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: Advanced Science

Article Title: NARFL Knockout Triggers Ferroptosis‐Driven Vascular Endothelial Dysfunction

doi: 10.1002/advs.202415580

Figure Lengend Snippet: Deletion of the Ciao3 Gene Leads to Embryonic Death and Vascular Development Disorder in Mice. A) The embryo morphology of the Ciao3 heterozygous offspring at 8.5, 10.5, 12.5 and 13.5 days; Ciao3 knockout embryos still existed at 12.5 days but were completely absorbed at 13.5 days and later. B) H&E staining morphology of wild type with 8.5‐day and 12.5‐day mouse embryos and Ciao3 −/− mice embryos with 10.5‐day and 12.5‐day, the development of Ciao3 knock‐out embryos was significantly slower than that of wild‐type mouse embryos and the development of the vascular system was blocked. C) The endothelial marker CD31 was detected by immunofluorescence staining in 12.5‐day mouse embryos. D) The endothelial progenitor cell marker CD34 was detected by immunofluorescence staining in 12.5‐day mouse embryos. E,F) The sections of 12.5‐day‐old mouse embryos were detected by 4‐HNE (E) and BODIPY (F) probes; the positive rate of 4‐HNE and BODIPY in Ciao3 − / − mouse embryo was significantly higher than in wild‐type mouse embryo. H) γ‐H2AX detection of 12.5‐day mouse embryos showed that the DNA damage of Ciao3 − / − mouse embryos was significantly enhanced compared with wild‐type embryos. G‐I) Western blotting showed that GPX4, xCT and FTL were significantly down‐regulated in Ciao3 knockout mouse embryos; the expression of TFR1 and IRP1 was significantly up‐regulated. Data were analyzed by an unpaired Student's t‐test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Primary antibodies for Ciao3 (sc‐514078, 1/1000) were obtained from santa, GPX4 (ab125066, 1/2000) and FTL (ab6990, 1/500) from Abcam, xCT (NB300‐318, 1/2000) and FTH1 (NBP1‐31944, 1/500) from Novus, TFR1 (13‐6800, 1/500) from Invitrogen, GAPDH (ab8245, 1/1000) from Abcam.

Techniques: Knock-Out, Staining, Marker, Immunofluorescence, Western Blot, Expressing

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: Oncotarget

Article Title: Targeting cancer stem cell propagation with palbociclib, a CDK4/6 inhibitor: Telomerase drives tumor cell heterogeneity

doi: 10.18632/oncotarget.14196

Figure Lengend Snippet: A-C . Mitochondrial inhibitors. Mitochondrial function is required for the efficient clonal expansion and anchorage-independent growth of CSCs. Indeed, oligomycin A, an inhibitor of the mitochondrial ATP synthase, inhibits 3D-sphere formation in the GFP(+) cell population isolated from A549 and SKOV3 cells ( panel A ). Complementary results were also obtained with two other well-established mitochondrial inhibitors, namely XCT790 ( panel B ) and doxycycline ( panel C ). XCT790 is a well-established ERRα inverse agonist that effectively blocks the function of PGC1A, the major mitochondrial transcription factor. Doxycycline functions as an inhibitor of mitochondrial biogenesis, by inhibiting mitochondrial protein synthesis at the level of the mito-ribosome. D . Inhibition of glycolysis. Glycolytic metabolism is important for 3D-spheroids. Note that treatment with the glycolytic inhibitor 2-deoxy-glucose (2-DG) significantly reduces sphere formation. Bar graphs show the mean ± SEM, t -test, two-tailed test. *p < 0.05, **p < 0.005, ***p < 0.001.

Article Snippet: XCT790 was purchased from Tocris.

Techniques: Isolation, Inhibition, Two Tailed Test

Journal: Oncotarget

Article Title: Targeting cancer stem cell propagation with palbociclib, a CDK4/6 inhibitor: Telomerase drives tumor cell heterogeneity

doi: 10.18632/oncotarget.14196

Figure Lengend Snippet: A . Cell migration. A549 and SKOV3 cells stably-transduced with the hTERT-eGFP reporter were subjected to flow cytometry to isolate the GFP(+) and GFP(-) cell populations. Then, the migratory capacity of these two cell populations was assessed using a modified “Boyden Chamber” assay. More specifically, the cells were allowed to migrate across an 8 μm pore uncoated membrane for 12-16 hours. Note that the GFP(+) cell population (derived from A549 or SKOV3 cells) shows a near 2-fold increase in migration, as compared with the GFP(-) cells. p<0.05 for the A549 and p<0.001 for the SKOV3 cells (Student's t-test). B . Effects of mitochondrial inhibitors. To assess whether mitochondrial function is involved in cancer cell migration, mitochondrial inhibitors (either oligomycin A or XCT790) were placed in both the upper and lower chambers. Note that oligomycin A and XCT790 both effectively inhibited migration in the GFP(+) cell population. Results are shown as the mean ± SEM, t -test, two-tailed test. *p < 0.05.

Article Snippet: XCT790 was purchased from Tocris.

Techniques: Migration, Stable Transfection, Transduction, Flow Cytometry, Modification, Boyden Chamber Assay, Membrane, Derivative Assay, Two Tailed Test

Journal: Oncotarget

Article Title: Targeting cancer stem cell propagation with palbociclib, a CDK4/6 inhibitor: Telomerase drives tumor cell heterogeneity

doi: 10.18632/oncotarget.14196

Figure Lengend Snippet: This schematic diagram summarizes the isolation of two distinct populations of cancer cells, based on telomerase transcriptional activity. A schematic diagram of the hTERT-Promoter-eGFP-Puro-R reporter construct is shown on the right. Note that the GFP(+) cell population shows significant increases in “stemness”, metabolic activity (both oxidative and glycolytic), cell migration and proliferation. As a consequence, the propagation of this cell population can be effectively targeted with metabolic inhibitors (oligomycin A, XCT790, doxycycline, 2-DG) or by using a CDK4/6 inhibitor (palbociclib).

Article Snippet: XCT790 was purchased from Tocris.

Techniques: Isolation, Activity Assay, Construct, Migration