Additional file 1 of 5mC and H3K9me3 of TRAF3IP2 promoter region accelerates the progression of translocation renal cell carcinoma
Creators: name: Yang, Lei
name: Chen, Yi
name: Liu, Ning
name: Lu, Yanwen
name: Li, Xin
name: Ma, Wenliang
name: Gan, Weidong
name: Li, Dongmei
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Developmental Biology
subject: Cancer
subject: Virology

description: Additionalfile 1: Figure S1. (A) The protein level of TRAF3IP2 after up-/down-regulated TRAF3IP2. (B) The effects of TRAF3IP2 knockdown on the proliferation of UOK109 and 786-O cells respectively were examined by CCK-8 assay. The data are presented as the mean ± SD, **P&lt;0.01, ***P&lt; 0.001. Figure S2. NOTCH1 pathway induces development of NONO-TFE3 tRCC. (A-E) The effects of NICD1 overexpression or NOTCH1 knockdown on the proliferation of  UOK109 and 786-O cells respectively were examined by CCK-8 assay (A-B), colony formation assays (C-D) and tumor sphere formation (E). (F) EdU assays were used to detect the proliferationrate of  UOK109 and 786-O cells after transfection for 48h. (G) Cell cycle was analyzed using flow cytometry after transfection for 48h. H Cell apoptosis was analyzed via flow cytometry using an Annexin V/PI kit after transfection for 48h. (I-K) Migration and invasion assays were performed with transfected cells using Transwell inserts. The data are presented as the mean ± SD, *P&lt; 0.05, **P&lt; 0.01, ***P&lt;0.001. FigureS3. Level of TRAF3IP2 mRNA detected by qRT-PCRafter MS2-RIP for GFP in UOK109 cells. AS1, NC and AS1-antisense correspond to TRAF3IP2-AS1, empty vector and TRAF3IP2-AS1-antisense. The data are presented as the mean ± SD. Figure S4. TRAF3IP2-AS1 down-regulates TRAF3IP2 by recruiting HNRNPK to TRAF3IP2 promoter. (A) UOK109 cells were lysed, then CHIRP were performed with TRAF3IP2-AS1 probe or LacZ probe. (B) The mRNA levels were detected by qRT-PCR after transfected with shRNA targeted the potential TRAF3IP2-AS1 binding proteins. (C) UOK109 cells were lysed, then ChIP were performed with anti-HNRNPK antibody. (D-E) UOK109 cells were transfected with indicated lentivirus and siRNAs, then the promoter region of TRAF3IP2 and TRAF3IP2-AS1 were enriched by TRAF3IP2-AS1 probe. (F) UOK109 cells were co-transfected with TRAF3IP2-AS1/ TRAF3IP2-AS1Mut and MS2-GFP, then MS2-RIP assay was performed. (G) The mRNA level of TRAF3IP2 were detected in UOK109 cellstransfected TRAF3IP2-AS1/TRAF3IP2-AS1Mut. The data are presented as the mean ± SD, **P&lt; 0.01, ***P&lt; 0.001. FigureS5. HNRNPK inhibits development of NONO-TFE3 tRCC by down-regulating the expression of TRAF3IP2. (A-E) The effects of HNRNPK on the proliferation of UOK109 and 786-O cells respectively were examined by CCK-8 assay (A-B), colony formation assays (C-D) and tumor sphere formation (E). (F) EdU assays were used to detect the proliferation rate of UOK109 and 786-O cells after transfection for 48h. (G) Cellcycle was analyzed using flow cytometry after transfection for 48h. (H) Cellapoptosis was analyzed via flow cytometry using an Annexin V/PI kit after transfection for 48h. (I-K) Migration and invasion assays were performed with transfected cells using Transwell inserts. The data are presented as the mean ± SD, **P&lt; 0.01, ***P&lt; 0.001. Figure S6. DNMT1 inhibits development of NONO-TFE3 tRCC by down-regulating the expression of TRAF3IP2. (A-E) The effects of DNMT1 on the proliferation of UOK109 cells respectively were examined by CCK-8 assay (A), colony formation assays (B-C) and tumor sphere formation (D-E). (F-G) EdU assays were used to detect the proliferation rate of UOK109 cells after transfection for 48h. (H-I) Cell cycle was analyzed using flow cytometry after transfection for 48h. (J-K) Cell apoptosis was analyzed via flow cytometry using an Annexin V/PI kit after transfection for 48h. (L-M) Migration and invasion assays were performed with transfected cells using Transwell inserts.The data are presented as the mean ± SD, *P&lt; 0.05, **P&lt; 0.01, ***P&lt;0.001. TableS1. Primers used for real-time PCR. Table S2. Primers used for ChIP/CHIRP/MS2-RIP assay. Table S3. Primary antibodies used in this study. Table S4. Primers used for MSP analysis. Table S5. Small-molecule inhibitors used in this study. Table S6. SiRNA, shRNA and ASOs used for silencing target genes. Table S7. Guide RNA used for CRISPR/dCas9 system. Table S8. Primers used for MSP analysis.