Journal: iScience

Article Title: IGF2BP2-Shox2 axis regulates hippocampal-neuronal senescence to alleviate microgravity-induced recognition disturbance

doi: 10.1016/j.isci.2024.109917

Figure Lengend Snippet:

Article Snippet: METTL3 (E3F2A) Rabbit mAb , Cell Signaling Technology , Cat# 86132; RRID:AB_2800072.

Techniques: Control, Recombinant, Immunoprecipitation, Staining, Reporter Assay, Virus, Software

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: HeLa cells were infected with rBPIV3-EGFP at an multiplicity of infection (MOI) of 1 and fixed at 48 h post-infection (hpi). The cells were costained with anti-BPIV3-N antibody (Ab) and anti-double-strand RNA (dsRNA) Ab (A) or m6A Ab (B). Representative images from three independent experiments are shown. White boxes in each panel indicate regions shown as magnified views in the corresponding lower-right insets. Cell nuclei were stained with DAPI. Manders’ colocalization coefficient for N with EGFP and with dsRNA (C), or with EGFP and with m6A (D), was calculated using the Coloc 2 plugin in Fiji/ImageJ software, based on analyses of eight cells for panel C and nine cells for panel D. The 293T cells were transfected with empty vector, METTL3, METTL14, ALKBH5, or FTO expression plasmids as m6A factors, and at 24 h post-transfection, the cells were infected with rBPIV3-EGFP at an MOI of 1. At 72 hpi, the culture supernatant was harvested, and the viral titer was determined by the TCID 50 method (E, F). METTL3-KD, METTL14-KD, or control-sh HeLa cells were infected with rBPIV3-EGFP at an MOI of 1. At 48 hpi, supernatants from infected cells were harvested, and viral titers in each cell line were determined using the TCID 50 method (G, H). HeLa cells were pre-treated with 7.5 µM STM2457 24 h before infection, and then the cells were infected with rBPIV3-EGFP at an MOI of 1 in the presence or absence (STM2457 + wash) of STM2457. At 72 hpi, supernatants from infected cells were harvested, and viral titers were determined in each cell line using the TCID 50 method (I, J). CelliterGlo was used to measure the cell viability of the STM2457-treated-infected cells (K). RLU: relative luminescence unit. Data are representative of three independent experiments (n = 3). Asterisks indicate significance (* p < 0.05); ns, not significant. Data represent the mean ± SD from n = 3 independent experiments. Schematic illustrations in panels E, G, and I were created with MS PowerPoint.

Article Snippet: The FLAG-tagged human METTL3 (Addgene, Cat. #53739), METTL14 (Addgene, Cat. #53740), ALKBH5 (Sino Biological, Cat. # HG24078-CF), and FTO (Sino Biological, Cat. # HG12125-NF) expression plasmids were purchased.

Techniques: Infection, Staining, Software, Transfection, Plasmid Preparation, Expressing, Control

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: HeLa cells were transfected with FLAG-METTL3 expression plasmid, and at 24 h post-transfection (hpt), the cells were infected with rBPIV3-EGFP at an MOI of 1. At 48 h post-infection (hpi), the cells were fixed and costained with anti-FLAG antibody (Ab) for METTL3 and anti-BPIV3-N Ab (A). Arrowheads indicate regions where N and METTL3 fluorescence signals colocalize within the cells. The bar graph shows the distribution of METTL3 subcellular localization. For classification-based quantification, more than 30 METTL3 single- or METTL3/N double-positive cells per condition were randomly selected and classified into two localization patterns (“nucleus only” or “cytoplasmic or nucleus”). The number of cells displaying each localization pattern was counted for each condition from three independent experiments, and the results are presented as the percentage of total cells analyzed (B). HeLa cells were transfected with FLAG-METTL3 plasmid and, 24 hpt, infected with rBPIV3-EGFP at an MOI of 1. At 48 hpi, cells were fixed and co-stained with antibodies against dsRNA and FLAG-METTL3 (C) or dsRNA and endogenous METTL3 (endo METTL3) (D). Representative images from three independent experiments are shown. Manders’ colocalization coefficient between dsRNA and FLAG-METTL3 (E) or dsRNA and endogenous METTL3 (F) fluorescence signals in panels C and D was calculated using the Coloc 2 plugin in Fiji/ImageJ software. The coefficient was determined for each cell, and results represent analyses of 6–7 individual cells per condition from three independent experiments. Bars indicate the mean ± SD. White boxes in panels C and D indicate regions shown as magnified views in the corresponding lower-right insets. Data are representative of three independent experiments (n = 3). Asterisks indicate significance (* p < 0.05); ns, not significant.

