Journal: Cell Death Discovery

Article Title: m6A modification promotes miR-133a repression during cardiac development and hypertrophy via IGF2BP2

doi: 10.1038/s41420-021-00552-7

Figure Lengend Snippet: Neonatal mouse cardiomyocytes (CMs) were transfected with miR-133a and Si-RNAs. A Representative western blotting (up) and quantification (down) showing the miR-133a repression on CDC42 protein level could be reversed by Igf2bp2 , but neither HuR nor Ythdf2 knockdown in CMs. B Western blotting assay indicating overexpression of IGF2BP2 could further decrease CDC42 with miR-133a transfection. C Luciferase reporter activities were quantitated in CMs and indicated miR-133a activity could increase by knockdown of Ago2 or I gf2bp2 , but neither HuR nor Ythdf2 . D Inhibiting Cdc42 mRNA decay by silencing Ago2 or Igf2bp2 in Actinomycin D treated CM cells. E RNA immunoprecipitated-qRT-PCR assay using IGF2BP2 antibody suggested that the enrichment of IGF2BP2 on Cdc42 mRNA was increased by Fto knockdown, but had no effect by Ago2 silencing. F AGO2-RNA immunoprecipitated-qRT-PCR assay showing the enrichment of AGO2 on Cdc42 was increased by Fto knockdown but decreased by Igf2bp2 knockdown. G Co-immunoprecipitation and western blotting showing the interaction of AGO2 and IGF2BP2 in CMs cells, representative of three independent experiments. H Carton model of IGF2BP2 binding to m6A-modified site promotes the miR-133a-AGO2-RISC complex accumulation on its targets, which would enhance the decreasing of their stability and translation. Data are mean ± SD ( n = 4 per group). * p < 0.05; ** P < 0.01 was determined by one-way ANOVA followed by Tukey’s test in ( A , C , E , and F ). * p < 0.05 was detected by Student’s t test in ( B ).

Article Snippet: The short interfering RNAs (siRNA, 50 nM) including si- Igf2bp2 (sc-146180), si- HuR (sc-35620), si- Ythdf2 (sc-155424), si -Fto (sc-75003), si- Ago2 (sc-44659), and si-RNA-A (sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Western Blot, Knockdown, Over Expression, Luciferase, Activity Assay, Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Modification

Journal: Cell Death & Disease

Article Title: LCAT1 is an oncogenic LncRNA by stabilizing the IGF2BP2-CDC6 axis

doi: 10.1038/s41419-022-05316-4

Figure Lengend Snippet: A , C Volcano plots showing enrichment of IGF2BP2 target genes that were altered in IGF2BP2 knockdown ( A ) or LCAT1 knockdown ( C ) Calu1 cells. The numbers of significantly downregulated (log2 FC < −1, P < 0.05, two-sided Student’s t -test) or upregulated (log2 FC > 1, P < 0.05, two-sided Student’s t -test) genes in the IGF2BP2 target group and non-target group are displayed. Vertical dashed lines indicate the cut-off of log2 FC of 1 or –1, while the horizontal dashed line indicates the cut-off of a P value of 0.05. FC, fold change. B , D Cumulative density of mRNA log2 FC in IGF2BP2 target and non-target genes upon IGF2BP2 ( B ) or LCAT1 ( D ) knockdown. E Venn diagram of the cross-comparison of three datasets: RNA-seq of IGF2BP2 knockdown; RNA-seq of LCAT1 knockdown; and IGF2BP2 targets. F Heatmap of target genes. G , H qPCR and western blot were performed to detect CDC6 expression in IGF2BP2 knockdown and control cells. I , J qPCR and western blot were performed to detect CDC6 expression in LCAT1 knockdown and control cells. K , L qPCR and western blot were conducted to determine the impacts of LCAT1 and IGF2BP2 silencing on CDC6 expression. M Scatter plot of IGF2BP2 and CDC6 mRNA expression in lung cancers from the TCGA. N Scatter plot of LCAT1 and CDC6 mRNA expression in lung cancers from the TCGA. Student’s two-sided t -tests were used. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The small interference RNAs (siRNAs) targeting IGF2BP2 and CDC6 (Supplementary Table ) were designed using the website: http://rnadesigner.thermofisher.com/ and synthesized by RiboBio (Guangzhou, China).

Techniques: Knockdown, Comparison, RNA Sequencing, Western Blot, Expressing, Control

Journal: Cell Death & Disease

Article Title: LCAT1 is an oncogenic LncRNA by stabilizing the IGF2BP2-CDC6 axis

doi: 10.1038/s41419-022-05316-4

Figure Lengend Snippet: A , B qPCR and western blot were taken to measure the CDC6 expression in A549 and Calu1 cells transfected with CDC6 siRNAs and negative control. C – G CCK-8 ( C ), colony formation ( D , E ), and EdU staining ( F , G ) assays were performed to evaluate the impacts of CDC6 depletion on lung cancer cell viability. H , I Transwell assays were conducted to determine the migration ability of CDC6 silenced and control cells. J , K Growth curve and colony formation assays were performed in A549 cells co-transfected with siLCAT1 and CDC6 overexpress vector. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The small interference RNAs (siRNAs) targeting IGF2BP2 and CDC6 (Supplementary Table ) were designed using the website: http://rnadesigner.thermofisher.com/ and synthesized by RiboBio (Guangzhou, China).

Techniques: Western Blot, Expressing, Transfection, Negative Control, CCK-8 Assay, Staining, Migration, Control, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: LCAT1 is an oncogenic LncRNA by stabilizing the IGF2BP2-CDC6 axis

doi: 10.1038/s41419-022-05316-4

Figure Lengend Snippet: A MeRIP-qPCR was performed to determine the enrichment of m 6 A-modified CDC6 RNA in anti-m 6 A and IgG groups. B RIP-qPCR was conducted to evaluate the enrichment of CDC6 in anti-IGF2BP2 antibody and IgG antibody groups. C Western blot of METTL3 knockout in A549 cells. D Dot blot assay indicated that knockout of METTL3 decreased global levels of m 6 A modification. E RIP-qPCR was performed in METTL3 knockout A549 cells using an anti-IGF2BP2 antibody or IgG antibody. F , G Depletion of IGF2BP2 ( F ) or LCAT1 ( G ) reduced CDC6 RNA stability in lung cancer cells. Cells were first transfected with IGF2BP2 siRNA or LCAT1 siRNA, then treated with 2 μg/ml actinomycin D at different time points. H Schematic diagram of the LCAT1-IGF2BP2-CDC6 axis in mediating lung cancer pathogenesis. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The small interference RNAs (siRNAs) targeting IGF2BP2 and CDC6 (Supplementary Table ) were designed using the website: http://rnadesigner.thermofisher.com/ and synthesized by RiboBio (Guangzhou, China).

Techniques: Modification, Western Blot, Knock-Out, Dot Blot, Transfection