Supplementary Figure 4 : AMPK is reactivated during mitotic arrest.

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

Supplementary Figure 4

(a) Schematic representation of the protocol followed for synchronization in human cells. Cells were treated with RO-3306 and this compound was washed-out 18 h later allowing mitotic entry in the presence of taxol and pro-TAME (an APC/C inhibitor) which mimic the arrest in metaphase imposed by Cdc20 ablation. (b) The levels of the indicated antigens were analyzed by immunoblot in HeLa or MDA-MB-231 cells. α-tubulin was included as a loading control and unprocessed original scans of blots are shown in Supplementary Fig. 7. (c) Changes in AMPK activity were monitored by using a FRET biosensor in Cdc20(lox/lox) cells in the absence of 4-OHT. Upon treatment of these control cells with 2-DG, a glucose analog, AMPK is activated giving an increase of about 2-fold in biosensor signal (indicated in red in the images after 2DG addition; bottom panel). This increase is not observed in cells treated with siRNAs against AMPK. Data are mean ± SD (n = 4 independent experiments). (d) AMPK activity was also monitored by the FRET biosensor in mitotic cells treated with vehicle (blue), 2DG (green) or Glucose (red). Data are represented as mean ± SD (n = 3 independent experiments) after scoring the indicated number of mitotic cells in each condition. (e) mRNA levels of APMKα1 and AMPKα2 48 h after nucleofection of specific siRNAs. Data are mean ± SD (n = 3 independent experiments) normalized against β-actin mRNA levels. (f) Levels of OCR (left) and ECAR (right) in Cdc20-null cells arrested in mitosis for 24 h and transfected with siRNAs against siAMPKα1 (siα1), siAMPKα2 (siα2) or the combination of both relative to Scrambled (Scr.) sequences. Data are mean ± SD (one experiment with 12 replicates). (g) Effect of siRNAs against AMPKα1 and/or AMPKα2 on mitotracker levels in Cdc20-null cells 24 h after the release from RO-3306. Representative FACS profiles are shown on the left, and the quantification of mitotracker signal mean ± SEM (n = 3 independent experiments) is represented in the histogram. In g, NS, no significant; , P < 0.05 (Student’s t-test). Source data for all the figures can be found in Supplementary Table 4.