Figure 6 ( A - F ) Cells were either untreated ( UT ) or treated with neocarzinostatin ( NCS ) in the presence or absence of PIKK inhibitors . Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM .

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Figure 6(A-F) Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from 3 independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.


Figure 1 . UMAP plot revealing the cell type annotations of three samples . B ) The cellular composition of three healthy kidney samples . Dotplot exhibition of canonical markers used for cell type classification . The gene expression patterns of ACE2 of all detected cell types in kidney .

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Figure 1.UMAP plot revealing the cell type annotations of three samples. B) The cellular composition of three healthy kidney samples.Dotplot exhibition of canonical markers used for cell type classification.The gene expression patterns of ACE2 of all detected cell types in kidney.


Figure 2 . cell type annotations visualized by UMAP . B ) The sample specific UMAP visualization of two samples . Heatmap of canonical markers applied for cell type assignment . Violin plot displaying the expression signals of kidney cell types .

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Figure 2.cell type annotations visualized by UMAP. B) The sample specific UMAP visualization of two samples.Heatmap of canonical markers applied for cell type assignment.Violin plot displaying the expression signals of kidney cell types.


Figure 3 . two - dimensional visualization of bladder cell atlas by UMAP . Expression pattern of canonical markers used for identifying bladder cell types . ACE2 expression across 12 cell subgroups in the bladder dataset .

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Figure 3.two-dimensional visualization of bladder cell atlas by UMAP.Expression pattern of canonical markers used for identifying bladder cell types.ACE2 expression across 12 cell subgroups in the bladder dataset.


Figure 1 ( A ) E7 . 0 whole embryo and transverse section ( TSs ) ( dashed lines ) through ( a1 ) extra - embryonic tissue showing expression in a ring of extra - embryonic ectoderm ( ee ) and extraembryonic mesoderm ( em ) and absence of transcripts in the allantois ( al ) ; ( a2 ) revealing expression in epiblast ( ep ) and in mesendoderm ( me ) emerging from the primitive streak ( ps ) , dashed box ( a2 ' ) defines higher magnification region shown in a2 . ( B ) E8 . 5 whole embryo dorsal view and TSs ( dashed lines ) showing expression in : ( b1 , b2 , b3 ) the open anterior neural plate ( future fore , mid and hindbrain ) including ( b3 ) neural crest ( nc ) emerging from hindbrain ; ( b4 ) in the dorsal neural tube ( prospective neural crest ) and dorsal somites ( s ) ; ( b5 ) in closing preneural tube neural ( pnt ) and epithelising somites ; ( b6 ) in caudal lateral epiblast ( cle ) adjacent to the node ( n ) at the anterior tip of the primitive streak , but not in presomitic mesoderm ( psm ) . White arrows indicate open anterior and posterior neural plate . ( C ) E9 . 5 whole embryo side view and TSs ( white dashed lines ) showing expression in : ( c1 ) forebrain ( fb ) and hindbrain , including optic vesicles ( opv ) , ( c1 ' ) seen at high magnification ( and indicated by box grey dashed line ) and otic vesicles ( ov ) ( c1 ' ' ) seen at high magnification ( box grey dashed line ) ; ( c2 ) high expression in dorsal hindbrain ( hb ) and ( c3 ) spinal cord ( sc ) and dermamyotome ( dm ) of the differentiating somite ; ( c5 ) throughout open posterior neural plate . ( D ) High magnification of E9 . 5 whole embryo showing expression in first branchial arch ( ba ) and forming cranial ganglia , which are just becoming apparent , ( V ) , ( VII ) , ( VIII ) ( IX ) and ( X ) . OV , otic vesicle . ( E ) Expression in mesenchyme cells underlying the apical ectodermal ridge ( aer ) in the progress zone ( pz ) and in the zone of polarising activity ( zpa ) of the forelimb ( fl ) . Data information : Images representative of n ≥ 4 embryos for each stage . Scale bars 200 µm , except sections 100 µm .

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Figure 1(A) E7.0 whole embryo and transverse section (TSs) (dashed lines) through (a1) extra-embryonic tissue showing expression in a ring of extra-embryonic ectoderm (ee) and extraembryonic mesoderm (em) and absence of transcripts in the allantois (al); (a2) revealing expression in epiblast (ep) and in mesendoderm (me) emerging from the primitive streak (ps), dashed box (a2') defines higher magnification region shown in a2. (B) E8.5 whole embryo dorsal view and TSs (dashed lines) showing expression in: (b1,b2,b3) the open anterior neural plate (future fore, mid and hindbrain) including (b3) neural crest (nc) emerging from hindbrain; (b4) in the dorsal neural tube (prospective neural crest) and dorsal somites (s); (b5) in closing preneural tube neural (pnt) and epithelising somites; (b6) in caudal lateral epiblast (cle) adjacent to the node (n) at the anterior tip of the primitive streak, but not in presomitic mesoderm (psm). White arrows indicate open anterior and posterior neural plate . (C) E9.5 whole embryo side view and TSs (white dashed lines) showing expression in: (c1) forebrain (fb) and hindbrain , including optic vesicles (opv), (c1') seen at high magnification (and indicated by box grey dashed line) and otic vesicles (ov) (c1'') seen at high magnification (box grey dashed line); (c2) high expression in dorsal hindbrain (hb) and (c3) spinal cord (sc) and dermamyotome (dm) of the differentiating somite; (c5) throughout open posterior neural plate. (D) High magnification of E9.5 whole embryo showing expression in first branchial arch (ba) and forming cranial ganglia, which are just becoming apparent, (V), (VII), (VIII) (IX) and (X). OV, otic vesicle. (E) Expression in mesenchyme cells underlying the apical ectodermal ridge (aer) in the progress zone (pz) and in the zone of polarising activity (zpa) of the forelimb (fl). Data information: Images representative of n≥ 4 embryos for each stage. Scale bars 200 µm, except sections 100 µm.


Figure 2 ( A - H ) Live wildtype littermate and Slc7a5 - null embryos imaged shortly after dissection . ( A - D ) Wildtype and ( E - H ) Slc7a5 - null E9 . 75 embryos from lateral ( A , D , E , H ) , frontal ( B , F ) or dorsal ( C , G ) views ; ( e1 - h1 ) higher magnification images of the Slc7a5 - null embryo . White arrowheads indicate the smaller limb bud ( compare B and F ) , open / reduced forebrain ( compare D and H ) , open neural tube at posterior ( compare C and E , e1 ) and anterior ( compare B and F , f1 ) regions . Black arrowheads indicate kinked neural tube ( compare C and G , g1 ) and apparently missing optic and smaller otic vesicles ( ov ) ( compare D and h1 ) . Data information : Scale bars 200 µmmRNA in situ hybridization and immunofluorescence in E9 . 5 or E10 . 5 wildtype and Slc7a5 - null embryos for key marker genes . ( I - L ) Fgf8 mRNA transcripts were detected in wildtype ( I - j1 ) and in Slc7a5 - null ( K - l1 ) E10 . 5 embryos ( n = 4 each ) ( white arrowheads indicate limb buds in K and the isthmus in L ) with ( j1 , l1 ) sections through isthmus at midbrain / hindbrain border and ( i1 , k1 ) lateral views of forelimb buds . Data information : Scale bars 200 µm , except sections 100 µm . mRNA in situ hybridization and immunofluorescence in E9 . 5 or E10 . 5 wildtype and Slc7a5 - null embryos for key marker genes . ( M - N ) Wholemount Tubulin - β - III ( Tuj1 ) immunofluorescence performed on ( M , m1 ) wildtype and ( N , n1 ) Slc7a5 - null E9 . 5 - E10 . 5 embryos ( n = 5 each condition ) . Dorsal root ganglia ( DRG ) and Sympathetic Chain Ganglia ( SCG ) are indicated with open arrowheads together with cranial ganglia IX , X and XI . Images in ( m1 , n1 ) show higher magnification of the cranial ganglia V , VII / VIII ( open arrowheads ) . Data information : Scale bars 200 µmmRNA in situ hybridization and immunofluorescence in E9 . 5 or E10 . 5 wildtype and Slc7a5 - null embryos for key marker genes . ( O - P ) Neurog2 expression in wildtype ( O - o4 ) and Slc7a5 - null embryos ( P - p4 ) ( n = 5 and n = 6 each ) , showing reduced expression in forebrain ( o2 , p2 ) , cranial ganglia ( o1 , o3 , p1 , p3 , arrowheads ) and in spinal cord at level of forelimb ( o4 , p4 , arrowheads indicate position of neural crest ) . Data information : Scale bars 200 µm , except sections 100 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-H) Live wildtype littermate and Slc7a5-null embryos imaged shortly after dissection. (A-D) Wildtype and (E-H) Slc7a5-null E9.75 embryos from lateral (A, D, E, H), frontal (B, F) or dorsal (C, G) views; (e1-h1) higher magnification images of the Slc7a5-null embryo. White arrowheads indicate the smaller limb bud (compare B and F), open/reduced forebrain (compare D and H), open neural tube at posterior (compare C and E, e1) and anterior (compare B and F, f1) regions. Black arrowheads indicate kinked neural tube (compare C and G, g1) and apparently missing optic and smaller otic vesicles (ov) (compare D and h1). Data information: Scale bars 200 µmmRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (I-L) Fgf8 mRNA transcripts were detected in wildtype (I-j1) and in Slc7a5-null (K-l1) E10.5 embryos (n=4 each) (white arrowheads indicate limb buds in K and the isthmus in L) with (j1, l1) sections through isthmus at midbrain/hindbrain border and (i1, k1) lateral views of forelimb buds. Data information: Scale bars 200 µm, except sections 100 µm.mRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (M-N) Wholemount Tubulin-β-III (Tuj1) immunofluorescence performed on (M, m1) wildtype and (N, n1) Slc7a5-null E9.5-E10.5 embryos (n=5 each condition). Dorsal root ganglia (DRG) and Sympathetic Chain Ganglia (SCG) are indicated with open arrowheads together with cranial ganglia IX, X and XI. Images in (m1, n1) show higher magnification of the cranial ganglia V, VII/VIII (open arrowheads). Data information: Scale bars 200 µmmRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (O-P) Neurog2 expression in wildtype (O-o4) and Slc7a5-null embryos (P-p4) (n= 5 and n=6 each), showing reduced expression in forebrain (o2, p2), cranial ganglia (o1, o3, p1, p3, arrowheads) and in spinal cord at level of forelimb (o4, p4, arrowheads indicate position of neural crest). Data information: Scale bars 200 µm, except sections 100 µm.


