Deparaffinized and rehydrated liver tissue sections were immersed in citrate buffer for antigen retrieval and then incubated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Next, sections were blocked with 10% goat serum albumin and incubated with a primary antibody targeting alpha-smooth muscle actin (α-SMA, 1:100; Abways, Shanghai, China), interleukin-1 beta (IL-1β, 1:100; ABclonal, Wuhan, China), YAP (1:100; Santa Cruz Biotechnology, Inc., California, USA), P-YAP (1:100; Abways, Shanghai, China), glutathione peroxidase 4（GPX4, 1:100; Abways, Shanghai, China), solute carrier family 7 members 11 (SLC7A11, 1:250; Proteintech, Wuhan, China), receptor-interacting serine-threonine kinase 3 (RIPK3, 1:100; Signalway antibody, College Park, USA), or caspase8 (1:200; Abcam, Cambridge, UK) at 4 °C overnight. After washing with phosphate-buffered saline (PBS) three times, sections were incubated with the corresponding horse radish peroxidase (HRP)-conjugated secondary antibody at room temperature for 30 min. After washing, sections were visualized with 3,3′-diaminobenzidine (DAB) and stained with hematoxylin. Images were captured and integral optical density (IOD) was determined using Image-Pro Plus 6.0 software. A total of ten non-overlapping fields were analyzed from each specimen.
Slc7a11
Manufactured by Proteintech
Sourced in China, United States
SLC7A11 is a transporter protein that is responsible for the uptake of cystine, a precursor of the antioxidant glutathione, into cells. It plays a critical role in maintaining cellular redox balance and protecting cells from oxidative stress.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Proteintech (12 mentions)
β actin, by Proteintech (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bcl 2, by Abcam (3 mentions)
Erastin, by Selleck Chemicals (3 mentions)
Acsl4, by Abcam (3 mentions)
Cleaved caspase 3, by Abcam (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Bcl2 associated x (bax), by Proteintech (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Flag tag (flag), by Proteintech (2 mentions)
Caspase 3, by Abcam (2 mentions)
E cadherin, by Proteintech (2 mentions)
Vimentin, by Proteintech (2 mentions)
β actin, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
34 protocols using slc7a11
1
Immunohistochemical Analysis of Liver Tissue
2
Comprehensive Western Blot Analysis
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Western blot was performed according to a standard protocol (Li et al., 2018 (link)). The primary antibodies used were the following: Cleaved-caspase3 (Abcam, Cambridge, MA, United States), Bcl-2 (Abcam, Cambridge, MA, United States), Bax (Abcam, Cambridge, MA, United States), RIPK1 (Proteintech, Wuhan, China), MLKL (Proteintech, Wuhan, China), SLC7A11 (Proteintech, Wuhan, China), GPX4 (Proteintech, Wuhan, China), LC3 (Proteintech, Wuhan, China), p62 (Proteintech, Wuhan, China), tubulin (Proteintech, Wuhan, China) and GAPDH (Proteintech, Wuhan, China) used a s the loading control.
3
Evaluating Antioxidant Defense Mechanisms
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IMCA was purchased from Tao Su Biochemical Technology Co. Ltd. (AE-848/32005043, Shanghai, China). GPX4, glutathione synthetase (GSS), and SLC7A11 antibodies were purchased from Proteintech Co. Ltd. (Wuhan, China). PCR primers were designed and synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The SYBR green PCR Master Mix was purchased from Thermo Scientific (Waltham, MA, USA). MTT cell proliferation and cytotoxicity detection kit, ROS detection kit, GSH detection kit, GSSG detection kit, and Cys detection kit were purchased from Solarbio Co. Ltd. (Beijing, China).
4
Investigating AhR and SLC7A11 in Cancer Cells
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We used PC9, SPC-A-1, and A549 cell lines and followed previously described maintenance methods 28 (link), 29 (link). The primary antibodies were β-actin (Cat# A5441; Sigma-Aldrich, St. Louis, MO, USA; 1:10000), AhR (Cat# sc-133088; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000), and SLC7A11 (Cat# 26864-1-AP; Proteintech, Wuhan, China; 1:1000).
We used previously published shRNA sequences 17 (link). Plasmid transfection was performed with Lipofectamine® 3000 following the manufacturer's instructions; colonies with stable expression were screened using puromycin (1.5 μg/mL). Vigene Biosciences designed and established the AhR overexpression plasmid (Vigene Biosciences, Shandong, China).
