Journal: Cancer medicine

Article Title: IGF2BP2 promotes pancreatic carcinoma progression by enhancing the stability of B3GNT6 mRNA via m6A methylation.

doi: 10.1002/cam4.5096

Figure Lengend Snippet: FIGURE 3 Expression of IGF2BP2 in pancreatic carcinoma. (A) Expression of IGF2BP2 mRNA in TCGA and GTEx database. (B) IHC (IGF2BP2)-stained paraffin-embedded sections verified the expression of IGF2BP2 protein in PC and normal tissue. (C) Quantification of overall m6A RNA methylation in PC tissues. (D) Relationship between different CNV types and IGF2BP2 expression level. (E) Relationship between IGF2BP2 DNA methylation and mRNA expression. (F) IGF2BP2 mRNA expression in si-NC and IGF2BP2-si groups. (G–H) IGF2BP2 protein expression in si-NC and IGF2BP2-si groups. (*p < 0.05).

Article Snippet: Protein separation under 80 V, 30 min and 120 V, 1 h electrophoresis conditions, followed by transferring to polyvinylidene difluoride membranes at 200 mA for 1.5 h. Then, membranes were blocked with 5% skim milk and incubated with IGF2BP2 primary antibody (OriGene, American) and B3GNT6 (Proteintech, American) primary antibody overnight at 4°C.

Techniques: Expressing, Staining, Methylation, DNA Methylation Assay

Journal: Cancer medicine

Article Title: IGF2BP2 promotes pancreatic carcinoma progression by enhancing the stability of B3GNT6 mRNA via m6A methylation.

doi: 10.1002/cam4.5096

Figure Lengend Snippet: FIGURE 4 IGF2BP2 KO suppresses pancreatic carcinoma proliferation and migration ability. (A)–(D) CCK-8 and EdU assay were used to detect cell proliferation ability. (E) Transwell experiment revealed PC cell migration ability with or without IGF2BP2 KO. (F) PC cell colony formation ability detection after IGF2BP2 KO. (*p < 0.05).

Article Snippet: Protein separation under 80 V, 30 min and 120 V, 1 h electrophoresis conditions, followed by transferring to polyvinylidene difluoride membranes at 200 mA for 1.5 h. Then, membranes were blocked with 5% skim milk and incubated with IGF2BP2 primary antibody (OriGene, American) and B3GNT6 (Proteintech, American) primary antibody overnight at 4°C.

Techniques: Migration, CCK-8 Assay, EdU Assay

Journal: Cancer medicine

Article Title: IGF2BP2 promotes pancreatic carcinoma progression by enhancing the stability of B3GNT6 mRNA via m6A methylation.

doi: 10.1002/cam4.5096

Figure Lengend Snippet: FIGURE 5 IGF2BP2 regulates B3GNT6 mRNA expression to promote pancreatic carcinoma. (A) Prediction of IGF2BP2 target genes, Venn diagram shows substantial and significant overlap among TCGA-GTEx, IGF2BP2-CLIP and DEGs between PA1 and PA2 subgroups. (B) Expression of predicted genes after IGF2BP2 KO. C m6A IP and IGF2BP2 IP verified the binding efficacy of target genes. (D) TCGA- GTEx reveals the high expression of B3GNT6 in PC. (E) Spearman correlation analysis reveals positive co-expression between IGF2BP2 and B3GNT6. (F–H) CCK-8 and EdU assay revealed the PC cell proliferation ability after B3GNT6 KO. (I) Transwell experiment revealed PC cell migration ability after B3GNT6 KO. (J) B3GNT6 KO inhibited colony formation ability in PC cells. (*p < 0.05).

Article Snippet: Protein separation under 80 V, 30 min and 120 V, 1 h electrophoresis conditions, followed by transferring to polyvinylidene difluoride membranes at 200 mA for 1.5 h. Then, membranes were blocked with 5% skim milk and incubated with IGF2BP2 primary antibody (OriGene, American) and B3GNT6 (Proteintech, American) primary antibody overnight at 4°C.