Figure 3 ( A - B ) Sox10 in situ hybridization in ( A ) wildtype and ( B ) Slc7a5 - null E9 . 5 embryos ( n = 4 each ) with TSs of ( a1 , b1 ) the hindbrain at level of trigeminal ganglion V , ( a2 , b2 ) otic vesicles and the neural tube at more posterior levels ( a3 , a4 , b3 , b4 ) . Scale bars 200 µm , except sections a1 - b4 , 100 µm . Closed white arrowheads in ( a3 , a4 ) indicate neural crest , open white arrowheads indicate depleted neural crest in ( b3 ) and position where neural crest should be in ( b4 ) . ( C - G ) Immunofluorescence on TSs caudal spinal cord of E9 . 5 wildtype littermates and Slc7a5 - null embryos ; Pax3 and FoxA2 were used as indicators for dorso - ventral organisation in ( C , F ) wildtype , n = 2 ( for FoxA2 ) and n = 4 ( For Pax3 ) and ( D , G ) Slc7a5 - null , n = 3 ( for FoxA2 ) and n = 3 for Pax3 ) neural tube . Arrowheads indicate border of Pax3 expression domain . Scale bars 50 µm . ( E ) Percentage of Pax3 expressing cells was determined by counting these cells and all DAPI labelled nuclei in the neural tube and comparison made between wildtype ( 4 embryos , 6 sections each ) and Slc7a5 - null ( 3 embryos , 6 sections each ) , each dot represents the average for one embryo , unpaired t - test , * p = 0 . 018 ( see original source data ) . Error bars indicate SEM .

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Figure 3(A-B) Sox10 in situ hybridization in (A) wildtype and (B) Slc7a5-null E9.5 embryos (n=4 each) with TSs of (a1, b1) the hindbrain at level of trigeminal ganglion V, (a2, b2) otic vesicles and the neural tube at more posterior levels (a3, a4, b3, b4). Scale bars 200 µm, except sections a1 - b4, 100 µm. Closed white arrowheads in (a3, a4) indicate neural crest, open white arrowheads indicate depleted neural crest in (b3) and position where neural crest should be in (b4).(C-G) Immunofluorescence on TSs caudal spinal cord of E9.5 wildtype littermates and Slc7a5-null embryos; Pax3 and FoxA2 were used as indicators for dorso-ventral organisation in (C, F) wildtype, n=2 (for FoxA2) and n= 4 (For Pax3) and (D, G) Slc7a5-null, n=3 (for FoxA2 ) and n = 3 for Pax3) neural tube. Arrowheads indicate border of Pax3 expression domain. Scale bars 50 µm. (E) Percentage of Pax3 expressing cells was determined by counting these cells and all DAPI labelled nuclei in the neural tube and comparison made between wildtype (4 embryos, 6 sections each) and Slc7a5-null (3 embryos, 6 sections each), each dot represents the average for one embryo, unpaired t-test, *p = 0.018 (see original source data). Error bars indicate SEM.


Figure 4 ( A - D ) Proliferation was assessed in the spinal cord ( forelimb level ) ( A , B ) using a phospho - Histone3 ( phospho - H3 ) antibody ( green ) to identify mitotic cells and DAPI ( blue ) to label nuclei ; cells were counted in 3 wildtype ( 18 sections ) and 3 Slc7a5 - null embryos ( 18 sections ) and mitotic index calculated ; ( C , D ) Proliferation was also assessed in the forebrain in 3 wildtype embryos ( 17 sections ) and 3 Slc7a5 - null embryos ( 16 sections ) . White dashed lines indicate midline . Data information All scale bars 50 µm . ( E , F ) Comparison of mitotic index in wildtype and Slc7a5 - null ( E ) spinal cord , p = 0 . 872 , and ( F ) forebrain * p = 0 . 049 , each dot represents the average for one embryo , unpaired t - test Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( G , H ) mTORC1 activity in the spinal cord ( hindlimb level ) was assessed by immunofluorescence using an antibody against the phospho - ribosomal protein S6 ( phosho - S6 Ser 235 / 236 ) . Data information All scale bars 50 µm . ( I ) Labelling intensity was measured and plotted relative to DAPI intensity ; 6 wildtype embryos ( 18 sections ) and 5 Slc7a5 - null embryos ( 19 sections ) . Each dot represents the average for one embryo , ( * p = 0 . 0457 ) unpaired t - test with Welch correction Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( J ) Western blot of individual E9 . 5 wildtype ( n = 3 ) and Slc7a5 - null ( n = 4 ) embryo lysates immunoblotted using an antibody against phospho - S6K ( Thr389 ) and total P70S6K for loading control . Band intensities were measured with FIJI and an unpaired t - test was performed for statistical analysis Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( K - L ) ( K ) Validation by qPCR of the targets identified by RNAseq ; qPCR was performed on individual E8 . 5 embryos of each genotype ( wildtype n = 5 , heterozygous n = 9 and Slc7a5 - null n = 7 ) . ( L ) qPCR was performed on individual E9 . 5 embryos ( wildtype n = 5 , heterozygous n = 3 and Slc7a5 - null n = 4 ) using primers specific for genes associated with the integrated stress response . A one - way Anova test with a Turkey post - test was performed . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 Error bars indicate SEM .

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Figure 4(A-D) Proliferation was assessed in the spinal cord (forelimb level) (A, B) using a phospho-Histone3 (phospho-H3) antibody (green) to identify mitotic cells and DAPI (blue) to label nuclei; cells were counted in 3 wildtype (18 sections) and 3 Slc7a5-null embryos (18 sections) and mitotic index calculated; (C, D) Proliferation was also assessed in the forebrain in 3 wildtype embryos (17 sections) and 3 Slc7a5-null embryos (16 sections). White dashed lines indicate midline. Data information All scale bars 50 µm.(E, F) Comparison of mitotic index in wildtype and Slc7a5-null (E) spinal cord, p = 0.872 ,and (F) forebrain *p = 0.049, each dot represents the average for one embryo, unpaired t-test Data information: *p<0.05, **p<0.01, ***p<0.001(G, H) mTORC1 activity in the spinal cord (hindlimb level) was assessed by immunofluorescence using an antibody against the phospho-ribosomal protein S6 (phosho-S6 Ser 235/236). Data information All scale bars 50 µm.(I) Labelling intensity was measured and plotted relative to DAPI intensity; 6 wildtype embryos (18 sections) and 5 Slc7a5-null embryos (19 sections). Each dot represents the average for one embryo, (*p = 0.0457) unpaired t-test with Welch correction Data information: *p<0.05, **p<0.01, ***p<0.001(J) Western blot of individual E9.5 wildtype (n=3) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-S6K (Thr389) and total P70S6K for loading control. Band intensities were measured with FIJI and an unpaired t-test was performed for statistical analysis Data information: *p<0.05, **p<0.01, ***p<0.001(K-L) (K) Validation by qPCR of the targets identified by RNAseq; qPCR was performed on individual E8.5 embryos of each genotype (wildtype n=5, heterozygous n=9 and Slc7a5-null n=7). (L) qPCR was performed on individual E9.5 embryos (wildtype n=5, heterozygous n=3 and Slc7a5-null n=4) using primers specific for genes associated with the integrated stress response. A one-way Anova test with a Turkey post-test was performed. Data information: *p<0.05, **p<0.01, ***p<0.001 Error bars indicate SEM.


Figure 5mRNA in situ hybridization was performed at E9 . 5 to detect Chac1 in ( A - a6 ) wildtype and ( B - b6 ) Slc7a5 - null littermate embryos ( n = 3 for each condition ) ; Chac1 expression at E10 in emerging limb buds assessed in ( C ) wildtype and ( D ) Slc7a5 - null littermate embryos ( n = 2 for each condition ) Scale bars 200 µm for whole embryo images and 100 µm for sections . mRNA in situ hybridization was performed at E9 . 5 to detect Trib3 mRNA detected in ( E - e6 ) wildtype and ( F - f6 ) Slc7a5 - null littermate embryos ( n = 3 for each condition ) and in emerging limb buds at E10 . 5 assessed in ( G ) wildtype and ( H ) Slc7a5 - null littermate embryos ( n = 2 for each condition ) . Scale bars 200 µm for whole embryo images and 100 µm for sections . mRNA in situ hybridization was performed at E9 . 5 Chac1 and Trib3 transcripts were also detected in wildtype E9 . 5 embryos after > 3 days in situ hybridisation reaction ( n = 3 and n = 6 respectively ) . Lateral view ( I and K ) , optic vesicles ( white arrowheads ) , branchial arches ( ba ) and otic vesicles ( ov ) . Dorsal view ( J and L ) , neural tube ( nt ) and limb buds ( white arrowheads ) . Scale bars 200 µm for whole embryo images and 100 µm for sections .

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Figure 5mRNA in situ hybridization was performed at E9.5 to detect Chac1 in (A-a6) wildtype and (B-b6) Slc7a5-null littermate embryos (n=3 for each condition); Chac1 expression at E10 in emerging limb buds assessed in (C) wildtype and (D) Slc7a5-null littermate embryos (n=2 for each condition) Scale bars 200 µm for whole embryo images and 100 µm for sections.mRNA in situ hybridization was performed at E9.5 to detect Trib3 mRNA detected in (E-e6) wildtype and (F-f6) Slc7a5-null littermate embryos (n=3 for each condition) and in emerging limb buds at E10.5 assessed in (G) wildtype and (H) Slc7a5-null littermate embryos (n=2 for each condition). Scale bars 200 µm for whole embryo images and 100 µm for sections.mRNA in situ hybridization was performed at E9.5 Chac1 and Trib3 transcripts were also detected in wildtype E9.5 embryos after >3 days in situ hybridisation reaction (n=3 and n=6 respectively). Lateral view (I and K), optic vesicles (white arrowheads), branchial arches (ba) and otic vesicles (ov). Dorsal view (J and L), neural tube (nt) and limb buds (white arrowheads). Scale bars 200 µm for whole embryo images and 100 µm for sections.


