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: bioRxiv

Article Title: Environmental cystine drives glutamine anaplerosis and sensitizes cells to glutaminase inhibition

doi: 10.1101/126631

Figure Lengend Snippet: The cystine/glutamate antiporter xCT/ SLC7A11 is necessary and sufficient for cystine induced glutamine anaplerosis and CB-839 sensitivity. (A) System x c - is a plasma membrane antiporter composed of two polypeptides, xCT (encoded by SLC7A11 ) and 4F2hc/CD98 (encoded by SLC3A2 ), that exchanges intracellular glutamate for extracellular cystine. (B) A549 cells were infected with lentiviruses encoding a SLC7A11 targeting shRNA or a control shRNA targeting GFP as indicated. These cells were then infected with retroviruses expressing either shRNA resistant SLC7A11 cDNA or empty vector (E.V.) as indicated. Shown is an immunoblot analysis of these modified cell lines for xCT protein expression with vinculin as a loading control. (C) The four cell lines from (B) were cultured for 8h in adult bovine serum or adult bovine serum with 208 μM cystine. Each medium included [U- 13 C 5 ]glutamine added to ∼33% enrichment (n=3). M+5 fractional labeling of α-ketoglutarate and glutamine for each cell line in each condition was determined. Shown is the difference in m+5 fractional label of α-ketoglutarate (normalized to m+5 fractional enrichment of glutamine) between adult bovine serum with 208 μM cystine and adult bovine serum. We define this as the ‘cystine induced increase of glutamine contribution to αGK’. (D) Immunoblot analysis of A549 cells infected with lentiviruses encoding SLC7A11 targeting shRNAs or a control shRNA targeting GFP as indicated. (E) Proliferation rates of cell lines from (D) cultured in adult bovine serum with 208 μM cystine with vehicle (DMSO) or 1 µM CB-839 is shown (n=3). (F) Multiple cell lines (see source data for identity of cell lines) were cultured for 8h in RPMI or RPMI with 10μM cystine. Each medium included [U- 13 C 5 ]glutamine added to ∼33% enrichment (n=2-3). M+5 fractional labeling of a-ketoglutarate and glutamine for each cell line in each condition was determined. Shown is ‘cystine induced increase of glutamine contribution to αKG’ defined as the difference of m+5 fractional label of α-ketoglutarate (normalized to m+5 fractional enrichment of glutamine) between RPMI and RPMI with 10μM cystine for a given cell line. This term is plotted against SLC7A11 mRNA expression data obtained from the cancer cell line encyclopedia (CCLE) . (G) SLC7A11 mRNA expression data from the CCLE is shown for breast cancer cell lines reported to be CB-839 resistant (IC50 > 1 μM) or CB-839 sensitive (IC50 < 1 μM) . Difference in SLC7A11 expression between the two groups was tested by two-tailed unpaired t-test with the p value for significance shown. (H) Indicated cell lines were infected with lentiviruses encoding SLC7A11 cDNA or empty vector (E.V.). Shown is an immunoblot analysis of xCT protein expression for these modified cell lines with vinculin expression presented as a loading control. (I) Cystine induced increase of glutamine contribution to αKG was determined as in (F) for the cell lines described in (H). (J) Proliferation rates for MCF7 cells without (E.V.) or with SLC7A11 expression cultured in RPMI or RPMI with 10μM cystine in the presence of vehicle (DMSO) or1μM CB-839 as indicated. For all panels, values represent the mean and the error bars represent ± SEM.

Article Snippet: To rescue xCT /SLC7A11 expression, cDNA encoding human SLC7A11 was obtained from Origene.

Techniques: Clinical Proteomics, Membrane, Infection, shRNA, Control, Expressing, Plasmid Preparation, Western Blot, Modification, Cell Culture, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Environmental cystine drives glutamine anaplerosis and sensitizes cells to glutaminase inhibition

doi: 10.1101/126631

Figure Lengend Snippet: Overexpression of xCT /SLC7A11 causes cystine-induced CB-839 sensitivity for MDA-MB-468 and AU565 breast cancer cell lines. Proliferation rates for MDA-MB-468 and AU565 cell lines overexpressing SLC7A11 or empty vector (from ) cultured in RPMI or RPMI with 10μM cystine in the presence of vehicle (DMSO) or 1μM CB-839 as indicated. The values represent the mean and the error bars represent ± SEM.

Article Snippet: To rescue xCT /SLC7A11 expression, cDNA encoding human SLC7A11 was obtained from Origene.

Techniques: Over Expression, Plasmid Preparation, Cell Culture

Journal: Heliyon

Article Title: Identification and validation of a disulfidptosis-related genes prognostic signature in lung adenocarcinoma

doi: 10.1016/j.heliyon.2023.e23502

Figure Lengend Snippet: Evaluation of SLC7A11, SLC3A2, NCKAP1 and GYS1 expression and prognosis value in LUAD. (A–D) Immunohistochemical staining of NCKAP1, GYS1, SLC7A11, SLC3A2 in clinical LUAD samples between tumor and adjacent tissues (scale bars, 20 μm). (E) Immunoblot of NCKAP1 in lung cancer cells. CCK-8 assay results showed the NCKAP1 knockdown cell proliferation in H460 cell lines, quantification of cell death after NCKAP1 knockdown by flow cytometry. (F) Immunoblot analysis of GYS1 in lung cancer cells. CCK-8 assay about GYS1 knockdown cell proliferation in H460 cell lines, quantification of cell death after NCKAP1 knockdown by flow cytometry. (G) NCKAP1 inhibited cell growth, migration and invasion in H460. Cell colony formation; wound-healing assay: Si -NCKAP1-1 and Si -NCKAP1-2; transwell invasion showed a decrease of invaded knockdown cells. Scale bar = 20 μm. (H) GYS1 inhibited cell growth, migration and invasion in H460. Cell colony formation; wound-healing: Si -GYS1-1 and Si -GYS1-2; transwell invasion assay results showed a decrease of invaded knockdown cells. Scale bar = 20 μm. (I) Correlation between GYS1 expression level and infiltration level of CSF1, quantitative analysis of CSF1 using real time PCR in GYS1 high and low expression groups. (J) IHC staining of GYS1 high/low expression and CD163 infiltration in LUAD tissue, scale bar = 20 μm.

Article Snippet: As previously described [ ], the paraffin-embedded microarray tumor tissues using IHC primary antibodies, including SLC7A11 (Proteintech 26864-1-AP), SLC3A2 (Proteintech 15193-1-AP), NCKAP1 (Proteintech, 12140-1-AP), and GYS1 (Proteintech 10566-1-AP) antibodies, along with their corresponding non-cancerous tissues.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, CCK-8 Assay, Knockdown, Flow Cytometry, Migration, Wound Healing Assay, Transwell Invasion Assay, Real-time Polymerase Chain Reaction, Immunohistochemistry

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