Techniques: Expressing, Binding Assay, CCK-8 Assay, EdU Assay, Migration

Journal: Cancer medicine

Article Title: IGF2BP2 promotes pancreatic carcinoma progression by enhancing the stability of B3GNT6 mRNA via m6A methylation.

doi: 10.1002/cam4.5096

Figure Lengend Snippet: FIGURE 7 Mechanism diagram of IGF2BP2 promoting B3GNT6 expression via m6A methylation.

Article Snippet: Protein separation under 80 V, 30 min and 120 V, 1 h electrophoresis conditions, followed by transferring to polyvinylidene difluoride membranes at 200 mA for 1.5 h. Then, membranes were blocked with 5% skim milk and incubated with IGF2BP2 primary antibody (OriGene, American) and B3GNT6 (Proteintech, American) primary antibody overnight at 4°C.

Techniques: Expressing, Methylation

Journal: Biochemistry

Article Title: Chemoproteomic Profiling of 8-Oxoguanosine-Sensitive RNA-Protein Interactions.

doi: 10.1021/acs.biochem.3c00461

Figure Lengend Snippet: Figure 1. Proteomic profiling of the 8OG-RNA interactome. (A) Structure of the 8OG or G oligo probes. Schematic for chemical proteomics workflow. (B) Volcano plot of protein enrichment ratios (8OG/G spectral counts) and p-values from label-free proteomics experiments (n = 3). To calculate enrichment ratios for proteins identified in only one of the two conditions (G or 8OG probe), we added 1 to all spectral count values. P- values were calculated based on Student’s t test. (C) Validation of protein hits from (B). Lysates were photo-cross-linked with 8OG (1) or G (2) oligo probe, and RBPs were detected by Western blotting after streptavidin enrichment. IGF2BP1 and hnRNPD were detected with corresponding antibodies against endogenous protein. Anti-FLAG M2 antibody was used to detect overexpressed epitope-tagged hnRNPDL, IGF2BP2, RBM4, and RBM4B. See Figure S1 for full Western blotting data.

Article Snippet: Plasmids encoding cDNA were obtained from Addgene: IGF2BP1 (no. 21659), IGF2BP2 (no. 91890), HNRNPD (no. 38066); purchased from Genscript: RBM4 (#OHu17642D), RBM4B (#OHu04653D); or purchased from Origene: HNRNPDL (#SC107613).

Techniques: Protein Enrichment, Biomarker Discovery, Western Blot

Journal: Biochemistry

Article Title: Chemoproteomic Profiling of 8-Oxoguanosine-Sensitive RNA-Protein Interactions.

doi: 10.1021/acs.biochem.3c00461

Figure Lengend Snippet: Figure 2. Biochemical characterization of the interaction between IGF2BP1/2 and 8OG-containing RNA oligos. (A) Electrophoretic mobility shift assay (EMSA) with IGF2BP1-KH (195−577) and fluorescein-labeled 8OG (5) or G (6) oligos. Oligos were mixed with various concentrations of IGF2BP1 and RNA−protein complexes were resolved on a TGE native gel and detected by using in-gel fluorescence. See Figure S3 for full EMSA data. Kd values were determined by fitting data to a sigmoidal dose−response curve. Values represent mean ± s.e. (n = 3). Asterisks represent statistically significant differences in binding affinity (**, p < 0.01). (B) Fluorescence anisotropy assay with IGF2BP1-KH and oligos 5 and 6. Oligos were mixed with various concentrations of protein, and binding was monitored by fluorescence polarization. Kd values were determined by fitting data to a sigmoidal dose−response curve. Values represent mean ± s.e. (n = 4). Asterisks represent statistically significant differences in binding affinity (****, p < 0.0001). (C) Photo-cross-linking of IGF2BP2 with 8OG (1) or G (2) diazirine-containing oligo probes. IGF2BP2 was mixed with various concentrations of probe and UV irradiated at 365 nm. Cross-linked RNA−protein complexes were detected by streptavidin Western blotting. See Figure S4 for full blot. EC50 values were determined by fitting data to a sigmoidal dose−response curve. Values represent mean ± s.e. (n = 4). Asterisks represent statistically significant differences in cross-linking (****, p < 0.0001). (D) Fluorescence polarization assay with IGF2BP2 and 8OG (5) or G (6) oligos. Experiment was performed as described in (B). Values represent mean ± s.e. (oligo 5, n = 4; oligo 6, n = 3;).