Figure 6Western blot of individual E9 . 5 wildtype ( n = 4 ) and Slc7a5 - null ( n = 4 ) embryo lysates immunoblotted using an antibody against phospho - GCN2 ( Thr899 ) and total GCN2 ( loading control ) , * * * p = 0 . 0001 ; band intensities measured with FIJI and analysed with unpaired t - test Data information : All error bars indicate SEM . Western blot of individual E9 . 5 wildtype ( n = 4 ) and Slc7a5 - null ( n = 4 ) embryo lysates immunoblotted using an antibody against phospho - eIF2α ( Ser51 ) and total eIFα ( loading control ) , * p = 0 . 0482 , band intensities measured with FIJI and analysed with unpaired t - test Data information : All error bars indicate SEM . ( C - F ) phosphorylated eIFα was assessed by immunofluorescence in E9 . 5 embryos in the forebrain , wildtype ( C , c1 ) ( n = 2 embryos , 0 / 14 sections ) and Slc7a5 - null ( D , d1 ) ( n = 2 embryos 16 / 16 sections ) , and in hindbrain / anterior spinal cord , wildtype ( E - e2 ) ( n = 4 embryos , 0 / 62 sections ) and Slc7a5 - null ( F - f2 ) ( n = 4 embryos , 54 / 54 sections ) including in the otic vesicle ( compare e2 , f2 ) . Scale bars in ( C - e1 , D - f1 ) 100 µm and in ( e2 and f2 ) 25µm . Arrowheads indicate ventral midline . ( G ) An increase in Trib3 protein was detected by Western blot in Slc7a5 - null embryos , each lane represents an E9 . 5 embryo lysate ( wildtype n = 3 , Slc7a5 - null , n = 4 ) with α - Tubulin loading control , unpaired t - test * p = 0 . 0153 Data information : All error bars indicate SEM . ( H - L ) TUNEL assay to detect apoptotic cells in wildtype ( n = 3 embryos , 32 sections ) or Slc7a5 - null ( n = 5 embryos , 47 sections ) ; ( H , I ) transverse sections through the head at level of the forebrain and ( J , K ) the spinal cord , ( H - K ) scale bars 50 µm , arrowheads indicate ventral midline . ( L ) Quantification of TUNEL positive cells within spinal cord sections was performed Each dot represents the average apoptosis cell count for a single embryo , Welch ' s correction unpaired t - test , p = 0 . 250 , F - test to compare variances , p = 0 . 0003 Data information : All error bars indicate SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-GCN2 (Thr899) and total GCN2 (loading control), ***p = 0.0001 ; band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM.Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-eIF2α (Ser51) and total eIFα (loading control), *p = 0.0482, band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM.(C-F) phosphorylated eIFα was assessed by immunofluorescence in E9.5 embryos in the forebrain, wildtype (C,c1) (n=2 embryos, 0/14 sections) and Slc7a5-null (D, d1) (n=2 embryos 16/16 sections), and in hindbrain/anterior spinal cord, wildtype (E-e2) (n= 4 embryos, 0/62 sections) and Slc7a5-null (F-f2) (n= 4 embryos, 54/54 sections) including in the otic vesicle (compare e2, f2). Scale bars in (C-e1, D-f1) 100 µm and in (e2 and f2) 25µm. Arrowheads indicate ventral midline.(G) An increase in Trib3 protein was detected by Western blot in Slc7a5-null embryos, each lane represents an E9.5 embryo lysate (wildtype n=3, Slc7a5-null, n=4) with α-Tubulin loading control, unpaired t-test *p = 0.0153 Data information: All error bars indicate SEM.(H-L) TUNEL assay to detect apoptotic cells in wildtype (n=3 embryos, 32 sections) or Slc7a5-null (n=5 embryos, 47 sections); (H, I) transverse sections through the head at level of the forebrain and (J, K) the spinal cord, (H-K) scale bars 50 µm, arrowheads indicate ventral midline. (L) Quantification of TUNEL positive cells within spinal cord sections was performed Each dot represents the average apoptosis cell count for a single embryo, Welch's correction unpaired t-test, p = 0.250, F-test to compare variances, p = 0.0003 Data information: All error bars indicate SEM.


Figure 7 ( A - B ) ( A , a1 ) Wnt β - catenin target Axin2 strongly expressed in littermate , ( a2 , a3 ) in TS , and ( B , b1 ) in Scl7a5 - null embryos ( b2 , b3 ) in TS ( n = 2 littermates , n = 6 / 6 Slc7a5 - null embryos ) . Data information : White dashed lines indicate position of sections . Scale bars 200 µm ( C , d1 ) Axin2 transcripts in embryo trunk explant exposed to DMSO ( C , c1 , n = 12 / 12 ) were reduced in Wnt - c59 exposed explants ( D , d1 , n = 14 / 14 ) . Images in ( c1 , d1 ) show TS through explants ( data from 3 independent embryo explant experiments ) . ( E , F ) Slc7a5 transcripts in embryo trunk explant exposed to DMSO ( E , e1 , n = 9 / 10 ) or Wnt - c59 ( F , f1 , n = 15 / 15 ) . Images in ( e1 , f1 ) show TS through explants ( data from 3 independent embryo explant experiments ) . Data information : Scale bars = 100 µm ( G , H ) Slc7a5 expression in control T - Cre ; Ctnnb1flLOF / Δ heterozygous littermate embryo ( G , g1 ) and in TS ( g2 , g3 ) , and in T - Cre ; Ctnnb1flLOF / Δ mutant embryo where Slc7a5 transcripts are absent in posterior neural tube ( the region of T - cre recombination , indicated with white arrowhead in H ) and shown in TS in ( h2 , h3 ) ( n = 0 / 6 littermate and n = 6 / 6 mutant embryos ) . Data information : White dashed lines indicate position of sections . Scale bars , 200 µm ( I , J ) Trib3 is lacking in T - Cre ; Ctnnb1flLOF / Δ heterozygous control embryos at E7 . 75 ( I ) , caudal region dissected from the same embryo ( white dashed box in I ) ( i1 ) and viewed in TS ( i2 ) ; Trib3 detected in homozygous T - Cre ; Ctnnb1flLOF / Δ embryo ( J ) , in caudal region dissected from the same embryo ( white dashed box in J ) ( j1 ) and shown in TS in caudal epiblast and remnant primitive streak ( ps ) ( j2 ) ( n = 3 / 3 T - Cre ; Ctnnb1flLOF / Δ embryos and 14 littermate controls ) . Trib3 was also detected in the more rostral forming neural tube at this early stage ( seen in J ) and so in cells not directly experiencing β - catenin loss ; this may reflect the failure to form paraxial mesoderm , which provides signals that promote and support neurogenesis . Data information : White dashed lines indicate position of sections . Scale bars all sections 50 µm . ( K - M ) At E9 . 5 Trib3 was not detected in T - Cre ; Ctnnb1flLOF / Δ heterozygous littermate embryos ( K , k1 ) and in TS ( k2 , k3 ) , but was detected in homozygous T - Cre ; Ctnnb1flLOF / Δ mutant embryos ( L - m3 ) in patches of cells in neural tube ( nt ) ( L - l2 ) , gut ( g ) ( l3 ) and somites ( s ) ( m2 , arrowheads ) ( n = 9 / 9 T - Cre ; Ctnnb1flLOF / Δ embryos and 12 littermate controls ) . Data information : White dashed lines indicate position of sections . Scale bars , all sections 50 µm .

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Figure 7(A-B) (A,a1) Wnt β-catenin target Axin2 strongly expressed in littermate, (a2, a3) in TS, and (B, b1) in Scl7a5-null embryos (b2, b3) in TS (n=2 littermates, n=6/6 Slc7a5-null embryos). Data information: White dashed lines indicate position of sections. Scale bars 200 µm(C, d1) Axin2 transcripts in embryo trunk explant exposed to DMSO (C, c1, n=12/12) were reduced in Wnt-c59 exposed explants (D, d1, n=14/14). Images in (c1, d1) show TS through explants (data from 3 independent embryo explant experiments). (E, F) Slc7a5 transcripts in embryo trunk explant exposed to DMSO (E, e1, n=9/10) or Wnt-c59 (F, f1, n=15/15). Images in (e1, f1) show TS through explants (data from 3 independent embryo explant experiments). Data information: Scale bars = 100 µm(G, H) Slc7a5 expression in control T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryo (G, g1) and in TS (g2, g3), and in T-Cre;Ctnnb1flLOF/Δ mutant embryo where Slc7a5 transcripts are absent in posterior neural tube (the region of T-cre recombination, indicated with white arrowhead in H) and shown in TS in (h2, h3) (n= 0/6 littermate and n=6/6 mutant embryos). Data information: White dashed lines indicate position of sections. Scale bars, 200 µm(I, J) Trib3 is lacking in T-Cre;Ctnnb1flLOF/Δ heterozygous control embryos at E7.75 (I), caudal region dissected from the same embryo (white dashed box in I) (i1) and viewed in TS (i2); Trib3 detected in homozygous T-Cre;Ctnnb1flLOF/Δ embryo (J), in caudal region dissected from the same embryo (white dashed box in J) (j1) and shown in TS in caudal epiblast and remnant primitive streak (ps) (j2) (n= 3/3 T-Cre;Ctnnb1flLOF/Δ embryos and 14 littermate controls). Trib3 was also detected in the more rostral forming neural tube at this early stage (seen in J) and so in cells not directly experiencing β-catenin loss; this may reflect the failure to form paraxial mesoderm, which provides signals that promote and support neurogenesis. Data information: White dashed lines indicate position of sections. Scale bars all sections 50 µm.(K-M) At E9.5 Trib3 was not detected in T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryos (K, k1) and in TS ( k2, k3), but was detected in homozygous T-Cre;Ctnnb1flLOF/Δ mutant embryos (L-m3) in patches of cells in neural tube (nt) (L - l2), gut (g) (l3) and somites (s) (m2, arrowheads) (n=9/9 T-Cre;Ctnnb1flLOF/Δ embryos and 12 littermate controls). Data information: White dashed lines indicate position of sections. Scale bars, all sections 50 µm.


