H , I , Immunostaining for HIF - 1α ( red ) and DAPI ( blue ) in control ( H , H ' , H ' ' ) and Gpr124KO ( I , I ' , I ' ' ) cortices . Panels H ' , H ' ' , I ' and I ' ' are magnifications of the boxed areas in panels I and J , showing HIF - 1α signal alone ( H ' , I ' ) or together with DAPI ( H ' ' , I ' ' ) . Asterisks indicate autofluorescent blood cells . The dashed line indicates the basal boundary of the VZ . Scale bar = 100 µm . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-TISSUE O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

H,I, Immunostaining for HIF-1α (red) and DAPI (blue) in control (H, H', H'') and Gpr124KO (I,I',I'') cortices. Panels H',H'',I' and I'' are magnifications of the boxed areas in panels I and J, showing HIF-1α signal alone (H',I') or together with DAPI (H'',I''). Asterisks indicate autofluorescent blood cells. The dashed line indicates the basal boundary of the VZ. Scale bar = 100 µm. Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


J , qRT - PCR analysis of HIF - 1 target gene expression in VZ cells from the cortex of control and Gpr124KO E13 . 5 embryos ( mean ± SEM ; N = 4 ; * * * p < 0 . 001 ) . SVZ , subventricular zone ; VZ , ventricular zone .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O

J, qRT-PCR analysis of HIF-1 target gene expression in VZ cells from the cortex of control and Gpr124KO E13.5 embryos (mean±SEM; N=4; *** p<0.001). SVZ, subventricular zone; VZ, ventricular zone.


A - F , Staining for Isolectin B4 ( A - C ) and oxidized thiols ( D - F ) in WT cortices at E10 . 5 ( A , D ) , E11 . 5 ( B , E ) and E12 . 5 ( C , F ) . Panels D - F are pseudocolor conversions of staining intensity ( from low [ purple ] to high [ red ] ) . Arrowheads denote the position of the vascular front .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F, Staining for Isolectin B4 (A-C) and oxidized thiols (D-F) in WT cortices at E10.5 (A,D), E11.5 (B,E) and E12.5 (C,F). Panels D-F are pseudocolor conversions of staining intensity (from low [purple] to high [red]). Arrowheads denote the position of the vascular front.


G - I , Immunostaining for HIF - 1α ( red ) and DAPI ( blue ) in WT cortices at E10 . 5 ( G - G ' ' ) , E11 . 5 ( H - H ' ' ) and E12 . 5 ( I - I ' ' ) . Panels G ' , G ' ' , H ' , H ' ' , I ' and I ' ' are magnifications of the boxed areas in their respective original panels . Arrows point to the border of lateral and dorsal cortex .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O

G-I, Immunostaining for HIF-1α (red) and DAPI (blue) in WT cortices at E10.5 (G-G''), E11.5 (H-H'') and E12.5 (I-I''). Panels G',G'',H',H'',I' and I'' are magnifications of the boxed areas in their respective original panels. Arrows point to the border of lateral and dorsal cortex.


J - L , Immunostaining for Glut1 of WT cortices at E10 . 0 ( J ) , E11 . 5 ( K ) and E12 . 5 ( L ) . Dashed lines indicate the apical border of the cortex . Scale bar : 100 µm ( D , F , G , H , I , J - L ) or 50 µm ( I ' ' ) , respectively .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-L, Immunostaining for Glut1 of WT cortices at E10.0 (J), E11.5 (K) and E12.5 (L). Dashed lines indicate the apical border of the cortex. Scale bar: 100 µm (D,F,G,H,I, J-L) or 50 µm (I''), respectively.


A , qRT - PCR analysis of HIF - 1 and HIF - 1 target gene expression in cortices from E13 . 5 WT and HIF - 1αCC + / - embryos ( mean ± SEM ; N = 8 for WT and N = 5 for HIF - 1αCC + / - ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, qRT-PCR analysis of HIF-1 and HIF-1 target gene expression in cortices from E13.5 WT and HIF-1αCC+/-embryos (mean±SEM; N=8 for WT and N=5 for HIF-1αCC+/-; * p<0.05, ** p<0.01, *** p<0.001).


B , C , Staining for EdU ( red ) and Ki67 ( green ) in WT ( B ) and HIF - 1αCC + / - ( C ) cortices at E13 . 5 , 24 hours after EdU injection . D , Quantification of Ki67 + NPCs in the in VZ ( blue ) , SVZ / CP ( grey ) and of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control and HIF - 1αCC + / - brains ( mean ± SEM ; N = 4 ; * p < 0 . 05 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-TISSUE O O O O B-TISSUE O B-TISSUE O O O O O B-GENEPROD O B-CELL_TYPE O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

B,C, Staining for EdU (red) and Ki67 (green) in WT (B) and HIF-1αCC+/- (C) cortices at E13.5, 24 hours after EdU injection. D, Quantification of Ki67+NPCs in the in VZ (blue), SVZ/CP (grey) and of Ki67-neurons (cyan) generated from EdU-labeled NPCs in control and HIF-1αCC+/-brains (mean±SEM; N=4; * p<0.05). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


E , F , Immunostaining for Tbr2 ( red ) and Ngn1 ( green ) in WT ( E ) or HIF - 1αCC + / - ( F ) cortices at E13 . 5 . G - I Quantification of neurogenic ( Ngn1 + ) RGs and BPs ( G ) , newborn BPs ( H ) or expanding ( Tbr2 - Ngn1 - ) RGs ( I ) in control and HIF - 1αCC + / - cortices at E13 . 5 ( mean ± SEM ; N = 5 ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

E,F, Immunostaining for Tbr2 (red) and Ngn1 (green) in WT (E) or HIF-1αCC+/- (F) cortices at E13.5. G-I Quantification of neurogenic (Ngn1+) RGs and BPs (G), newborn BPs (H) or expanding (Tbr2-Ngn1-) RGs (I) in control and HIF-1αCC+/-cortices at E13.5 (mean±SEM; N=5; * p<0.05, ** p<0.01, *** p<0.001). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


J , Whole mount image of brains from WT or HIF - 1αCC + / - mice at P5 . K Quantification of cortex area in brain sections from P5 WT or HIF - 1αCC + / - mice ( mean ± SEM ; N = 8 ( WT ) or N = 6 ( HIF - 1αCC + / - ) ; * p < 0 . 05 ) .

O O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

J, Whole mount image of brains from WT or HIF-1αCC+/-mice at P5. K Quantification of cortex area in brain sections from P5 WT or HIF-1αCC+/-mice (mean±SEM; N=8 (WT) or N=6 (HIF-1αCC+/-); * p<0.05).


