Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB triggered GSH synthesis. (A,B) The GSE101586, GSE112214, and GSE101684 data sets were re-analyzed to identify the circRNAs that were differentially expressed between the LUAD and adjacent normal tissues; (C) P4HB expression levels in LUAD tissue samples were analyzed using TCGA data; (D) the prognosis of P4HB expression was shown by Kaplan-Meier plotter curves; (E,F) the pathway enrichment of those metabolites with a high P4HB level was predicted; (G) circP4HB expression levels in LUAD tissue samples were detected by RT-qPCR assays; (H,I) the GSH levels and GSH/GSSG ratios of circP4HB overexpression H1299 and SPCA1 cells were measured. **, P<0.01; ***, P<0.001. LUAD, lung adenocarcinoma; GSH, glutathione; GSSG, oxidized glutathione disulfide.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Over Expression

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB protected LUAD from ferroptosis through promoting GSH synthesis. (A) The effect of erastin (10 µm 24 h) on the viability of LUAD and 16HBE cells was evaluated by CCK-8. DMSO was used as a control; (B,C) ferroptosis markers, Ptgs2 and MDA, were examined in cell lines in response to erastin (10 µm, 24 h); (D) the effect of erastin on the viability of H1299 cells overexpressing circP4HB was measured by CCK-8; (E,F) GSH levels and GSH/GSSG ratios of the circP4HB-overexpressing cells were measured; (G,H) Ptgs2 mRNA and MDA levels in circP4HB-overexpressing cells were examined in response to erastin; (I,J) lipid ROS and JC-1 levels were measured following immunofluorescence staining of H1299 cells following treatment with erastin. *, P<0.05; **, P<0.01; ***, P<0.001; ns, non-significant. LUAD, lung adenocarcinoma; Ptgs2, prostaglandin-endoperoxide synthase 2; MDA, malondialdehyde; DMSO, dimethyl sulfoxide; CCK-8, cell counting kit-8; GSH, glutathione; lipid ROS, lipid reactive oxygen species.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: CCK-8 Assay, Control, Immunofluorescence, Staining, Cell Counting

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: Validation of the interactions between circP4HB, miR-1184, and SLC7A11. (A) The relative abundance of circP4HB in the nucleus and cytoplasm was investigated via nuclear mass separation assays; (B) bioinformatic predictions https://circinteractome.nia.nih.gov and circNet, identified miR-1184 as a target of circP4HB; (C) FISH assay showed circP4HB and miR-1184 probes co-localized in the cytoplasm; (D) the interaction between circP4HB and miR-1184 was determined by using dual-luciferase reporter assays; (E) RIP assays were performed on the cells using AGO2 antibodies; (F) RNA pull-down assays and qRT-PCR assays were used to confirm the binding of circP4HB to miR-1184; (G) predictions using TargetScan and FerrDb, SLC7A11 was taken forward as a target of interest; (H,I) dual-luciferase reporter assays were carried out using HEK-293 T cells co-infected with a plasmid containing the SLC7A11 3'-UTR and miR-1184 mimics; (J,K) miR-1184 and SLC7A11 levels were measured in knockdown circP4HB cells. Negative controls (NC) were also used. **, P<0.01; ***, P<0.001; ns, non-significant. SLC7A11, solute carrier family 7 member 11; AGO2, Argonaute 2; RIP, RNA immunoprecipitation; FISH, fluorescence in situ hybridization.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: Biomarker Discovery, Luciferase, Quantitative RT-PCR, Binding Assay, Infection, Plasmid Preparation, Knockdown, RNA Immunoprecipitation, Fluorescence, In Situ Hybridization

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB inhibits ferroptosis by regulating miR-1184/SLC7A11-mediated GSH synthesis. (A,B) Lipid ROS and JC-1 levels were measured using Lipid ROS and JC-1 staining; (C,D) GSH levels and the GSH/GSSG ratios were detected; (E) protein levels of SLC7A11 were tested by western blot analysis; (F) mRNA levels of downstream effectors (Gclc, Gclm, Gsr, Gss, and Nqo1) were examined by qRT-PCR. +, transfected with; −, transfected without. **, P<0.01; ***, P<0.001. SLC7A11, solute carrier family 7 member 11; GSH, glutathione; lipid ROS, lipid reactive oxygen species; GSSG, oxidized glutathione disulfide.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: Staining, Western Blot, Quantitative RT-PCR, Transfection

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: CircP4HB promotes LUAD growth and inhibits ferroptosis in vivo. (A-C) Tumor size and, weight, and volume of the mice were measured. (D,E) Gene expression levels of circP4HB and SLC7A11 were measured by in harvested tumor tissues by qRT-PCR. (F) SLC7A11 levels were detected by IHC assays. (G) SLC7A11 mRNA expression levels in LUAD tissues and normal samples from TCGA data. (H) Kaplein-Meier plotter showing prognostic significance of SLC7A11 gene expression in LUAD patients. The prognosis of abnormal SLC7A11 expression in LUAD patients from TCGA data. (I) The SLC7A11 mRNA levels in 50 cases (tumor tissues and para-tumor samples) were detected by qRT-PCR. (J) Protein levels of SLC7A11 in LUAD tissues and para-tumor were tested by IHC. *, P<0.05; **, P<0.01; ***, P<0.001; ns non-significant. LUAD, lung adenocarcinoma; SLC7A11, solute carrier family 7 member 11; TCGA, The Cancer Genome Atlas, IHC, immunohistochemistry.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: In Vivo, Gene Expression, Quantitative RT-PCR, Expressing, Paraffin-embedded Immunohistochemistry, Immunohistochemistry

Journal: Translational Lung Cancer Research

Article Title: CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma

doi: 10.21037/tlcr-22-138

Figure Lengend Snippet: Schematic representation of a proposed mechanism by which circP4HB protects lung adenocarcinoma cells from ferroptosis via SLC7A11-mediated glutathione synthesis. SLC7A11, solute carrier family 7 member 11; GSH, glutathione; GSSG, oxidized glutathione disulfide; ROS, reactive oxygen species.

Article Snippet: The random oligo probe and biotin labelled probe specifically targeting circP4HB (RiboBio, Guangzhou, China) were obtained and were incubated with M280 streptavidin Dynabeads (Invitrogen, USA) for 2 h at room temperature.

Techniques: