Supplementary Figure 3 : Autophagosomes and ROS accumulate during mitotic arrest.

From: AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest

Supplementary Figure 3

(a) Representative profiles of mitotracker (left) and Dihydroethidium (DHE; right) in Cdc20-null (green) or control (grey) cells expressing CreERT, at the indicated time points after the release from RO-3306 (all the cells were previously treated 18 h with 4OHT). (b) Immunodetection of the indicated mitochondrial proteins at 24 h after the release from RO-3306 (all the cells were previously treated 18 h with 4OHT) in control-CreERT2 (left) and Cdc20-null cells treated with vehicle or the autophagy inhibitor 3MA. Unprocessed original scans of blots are shown in Supplementary Fig. 7. β-actin was used as a loading control. (c) Effect of 3MA and Cyclosporin A (CsA) on mitotracker levels in control-CreERT2 (left panels) and Cdc20-null cells 24 h after the release from RO-3306 (all the cells were previously treated 18 h with vehicle or 4OHT). Representative FACS profiles are shown and quantification of mitotracker signal is represented in the bottom graphs. Data are mean ± SEM (n = 3 independent experiments). (d) Quantification of ROS (mean ± SEM; n = 3 independent experiments) in Cdc20-null cells as determined by the OxiSelect TM Intracellular ROS assay kit. The effect of NAC treatment in the accumulation of ROS (mean ± SEM; n = 3 independent experiments) is also shown at the indicated times. (e) Quantification of ROS (mean ± SEM; n = 3 independent experiments) in Cdc20-null cells or Cdc20(lox/lox) cells arrested in mitosis after 24 h with taxol and pro-TAME, as determined by the OxiSelect TM Intracellular ROS assay kit. (f) Duration of mitosis until cell death in Cdc20-null and HeLa cells treated with the antioxidant NAC or the caspase inhibitor ZVAD after synchronization with nocodazole and release in MG132. The mean is indicated by red bars and data represent two separate experiments. In d,e,f the number of cells analyzed (n) is indicated in each condition. , P < 0.05; , P < 0.01; , P < 0.001 (Student’s t-test). Source data can be found in Supplementary Table 4.