L , M , Staining with Hoechst 33248 in P5 WT ( L ) or HIF - 1αCC + / - ( M ) cortices . N , Quantification of cortical thickness of WT and HIF - 1αCC + / - cortices . The double arrow denotes the measured thickness of the cortex . N , Quantification of cortical thickness of WT and HIF - 1αCC + / - cortices ( mean ± SEM ; N = 8 ( WT ) or N = 6 ( HIF - 1αCC + / - ) ; * * * p < 0 . 001 ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

L,M, Staining with Hoechst 33248 in P5 WT (L) or HIF-1αCC+/- (M) cortices. N, Quantification of cortical thickness of WT and HIF-1αCC+/-cortices. The double arrow denotes the measured thickness of the cortex. N, Quantification of cortical thickness of WT and HIF-1αCC+/-cortices (mean±SEM; N=8 (WT) or N=6 (HIF-1αCC+/-); *** p<0.001).


O - Q , Epifluorescent images of the E16 . 5 cortex , 3 days after in utero electroporation ( IUE ) with EGFP alone ( ctrl , O ) , wild type HIF - 1 + EGFP ( P ) or mutant HIF - 1ΔC + EGFP ( Q ) . O ' , P ' and Q ' are magnifications of the boxed area in their respective original panel . R , Quantification of EGFP + cells in the cortical zones VZ / SVZ , IZ and CP shown in O - Q ( mean ± SEM ; N = 5 ; * * p < 0 . 01 , * p < 0 . 05 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O 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-GENEPROD O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

O-Q, Epifluorescent images of the E16.5 cortex, 3 days after in utero electroporation (IUE) with EGFP alone (ctrl, O), wild type HIF-1+EGFP (P) or mutant HIF-1ΔC+EGFP (Q). O',P' and Q' are magnifications of the boxed area in their respective original panel. R, Quantification of EGFP+ cells in the cortical zones VZ/SVZ, IZ and CP shown in O-Q (mean±SEM; N=5; ** p< 0.01,* p<0.05). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


S - U , qRT - PCR analysis of NSC marker ( S ; mean ± SEM ; N = 5 ; * P < 0 . 05 ) , neurogenic markers ( T ; mean ± SEM ; N = 5 ; * * * p < 0 . 001 , * p < 0 . 05 ) , and HIF target genes ( U ; mean ± SEM ; N = 5 ; * * p < 0 . 01 , * p < 0 . 05 ) in expanding NPCs ( eNPCs ) and neurogenic NPCs ( nNPCs ) . Gene expression was normalized to the levels in eNPCs .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O 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-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O

S-U, qRT-PCR analysis of NSC marker (S; mean±SEM; N=5; * P<0.05), neurogenic markers (T; mean±SEM; N=5; *** p< 0.001, * p<0.05), and HIF target genes (U; mean±SEM; N=5; ** p< 0.01,* p<0.05) in expanding NPCs (eNPCs) and neurogenic NPCs (nNPCs). Gene expression was normalized to the levels in eNPCs.


B - E , Staining for pimonodazole ( PIMO , green ) and Isolectin B4 ( IB4 , red ) in Gpr124KO ( B , C ) and control ( D , E ) cortices after exposure of the pregnant dams to 21 % O2 ( B , D ) or 80 % O2 ( C , E ) . Compared to the pimonidazole staining in panels B , C , the intensity of this staining is much weaker in panels D , E . For reasons of clarity , the pimonidazole staining in panels D , E was therefore enhanced , equally in both panels .

O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O

B-E, Staining for pimonodazole (PIMO, green) and Isolectin B4 (IB4,red) in Gpr124KO (B,C) and control (D,E) cortices after exposure of the pregnant dams to 21% O2 (B,D) or 80% O2 (C,E). Compared to the pimonidazole staining in panels B,C, the intensity of this staining is much weaker in panels D,E. For reasons of clarity, the pimonidazole staining in panels D,E was therefore enhanced, equally in both panels.


F , G , Immunostaining for HIF - 1 ( red ) and DAPI ( blue ) in Gpr124KOcortices after exposure of the pregnant dams to 21 % O2 ( F ) or 80 % O2 ( G ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

F,G, Immunostaining for HIF-1 (red) and DAPI (blue) in Gpr124KOcortices after exposure of the pregnant dams to 21% O2 (F) or 80% O2 (G). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


H , I , Immunostaining for Tbr2 ( red ) and Ngn1 ( green ) in Gpr124KO cortices after exposure of the pregnant dams to 21 % O2 ( H ) or 80 % O2 ( I ) . J - L Quantification of neurogenic ( Ngn1 + ) RGs and BPs ( J ) , newborn BPs ( K ) or expanding ( Tbr2 - Ngn1 - ) RGs ( L ) in Gpr124KO cortices after exposure of the pregnant dams to 21 % O2 or 80 % O2 ( mean ± SEM ; N = 4 for 21 % O2 and N = 5 for 80 % O2 ; * p < 0 . 05 , * * p < 0 . 01 ) . M , N , Immunostaining for Tbr2 ( red ) and Ngn1 ( green ) in control cortices after exposure of the pregnant dams to 21 % O2 ( M ) or 80 % O2 ( N ) . O - Q Quantification of neurogenic ( Ngn1 + ) RGs and BPs ( O ) , newborn BPs ( P ) or expanding ( Tbr2 - Ngn1 - ) RGs ( Q ) in control cortices after exposure of the pregnant dams to 21 % O2 or 80 % O2 ( mean ± SEM ; N = 5 ; * * p < 0 . 01 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD B-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD B-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O B-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

H,I, Immunostaining for Tbr2 (red) and Ngn1 (green) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 (H) or 80% O2 (I). J-L Quantification of neurogenic (Ngn1+) RGs and BPs (J), newborn BPs (K) or expanding (Tbr2- Ngn1-) RGs (L) in Gpr124KO cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=4 for 21% O2 and N=5 for 80% O2; * p<0.05, ** p<0.01). M,N, Immunostaining for Tbr2 (red) and Ngn1 (green) in control cortices after exposure of the pregnant dams to 21% O2 (M) or 80% O2 (N). O-Q Quantification of neurogenic (Ngn1+) RGs and BPs (O), newborn BPs (P) or expanding (Tbr2-Ngn1-) RGs (Q) in control cortices after exposure of the pregnant dams to 21% O2 or 80% O2 (mean±SEM; N=5; ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A , qRT - PCR analysis of glycolytic enzymes in Prom1 + VZ cells and their Prom1 - differentiated progeny ( BPs and neurons ) in WT cortices at E14 . 5 ( mean ± SEM ; N = 3 ( Hk2 , Gapdh ) ; N = 6 ( Eno1 ; Ldha ; * * p < 0 . 01 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-TISSUE O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

A, qRT-PCR analysis of glycolytic enzymes in Prom1+ VZ cells and their Prom1- differentiated progeny (BPs and neurons) in WT cortices at E14.5 (mean±SEM; N=3 (Hk2, Gapdh); N=6 (Eno1; Ldha; ** p<0.01).