Article Snippet: The FLAG-tagged human METTL3 (Addgene, Cat. #53739), METTL14 (Addgene, Cat. #53740), ALKBH5 (Sino Biological, Cat. # HG24078-CF), and FTO (Sino Biological, Cat. # HG12125-NF) expression plasmids were purchased.

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Fluorescence, Staining, Software

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: HeLa cells were cotransfected with METTL3 and M expression plasmid or an empty vector. At 48 h post-transfection (hpt), the cells were fixed and costained with anti-BPIV3-M antibody (Ab) and anti-FLAG Ab for METTL3 (A). HeLa cells were transfected with METTL3 expression plasmid, and at 24 hpt, the cells were infected with rBPIV3-EGFP at an MOI of 1. At 48 h post-infection (hpi), the cells were fixed and costained with anti-FLAG antibody (Ab) for METTL3 and anti-BPIV3-M Ab (B). Cell nuclei were stained with DAPI. Insets represent an enlargement of the areas indicated by a small square. Arrowheads indicate cytoplasmic colocalization of M with METTL3. Representative images from three independent experiments are shown. For classification-based quantification, more than 20 METTL3 single-positive or METTL3/M double-positive cells per condition were randomly selected and categorized into two subcellular localization patterns (“nucleus only” or “cytoplasmic or nucleus”). The number of cells in each category was counted, and the data are presented as the percentage of total cells analyzed from n = 3 independent experiments. (C and D). Asterisks indicate statistically significant differences (* p < 0.05); ns, not significant.

Article Snippet: The FLAG-tagged human METTL3 (Addgene, Cat. #53739), METTL14 (Addgene, Cat. #53740), ALKBH5 (Sino Biological, Cat. # HG24078-CF), and FTO (Sino Biological, Cat. # HG12125-NF) expression plasmids were purchased.

Techniques: Expressing, Plasmid Preparation, Transfection, Infection, Staining

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: (A) Schematic representation of BPIV3-M point mutants. BPIV3-M contains a nuclear export signal (NES) at L106 and L107 and a ubiquitination site (Ubi site) at K258. L106 and L107, or K258 in BPIV3-M, were substituted with alanine (M-L106A/L107A) or arginine (M-K258R) residues as indicated, respectively, and the substituted residue is shown as a red letter. (B) HeLa cells were cotransfected with expression plasmids of wt-BPIV3-M, M-L106A/L107A, and K258R along with FLAG-METTL3 expression plasmid. At 48 h post-transfection (hpt), the cells were costained with anti-M antibody (Ab) and anti-FLAG Ab for METTL3. (C) HeLa cells were transfected with the METTL3 expression plasmid. At 24 hpt, the cells were infected with rBPIV3-EGFP at an MOI of 1. At 4 h post-infection (hpi), DMSO or 5 ng/mL Leptomycin B (LMB) was added, and the cells were incubated for 48 h in the presence of LMB. Then, the cells were fixed, and METTL3 and BPIV3-N were costained. (D) HeLa cells were transfected with siRNA targeting Exportin-1 (si-Exportin-1) or control siRNA (si-Control). At 48 hpt, cells were lysed and subjected to immunoblotting with anti–Exportin-1 antibody to confirm knockdown efficiency. (E) HeLa cells were cotransfected with siRNA for Exportin-1 and METTL3 expression plasmid. At 24 hpt, the cells were infected with rBPIV3-EGFP at an MOI of 1. The cells were fixed at 48 hpi and costained for BPIV3-N and METTL3. Representative images from three independent experiments are shown. For classification-based quantification, more than 20 METTL3/M double-positive cells (F), METTL3/N double-positive cells (G) , or METTL3/N double-positive cells under siRNA conditions (H) per condition were randomly selected and categorized into two subcellular localization patterns (“nucleus only” or “cytoplasmic or nucleus”). The number of cells in each category was counted, and the data are presented as the percentage of total cells analyzed from n = 3 independent experiments. Asterisks indicate statistically significant differences (* p < 0.05).

Article Snippet: The FLAG-tagged human METTL3 (Addgene, Cat. #53739), METTL14 (Addgene, Cat. #53740), ALKBH5 (Sino Biological, Cat. # HG24078-CF), and FTO (Sino Biological, Cat. # HG12125-NF) expression plasmids were purchased.

