11601 1 ap  (Proteintech)

Journal: Non-coding RNA Research

Article Title: The regulatory role of circGDI2 in hepatocellular carcinoma proliferation and glycolysis with the involvement of m6A modification

doi: 10.1016/j.ncrna.2025.11.006

Figure Lengend Snippet: Silencing circGDI2 inhibits proliferation and glycolysis and PKM2 expression through IGF2BP2 in HCC cells. (A) Western blot was used to analyze IGF2BP2 expression in Li-7 and Huh-7 cell lines after circGDI2 knockdown. (B) The expression level of IGF2BP2 was examined in clinical HCC tissues and adjacent normal tissues using RT-qPCR. To clarify if circGDI2 regulated HCC cell proliferation and glycolysis through IGF2BP2, sh-circGDI2 and OE-IGF2BP2 were co-transfected into Li-7 and Huh-7 cells. (C) Western blot was used to assess the effect of IGF2BP2 on the expression of PKM2. (D) CCK-8 assay was used to assess cell proliferation. (E) Glucose consumption level and lactate production were detected using the Glucose Assay Kit with O-toluidine and Lactate Assay Kit. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. the sh-NC group, or the Adjacent group. # P < 0.05, ## P < 0.01, ### P < 0.001, vs. the sh-circGDI2+OE-NC group.

Article Snippet: IGF2BP2 , IHC , Rabbit , 1:500 , Proteintech , 11601-1-AP.

Techniques: Expressing, Western Blot, Knockdown, Quantitative RT-PCR, Transfection, CCK-8 Assay, Glucose Assay, Lactate Assay

Journal: Non-coding RNA Research

Article Title: The regulatory role of circGDI2 in hepatocellular carcinoma proliferation and glycolysis with the involvement of m6A modification

doi: 10.1016/j.ncrna.2025.11.006

Figure Lengend Snippet: Silencing circGDI2 inhibits HCC tumor growth and PKM2 expression through IGF2BP2. To verify the effect of circGDI2 and IGF2BP2 on HCC tumor growth, a xenograft mouse model was constructed. (A) Pictures of the isolated tumors of the indicated group. (B) The tumor volume and weight were recorded. (C) HE staining, Tunel and Ki-67 staining were performed to observe the histological characteristics and cell proliferation in the tumor tissues (scale bar = 100 μm). (D) RT-qPCR was used to detect circGDI2, IGF2BP2 and PKM2 levels. (E) IHC was used to detect IGF2BP2 and PKM2 levels (scale bar = 100 μm). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. the sh-NC group. # P < 0.05, ## P < 0.01, ### P < 0.001, vs. the sh-circGDI2+OE-NC group.

Article Snippet: IGF2BP2 , IHC , Rabbit , 1:500 , Proteintech , 11601-1-AP.

Techniques: Expressing, Construct, Isolation, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: Non-coding RNA Research

Article Title: The regulatory role of circGDI2 in hepatocellular carcinoma proliferation and glycolysis with the involvement of m6A modification

doi: 10.1016/j.ncrna.2025.11.006

Figure Lengend Snippet: Silencing FTO inhibits HCC tumor growth and decreases circRNA, IGF2BP2 and PKM2 levels. To investigate the biological role of FTO on HCC tumor growth, the xenograft tumor models of HCC cells in the sh-NC and sh-FTO groups were established. (A) Pictures of the isolated tumors of the indicated group. (B) The tumor volume and weight were recorded. (C) HE staining, Ki-67 staining and Tunel stainning were performed to observe the histological characteristics and cell proliferation in the tumor tissues (scale bar = 100 μm). (D) RT-qPCR was used to detect circGDI2 and FTO levels. (E) IHC was used to detect IGF2BP2 and PKM2 levels (scale bar = 100 μm). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. the sh-NC group.

Article Snippet: IGF2BP2 , IHC , Rabbit , 1:500 , Proteintech , 11601-1-AP.