Article Snippet: Plasmids encoding cDNA were obtained from Addgene: IGF2BP1 (no. 21659), IGF2BP2 (no. 91890), HNRNPD (no. 38066); purchased from Genscript: RBM4 (#OHu17642D), RBM4B (#OHu04653D); or purchased from Origene: HNRNPDL (#SC107613).

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Fluorescence, Binding Assay, Irradiation, Western Blot

Journal: Cancer Research

Article Title: Oncogenic NRAS, Required for Pathogenesis of Embryonic Rhabdomyosarcoma, Relies upon the HMGA2–IGF2BP2 Pathway

doi: 10.1158/0008-5472.can-12-3947

Figure Lengend Snippet: Figure 4. HMGA2 regulates IGF2BP2, which is necessary for ERMS growth. A, heatmap of microarray on HMGA2 knockdown samples illustrates HMGA2- regulated genes in rhabdomyosarcoma cells. B, genes significantly downregulated by HMGA2 knockdown are enriched for those controlling cell death, growth, and proliferation, including NRAS. Gene sets were analyzed by IPA software. C, knockdown of HMGA2 reduces IGF2BP2 mRNA. , P < 0.05, Student t test, sh-HMGA2 sample versus sh-NT sample. D, knockdown of HMGA2 reduces IGF2BP2 protein. E, HMGA2 and IGF2BP2 mRNA levels are correlated in cancer cell lines. F, knockdown of IGF2BP2 using lentiviral-based shRNAs. , P < 0.01, Student t test, sh-IGF2BP2 sample versus sh-NT sample. G, knockdown of IGF2BP2 in ERMS cells reduces proliferation and induces cell death. Images were taken 96 hours after infection. Scale bar, 100 mm. H, knockdown of IGF2BP2 using doxycycline-inducible shRNAs in rhabdomyosarcoma cells inhibits colony formation.

Article Snippet: Human IGF2BP2 (Origene, # SC324486) was subcloned using a similar strategy.

Techniques: Microarray, Knockdown, Software, Infection

Journal: Cancer Research

Article Title: Oncogenic NRAS, Required for Pathogenesis of Embryonic Rhabdomyosarcoma, Relies upon the HMGA2–IGF2BP2 Pathway

doi: 10.1158/0008-5472.can-12-3947

Figure Lengend Snippet: Figure 5. IGF2BP2 binds to mRNAs and regulates multiple oncogenic genes, including NRAS. A, IGF2BP2 binds to mRNAs of Igf2, Ccnd2, NRas, Ran, RhoA, and Sp1, but not GAPDH, MYOD, and HMGA2. , P < 0.01, Student t test, IGF2BP2 antibody versus IgG control. B, IGF2BP2 binds to the 30UTR of NRAS mRNA. C, IGF2BP2 regulates the stability of NRAS mRNA. After IGF2BP2 shRNA treatment, NRAS mRNA half-life (T1/2) is reduced from 6.3 to 3.9 hours. HMGA2 shRNA reduces half-life to 3.5 hours. D, knockdown of IGF2BP2 reduces NRAS protein levels. Five different shRNAs were used to knock down IGF2BP2; NRAS protein levels correlate well with IGF2BP2 knockdown efficacy. E, knockdown of HMGA2 reduces the NRAS protein level in ERMS cells. F, IGF2BP2 stabilizes NRAS-30UTR-luciferase protein. Luciferase assays show a dose-dependent increase of luciferase activity with IGF2BP2 overexpression. , P < 0.01, Student t test.

Article Snippet: Human IGF2BP2 (Origene, # SC324486) was subcloned using a similar strategy.