Techniques: Ubiquitin Proteomics, Residue, Expressing, Plasmid Preparation, Transfection, Infection, Incubation, Control, Western Blot, Knockdown

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: (A) 293T cells were cotransfected with M expression plasmid together with FLAG-METTL3 expression plasmid. For infected cells, the cells were transfected with FLAG-METTL3 expression plasmid and infected with rBPIV3-EGFP at an MOI of 1 at 24 h post-transfection (hpt). At 72 hpt, the cells were harvested and subjected to coimmunoprecipitation with anti-FLAG antibody (Ab). The precipitates were analyzed by western blotting using anti-BPIV3-M Ab. (B) Schematic representation of human METTL3. ZF1 and ZF2: two zinc finger domains, MTD: methyltransferase domain, DPPW: DPPW motif. The 1-400, 1-380, 1-200, and 161-580 deletion mutants lacking each domain are shown. 293T cells were cotransfected with the deletion mutants along with BPIV3-M. At 48 hpt, the cells were harvested and subjected to coimmunoprecipitation with anti-FLAG Ab as described in the legend of . (C) Schematic representation of FLAG- and GST-tagged deletion mutants of the BPIV3 M protein used to map the METTL3 interaction domain. Cells were transfected with a FLAG-METTL3 expression plasmid, and cells were harvested at 48–72 h post-transfection. Deletion mutants of the M protein fused with FLAG and GST tags were synthesized using a wheat germ cell-free expression system. Each FLAG-GST-tagged M deletion mutant was incubated with glutathione–sepharose beads at 4°C, followed by the addition of lysates containing FLAG-METTL3. After incubation, the beads were extensively washed, and bound proteins were analyzed by western blotting. Both METTL3 and M deletion mutants were detected using an anti-FLAG antibody. Each experiment was performed at least twice.

Article Snippet: The FLAG-tagged human METTL3 (Addgene, Cat. #53739), METTL14 (Addgene, Cat. #53740), ALKBH5 (Sino Biological, Cat. # HG24078-CF), and FTO (Sino Biological, Cat. # HG12125-NF) expression plasmids were purchased.

Techniques: Expressing, Plasmid Preparation, Infection, Transfection, Western Blot, Synthesized, Mutagenesis, Incubation

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: (A) A schematic diagram of the RNA immunoprecipitation (RIP) assay used to detect METTL3-associated N mRNA. (B) 293T cells were transfected with METTL3 expression plasmids. At 24 h post-transfection (hpt), the cells were infected with rBPIV3-EGFP at an MOI of 1 for 48 h. The infected cells were lysed, and the extracts were subjected to RNA immunoprecipitation assay with anti-FLAG antibody (Ab). After immunoprecipitation, cDNA of BPIV3-N mRNA captured by METTL3 was synthesized and the amount of N mRNA was quantified by qPCR. Followed by normalization to input RNA values, relative enrichment values of N mRNA were expressed relative to control IgG values using the ΔΔCt method. (C) SRAMP was used to predict the m6A site of the N gene derived from the BPIV3 genome. The vertical axis shows the m6A score and the horizontal axis shows the position number of the base of the N gene where N6-methyladenosine is present within the m6A motif. A yellow bar indicates low-scoring m6A site, N327, and red bars indicate high-scoring m6A sites, N872, N1146, N1372, N1427, and N1443. (D) Schematic representation of the MeRIP (m6A RNA immunoprecipitation) assay. (i) The m6A-containing RNA fragments were bound by an m6A-specific Ab. (ii) Viral N mRNA containing m6A modifications was fragmented. (iii) Antibody–RNA complexes were captured using protein affinity beads. (iv) RNA was eluted, reverse transcribed into cDNA, and amplified by qPCR using primers specific for the indicated N gene regions (N327, N872, N1146, and N1372–1443). (E) 293T cells were infected with rBPIV3-EGFP at an MOI of 1. At 72 h post-infection, the infected cells were harvested, and only mRNA from total RNA was purified. The m6A-modified mRNA was immunoprecipitated with m6A-specific Ab and magnetic beads. Followed by cleavage of the captured mRNA with the cleavage enzyme, the m6A-specific Ab-captured mRNA was eluted from magnetic beads and purified. cDNA was synthesized by reverse transcription reaction, and the m6A sites in N mRNA were then identified by qPCR. The relative value of each to the mRNA value of input RNA was calculated using the ΔΔCt method. Data represent the mean ± SD from n = 3 independent experiments. Asterisks represent statistically significant differences (* p < 0.05). ns; not significant.