B , Measurement of lactate secretion from proliferating NSCs , and of NSCs differentiating for 3 days ( switch to neurogenesis ) or for 7 days ( fully differentiated ) , normalized to cellular protein content ( mean ± SEM ; N = 6 ; * * * p < 0 . 001 ) .

O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O 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, Measurement of lactate secretion from proliferating NSCs, and of NSCs differentiating for 3 days (switch to neurogenesis) or for 7 days (fully differentiated), normalized to cellular protein content (mean±SEM; N=6; *** p<0.001).


C , qRT - PCR analysis of Pfkfb3 expression in proliferating NSCs , transduced with scr or Pfkfb3shRNA # 1 ( mean ± SEM ; N = 4 ; * p < 0 . 05 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

C, qRT-PCR analysis of Pfkfb3 expression in proliferating NSCs, transduced with scr or Pfkfb3shRNA#1 (mean±SEM; N=4; * p<0.05).


D , Measurement of lactate secretion from proliferating NSCs transduced with scr or Pfkfb3shRNA # 1 , normalized to cellular protein content ( mean ± SEM ; N = 3 ; * p < 0 . 05 ) .

O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

D, Measurement of lactate secretion from proliferating NSCs transduced with scr or Pfkfb3shRNA#1, normalized to cellular protein content (mean±SEM; N=3; * p<0.05).


E , Measurement of mitochondrial respiration rate ( oxygen consumption rate , OCRMITO ; see supplemental methods ) in proliferating NSCs , transduced with scr or Pfkfb3shRNA # 1 , normalized to cellular protein content ( mean ± SEM ; N = 3 ; N . S . , not significant ) .

O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

E, Measurement of mitochondrial respiration rate (oxygen consumption rate, OCRMITO; see supplemental methods) in proliferating NSCs, transduced with scr or Pfkfb3shRNA#1, normalized to cellular protein content (mean±SEM; N=3; N.S., not significant).


F - H , Epifluorescent images of the E15 . 5 cortex , 3 days after in utero electroporation ( IUE ) with EGFP + scr shRNA ( F ) , EGFP + Pfkfb3 shRNA # 1 ( G ) or EGFP + Pfkfb3 shRNA # 2 ( H ) . J , Quantification of EGFP + cells in the cortical zones VZ / SVZ , IZ and CP shown in panels F - H ( mean ± SEM ; N = 4 for scr and N = 5 for Pfkfb3 shRNAs ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . K - M , Epifluorescent images of the mouse cortex after in utero electroporation ( IUE ) with HIF - 1α and scr shRNA ( K ) or with HIF - 1α and two different Pfkfb3 shRNAs ( L , M ) at E16 . 5 , 3 days after transfection . N , Quantification of EGFP + cells in the cortical zones VZ / SVZ , IZ and CP shown in K - M ( mean ± SEM ; N = 5 ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . Full , dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O

F-H, Epifluorescent images of the E15.5 cortex, 3 days after in utero electroporation (IUE) with EGFP + scr shRNA (F), EGFP + Pfkfb3 shRNA#1 (G) or EGFP + Pfkfb3 shRNA#2 (H). J, Quantification of EGFP+ cells in the cortical zones VZ/SVZ, IZ and CP shown in panels F-H (mean±SEM; N=4 for scr and N=5 for Pfkfb3 shRNAs; * p<0.05, ** p< 0.01, *** p< 0.001). K-M, Epifluorescent images of the mouse cortex after in utero electroporation (IUE) with HIF-1α and scr shRNA (K) or with HIF-1α and two different Pfkfb3 shRNAs (L,M) at E16.5, 3 days after transfection. N, Quantification of EGFP+ cells in the cortical zones VZ/SVZ, IZ and CP shown in K-M (mean±SEM; N=5; * p<0.05, ** p< 0.01, *** p< 0.001). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A Affinity measurements by biolayer interferometry ( BLI ) of bipNbs NM1267 and NM1268 . NM1267 and NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM and 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD derived from SARS - CoV - 2 B . 1 ( RBDB . 1 ) . Global 1 : 1 fits are illustrated as dashed lines . Global 1 : 1 fits are illustrated as dashed lines and binding affinity ( KD ) , association ( kon ) and dissociation constant ( koff ) determined for the individual bipNbs are summarized .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

A Affinity measurements by biolayer interferometry (BLI) of bipNbs NM1267 and NM1268. NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD derived from SARS-CoV-2 B.1 (RBDB.1). Global 1:1 fits are illustrated as dashed lines. Global 1:1 fits are illustrated as dashed lines and binding affinity (KD), association (kon) and dissociation constant (koff) determined for the individual bipNbs are summarized.


A - P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B . 1 . 1 . 7 ( Alpha ) ( A , I ) , B . 1 . 351 ( Beta ) ( B , J ) , P1 ( Gamma ) ( C , K ) , B . 1 . 617 . 2 ( Delta ) ( D , L ) , B . 1 . 429 ( Epsilon ) ( E , M ) , P3 ( Theta ) ( F , N ) , B . 1 . 617 . 1 ( Kappa ) ( G , O ) and A . 23 . 1 ( H , P ) . NM1267 and NM1268 were applied with concentrations ranging from 5 - 0 . 625 nM and 20 ‑ 2 . 5 nM , respectively ( illustrated with gradually lighter shades ) on immobilized RBD variants . Global 1 : 1 fits are illustrated as dashed lines .

O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-P Affinity measurements by BLI of bipNb NM1267 and NM1268 on recently identified RBD variants B.1.1.7 (Alpha) (A, I), B.1.351 (Beta) (B, J), P1 (Gamma) (C, K), B.1.617.2 (Delta) (D, L), B.1.429 (Epsilon) (E, M), P3 (Theta) (F, N), B.1.617.1 (Kappa) (G, O) and A.23.1 (H, P). NM1267 and NM1268 were applied with concentrations ranging from 5-0.625 nM and 20‑2.5 nM, respectively (illustrated with gradually lighter shades) on immobilized RBD variants. Global 1:1 fits are illustrated as dashed lines.


