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: 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

Journal: Journal of Cellular and Molecular Medicine

Article Title: Therapeutic potential of melatonin in the intervertebral disc degeneration through inhibiting the ferroptosis of nucleus pulpous cells

doi: 10.1111/jcmm.17818

Figure Lengend Snippet: The list of primers was utilized for qPCR.

Article Snippet: The following primary polyclonal antibodies were used: rabbit anti‐mouse IL‐6 (Cat. No. PAA079Mu01), rabbit anti‐mouse IL‐1β (Cat. No. PAA563Mu01), rabbit anti‐mouse TNF‐α (Cat. No. PAA133Mu01), rabbit anti‐mouse iNOS (Cat. No. PAA837Mu01), rabbit anti‐mouse COX2 (Cat. No. PAA699Mu01), rabbit anti‐mouse ACAN (Cat. No. PAB908Mu01), rabbit anti‐mouse COL2A1 (Cat. No. PAD194Mu01), rabbit anti‐mouse MMP‐13 (Cat. No. PAA099Mu01), rabbit anti‐mouse GPX4 (Cat. No. PAC994Mu01), rabbit anti‐mouse LPCAT3 (Cat. No. PAG531Mu01) that from the Cloud Clone Corporation, Wuhan, China; rabbit anti‐mouse ADAMTS‐4 (Cat. No. ab185722), rabbit anti‐mouse ADAMTS‐5 (Cat. No. ab41037) that from the Abcam, Cambridge, England; rabbit anti‐mouse anti‐ACSL4 (Cat. No. ab2844946) that from the Affbiotech, Jiangsu, China; and rabbit anti‐mouse SLC7A11 (Cat. No. BM5318) that from the Boster Biological Technology Co., Ltd, Wuhan, China.

Techniques:

Journal: MedComm

Article Title: Slc7a11‐Mediated Cystine/Glutamate Antiport Reprograms Macrophage Polarization and Ameliorates Atherosclerosis

doi: 10.1002/mco2.70646

Figure Lengend Snippet: Slc7a11 expression is enhanced in the macrophages of atherosclerotic lesions. (A) Pathway enrichment analysis of RNA‐sequencing data from foamy and nonfoamy macrophages isolated from atherosclerotic intima. (B) Heatmap presenting differentially expressed genes in cellular response to oxidative stress, ferroptosis, glutathione metabolic process, and amino acid transmembrane transport pathways. (C) Real‐time PCR analysis of Slc7a11 mRNA level in bone marrow‐derived macrophage (BMDMs) treated with oxidized low‐density lipoprotein (oxLDL, 50 or 100 µg/mL) for 0, 6, 12, and 24 h ( n = 3 biological replicates per group). (D and E) Western blot analysis of Slc7a11 protein expression level in BMDMs treated with oxLDL (100 µg/mL) for 0, 12, 24, and 36 h ( n = 3 biological replicates per group). (F and G) Western blot analysis of Slc7a11 protein expression level in BMDMs treated with 0, 50, 100, and 200 µg/mL oxLDL for 24 h ( n = 3 biological replicates per group). (H) Quantification of flow cytometric analysis for Slc7a11 expression in BMDMs treated with oxLDL (100 or 200 µg/mL) for 36 and 48 h, respectively. (I) Slc7a11 mRNA expression level in BMDMs treated with 50 µg/mL oxLDL for 24 h in the presence or absence of Nrf2‐inhibitor Nrf‐In‐1 (2 µM) ( n = 3 biological replicates per group). (J) Representative images of immunofluorescence staining for Slc7a11 (red), Mac‐3 (macrophage marker, green), and 4′,6‐diamidino‐2‐phenylindole (DAPI, cell nuclei, blue) in the atherosclerotic plaques from ApoE –/– mice fed a western diet for 16 weeks. Images are representative of n = 3 independent experiments. Scale bar, 50 µm. Student's t ‐test was used to compare the two groups. One‐way ANOVA test was used to compare multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001. The molecular weight (in kDa) was indicated to the right of each band.

