(A and B) Parental and the indicated U87 cells with ACSS2 S659A, R664/665A, or T363K knock-in were deprived of glucose for 10 h. Immunofluorescent analyses were performed with anti-LAMP1 (A) and anti-LC3B (B) antibodies. The immunofluorescence intensity in 100 cells (A) or the numbers of LC3B puncta in 20 cells (B) was quantitated using the ImageJ software program.
(C) Parental and the indicated U87 cells with knock-in of ACSS2 R664/665A or T363K mutant were deprived of glucose for 10 h. Electron microscopic analyses were performed. The blue and red arrows point to the lysosome and autophagosome, respectively (left panel). The lysosomes and autophagosomes in 10 cells in each group were quantitated (right panel).
(D) Parental and the indicated U87 cells with knock-in of ACSS2 S659A, ACSS2 R664/665A, or T363K mutant were deprived of glucose for 24 h and stained with trypan blue (0.5%). The viable cells were counted.
See also Figure S6.