A - F Neutralization potency of NM1267 and NM1268 were analyzed in Caco ‑ 2 cells using the SARS - CoV - 2 B . 1 ( A , D ) SARS - CoV - 2 B . 1 . 351 ( Beta ) ( B , E ) and SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) ( C , F ) . Infection normalized to virus - only infection control is illustrated as percent of infection ( infection [ % ] ) . Data are presented as mean ± SEM of three ( n = 3 ) biological replicates .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F Neutralization potency of NM1267 and NM1268 were analyzed in Caco‑2 cells using the SARS-CoV-2 B.1 (A, D) SARS-CoV-2 B.1.351 (Beta) (B, E) and SARS-CoV-2 B.1.617.2 (Delta) (C, F). Infection normalized to virus-only infection control is illustrated as percent of infection (infection [%]). Data are presented as mean ± SEM of three (n = 3) biological replicates.


C Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 15 ) or NM1267 ( n = 12 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . survival were monitored for 14 days . , * * * * P < 0 . 0001 , by log - rank

O O B-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

C Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 15) or NM1267 (n = 12) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1. survival were monitored for 14 days. , ****P < 0.0001, by log-rank test


D Nasal swabs were collected from six mice per group ( n = 6 ) at the indicated time points and viral load was determined by plaque - assay . Bars represent mean ± SEM . * * P < 0 . 01 , by unpaired t

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D Nasal swabs were collected from six mice per group (n = 6) at the indicated time points and viral load was determined by plaque-assay. Bars represent mean ± SEM. **P < 0.01, by unpaired t test


Histopathological analysis of mice , which were intranasally treated with bipNb NM1267 or control Nb NM1251 and subsequently infected with 3 * 103 PFU SARS - CoV - 2 B . 1 . A - B Serial tissue sections revealed severe inflammation ( H & E ) and numerous widespread SARS - CoV - 2 RNA positive alveolar epithelia cells and macrophages ( in situ hybridization ( ISH ) ) in lungs of infected , control - treated mice . In infected and NM1267 - treated animals no inflammation or only small focal areas with inflammation and a few SARS - CoV - 2 RNA positive cells were observed . Scale bars represent 1 mm and 200 µm , respectively .

B-EXP_ASSAY O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-TISSUE O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O

Histopathological analysis of mice, which were intranasally treated with bipNb NM1267 or control Nb NM1251 and subsequently infected with 3*103 PFU SARS-CoV-2 B.1. A-B Serial tissue sections revealed severe inflammation (H&E) and numerous widespread SARS-CoV-2 RNA positive alveolar epithelia cells and macrophages (in situ hybridization (ISH)) in lungs of infected, control-treated mice. In infected and NM1267-treated animals no inflammation or only small focal areas with inflammation and a few SARS-CoV-2 RNA positive cells were observed. Scale bars represent 1 mm and 200 µm, respectively.


B , Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 8 ) , NM1267 ( n = 9 ) or NM1268 ( n = 9 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) . Weight loss were monitored for 14 days . Dashed line indicates humane endpoint and symbols represent mean ± SEM * * * * P < 0 . 0001 in orange between NM1251 and NM1267 and in purple between NM1251 and NM1268 , * P < 0 . 1 in black between NM1267 and NM1268 , by two - way ANOVA with Sidak ' s multiple comparison

O O O B-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O

B, Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta). Weight loss were monitored for 14 days. Dashed line indicates humane endpoint and symbols represent mean ± SEM ****P < 0.0001 in orange between NM1251 and NM1267 and in purple between NM1251 and NM1268, *P < 0.1 in black between NM1267 and NM1268, by two-way ANOVA with Sidak's multiple comparison test


C Hemizygous K18 - hACE2 mice were treated intranasally with 20 µg of NM1251 ( n = 8 ) , NM1267 ( n = 9 ) or NM1268 ( n = 9 ) seven hours prior to infection with 3 * 103 PFU SARS - CoV - 2 B . 1 . 617 . 2 ( Delta ) . survival were monitored for 14 days . * * * P < 0 . 001 in orange between NM1251 and NM1267 and * * * * P < 0 . 0001 in purple between NM1251 and NM1268 , by log - rank

O O B-GENEPROD O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

C Hemizygous K18-hACE2 mice were treated intranasally with 20 µg of NM1251 (n = 8), NM1267 (n = 9) or NM1268 (n = 9) seven hours prior to infection with 3*103 PFU SARS-CoV-2 B.1.617.2 (Delta). survival were monitored for 14 days. ***P < 0.001 in orange between NM1251 and NM1267 and ****P < 0.0001 in purple between NM1251 and NM1268, by log-rank test


B , C Relative abundance of sgRNAs in cell populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for ( B ) p62 or ( C ) NDP52 . Entire data is reported in Dataset EV1

O O O O O O B-GENEPROD O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O

B, C Relative abundance of sgRNAs in cell populations with high versus low signal were visualized as log2 fold chances for basal as well as activated autophagy conditions for (B) p62 or (C) NDP52. Entire data is reported in Dataset EV1


D - F Validation of TMEM41B . H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non - targeting ( NT ) control , treated with 500 nM AZD8055 , 50nM Bafilomycin A1 ( BafA1 ) or DMSO vehicle control for 24 h , and analyzed by immunoblotting . ( E , F ) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells ( E ) , or as flux by calculating the ratio in BafA1 - treated cells versus vehicle control ( F ) . Data are presented as mean ± SD ( n = 6 independent experiments ) with paired t - test values

O O O O O B-GENEPROD O B-CELL_LINE B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F Validation of TMEM41B. H4 Cas9 cells were infected with sgRNAs targeting TMEM41B or a non-targeting (NT) control, treated with 500 nM AZD8055, 50nM Bafilomycin A1 (BafA1) or DMSO vehicle control for 24 h, and analyzed by immunoblotting. (E, F) p62 and NDP52 band intensities are depicted as total levels relative to vehicle control in H4 Cas9 NT cells (E), or as flux by calculating the ratio in BafA1-treated cells versus vehicle control (F). Data are presented as mean ± SD (n=6 independent experiments) with paired t-test values


G LAMP1 and p62 were co - stained and imaged using an LSM700 confocal microscope . Arrowheads point at p62 - filled and empty LAMP1 - positive organelles . Scale bar : 5 µm

O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O

G LAMP1 and p62 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at p62-filled and empty LAMP1-positive organelles. Scale bar: 5 µm


H LAMP1 - positive organelles were manually quantified for containing a p62 - filled or empty lumen . A total of 50 - 60 LAMP1 - positive organelles were analyzed in each experiment . Data are presented as mean ± SD ( n = 3 independent experiments ) with paired t - test values

O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H LAMP1-positive organelles were manually quantified for containing a p62-filled or empty lumen. A total of 50-60 LAMP1-positive organelles were analyzed in each experiment. Data are presented as mean ± SD (n=3 independent experiments) with paired t-test values


I HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase - expressing S . typhimurium . Luciferase readings were taken up to 8 . 5 h post - infection . Results from one representative experiment are presented as mean ± SD ( n = 8 technical replicates )

O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I HeLa cells were transduced with Cas9 and TMEM41B or NT sgRNAs followed by infection with luciferase-expressing S. typhimurium. Luciferase readings were taken up to 8.5 h post-infection. Results from one representative experiment are presented as mean ± SD (n=8 technical replicates)


A H4 Cas9 cells were infected with sgRNAs targeting ATG7 , TMEM41B or NT control , treated with 500 nM AZD8055 or vehicle control for 24 h , and analyzed by immunoblotting . B Ratio of LC3 - II to LC3 - I band intensities are depicted as mean ± SD ( n = 6 independent experiments ) with Wilcoxon test values

O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O

A H4 Cas9 cells were infected with sgRNAs targeting ATG7, TMEM41B or NT control, treated with 500 nM AZD8055 or vehicle control for 24 h, and analyzed by immunoblotting. B Ratio of LC3-II to LC3-I band intensities are depicted as mean ± SD (n=6 independent experiments) with Wilcoxon test values


C H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm . D The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software ( YAS ) and depicted as mean ± SD ( n = 3 technical replicates )

O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

C H4 Cas9 TMEM41B and NT control cells were probed by LC3B immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. D The number of LC3 puncta per cell was quantified using Yokogawa Analysis Software (YAS) and depicted as mean ± SD (n=3 technical replicates)


E LC3 and WIPI2 were co - stained and imaged using an LSM700 confocal microscope . Arrowheads point at co - localized LC3 and WIPI2 puncta . Scale bar : 5 µm

O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O O

E LC3 and WIPI2 were co-stained and imaged using an LSM700 confocal microscope. Arrowheads point at co-localized LC3 and WIPI2 puncta. Scale bar: 5 µm


F The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD ( n = 5 technical replicates ) . G The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope . Data are depicted as mean ± SD ( n = 3 technical replicates )

O O O O B-GENEPROD B-SUBCELLULAR O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

F The number of WIPI2 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=5 technical replicates). G The total number of WIPI2 puncta per cell was quantified using the Harmony software on images acquired with an automated Operetta microscope. Data are depicted as mean ± SD (n=3 technical replicates)


H H4 Cas9 TMEM41B and NT control cells were infected with a RFP - DFCP1 expression construct for 72h , fixed , stained with LC3 antibodies and imaged with an automated CV7000 confocal microscope . Arrowheads point at co - localized LC3 and RFP - DFCP1 puncta . Scale bar : 5 µm . I The number of RFP - DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD ( n = 10 technical replicates )

O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O

H H4 Cas9 TMEM41B and NT control cells were infected with a RFP-DFCP1 expression construct for 72h, fixed, stained with LC3 antibodies and imaged with an automated CV7000 confocal microscope. Arrowheads point at co-localized LC3 and RFP-DFCP1 puncta. Scale bar: 5 µm. I The number of RFP-DFCP1 puncta positive for LC3 was quantified using ImageJ and depicted as mean ± SD (n=10 technical replicates)


J mCherry - GFP - LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h , fixed and imaged . Scale bar : 20 μm . The number of mCherry - and GFP - positive puncta per cell was quantified using YAS and are depicted as mean ± SD ( n = 3 technical replicates )

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

J mCherry-GFP-LC3 was expressed in H4 Cas9 TMEM41B KO and NT control cells which were treated with 50nM BafA1 or vehicle control for 24 h, fixed and imaged. Scale bar: 20 μm. The number of mCherry- and GFP-positive puncta per cell was quantified using YAS and are depicted as mean ± SD (n=3 technical replicates)


A H4 Cas9 cells stably expressing NT , TMEM41B or ATG7 sgRNAs were stained with BODIPY 493 , NBD cholesterol or BODIPY FL C12 probes for 2 h at 37 ° C and imaged live with an automated CV7000 confocal microscope . Puncta area per cell was quantified using YAS and depicted as mean ± SD ( n = 4 technical replicates ) . Scale bar : 20 μm

O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A H4 Cas9 cells stably expressing NT, TMEM41B or ATG7 sgRNAs were stained with BODIPY 493, NBD cholesterol or BODIPY FL C12 probes for 2 h at 37°C and imaged live with an automated CV7000 confocal microscope. Puncta area per cell was quantified using YAS and depicted as mean ± SD (n=4 technical replicates). Scale bar: 20 μm


B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA - conjugated oleic acid or 0 . 1 % BSA as vehicle control , stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope . Scale bar : 20 μm

O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O

B H4 Cas9 TMEM41B KO and NT control cells were treated overnight with 400 μM BSA-conjugated oleic acid or 0.1% BSA as vehicle control, stained for 2 h with HCS LipidTox Green Neutral Lipid Stain and imaged with an automated Operetta microscope. Scale bar: 20 μm


C Lipid droplet size , number and mean fluorescence intensity was quantified using Harmony software . Data are presented as mean ± SD ( n = 3 technical replicates ) . An average of 1500 cells was analyzed per replicate

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C Lipid droplet size, number and mean fluorescence intensity was quantified using Harmony software. Data are presented as mean ± SD (n=3 technical replicates). An average of 1500 cells was analyzed per replicate


D H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm . E Size of ADRP droplets was quantified with YAS and depicted as mean ± SD ( n = 3 technical replicates )

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D H4 Cas9 TMEM41B KO and NT control cells were probed by ADRP immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm. E Size of ADRP droplets was quantified with YAS and depicted as mean ± SD (n=3 technical replicates)


F Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells . G ADRP band intensities were quantified and depicted as mean ± SD ( n = 4 independent experiments ) with paired t - test values

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F Protein level of ADRP was probed by immunoblotting in H4 Cas9 TMEM41B KO and NT control cells. G ADRP band intensities were quantified and depicted as mean ± SD (n=4 independent experiments) with paired t-test values


A H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12 , chased in complete medium or upon serum - deprivation , and stained with MitoTracker . Cells were imaged live with an automated CV7000 confocal microscope . Scale bar : 20 μm

O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O O O

A H4 Cas9 TMEM41B KO and NT control cells were pulsed with the fatty acid analog Red C12, chased in complete medium or upon serum-deprivation, and stained with MitoTracker. Cells were imaged live with an automated CV7000 confocal microscope. Scale bar: 20 μm