Techniques: Isolation, Staining, TUNEL Assay, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: IGF2BP2 was aberrantly expressed in THCA, especially in ATC, and related to poor prognosis. (A) Relative expression of 'm6A regulators' in thyroid carcinoma and normal thyroid tissue in the TCGA-THCA databases. (B) Quantitative real-time PCR was used to measure the mRNA level of IGF2BP2 in normal and thyroid cancer tissues. (C) Kaplan-Meier survival analysis performed using the KM Plotter online tool showed that upregulation of IGF2BP2 was significantly associated with shorter RFS in THCA patients. (D) The expression of IGF2BP2 in human normal thyroid cells, PTC cells, PDTC cells, and ATC cells was mined from the GEO datasets ( GSE29265 , GSE65144 , GSE76039 , and GSE33630 ). (E) IGF2BP2 expression in normal thyroid tissues, PTC tissues, PDTC and ATC tissues was observed by immunohistochemistry staining. Scale bars, 500 μm. (F) IHC H-scores for IGF2BP2 expression are quantified and presented as the mean ± SD in the adjacent violin graphs. Scale bar, 500 μm (applicable to all images). (G-H) The expression levels of IGF2BP2 were examined by qRT-PCR (G), and western blot (H) in human normal thyroid cells, PTC cells, and ATC cells. Intergroup differences were analyzed using unpaired Student's t-test (parametric data) or Mann-Whitney U test (nonparametric data). For multi-group comparisons, one-way ANOVA with Tukey's post hoc test was applied. All data: mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Briefly, cells were lysed with RIP Buffer supplemented with RNase and protease inhibitors and immunoprecipitated with 5 μg of anti-IGF2BP2 antibody (Proteintech, China, 11601-1-AP) or rabbit IgG (Beyotime, China, A7016) on a 4 °C rocker overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining, Quantitative RT-PCR, Western Blot, MANN-WHITNEY

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: IGF2BP2 was closely correlated with dedifferentiation of THCA. (A) The correlation between IGF2BP2 expression and TDS. (B) The lollipop chart summarizes the correlation between IGF2BP2 and thyroid differentiation genes in GEO datasets. (C) tSNE demonstrates the 12 cell clusters using scRNA-seq data. (D) Identification of thyroid cells using scRNA-seq data. (E) The expression pattern of IGF2BP2 in identified normal follicular epithelium, PTC and ATC cells. (F) Single-cell trajectory analysis depicting the expression of IGF2BP2 during thyroid cancer progression. Color scale represents expression level. (G) The efficiency of stable IGF2BP2 overexpression and knockdown cell lines verified by Western blot. (H-I) The mRNA levels of differentiation markers were evaluated by RT-qPCR in IGF2BP2 -overexpressed PTC cell lines. (J) The changes of differentiation markers in PTC cell lines determined by Western blot. (K-L) The mRNA levels of differentiation markers were evaluated by RT-qPCR in IGF2BP2 knockdown ATC cell lines. (M) The changes of differentiation markers in ATC cell lines were determined by Western blot. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Briefly, cells were lysed with RIP Buffer supplemented with RNase and protease inhibitors and immunoprecipitated with 5 μg of anti-IGF2BP2 antibody (Proteintech, China, 11601-1-AP) or rabbit IgG (Beyotime, China, A7016) on a 4 °C rocker overnight.

