Journal: Discover. Oncology

Article Title: METTL3 dual regulation of the stability of LINC00662 and VEGFA RNAs promotes colorectal cancer angiogenesis

doi: 10.1007/s12672-022-00557-3

Figure Lengend Snippet: METTL3 dual regulation of LINC00662 and VEGFA RNAs stability. A RIP experiments verified the enrichment levels of LINC00662 and m6A enzymes. B Expression of LINC00662 RNA at 0 h, 1 h, 3 h, 6 h and 9 h after interference and overexpression of METTL3. C Validation of m6A enrichment level of LINC00662 RNA by MeRIP assay. D Validation of Mettl3 enrichment level of LINC00662 RNA by MeRIP assay. E Expression of VEGFA mRNA at 0 h, 1 h, 3 h, 6 h and 9 h after interference and overexpression of METTL3. F Validation of m6A enrichment level of VEGFA mRNA by MeRIP assay. G Validation of METTL3 enrichment level of VEGFA mRNA by MeRIP assay. H Expression of LINC00662 RNA at 0 h, 1 h, 3 h, 6 h and 9 h after interference and overexpression of IGF2BP1. I Expression of VEGFA mRNA at 0 h, 1 h, 3 h, 6 h and 9 h after interference and overexpression of IGF2BP1

Article Snippet: Approximately 160 μl of fragmented RNA was diluted with the MeRIP buffer (150 mM KCl, 25 mM Tris, 5 mM EDTA, 0.5% Triton X-100, 0.5 mM DTT, protease inhibitor (1:100) (Invitrogen, Carlsbad, California, USA), and RNAase inhibitor (1:1000) (ABclonal, Wuhan, China)) and divided into two tubes that were incubated with the anti-m6A antibody (ABclonal, Wuhan, China) or the control IgG antibody with protein A/G conjugated magnetic beads (MCE, Monmouth Junction, NJ, USA) in 900 μl of the RNA binding protein immunoprecipitation (RIP) lysis buffer at 4 °C for 4 h. In total, 20 ul of fragmented RNA was collected.

Techniques: Expressing, Over Expression, Biomarker Discovery

Journal: Clinical and Translational Medicine

Article Title: Loss of m6A demethylase ALKBH5 promotes post‐ischemic angiogenesis via post‐transcriptional stabilization of WNT5A

doi: 10.1002/ctm2.402

Figure Lengend Snippet: MeRIP‐seq combined with RNA‐seq revealed potential target genes of ALKBH5. (A ) Venn diagram of MeRIP‐seq data showing the common and unique peaks of m6A RNA methylation in control (siCtrl) and ALKBH5‐knockdown (siALKBH5) groups (upper panel). Bar chart showing the numbers of differentially methylated peaks between the siCtrl and siALKBH5 groups (lower panel). ( B ) Distribution density of hypo‐methylated peaks (upper panel) and hyper‐methylated peaks (lower panel) across mRNA transcripts. ( C ) Pie chart showing the differential distribution of hypo‐methylated peaks (upper panel) and hyper‐methylated peaks (lower panel) across mRNA transcripts. ( D ) Representative motif analysis of hypo‐methylated and hyper‐methylated peaks. ( E ) Venn diagram of RNA‐seq data showing the common and unique genes between the siCtrl and siALKBH5 groups (upper panel). Bar chart showing the differentially expressed genes between the siCtrl and siALKBH5 groups (lower panel). ( F ) Integrated genome browser views of the hypo‐methylated NPPC gene (left panel) and hyper‐methylated FBXW5 gene (right panel). ( G ) Gene ontology analysis of hypo‐methylated and hyper‐methylated genes. ( H ) Screening strategy of ALKBH5 target genes

Article Snippet: Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) service was provided by Cloudseq Biotech Inc. (Shanghai, China).