C - E H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96 . ( C ) Basal oxygen consumption rates ( OCR ) and extra cellular acidification rates ( ECAR ) were measured in cells grown in complete medium . OCR / ECAR ratio is also reported . Data are presented as mean ± SEM ( n = 30 - 32 technical replicates ) from one representative experiment

O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E H4 Cas9 TMEM41B KO and NT control cells were plated in XF96 plates and analyzed using a Seahorse Bioscience XF96. (C) Basal oxygen consumption rates (OCR) and extra cellular acidification rates (ECAR) were measured in cells grown in complete medium. OCR/ECAR ratio is also reported. Data are presented as mean ± SEM (n=30-32 technical replicates) from one representative experiment


( D ) OCR was measured in cells grown in substrate - limited medium and treated in the absence or presence of Etomoxir ( Eto ) . Oligomycin ( OA ) , FCCP ( FC ) and a mixture of rotenone and antimycin A ( RA ) was added as indicated . Data are shown as mean ± SEM ( n = 69 technical replicates )

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(D) OCR was measured in cells grown in substrate-limited medium and treated in the absence or presence of Etomoxir (Eto). Oligomycin (OA), FCCP (FC) and a mixture of rotenone and antimycin A (RA) was added as indicated. Data are shown as mean ± SEM (n= 69 technical replicates)


( E ) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto - treated cells from untreated cells . Data are shown as mean ± SEM ( n = 69 technical replicates )

O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

(E) Endogenous FA utilization was calculating by subtracting basal normalized OCR values of Eto-treated cells from untreated cells. Data are shown as mean ± SEM (n= 69 technical replicates)


A H4 Cas9 cells stably expressing N - terminally ( Myc - TMEM41B ) or C - terminally Myc - tagged TMEM41B ( TMEM41B - Myc ) were probed by calnexin ( CANX ) and Myc tag immunostaining and imaged with an automated CV7000 confocal microscope . Scale bar : 20 μm

O O B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O

A H4 Cas9 cells stably expressing N-terminally (Myc-TMEM41B) or C-terminally Myc-tagged TMEM41B (TMEM41B-Myc) were probed by calnexin (CANX) and Myc tag immunostaining and imaged with an automated CV7000 confocal microscope. Scale bar: 20 μm


B - D Endogenous TMEM41B locus was tagged with C - terminal Myc using CRISPR - directed homologous recombination in HeLa cells . HeLa knock - in ( KI ) cells were compared to parental HeLa ( - ) and HeLa cells stably transduced with a TMEM41B - Myc over - expression ( OE ) construct . ( B ) Confocal micrographs of cells stained with antibodies against CANX and Myc confirming ER localization of both KI and OE TMEM41B - Myc . Scale bar represents 20 μm

O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O

B-D Endogenous TMEM41B locus was tagged with C-terminal Myc using CRISPR-directed homologous recombination in HeLa cells. HeLa knock-in (KI) cells were compared to parental HeLa (-) and HeLa cells stably transduced with a TMEM41B-Myc over-expression (OE) construct. (B) Confocal micrographs of cells stained with antibodies against CANX and Myc confirming ER localization of both KI and OE TMEM41B-Myc. Scale bar represents 20 μm


μm . ( C ) Immunoblot analysis confirming expression of both KI and OE TMEM41B -

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μm. (C) Immunoblot analysis confirming expression of both KI and OE TMEM41B-Myc


( D ) PCR analysis with primers binding to TMEM41B last intron and to Myc tag confirming successful targeting of the endogenous TMEM41B locus with Myc ( band labeled with asterisk ) . M = size

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

(D) PCR analysis with primers binding to TMEM41B last intron and to Myc tag confirming successful targeting of the endogenous TMEM41B locus with Myc (band labeled with asterisk). M=size marker


E H4 Cas9 cells and H4 Cas9 cells stably expressing Myc - TMEM41B or TMEM41B - Myc were lysed and subjected to anti - Myc IP . Eluates were analyzed by mass spectrometry . Enrichment of proteins in IPs from Myc - TMEM41B or TMEM41B - Myc cells versus H4 Cas9 cells is depicted as log2 fold changes . Entire data is reported in Dataset EV2

O O B-CELL_LINE O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O

E H4 Cas9 cells and H4 Cas9 cells stably expressing Myc-TMEM41B or TMEM41B-Myc were lysed and subjected to anti-Myc IP. Eluates were analyzed by mass spectrometry. Enrichment of proteins in IPs from Myc-TMEM41B or TMEM41B-Myc cells versus H4 Cas9 cells is depicted as log2 fold changes. Entire data is reported in Dataset EV2


( a ) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis . The increase in cell number following each mitosis is shown by an overlaid curve ( in orange ; mean and s . d . of n = 3 ) .

O O O B-EXP_ASSAY O O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Synchronization dynamics of a population of HeLa cells within almost 3 complete cell cycles measured via PI staining followed by FACS analysis. The increase in cell number following each mitosis is shown by an overlaid curve (in orange; mean and s.d. of n=3).


( b ) Computational modelling of the synchronization loss , considering 11 % cell - cell variation in doubling time , shows that the simulated fraction of the cells in G1 , S , and G2 / M in the synchronized cells throughout the cell cycle ( straight lines ) match experimental measurements ( marked with asterisk ) .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Computational modelling of the synchronization loss, considering 11% cell-cell variation in doubling time, shows that the simulated fraction of the cells in G1, S, and G2/M in the synchronized cells throughout the cell cycle (straight lines) match experimental measurements (marked with asterisk).


( c ) The measured average cell volume in the synchronized cell population ( red ; mean and s . d . of n = 3 ; in Equation 6 ) , the deconvoluted signal ( in case of no synchronization loss ; green ; in Equation 6 ) , and the simulated average cell volume considering the loss in synchronization ( black ; matching the measured concentration data ; Equation 6 ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O

(c) The measured average cell volume in the synchronized cell population (red; mean and s.d. of n=3; in Equation 6), the deconvoluted signal (in case of no synchronization loss; green; in Equation 6), and the simulated average cell volume considering the loss in synchronization (black; matching the measured concentration data; Equation 6).


( d ) The measured concentration of CTP in synchronized cells shown in red ( mean and s . d . of n = 5 ; ) in Equation 7 ; the deconvoluted concentration dynamics , in case of no synchronization loss ( green ; in Equation 7 ) ; and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization , matching the measured concentrations ( black ; Equation 7 ) . The measured concentrations converge towards the steady - state concentrations measured in non - synchronized cells ( horizontal blue line ) .

