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Supplementary Data
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Supplementary Figure Legends
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Supplementary Figure 1. L-Pro modulates AAR and controls ESC behaviour
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(a-b) ESCs were incubated without NEAAs or supplemented with each single NEAA (0.5
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mM), and the expression of AAR-related genes Atf4, Asns, and Nupr1 (a) and the TGF-
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related gene Lefty2 (b) was analysed by qPCR. The gene expression values are presented
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as fold change compared to control cells after normalisation to Gapdh. (c) Effect of NEAAs
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on the ESC to PiC transition evaluated by the colony phenotype assay. ESCs were grown for
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5 days in complete ESC medium with or without NEAAs, fixed, stained with crystal violet and
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photographed. Scale bar, 250 μm. (d-e) ESCs were either untreated as Control or treated
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with either NAC (0.625, 1.25, 2.5 and 5 mM) or GSH (0.04, 0.2, 1 and 5 mM) and +/- L-Pro;
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proliferation (d) and expression of Atf4 and Nupr1 (e) were measured at 48 hrs after plating
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by qPCR and CyQuantR assay, respectively. (f) The intracellular concentration of free L-Pro
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was measured in ESCs, PiCs, and mouse embryonic fibroblasts (MEFs). Data are expressed
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as relative to ESCs and represent the mean  SD from (a, b and f) three and (d and e) two
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independent experiments, *p<0.001.
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Supplementary Figure 2. Halofuginone (HF) blocks L-Pro-induced esMT and rapidly reverts
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the PiC phenotype.
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(a) Effect of HF on L-Pro-mediated regulation of Lefty1, Asns, and Slc1a4 expression. ESCs
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were incubated with L-Pro (0.2 mM) or with L-Pro (0.2 mM)/HF (8 nM). The mRNA levels
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were measured by qPCR analysis and the results expressed as the fold change compared to
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control cells. (b-c) Effect of Histidinol and Cycloheximide. ESCs were left untreated as
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control or treated with increasing concentrations of either Histidinol or Cycloheximide +/- L-
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Pro; ESC proliferation (b) and expression of Atf4, Nupr1 and Trib3 (c) were measured at 36
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hrs by CyQuantR and qPCR assay, respectively. (d) L-Pro but not L-Orn counteracts the
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antiproliferative effect of HF. ESCs were left untreated as control or treated with either L-Pro
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or L-Orn (2 mM) +/- HF (4 nM), and proliferation was measured at 36 hrs by CyQuantR assay
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and expressed as RFU. (e-h) Effect of HF on L-Pro-induced esMT. (e-f) ESCs were
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incubated with or without L-Pro (0.5 mM) plus increasing concentrations of HF (10, 15 or 20
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nM). Five days post-seeding, the resulting cell colonies were fixed, stained with crystal violet
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and analysed. (e) Colony type frequency (% of domed vs flat colonies); n=300 colonies
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scored/each condition. (f) Representative photomicrographs of colonies displaying either a
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round shaped three-dimensional (domed) or an irregular (flat) morphology. Scale bar, 75 m.
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(g) HF and VitC inhibit the L-Pro-induced esMT process with similar efficiency.
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Representative frames of time-lapse series from ESCs untreated and treated with different
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combinations of the follwoing compounds: L-Pro (0.2 mM), HF (8 nM) and VitC (0.5 mM).
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Images (20x) were captured beginning on day 3 post-plating. (h) Schematic representation of
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the experimental strategy (left panel). Freshly generated PiCs were plated at low density
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(500 cells/cm2) and grown in complete medium either without L-Pro (0.5 mM), or in the
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presence of L-Pro ± Halofuginone (HF; 10 nM). At day 5 after plating, the resulting colonies
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were stained with crystal violet and analysed (right panels). Data represent the percentage of
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colony type frequency (domed or flat) in the different culture conditions (~300 colonies
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scored/condition). The number of colonies was comparable in the different culture conditions.
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Data represent the mean  SD from (a, e, and h) three and (b, c and d) two independent
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experiments, *p<0.001.