Techniques: Control, shRNA, Knockdown, Luciferase, Activity Assay, Over Expression

Journal: International journal of molecular sciences

Article Title: Dual Disruption of EGFR/PI3K Signaling: IGF2BP2 Targeting Reverses Anti-EGFR Resistance in CAFs-Infiltrated Oral Squamous Cell Carcinoma.

doi: 10.3390/ijms26093941

Figure Lengend Snippet: Figure 1. IGF2BP2 is the most markedly upregulated RNA-binding protein (RBP) in OSCC cells and CAFs, and is associated with poor clinical prognosis. (a) Dysregulated RBPs in OSCC tissues identified by RNA-seq. Heat map illustrated the expression of co-dysregulated RBPs. (b) Expression of five RBPs in HNSCC based on the TCGA database. (c,d) Expression of five RBPs in different cell types based on scRNA-seq. (e) Uniform manifold approximation and projection (UMAP) visualization of primary and metastatic HNSCC highlighted different IGF2BP2 expression. (f) Representative images of IGF2BP2 staining in various OSCC tissues. LM: lymph node metastasis. (g) Results of hierarchical clustering analysis visualized IGF2BP2 expression patterns across different tissues. (h) Histoscores of IGF2BP2 in different tissues. n = 196. (i,j) IGF2BP2 expression was associated with tumor grade (n = 195) and lymph node metastasis (n = 196). G1/G2/G3: grade 1/grade 2/grade 3. (k) Kaplan–Meier survival analysis of OSCC patients. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns p > 0.05.

Article Snippet: The Igf2bp2 depletion transgenic mouse was generated through CRISPR/Cas9 technology by Cyagen (detailed in Figure S4).

Techniques: RNA Binding Assay, RNA Sequencing, Expressing, Staining

Journal: International journal of molecular sciences

Article Title: Dual Disruption of EGFR/PI3K Signaling: IGF2BP2 Targeting Reverses Anti-EGFR Resistance in CAFs-Infiltrated Oral Squamous Cell Carcinoma.

doi: 10.3390/ijms26093941

Figure Lengend Snippet: Figure 2. Impaired IGF2BP2 expression inhibits tumor progression in OSCC. (a) Inhibition of IGF2BP2 reduced cell growth in HSC3 and Cal-27 cells. n = 3. (b) Stable IGF2BP2 knockout in HSC3 cells markedly inhibited cell growth. n = 3. (c) Images of tumors from nude mice bearing OSCC xenografts. (d,e) Growth curves, volumes, and weights of xenograft tumors. n = 5. (f) Cell invasion was suppressed in HSC3 and Cal-27 cells after IGF2BP2 knockdown. n = 3. (g–j) Images and weights of lung tissues, mice body weights, and survival rates from nude mice injected with HSC3–GV493- con/IGF2BP2-sh1 cells. n = 5. (k) Schematic diagram of the mouse TSCC model induction and primary mouse TSCC cell culture methodology. (l) Images of tongues from 4NQO-treated Igf2bp2wt

Article Snippet: The Igf2bp2 depletion transgenic mouse was generated through CRISPR/Cas9 technology by Cyagen (detailed in Figure S4).

Techniques: Expressing, Inhibition, Knock-Out, Knockdown, Injection, Cell Culture

Journal: International journal of molecular sciences

Article Title: Dual Disruption of EGFR/PI3K Signaling: IGF2BP2 Targeting Reverses Anti-EGFR Resistance in CAFs-Infiltrated Oral Squamous Cell Carcinoma.