Techniques: RNA Sequencing, Methylation, Control, Knockdown

Journal: Clinical and Translational Medicine

Article Title: Loss of m6A demethylase ALKBH5 promotes post‐ischemic angiogenesis via post‐transcriptional stabilization of WNT5A

doi: 10.1002/ctm2.402

Figure Lengend Snippet: ALKBH5 regulated the stability and decay of WNT5A mRNA. (A ) Integrated genome browser views of SKP2 , WNT5A , and FGF18 genes. ( B ) MeRIP‐qPCR quantitative analysis of the fold enrichment of SKP2, WNT5A, and FGF18 m6A level by immunoprecipitation with specific m6A antibody after transfection with control siRNA (siCtrl) or ALKBH5 siRNA (siALKBH5). ( C ) RT‐qPCR validation of target genes expression after siCtrl or siALKBH5 transfection. ( D ) Western blot validation of target gene protein expression after siCtrl or siALKBH5 transfection. ( E ) RT‐qPCR showing the half‐life of WNT5A mRNA by monitoring the transcript abundance after transcriptional inhibition with actinomycin D at different time points after siCtrl or siALKBH5 transfection (non‐linear regression). ( F ) RT‐qPCR quantification (left panel) and representative Western blot validation (right panel) of WNT5A expression after the control adenovirus (adCtrl) or ALKBH5‐overexpression adenovirus (adALKBH5) infection. ( G ) RT‐qPCR showing the half‐life of WNT5A mRNA after infection with adCtrl or adALKBH5 (non‐linear regression). ( H ) Representative image (left panel) and quantification (right panel) of RIP‐qPCR verifying the binding of ALKBH5 protein to WNT5A mRNA. ( I ) CCK‐8 assay showing the effect of Foxy5 on CMECS proliferation after adCtrl or adALKBH5 infection (one‐way ANOVA). ( J‐L ) Representative EdU (scale bar = 200 μm), scratch (scale bar = 100 μm), transwell (scale bar = 200 μm), and tube formation (scale bar = 200 μm) images showing the effect of Foxy5 on CMECS proliferation, migration, and tube formation after adCtrl or adALKBH5 infection (one‐way ANOVA). All images were acquired from three random microscopic fields per group and were analyzed using NIH ImageJ software. All data are from three independent experiments and are presented as the mean ± SD. No significant difference is indicated by ns. Significant differences are presented as *( p < 0.05), **( p < 0.01) and determined by Student's t ‐test unless specified

Article Snippet: Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) service was provided by Cloudseq Biotech Inc. (Shanghai, China).

Techniques: Immunoprecipitation, Transfection, Control, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot, Inhibition, Over Expression, Infection, Binding Assay, CCK-8 Assay, Migration, Software

Journal: Frontiers in Genetics

Article Title: Alteration of mRNA 5-Methylcytosine Modification in Neurons After OGD/R and Potential Roles in Cell Stress Response and Apoptosis

doi: 10.3389/fgene.2021.633681

Figure Lengend Snippet: MeRIP qRT-PCR primer information.

Article Snippet: Methylated RNA immunoprecipitation (MeRIP) was performed by Cloudseq Biotech, Inc. (Shanghai, China).

Techniques:

Journal: Frontiers in Genetics

Article Title: Alteration of mRNA 5-Methylcytosine Modification in Neurons After OGD/R and Potential Roles in Cell Stress Response and Apoptosis

doi: 10.3389/fgene.2021.633681

Figure Lengend Snippet: verification of m 5 C methylation positions. (A) Illustration of the MeRIP approach. RNA was extracted from neurons, chemically lysed and then divided into the IP group and input group. The IP group was incubated with anti-5-methylcytosine antibody. Finally, the expression level of candidate transcripts was analyzed by qRT-PCR. The blue line represents the hypermethylated transcripts, the purple line represents the hypomethylated transcripts, and the black line indicates the enrichment of the input group. (B) m 5 C level changes in 5 candidate hypermethylated transcripts and 5 candidate hypomethylated transcripts in the control and OGD/R groups (fold change > 2, p < 0.01). ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Methylated RNA immunoprecipitation (MeRIP) was performed by Cloudseq Biotech, Inc. (Shanghai, China).

Techniques: Methylation, Incubation, Expressing, Quantitative RT-PCR, Control