Fig. 3.

NS-KO-derived tsRNAs alleviate liver injury. a NSun2-knockdown HL-7702 cells were rescued with wild-type NSun2 (NS), two catalytically dead mutants (K190M, C271A), or the empty vector. Relative levels of EdU-positive cells were determined. n = 3 replicates. b Cell proliferation assay (CCK-8) of NSun2-knockdown HL-7702 rescued by enzymatic-dead NSun2. Detection of OD450 at the indicated times (day 1, day 2, day 3, day 4, day 5) under H₂O₂ stress, **P < 0.01, versus NS-KD at day 5. c Cell viability detection of NSun2-knockdown HL-7702 rescued by enzymatic-dead NSun2. Relative cell viability was assessed by CCK-8 assay. n = 3 replicates. The data in Fig. 3c and Fig. 1g are from the same batch of experiments. d NSun2 expression was measured to verify the rescue effect of NSun2 by western blotting in (a–c). e tRNA abundance in WT and NS-KO mice. Data indicate the tRNA abundance relative to WT, n = 3 mice. f, g Liquid chromatography-mass spectrometry showing the differences of tRNA m⁵U (f) and m⁵C (g) modifications in WT and NS-KO mice. Data are shown as a relative value (tRNA modification/total tRNA), n = 2 mice. h Schematic for isolating small liver RNAs. i EdU assay showing the proliferation of isolated fragments. EdU-positive cells were analyzed as data relative to WT. Scale bar, 100 μm. n = 3 replicates. j CCK-8 assay showing the survival of isolated fragments. Relative cell viability (CCK-8 assay) of 14–50 nt RNAs transfection was assessed by detecting the OD450 value. n = 3 replicates. For all data in this figure representative images are shown. All data are presented as the mean ± SD. *P < 0.05, **P < 0.01, Student’s t-test