1. FIGURE S1
S1.c
Scr.
iPARG 30 nM
H2O2
PAR
iPARP-1 60 nM
DAPI
S1.a
S1.b
PAR
MCF7 GFPLC3
control
Starved 60 min
H2O2 10 mM
β actin
H2O2 + PJ34 10 µM
MCF7 GFPLC3
control
30 min
60 min 5 10 15 20 25 30 35 40 45
% LC3 punctated cells
Untreated
DPQ 40 μM
Olaparib 10 μM ** *
Starvation
S1.d 1 2 3 4 5 6 7 8 9 10 11 12 25 50 75
100
125
Scr. 30 nM
siPARG 30 nM
Starvation ( hours )
MTT % Survival
MCF7 GFPLC3 ** * # ##
Figure S1.a. Western Blot analysis of PAR production during starvation. MCF7 GFPLC3 cells were starved for 60 minutes with HANK Buffer. Whole cell extracts were subjected to SDS-PAGE and PAR polymer was measured with a specific antibody. Where indicated, cells were pre-treated with PJ34, PARPs inhibitor, during 1,5 hours and maintained during starvation. 10 mM H2O2 for 10 minutes was used as positive control of PARylation. -tubulin was used as loading control. Similar results were obtained in three independent experiments.
Figure S1.b. DPQ and Olaparib had the same inhibitory effect on starvation-induced autophagy. MCF7 GFPLC3 cells were pre-treated with DPQ 40 M and Olaparib 10 M for 1.5 hours. Both PARPs inhibitors were maintained during starvation treatment with HANK Buffer for 30 and 60 minutes. The number of cells with the green GFPLC3 pattern was counted under fluorescence microscopy. Approximately 250 cells were counted in a fluorescent microscope in four independent experiments. *p<0.05, **p<0.01 by t-test comparing between starved and untreated MCF7 GFPLC3 cells.
Figure S1.c. IF of PAR polymer shows the effect of PARP-1 and PARG silencing. MCF7 GFPLC3 were transfected with PARP-1 siRNA (60nM), PARG siRNA (30nM). Scrambled or non-specific siRNA was used as negative control, using the same protocol as for siRNA transfection. 10 mM H2O2 for 10 minutes was used as positive control of PARylation. Similar results were obtained in three independent experiments.
Figure S1.d. Silencing of PARG and PAR accumulation didn’t affect cell survival during starvation. MCF7 GFPLC3 cells were transfected with PARG siRNA (30nM) and non-specific siRNA or scrambled (30nM) and treated with HANK Buffer for different times. Percentage of cellular viability was measured by MTT assay and referred to the control. Similar results were obtained in four independent assays. * p<0.05, ** p<0.01 comparing starved scrambled MCF7 GFPLC3 and untreated scrambled MCF7 GFPLC3 cells. # p<0.05, ## p<0.01 by t-test comparing starved PARG silenced MCF7 GFPLC3 and untreated silenced MCF7 GFPLC3.

2. FIGURE S1 S1.e Scrambled PJ34 iPARP1 iPARG MCF7 GFPLC3
Starvation 60 min
iPARG + Starvation 60 min
Starvation * **
MCF7 GFPLC3
S1.f
Figure S1.e. Effect of PARPs inhibitor, silencing of PARP-1 and accumulation of PAR polymer (iPARG) on starvation-induced autophagy. scrambled or non-specific siRNA was used as negative control, using the same protocol as for siRNA transfection. 48 hours later were starved with HANK Buffer for 60 min. At least 250 cells were counted in a Zeiss fluorescent microscope in 3 independent experiments.
Figure S1.f. Effect of autophagy and PARylation inhibitors on starvation-induced autophagy. MCF7 GFPLC3 cells were starved with HANK Buffer for 30 and 60 min. 10 µM PJ34, 10 CQ µM, 5 mM 3MA and 0,2 µM Bafilomycin A1 were used as co-treatment with HANK Buffer. * p<0.05, **p < 0.01 versus the indicated group in MCF7 GFPLC3 cells by t-test. Similar results were obtained in three independent experiments.

