Figure 3.

SNEV Is Required for Repair of Oxidative DNA Damage during Adipogenic Differentiation of hASCs

hASCs were transfected with siSNEV or siControl, treated with 500 μM H₂O₂ for 90 and 60 min recovery, and submitted to comet assay.

(A) Transfection with siSNEV results in 90% reduction of SNEV mRNA expression. The average of four technical replicates of cells from donor 812 is shown. Error bars indicate SD.

(B) Representative images of comet assays. Scale bar, 100 μm.

(C) In hASCs transfected with siSNEV and treated with H₂O₂, the percentage of cells with high levels of DNA damage triples, while the percentage of cells with low DNA damage drops to one-third compared with control cells after H₂O₂ treatment. Numbers indicate the percentage of cells in the respective category. Pooled data from three biological replicates are shown (donors 803, 812, and 851). A minimum of 150 cells per condition and replicate were analyzed. Chi-square test was performed to compare results from control and SNEV knockdown: ^∗∗∗p < 0.001.

(D) Schematic representation of experimental design. hASC were transfected with SNEV or control siRNAs and submitted to adipogenic differentiation. RNA samples to monitor knockdown and ROS measurements were taken on days 0, 8, and 11 of differentiation. Comet assays were performed on days 0 and 11.

(E) RT-qPCR shows stable knockdown over the course of differentiation.

(F) ROS formation was quantified by H₂DCFDA staining. Mean values of three independent differentiation experiments are shown (2× donor 812 and 1× donor 803). Error bars indicate SD. Unpaired two-sided Student's t tests were performed to compare control and siSNEV treated samples and did not reveal statistical differences for any condition.

(G) Upper panel, 48 hr post-transduction, before adipogenic differentiation is induced, Comet assays reveal that DNA damage levels are slightly higher in siControl than siSNEV transfected ASCs. Lower panel, after adipogenic differentiation, SNEV knockdown leads to significant increase of DNA damage. Numbers indicate percentage of cells in the respective category. Pooled data from three independent differentiation experiments are shown (donors 803, 812, and 851). A minimum of 200 cells per condition and replicate were analyzed. Chi-square test was performed to compare results of control and SNEV knockdown: ^∗∗∗p < 0.001, ^∗p < 0.05. See also Figures S3 and S4.