Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Reply to the Comment on "The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ".

doi: 10.1002/advs.202201452

Figure Lengend Snippet: Figure 1. IGF2BP2 expression in LPS activated human macrophages. A) IGF2BP2 mRNA level in HMDMs treated by saline (Control) or LPS (100 ng mL−1) at indicated time. B) IGF2BP2 protein expression in HMDMs treated by normal saline or LPS (100 ng mL−1) for 4 and 8 h. C) Statistically analysis of (B). D) IGF2BP2 protein expression in HMDMs treated by saline or LPS (100 ng mL−1) for 24, 36, and 48 h. E) Statistically analysis of (D). F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP). The mRNA levels in the Control group were set to 1 and levels in others experimental conditions were relative to that after normalization with GAPDH. The dates were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, versus the control group. p-values were determined by using two-way analysis of variance (ANOVA).

Article Snippet: F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP).

Techniques: Expressing, Saline, Control, Quantitative RT-PCR, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Reply to the Comment on "The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ".

doi: 10.1002/advs.202201452

Figure Lengend Snippet: Figure 2. IGF2BP2 expression in LPS activated mouse macrophages. A) IGF2BP2 mRNA level in BMDMs treated by saline (Control) or LPS (100 ng mL−1) at the indicated time. B) IGF2BP2 protein expression in BMDMs treated by normal saline or LPS (100 ng mL−1) at the indicated time points, anti-IGF2BP2 antibody was purchased from Proteintech (11601- 1-AP); qPCR and western blot biological replicates from three different donors; IGF2BP2 mRNA levels in Control group were set to 1 and levels in others experimental conditions were relative to that after normalization with GAPDH. The dates were shown as mean ± SEM. *p < 0.05, **p < 0.01, versus the Control group. p-values were determined by using a two- way analysis of variance (ANOVA).

Article Snippet: F–I) Inflammatory cytokine and TSC1 mRNA level in HMDMs determined by RT-qPCR after normal saline or LPS treatment at indicated time points; qPCR biological replicates come from six different donors; western blot biological replicates come from three different donors; anti-IGF2BP2 antibody was purchased from Proteintech (11601-1-AP).

Techniques: Expressing, Saline, Control, Western Blot

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 1 The expression of IGF2BP3 was related to progression and COG risk in NB patients. A Differential expression of m6A methylation regulators between subgroups stratified by progression (N: non-progression; P: progression) in dataset GSE49711. B Differential expression of m6A methylation regulators between subgroups stratified by COG risk (Children’s Oncology Group risk stratification) in dataset GSE49711. C Univariate Cox regression analysis evaluating independently predictive ability of m6A methylation regulators for overall survival of NB patients in TARGET databases. D IGF2BP3 expression level in progression and non-progression NB patients of dataset GSE49711. E IGF2BP3 expression level in high COG risk and nonhigh COG risk NB patients of dataset GSE49711. F The expression levels of IGF2BP3 and MYCN were positively correlated.

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Expressing, Quantitative Proteomics, Methylation

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 2 IGF2BP3 is upregulated in MYCN-amplified NB tissues and cell lines. A Immunohistochemistry images of IGF2BP3 in MYCN-amplified and non-MYCN-amplified NB tissues (n = 35). B The expression level of IGF2BP3 in MYCN-amplified tissues was higher than that in non-MYCN- amplified tissues (n = 35). C qRT-PCR analysis of the expression level of IGF2BP3 in NB cell lines. D Western blot analysis of the expression level of IGF2BP3 in NB cell lines (n = 3).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 3 Knockdown of IGF2BP3 inhibited N-myc expression in MYCN-amplified NB cells. A qRT-PCR analysis of the expression level of IGF2BP3 mRNA in SK-N-BE(2) (n = 3); B qRT-PCR analysis of the expression level of MYCN mRNA in SK-N-BE(2) (n = 3); C qRT-PCR analysis of the expression level of IGF2BP3 mRNA in BE(2)-C (n = 3); D qRT-PCR analysis of the expression level of MYCN mRNA in BE(2)-C (n = 3); E RNA-FISH and immunofluorescence analysis of the expression level of MYCN mRNA and IGF2BP3 in SK-N-BE(2) (Green: MYCN mRNA; Red: IGF2BP3 protein; Blue: DAPI); F RNA-FISH and immunofluorescence analysis of the expression level of MYCN mRNA and IGF2BP3 in BE(2)-C (Green: MYCN mRNA; Red: IGF2BP3 protein; Blue: DAPI).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Knockdown, Expressing, Quantitative RT-PCR

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 4 Knockdown of IGF2BP3 repressed proliferation of MYCN-amplified NB cells. A Monolayer colonies were detected in SK-N-BE(2) and BE(2)-C (n = 3); B, C Xenograft formation of SK-N-BE(2) cells (n = 6); D, E Xenograft formation of BE(2)-C cells (n = 6).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Knockdown