Figure 1B Representative examples of chromatin accessibility and gene expression of KRT5 and KRT10 , two key epidermal differentiation - related genes , in the main keratinocyte populations . C Co - accessibility at the human epidermal differentiation complex ( EDC ) in undifferentiated ( cluster 2 ) and differentiated ( cluster 3 ) keratinocytes . Only connections ( arcs ) with a co - accessibility score above 0 . 25 are plotted . Grey boxes below tracks represent scATAC - seq peaks . D Left : Heatmap displaying the differentially accessible peaks between undifferentiated ( cluster 2 ) and differentiated ( cluster 3 ) keratinocytes . Number of Tn5 insertion sites in each region were scaled by row . Right : DNA sequence motifs for the top six overrepresented transcription factor ( TF ) motifs in undifferentiated ( cluster 2 ) and differentiated ( cluster 3 ) keratinocyte - specific accessible peaks . E Heatmaps displaying the predicted top five transcription factors in each cell cluster using chromVAR motif activity ( left ) and gene expression ( right ) . F chromVAR deviations ( in quantiles ) and gene expression for representative enriched TF projected onto the joint UMAP plot of keratinocytes from the single - cell multi - omics dataset . G Heatmap showing the top five active regulons in each cell cluster , based on the expression data of the multi - omics dataset . TF regulons also identified by other approaches are highlighted .

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Figure 1B Representative examples of chromatin accessibility and gene expression of KRT5 and KRT10, two key epidermal differentiation-related genes, in the main keratinocyte populations.C Co-accessibility at the human epidermal differentiation complex (EDC) in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Only connections (arcs) with a co-accessibility score above 0.25 are plotted. Grey boxes below tracks represent scATAC-seq peaks.D Left: Heatmap displaying the differentially accessible peaks between undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Number of Tn5 insertion sites in each region were scaled by row. Right: DNA sequence motifs for the top six overrepresented transcription factor (TF) motifs in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocyte-specific accessible peaks.E Heatmaps displaying the predicted top five transcription factors in each cell cluster using chromVAR motif activity (left) and gene expression (right). F chromVAR deviations (in quantiles) and gene expression for representative enriched TF projected onto the joint UMAP plot of keratinocytes from the single-cell multi-omics dataset.G Heatmap showing the top five active regulons in each cell cluster, based on the expression data of the multi-omics dataset. TF regulons also identified by other approaches are highlighted.


Figure 2A Average histone modification profiles of undifferentiated and differentiated keratinocyte - specific peaks using previously published data generated on NHEK cells ( ENCODE ) . The normalized signal of H3K27ac , H3K4me1 / me2 / me3 and H3K27me3 were measured in a window of ± 10 , 000 base - pair ( bp ) . B Fractions of CpGs located within epigenomic substructures for the 4 , 351 InfiniumEPIC CpG probes found within undifferentiated and differentiated keratinocyte - specific peaks . C Unsupervised hierarchical clustering of 12 healthy , 20 AK and 35 cSCC epidermal samples based on the methylation status at undifferentiated and differentiated keratinocyte - specific peaks . Each row represents the average methylation value of all CpGs contained in a particular peak .

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Figure 2A Average histone modification profiles of undifferentiated and differentiated keratinocyte-specific peaks using previously published data generated on NHEK cells (ENCODE). The normalized signal of H3K27ac, H3K4me1/me2/me3 and H3K27me3 were measured in a window of ±10,000 base-pair (bp).B Fractions of CpGs located within epigenomic substructures for the 4,351 InfiniumEPIC CpG probes found within undifferentiated and differentiated keratinocyte-specific peaks.C Unsupervised hierarchical clustering of 12 healthy, 20 AK and 35 cSCC epidermal samples based on the methylation status at undifferentiated and differentiated keratinocyte-specific peaks. Each row represents the average methylation value of all CpGs contained in a particular peak.


Figure 3B , C Average DNA methylation in the 15 chromatin states defined by ChromHMM in NHEK for ( B ) both EpSC and Keratinocytes from the sci - MET single - cell dataset , or for ( C ) EpSC - like and keratinocyte - like AK / cSCC from the EPIC dataset . ChromHMM state 1 : active promoter ; state 2 : weak promoter ; state 3 : inactive / poised promoter ; state 4 : strong enhancer ; state 5 : strong enhancer ; state 6 : weak / poised enhancer ; state 7 : weak / poised enhancer ; state 8 : insulator ; state 9 : transcriptional transition ; state 10 : transcriptional elongation ; state 11 : weak transcription ; state 12 : polycomb - repressed ; state 13 : heterochromatin / low signal ; state 14 : repetitive / copy number variation ; state 15 : repetitive / copy number variation . AK : actinic keratosis , cSCC : cutaneous squamous cell carcinoma . Data information : Statistical analyses in B and C were performed using a Wilcoxon Rank Sum test comparing the average methylation values in genomic states 4 and 5 between EpSC ( n = 6 ) and keratinocytes ( n = 548 ) ( B ) or EpSC - like ( n = 22 ) and keratinocyte - like ( n = 33 ) AK / cSCC samples ( C ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . D Heatmaps displaying the gene expression ( upper ) and the imputed promoter DNA methylation ( lower ) reference matrices for keratinocyte populations involved in terminal differentiation , calculated by EpiSCORE . The weight of each gene in the reference DNA matrix is also depicted .

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Figure 3B, C Average DNA methylation in the 15 chromatin states defined by ChromHMM in NHEK for (B) both EpSC and Keratinocytes from the sci-MET single-cell dataset, or for (C) EpSC-like and keratinocyte-like AK/cSCC from the EPIC dataset. ChromHMM state 1: active promoter; state 2: weak promoter; state 3: inactive/poised promoter; state 4: strong enhancer; state 5: strong enhancer; state 6: weak/poised enhancer; state 7: weak/poised enhancer; state 8: insulator; state 9: transcriptional transition; state 10: transcriptional elongation; state 11: weak transcription; state 12: polycomb-repressed; state 13: heterochromatin/low signal; state 14: repetitive/copy number variation; state 15: repetitive/copy number variation. AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma. Data information: Statistical analyses in B and C were performed using a Wilcoxon Rank Sum test comparing the average methylation values in genomic states 4 and 5 between EpSC (n= 6) and keratinocytes (n= 548) (B) or EpSC-like (n= 22) and keratinocyte-like (n= 33) AK/cSCC samples (C). *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.D Heatmaps displaying the gene expression (upper) and the imputed promoter DNA methylation (lower) reference matrices for keratinocyte populations involved in terminal differentiation, calculated by EpiSCORE. The weight of each gene in the reference DNA matrix is also depicted.


Figure 4C Upper : Heatmaps displaying the unsupervised clustering of 67 AK / cSCC and healthy controls based on the methylation patterns of the MIR200C / 141 cluster and the MIR205 gene . Lower : Bar plots showing the quantification of the methylation values at CpGs located at promoter regions in each cell - of - origin - based subclass . For the MIR200C / 141 cluster , probes located at the regulatory CpG island are depicted with green arrows . For the MIR205 gene , all probes shown in the heatmap are part of the promoter region and were used for quantification . Data information : AK : actinic keratosis , cSCC : cutaneous squamous cell carcinoma . D Left : Representative images of EpSC - like ( n = 5 ) and Keratinocyte - like ( n = 5 ) cSCC tumors stained for the EMT - marker ZEB2 ( green ) and the keratinocyte marker TP63 ( red ) . Nuclei were counterstained with DAPI . Images are shown at 40x original magnification . Scale bar , 50 µm . Right : Quantification of ZEB2 - positive tumor cells ( TP63 - positive ) in three independent regions per sample . Each dot represents a region with at least 500 tumor cells ( TP63 - positive ) counted . Data information : Statistical analysis in D was performed using an unpaired two - sided t - test ( EpSC - like : n = 5 samples , 15 technical replicates ; keratinocyte - like : n = 5 samples , 15 technical replicates ) . Central bar represents the mean and error bars represent the standard deviation . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Figure 4C Upper: Heatmaps displaying the unsupervised clustering of 67 AK/cSCC and healthy controls based on the methylation patterns of the MIR200C/141 cluster and the MIR205 gene. Lower: Bar plots showing the quantification of the methylation values at CpGs located at promoter regions in each cell-of-origin-based subclass. For the MIR200C/141 cluster, probes located at the regulatory CpG island are depicted with green arrows. For the MIR205 gene, all probes shown in the heatmap are part of the promoter region and were used for quantification. Data information: AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma.D Left: Representative images of EpSC-like (n=5) and Keratinocyte-like (n=5) cSCC tumors stained for the EMT-marker ZEB2 (green) and the keratinocyte marker TP63 (red). Nuclei were counterstained with DAPI. Images are shown at 40x original magnification. Scale bar, 50 µm. Right: Quantification of ZEB2-positive tumor cells (TP63-positive) in three independent regions per sample. Each dot represents a region with at least 500 tumor cells (TP63-positive) counted. Data information: Statistical analysis in D was performed using an unpaired two-sided t-test (EpSC-like: n= 5 samples, 15 technical replicates; keratinocyte-like: n= 5 samples, 15 technical replicates). Central bar represents the mean and error bars represent the standard deviation. *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.