Article Snippet: The FLAG-tagged human METTL3 (Addgene, Cat. #53739), METTL14 (Addgene, Cat. #53740), ALKBH5 (Sino Biological, Cat. # HG24078-CF), and FTO (Sino Biological, Cat. # HG12125-NF) expression plasmids were purchased.

Techniques: RNA Immunoprecipitation, Transfection, Expressing, Infection, Immunoprecipitation, Synthesized, Control, Derivative Assay, Reverse Transcription, Amplification, Purification, Modification, Magnetic Beads

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: The newly synthesized paramyxovirus matrix (M) protein enters the nucleus and binds to METTL3 and METTL14 methyltransferase complexes. Exportin-1 recognizes M protein, facilitating the export of M-writer complexes to the cytoplasm. These complexes are then recruited into cytoplasmic inclusion bodies, which serve as viral RNA synthesis sites. Within these structures, m6A writers modify viral RNA, particularly N mRNA. The nuclear efflux of METTL3, driven by M protein, depletes nuclear m6A writers, leading to abnormal m6A modification of host mRNAs. Interferon-beta (IFN-β) mRNA is especially affected, showing reduced m6A levels. Consequently, reduced IFN-β mRNA levels result in attenuated antiviral and immune responses, allowing for more efficient viral replication. This process represents a sophisticated viral strategy to enhance replication while simultaneously suppressing host defense mechanisms, illustrating how paramyxoviruses manipulate cellular m6A machinery to their advantage. Schematic illustration in this Fig was created with MS PowerPoint.

Article Snippet: The FLAG-tagged human METTL3 (Addgene, Cat. #53739), METTL14 (Addgene, Cat. #53740), ALKBH5 (Sino Biological, Cat. # HG24078-CF), and FTO (Sino Biological, Cat. # HG12125-NF) expression plasmids were purchased.

Techniques: Synthesized, Modification

Journal: Retrovirology

Article Title: YTHDC1 regulates distinct post-integration steps of HIV-1 replication and is important for viral infectivity

doi: 10.1186/s12977-022-00589-1

Figure Lengend Snippet: Antibodies

Article Snippet: Anti-METTL3 , Bethyl A301-567A , Western Blot.

Techniques: Immunoprecipitation, Western Blot

Journal: Cell Proliferation

Article Title: Cross talk between RNA N6‐methyladenosine methyltransferase‐like 3 and miR‐186 regulates hepatoblastoma progression through Wnt/β‐catenin signalling pathway

doi: 10.1111/cpr.12768

Figure Lengend Snippet: High expression level of METTL3 is correlated with poor prognosis in HB. A, The univariate and multivariate Cox regression analyses were performed to depict the correlations between the indicated clinical criteria and the expression levels of m6A‐related genes. B, C, Representative IHC staining and scoring of METTL3 in HB tissues and normal non‐tumour tissues of TMA (ZZU cohort). Scale bars, 200 μm. D‐G, The correlation between the expression of METTL3 and vascular invasion (D), distant metastasis (E), recurrence (F) and COG stage (G). *** P < .001

Article Snippet: HuH‐6, Hepa1‐6 and HCCLM9 cells (1 × 10 5 ) were transduced with lentivirus encoding miR‐186 mimics, miR‐186 inhibitor, METTL3 overexpression plasmid, METTL3 shRNA or an empty lentivirus for 96 hours and then selected by puromycin treatment (Santa Cruz Biotechnology) for 4 weeks.

Techniques: Expressing, Immunohistochemistry

Journal: Cell Proliferation

Article Title: Cross talk between RNA N6‐methyladenosine methyltransferase‐like 3 and miR‐186 regulates hepatoblastoma progression through Wnt/β‐catenin signalling pathway

