Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 1 | Design strategies for endocytosis-triggering EndoTags. a, Schema of designed endocytosis mechanisms. Top, design of binding to constitutively cycling receptors at sites that do not overlap with binding sites for natural ligands to avoid competition. Middle, design of binders that trigger endocytosis by eliciting conformational changes in the receptor. The EndoTag binds at two distinct epitopes on the target and actively triggers the conformational change. Bottom, designed endocytosis via receptor clustering. The multivalent EndoTag clusters multiple copies of the target receptor and induces endocytosis. b, Design strategy for sortilin48 and TfR49 EndoTags. c, Cellular uptake of 100 nM AF647-labelled Sort_EndoTags, TfR-EndoTags or LHDB22 scaffold control for 2 h in U-251MG cells. Data were normalized to the 100 nM AF647-labelled LHDB group (no endocytosis). MFI, mean fluorescence intensity. d, Confocal imaging of Sort_EndoTag (red) and lysosomal marker (green, AF488-labelled LysoTracker) after 24 h incubation in U-251MG cells. e, Design strategy for

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Binding Assay, Control, Fluorescence, Imaging, Marker, Incubation

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 3 | Clearance of soluble proteins by IGF2R pLYTACs. a, Schema for the use of soluble pLYTACs with IGF_EndoTags. b, Cellular uptake of LHDB–AF647 via LHDA–IGF_EndoTags in Jurkat cells. Cells were incubated with 33 nM LHDB– AF647 with or without 1 μM LHDA–IGF_EndoTags for 24 h, washed twice with cold PBS and analysed by flow cytometry. c, Remaining supernatant LHDB– AF647 levels in Jurkat cells. Jurkat cells were incubated with 100 nM LHDB– AF647 with or without 500 nM LHDA–IGF_EndoTags. At timepoints 24 h and 48 h, the cells were pelleted down, and IgG in the supernatant was quantified using a Neo2 plate reader. IgG level was normalized to the IgG-alone control group. d, Cellular uptake of IgG–AF647 via protein G–IGF_EndoTags in K562 cells. Cells were incubated with 33 nM IgG–AF647 with or without 1 μM protein G–IGF_EndoTag3 for 24 h, washed twice with cold PBS and analysed by flow cytometry. The fold change in MFI was calculated by normalizing to the

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Incubation, Flow Cytometry, Control

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: 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