Figure 1iPSC - derived HE , EHT and HSC - like cells were sorted , cultured for 1 day and analyzed by scRNAseq . ( d ) Heatmap showing expression levels of endothelial and hematopoietic genes in AEC / Hem cluster cells which have mapped to the HE , EHT and HSC - like

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Figure 1iPSC-derived HE, EHT and HSC-like cells were sorted, cultured for 1 day and analyzed by scRNAseq. (d) Heatmap showing expression levels of endothelial and hematopoietic genes in AEC/Hem cluster cells which have mapped to the HE, EHT and HSC-like populations


Figure 2 ( b ) Dot plots showing gene expression levels of glycolytic enzymes detected by scRNAseq and based on percent expressed ( size of the dots ) and average level of expression ( color intensity ) . ( g ) Oxygen consumption rate ( OCR ) was measured in HE and EHT cells ( n = 3 biological replicates with 2 - 3 technical replicates each ) and oxidative phosphorylation was assessed by extracellular flux analysis . Bar graphs show relative levels ± SEM of the indicated processes ( from 3 independent experiments ; unpaired t - tests ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( j ) Live cell imaging of HE and HSC - like cells stained with TMRE ( red ) at day 3 of subculture . Representative merged brightfield / TMRE and TMRE images are shown . Scale bars , 100 µm . Bar graphs show means of TMRE staining intensity from all replicate wells across all experiments ( HE spindle and HE round , n = 3 biological replicates with 3 technical replicates each ; HSC - like , n = 2 biological replicates with 3 technical replicates each , Kruskal - Wallis test with multiple comparisons ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( k ) Dot plots showing gene expression levels of TCA cycle enzymes detected by scRNAseq and based on percent expressed ( size of the dots ) and average level of expression ( color intensity ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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Figure 2(b) Dot plots showing gene expression levels of glycolytic enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).(g) Oxygen consumption rate (OCR) was measured in HE and EHT cells (n=3 biological replicates with 2-3 technical replicates each) and oxidative phosphorylation was assessed by extracellular flux analysis. Bar graphs show relative levels ± SEM of the indicated processes (from 3 independent experiments; unpaired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(j) Live cell imaging of HE and HSC-like cells stained with TMRE (red) at day 3 of subculture. Representative merged brightfield/TMRE and TMRE images are shown. Scale bars, 100 µm. Bar graphs show means of TMRE staining intensity from all replicate wells across all experiments (HE spindle and HE round, n=3 biological replicates with 3 technical replicates each; HSC-like, n=2 biological replicates with 3 technical replicates each, Kruskal-Wallis test with multiple comparisons). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(k) Dot plots showing gene expression levels of TCA cycle enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001


Figure 3 ( b - e ) FACS - sorted HE cells were subcultured with or without UK5099 ( 10 µM ) or DCA ( 3 mM ) . Subculture day 3 representative GPA / CD43 plots ( b , d ) and subculture day 6 representative CD45 / CD43 plots ( c , e ) are shown Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( g ) Fold change in the expression of HBE1 or HBG1 - 2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 ( 10 µM ) or DCA ( 3 mM ) relative to non - treated cells ( n = 3 biological replicates , paired t - test ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( i - k ) Pregnant mice were injected with UK5099 or DCA at E9 . 5 and fetal livers were analyzed at E14 . 5 by flow cytometry . FL , fetal liver . Levels of T and B cells ( i ) and LT - HSCs ( j ) as percentages in fetal liver are shown for control ( n = 10 biological replicates ) , UK5099 - treated ( n = 14 biological replicates ) and DCA - treated ( n = 16 biological replicates ) conditions ( one - way ANOVA test ) . ( k ) The ratio of BFU - E to CFU - GM colonies obtained from sorted LT - HSCs are shown CFU , colony forming unit ; BFU , burst forming unit ; E , erythroid ; M , macrophage ; G , granulocyte . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( l - p ) HE cells co - cultured with OP9 - DL1 stroma were treated with DCA for 3 days and transplanted into irradiated NSG mice . Bone marrow ( BM ) and thymi were harvested on week 12 . ( l ) The percentages ± SEM of human CD4 + CD8 + double positive thymocytes in huCD45 + cells from the thymus are shown ( Control , n = 6 biological replicates ; DCA , n = 7 biological replicates ; unpaired t tests ) . The percentages ± SEM of human B cells ( m ) , CLPs ( n ) from the BM at week 12 , as well as myeloid cells from PB at week 8 ( o ) and myeloid cells from BM at week 12 ( p ) in huCD45 + cells are shown ( Control , n = 6 biological replicates ; DCA , n = 7 biological replicates ; unpaired t tests ) . PB , peripheral blood . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(b-e) FACS-sorted HE cells were subcultured with or without UK5099 (10 µM) or DCA (3 mM). Subculture day 3 representative GPA/CD43 plots (b, d) and subculture day 6 representative CD45/CD43 plots (c, e) are shown Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001(g) Fold change in the expression of HBE1 or HBG1-2 transcripts ± SEM normalized to KLF1 in CFUs obtained from HE cells treated with UK5099 (10 µM) or DCA (3 mM) relative to non-treated cells (n=3 biological replicates, paired t-test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001(i-k) Pregnant mice were injected with UK5099 or DCA at E9.5 and fetal livers were analyzed at E14.5 by flow cytometry. FL, fetal liver. Levels of T and B cells (i) and LT-HSCs (j) as percentages in fetal liver are shown for control (n=10 biological replicates), UK5099-treated (n=14 biological replicates) and DCA-treated (n=16 biological replicates) conditions (one-way ANOVA test). (k) The ratio of BFU-E to CFU-GM colonies obtained from sorted LT-HSCs are shown CFU, colony forming unit; BFU, burst forming unit; E, erythroid; M, macrophage; G, granulocyte. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001(l-p) HE cells co-cultured with OP9-DL1 stroma were treated with DCA for 3 days and transplanted into irradiated NSG mice. Bone marrow (BM) and thymi were harvested on week 12. (l) The percentages ± SEM of human CD4+CD8+ double positive thymocytes in huCD45+ cells from the thymus are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). The percentages ± SEM of human B cells (m), CLPs (n) from the BM at week 12, as well as myeloid cells from PB at week 8 (o) and myeloid cells from BM at week 12 (p) in huCD45+ cells are shown (Control, n=6 biological replicates; DCA, n=7 biological replicates; unpaired t tests). PB, peripheral blood. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001


Figure 4 ( d ) Dot plots show expression levels of the indicated hematopoietic transcription factors in clusters 6 and 7 for HE ctrl , HE + UK5099 and HE + DCA conditions , detected by scRNAseq and based on percent expressed ( size of the dots ) and average level of expression ( color intensity ) .

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Figure 4(d) Dot plots show expression levels of the indicated hematopoietic transcription factors in clusters 6 and 7 for HE ctrl, HE + UK5099 and HE + DCA conditions, detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).


Figure 5 ( d ) HE cells were transduced with shScrambled ( shScr ) or shLSD1 with or without UK5099 ( 10 µM ) the day after the sort and day 3 CD43 + / GPA + cell frequencies ± SEM relative to shScr are presented ( n = 3 biological replicates 1 - 2 technical replicates each , one - way ANOVA test ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( f - g ) FACS - sorted HE cells were subcultured with ACSS2i ( 5 µM ) , DCA ( 3 mM ) , or both ( n = 5 biological replicates , one - way ANOVA test ) ( f ) or with C646 ( 10 µM ) , DCA ( 3 mM ) , or both ( n = 3 biological replicates , one - way ANOVA test ) ( g ) and day 6 CD43 + CD45 + cell frequencies ± SEM are shown . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( j ) FACS - sorted HE cells were subcultured with CP ( 5 µM ) , DCA ( 3 mM ) or both and day 6 CD43 + CD45 + cell frequencies ± SEM relative to the control are shown ( n = 4 biological replicates , one - way ANOVA test ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( k ) Cholesterol content ± SEM in HE cells was measured by confocal microscopy at day 2 of DCA ( 3 mM ) treatment by filipin III staining ( n = 3 biological replicates , paired t test ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( m ) FACS - sorted HE cells were subcultured with Ato ( 0 . 5 µM ) , DCA ( 3 mM ) or both and day 3 CD43 + CD45 + cell frequencies ± SEM relative to the control are shown ( n = 3 biological replicates for control / DCA , n = 2 biological replicates with 2 technical replicates for Ato / Ato + DCA , one - way ANOVA test ) . Data information : ns , not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( n ) Dot plots showing gene expression levels of NOTCH1 , JAG1 and HES1 detected by scRNAseq and based on percent expressed ( size of the dots ) and average level of expression ( color intensity ) .

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Figure 5(d) HE cells were transduced with shScrambled (shScr) or shLSD1 with or without UK5099 (10 µM) the day after the sort and day 3 CD43+/GPA+ cell frequencies ± SEM relative to shScr are presented (n=3 biological replicates 1-2 technical replicates each, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(f-g) FACS-sorted HE cells were subcultured with ACSS2i (5 µM), DCA (3 mM), or both (n=5 biological replicates, one-way ANOVA test) (f) or with C646 (10 µM), DCA (3 mM), or both (n=3 biological replicates, one-way ANOVA test) (g) and day 6 CD43+CD45+ cell frequencies ± SEM are shown. Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(j) FACS-sorted HE cells were subcultured with CP (5 µM), DCA (3 mM) or both and day 6 CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=4 biological replicates, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(k) Cholesterol content ± SEM in HE cells was measured by confocal microscopy at day 2 of DCA (3 mM) treatment by filipin III staining (n=3 biological replicates, paired t test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(m) FACS-sorted HE cells were subcultured with Ato (0.5 µM), DCA (3 mM) or both and day 3 CD43+CD45+ cell frequencies ± SEM relative to the control are shown (n=3 biological replicates for control/DCA, n=2 biological replicates with 2 technical replicates for Ato/Ato+DCA, one-way ANOVA test). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001(n) Dot plots showing gene expression levels of NOTCH1, JAG1 and HES1 detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity).