doi: 10.3390/ijms26093941

Figure Lengend Snippet: Figure 3. IGF2BP2 regulates EGFR and PIK3R1 in an m6A-dependent manner. (a) Results of GSEA analysis of RNA-seq revealed the enrichment of REACTOME_SIGNALING_BY_ EGFR_IN_CANCER pathway-related genes correlating with IGF2BP2 expression. (b) Venn diagram illustrating the overlap between pathway-enriched genes, RNA-seq, and anti-IGF2BP2 RIP-seq datasets. (c) RNA expression levels of EGFR, PIK3R1, KRAS, and HSP90AA1 in HSC3 and Cal-27 cells after IGF2BP2 knockdown. The data were normalized to the mRNA expression of β-actin. n = 3. (d) The protein expression of EGFR and PIK3R1 were suppressed by IGF2BP2 downregulation. The data were normalized to the protein expression of β-actin. n = 3. (e) RIP-qPCR assays confirmed the binding sites between IGF2BP2 and 3′UTR of EGFR and PIK3R1 mRNA. n = 3. (f) Schematics illustrated the EGFR-RIP sequence and PIK3R1-RIP sequence with very high-confidence m6A sites predicted by SRAMP software (version 2016). (g) Results of MeRIP-qPCR analysis confirmed the m6A sites. n = 3. (h,i) Luciferase reporter assays measured the luciferase activities of 3′UTR-wt or 3′UTR-mut of EGFR and PIK3R1 in HSC3 cells with IGF2BP2 knockout. n = 4. (j,k) EGFR and PIK3R1 mRNA decay rates in HSC3-IGF2BP2-ko cells. n = 3. (l) Protein expression levels of EGFR, p-EGFR, PIK3R1, AKT, and p-AKT in HSC3-con/HSC3-IGF2BP2-ko cells. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05.

Article Snippet: The Igf2bp2 depletion transgenic mouse was generated through CRISPR/Cas9 technology by Cyagen (detailed in Figure S4).

Techniques: RNA Sequencing, Expressing, RNA Expression, Knockdown, Binding Assay, Sequencing, Software, Luciferase, Knock-Out

Journal: International journal of molecular sciences

Article Title: Dual Disruption of EGFR/PI3K Signaling: IGF2BP2 Targeting Reverses Anti-EGFR Resistance in CAFs-Infiltrated Oral Squamous Cell Carcinoma.

doi: 10.3390/ijms26093941

Figure Lengend Snippet: Figure 4. IGF2BP2 inhibitor exhibits potent anti-OSCC efficacy in both cetuximab-sensitive and -resistant cells. (a,b) Cell growth of HSC3, Cal-27, and SCC25 treated with cetuximab or CWI1-2. n = 3. (c,d) Protein expression levels of EGFR, p-EGFR, PIK3R1, p-AKT, and AKT in HSC3 and Cal-27 cells treated with cetuximab or CWI1-2 for 48 h. n = 3. (e) Image of xenograft tumors of Cal-27 from mice treated with saline, cetuximab, or CWI1-2. n = 8. (f,g) Growth curves, volumes, and weights of xenograft tumors. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05.

Article Snippet: The Igf2bp2 depletion transgenic mouse was generated through CRISPR/Cas9 technology by Cyagen (detailed in Figure S4).

Techniques: Expressing, Saline

Journal: International journal of molecular sciences

Article Title: Dual Disruption of EGFR/PI3K Signaling: IGF2BP2 Targeting Reverses Anti-EGFR Resistance in CAFs-Infiltrated Oral Squamous Cell Carcinoma.

doi: 10.3390/ijms26093941

Figure Lengend Snippet: Figure 5. CWI1-2 overcomes CAFs-mediated cetuximab resistance in OSCC. (a,b) Growth of CAFs treated with cetuximab or CWI1-2. n = 3. (c,d) Protein expression of α-SMA, EGFR, p-EGFR, PIK3R1, p-AKT, AKT, and IGF2BP2 in NFs and CAFs (c), and in CAFs with IGF2BP2 knockdown (d). n = 3. (e) Cell growth in CAF-con/CAF-IGF2BP2-shs cells. n = 3. (f). Conditioned medium from CAFs (CM- CAFs) enhanced the proliferation of HSC3 cells. n = 3. (g,h) Therapeutic efficacy of cetuximab (g) or CWI1-2 (h) in HSC3 cells in the presence of CM-CAFs. n = 3. (i,j) Representative immunofluorescence images of HSC3 cells monoculture (i) or co-cultured with CAFs (j) in the presence of saline, cetuximab, or CWI1-2. Bar charts depicted cell numbers in each group. n = 3. (k,l) Representative images of tumor spheres from each group are given. Bar charts displayed the sphere diameter across groups. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05.

Article Snippet: The Igf2bp2 depletion transgenic mouse was generated through CRISPR/Cas9 technology by Cyagen (detailed in Figure S4).

Techniques: Expressing, Knockdown, Drug discovery, Immunofluorescence, Cell Culture, Saline