For p53 phospho/acetyl-flow, cells were washed in PBS, fixed in BD Cytofix Fixation buffer (BD Biosciences, San Jose, CA, USA), and permeabilized in 90% methanol in PBS. Cells were washed in 0.5% BSA in PBS and stained with FITC Mouse Anti-Human p53 (BD), Phospho-p53 (Ser15) (16G8) Mouse mAb Alexa Fluor 488 conjugate (Cell Signaling Technologies, Beverly, MA, USA), Alexa Fluor 647 Mouse anti-p53 (pS37) (BD), or Acetyl-p53 (Lys382) Antibody (Cell Signaling Technology, Danvers, MA, USA) followed by secondary Alexa Fluor 647 F(ab’)2 fragment of goat anti-rabbit IgG (H+L) (Invitrogen Corp., Carlsbad, CA, USA).
Lyso Tracker® Red DND-99 (Invitrogen) was used to look for features of autophagic cell death. Lyso Tracker was diluted to working solution as indicated by the supplier and incubated with the cells for 30 min. Cells were washed in PBS and fixed in 1% PFA. Red fluorescence was measured by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA, USA). Data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Results are shown as mean fluorescence relative to the control samples.
Lyso Tracker® Red DND-99 (Invitrogen) was used to look for features of autophagic cell death. Lyso Tracker was diluted to working solution as indicated by the supplier and incubated with the cells for 30 min. Cells were washed in PBS and fixed in 1% PFA. Red fluorescence was measured by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA, USA). Data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Results are shown as mean fluorescence relative to the control samples.