Figure 1 ( A , B ) Immunoblot analysis of ATG5 - ATG12 protein levels in cortical brain lysates of 13 - week - old Atg5flox : CamKIIα - Cre WT / KO mice ( WT set to 100 % , KO : 28 . 13 ± 8 . 18 % ; p = 0 . 0005 , nWT = 3 , nKO = 3 , one - tailed unpaired t - test ) . ( C , D ) Immunoblot analysis of ATG5 - ATG12 protein level in striatal brain lysates of 13 - week - old Atg5flox : Slc32a1 - Cre WT / KO mice ( WT set to 100 % , KO : 21 . 71 ± 0 . 41 % ; p < 0 . 0001 , nWT = 3 , nKO = 3 , one - tailed unpaired t - test ) . Protein levels of p62 are significantly increased in cortical lysates of Atg5flox : CamKIIα - Cre KO mice compared to the WT set to 100 % ( KO : 4855 ± 587 . 4 % ; p < 0 . 0001 , n = 4 , one - tailed unpaired t - test ) . Protein levels of p62 are significantly increased in cortical lysates of Atg5flox : CamKIIα - Cre KO mice compared to the WT set to 100 % ( KO : 4855 ± 587 . 4 % ; p < 0 . 0001 , n = 4 , one - tailed unpaired t - test ) . ( G , H ) Protein levels of p62 are significantly increased in striatal lysates of Atg5flox : Slc32a1 - Cre KO mice compared to the WT set to 100 % ( KO : 2956 ± 839 . 3 % ; p = 0 . 0072 , n = 4 , one - tailed unpaired t - test ) .

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Figure 1(A, B) Immunoblot analysis of ATG5-ATG12 protein levels in cortical brain lysates of 13-week-old Atg5flox:CamKIIα-Cre WT/KO mice (WT set to 100%, KO: 28.13±8.18%; p=0.0005, nWT=3, nKO=3, one-tailed unpaired t-test).(C,D) Immunoblot analysis of ATG5-ATG12 protein level in striatal brain lysates of 13-week-old Atg5flox:Slc32a1-Cre WT/KO mice (WT set to 100%, KO: 21.71±0.41%; p<0.0001, nWT=3, nKO=3, one-tailed unpaired t-test).Protein levels of p62 are significantly increased in cortical lysates of Atg5flox:CamKIIα-Cre KO mice compared to the WT set to 100% (KO: 4855± 587.4%; p<0.0001, n=4, one-tailed unpaired t-test).Protein levels of p62 are significantly increased in cortical lysates of Atg5flox:CamKIIα-Cre KO mice compared to the WT set to 100% (KO: 4855± 587.4%; p<0.0001, n=4, one-tailed unpaired t-test).(G, H) Protein levels of p62 are significantly increased in striatal lysates of Atg5flox:Slc32a1-Cre KO mice compared to the WT set to 100% (KO: 2956± 839.3%; p=0.0072, n=4, one-tailed unpaired t-test).


Figure 2 ( F , G ) PKA R1α ( F ) and PKA R1β ( G ) protein levels are significantly increased in cortical brain lysates from 13 - week - old Atg5flox : CamKIIα - Cre KO mice compared to the WT set to 100 % ( KOPKA R1α : 286 . 1 ± 50 . 53 % , p = 0 . 005 , KOPKA R1β : ( H , I ) PKA R1α ( H ) and PKA R1β ( I ) protein levels are significantly increased in striatal lysates of Atg5flox : Slc32a1 - Cre KO mice compared to the WT set to 100 % ( KOPKA R1α : 524 . 1 ± 8 . 35 % , p < 0 . 0001 , KOPKA R1β : ( M , N ) Immunohistochemistry analysis of PKA R1α levels in NeuN - posititve neurons in the hippocampus of Atg5flox : CamKIIα - Cre WT / KO mice . Scale bar : 50µm , 20µm ( small panels ) . WT : 54 . 70 ± 0 . 693 , KO : 89 . 79 ± 1 . 831 ; p < 0 . 0001 , two - tailed unpaired t - test . nWT = 64 ; nKO = 71 neurons from n = 3 mice per genotype . Immunohistochemistry analysis of PKA R1α levels in GFAP - positive cells in the hippocampus of Atg5flox : CamKIIα - Cre WT / KO mice . Scale bar : 50µm , 30µm ( small panels ) . WT : 63 . 86 ± 2 . 477 , KO : 61 . 71 ± 2 . 653 ; p = 0 . 5549 , two - tailed unpaired t - test . nWT = 45 ; nKO = 40 cells from n = 3 mice per genotype . Immunohistochemistry analysis of PKA R1α levels in GFAP - positive cells in the hippocampus of Atg5flox : CamKIIα - Cre WT / KO mice . Scale bar : 50µm , 30µm ( small panels ) . WT : 63 . 86 ± 2 . 477 , KO : 61 . 71 ± 2 . 653 ; p = 0 . 5549 , two - tailed unpaired t - test . nWT = 45 ; nKO = 40 cells from n = 3 mice per genotype . ( Q , R ) Immunohistochemistry analysis of PKA R1α in GFAP - positive cells in the hippocampus of Atg5flox : Slc32a1 - Cre WT and KO mice . Scale bar : 50µm , 30µm ( small panels ) . WT : 45 . 28 ± 1 . 018 , KO : 48 . 50 ± 1 . 440 ; p = 0 . 0636 , two - tailed unpaired t - test . nWT = 61 ; nKO = 48 cells from n = 3 mice per genotype .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(F, G) PKA R1α (F) and PKA R1β (G) protein levels are significantly increased in cortical brain lysates from 13-week-old Atg5flox:CamKIIα-Cre KO mice compared to the WT set to 100% (KOPKA R1α: 286.1± 50.53%, p=0.005, KOPKA R1β:(H, I) PKA R1α (H) and PKA R1β (I) protein levels are significantly increased in striatal lysates of Atg5flox:Slc32a1-Cre KO mice compared to the WT set to 100% (KOPKA R1α: 524.1± 8.35%, p<0.0001, KOPKA R1β:(M, N) Immunohistochemistry analysis of PKA R1α levels in NeuN-posititve neurons in the hippocampus of Atg5flox:CamKIIα-Cre WT/KO mice. Scale bar: 50µm, 20µm (small panels). WT: 54.70±0.693, KO: 89.79±1.831; p<0.0001, two-tailed unpaired t-test. nWT= 64; nKO= 71 neurons from n=3 mice per genotype.Immunohistochemistry analysis of PKA R1α levels in GFAP-positive cells in the hippocampus of Atg5flox:CamKIIα-Cre WT/KO mice. Scale bar: 50µm, 30µm (small panels). WT: 63.86±2.477, KO: 61.71±2.653; p=0.5549, two-tailed unpaired t-test. nWT= 45; nKO= 40 cells from n=3 mice per genotype.Immunohistochemistry analysis of PKA R1α levels in GFAP-positive cells in the hippocampus of Atg5flox:CamKIIα-Cre WT/KO mice. Scale bar: 50µm, 30µm (small panels). WT: 63.86±2.477, KO: 61.71±2.653; p=0.5549, two-tailed unpaired t-test. nWT= 45; nKO= 40 cells from n=3 mice per genotype.(Q, R) Immunohistochemistry analysis of PKA R1α in GFAP-positive cells in the hippocampus of Atg5flox:Slc32a1-Cre WT and KO mice. Scale bar: 50µm, 30µm (small panels). WT: 45.28±1.018, KO: 48.50±1.440; p=0.0636, two-tailed unpaired t-test. nWT= 61; nKO= 48 cells from n=3 mice per genotype.


Figure 3 ( G ) Immunohistochemistry for PKA R1α and postsynaptic density marker PSD95 on horizontal brains sections from Atg5flox : CamKIIα - Cre WT / KO mice . White rectangular boxes indicate areas magnified below with arrows indicating PKA R1α and PSD95 colocalization . ( H ) Mander ' s overlap coefficient of PKA R1α and PSD95 colocalization in Atg5flox : CamKIIα - Cre WT / KO hippocampus ( WT : 0 . 29 ± 0 . 009 , KO : 0 . 40 ± 0 . 012 ; p < 0 . 0001 , two - tailed unpaired t - test ) . nWT = 31 ; nKO = 38 from n = 3 mice . ( I ) Electron microscopy - based analysis of immunogold - labeled PKA R1α / β on Tokuyasu cryosections of the CA1 neuropil area of the hippocampus of Atg5flox : CamKIIα - Cre KO mice . No immunogold labelling was detected in samples where the PKA R1α / β antibody were omitted ( negative control ) . Scale bar : upper row 50 nm , 500 nm lower picture . ( J ) PKA R1α / β is enriched at mitochondria ( WT : 1 . 10 ± 0 . 266 , KO : 2 . 73 ± 0 . 515 ; p = 0 . 0082 , two - tailed unpaired t - test ) and synapses ( WT : 0 . 52 ± 0 . 131 , KO : 1 . 14 ± 0 . 221 ; p = 0 . 0234 , two - tailed unpaired t - test ) of KO mice . Values display the mean number of gold particles counted per structure in nWT = 21 and n KO = 22 pictures per genotype .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(G) Immunohistochemistry for PKA R1α and postsynaptic density marker PSD95 on horizontal brains sections from Atg5flox:CamKIIα-Cre WT/KO mice. White rectangular boxes indicate areas magnified below with arrows indicating PKA R1α and PSD95 colocalization.(H) Mander's overlap coefficient of PKA R1α and PSD95 colocalization in Atg5flox:CamKIIα-Cre WT/KO hippocampus (WT: 0.29±0.009, KO: 0.40±0.012; p<0.0001, two-tailed unpaired t-test). nWT= 31; nKO= 38 from n=3 mice.(I) Electron microscopy-based analysis of immunogold-labeled PKA R1α/β on Tokuyasu cryosections of the CA1 neuropil area of the hippocampus of Atg5flox:CamKIIα-Cre KO mice. No immunogold labelling was detected in samples where the PKA R1α/β antibody were omitted (negative control). Scale bar: upper row 50 nm, 500 nm lower picture.(J) PKA R1α/β is enriched at mitochondria (WT: 1.10± 0.266, KO: 2.73± 0.515; p=0.0082, two-tailed unpaired t-test) and synapses (WT: 0.52± 0.131, KO: 1.14± 0.221; p=0.0234, two-tailed unpaired t-test) of KO mice. Values display the mean number of gold particles counted per structure in nWT=21 and n KO=22 pictures per genotype.


