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: After blocked with QuickBlockTM Blocking Buffer (Beyotime, China, P0252) for 15min, membranes were incubated with primary antibodies overnight at 4°C and secondary antibodies (1:5000, Cell Signaling Technology, USA, 7074P2) at room temperature for 2 h. The primary antibodies used were as follows: IGF2BP2 (1:1000, Proteintech, China, 11601-1-AP), FOXE1 (1:1000, UpingBio, China, YP-Ab-01724), PAX8 (1:1000, UpingBio, China, YP-Ab-15794), NKX2.1 (1:1000, UpingBio, China, YP-Ab-15626), TSHR (1:1000, UpingBio, China, YP-Ab-07557), TPO, SLC5A5 (1:1000, UpingBio, China, YP-Ab-17263), SLC26A4 (1:1000, UpingBio, China, YP-Ab-07718), CD133 (1:1000, UpingBio, China, YP-Ab-13976), NANOG (1:1000, UpingBio, China, YP-Ab-15732), SOX2 (1:1000, UpingBio, China, YP-Ab-01022), OCT4 (1:1000, UpingBio, China, YP-Ab-15741), STAT1 (1:1000, Proteintech, China, 66545-1-IG), and GAPDH (1:2000, Proteintech, China, 60004-1-IG).

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: 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: After blocked with QuickBlockTM Blocking Buffer (Beyotime, China, P0252) for 15min, membranes were incubated with primary antibodies overnight at 4°C and secondary antibodies (1:5000, Cell Signaling Technology, USA, 7074P2) at room temperature for 2 h. The primary antibodies used were as follows: IGF2BP2 (1:1000, Proteintech, China, 11601-1-AP), FOXE1 (1:1000, UpingBio, China, YP-Ab-01724), PAX8 (1:1000, UpingBio, China, YP-Ab-15794), NKX2.1 (1:1000, UpingBio, China, YP-Ab-15626), TSHR (1:1000, UpingBio, China, YP-Ab-07557), TPO, SLC5A5 (1:1000, UpingBio, China, YP-Ab-17263), SLC26A4 (1:1000, UpingBio, China, YP-Ab-07718), CD133 (1:1000, UpingBio, China, YP-Ab-13976), NANOG (1:1000, UpingBio, China, YP-Ab-15732), SOX2 (1:1000, UpingBio, China, YP-Ab-01022), OCT4 (1:1000, UpingBio, China, YP-Ab-15741), STAT1 (1:1000, Proteintech, China, 66545-1-IG), and GAPDH (1:2000, Proteintech, China, 60004-1-IG).

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: 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: After blocked with QuickBlockTM Blocking Buffer (Beyotime, China, P0252) for 15min, membranes were incubated with primary antibodies overnight at 4°C and secondary antibodies (1:5000, Cell Signaling Technology, USA, 7074P2) at room temperature for 2 h. The primary antibodies used were as follows: IGF2BP2 (1:1000, Proteintech, China, 11601-1-AP), FOXE1 (1:1000, UpingBio, China, YP-Ab-01724), PAX8 (1:1000, UpingBio, China, YP-Ab-15794), NKX2.1 (1:1000, UpingBio, China, YP-Ab-15626), TSHR (1:1000, UpingBio, China, YP-Ab-07557), TPO, SLC5A5 (1:1000, UpingBio, China, YP-Ab-17263), SLC26A4 (1:1000, UpingBio, China, YP-Ab-07718), CD133 (1:1000, UpingBio, China, YP-Ab-13976), NANOG (1:1000, UpingBio, China, YP-Ab-15732), SOX2 (1:1000, UpingBio, China, YP-Ab-01022), OCT4 (1:1000, UpingBio, China, YP-Ab-15741), STAT1 (1:1000, Proteintech, China, 66545-1-IG), and GAPDH (1:2000, Proteintech, China, 60004-1-IG).

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