Article Snippet: Suspension cells were incubated with Alexa‐488 labeled Slc7a11 antibody (Novus; Cat. No. NB300‐318AF488, USA) in the dark for 30 min at 4°C.

Techniques: Expressing, RNA Sequencing, Isolation, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot, Immunofluorescence, Staining, Marker, Molecular Weight

Journal: MedComm

Article Title: Slc7a11‐Mediated Cystine/Glutamate Antiport Reprograms Macrophage Polarization and Ameliorates Atherosclerosis

doi: 10.1002/mco2.70646

Figure Lengend Snippet: Macrophage‐specific Slc7a11 overexpression attenuates atherosclerotic lesions and increases the plaque stability. Eight‐week‐old ApoE – /– and ApoE –/– Slc7a11 MOE mice were fed a western diet for 16 weeks. (A) Hematoxylin and eosin (H&E) staining of aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice. Scale bar, 200 µm. (B) Lesion area of aortic plaques across a 400 µm distance of the aortic root from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group). (C) Necrotic core areas of aortic plaques across a 400 µm distance of the aortic root from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group). (D) The proportion of early, moderate, and advanced plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice based on the histological staining ( n = 10 mice in each group). (E) Representative images of immunofluorescence staining for smooth muscle cells (α‐SMA, red) and 4′,6‐diamidino‐2‐phenylindole (DAPI, cell nuclei, blue) in the aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice. Scale bar, 50 µm. (F) Quantification of the smooth muscle cells area in the aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group, and each mouse had two sections). (G) Representative images of immunofluorescence staining for Mac‐3 (macrophage marker, green) and DAPI (cell nuclei, blue) in the aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice. Scale bar, 50 µm. (H) Quantification of macrophages numbers in the aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group, and each mouse had two sections). (I) Representative images of Masson trichrome staining of aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice. Scale bar, 200 µm. (J) Quantification of Masson trichrome staining in aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group, and each mouse had two sections). (K, L, and M) Glutathione (GSH), glutathione peroxidase (GSH‐PX), and malondialdehyde (MDA) levels in serum from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group). (N) Representative images of immunofluorescence staining for 4‐hydroxynonenal (4‐HNE, lipid oxidation product, red), Mac‐3 (macrophage marker, green), and DAPI (cell nuclei, blue) in the aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice. Scale bar, 50 µm. (O) Quantification of 4‐HNE fluorescence intensity in the aortic plaques from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group, and each mouse had two sections). Student's t ‐test was used to compare the two groups. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Suspension cells were incubated with Alexa‐488 labeled Slc7a11 antibody (Novus; Cat. No. NB300‐318AF488, USA) in the dark for 30 min at 4°C.

Techniques: Over Expression, Western Blot, Staining, Immunofluorescence, Marker, Fluorescence

Journal: MedComm

Article Title: Slc7a11‐Mediated Cystine/Glutamate Antiport Reprograms Macrophage Polarization and Ameliorates Atherosclerosis

