Journal: Molecular Medicine Reports

Article Title: Reoxygenation induces reactive oxygen species production and ferroptosis in renal tubular epithelial cells by activating aryl hydrocarbon receptor

doi: 10.3892/mmr.2020.11679

Figure Lengend Snippet: AhR activation status does not affect Nrf2 activation or transcriptional activity. RPTECs were cultured under ctrl conditions or subjected to Reox with or without the AhR inhibitor CH223191. (A) Representative western blots of Nrf2 levels (corresponding to its activation status) and the expression of the Nrf2 transcriptional targets xCT (SLC7A11) and SOD-3. (B-D) Statistical analysis of the western blots. Neither Reox nor CH223191 affects Nrf2 activity, or the expression of xCT and SOD-3. Data are presented as the mean ± SEM of six independent experiments. AhR, arylhydrocarbon receptor; Nrf2, nuclear factor erythroid 2-related factor 2; xCT, cystine-glutamate antiporter; SOD-3, superoxide dismutase; RPTEC, renal proximal tubular epithelial cell; ctrl, control; Reox, reoxygenation.

Article Snippet: Primary antibodies were specific for AhR (1:200; cat. no. sc-133088; Santa Cruz Biotechnology, Inc.), cytochrome P450 family 1 subfamily A member 1 (CYP1A1; 1:500; cat. no. sc-25304; Santa Cruz Biotechnology, Inc.), Nrf2 (1:1,000; cat. no. TA343586; OriGene Technologies, Inc.), superoxide dismutase 3 (SOD-3; 1:100; cat. no. sc-271170; Santa Cruz Biotechnology, Inc.), cystine-glutamate antiporter (xCT, also known as SLC7A11; 1:1,000; cat. no. ANT-111; Alomone Labs), HIF-1α (1:500; cat. no. sc-10790; Santa Cruz Biotechnology, Inc.), LDH-A (1:1,000; cat. no. 2012; Cell Signaling Technology, Inc.), activated cleaved caspase-3 (CC3; 1:1,000; cat. no. ab13847; Abcam) and β-actin (1:2,500; cat. no. 4967; Cell Signaling Technology, Inc.).

Techniques: Activation Assay, Activity Assay, Cell Culture, Western Blot, Expressing, Control

Journal: MedComm

Article Title: ATR‐dependent ubiquitin‐specific protease 20 phosphorylation confers oxaliplatin and ferroptosis resistance

doi: 10.1002/mco2.463

Figure Lengend Snippet: USP20 de‐polyubiquitylates SLC7A11. (A) Huh‐7 cells transfected with the indicated shRNA were treated with MG132 for 6 h before collection. SLC7A11 was immunoprecipitated with anti‐SLC7A11 and immunoblotted with anti‐HA. (B) Immunoblotting was used to detect the ubiquitination of SLC7A11 in HEK293T cells cotransfected with Myc‐SLC7A11, HA‐ubiquitin and Flag‐USP20 (wild type or C154A). (C) USP20 removed the ubiquitin chain of SLC7A11 in a time‐ and dose‐dependent manner. (D) HA‐WT, K6, K11, K27, K29, K33, K48, or K63 Ub were cotransfected with Myc‐SLC7A11 and Flag‐USP20 into HEK293T cells. After treatment with 10 μM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of SLC7A11 detected by HA antibody. (E). Immunoblotting to detect the ubiquitination of the SLC7A11 deletion mutants (FL, ΔN, and ΔN) in HEK293T cells cotransfected with Myc‐SLC7A11 (FL, ΔN, and ΔN), HA‐Ub and USP20. (F) A schematic diagram of SLC7A11 and its mutants. (G) Immunoblotting to detect the ubiquitination of SLC7A11 mutants (K4, K12, K30, K37, K41, and K43) in HEK293T cells cotransfected with Myc‐SLC7A11 mutants, USP20 and HA‐Ub.

Article Snippet: Briefly, HA‐Ub, Myc‐SLC7A11, Flag‐USP20, or Flag‐USP20 C154A plasmid were transfected into HEK293T cells for 48 h and treated with 10 μM MG132 (MCE) for another 6 h. Cells were harvested at 4°C using RIPA extraction reagent (Meilun) after washed three times with prechilled PBS.

Techniques: Transfection, shRNA, Immunoprecipitation, Western Blot, Ubiquitin Proteomics