Figure 3.
Melatonin attenuates SASP by upregulating YTHDF2. HOSEpiCs were transfected with a vector containing H-RasV12 or an empty control vector and treated with or without 1 μM melatonin. (A) Western blotting revealed the expression levels of m6A reader proteins in cells. (B) Relative mRNA levels of m6A readers were determined using RT-qPCR. (C) Representative fluorescent images of ROS in HOSEpiCs. (D) Relative mRNA levels of YTHDF2 from HOSEpiCs subjected to retrovirus-mediated expression of H-RasV12 (+) for OIS or empty vector (−) as a control and, where indicated, treated with 10 mM of the antioxidant NAC (+) or DMSO (−) for 8 days. Statistical values are presented as derived from ten independent experiments. (E) Immunofluorescence images of YTHDF2 measured by confocal microscopy. (F) The interference efficiency of YTHDF2 and the effect of YTHDF2 overexpression shown by Western blotting. HOSEpiCs were transfected with scramble as a control. (G) The expression of typical SASP genes in H-RasV12-transfected senescent HOSEpiCs transfected with either siYTHDF2 or oeYTHDF2 in the presence or absence of melatonin. Statistical values are presented as the mean ± standard error of the mean of three independent experiments. A P < 0.05 was considered statistically significant using one-way analysis of variance. ∗, P < 0.05.