AhR agonist V, VAF347 was purchased from Calbiochem (Cat# 182690; Calbiochem/EMD Chemicals, San Diego, CA, USA), and Erastin (Cat# S7242) and Ferrostatin-1 (Cat# S7243) were purchased from Selleck Chemicals (Houston, TX, USA).
We used previously published shRNA sequences 17 (link). Plasmid transfection was performed with Lipofectamine® 3000 following the manufacturer's instructions; colonies with stable expression were screened using puromycin (1.5 μg/mL). Vigene Biosciences designed and established the AhR overexpression plasmid (Vigene Biosciences, Shandong, China).
AhR agonist V, VAF347 was purchased from Calbiochem (Cat# 182690; Calbiochem/EMD Chemicals, San Diego, CA, USA), and Erastin (Cat# S7242) and Ferrostatin-1 (Cat# S7243) were purchased from Selleck Chemicals (Houston, TX, USA).
5
Western Blot Analysis of Protein Expressions
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Western blot analysis was performed as previously described [51 (link)]. Specifically, NHA cells were lysed using ice-cold lysis buffer (Beyotime, Shanghai, China). Concentration was measured with BCA Protein Assay kit (New Cell and Molecular Biotech, Suzhou, China). After target proteins were loaded onto 10% SDS polyacrylamide gel, they were transferred from gel to PVDF membranes. Subsequently, membranes were blocked with 5% skim milk for 2 h in room temperature and then incubated overnight with GAPDH (Proteintech, Wuhan, China), HSPB1 (Proteintech, Wuhan, China), FTH1 (Immunoway, Suzhou, China), CD44 (Proteintech, Wuhan, China), SAT1 (Proteintech, Wuhan, China), ZFP36 (Proteintech, Wuhan, China), GPX4 (Proteintech, Wuhan, China), SLC7A11 (Proteintech, Wuhan, China), Caspase3 (Proteintech, Wuhan, China), Cleaved-Caspase3 (Proteintech, Wuhan, China), Bcl-2 (Abcam, Cambridge, MA, USA), Bax (Proteintech, Wuhan, China), and Cyt-c (Proteintech, Wuhan, China). Afterwards, membranes were incubated with HRP-linked secondary antibody (Abcam, Cambridge, MA, USA) for 1 h. Bands were analyzed with chemiluminescence Western blotting detection system (Tanon, Shanghai, China) [52 (link)].
6
Protein Expression Analysis in Cells
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The cells were lysed in RIPA buffer containing a protease inhibitor cocktail for 15 min on ice, followed by centrifugation at 12,000 g. The total protein concentration was determined using the BCA protein assay kit (Beyotime, China). A total of 20 micrograms of protein was loaded onto a SDS-PAGE gel and separated. The separated proteins were then transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% BSA, then incubated with primary antibodies. TFR1(#ab84036) and HIF-1α(#ab179438) were purchased from Abcam. BNIP3(#3769S), IL-6 (#12,912), IL-1β(#12,242), iNOS (#sc-7271), COX2 (#4842), p65 (#8242), Phospho-p65 (#3033), SOX9 (#A00177-2) were purchased form CST. MMP3 (#17873-1-AP), COL2 (#28459-1-AP), MMP13 (#18,165- 1-AP), SLC7A11 (#26864-1-AP), GPX4 (#67763-1-Ig), cGAS(#A8335), STING(#19851-1-AP), DRP1(#12957-1-AP), MFF(#17090-1-AP), GAPDH (#10494-1-AP) were purchased from Proteintech. COL10 (#BA 2023, Boster) was purchased from Boster. Following an overnight incubation at 4 °C, the membranes were washed three times with Tris-buffered saline with Tween (TBST) and then incubated with the appropriate anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature. The signal intensity on the membranes was visualized using a Bio-Rad scanner (Bio-Rad, Hercules, CA).