O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O 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-EXP_ASSAY 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-EXP_ASSAY O O 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-EXP_ASSAY O O O O O O O

(d) The measured concentration of CTP in synchronized cells shown in red (mean and s.d. of n=5; ) in Equation 7; the deconvoluted concentration dynamics, in case of no synchronization loss (green; in Equation 7); and the expected concentration dynamics based on the deconvoluted concentrations and considering the loss in synchronization, matching the measured concentrations (black; Equation 7). The measured concentrations converge towards the steady-state concentrations measured in non-synchronized cells (horizontal blue line).


Figure 2 : Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells . The figure shows metabolites found to significantly oscillate throughout the cell cycle ( concentrations normalized per metabolite ; maximal concentration in red ; minimal concentration in blue ) . The amplitude of the oscillations is shown on the right . Metabolites are color - coded according to metabolic pathways : Energy / redox cofactors ( purple ) ; amino acids ( blue ) ; glycolytic metabolites ( red ) ; TCA cycle metabolites ( green ) .

O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O

Figure 2: Oscillation in metabolite concentrations throughout the cell cycle in HeLa cells. The figure shows metabolites found to significantly oscillate throughout the cell cycle (concentrations normalized per metabolite; maximal concentration in red; minimal concentration in blue). The amplitude of the oscillations is shown on the right. Metabolites are color-coded according to metabolic pathways: Energy/redox cofactors (purple); amino acids (blue); glycolytic metabolites (red); TCA cycle metabolites (green).


( c - e ) Measured relative fraction of the m + 2 labeling of TCA cycle intermediates after feeding [ U - 13C ] - glucose ( red ; mean and s . d . of n = 3 ) , the deconvoluted signal ( green ) , and the expected labeling dynamics considering the loss in synchronization ( black ; representing TCA cycle oxidation of glucose - derived acetyl - CoA ) .

O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O 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-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(c-e) Measured relative fraction of the m+2 labeling of TCA cycle intermediates after feeding [U-13C]-glucose (red; mean and s.d. of n=3), the deconvoluted signal (green), and the expected labeling dynamics considering the loss in synchronization (black; representing TCA cycle oxidation of glucose-derived acetyl-CoA).


( f ) Oscillations in citrate m + 4 labeling after feeding [ U - 13C ] - glutamine ( mean and s . d . of n = 3 ) .

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(f) Oscillations in citrate m+4 labeling after feeding [U-13C]-glutamine (mean and s.d. of n=3).


( g ) Oscillations in the total citrate concentration throughout the cell cycle ( mean and s . d . of n = 5 ) .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(g) Oscillations in the total citrate concentration throughout the cell cycle (mean and s.d. of n=5).


( h ) The measured lactate secretion flux in synchronized cells shown in red ( mean and s . d . of n = 5 ; in Equation 9 ; ) ; the deconvoluted secretion flux dynamics , in case of no synchronization loss ( green ; in Equation 10 ) ; and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization , matching the measured fluxes ( black ; Equation 10 ) .

O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O 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-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O O O O

(h) The measured lactate secretion flux in synchronized cells shown in red (mean and s.d. of n=5; in Equation 9;); the deconvoluted secretion flux dynamics, in case of no synchronization loss (green; in Equation 10); and the expected secretion flux based on the deconvoluted fluxes and considering the loss in synchronization, matching the measured fluxes (black; Equation 10).


Oscillations in aspartate ( a ) concentrations throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O

Oscillations in aspartate (a)concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


Oscillations in malate ( b ) concentrations throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing oxidative TCA cycle activity ) .

Oscillations in malate (b) concentrations throughout the cell cycle when feeding [U-13C]-glutamine (representing oxidative TCA cycle activity).


( c ) Uniform malate m + 4 labeling throughout the cell cycle ( combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase ) .

O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(c) Uniform malate m+4 labeling throughout the cell cycle (combined with the increase in malate concentration in S phase representing increased oxidative TCA cycle flux in S phase).


( d ) Oscillations in pyrimidines m + 3 labeling throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing de novo pyrimidine biosynthesis ) .

O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O

(d) Oscillations in pyrimidines m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing de novo pyrimidine biosynthesis).


( e ) Oscillations in lactate m + 3 labeling throughout the cell cycle when feeding [ U - 13C ] - glutamine ( representing malic enzyme activity ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O

(e) Oscillations in lactate m+3 labeling throughout the cell cycle when feeding [U-13C]-glutamine (representing malic enzyme activity).


( f ) Oscillations in malate m + 3 throughout the cell cycle when feeding [ U - 13C ] - glucose ( representing pyruvate carboxylase activity ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O

(f) Oscillations in malate m+3 throughout the cell cycle when feeding [U-13C]-glucose (representing pyruvate carboxylase activity).


( g ) Oscillations in citrate m + 5 when feeding [ U - 13C ] - glutamine throughout the cell cycle ( representing reductive IDH flux ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O

(g) Oscillations in citrate m+5 when feeding [U-13C]-glutamine throughout the cell cycle (representing reductive IDH flux).


( h ) Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glucose , representing oxidative glucose metabolism .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O

(h) Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glucose, representing oxidative glucose metabolism.


Oscillations in acetyl - CoA m + 2 when feeding [ U - 13C ] - glutamine , representing reductive glutamine metabolism ( i ) . For measurements of metabolite concentrations ( a - b ) , red marks represent mean and s . d . of n = 5 . For measurements of fractional isotopic labeling ( c - i ) , red marks represent mean and s . d . of n = 3 .

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Oscillations in acetyl-CoA m+2 when feeding [U-13C]-glutamine, representing reductive glutamine metabolism (i). For measurements of metabolite concentrations (a-b), red marks represent mean and s.d. of n=5. For measurements of fractional isotopic labeling (c-i), red marks represent mean and s.d. of n=3.


Figure 5 : Complementary oscillations of glucose versus glutamine - derived fluxes in TCA cycle . ( a - i ) Oscillations in metabolic flux throughout the cell cycle ( in mM / h ) , computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling ( red and green marks represent optimal estimates of transient flux with 95 % c . i . ) . Blue lines represent average fluxes inferred in a non - synchronized cell population . As shown , glucose - derived flux into TCA cycle peak in late G1 phase , while oxidative and reductive glutamine metabolism dominates S phase .

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Figure 5: Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle. (a-i) Oscillations in metabolic flux throughout the cell cycle (in mM/h), computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling (red and green marks represent optimal estimates of transient flux with 95% c.i.). Blue lines represent average fluxes inferred in a non-synchronized cell population. As shown, glucose-derived flux into TCA cycle peak in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase.


( a - b ) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m + 2 ( a ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in citrate m+2 (a) from isotopic glucose (representing glycolytic flux into TCA cycle).


( a - b ) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl - CoA m + 2 ( b ) from isotopic glucose ( representing glycolytic flux into TCA cycle ) .

