Hmox1

CCK-8 was obtained from Dojindo (Kumamoto, Japan). Antioxidants including NAC, PDTC, and AGI-1067, as well as puromycin, neomycin, polybrene, and Matrigel were provided by Sigma-Aldrich Chemicals (St. Louis, MO, USA). Cell culture reagents, including fetal bovine serum (FBS), DMEM, and antibiotics, were obtained from Hyclone (Logan, UT, USA). Antibodies of MAFG, HMOX1, and tubulin were provided by Cell Signaling Technology (Beverly, MA, USA).

Cells were washed in cold PBS and lysed with 2× SDS reducing buffer with protease inhibitors to make whole cell lysates. The protein concentration was measured by a Micro BCA Protein Assay kit (Thermo Scientific). Lysates were then subjected to SDS‐PAGE; and immunoblotting was performed as described before.
²² (link) Densitometric analysis was done using ImageJ software. Antibodies against EZH2 (Cell signalling, Cat# 5246), H3K27Me3 (Cell Signalling, Cat# 9733), Cox2 (Abcam, Cat# ab15191), Hmox1 (Cell signalling, Cat# 5853) were used, and the signals were detected using an enhanced chemiluminescence system and imaged with an Amersham Biosciences 600 Imager (GE Healthcare).

MCF-10A cells were seeded in 6-well tissue culture plates at a density of 5 × 10⁵ cells per well. Cells were then treated with SWT or z-LIG for 24 hours. 293 (HEK) cells treated with 50 μM Arsenic for 8 hours (lysate provided by Cell Signaling) was used as a positive control. The protein concentration was determined using the BCA protein assay kit (Pierce Chemical Company) according to manufacturer’s protocol. Primary antibodies were prepared in either 5% BSA or milk and blots were incubated for 18 hours in 4°C. The following concentrations for primary antibodies were used: β-actin 1:500 (Millipore), Nrf2 1:1000 (Cell Signaling) and HMOX-1 1:200 (Cell Signaling). Blot was incubated with secondary antibody for 90 minutes at a 1:2000 or 1:3000 dilution prepared in 5% milk. Western blot Luminol Reagent (Santa Cruz) was used for chemiluminescence detection.

Expression of key biomarkers was assessed at T1, T5, and PT7 by immunoblotting. Cultures were lysed in a SIGMAFAST™-supplemented M-PER Mammalian Cell Lysis Buffer (Pierce, Waltham, MA, USA). Denatured proteins were separated on a 4–12% Nu-PAGE^® Novex^® Bis-Tris gradient gel (Life Technologies, Carlsbad, CA, USA) and transferred onto a nitrocellulose membrane (LI-COR, Lincoln, NE, USA) by following the manufacturer’s instructions. Primary antibodies used in this study included HMOX-1 (Cell Signaling Technology, Danvers, MA, USA), NQO1 (Santa Cruz Biotechnology, Dallas, TX, USA), AKR1B10 (Sigma-Aldrich), GCS (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), acetylated α-tubulin (Sigma-Aldrich), DNAI1 (Sigma-Aldrich), CDC20B (Thermo Fisher Scientific, Waltham, MA, USA), Forkhead-box J1 (FoxJ1) (Santa Cruz Biotechnology), and involucrin (Neomarkers, Fremont, CA, USA). IRDye-conjugated secondary antibodies were obtained from LI-COR (Lincoln, NE, USA). The density of the protein bands was quantified using a LI-COR Odyssey imaging system and Image Studio Software (version 5.0).

Protein expression was determined by western blot analysis. Equal amount of protein from each sample was run in Tris-glycine SDS-PAGE gel, followed by transfer to PVDF membrane. After blocking the membrane with 5% milk for 1 h at room temperature, the membrane was incubated further for 2 h with antibodies specific for target proteins: NRF2 (NBP1-32822, 1/1000 dilution), pNRF2 (S40) (NB100-80012, 1/1000 dilution) from Novus Biologicals (Littleton, CO); KEAP1 (4617, 1/500 dilution), HMOX1 (5061, 1/500 dilution), NQO1 (3187, 1/1000 dilution), HIF1Aα (3716, 1/1000 dilution), HSF1 (4356, 1/1000 dilution), and lamin B1 (12586, 1/1000 dilution) from Cell Signaling (Danvers, MA); GAPDH (sc-47724, 1/5000 dilution) from Santa Cruz (Dallas, TX); β-Actin (MA5-15739-HRP, 1/2000 dilution) from Thermo Fisher (Waltham, MA) and GCLC (ab190685, 1/1000 dilution), GCLM (ab124827, 1/1000 dilution), and TXN (ab26320, 1/1000 dilution) from Abcam (Cambridge, MA). The membrane was subsequently incubated with species-specific HRP-conjugated secondary antibody followed by incubation with chemiluminescence substrate and imaging. The band intensity of each of the target proteins was quantified using either ImageQuant or Image J software (GE Healthcare, Sweden).

Protein analysis and immunoblotting were performed as previously described [13 (link), 14 (link)]. The following primary antibodies were used: anti-β-ACTIN Sigma-Aldrich (Castle Hill, NSW, AUS), anti-NRF2 (Merck KGaA, Darmstadt, Germany), and antibodies against phospho-histone H2A.X, cleaved PARP and HMOX1 (Cell Signaling Technology, Inc., MA, USA).