Fig. 5: NSUN2 promotes the stability of FABP5 mRNA via m⁵C.

A The schematic diagram showed that two cysteines of NSUN2 (C271 and C321) were mutated into alanine. The sh-NSUN2 group means the cells were stably transfected with sh-NSUN2#1. B An m⁵C dot blot assay was used to detect the m⁵C levels of mRNA extracted from stably transfected 143b and U2-OS cells. RNA was serially diluted and loaded equally at 400 ng, 200 ng, and 100 ng. Methylene blue staining (below) was used to detect the amount of RNA loaded, while the intensity of dot immunoblotting (above) represented the level of m⁵C modification. C, D The results of methylated-RIP for 143b and U2 cells. The relative m⁵C enrichment of FABP5 mRNA for each group was normalized to the Input. The low concentration of cycloleucine was 10 mM, the middle was 20 mM, and the high was 40 mM. E, F The results of RT–qPCR showing the expression of NSUN2 mRNA and FABP5 mRNA in 143b and U2 cells. G, H The curve of FABP5 mRNA remaining versus time after ActD treatment in 143b and U2 cells. I, J The results of western blot showing the expression of FABP5 protein and NSUN2 protein. The result of RT-qPCR was statistical analyzed according to the data of three independent experiments.