Fig 2: Knockdown of IGF2BP3 decreases AML cell viability in vivo.a Schematic diagram showing the schedule of nude mouse xenograft assays. b Subcutaneous tumors were observed at 36 days in two different groups (the black arrows indicate xenograft tumors). c The xenograft growth curves for the shIGF2BP3#1, shIGF2BP3#2, and shNC groups were plotted by measuring the tumor size (width2 × length × π/6) with a Vernier caliper every four days. d Nude mice were sacrificed, and xenografts were harvested and weighed. e–i Representative images of immunohistochemical staining for Caspase-3, cleaved Caspase-3, Ki-67, and PCNA in tumors excised from xenograft model mice. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig 3: Exosomal miR-221-3p targets the PTEN/Akt signaling pathway after myocardial injury. (A,B) Western blot images showed the expression of proteins associated with the PTEN/Akt pathway in H9c2 (A) cells and HUVECs (B). (C) Quantitative analysis of the levels of Akt, p-Akt, Cleaved caspase-3, Bcl-2, PTEN in H9c2 cells. (n = 3). (D) Quantitative analysis of the levels of Akt, p-Akt, Cleaved caspase-3, Bcl-2, PTEN, VEGF in HUVECs. (n = 3). (E) A proposed working model of this study. All miR-221 mimic groups were normalized to the relevant mimic negative control group. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: Knockdown of IGF2BP3 significantly inhibits AML progression in vitro.a The protein expression level of IGF2BP3 in various hematologic tumor cell lines was measured by western blotting. b The knockdown efficiency of IGF2BP3 shRNAs (shIGF2BP3#1 and shIGF2BP3#2) delivered via lentiviral vectors in HL-60 and KG-1 cell lines was confirmed by western blotting. GAPDH was used as the internal reference. c Cell proliferation was measured by a CCK-8 assay at different time points (0, 24, 48, 72, and 96 h) in HL-60 and KG-1 cells after shRNA transduction. d The transduction efficiency after puromycin selection was evaluated by GFP fluorescence imaging in both cell lines. e Flow cytometry (representative images are presented) was used to confirm the induction of apoptosis by IGF2BP3 knockdown. f Western blotting was used to explore apoptosis-related protein levels. The levels of cleaved caspase-3 and Bax were increased but the level of Bcl-2 was decreased under shIGF2BP3 treatment compared with control treatment. g Flow cytometry (representative images are presented) was used to analyze the cell cycle distribution. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: IGF2BP3 regulates RCC2 expression in an m6A-dependent manner.a, b RCC2 expression was positively correlated with IGF2BP3 expression in the GSE37642 and TARGET datasets. c, d Kaplan–Meier survival analysis revealed that high RCC2 expression indicated a poor prognosis in AML patients. e, f Protein expression level of RCC2 following knockdown or overexpression of IGF2BP3 in HL-60 and KG-1 cells. g, h The interference efficiency of the siRNAs was evaluated to confirm the feasibility of the siRNAs, and si-RCC2#2 was found to be effective in reducing RCC2 expression. i, j Apoptosis was detected by flow cytometry. RCC2 deficiency promoted the induction of apoptosis by IGF2BP3 overexpression. k The mRNA of RCC2 was enriched by the anti-IGF2BP3 antibody compared to IgG in the HL-60 and KG-1 cell lines. l The mRNA of RCC2 was enriched by the m6A-specific antibody compared to IgG in the HL-60 and KG-1 cell lines. m Overexpression of IGF2BP3 restored the increases in the levels of proapoptotic proteins (Bax and cleaved Caspase 3) caused by silencing RCC2, and the level of the antiapoptotic protein Bcl-2 was slightly decreased. n The potential m6A sites in RCC2 were predicted by SRAMP. The different colored lines indicate different confidence levels. o, p Loss of IGF2BP3 reduced RCC2 stability in HL-60 and KG-1 cells. Transfected cells were treated with 5 µg/ml actinomycin D for 0 h, 3 h, or 6 hours prior to RNA extraction. *P < 0.05; **P < 0.01; ***P < 0.001.