Liproxstatin 1

Once primary breast tumors reached approximately 0.8 cm in diameter, AAV virus particles (5 × 10⁹ genome copies) suspended in 50 μl of sterile phosphate-buffered saline (PBS) were injected intratumorally. Tumors were injected with AAV on days 0, 3, and 8. For the liproxstatin-1 rescue experiment, animals were intraperitoneally injected with liproxstatin-1 (25 mg/kg; 17730, Cayman Chemicals) daily for 2 weeks. liproxstatin-1 injections started on the same day as AAV injections.
To determine the functional significance of Mgst3 and Prdx6, P²⁴⁵CC (1128) cells expressing shRNA targeting Mgst3, Prdx6, or empty vector were grown in nude animals. A total of 5 × 10⁵ cancer cells suspended in 30 μl of PBS were injected into the mammary fat pad. Once tumors reached approximately 6 to 7 mm in diameter, all tumors were injected intratumorally with RSL3 (100 mg/kg; 19288, Cayman Chemicals), on days 0, 6, and 12. Tumor growth was monitored with caliper measurements of the length and width of the tumor. To determine the functional significance of MGST3 and PRDX6, human cell line MDA-MB-231 expressing shRNA targeting MGST3, PRDX6, or empty vector were grown in nude animals. MDA-MB-231 (5 × 10⁵ or 3 × 10⁶) was suspended in 30 or 50 μl of PBS, respectively, and was injected into the mammary fat pad. Tumor growth was monitored with caliper measurements of the length and width of the tumor.

Hepa1 and Huh7 hepatocytes were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum (FBS), 100 units mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin. Primary hepatocytes were isolated from mice using type II collagenase (Worthington Biochem, Lakewood, NJ), as described.⁽²⁸ (link)
⁾ Primary hepatocytes were grown on William’s medium E (Sigma) supplemented with 2% FBS, 100 units mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin and transduced with β‐galactosidase (β‐gal) or NIK adenoviral vectors. After 12‐14 hours of growth, hepatocytes were treated with APAP for 2‐24 hours in the presence or absence of NAC, ferrostatin‐1 (Item No. 17729, CAS No. 347174‐05‐4; Cayman Chemicals), or liproxstatin‐1 (Item No. 17730 CAS No. 950455‐15‐9, Cayman Chemicals). Hepatocyte viability was measured using colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays (DOT Scientific Inc., Burton, MI).