Figure 4 ( A ) Western Blot analysis of PKA R1α protein levels in NSC34 cells , treated with 250 nM Torin1 or deprived of amino - acids and serum for 16 hours . Lysosomal degradation was blocked in the last 4 hours using 67 nM of Bafilomycin A ( BafA ) . ( B ) Starvation significantly reduced PKA R1α protein levels in NSC34 cells ( vehicle set to 100 % , starvation : 31 . 19 ± 5 . 085 % , p < 0 . 0001 , one - tailed unpaired t - test ) . n = 5 . ( C ) Application of 67 nM BafA for 4 hours before harvesting was sufficient to stabilize the PKA R1α protein levels in starved NSC34 cells ( starvation set to 100 % , starvation + BafA : 155 . 5 ± 13 . 93 % , p = 0 . 002 , one - tailed unpaired t - test ) . 16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2 - positive cultured primary neurons ( Vehicle : 212 . 4 ± 5 . 619 , Starvation : 255 . 1 ± 5 . 715 ; p < 0 . 0001 , two - tailed paired t - test ) . nVehicle = 314 , nStarvation = 306 neurons from n = 3 . 16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2 - positive cultured primary neurons ( Vehicle : 212 . 4 ± 5 . 619 , Starvation : 255 . 1 ± 5 . 715 ; p < 0 . 0001 , two - tailed paired t - test ) . nVehicle = 314 , nStarvation = 306 neurons from n = 3 . ( F , G ) Phosphorylation state of proteins containing PKA substrate RRXS / T motif was increased upon 16 hours of amino - acid and serum starvation in primary cortico - hippocampal neurons at DIV14 and was suppressed by 1 µM H89 supplementation in the media ( Vehicle set to 100 % , Starvation : 326 . 60 ± 94 . 53 % , H89 : 214 . 80 ± 56 . 87 % , p = 0 . 033 , one - way ANOVA with Dunn ' s multiple comparison test ) . n = 4 . The pPKA substrate levels were normalized to the total protein amount stained with Ponceau S . Treatment of NSC34 cells with BafA ( 67nM ) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO - treated group set to 100 % ( BafA : 198 . 9 ± 28 . 44 % , p = 0 . 003 , one - tailed unpaired t - test ) . Treatment of NSC34 cells with BafA ( 67nM ) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO - treated group set to 100 % ( BafA : 198 . 9 ± 28 . 44 % , p = 0 . 003 , one - tailed unpaired t - test ) . n = 6 . ( J ) Western blot analysis of purified autophagosomes ( AVs ) ( 50μg AVs / lane ) with and without Proteinase K ( PK ) treatment . TX - 100 ( 1 % final ) was used as a positive control for the activity of PK . Synaptosome lysates ( Syn , 30μg / lane ) were used as a positive control for the signal of antibodies . ( M , N ) Representative fluorescent images and subsequent analysis of PKA R1 - α / p62 colocalization ( Pearson ' s correlation coefficient ) in primary cortico - hippocampal neurons , which have undergone 16 hours amino - acids and serum starvation and were additionally treated with BafA ( 67nM ) to visualize the lysosomes ( control : 0 . 68 ± 0 . 019 , starvation : 0 . 58 ± 0 . 015 , starvation + BafA : 0 . 83 ± 0 . 014 ; pcontrol vs starvation < 0 . 0001 , pcontrol vs starvation + BafA < 0 . 0001 , pstarvation vs starvation + BafA < 0 . 0001 Ordinary one - way ANOVA with Tukey ' s multiple comparison test ) . ncontrol = 36 , nstarvation = 38 , nstarvation + BafA = 36 neurons from n = 3 . Co - immunoprecipitation of endogenous PKA R1 - β with PKA - Cα from Atg5flox : CamKIIα - Cre WT / KO mouse brain lysates ( WT set to 100 % , KO : 170 . 3 ± 14 . 88 % , p = 0 . 005 , one - tailed unpaired t - test , n = 3 ) . Input , 1 . 5 % of the total lysate was added to the assay . Co - immunoprecipitation of endogenous PKA R1 - β with PKA - Cα from Atg5flox : CamKIIα - Cre WT / KO mouse brain lysates ( WT set to 100 % , KO : 170 . 3 ± 14 . 88 % , p = 0 . 005 , one - tailed unpaired t - test , n = 3 ) . Input , 1 . 5 % of the total lysate was added to the assay .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Western Blot analysis of PKA R1α protein levels in NSC34 cells, treated with 250 nM Torin1 or deprived of amino-acids and serum for 16 hours. Lysosomal degradation was blocked in the last 4 hours using 67 nM of Bafilomycin A (BafA). (B) Starvation significantly reduced PKA R1α protein levels in NSC34 cells (vehicle set to 100%, starvation: 31.19± 5.085%, p<0.0001, one-tailed unpaired t-test). n=5. (C) Application of 67 nM BafA for 4 hours before harvesting was sufficient to stabilize the PKA R1α protein levels in starved NSC34 cells (starvation set to 100%, starvation+BafA: 155.5± 13.93%, p=0.002, one-tailed unpaired t-test).16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2- positive cultured primary neurons (Vehicle: 212.4±5.619, Starvation: 255.1±5.715; p<0.0001, two-tailed paired t-test). nVehicle=314, nStarvation=306 neurons from n=3.16 hours starvation significantly increased intracellular cAMP level measured as mean intensity in MAP2- positive cultured primary neurons (Vehicle: 212.4±5.619, Starvation: 255.1±5.715; p<0.0001, two-tailed paired t-test). nVehicle=314, nStarvation=306 neurons from n=3.(F, G) Phosphorylation state of proteins containing PKA substrate RRXS/T motif was increased upon 16 hours of amino-acid and serum starvation in primary cortico-hippocampal neurons at DIV14 and was suppressed by 1 µM H89 supplementation in the media (Vehicle set to 100%, Starvation: 326.60±94.53%, H89: 214.80±56.87%, p=0.033, one-way ANOVA with Dunn's multiple comparison test). n=4. The pPKA substrate levels were normalized to the total protein amount stained with Ponceau S.Treatment of NSC34 cells with BafA (67nM) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO-treated group set to 100% (BafA: 198.9± 28.44%, p=0.003, one-tailed unpaired t-test).Treatment of NSC34 cells with BafA (67nM) for 6 hours significantly increased PKA R1α protein levels compared to the DMSO-treated group set to 100% (BafA: 198.9± 28.44%, p=0.003, one-tailed unpaired t-test). n=6.(J) Western blot analysis of purified autophagosomes (AVs) (50μg AVs/lane) with and without Proteinase K (PK) treatment. TX-100 (1% final) was used as a positive control for the activity of PK. Synaptosome lysates (Syn, 30μg/lane) were used as a positive control for the signal of antibodies.(M, N) Representative fluorescent images and subsequent analysis of PKA R1- α/p62 colocalization (Pearson's correlation coefficient) in primary cortico-hippocampal neurons, which have undergone 16 hours amino-acids and serum starvation and were additionally treated with BafA (67nM) to visualize the lysosomes (control: 0.68± 0.019, starvation: 0.58± 0.015, starvation+BafA: 0.83± 0.014; pcontrol vs starvation<0.0001, pcontrol vs starvation+BafA<0.0001, pstarvation vs starvation+BafA<0.0001 Ordinary one-way ANOVA with Tukey's multiple comparison test). ncontrol=36, nstarvation=38, nstarvation+BafA=36 neurons from n=3.Co-immunoprecipitation of endogenous PKA R1-β with PKA-Cα from Atg5flox:CamKIIα-Cre WT/KO mouse brain lysates (WT set to 100%, KO: 170.3± 14.88%, p=0.005, one-tailed unpaired t-test, n=3). Input, 1.5% of the total lysate was added to the assay.Co-immunoprecipitation of endogenous PKA R1-β with PKA-Cα from Atg5flox:CamKIIα-Cre WT/KO mouse brain lysates (WT set to 100%, KO: 170.3± 14.88%, p=0.005, one-tailed unpaired t-test, n=3). Input, 1.5% of the total lysate was added to the assay.


