cell ferrous ion fluorescence assay kit  (Elabscience Biotechnology)

Journal: International Journal of Biological Sciences

Article Title: LncRNA SLC7A11AR promotes lung adenocarcinoma progression by inhibiting ferroptosis via promoting SLC7A11 expression

doi: 10.7150/ijbs.112233

Figure Lengend Snippet: MiR-150-5p promotes ferroptosis in lung adenocarcinoma. a Screening potential microRNAs that may bind to both SLC7A11AR and SLC7A11 using multiple databases: Annolnc dataset (Blue); SLC7A11AR as target gene in Starbase dataset (Red); miRDB dataset (Green); SLC7A11 as target gene in Starbase dataset (Yellow); TargetScan dataset (Brown). b Correlation analysis of SLC7A11AR with miR-150-5p using the TCGA-LUAD dataset. c Relative expression levels of miR-150-5p in TCGA-LUAD (Normal: 20 cases; Tumor: 512 cases). d Relative expression levels of miR-150-5p in LUAD cell lines (H358, SPC-A1, A549, HCC827, H1299, H1975, PC9, H1650) compared to the normal bronchial epithelial cell line BEAS-2B, assessed by Real-time RT-PCR. e The expression of miR-150-5p was detected in paired clinical tissue samples from LUAD patients by Real-time RT-PCR (n=17). f Relative SLC7A11AR expression levels in H1299, H1975, and SPC-A1 cells transfected with miR-150-5p mimics compared with miR-NC group were evaluated by Real-time RT-PCR. g Relative miR-150-5p expressions after SLC7A11AR knockdown, were evaluated by Real-time RT-PCR. h-i Binding sites of miR-150-5p to SLC7A11AR predicted by miRDB, with the schematic of wild-type (WT) and mutant (MUT) luciferase reporter plasmids shown ( h ). HEK-293T cells co-transfected with specific plasmids and miR-NC or miR-150-5p mimics were collected to detect luciferase activity ( i ). j Transfection efficiency of miR-150-5p mimics and inhibitors in H1299 cells. k The RNA (top) and protein (bottom) expressions of SLC7A11 were detected by Real-time RT-PCR and Immunoblots, respectively. l-p Cell proliferation assay ( l ), colony formation assay ( m ), and trans-well migration assay ( n ) demonstrated that miR-150-5p regulates H1299 cell proliferation and migration. ( o-p ) is the statistical result for ( m-n ). Scale bar = 50μm. q - r Flow cytometry assay detecting lipid peroxidation levels ( q ) and reactive oxygen species (ROS) levels ( r ) in H1299 cells. Statistical results were also presented. s Relative levels of glutathione (GSH), ferrous ions (Fe² + ), and malondialdehyde (MDA) in H1299 cells after forced expression of miR-150-5p mimics compared with miR-NC group were detected. t-v Xenograft tumor formation for H1299 cells treated with miR-150-5p-mimics or miR-NC were examined. Tumor images ( t ), volumes ( u ), and tumor masses ( v ) were presented, respectively. w-x Results of immunohistochemical staining( w ) including quantification( x ) of positive signals for Ki67, Cleaved Caspase 3 (CC3), and SLC7A11. Scale bar = 50μm. * P < 0.05, ** P < 0.01, *** P < 0.001. HPF = high power field.

Article Snippet: Glutathione (GSH) (Reduced Glutathione Colorimetric Assay Kit, Cat. E-BC-K030-M), malondialdehyde (MDA) (Malondialdehyde Colorimetric Assay Kit, Cat. E-BC-K028-M), and ferrous ion (Fe2+) (Cell Ferrous Iron Colorimetric, Cat. E-BC-K881-M) level validations were according to the manual provided by Elabscience.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Knockdown, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Western Blot, Proliferation Assay, Colony Assay, Migration, Flow Cytometry, Immunohistochemical staining, Staining