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Supplementary Figure 3. Atf4 modulation (LOF & GOF) alters L-Pro activity in ESCs
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(a-b) Molecular characterisation of Atf4 KD ESCs. Atf4 mRNA levels were analysed by qPCR
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on four different Atf4 KD ESC lines (a). The results are expressed as the fold change
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compared to control ESCs (NT), normalised to Gapdh. (b) Atf4 protein levels were measured
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by western blot analysis. Densitometric analysis is expressed in ADU as the Atf4/Gadph
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ratio. (c) Atf4 mRNA levels in shNT and shATF4 ESCs was measured by qPCR in cells
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incubated with or without L-Pro. (d) Expression of AAR markers (Trib3, Slc1a4, and Nupr1)
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and of AAR unrelated genes, such as the pluripotency markers Oct4 and Nanog, in control
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and Atf4 KD ESCs, measured by qPCR. (e) shATF4 ESCs developed colonies significantly
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smaller (~2 times lower diameter values, at right) compared to the control (~350 colonies
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scored/condition). Representative images of the colonies raised by shNT and shATF4 ESCs.
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Scale bar, 200 m. (f-i) Effect of Atf4 overexpression on ESCs treated with L-Pro. (f) Atf4
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protein level was measured by Western blot analysis on ESCs at two different time points (24
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hrs and 48 hrs +/-Tet). Densitometric analysis is expressed in arbitrary units (ADU) as the
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Atf4/Gadph ratio. (g) Expression of AAR markers (Trib3, Nupr1, and Slc1a4) and of AAR
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unrelated genes (Oct4 and Nanog) measured by qPCR in ESCs incubated without or with
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Tet. (h) Quantification of crystal violet staining of cell colonies generated in the different
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culture conditions. (i) Representative photomicrographs of ESCs incubated  Tet and left
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untreated as control or treated with increasing amounts of L-Pro. (j) qPCR analysis of
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Aldh18a1 and Pycr1 in ESCs  HF (8 nM) either alone or treated with L-Pro (0.2 mM). Data
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represent the mean  SD from three (a, b, c, d, e, f, g and j) or five (h and i) independent
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experiments, *p<0.001, **p<0.01.
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Supplementary Figure 4. L-Pro regulates autophagy in ESCs
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(a) Intracellular localisation of LC3-GFP fusion was monitored at different time points in
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ESCs stably expressing LC3-GFP wild-type fusion protein (LC3_wt). Representative
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photomicrographs showing LC3-GFP localisation. Scale bar, 25 m. (b) Effect of NEAAs on
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LC3-GFP localisation. Representative photomicrographs of LC3_wt cells incubated without
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NEAAs or supplemented with each single NEAA (0.5 mM). Scale bar, 25 m. (c-d) Effect of
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Histidinol on LC3-GFP localisation. Representative photomicrographs of LC3_wt ESCs
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incubated with L-Pro (2 mM) Histidinol (1.25 mM) or untreated as a control (c). Quantification
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of LC3 punctuated ESCs in the different culture conditions (c) at 24 hrs after plating (d). (e)
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Punctate
LC3
pattern
in
untreated
or
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L-Pro
treated
colonies.
Representative
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photomicrographs of 5-day-old colonies developed from LC3_wt ESCs incubated with or
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without supplemental L-Pro (0.2 mM). (f) LC3_wt and LC3_mut cells were plated with L-Pro
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(0.2 mM), and the intracellular localisation of LC3-GFP was monitored at days 3 and 4 after
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plating. Scale bar, 20 m. (g) Representative TEM images showing multivesicular structures
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(left panels) and multilamellar bodies (right panels) in L-Pro-induced cells.