Figure 5 ( A - B ) Western Blot analysis of pCREB protein levels in Atg5flox : CAG - CreTmx WT / KO cultured neurons at DIV14 , treated with forskolin ( WTDMSO set to 100 % , WTForskolin : 214 . 17 ± 48 . 87 % , KODMSO : 63 . 26 ± 15 . 85 % , KOForskolin : 33 . 50 ± 10 . 84 % , pWT DMSO / WT Forskolin = 0 . 042 , pWT Forskolin / KO Forskolin = 0 . 002 , two - way ANOVA with Tukey ' s multiple comparisons ) . n = 4 . The same lysates were run in parallel to detect pCREB and CREB and normalized to their respective loading control before calculating the pCREB / CREB ratio . ( I , J ) Representative fluorescent images and subsequent analysis of primary cortico - hippocampal neurons containing nuclear pCREB either under DMSO - treated condition or under condition when amino acids and serum starvation was induced for 16 hours in the absence or presence of 1µM of PKA inhibitor H89 . Neurons were identified by their polarized morphology in DIC . White rectangular boxes indicate panel magnified to the right . In the inserts , LUT gradient was added to highlight the pCREB intensity in the nucleus . Scale bar : 50 µm . DMSO : 52 . 50 ± 2 . 757 , starvation : 70 . 00 ± 2 . 697 , starvation + H89 : 53 . 92 ± 3 . 147 ; pDMSO / starvation < 0 . 0001 , pstarvation / starvation & H89 = 0 . 0004 , one - way ANOVA with Tukey ' s multiple comparisons test , n = 30 images from n = 3 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-B) Western Blot analysis of pCREB protein levels in Atg5flox:CAG-CreTmx WT/ KO cultured neurons at DIV14, treated with forskolin (WTDMSO set to 100%, WTForskolin: 214.17± 48.87%, KODMSO: 63.26± 15.85%, KOForskolin: 33.50± 10.84%, pWT DMSO/WT Forskolin=0.042, pWT Forskolin/KO Forskolin=0.002, two-way ANOVA with Tukey's multiple comparisons). n=4. The same lysates were run in parallel to detect pCREB and CREB and normalized to their respective loading control before calculating the pCREB/CREB ratio.(I, J) Representative fluorescent images and subsequent analysis of primary cortico-hippocampal neurons containing nuclear pCREB either under DMSO-treated condition or under condition when amino acids and serum starvation was induced for 16 hours in the absence or presence of 1µM of PKA inhibitor H89. Neurons were identified by their polarized morphology in DIC. White rectangular boxes indicate panel magnified to the right. In the inserts, LUT gradient was added to highlight the pCREB intensity in the nucleus. Scale bar: 50 µm. DMSO: 52.50±2.757, starvation: 70.00±2.697, starvation+H89: 53.92±3.147; pDMSO/starvation<0.0001, pstarvation/starvation&H89=0.0004, one-way ANOVA with Tukey's multiple comparisons test, n=30 images from n=3 independent experiments.


Figure 6 ( A - B ) Western Blot analysis of PKA substrates carrying RRXS / T motif in primary cortico - hippocampal Atg5flox : CAG - CreTmx WT / KO neurons at DIV14 , treated with Forskolin ( WTDMSO set to 100 % , WTForskolin : 291 . 85 ± 17 . 02 % , KODMSO : 152 . 97 ± 25 . 26 % , KOForskolin : 182 . 56 ± 31 . 89 % , pWTDMSO / WTForskolin : 0 . 001 , pWTForskolin / KOForskolin : 0 . 033 , two - way ANOVA with Tukey ' s multiple comparisons ) . ( K - M ) Representative STED images and Pearson ' s colocalization coefficient analysis of SHANK3 ( K ) and CASKIN1 ( L ) and PSD95 in Atg5flox : CamKIIα - Cre : Ai9 WT / KO neurons . Scale bar : 5µm , 1µm ( small panels ) . WTSHANK3 : 0 . 324 ± 0 . 022 , KOSHANK3 : 0 . 415 ± 0 . 024 ; p = 0 . 0067 , two - tailed unpaired t - test . nWT = 46 , nKO = 46 . WTCASKIN1 : 0 . 255 ± 0 . 024 , KO CASKIN1 : 0 . 326 ± 0 . 025 ; p = 0 . 0432 , two - tailed unpaired t - test . nWT = 39 , nKO = 43 from n = 2 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A-B) Western Blot analysis of PKA substrates carrying RRXS/T motif in primary cortico-hippocampal Atg5flox:CAG-CreTmx WT/KO neurons at DIV14, treated with Forskolin (WTDMSO set to 100%, WTForskolin: 291.85± 17.02%, KODMSO: 152.97± 25.26%, KOForskolin: 182.56± 31.89%, pWTDMSO/WTForskolin: 0.001, pWTForskolin/KOForskolin: 0.033, two-way ANOVA with Tukey's multiple comparisons).(K-M) Representative STED images and Pearson's colocalization coefficient analysis of SHANK3 (K) and CASKIN1 (L) and PSD95 in Atg5flox:CamKIIα-Cre:Ai9 WT/KO neurons. Scale bar: 5µm, 1µm (small panels). WTSHANK3: 0.324±0.022, KOSHANK3: 0.415±0.024; p=0.0067, two-tailed unpaired t-test. nWT=46, nKO=46. WTCASKIN1: 0.255±0.024, KO CASKIN1: 0.326±0.025; p=0.0432, two-tailed unpaired t-test. nWT=39, nKO=43 from n=2 independent experiments.


Figure 7 ( C , D ) Analysis of GLUR1 fluorescent levels at the spines of 10 - 11 week - old Atg5wt / wt : Ai9 and Atg5flox / flox : Ai9 mice injected with CamKIIα - Cre AAV into the hippocampus . Scale bar : 5µm . GLUR1 level at PSD95 - positve spines were assessed measuring mean gray values after background subtraction ( WT : 64 . 55 ± 7 . 988 % , KO : 99 . 14 ± 6 . 863 % ; p = 0 . 0029 , two - tailed unpaired t - test ) . nWT = 13 , nKO = 14 images from n = 3 mice ( > 31 dendritic spines analyzed per each mouse ) . sp , spine . ( G ) Representative fluorescence images of Atg5flox : CAG - CreTmx WT / KO neurons transfected with eGFP or eGFP - ATG5 and immunostained for GLUR1 , PSD95 and GFP . ( H ) The overexpression of eGFP - ATG5 in Atg5flox : CAG - CreTmx KO neurons diminished the of GLUR1 / PSD95 colocalization compared to the eGFP - overexpressing KO neurons ( KOeGFP : 70 . 07 ± 1 . 473 % , KOeGFP - ATG5 : 65 . 42 ± 1 . 721 % , ; p = 0 . 048 , two - tailed unpaired t - test ) . nKO + GFP = 42 , nKO + GFP - ATG5 = 50 images from n = 5 . ( I , J ) The coexpression of eGFP - PKA R1α and eGFP - PKA R1β in cultured Atg5flox : CAG - CreTmx WT neurons results in significantly increased colocalization between GLUR1 and PSD95 compared to the eGFP - expressing neurons ( WT : 42 . 30 ± 2 . 453 % , KO : 51 . 32 ± 2 . 624 % , p = 0 . 014 , two - tailed unpaired t - test ) . nWT + GFP = 46 , nWTGFP + PKA R1 OE = 46 images from n = 3 . Analysis in H , J is performed using a unbiased custom - written Plugin for ImageJ . ( K - M ) GCAMP7f responses to tetanic stimulation ( four tetani , 100 APs at 100Hz , 3s interval ) in cell bodies of CamKIIα - Cre - transduced primary cortico - hippocampal Atg5wt : wt / Ai9 ( WT ) and Atg5flox : flox / Ai9 ( KO ) neurons expressing mCamKIIα - jGCaMP7f and treated either with DMSO ( K ) or with 10 µM CNQX for 5 min ( L ) . Autophagy deficient cells showed significantly increased facilitation of GCAMP7f signal to electrical stimulation ( ΔF / Fpeak1 ) compared to the WT , a phenotype which was blocked by the application of CNQX ( WTDMSO : 0 . 28 ± 0 . 12 , WTCNQX : - 0 . 07 ± 0 . 03 , KODMSO : 3 . 74 ± 0 . 86 , KOCNQX : - 0 . 014 ± 0 . 13 , p WT DMSO / KO DMSO < 0 . 0001 , pKO DMSO / KO CNQX < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparisons test ) . nWT DMSO = 25 , nWT CNQX = 27 , nKO DMSO = 33 , nKO CNQX = 30 neurons from n = 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(C,D) Analysis of GLUR1 fluorescent levels at the spines of 10-11 week-old Atg5wt/wt:Ai9 and Atg5flox/flox:Ai9 mice injected with CamKIIα-Cre AAV into the hippocampus. Scale bar: 5µm. GLUR1 level at PSD95-positve spines were assessed measuring mean gray values after background subtraction (WT: 64.55±7.988%, KO: 99.14±6.863%; p=0.0029, two-tailed unpaired t-test). nWT=13, nKO=14 images from n=3 mice (>31 dendritic spines analyzed per each mouse). sp, spine.(G) Representative fluorescence images of Atg5flox:CAG-CreTmx WT/KO neurons transfected with eGFP or eGFP-ATG5 and immunostained for GLUR1, PSD95 and GFP. (H) The overexpression of eGFP-ATG5 in Atg5flox:CAG-CreTmx KO neurons diminished the of GLUR1/PSD95 colocalization compared to the eGFP-overexpressing KO neurons (KOeGFP: 70.07±1.473%, KOeGFP-ATG5: 65.42±1.721%,; p=0.048, two-tailed unpaired t-test). nKO+GFP=42, nKO+GFP-ATG5=50 images from n=5. (I, J) The coexpression of eGFP-PKA R1α and eGFP-PKA R1β in cultured Atg5flox:CAG-CreTmx WT neurons results in significantly increased colocalization between GLUR1 and PSD95 compared to the eGFP-expressing neurons (WT: 42.30±2.453%, KO: 51.32±2.624%, p=0.014, two-tailed unpaired t-test). nWT+GFP=46, nWTGFP+PKA R1 OE=46 images from n=3. Analysis in H, J is performed using a unbiased custom-written Plugin for ImageJ.(K-M) GCAMP7f responses to tetanic stimulation (four tetani, 100 APs at 100Hz, 3s interval) in cell bodies of CamKIIα-Cre-transduced primary cortico-hippocampal Atg5wt:wt/Ai9 (WT) and Atg5flox:flox/Ai9 (KO) neurons expressing mCamKIIα-jGCaMP7f and treated either with DMSO (K) or with 10 µM CNQX for 5 min (L). Autophagy deficient cells showed significantly increased facilitation of GCAMP7f signal to electrical stimulation (ΔF/Fpeak1) compared to the WT, a phenotype which was blocked by the application of CNQX (WTDMSO: 0.28± 0.12, WTCNQX: -0.07± 0.03, KODMSO: 3.74± 0.86, KOCNQX: -0.014± 0.13, p WT DMSO/ KO DMSO<0.0001, pKO DMSO/KO CNQX<0.0001, two-way ANOVA with Tukey's multiple comparisons test). nWT DMSO= 25, nWT CNQX= 27, nKO DMSO= 33, nKO CNQX= 30 neurons from n=3.