytometry (B). Figure S9. S9 downregulates Cdc25c independent of p53. U2OS p53+/+ and p53−/− cells were treated with described concentration of S9 for 24 h, MDM2 and Cdc25c protein levels were detected by western blot analysis. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