doi: 10.1111/cpr.12768

Figure Lengend Snippet: Knockdown of METTL3 inhibits proliferation, migration and invasion of HB cells in vitro. A, The protein expression levels of METTL3 in HB cell lines (HepG2 and HuH‐6) and normal liver cell lines (L02 and Chang liver) were determined by Western blot analysis. HepG2 or HuH‐6 cells were transfected with negative control (NC) or different concentrations of METTL3 siRNA. B, The protein expression levels of METTL3 were analysed by Western blot 48 h later. C, D, CCK‐8 assay, (E) EDU staining (scale bars, 50 μm) and (F) colony formation assays (scale bars, 8 mm) were performed to determine the cell proliferation, DNA synthesis and colony formation in HepG2 or HuH‐6 cells. G, Wound‐healing assay (scale bars, 500 μm) and (H) Transwell assay (scale bars, 50 μm) were performed to assess the cell migration and invasion capacity. I, Representative micrographs and quantification of TUNEL‐positive signalling in the indicated assay (scale bars, 50 μm). J, Western blot analysis of Bcl‐2, Bax, Bak and cleaved caspase‐3 proteins in HepG2 and HuH‐6 cells. *P < .05, ** P < .01, *** P < .001

Article Snippet: HuH‐6, Hepa1‐6 and HCCLM9 cells (1 × 10 5 ) were transduced with lentivirus encoding miR‐186 mimics, miR‐186 inhibitor, METTL3 overexpression plasmid, METTL3 shRNA or an empty lentivirus for 96 hours and then selected by puromycin treatment (Santa Cruz Biotechnology) for 4 weeks.

Techniques: Knockdown, Migration, In Vitro, Expressing, Western Blot, Transfection, Negative Control, CCK-8 Assay, Staining, DNA Synthesis, Wound Healing Assay, Transwell Assay, TUNEL Assay

Journal: Cell Proliferation

Article Title: Cross talk between RNA N6‐methyladenosine methyltransferase‐like 3 and miR‐186 regulates hepatoblastoma progression through Wnt/β‐catenin signalling pathway

doi: 10.1111/cpr.12768

Figure Lengend Snippet: miR‐186 directly targets METTL3 and negatively regulates METTL3 expression. A, A total of 6 miRNA (miR‐520b, miR‐186, miR‐372, miR‐302d, miR‐600 and miR‐34c‐3p)‐targeted METTL3 were selected. B, A total of 6 miRNAs (miR‐520b, miR‐186, miR‐372, miR‐302d, miR‐600 and miR‐34c‐3p) were transfected into HuH‐6 cells, and the inhibitory effect on METTL3 expression was evaluated by qRT‐PCR. C, The protein expression levels of METTL3 were assessed by Western blot. D, Bioinformatics analysis predicted miR‐186 binding sites located in the 3’‑UTR of METTL3. E, The mRNA expression levels of METTL3 were assessed by qRT‐PCR. F, WT 3’‐UTR or mutated 3’‐UTR of METTL3 was cloned and constructed into luciferase reporter vector, and then co‐transfected with miR‐186 mimics or NC control into HEK293 cells. Relative luciferase activity was assessed 48 h after transfection. G, The expression of miR‐186 in HB tissues and non‐tumour tissues in GEO database was analysed. H, The expression of miR‐186 in HB tissues and non‐tumour tissues in ZZU cohort was assessed by qRT‐PCR. I, Pearson correlation analysis of the relationship between METTL3 expression level and miR‐186 expression level in ZZU cohort. * P < .05, ** P < .01, *** P < .001

Article Snippet: HuH‐6, Hepa1‐6 and HCCLM9 cells (1 × 10 5 ) were transduced with lentivirus encoding miR‐186 mimics, miR‐186 inhibitor, METTL3 overexpression plasmid, METTL3 shRNA or an empty lentivirus for 96 hours and then selected by puromycin treatment (Santa Cruz Biotechnology) for 4 weeks.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Binding Assay, Clone Assay, Construct, Luciferase, Plasmid Preparation, Control, Activity Assay

Journal: Cell Proliferation

Article Title: Cross talk between RNA N6‐methyladenosine methyltransferase‐like 3 and miR‐186 regulates hepatoblastoma progression through Wnt/β‐catenin signalling pathway

doi: 10.1111/cpr.12768

Figure Lengend Snippet: Overexpression of miR‐186 suppresses HB cell proliferation and invasion by targeting of METTL3. HepG2 or HuH‐6 cells were transfected with NC, miR‐186 mimics, and miR‐186 mimics & METTL3 overexpression plasmid. A, Western blot analysis of METTL3 protein levels in different groups. B, C, CCK‐8 assay, (D) EDU staining (scale bars, 50 μm) and (E) colony formation assays (scale bars, 8 mm) were performed to evaluate cell proliferation, DNA synthesis and colony formation of HepG2 or HuH‐6 cells. F, Cell invasion of HepG2 or HuH‐6 cells in different groups was analysed by Transwell assay. Scale bars, 50 μm (G) Cell migration capability of HepG2 or HuH‐6 cells in different groups was analysed by wound‐healing assay. Scale bars, 500 μm. *P < .05. ** P < .01, *** P < .001