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Supplementary Figure 5. The L-Pro-AAR-Atf4 axis regulates apoptosis during esMT
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(a) Identification of apoptotic cells via staining for annexin V and PI followed by FACS
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analysis. Quadrants define 4 cell populations: PI-/A- (intact living cells); PI-/A+ (intact
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apoptotic cells); PI+/A+ (necrotic/late-apoptotic cells); damaged cells (PI+/A-). Data represent
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the percentage of PI-/A+ cells in a representative experiment. (b) Western blot analysis
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showing the full-length form (116 KDa) and caspase-specific proteolytic fragment (89 kDa) of
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poly (ADP-ribose) polymerase (PARP) (left panels); densitometric analysis is expressed in
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ADU as the PARP/Gapdh ratio (right panel). (c) Quantitative determination of DNA laddering
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by a photometric immunoassay. ESCs were plated without or with supplemental L-Pro (0.5
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mM) and analysed at days 3, 4 and 5. (d) Atf4 KD and Atf4 overexpression have opposite
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effects on apoptosis during esMT. Control (shNT) and Atf4 KD (shATF4) ESCs were
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incubated (5 days) with or without supplemental L-Pro. DNA-laddering values are relative to
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untreated controls (left panel). Atf4 Tet-OFF ESCs were treated (5 days) +/- Tet and with L-
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Pro (1 mM). DNA-laddering values are relative to control (+Tet/+ L-Pro) ESCs (right panel).
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(e) Atf4 overexpression reduces autophagy in esMT. Control (+Tet) and Atf4 GOF (-Tet)
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ESCs were treated with L-Pro (1 mM) and stained with the red-fluorescent acidic dye
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(LysoTracker Red) at day 5 after the esMT. Data show the quantification of Lysotracker
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positive cells, as the percentage of the total number of cells (~300 cells scored/condition). (f)
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Z-VAD prevents the acquisition of the motile mesenchymal-like phenotype in esMT.
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Representative photomicrographs of cell colonies grown in the presence of L-Pro and +/- Z-
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VAD (0.1 mM). Scale bar, 200 m. (g-i) Addition of either VitC (0.5 mM) or
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PD0325901/CHIR99021 (1 M / 3 M) inhibitors prevents L-Pro (0.2 mM)-induced esMT,
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inhibits apoptosis (DNA laddering), and improves cell survival. (g) The colony-type frequency
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(domed vs flat colonies) was measured (300 colonies scored/condition). (h) DNA-laddering
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values are relative to untreated controls. (i) Crystal violet quantification values are relative to
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untreated controls. Data represent the mean  SD from (b, c, g and h) two, (d and e) three
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or (i) five independent experiments, *p<0.001, **p<0.01.
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Supplementary Table 2
Gene
Primer (sense)
Primer (antisense)
Atf4
TCCACTCCAGAGCATTCCTT
AAGCAGCAGAGTCAGGCTTC
Asns
CCTGTAGCATGTGTGGCATC
CGATCTTCATCGCACTCAGA
Nupr1
GAGAAGCTGCTGCCAATACC
GGTGTGGTGTCTGTGGTCTG
Trib3
GCTGGCAGATACCCATTCCA
GGCCGTGATCCTCTCTCAAC
Slc1a4
TCCAAGGAGTCAGTGCTGTG
AAAGTGTATGAGGGCCATGC
Lefty1
AGCTCAAGGCAATTGTGACC
TCATCTCTGAGGCGACACAC
Lefty2
GGACATGGCTATGCAGTGAA
TTGATTCCCCTGTTCTCACC
Fgf5
CAAAGTCAATGGCTCCCACGAAG
CTACAATCCCCTGAGACACAGCAAATA
Brachyury (T)
GAACCTCGGATTCACATCGTGAGA
ATCAAGGAAGGCTTTAGCAAATGGG
Pitx2
III-tubulin
MHC
Oct 3/4
Nanog
Aldh18a1
Pycr1
Gapdh
AGCTGTGCAAGAATGGCTTT
CACCATGCTGGACGACATAC
GAATGACCTGGTGTCCGAGT
TGAAAACGGAAAGACGGTGA
CCGATTCCTCGTCATCATCT
TCCTTGAGGTTGTACAGCACA
GAAGCAGAAGAGGATCACCTTG
TTCTTAAGGCTGAGCTGCAAG
AAGTACCTCAGCCTCCAGCA
CAGCTGGTCAGAGACATCCA
GTGCTGAGCCCTTCTGAATC
GCAGATCTCGGTGGATAAGC
tTAUP2
ATGAGCGTAGGCTTCATCG
GACCGTAGCTTGGTCCATGT
TGCACCACCAACTGCTTAGC
TCTTCTGGGTGGCAGTGATG
GCATCAAGTCGCTAAAGAAGAAAG
3’HygLW2
GAGTGCTGGGGCGTCGGTTTCC
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