Fig. 3

Propofol increases Oct4 expression via the downregulation of the methylation levels of its promoter. A The mRNA levels of Oct4 in U118-derived GSCs treated with or without propofol and EZH2 inhibitor, tazemetostat were analyzed by RT-PCR (n = 5 in each group). β-actin was used as the loading control. B The DNA methylation levels of the Oct4 promoter in U118-derived GSCs treated with or without propofol were determined by bisulfite genomic sequencing (n = 7). Black and white circles represent methylated and unmethylated sites, respectively. C The mRNA levels of Oct4 in U118-derived GSCs transfected with control, Dnmt1, Dnmt2, Dnmt3A, or Dnmt3B siRNA (n = 5 in each group). β-actin was used as the loading control. D Luciferase reporter assays with the wild-type Oct4 reporter vector and DNMT3A, EZH2 (or EZH2 palmitoylation mutants), and ZDHHC5, alone or in combination, in U118-derived GSCs. The luciferase values were relative to Renilla luciferase activity (n = 7 in each group). E Lysates from 293 T cells expressing Flag-EZH2 palmitoylation mutants (EZH2 C571, 576A), HA-ZDHCH5, and Myc-DNMT3A were subjected to immunoprecipitation, followed by immunoblotting with anti-Flag, anti-Myc, and anti-HA antibodies. Ctrl, control; Taz, tazemetostat