Supplementary information:
Included are seven supplementary figures, S1-S6.
Figure S1. Statistical analysis of the autophagic flux and mTORC1 activity
during thapsigargin treatment and depletion of DAPK2, related to Fig. 1. (A)
Statistical analysis of quantification of western blots in Fig. 1C. LC3II proteins levels
were quantitated by densitometry and normalized to actin. Graph represents the foldinduction upon TG treatment compared to DMSO, with control siRNA set at 1. SD is
calculated from the mean of 3 independent experiments. ** indicates p<0.01. (B)
HEK293T cells were transiently transfected with ctrl siRNA or DAPK2 siRNA. After
72 hours, the cells were treated with 100nM thapsigargin (TG) for an additional 24
hours, and then treated with 20nM BafA1 for 1 hour, and lysates blotted for the
indicated proteins. (C-D) Statistical analysis of quantification of protein levels on
western blots Figs. 1D and 1E, respectively. Levels of phosphorylated and nonphosphorylated protein were quantitated by densitometry and normalized to total
protein levels. Graphs represent the fold-induction upon TG treatment compared to
DMSO, with control siRNA set at 1. SD is calculated from the mean of 3 independent
experiments. * indicates p<0.05.
Figure S2. Statistical analysis of autophagic levels and mTORC1 activity during
amino-acid starvation and depletion of DAPK2, related to Fig. 2. (A) Statistical
analysis of quantification of western blots in Fig. 2C. LC3II proteins levels were
quantitated by densitometry and normalized to actin. Graph represents the foldinduction upon EBSS treatment compared to DMEM, with control siRNA set at 1. SD
is calculated from the mean of 3 independent experiments. ** indicates p<0.01. (B-D)
Statistical analysis of quantification of protein levels on western blots Figs. 2D-2F,
respectively. Levels of phosphorylated and non-phosphorylated proteins were
quantitated by densitometry and normalized to total protein levels. Graphs represent
the fold-induction upon EBSS treatment compared to DMSO, with control siRNA set
at 1. SD is calculated from the mean of 3 independent experiments. * indicates
p<0.05, ** indicates p<0.01.
Figure S3. Depletion of DAPK2 reduces amino acid starvation-induced
autophagy and relieves mTOR inhibition in HEK293T cells, related to Fig. 2.
HEK293T cells were transiently co-transfected with GFP-LC3 and ctrl siRNA or
DAPK2 siRNA. After 48 hours, the cells were replated and treated with EBSS for 2
hours. (A) Fluorescent micrographs of representative cells showing diffuse or
punctate GFP-LC3. SD is calculated from the mean of 3 replicates. * indicates p<0.05
(B-D) Samples of cellular extracts were analyzed by western blot using the indicated
antibodies. *, non-specific band.
Figure S4. Statistical analysis of 4E-BP1 phosphorylation levels during
incubation with rapamycin and depletion of DAPK2, related to Fig. 3. Statistical
analysis of quantification of western blot in Fig. 3C. non-phosphorylated 4E-BP1
proteins levels were quantitated by densitometry and normalized to total 4E-BP1.
Graph represents the fold-induction upon rapamycin treatment compared to DMSO,
with control siRNA set at 1. SD is calculated from the mean of 3 independent
experiments. * indicates p<0.05.
Figure S5. DAPK2 depletion affects mTORC1 activity in basal growing
conditions, but not the interaction between mTOR and raptor, or the interaction
between raptor and its substrate in vitro, related to Fig. 4. (A) A549 cells were
transfected with ctrl siRNA or DAPK2 siRNA. After 72 hours, western blot analysis
was done using the indicated antibodies. *, non-specific band. (B) Protein lysate was
prepared from HEK293T cells stably expressing shRNA against HcRed or DAPK2.
Western blot analysis was done using the indicated antibodies. *, non-specific band.
(C) HEK293T cells were transfected with control (ctrl) or DAPK2 siRNA. After 72h,
protein lysates were subjected to immunoprecipitation using anti-mTOR antibodies.
Protein levels were detected by western blot. (D) HEK293T cells were co-transfected
with the indicated siRNAs and DNA plasmids for 48h. The amount of GST-4E-BP1
bound to raptor was examined by far-western assay.
Figure S6: DAPK2 does not phosphorylate mTOR and ULK1, related to Fig. 6.
(A-B) myc-tagged kinase dead mTOR mutant (D2357E/V2364I) (A) or FLAG-tagged
kinase dead ULK1 mutant (K46I). (B) were immunopurified from HEK293T cells
and incubated with immunopurified DAPK2-FLAG in a kinase reaction mixture
containing 33P-labeled-ATP. Reactions were resolved by SDS-PAGE and exposed to
X-ray film. (C) Schematic representation of raptor functional domains and known
phosphorylation sites (taken from PhosphoSitePlus database). The black arrows
indicate the area that was chosen to produce a recombinant fragment of raptor (690931).