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 6 IGF2BP3 regulated NB cell proliferation via reading m6A modification of MYCN. A IGF2BP3 could bind to MYCN RNA predicted by RBPsuite website tools; B The enrichment of IGF2BP3 in the mRNA of MYCN performed by RIP-qPCR assay in SK-N-BE(2) (n = 3); C The enrichment of IGF2BP3 in the mRNA of MYCN performed by RIP-qPCR assay in BE(2)-C (n = 3); D, E The m6A modification site of MYCN predicted by SRAMP website tools based on sequence-derived features, and primers designed for MeRIP-qPCR assay. F Obvious m6A modification of MYCN confirmed by MeRIP-qPCR, and knockdown of m6A writer METTL3 repressed MYCN m6A modification in SK-N-BE(2) (n = 3); G Obvious m6A modification of MYCN confirmed by MeRIP-qPCR, and knockdown of m6A writer METTL3 repressed MYCN m6A modification in BE(2)-C (n = 3); H The mRNA stability and degradation halftime of MYCN in SK-N-BE(2) treated by Actinomycin D (n = 3); I The mRNA stability and degradation halftime of MYCN in BE(2)-C treated by Actinomycin D (n = 3).

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Sequencing, Derivative Assay, Knockdown

Journal: Cell death discovery

Article Title: RNA N6-methyladenosine reader IGF2BP3 interacts with MYCN and facilitates neuroblastoma cell proliferation.

doi: 10.1038/s41420-023-01449-3

Figure Lengend Snippet: Fig. 7 N-myc directly promotes the IGF2BP3 expression in NB cells. A qRT-PCR analysis of the expression level of IGF2BP3 mRNA in SK-N- BE(2) (n = 3); B qRT-PCR analysis of the expression level of MYCN mRNA in SK-N-BE(2) (n = 3); C qRT-PCR analysis of the expression level of IGF2BP3 mRNA in BE(2)-C (n = 3); D qRT-PCR analysis of the expression level of MYCN mRNA in BE(2)-C (n = 3); E Schematic illustration of E-boxes (CANNTG) element in IGF2BP3 promoter; F, G Analysis of the effect of N-myc on IGF2BP3 promoter by ChIP-qPCR (n = 3); H Analysis of the targeting effect of N-myc on IGF2BP3 promoter by luciferase reporter assay (n = 3); I Schematic diagram of a positive feedback loop between RNA N6-methyladenosine reader IGF2BP3 and MYCN facilitates NB proliferation.

Article Snippet: Subsequently, the corresponding antibodies were used to label IGF2BP3 (1:100, Cat No. 14642-1-AP, Proteintech Group, Inc.) or METTL3 (1:200, Cat No. 15073-1-AP, Proteintech Group, Inc.).

Techniques: Expressing, Quantitative RT-PCR, ChIP-qPCR, Luciferase, Reporter Assay

Journal: Discover Oncology

Article Title: Novel N 6 -methyladenosine (m 6 A) writer METTL16 promotes the cervical cancer tumorigenesis by targeting FTH1-dependent ferroptosis

doi: 10.1007/s12672-026-04403-8

Figure Lengend Snippet: FTH1 acted as the target of METTL16. A The immunofluorescence assay showed the subcellular localization of METTL16 and FTH1. B The binding motif sites of METTL16 for its target genes (GGAAC). C The FTH1 level in cervical cancer cells was test by the RT-PCR. D RNA immunoprecipitation (RIP) assay was conducted for the binding within METTL16 and FTH1 in HeLa cells. E RIP assay using anti-m 6 A conducted for the m 6 A modification on FTH1 mRNA. * p < 0.05, ** p < 0.01

Article Snippet: Cervical cancer cells were lysed by immunoprecipitation buffer (protease, phosphatase inhibitors, MCE) containing magnetic beads conjugated to specific antibodies (anti-IGF2BP2, anti-METTL16, anti-m 6 A).

Techniques: Immunofluorescence, Binding Assay, Reverse Transcription Polymerase Chain Reaction, RNA Immunoprecipitation, Modification

Journal: Discover Oncology

Article Title: Novel N 6 -methyladenosine (m 6 A) writer METTL16 promotes the cervical cancer tumorigenesis by targeting FTH1-dependent ferroptosis

doi: 10.1007/s12672-026-04403-8

Figure Lengend Snippet: IGF2BP2 promoted the stability of FTH1 mRNA. A The immunofluorescent staining showed the subcellular localization of IGF2BP2 and FTH1 in HeLa cells. B The positive correlation within IGF2BP2 and FTH1 in clinical samples ( http://gepia.cancer-pku.cn/index.html ). C RNA immunoprecipitation (RIP) assay showed the immunoprecipitated FTH1 mRNA by anti-IGF2BP2 antibody. D RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with IGF2BP2 overexpression. E RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with METTL16 silencing (sh-METTL16-1, sh-METTL16-2). F RNA decay analysis reported the stability of FTH1 mRNA in cervical cancer cells with IGF2BP2 silencing (si-IGF2BP2) and METTL16 overexpression. * p < 0.05, ** p < 0.01

Article Snippet: Cervical cancer cells were lysed by immunoprecipitation buffer (protease, phosphatase inhibitors, MCE) containing magnetic beads conjugated to specific antibodies (anti-IGF2BP2, anti-METTL16, anti-m 6 A).

Techniques: Staining, RNA Immunoprecipitation, Immunoprecipitation, Over Expression