3. FIGURE S2
S2.a
Starvation
% LC3 punctated cells **
A549
control
30 min
60 min 25 50 75
100
125
shVector
shVector Baf A1
shPARG
shPARG Baf A1
A549
Starvation
*** **
scrambled
shPARG
shVector
A549
β actin
LAMP1
iLAMP1
S2.b
Starvation
A549
control
30 min
60 min 50
100
% LC3 punctated cells * **
***
sh PARG iATG7
Vector sh scrambled
Vector + iATG7
sh PARG scrambled
scrambled
β actin
ATG7
iATG7
shPARG
shVector
A549
shPARG A549
Control
iATG7
Starved 30 min
control
Untreated
LC3-I/II
α tubulin
Figure S2.a. Effect of Bafilomycin A1 and LAMP1 silencing on starvation-induced autophagy. shVector and shPARG A549 cells were transfected with LAMP siRNA (60nM) or treated with Bafilomycin A1 0.2 µM during 1 hour. In all the cases shVector and shPARG were starved for 30 and 60 min with HANK Buffer. Cells with the typical pattern of LC3 starvation-induced punctated were counted by fluorescence microscopy. **p < 0.01, *** p<0.001 versus the indicated group by t-test.
Figure S2.b Effect of ATG7 silencing on starvation-induced autophagy. shVector and shPARG A549 cells were transfected with ATG7 siRNA (60nM). Scrambled or non-specific siRNA was used as negative control, using the same protocol as for siRNA transfection. 48 hours later were starved for 30 and 60 minutes with HANK Buffer. ATG7 silencing was confirmed using a specific antibody against ATG7 protein. The right panel shows the effect of ATG7 silencing on the endogenous starvation-induced LC3-II conversion in shPARG A549 cells. -tubulin was used as loading control. Similar results were obtained in three independent experiments. *p<0.05, **p < 0.01, *** p<0.001 versus the indicated group by t-test.

4. FIGURE S3
S3.a
S3.b 10 20 30 40 50 60 70 80 90
100
110
120
Starvation ( min)
Intracellular ATP Levels
( % of control )
MCF7 GFPLC3 ** *
p-AMPk
Control
30 min
60 min
120 min
240 min
15 min
Starvation
MCF7 GFPLC3
GAPDH
AMPk
p-ACC
scrambled
β actin
p-AMPk
AMPk
MCF7 GFPLC3
Starvation 30 min
PJ34 + Baf A1
iPARG 30 nM
PJ34
PJ MA
iPARG + Baf A1
iPARG + 3-MA
Figure S3.a. ATP levels during starvation-induced autophagy. MCF7 GFPLC3 cells were starved for 15, 30, 60 and 90 minutes with HANK Buffer. Concentrations of ATP were normalized with total proteins in each sample and referred to the control (100%). Similar results were obtained in three independent experiments. * p<0.05, **p<0.01 comparing between control and starved MCF7 GFPLC3 cells by t-test. Immunoblot analysis of AMPk activation at different times of starvation. Activation of AMPk kinase was measured using a specific antibody against the phosphorylated substrate ACC and phospho-AMPk, after HANK buffer treatment. Total AMPk was used to normalize for the non-phosphorylated protein and GAPDH was used as loading control. Similar results were obtained in three independent experiments.
Figure S3.b. AMPk activity in PARG silenced and after PARylation inhibition in MCF7 GFLC3 cells during starvation-induced autophagy. Cells were starved for 30 min with HANK Buffer and co treated with Bafilomycin A1 0.2 µM or 3 MA 5 mM, both drugs were maintained during starvation. Total AMPk was used to normalize for the non-phosphorylated protein and β actin was used as loading control. Similar results were obtained in three independent experiments.