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: IGF2BP2 maintained stemness of THCA. (A) IGF2BP2 overexpression promoted sphere formation ability of TPC1 and BCPAP cells. (Scale bar, 50 μm). (B) Flow cytometry analysis and mean fluorescence intensity of CD133 expression following IGF2BP2 overexpression in TPC1 and BCPAP. The mRNA (C-D) and protein (E) levels of stemness markers were assessed by RT-qPCR and western blot following IGF2BP2 overexpression in TPC1 and BCPAP, respectively. (F) IGF2BP2 knockdown inhibited sphere formation ability of CAL62 and C643 cells. (Scale bar, 50 μm). (G) Flow cytometry analysis and mean fluorescence intensity of CD133 expression upon IGF2BP2 knockdown in CAL62 and C643. The mRNA (H-I) and protein (J) levels of stemness markers were assessed by RT-qPCR and Western blot following IGF2BP2 knockdown in CAL62 and C643, respectively. (K-M) The tumor growth curve, and tumor weight of CAL62 xenograft in nude mice. (N) Representative immunohistochemical (IHC) staining of IGF2BP2, SLC5A5, and CD133 in xenograft tumors from each experimental group. Scale bar, 200 μm (applicable to all images). (O) IHC H-scores for SLC5A5 and CD133 expression are quantified and presented as the mean ± SD in the adjacent bar graphs. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Briefly, cells were lysed with RIP Buffer supplemented with RNase and protease inhibitors and immunoprecipitated with 5 μg of anti-IGF2BP2 antibody (Proteintech, China, 11601-1-AP) or rabbit IgG (Beyotime, China, A7016) on a 4 °C rocker overnight.

Techniques: Over Expression, Flow Cytometry, Fluorescence, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Immunohistochemical staining, Immunohistochemistry, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: Identification of the IGF2BP2 targets in thyroid cancer in various differentiation states. (A) Heatmap showing differentially expressed genes (DEGs) between Vector group and IGF2BP2 group in TPC1 cells as determined by RNA-sequencing. (B) Heatmap depicting DEGs between shNC group and sh IGF2BP2 group in CAL62 cells identified by RNA-sequencing. (C-D) GO enrichment analysis of DEGs. (E) Transcriptomic intersection analysis reveals the potential targets of IGF2BP2 in dedifferentiation.

Article Snippet: Briefly, cells were lysed with RIP Buffer supplemented with RNase and protease inhibitors and immunoprecipitated with 5 μg of anti-IGF2BP2 antibody (Proteintech, China, 11601-1-AP) or rabbit IgG (Beyotime, China, A7016) on a 4 °C rocker overnight.

Techniques: Plasmid Preparation, RNA Sequencing

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: STAT1 was a m6A target of IGF2BP2 in thyroid cancer associated with the dedifferentiation. (A) Distribution of IGF2BP2-binding peak of RIP-seq. (B) The upset plot shows the intersection of RNA-seq, RIP-seq, and MERIP seq ( GSE199205 ). (C) IGF2BP2 binding peaks in STAT1 transcripts visualized by IGV. Relative RNA level of STAT1 in PTC following IGF2BP2 overexpression (D) and ATC upon IGF2BP2 knockdown (E). (F) Western blot detected the protein level of STAT1 in PTC cells transfected with overexpressing lentiviruses carrying IGF2BP2. (G) The protein levels of STAT1 were measured by western blot analysis in ATC cells transfected with lentiviruses carrying sh- IGF2BP2 . (H) Kaplan-Meier analysis of overall survival of THCA patients using the KM Plotter online tool. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Briefly, cells were lysed with RIP Buffer supplemented with RNase and protease inhibitors and immunoprecipitated with 5 μg of anti-IGF2BP2 antibody (Proteintech, China, 11601-1-AP) or rabbit IgG (Beyotime, China, A7016) on a 4 °C rocker overnight.

