Fig 1: Upregulation of IGF2BP2 reverses the regulatory effect of knockdown of NT5DC2 on the proliferation and apoptosis of diffuse large B-cell carcinoma cells. (A) Viability of OCI-Ly7 cells transfected with shRNA-NT5DC2 and Oe-IGF2BP2 was analyzed by Cell Counting Kit-8 assay. (B) Proliferation of OCI-Ly7 cells transfected with shRNA-NT5DC2 and Oe-IGF2BP2 was observed by BrdU staining. (C) Cell cycle analysis and (D) apoptosis of OCI-Ly7 cells transfected with shRNA-NT5DC2 and Oe-IGF2BP2 was detected by flow cytometry. (E) Expression levels of apoptosis-associated proteins were analyzed by western blotting. *P<0.05, **P<0.01 and ***P<0.001 vs. shRNA-NC; #P<0.05, ##P<0.01 and ###P<0.001 vs. shRNA-NT5DC2 + Oe-NC. IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; NT5DC2, 5′-nucleotidase domain-containing 2; shRNA, short hairpin RNA; oe, overexpression; BrdU, 5-bromo-2-deoxyuridine; NC, negative control; cyto-c, cytochrome c; PARP, poly-ADP ribose polymerase; OD, optical density.
Fig 2: IGF2BP2 is upregulated in diffuse large B-cell carcinoma cells and is the RNA-binding protein of NT5DC2. (A) mRNA and protein expression levels of IGF2BP2 in human B lymphocyte (GM12878) and OCI-Ly7 cells were detected by RT-qPCR and western blotting. ***P<0.001 vs. GM12878. Combination of IGF2BP2 and NT5DC2 was confirmed by RNA (B) immunoprecipitation and (C) pull-down assay. ***P<0.001 vs. NT5DC2. (D) mRNA and protein expression levels of IGF2BP2 in OCI-Ly7 cells transfected with Oe-IGF2BP2 were detected by RT-qPCR and western blotting. **P<0.01 and ***P<0.001 vs. Control; ##P<0.01 and ###P<0.001 vs. Oe-NC. (E) mRNA and protein expression levels of NT5DC2 in OCI-Ly7 cells transfected with shRNA-NT5DC2 and Oe-IGF2BP2 were detected by RT-qPCR and western blotting. ***P<0.001 vs. Control; ###P<0.001 vs. shRNA-NC; ∆∆P<0.01 vs. shRNA-NT5DC2; &&P<0.01 vs. shRNA-NT5DC2 + Oe-NC. IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; NT5DC2, 5′-nucleotidase domain-containing 2; RT-qPCR, reverse transcription-quantitative PCR; oe, overexpression; NC, negative control; shRNA, short hairpin RNA.
Fig 3: VIM-AS1 interacts with the RNA-binding protein IGF2BP2. (A) Relative VIM-AS1 levels in the nucleus and cytoplasm of C4-2 cells. GAPDH and U6 RNA were used as the loading controls for the cytoplasmic and nuclear fractions, respectively. (B) VIM-AS1 intracellular localization in C4-2 cells as detected by fluorescence in situ hybridization. GAPDH and U6 RNA served as positive controls for the cytoplasmic and nuclear fractions, respectively. Scale bar, 20 µm. (C) Potential binding proteins purified from RNA pull-down assays using a biotinylated VIM-AS1 probe or normal control probe. (D) Analysis flow chart for identification of steroid biosynthesis-related proteins that interact with VIM-AS1. (E) An interaction between VIM-AS1 and IGF2BP2 was identified using RNA pulldown and western blotting assays in LNCaP and C4-2 cells. (F) Enrichment of VIM-AS1 with IGF2BP2 antibody as detected by RNA immunoprecipitation. IgG was used as the control antibody. (G) Effect of VIM-AS1 knockdown or overexpression on the protein expression levels of IGF2BP2 in prostate cancer cells. (H) Effects of VIM-AS1 knockdown or overexpression on the mRNA expression levels of IGF2BP2 in prostate cancer cells. Data are presented as the mean ± SD of three independent repeats. *P<0.05 vs. IgG. IGF2BP2, insulin like growth factor 2 mRNA binding protein 2; NC, normal scrambled control probe; ns, not significant; sh-NC, normal scramble short hairpin RNA.
Fig 4: Graphical illustration of the mechanism by which the VIM-AS1/IGF2BP2/HMGCS1 axis enhances proliferation, increases enzalutamide resistance and promotes the progression from HSPC to CRPC. 3′-UTR, 3′-untranslated region; CRPC, castration-resistant prostate cancer; HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; HSPC, hormone-sensitive prostate cancer; IGF2BP2, insulin like growth factor 2 mRNA binding protein 2; m6A, N6-methyladenosine.
Fig 5: VIM-AS1 enhances HMGCS1 mRNA stability by binding with IGF2BP2. (A) Effect of VIM-AS1 overexpression and/or IGF2BP2/HMGCS1 knockdown on the protein expression levels of HMGCS1 in LNCaP cells. (B) Effect of VIM-AS1 overexpression and/or IGF2BP2/HMGCS1 knockdown on the mRNA expression levels of HMGCS1 in LNCaP cells. *P<0.05 (VIM-AS1 OE vs. Vector). (C) Effect of VIM-AS1 knockdown and/or IGF2BP2/HMGCS1 overexpression on the protein expression levels of HMGCS1 in C4-2 cells. (D) Effect of VIM-AS1 knockdown and/or IGF2BP2/HMGCS1 overexpression on the mRNA expression levels of HMGCS1 in C4-2 cells. *P<0.05 (sh-VIM-AS1 vs. sh-NC). (E) Effect of VIM-AS1 overexpression and IGF2BP2 knockdown on the mRNA stability of HMGCS1in LNCaP cells treated with actinomycin D for different lengths of time. *P<0.05 (VIM-AS1 OE vs. Vector). (F) Effect of VIM-AS1 knockdown and IGF2BP2 overexpression on the mRNA stability of HMGCS1 in C4-2 cells treated with actinomycin D for the indicated periods of time. *P<0.05 (sh-VIM-AS1 vs. sh-NC). Data are presented as the mean ± SD of three independent repeats. HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase 1; IGF2BP2, insulin like growth factor 2 mRNA binding protein 2; NC, negative control; sh, short hairpin RNA; si, small interfering RNA.
Supplier Page from Abcam for Anti-IGF2BP2/IMP-2 antibody