7
Western Blot Analysis of Protein Expression
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Frozen heart tissues were lysed mechanically in cold RIPA buffer. Proteins were extracted and quantified using a BCA protein assay kit (Best Bio, Shanghai, China). The proteins were electrophoretically separated using SDS-PAGE and transferred to a polyvinylidene difluoride membranes (Millipore, United States). The membranes were blocked with 3% BSA for 1.5 h at room temperature, and antigens were detected using the following antibodies at 4°C overnight, CSE (1:1,000, Proteintech, United States), CBS (1:1,000, Proteintech, United States), 3-MST (1:5,000,SantaCruz, United States), P53 (1:2000, Proteintech, United States), P21 (1:2000, Proteintech, United States), NRF2 (1:2000, Proteintech, United States), SLC7A11 (1:2000, Proteintech, United States), ACSL4 (1:2000, Abcam, United States), GPX4 (1:2000, Proteintech, United States), FPN1 (1:1,000, Proteintech, United States), FTH (1:1,000, Immunoway, United States), TRF1 (1:1,000, Abcam, United States), and GAPDH (1:5,000, Proteintech, United States). The blots were incubated with a horseradish peroxidase-conjugated anti-rabbit (Proteintech, United States) or anti-mouse (Proteintech, United States) secondary antibody at room temperature for 1.5 h. All antibodies were diluted with TBST. Blot bands were visualized by enhanced chemiluminescence and detected by ImageJ software (1.52, NIH, United States).
8
Western Blot Analysis of Ferroptosis Markers
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Cells or tissue lysates were added to a lysis buffer supplemented with protease and phosphatase inhibitors on ice for 30 min. Then, cell lysates were centrifuged at a speed of 12,000 rpm for 15 min at 4 °C and quantified using the Bicinchoninic Acid (BCA) method (Beyotime, Shanghai, China). After that, samples were diluted in a loading buffer separated on SDS-PAGE gels and then electro-blotted to polyvinylidene difluoride (PVDF, Roche, IN, USA) membranes and blocked with 5% milk in 0.1% Tween-TBS for 1.5 h at room temperature and then incubated with the primary antibodies overnight at 4 °C. The following antibodies were used: SLC7A11 (1:1000, Proteintech, Wuhan, China), GPX4 (1:1000, Proteintech, Wuhan, China), GAPDH (1:1000, Proteintech, Wuhan, China). PVDF membranes were washed three times using TBST and then immersed in the secondary antibody (1:10,000, Wuhan Boster, China) and incubated for 2 h at room temperature. The results were detected with an ECL system (Amersham Biosciences, Slough, UK) using an image reader. GAPDH was served as an internal control.
9
Oridonin and Ferrostatin-1 Protocols
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Oridonin (HPLC ≥98%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (#B20310), then the drug was dissolved in DMSO and stored in − 20 °C for usage. Ferrostatin-1 (Fer-1) was purchased from MedChemExpress (#HY-100579, MCE), and stored in − 20 °C for usage. All primary antibody used in the experiments included Actin (1:10000, #60008–1-Ig, Proteintech), Bcl-2 (1:1000, #61193, Proteintech), BAX (1:5000, #50599–2-Ig, Proteintech), cl-caspase3 (1:10000, #19677–1-AP, Proteintech), Tublin (1:1000, #66031–1-Ig, Proteintech), GAPDH (1:2000, #CL594–60004, Proteintech), GPX4 (1:5000, #67763–1-IG, Proteintech), SLC7A11 (1:1000, #26864–1-AP, Proteintech), FTH1 (1:5000, #11682–1-AP, Proteintech), and ACSL4 (1:10000, #81196–1-RR, Proteintech), they were stored in − 20 °C for usage. Ferrostatin-1 (Fer-1) (#HY-100579, MCE), an effective ferroptosis inhibitor [32 (link)], was dissolved in dimethylsulfoxide (DMSO) to create stock solutions and stored at − 20 °C.
10
Western Blot Analysis of SLC7A11, USP20 Proteins
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Western blot analysis was performed as we previously described.
76 (link) The primary antibodies used for western blot analysis are listed as follows: SLC7A11 (Proteintech; 26864‐1‐AP; HUABIO, HA600098), USP20 (Proteintech; 17491‐1‐AP), GAPDH (Proteintech; 60004‐1‐Ig), Flag (Proteintech; 66008‐4‐Ig), Myc (Proteintech; 60003‐2‐Ig), HA (Proteintech; 51064‐2‐AP) antibodies. ECL (Meilun) and ChemiDocMP imager (Bio‐Rad) were used to detect and visualize the signals of bands.
76 (link) The primary antibodies used for western blot analysis are listed as follows: SLC7A11 (Proteintech; 26864‐1‐AP; HUABIO, HA600098), USP20 (Proteintech; 17491‐1‐AP), GAPDH (Proteintech; 60004‐1‐Ig), Flag (Proteintech; 66008‐4‐Ig), Myc (Proteintech; 60003‐2‐Ig), HA (Proteintech; 51064‐2‐AP) antibodies. ECL (Meilun) and ChemiDocMP imager (Bio‐Rad) were used to detect and visualize the signals of bands.
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