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. 2020 Sep 17;23(10):101569. doi: 10.1016/j.isci.2020.101569

Figure 4.

Figure 4

TG/LDs are Hydrolyzed by Autophagy to Support GBM Cell Survival upon Glucose Deprivation

(A) Representative fluorescence images of U87 cells stained with BODIPY 493/503 (green) and anti-LC3 antibody (red) in the absence or presence of glucose (25 mM) and chloroquine (CQ, 5 μM) for 6 h. The co-localization was quantified by ImageJ (mean ± SEM) in over 30 cells.

(B and C) Representative fluorescence images of U87 cells co-stained with BODIPY 493/503 (green) and LysoTracker (red) (B) or anti-LAMP1 antibody (red) (C) in the presence or absence of glucose (25 mM) and CQ (5 μM) for 6 h. The co-localization was quantified by ImageJ (mean ± SEM, n = 30).

(D–F) Representative fluorescence images of U87 cells stained with BODIPY 493/503 (green) and Hoechst 33342 (blue) in the absence or presence of glucose (25 mM) and CQ (5 μM) for 8 h. LDs/cell were quantified in 30 cells (mean ± SEM, n = 30) (D). Their total lipids were analyzed by TLC (mean ± SD, n = 3) (E). Cell death was determined after trypan blue staining (mean ± SD, n = 3) (F).

(G–J) U87 cells were transfected with ATG5 shRNA-expressing lentivirus for 24 h and then cultured in medium without/with glucose (25 mM) for 8 h. Cell lysates were subjected to western blotting analysis using the indicated antibodies (G). Cells were stained with BODIPY 493/503 (green) and LysoTracker (red) and observed by confocal microscopy (H). Total cellular lipids were analyzed by TLC (I). Cell death was determined after trypan blue staining (J). Quantification and significance determination of LD number, relative TG levels, and cell death were same as in panels D–F.

Statistical significance was determined by one-way ANOVA for this entire figure; ∗∗p < 0.01, ∗p < 0.05. Please also see Figures S3 and S4, and Videos S1, S2, S3, and S4.