Techniques: Binding Assay, RNA Sequencing, Over Expression, Knockdown, Western Blot, Transfection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: IGF2BP2 regulated stabilization of STAT1 transcript via an m6A-dependent manner. (A-B) TPC1-OE and CAL62-KD cells were treated with 5 μg/mL actinomycin D (ActD) for 0, 2, 4, 6, 8, and 10 h, followed by RT-qPCR. (C-F) TPC1 and CAL62 cell lysates were immunoprecipitated with IGF2BP2 or m6A antibody and control immunoglobulin G (IgG) to detect STAT1 mRNA expression and validated by agarose electrophoresis. (G) Sketch map shows the m6A -enriched sites of STAT1 transcript. (H-I) Dual-luciferase assay of STAT1 reporter activity driven by STAT1-A, B, and C transfection in vector and IGF2BP2 overexpression TPC1 cells as well as IGF2BP2 knockdown CAL62 cells. Data are relative to Renilla luciferase activity. n = 3. (J) Sketch map shows the dual-luciferase reporter plasmid construction for STAT1 m6A site validation. (K-L) Dual-luciferase assay of STAT1 reporter activity driven by STAT1-A mutant, and B mutant transfection in vector and IGF2BP2 overexpression TPC1 cells as well as IGF2BP2 knockdown CAL62 cells. Data are relative to Renilla luciferase activity. n = 3. (M-N) Co-IP and Western blot analysis of TPC1 (M) and CAL62 (N) cells demonstrate that IGF2BP2 is physically associated with CNOT1. (O-P) The relative STAT1 mRNA abundance and protein expression in si- CNOT1 TPC1 (O) and CAL62 cells (P). (Q-R) TPC1 (Q) and CAL62 (R) si- CNOT1 cells were treated with 5 μg/mL actinomycin D (ActD) for 0, 2, 4, 6, 8, and 10 h, followed by RT-qPCR. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001). * Method note: TPC1 and CAL62 RIPs were performed in separate batches with different starting cell numbers (10 × 10⁶ vs 6 × 10⁶). Equal amounts of eluted mRNA (500 ng) were reverse-transcribed; qPCR revealed 3.1 cycles higher Cp in CAL62, indicating ~8.6-fold lower template abundance. 10 µL PCR product was loaded for TPC1 and 5 µL for CAL62 (same cycle number). Bar graphs display within-cell-line IP/IgG enrichment ratios, absolute abundance cannot be compared across lines.

Article Snippet: Briefly, cells were lysed with RIP Buffer supplemented with RNase and protease inhibitors and immunoprecipitated with 5 μg of anti-IGF2BP2 antibody (Proteintech, China, 11601-1-AP) or rabbit IgG (Beyotime, China, A7016) on a 4 °C rocker overnight.

Techniques: Quantitative RT-PCR, Immunoprecipitation, Control, Expressing, Electrophoresis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Knockdown, Biomarker Discovery, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Two Tailed Test, Reverse Transcription

Journal: International Journal of Biological Sciences

Article Title: IGF2BP2 Drives Thyroid Cancer Dedifferentiation Through m6A-Dependent STAT1 mRNA Destabilization

doi: 10.7150/ijbs.121503

Figure Lengend Snippet: STAT1 reversed the vicious dedifferentiation, and stemness promoted by IGF2BP2 in thyroid cancer. (A) The TPC1-OE cells were transfected with pLvx-STAT1 plasmids confirmed by Western blotting. (B-C) Thyroid differentiation factor expressions were measured by qRT-PCR (B) and Western blot (C) in TPC1 cell lines. (D) CAL62-KD cells were interfered with si- STAT1 , the transfection efficiency was confirmed by Western blotting. (E-F) Thyroid differentiation factor expressions were measured by qRT-PCR (E) and Western blot (F) in TPC1 cell lines. (G) The CSCs CD133 features were measured by were assessed by flow cytometry in TPC1 cells. (H-I) The RNA (H) and protein (I) levels of stemness markers in TPC1 cells. (J) The CSCs CD133 features were measured by were assessed by flow cytometry in CAL62 cells. (K-L) The mRNA (K) and protein (L) levels of CSCs markers were measured by western blot analysis. (M-O) Growth curve and tumor weight of subcutaneous xenografts models with CAL62 cells. (P) Representative immunohistochemical (IHC) staining of IGF2BP2, STAT1, SLC5A5, and CD133 in xenograft tumors from each experimental group. Scale bar, 200 μm (applicable to all images). (Q) IHC H-scores for SLC5A5 and CD133 expression are quantified and presented as the mean ± SD in the adjacent bar graphs. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Briefly, cells were lysed with RIP Buffer supplemented with RNase and protease inhibitors and immunoprecipitated with 5 μg of anti-IGF2BP2 antibody (Proteintech, China, 11601-1-AP) or rabbit IgG (Beyotime, China, A7016) on a 4 °C rocker overnight.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Flow Cytometry, Immunohistochemical staining, Immunohistochemistry, Expressing, Two Tailed Test