Journal: Journal of Hematology & Oncology

Article Title: LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC

doi: 10.1186/s13045-021-01123-0

Figure Lengend Snippet: LCAT3 is upregulated in lung tumor tissues and associated with poor prognosis of LUAD. A Boxplot showing the relative expression of LCAT3 in lung tumors ( n = 485) and adjacent normal ( n = 56) tissues in a TCGA LUAD cohort. LCAT3 expression was quantified by FPKM in the RNA-seq data. B LCAT3 expression validated by qRT-PCR in an independent cohort of 13 paired samples of LUAD tissues and adjacent normal tissues. LCAT3 expression was normalized to the expression of β-actin. C , D Kaplan–Meier curves of overall survival ( C ) and disease-free survival ( D ) of lung cancer patients with high versus low expressions of LCAT3. E , F The protein coding potential of LCAT3 evaluated by the Coding Potential Assessment Tool (CPAT) and Coding Potential Calculator 2 (CPC2). Two verified lncRNAs, XIST and HORAIR, served as controls for the prediction. G , H Subcellular localization of LCAT3. Real-time PCR analysis confirmed the nuclear and cytoplasmic fraction of LCAT3 transcript in Calu1 and Hop62 cells; U6 and GAPDH served as positive controls for the nuclear and cytoplasmic fractions, respectively

Article Snippet: The human LUAD cell lines (A549, Calu1 and Hop62) and a human embryonic kidney cell line (HEK-293T) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal of Hematology & Oncology

Article Title: LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC

doi: 10.1186/s13045-021-01123-0

Figure Lengend Snippet: LCAT3 is upregulated by METTL3 through m6A modification. A METTL3 is upregulated in LUAD. Boxplot showing the relative expression of METTL3 in lung tumors ( n = 480) and adjacent normal tissues ( n = 56) in TCGA LUAD cohort. METTL3 expression was quantified by FPKM in the RNA-seq data. B Western blot assays to detect METTL3 knockout efficiency in A549 and Calu1 cells. C , D qRT-PCR analysis of LCAT3 expression in sg-lacZ and sg-METTL3 lung cancer cells. E MeRIP-seq of sgLacZ (top) and sgMETTL3 (bottom) A549 cells showed that depletion of METTL3 reduces the m6A modification on LCAT3. Red line represents input group while blue line represents IP group. F MeRIP-RT-qPCR confirmed the change in m6A levels in METTL3-knockout A549 cells. G , H Half-life of LCAT3 in sg-lacZ and sg-METTL3 lung cancer cells treated with 5 μg/mL Actinomycin D

Article Snippet: The human LUAD cell lines (A549, Calu1 and Hop62) and a human embryonic kidney cell line (HEK-293T) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Modification, Expressing, RNA Sequencing, Western Blot, Knock-Out, Quantitative RT-PCR

Journal: Journal of Hematology & Oncology

Article Title: LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC

doi: 10.1186/s13045-021-01123-0

Figure Lengend Snippet: LCAT3 physically interacts with FUBP1. A Proteins interacting with LCAT3 were identified by pull down assay followed by mass spectrometry analysis. B Western blot analysis of FUBP1 in beads only, negative control, sense and antisense LCAT3 pull-down fractions. GAPDH served as a negative control. C . FUBP1 RIP assays in Calu1 cells. A western blot assay confirmed FUBP1 immunoprecipitation (Up); The relative fold enrichment of LCAT3 between FUBP1 and IgG RIP fractions was determined by qRT-PCR (Down). D LCAT3 sense and 208–342 nt fragments were performed with RNA pull down and western blotting assays. E The schematic structure of full-length FUBP1 proteins (FL: 1–644) and three domain-deleted mutants (MUT1: 100–644; MUT2: 1–100 and 447–644; MUT3: 1–511) of FUBP1 variants used in this study. The blue box is the inhibitory domain, the orange box is the DNA and RNA binding domain, and the purple box is the transcription domain. F RIP assays were performed using anti-FLAG antibodies in Calu1 cells that were transfected with vectors expressing the FLAG-tagged FL and the deleted mutants (MUT1-3) of FUBP1. G Relative mRNA expression of FUBP1 primary tumors ( n = 515) and in adjacent normal tissues ( n = 59) from TCGA LUAD samples. H Relative protein expression of FUBP1 in primary tumors ( n = 111) and adjacent normal tissue ( n = 111) from Clinical Proteomic Tumor Analysis Consortium (CPTAC) LUAD samples. I Kaplan–Meier survival analysis of overall survival of lung cancer patients using FUBP1 expression. The data were downloaded from the study of Takeuchi et al. (GEO accession: GSE11969; No. patients: 90)

Article Snippet: The human LUAD cell lines (A549, Calu1 and Hop62) and a human embryonic kidney cell line (HEK-293T) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Pull Down Assay, Mass Spectrometry, Western Blot, Negative Control, Immunoprecipitation, Quantitative RT-PCR, RNA Binding Assay, Transfection, Expressing