Normal rabbit igg

CHIP-seq data (GSE74812; BED files) of H3K79me2 and H3K79me3 in human t(4;11) cell line [44 (link)] was downloaded from GEO and promoter enrichment region was analyzed by using the IGV 2.6.3 software. Chromatin immunoprecipitation (ChIP) assay was performed in HCT116 and SW480 cells by using the EZ CHIP^TM kit as according to the manufacturer’s protocol. The sequences of c-Myc promoter were downloaded from the UCSC website [45 ]. Antibodies that targeted H3K79me1, H3K79me2, H3K79me3, and DOT1L were purchased from Abcam and normal rabbit IgG was obtained from Beyotime (Taicang, Jiangsu, China). Primers used were shown as below:
c-Myc~-2682~-1682-F: 5′-CCTCCAGTAACTCCTCTTTCTTCG-3′;
c-Myc~-2682~-1682-R: 5′-TCTTCTCATCCTTGGTCCCTCA-3′;
c-Myc~-1682~-682-F: 5′-GCAATGCGTTGCTGGGTT-3′;
c-Myc~-1682~-682-R: 5′-CGTTCAGAGCGTGGGATGTT-3′;
c-Myc~-682~+284-F: 5′-TGCCTCTATCATTCCTCCCTATC-3′;
c-Myc~-682~+284-R: 5′-TCGGGTGTTGTAAGTTCCAGTG-3′;
c-Myc~+284~+1284-F: 5′-TTCGGCTCACCGCATTTC-3′
c-Myc~+284~+1284-R: 5′-CAACACCACGTCCTAACACCTCT-3′;
c-Myc~+1284~+2284-F: 5′-AAGAAGAAAAGCTGGCAAAAGG-3′;
c-Myc~+184~+2284-R: 5′-CCAAAATCCAAGGCACAAAGT-3′.

ChIP assays were performed with HCT116 cells as described previously^{45 (link)}. Chromatin fractions from HCT116 cells were immunoprecipitated with specific antibodies as indicated in the text. Normal rabbit IgG (A7016, Beyotime) served as a control. The final ChIP DNAs were analyzed by quantitative real-time PCR with primers that encompassed the promoter region of genes of interest. The primer sequences are listed in Supplementary Table 3.

ChIP assays were performed with ISK cells as described previously [17 (link)]. Antibodies used for ChIP were: anti-H3K27me3 (Abcam Cat# ab6002), and anti-H3K27me2 (Sigma-Aldrich Cat# 17-10108). Normal rabbit IgG (Beyotime Cat# A7016) and mouse IgG (Beyotime Cat# A7028) served as controls; the final ChIP DNAs were then used as templates in qPCR reactions, using primers that encompass the promoter region of interest. See Supplementary Table S5 for ChIP-qPCR primer sequences.

ChIP assay was conducted using EZ ChIP kit (Millipore, Billerica, MA, USA) and NFAT1 antibody (ab2722, Abcam) or ER-α antibody (#8644, Cell Signaling Technology) according to the instruction of the manufacturer. The primers specific to HERV-E clone 4–1 5′ LTR were: 5′-CTCCCCAACCTCCCCTTTTC-3′ and 5′-TGAGAAACATGACTGGGGGC-3′. Normal rabbit IgG (A7016, Beyotime, Shanghai, China) was used to control the nonspecific immunoprecipitation.

Four micrograms normal rabbit IgG (Beyotime, China, A7016) or IP antibody, 40 μl suspended IP Matrix (Santa Cruz, USA, sc45039) and 500 μl PBS were incubated at 4 °C on a rotator for at least an hour. The mixture was then centrifuged and washed three times with PBS in the presence of a protease inhibitor mixture (protease inhibitor, phosphatase inhibitor, PMSF; KangChen, Shanghai, China) and then discard supernatant carefully. Cells that transfected for 48 h were lysed and transferred to the matrix, incubating at 4 °C on a rotator overnight. Next, the matrix was centrifuged and washed for five times. SDS-PAGE sample loading buffer (Beyotime, China, P0015) was added to the immunoprecipitates followed by boiling for 10 min at 100 °C. The IP and input proteins were detected by western blot.

Cells were cotransfected with HA-Ub and FLAG-FUS plasmids using Lipofectamine 3000 (Promega, Madison, WI, USA), according to the manufacturer’s protocol. After 24 h of transfection, 20 μM of MG132 (Sigma) was added to the medium for 6 h, followed by cell lysis for IP. Cell lysate was prepared by briefly sonicating ten million cells in 1 ml ice precooling IP LYSIS/WASH buffer (0.025 M Tris [pH 7.4], 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol) supplemented with 1 × complete protease inhibitors [Roche]). The ubiquitination level of FLAG-FUS protein was detected by IP. IP was performed using Pierce classic IP kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. The cell lysates were incubated with 5 μg anti-HA antibody (Beyotime) or normal rabbit IgG (Beyotime) overnight at 4 °C. The ubiquitinated proteins were retrieved, washed, and eluted in elution buffer and subjected to western blot using the anti-FLAG antibody (Beyotime) to detect ubiquitinated FLAG-FUS.

Transfection reagent (Lipofectamine 2000) was obtained from Invitrogen Corporation (Carlsbad, CA). In vitro angiogenesis assay kit (ECM625) was from Millipore (Temecula, CA, USA). Mouse anti-human HIF-1α monoclonal antibody was from BD Transduction Laboratories (San Diego, CA, USA). Mouse anti-human β-actin antibody was purchased from Beyotime Biotechnology Corporation, Shanghai (Shanghai, China). One Step SYBR PrimeScript RT-PCR (No.DRR086A) was purchased from TaKaRa Biotechnology (Dalian) Co., LTD (Dalian, China). Total and phosphorylated Akt (Ser⁴⁷³), P70S6K (Thr³⁸⁹ and Thr⁴²¹), P85S6K, mTOR (Ser²⁴⁸¹), JNK (Thr¹⁸³/Tyr¹⁸⁵), and c-Jun (Ser⁶³) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-VHL antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). G418 was from Sigma (St. Louis, MO, USA). MG132, Cycloheximide (CHX), LY294002, SP6002125, normal rabbit IgG, protein A garose beads, and Immunol Fluorence Staining Kit were from Beyotime Biotechnology Corporation, Shanghai (Shanghai, China). Human VEGF and IL-8 Enzyme-linked immunosorbent assay (ELISA) reagent kits were purchased from Wuhan Boster Bio-engineering limited company (Wuhan, China).