Crl1830

Hepa1-6 (Hepa) murine hepatoma (ATCC: CRL1830) and Hep3B human HCC (HB-8064) cell lines, authenticated by short-tandem repeat profiling, were purchased from ATCC. The two lines, Hepa1-6 and Hep3B, do not appear in the misidentified cell line list maintained by the International Cell Line Authentication Committee (ICLAC; https://iclac.org/databases/cross-contaminations/; version 12; accessed 1 June 2023). The establishment of stable lines of Hepa and Hep3B cells expressing SOCS1 (Hepa-SOCS1; Hep3B-SOCS1) or the control vector (Hepa-Vector; Hep3B-Vector) were previously described [12 (link),13 (link)]. Hepa cells were transduced with lentiviral vector pWPT carrying N-terminal HA-tagged SOCS1 gene or empty vector, and stable lines were established after sorting for the human CD8 marker in the vector [12 (link)]. Hep3B cells were transfected with pCDNA3.1 vector carrying N-terminal Myc tagged SOCS1 gene or control vector and stable lines were established by G418 selection [13 (link)]. Both cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were treated with the indicated concentrations of cisplatin (cis-diamino-dichloroplatinum; cis-Pt; Sigma-Aldrich (St. Louis, MO, USA), Cat# C2210000) or tert-butyl hydroperoxide (t-BHP; Sigma-Aldrich, Cat# 458139) to induce oxidative stress for the indicated periods of time.

Spinal cord neuron-neuroblastoma (NSC-34) (CED-CLU140, Biozol, Eching, Germany), mouse embryonic fibroblasts (MEFs) (American Type Culture Collection, Manassas, VA), and mouse hepatoma (liver cancer, Hepa 1–6) (CRL-1830, American Type Culture Collection) cell lines were cultured and proteins prepared as previously described (28 (link)). Briefly, the cells were lysed in lysis buffer (4% SDS, 10 mm Hepes, pH 8.0) during sonication for 15 min (level 5, Bioruptor; Diagenode, Seraing (Ougrée) - Belgium). Cell lysis was followed by reduction of disulfide bonds with 10 mm DTT for 30 min and subsequent alkylation with 55 mm IAA for 45 min. To remove the detergent, cold acetone (−20 °C) was added to 100 μg of proteins to a final concentration of 80% v/v, and proteins were precipitated for at least 2 h at −20 °C. The suspension was centrifuged for 15 min (4 °C, 16,000 × g) and the precipitate was washed with 80% acetone (−20 °C) prior to re-suspension in 50 μl of 6 m urea/2 m thiourea, 10 mm Hepes, pH 8.0. An initial digestion step (3 h) was carried out after the addition of 1 μg of LysC, followed by dilution with four volumes of 50 mm ammonium bicarbonate and the final digestion with 1 μg of trypsin overnight at room temperature. The resulting peptide mixtures were desalted on SDB-RPS StageTips (29 (link)) and subjected to single shot LC-MS/MS analysis.

Hepa 1–6 (CRL-1830), the murine HCC cell line, was obtained from American Type Culture Collection (ATCC, USA) and maintained in DMEM (Biowest) supplemented with 10% FBS (Biowest). C57BL/6 J mice (6 to 8 weeks of age) were purchased from the Chinese Academy of Science and housed at the Animal Maintenance Facility of the Shanghai Medical College, Fudan University. All animal experiments were performed in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Care of Experimental Animals Committee of Fudan University approved all animal protocols (Permit Number: SYXK 2009–0082).
Hepa 1–6 tumor bearing mice were established as following: mice were injected subcutaneously at the left flank with 3 × 10⁶ Hepa 1–6 cells in 200 μL RPMI 1640 (Biowest) or 200 μL RPMI 1640 alone as control. Three groups of tumor bearing mice and control mice were established (12 mice in each group). Two weeks after inoculation, the mice with visible tumor were sacrificed and spleens were collected for isolation of Tregs using mouse regulatory T cell isolation kit (Miltenyi). In brief, CD4⁺ T cells were enriched by negative selection and then went through positive selection for CD25⁺ T cells. The purity of Tregs was monitored via FACS.