5. FIGURE S3
S3.c **
MEFs
parp-1 WT
parp-1 KO 10 20 30 40 50 60 70 80 90
Basal
Starved 30 min
Starved + PJ34
Starved + PJ34 + Baf A1
% LC3 punctated cells
MEFs (Starvation 30 min)
parp – 1-/- MEFs
Control
30 min
30 min + PJ34
30 min + PJ34 + Baf. A1
 actin
LC3-I/II
parp – 1+/+ MEFs
Figure S3.c. Delay of autophagy in parp-1-/- 3T3 MEFs during nutrient deprivation. parp-1+/+ and parp-1-/- MEFs transfected with GFPLC3 cells were starved with HANK Buffer for 30 minutes and the number of vesicles positive to GFP were counted under fluorescence microscopy. Both cell lines were co treated with PJ34 10 µM and Bafilomycin A1 0.2 µM. Endogenous LC3-II translocation was associated to starvation-induced autophagy in these cells. β actin was used as loading control Similar results were obtained in three independent experiments. **p < 0.01 versus the indicated group by t-test. Approximately 250 cells were counted in a fluorescent microscope in four independent experiments.
Figure S3.d.Treatment with AICAR increased the number of GFP-LC3 positive vesicles in parp-1 -/- 3T3 MEFs during starvation. parp-1+/+ and parp-1-/- MEFs 3T3 transfected with GFPLC3 cells were starved with HANK Buffer for 30, 60, 120 and 240 minutes and the number of vesicles positive to GFP were counted under fluorescence microscopy. Similar results were obtained in three independent experiments. *p<0.05, **p<0.01 versus the indicated group by t-test.
S3.d **
control
30min
60 min
120 min
240 min 10 20 30 40
parp-1+/+ MEFs
parp-1-/- MEFs
Nº GFP-LC3 puncta / cell
parp-1-/- AICAR
Starvation *

6. FIGURE S4
S4.a
S4.b
Control
15 min
COS 1
Starvation
p-AMPk
AMPk
 actin
30 min
60 min
p-p70S6k
p70S6k
p-ULK1
ULK1
LC3-II
NAD+ Binding Domain
523
1014
ACTIVE SITE
WGR N C
h PARP-1 FI
FII
NLS
DNA Binding Domain (DBD) 1-371
DEVD 214 21 53
125
162
207
226
FIII
Automodification Domain
372
522
BRCT LZ
pBC-ABC
pBC-D
pBC-EF
1014 Aa
S4.c
HeLa shControl
Control
15 min
30 min
60 min
p-AMPk
AMPk
β actin
Starvation
HeLa shPARG
HeLa shControl
Control
15 min
30 min
60 min
PAR
β actin
Starvation
HeLa shPARG
HU 2 mM 24 h
Figure S4.a. Mapping of human PARP-1 to observe interaction PARP-1/AMPk. All the constructs are previously published by Francoise Dantzer (Dantzer et al 2004). ABC: DNA Binding Domain, D: Automodification Domain, EF: Catalytic Domain.
Figure S4.b. Activation of AMPk/mTORC1/ULK1 pathway in COS1 cells during starvation. COS1 cells were starved for 15, 30 and 60 minutes with HANK Buffer. Immunoblot shows AMPk activity, mTORC1 inhibition (p-p70S6k) endogenous LC3-II translocation and ULK1 activation. β actin was used as loading control. Similar results were obtained in two independent experiments.
Figure S4.c. AMPk activity and PARylation levels in HeLa shPARG during starvation-induced autophagy. HeLa shControl and shPARG cells were starved for 15, 30 and 60 min with HANK Buffer. Hydroxyurea 2 mM was used as positive control of PARylation. β actin was used as loading control. Similar results were obtained in three independent experiments.

7. FIGURE S5
S5a
Control
Starved 30’
INPUT
IP PAR
WB FLAG
WB PAR
Untreated KU
AMPkα PARdef1
FLAG AMPk
FigureS5a: AMPkα mutated in PARylation sites (AMPKPARdef1) completely abolished AMPK modification by PARylation, as shown also in figure 4. Interaction was determined by IP of PAR polymer in shPARG HeLa cells transfected with FLAG-AMPk1 wild type or AMPKPARdef1. Transfected HeLa cells were pretreated with 100 nM KU for 2 hours.