doi: 10.1002/mco2.70646

Figure Lengend Snippet: Slc7a11 overexpression in macrophages inhibits classical M1 polarization and promotes alternative M2 polarization. (A) Classical M1 marker gene expressions in the aorta arteries from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group). (B) Alternative M2 marker gene expressions in the aorta arteries from ApoE – /– and ApoE –/– Slc7a11 MOE mice ( n = 10 mice in each group). (C) Western blot analysis of p‐Stat1 and Stat1 in bone marrow‐derived macrophage (BMDMs) from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with liposaccharides (LPS, 1 µg/mL) and interferon‐γ (IFN‐γ, 50 ng/mL) for 0, 6, 12, and 24 h ( n = 3 biological replicates in each group). (D) Classical M1 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h ( n = 3 biological replicates in each group). (E) Western blot analysis of p‐Stat1 and Stat1 in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 0, 6, 12, and 24 h ( n = 3 biological replicates in each group). (F) Classical M1 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h ( n = 3 biological replicates in each group). (G) Western blot analysis of p‐Stat1 and Stat1 in BMDMs treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 0, 6, 12, and 24 h in the presence and absence of erastin (15 µM) ( n = 3 biological replicates in each group). (H) Classical M1 marker gene expressions in BMDMs treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h in the presence and absence of erastin (15 µM) ( n = 3 biological replicates in each group). (I) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with interleukin‐4 (IL‐4, 30 ng/mL) for 0, 15, 30, 60, 120, and 180 min ( n = 3 biological replicates per group). (J) Alternative M2 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with IL‐4 (30 ng/mL) for 24 h ( n = 3 biological replicates per group). (K) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL‐4 (30 ng/mL) for 0, 15, 30, 60, 120, and 180 min ( n = 3 biological replicates in each group). (L) Alternative M2 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL‐4 (30 ng/mL) for 24 h ( n = 3 biological replicates in each group). (M) Western blot analysis of p‐Stat6 and Stat6 treated with IL‐4 (30 ng/mL) for 0, 15, 30, 60, 120, and 180 min in the presence and absence of erastin (15 µM) ( n = 3 biological replicates in each group). (N) Alternative M2 marker gene expressions in BMDMs treated with IL‐4 (30 ng/mL) for 24 h in the presence and absence of erastin (15 µM) ( n = 3 biological replicates in each group). Student's t ‐test was used to compare the two groups. One‐way ANOVA test was used to compare multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001. The molecular weight (in kDa) was indicated to the right of each band.

Article Snippet: Suspension cells were incubated with Alexa‐488 labeled Slc7a11 antibody (Novus; Cat. No. NB300‐318AF488, USA) in the dark for 30 min at 4°C.

Techniques: Over Expression, Marker, Western Blot, Derivative Assay, Molecular Weight

Journal: MedComm

Article Title: Slc7a11‐Mediated Cystine/Glutamate Antiport Reprograms Macrophage Polarization and Ameliorates Atherosclerosis

doi: 10.1002/mco2.70646

Figure Lengend Snippet: Slc7a11‐mediated cystine uptake and glutathione synthesis regulate classical M1 macrophages polarization. (A) Western blot analysis of p‐Stat1 and Stat1 in bone marrow‐derived macrophage (BMDMs) treated with liposaccharides (LPS, 1 µg/mL) and interferon‐γ (IFN‐γ, 50 ng/mL) for 0, 6, 12, and 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (B) Western blot analysis of p‐Stat1 and Stat1 in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (C) Western blot analysis of p‐Stat1 and Stat1 in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (D) Classical M1 marker gene expressions in BMDMs treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (E) Classical M1 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (F) Classical M1 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (G) Western blot analysis of p‐Stat1 and Stat1 in BMDMs treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 0, 6, 12, and 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (H) Western blot analysis of p‐Stat1 and Stat1 in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (I) Western blot analysis of p‐Stat1 and Stat1 in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (J) Classical M1 marker gene expressions in BMDMs treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (K) Classical M1 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (L) Classical M1 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (M) Western blot analysis of p‐Stat1 and Stat1 in BMDMs treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 0, 6, 12, and 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (N) Western blot analysis of p‐Stat1 and Stat1 in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (O) Western blot analysis of p‐Stat1 and Stat1 in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (P) Classical M1 marker gene expressions in BMDMs treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (Q) Classical M1 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (R) Classical M1 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with LPS (1 µg/mL) and IFN‐γ (50 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). Student's t ‐test was used to compare the two groups. One‐way ANOVA test was used to compare multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001. The molecular weight (in kDa) was indicated to the right of each band.

Article Snippet: Suspension cells were incubated with Alexa‐488 labeled Slc7a11 antibody (Novus; Cat. No. NB300‐318AF488, USA) in the dark for 30 min at 4°C.

