Fig 2: EEF1A2 overexpression and Akt signaling pathway activator SC79 can counterbalance the inhibition of NSUN6 deficiency on OS progression. (A) Results of reverse transcription-quantitative PCR showing the expression level of EEF1A2 mRNA in 143b cells of different groups. Results of (B) Cell Counting Kit-8, (C) Colony formation, (D) Transwell (scale bar, 100 µM) and (E) Wound-healing assays (scale bar, 200 µM). Statistical charts of (F) Colony formation, (G) Transwell, and (H) Wound-healing assay. ***P#x003C;0.001. NSUN6, NOP2/Sun RNA methyltransferase family member 6; EEF1A2, eukaryotic elongation factor 1 α-2; sh, short-hairpin; OE, overexpression; ctrl, control.

Fig 3: EEF1A2 is the potential target of NSUN6 in human OS. (A) Volcano plot showing that expression difference of EEF1A2, PRAME, FGFBP2 and TP53 was the largest between high- and low-NSUN6 groups. Correlation of NSUN6 with (B) EEF1A2 (P=0.0017), (C) PRAME (P=0.028), (D) FGFBP2 (P=0.028) and (E) TP53 (P=0.086) in human OS. Expression of EEF1A2 mRNA in sh-ctrl, sh-NSUN6#1 group and sh-NSUN6#2 groups of (F) 143b and (G) U2-OS cells. RIP assay results in (H) 143b and (I) U2-OS cells. IgG was used as a negative control. ***P#x003C;0.001. NSUN6, NOP2/Sun RNA methyltransferase family member 6; OS, osteosarcoma; EEF1A2, eukaryotic elongation factor 1 α-2; PRAME, melanoma antigen preferentially expressed in tumors; FGFBP2, fibroblast growth factor binding protein 2; sh, short-hairpin; ctrl, control.

Fig 4: NSUN6 deficiency significantly inhibits OS progression in vitro. (A) The expression of NSUN6 mRNA in sh-ctrl, sh-NSUN6#1 and sh-NSUN6#2 groups of 143b and U2-OS cells. Results of (B) Cell Counting Kit-8, (C) Colony formation, (D) Transwell (scale bar, 100 µM) and (E) Wound-healing (scale bar, 200 µM) assays. ***P#x003C;0.001. NSUN6, NOP2/Sun RNA methyltransferase family member 6; OS, osteosarcoma; sh, short-hairpin; ctrl, control; OD, optical density.

Fig 5: NSUN6 regulates the stability of EEF1A2 mRNA via m5C modification. Dot Blot assay of (A) 143b and (B) U2-OS cells. Methylene blue staining (lower image) was used to detect the amount of RNA loaded, while the intensity of dot immunoblotting (upper image) represented the level of m5C modification. Methylated-RIP was used to detect the m5C modification level of EEF1A2 mRNA of (C) 143b and (D) U2-OS cells. Curves of EEF1A2 mRNA remaining vs. time after ActD treatment in (E) 143b and (F) U2-OS cells. ***P#x003C;0.001. NSUN6, NOP2/Sun RNA methyltransferase family member 6; OS, osteosarcoma; EEF1A2, eukaryotic elongation factor 1 a-2; RIP, RNA immunoprecipitation; m5C, 5-methylcytidine; sh, short-hairpin; ctrl, control.