Article Snippet: HuH‐6, Hepa1‐6 and HCCLM9 cells (1 × 10 5 ) were transduced with lentivirus encoding miR‐186 mimics, miR‐186 inhibitor, METTL3 overexpression plasmid, METTL3 shRNA or an empty lentivirus for 96 hours and then selected by puromycin treatment (Santa Cruz Biotechnology) for 4 weeks.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, CCK-8 Assay, Staining, DNA Synthesis, Transwell Assay, Migration, Wound Healing Assay

Journal: Cell Proliferation

Article Title: Cross talk between RNA N6‐methyladenosine methyltransferase‐like 3 and miR‐186 regulates hepatoblastoma progression through Wnt/β‐catenin signalling pathway

doi: 10.1111/cpr.12768

Figure Lengend Snippet: miR‐186 suppresses tumour growth and metastasis in vivo . A, Representative image of Luciferase signal emission in Lenti‐miR‐186 group and Lenti‐NC group. B, Relative photon flux in the Lenti‐miR‐186 group and Lenti‐NC group was quantified and analysed using the IVIS imaging system 5 wk after implantation. C, Growth curves of tumour volumes in xenografts of nude mice were determined based on tumour volume measured every week. D, Representative images of tumour and quantitative analysis of xenograft tumour weight in Lenti‐NC and Lenti‐miR‐186 groups. E‐G, Representative immunohistochemical staining images and relative expression levels of Ki‐67 and METTL3 in tumours from Lenti‐miR‐186 group and Lenti‐NC group. Scale bars, 200 μm. H, I, Representative results of gross and H&E staining of metastatic lung nodules in different groups. Scale bars, 200 μm. J, K, Relative photon flux of orthotopic transplanted tumour in Lenti‐miR‐186 and Lenti‐NC groups was quantified and analysed. L, Representative images of orthotopic tumours in the liver of Lenti‐miR‐186 and Lenti‐NC groups after dissection. M, The ratio of liver weight/body weight in orthotopical mice from Lenti‐miR‐186 and Lenti‐NC groups.* P < .05, ** P < .01, *** P < .001

Article Snippet: HuH‐6, Hepa1‐6 and HCCLM9 cells (1 × 10 5 ) were transduced with lentivirus encoding miR‐186 mimics, miR‐186 inhibitor, METTL3 overexpression plasmid, METTL3 shRNA or an empty lentivirus for 96 hours and then selected by puromycin treatment (Santa Cruz Biotechnology) for 4 weeks.

Techniques: In Vivo, Luciferase, Imaging, Immunohistochemical staining, Staining, Expressing, Dissection

Journal: Cell Proliferation

Article Title: Cross talk between RNA N6‐methyladenosine methyltransferase‐like 3 and miR‐186 regulates hepatoblastoma progression through Wnt/β‐catenin signalling pathway

doi: 10.1111/cpr.12768

Figure Lengend Snippet: miR‐186/METTL3 axis regulates Wnt/β‐catenin signalling in human HB. A, GO and (B) KEGG analysis showed the enriched biological functions of differentially expressed gene profiles. C, GSVA showed the correlations between METTL3 mRNA levels and signalling pathways. D, E, The protein levels of Wnt/β‐catenin pathway‐related targets in HepG2 or HuH‐6 cells transfected with miR‐186 mimics, METTL3 plasmid, miR‐186 inhibitor, METTL3 siRNA or relative NC. F, Representative immunohistochemical staining images and relative expression levels of Wnt/β‐catenin pathway‐associated proteins in tumours from Lenti‐miR‐186 group and Lenti‐NC group. Scale bars, 200 μm. G, The proposed function model of miR‐186/METTL3 axis in HB

Article Snippet: HuH‐6, Hepa1‐6 and HCCLM9 cells (1 × 10 5 ) were transduced with lentivirus encoding miR‐186 mimics, miR‐186 inhibitor, METTL3 overexpression plasmid, METTL3 shRNA or an empty lentivirus for 96 hours and then selected by puromycin treatment (Santa Cruz Biotechnology) for 4 weeks.

