Supplementary Figure 2 : Induction of autophagy during mitotic arrest.

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

Supplementary Figure 2

(a) Mitotic entry in Cdc20-null cells after release from RO-3306. Cdc20(lox/lox) cells were treated with 4-OHT to eliminate Cdc20 12 h before the addition of the Cdk1 inhibitor RO-3306 to trigger G2 arrest. This compound was washed-out 18 h later allowing the accumulation of mitotic cells over time (left panel). Representative images are shown in the right panel. Scale bars, 50 μm. (b) LC3 lipidation was analyzed in MDA-MB 231 cells arrested by treatment with taxol + pro-TAME, in the presence or absence of E64D/PA. Two different time exposures for LC3 are shown. (c) Colocalization of LC3 and Annexin V (mean ± SD; n = 3 independent experiments) in Cdc20-null cells at the time points of mitotic arrest indicated in the graph. Images are representative for the double staining at 24 h. Scale bar, 10 μm. (d) mRNA levels (mean ± SD; n = 3 independent experiments) of the indicated transcripts 48 h after siRNA nucleofection. Data were normalized against the levels of β-actin transcripts. (e) Duration of mitosis (from metaphase until cell death) in Cdc20-null cells treated with ZVAD, the indicated inducers of autophagy (mTOR inhibitors PP242, temsirolimus, BEZ235), or transfected with siRNAs against Raptor (Rap.). (f) Duration of mitosis in Hela and MDA-MB 231 cells synchronized by the taxol + pro-TAME protocol and treated with the indicated inhibitors. (g) Duration of mitosis in Cdc20-null cells treated with the indicated PI3K/Akt inhibitors. (h) Immunoblot of LC3 after mTOR inhibition with PP242 or BEZ235 in Cdc20-null cells. Cdc27 band-shift (indicating phosphorylation) was used to confirm mitotic arrest. The samples were processed simultaneously and run in the same blot. (i) Immunoblot of LC3 after mTOR inhibition with PP242 in MDA-MB-231 cells synchronized by the taxol + pro-TAME protocol. (j) Duration of mitosis in Atg5-null cells treated or not with the caspase inhibitor ZVAD (left) and Bax/Bak double mutant cells treated or not with the autophagy inhibitor 3MA (right), in both cases using the taxol + pro-TAME protocol. (k) LC3 lipidation and PARP cleavage after treatment with caspase (ZVAD) and autophagy (E64d/PA) inhibitors in Cdc20-null cells. Cdc27 phosphorylation (retarded motility) indicates mitotic cells. In b,h,i,k, α-tubulin is included as a loading control. In eg; j dots represent individual cells and the mean are indicated by red lines. The number of cells analyzed (n) is indicated in each condition, and data are representative of three (e), two (f), six (g) or one (j) separate experiments. Green or red backgrounds indicate a significant delay or premature cell death in mitosis, respectively. , P < 0.05; , P < 0.01; , P < 0.001; Student’s t-test. For b,h,i,k Unprocessed original scans of blots are shown in Supplementary Fig. 7, and α-tubulin is included as a loading control. Source data can be found in Supplementary Table 4.