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: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

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: STAT1 activated the transcription of thyroid differentiation genes in thyroid cancer and mediated the differentiation and stemness. (A-B) Relative mRNA (A) and protein (B) level of thyroid differentiation-related genes measured by qRT-PCR in STAT1 -knockdown TPC1 cells. (C-D) The mRNA (C) and protein (D) variation of dedifferentiation molecules was measured by western blot in CAL62 cells. (E) Layout and MFI of CD133 expression in TPC1 cells determined by flow cytometry. (F-G) The transcript (F) and protein (G) levels of stemness markers in si- STAT1 TPC1 cells. (H) Layout and MFI of CD133 expression in CAL62 cells determined by flow cytometry. (I-J) The transcript (I) and protein (J) levels of stemness markers in STAT1 -OE CAL62 cells. (K) CUT&Tag was performed with STAT1 antibody in TPC1 and CAL62 cells. Heatmap showing the genomic distribution of STAT1 flanking TSSs in TPC1 and CAL62 cells. (L) IGV tracks for thyroid differentiation genes from CUT&Tag. (M-N) Dual-luciferase reporter assay of p GL3-basic, TSHR, SLC26A4, SLC5A5, TPO, PAX8, FOXE1, and NKX2.1 promoter activity driven by STAT1 or pcDNA3.1 transfection in wild-type TPC1 and CAL62 tumor cells. Data are relative to Renilla luciferase activity. n = 3. P values were determined using a two-tailed unpaired Student's test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Flow Cytometry, Luciferase, Reporter Assay, Activity Assay, 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: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

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: Anti-STAT1 primary antibodies (Cell Signaling Technology, USA,14994T) were diluted in antibody buffer (1% BSA, protease inhibitors) and incubated overnight at 4 °C, followed by species-matched secondary antibodies (ChiTag Goat anit-Rabbit IgG antibody, N269) conjugated to Protein A/G (1:500, Thermo Fisher, USA).

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