Techniques: Transfection, Plasmid Preparation, Immunohistochemical staining, Staining, Expressing

Journal: bioRxiv

Article Title: Differential requirement of m 6 A reader proteins, IGF2BP2 and HNRNPA2B1 for the processing of N 6 -methyladenosine modified H19 lncRNA: Stability versus miR-675 biogenesis

doi: 10.1101/2024.08.07.606971

Figure Lengend Snippet: A. The schematic represents the H19 transcript and cognate miR-675 derived from its first exon (the red-colored sequences represent miR-675-5p). B. m 6 A pulldown, followed by qRT-PCR measured the enrichment of m 6 A in the H19 transcript. m 6 A enrichment in H19 transcripts in control and METTL3-silencing cells using m6A RIP-qPCR. C. The METTL3 binding to the H19 and SOX2 transcripts was quantified by METTL3-RIP-PCR. D. METTL3 expression in LN229 cells transfected with control, or METTL3 shRNA was detected by immunoblotting. GAPDH served as the control. E & F. The expression of H19 and miR-675-5p were measured by qRT-PCR in METTL3 silenced LN229 cells. G. The stability of the H19 transcript was measured at indicated time points post Actinomycin D (5 μg/ml) treatment in shNT and shMETTL3-transfected LN229 cells by qRT-PCR. The log2 ratio of the remaining H19 was plotted using linear regression after normalizing to the 0th hour of the respective condition. H. METTL3 expression in METTL3 overexpressed LN229 cells was detected by immunoblotting. GAPDH served as the control. I & J. The expression of H19 and miR-675-5p were measured by qRT-PCR in METTL3 overexpressing LN229 cells. K & L. The expression levels of H19 and miR-675-5p were measured by qRT-PCR in H19-overexpressing LN229 cells. M. The expression of METTL3 were measured by immunoblotting in shNT and shMETTL3 LN229 cells. N & O. H19 and miR-675-5p were measured in stable H19 overexpressed with METTL3 silenced cells. Data are shown as mean ± SD (n=3). Asterisks indicate statistical significance: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001, and ‘ns’ for non-significant differences.

Article Snippet: The over-expression constructs of METTL3 were obtained from Origene (#RC200869, U.S.A), and the over-expression constructs of H19 were obtained from Addgene (#122473, U.S.A).

Techniques: Derivative Assay, Quantitative RT-PCR, Control, Binding Assay, Expressing, Transfection, shRNA, Western Blot

Journal: bioRxiv

Article Title: Differential requirement of m 6 A reader proteins, IGF2BP2 and HNRNPA2B1 for the processing of N 6 -methyladenosine modified H19 lncRNA: Stability versus miR-675 biogenesis

doi: 10.1101/2024.08.07.606971

Figure Lengend Snippet: A. The Venn diagram depicts the number of RBPs that are (1) positively correlated with H19 in the TCGA RNA-Seq cohort, (2) experimentally validated to bind H19 in humans, and (3) experimentally validated to bind H19 in mice, as obtained from the ENCORI database. HNRNPA2B1 and IGF2BP2 are highlighted as the only two RBPs that bind to H19 and are conserved in humans and mice. B. The scatter plot shows log2-transformed H19 expression on the x-axis and IGF2BP2 expression on the y-axis. The regression line with a 95% confidence interval indicates the overall correlation trend, with Pearson’s correlation coefficient (r) and p-value (p) provided to quantify the strength and statistical significance of the correlation. C. The scatter plot shows log2-transformed H19 expression on the x-axis and HNRNPA2B1 expression on the y-axis. The regression line with a 95% confidence interval indicates the overall correlation trend, with Pearson’s correlation coefficient (r) and p-value (p) provided to quantify the strength and statistical significance of the correlation. D. The schematic represents 4s1m-H19 transcript structure for pulldown to validate the interaction between H19 and reader proteins. E. We performed a pulldown of 4s1m-H19 using streptavidin magnetic beads, followed by quantification of H19 using qRT-PCR. F. We conducted the pulldown of 4s1m-H19, followed by protein elution to demonstrate the interaction between H19 and reader proteins (HNRNPA2B1 and IGF2BP2) using immunoblotting. G. We conducted immunoprecipitation using antibodies against IGF2BP2 and HNRNPA2B1, and the eluted proteins were subsequently subjected to SDS-PAGE, followed by immunoblotting using antibodies against HNRNPA2B1 and IGF2BP2. H. We performed immunoprecipitation using antibodies against IGF2BP2 and HNRNPA2B1, and the eluted RNA was subsequently analyzed using qRT-PCR. I. The protein levels of HNRNPA2B1 and IGF2BP2 were assessed by immunoblotting in HNRNPA2B1 and IGF2BP2 silenced LN229 cells compared to control cells. J & K. We conducted qRT-PCR for H19 and miR-675 in HNRNPA2B1 and IGF2BP2 silenced LN229 cells. L. The stability of the H19 transcript was measured at indicated time points post Actinomycin D (5 μg/ml) treatment in shHNRNPA2B1 and shIGF2BP2-transfected LN229 cells by qRT-PCR. The log2 ratio of the remaining H19 was plotted using linear regression after normalizing to the 0th hour of the respective condition. M & N. H19 and miR-675-5p levels were assessed via qRT-PCR in METTL3 overexpressed cells where HNRNPA2B1 and IGF2BP2 were silenced. O & P. H19 and miR-675-5p levels were assessed via qRT-PCR in H19 overexpressed LN229 cells where HNRNPA2B1 and IGF2BP2 were silenced. Data are shown as mean ± SD (n=3). Asterisks indicate statistical significance: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001, and ‘ns’ for non-significant differences.

