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. 2020 Feb 19;13(3):100748. doi: 10.1016/j.tranon.2020.100748

Figure 2.

Figure 2

Oxidative stress and lipid peroxidation after TMZ treatment

To investigate ROS production in ALDH1A3 WT and KO cells, in vitro ROS/RNS assay was conducted based on the oxidation of DCFH to highly fluorescent 2′,7′-dichlorodihydrofluorescein. The OxiSelect TBARS assay based on MDA-TBA adducts was used to investigate lipid peroxidation levels in cells. Quantitative results of ROS/RNS assay (A) and TBARS assay (B). TMZ treatment led to significant increase of ROS production (A) and MDA content (B) in all three cell lines. No difference of ROS was observed between ALDH1A3 WT and KO cells, but the difference of MDA content was significant in all three cell lines. The addition of NAC dramatically counteracted the effect of TMZ and eliminated the difference between ALDH1A3 WT and KO cells. Each bar indicates the mean ± SD. *, P < .05; **, P < .01;***, P < .001.

(C) Western blots using antibodies against ALDH1A3 and HNE-modified proteins. Vinculin served as loading control. The cells were treated with 100 μM TMZ for 5 days. The proteins were isolated 30 minutes after last treatment; 30-μM HNE treatment for 1 hour served as positive control. More HNE-modified proteins could be observed in ALDH1A3KO cells than WT cells.