Techniques: Western Blot, Derivative Assay, Incubation, Marker, Molecular Weight

Journal: MedComm

Article Title: Slc7a11‐Mediated Cystine/Glutamate Antiport Reprograms Macrophage Polarization and Ameliorates Atherosclerosis

doi: 10.1002/mco2.70646

Figure Lengend Snippet: Slc7a11‐mediated cystine uptake and glutathione synthesis control alternative M2 macrophages polarization. (A) Western blot analysis of p‐Stat6 and Stat6 in bone marrow‐derived macrophage (BMDMs) treated with interleukin‐4 (IL‐4, 30 ng/mL) for 0, 15, 30, 60, 120, and 180 min after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (B) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL‐4 (30 ng/mL) for 30 min after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (C) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with IL‐4 (30 ng/mL) for 30 min after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (D) Alternative M2 marker gene expressions in BMDMs treated with IL‐4 (30 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (E) Alternative M2 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL‐4 (30 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (F) Alternative M2 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with IL‐4 (30 ng/mL) for 24 h after incubation with or without glutathione (1 mM) for 6 h ( n = 3 biological replicates per group). (G) Western blot analysis of p‐Stat6 and Stat6 in BMDMs treated with IL‐4 (30 ng/mL) for 0, 15, 30, 60, 120, and 180 min after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (H) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL‐4 (30 ng/mL) for 30 min after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (I) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with IL‐4 (30 ng/mL) for 30 min after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (J) Alternative M2 marker gene expressions in BMDMs treated with IL‐4 (30 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (K) Alternative M2 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL4 (30 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (L) Alternative M2 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with IL‐4 (30 ng/mL) for 24 h after cystine deprivation or none deprivation for 6 h ( n = 3 biological replicates per group). (M) Western blot analysis of p‐Stat6 and Stat6 in BMDMs treated with IL‐4 (30 ng/mL) for 0, 15, 30, 60, 120, and 180 min after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (N) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL‐4 (30 ng/mL) for 30 min after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (O) Western blot analysis of p‐Stat6 and Stat6 in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with IL‐4 (30 ng/mL) for 30 min after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (P) Alternative M2 marker gene expressions in BMDMs treated with IL‐4 (30 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (Q) Alternative M2 marker gene expressions in BMDMs from wildtype or Slc7a11 deficiency mice treated with IL‐4 (30 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). (R) Alternative M2 marker gene expressions in BMDMs from ApoE – /– or ApoE –/– Slc7a11 MOE mice treated with IL‐4 (30 ng/mL) for 24 h after incubation with or without glutamate (600 µM) for 6 h ( n = 3 biological replicates per group). Student's t ‐test was used to compare the two groups. One‐way ANOVA test was used to compare multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001. The molecular weight (in kDa) was indicated to the right of each band.

Article Snippet: Suspension cells were incubated with Alexa‐488 labeled Slc7a11 antibody (Novus; Cat. No. NB300‐318AF488, USA) in the dark for 30 min at 4°C.

Techniques: Control, Western Blot, Derivative Assay, Incubation, Marker, Molecular Weight

Journal: MedComm

Article Title: Slc7a11‐Mediated Cystine/Glutamate Antiport Reprograms Macrophage Polarization and Ameliorates Atherosclerosis

doi: 10.1002/mco2.70646

Figure Lengend Snippet: Slc7a11‐mediated cystine/glutamate antiport regulates macrophage polarization and atherosclerosis development. Slc7a11‐mediated cystine uptake and glutathione synthesis inhibits the classically activated macrophage polarization (M1) by reducing the Stat1 phosphorylation and classical M1 gene expressions ( Tnfa , Nos2 , Il6 , Il1b ), and promotes alternatively activated macrophage polarization (M2) by enhancing Stat6 phosphorylation and alternative M2 gene expressions ( Arg1 , Mrc1 , Irf4 , Retnla ). In contrast, glutamate treatment or glutathione depletion reverses the phenotypic switch of macrophage from M1 to M2. Macrophage‐specific Slc7a11 overexpression or macrophage‐targeting lipid nanoparticles loading with ferrostatin‐1 (LNP–Fer‐1) promotes M2 macrophage polarization, decreases atherosclerotic macrophage content, and attenuates the development of atherosclerosis.

Article Snippet: Suspension cells were incubated with Alexa‐488 labeled Slc7a11 antibody (Novus; Cat. No. NB300‐318AF488, USA) in the dark for 30 min at 4°C.

Techniques: Phospho-proteomics, Over Expression