Article Snippet: The over-expression constructs of METTL3 were obtained from Origene (#RC200869, U.S.A), and the over-expression constructs of H19 were obtained from Addgene (#122473, U.S.A).

Techniques: RNA Sequencing, Transformation Assay, Expressing, Magnetic Beads, Quantitative RT-PCR, Western Blot, Immunoprecipitation, SDS Page, Control, Transfection

Journal: bioRxiv

Article Title: Differential requirement of m 6 A reader proteins, IGF2BP2 and HNRNPA2B1 for the processing of N 6 -methyladenosine modified H19 lncRNA: Stability versus miR-675 biogenesis

doi: 10.1101/2024.08.07.606971

Figure Lengend Snippet: A. This figure shows box-violin plots comparing CALN1 expression levels in GBM (red) and control (green) samples across 22 cancer types obtained from the TCGA Pan-Cancer Atlas Project. The y-axis displays RSEM-normalized gene expression values. Statistical differences between GBM and control groups were assessed with the Student’s t-test, and p-values are shown in the panels. B. Comparative analysis of CALN1 expression in GBM and control samples across nine independent cohorts. The box-violin plots illustrate the gene expression distribution for each group within each cohort, with GBM samples depicted in red and control samples in green. The y-axis is scaled to represent the log2-transformed normalized gene expression values. Statistical significance between GBM and control groups within each cohort was assessed using the Student’s t-test, with p-values indicated in the panels. C & D. CALN1 expression was quantified using qRT-PCR in shMETTL3, shHNRNPA2B1, and shIGF2BP2 cells compared to the control. E. LN229 cells with silenced METTL3 were transfected with either a miR-675-5p mimic or negative control (NC), followed by qRT-PCR analysis to measure miR-675-5p expression. F & G. The Boyden chamber invasion and migration assays were quantified on LN229 cells with silenced METTL3 and a miR-675 mimic. H. The migratory capacity of cells with silenced IGF2BP2 and HNRNPA2B1 transfected with a miR-675-5p mimic was evaluated using a scratch wound assay. Data are shown as mean ± SD (n=3). Significance is denoted by asterisks: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, **** for p < 0.0001, and ‘ns’ for non-significant differences.

Article Snippet: The over-expression constructs of METTL3 were obtained from Origene (#RC200869, U.S.A), and the over-expression constructs of H19 were obtained from Addgene (#122473, U.S.A).

Techniques: Expressing, Control, Gene Expression, Transformation Assay, Quantitative RT-PCR, Transfection, Negative Control, Migration, Scratch Wound Assay Assay

Journal: bioRxiv

Article Title: Differential requirement of m 6 A reader proteins, IGF2BP2 and HNRNPA2B1 for the processing of N 6 -methyladenosine modified H19 lncRNA: Stability versus miR-675 biogenesis

doi: 10.1101/2024.08.07.606971

Figure Lengend Snippet: Proposed working model of this study. Decoding the role of METTL3 mediated m 6 A modification on H19 (created with BioRender.com ).

Article Snippet: The over-expression constructs of METTL3 were obtained from Origene (#RC200869, U.S.A), and the over-expression constructs of H19 were obtained from Addgene (#122473, U.S.A).

Techniques: Modification