O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(a-b) One hour treatment of synchronized cells with DCA inhibits the oscillations in acetyl-CoA m+2 (b) from isotopic glucose (representing glycolytic flux into TCA cycle).


It further inhibits oscillations in citrate m + 5 / m + 4 ratio ( c ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

It further inhibits oscillations in citrate m+5/m+4 ratio (c) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


It further inhibits oscillations in acetyl - CoA m + 2 ( d ) from isotopic glutamine ( representing oxidative versus reductive TCA cycle flux ) .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

It further inhibits oscillations in acetyl-CoA m+2 (d) from isotopic glutamine (representing oxidative versus reductive TCA cycle flux).


( e ) The fraction of cells in G1 , S , and G2 / M phases in synchronized HeLa cells after 3 hour treatment with DCA ( normalized by measurements in untreated control cells ) .

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(e) The fraction of cells in G1, S, and G2/M phases in synchronized HeLa cells after 3 hour treatment with DCA (normalized by measurements in untreated control cells).


( f ) The fraction of cells in G1 , S , and G2 / M phases in non - synchronized HeLa and HCT - 116 cells after 24 hour treatment with DCA ( normalized by measurements in untreated control cells ) . As shown , DCA significantly increases the fraction of cells in S phase , inhibiting cellular progression into G2 phase ; showing mean and s . d . of n = 3 for all isotopic labeling forms and FACS measurements ( a - f ) . Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two - tailed , unequal variance t - test ( e - f ) .

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(f) The fraction of cells in G1, S, and G2/M phases in non-synchronized HeLa and HCT-116 cells after 24 hour treatment with DCA (normalized by measurements in untreated control cells). As shown, DCA significantly increases the fraction of cells in S phase, inhibiting cellular progression into G2 phase; showing mean and s.d. of n=3 for all isotopic labeling forms and FACS measurements (a-f). Statistical significance of changes in the fraction of cells in each cell cycle phase following DCA treatment is calculated based on two-tailed, unequal variance t-test (e-f).


B - D BSP amyloid aggregation in vitro is pH - dependent in 10 mM EDTA ( B ) , protein concentration - dependent in 10 mM EDTA and at pH 4 . 5 ( C ) and strongly inhibited by millimolar Ca2 + at various protein concentrations ( D ) .

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B-D BSP amyloid aggregation in vitro is pH-dependent in 10 mM EDTA (B), protein concentration-dependent in 10 mM EDTA and at pH 4.5 (C) and strongly inhibited by millimolar Ca2+ at various protein concentrations (D).


E - H TEM images showing fibril morphology of BSP12 ( E ) , BSP123 ( F ) , BSP3 ( G ) , BSP45 ( H ) .

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E-H TEM images showing fibril morphology of BSP12 (E), BSP123 (F), BSP3 (G), BSP45 (H).


I - J Amyloid aggregation by BSP subconstructs . BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4 . 5 ( I ) . The conjunction of module 3 with modules 1 and 2 promotes aggregation ( J ) .

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I-J Amyloid aggregation by BSP subconstructs. BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4.5 (I). The conjunction of module 3 with modules 1 and 2 promotes aggregation (J).


B Representative fluorescence microscopy images of BSP and its subconstructs ( 20 μM ) at varying pHs . PEG 8K concentration is 2 % . Scale bar , 20 μm .

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B Representative fluorescence microscopy images of BSP and its subconstructs (20 μM) at varying pHs. PEG 8K concentration is 2 %. Scale bar, 20 μm.


E Quantification of droplet area is shown for each protein under various Ca2 + concentration conditions . Each sample is quantified by total area ( left ) and mean area ( right ) . Each experiment was repeated three times , data shown as mean ± SD .

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E Quantification of droplet area is shown for each protein under various Ca2+ concentration conditions. Each sample is quantified by total area (left) and mean area (right). Each experiment was repeated three times, data shown as mean ± SD.


( A ) Identified acetylated histone peptides plotted against their log2 SILAC ratio ( 1h after 16 J / m2 UV / mock ) , ranked by SILAC

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY

(A) Identified acetylated histone peptides plotted against their log2 SILAC ratio (1h after 16 J/m2 UV/mock), ranked by SILAC ratio


( C ) Western blot of acid extracted histones and WCE from HeLa cells , treated with HATi ( CTK7A , 100 μM and CPTH2 , 50 μM ) or HDACi ( TSA , 1 μM ) for 4h or mock treated as

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated


( D ) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV - irradiation ( 16 J / m2 ) . Blots were stained with α - acetyl - lysine ( top panels , high and low exposure ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel )

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(D) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV-irradiation (16 J/m2). Blots were stained with α-acetyl-lysine (top panels, high and low exposure), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( F ) Representative western blots of HeLa WCE from different time points after UV - C irradiation ( 16J / m2 ) using the indicated histone modification specific antibodies . Histone H4 and tubulin were used as loading

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O

(F) Representative western blots of HeLa WCE from different time points after UV-C irradiation (16J/m2) using the indicated histone modification specific antibodies. Histone H4 and tubulin were used as loading controls


( A ) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation ( 16 J / m2 , left panel ) , α - amanitin ( second panel , 100 µg / ml ) , THZ1 ( third panel , 1 μM ) or flavopiridol ( right panel , 1 μM ) treatment . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel )

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(A) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation (16 J/m2, left panel), α-amanitin (second panel, 100 µg/ml), THZ1 (third panel, 1 μM) or flavopiridol (right panel, 1 μM) treatment. Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative western blots of histone acetylation levels of HeLa cells pre - treated with transcription inhibitors ( mock ( left ) , THZ1 ( middle , 1 μM ) or flavopiridol ( right , 1 μM ) an hour before UV irradiation and lysed at the indicated time points after UV ( 16J / m2 ) . Blots were stained with the indicated

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative western blots of histone acetylation levels of HeLa cells pre-treated with transcription inhibitors (mock (left), THZ1 (middle, 1 μM) or flavopiridol (right, 1 μM) an hour before UV irradiation and lysed at the indicated time points after UV (16J/m2). Blots were stained with the indicated antibodies


( A ) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation ( 16 J / m2 ) , HU / AraC treatment ( 100 mM / 10 μM ) or MMC treatment ( 10 µg / ml ) . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel )

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(A) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation (16 J/m2), HU/AraC treatment (100 mM/10 μM) or MMC treatment (10 µg/ml). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative Western blots , stained with the indicated antibodies of HeLa cells pre - treated o / n with an S - phase inhibitor ( PHA 767491 hydrochloride , 10 μM ) and lysed at indicated time points after UV ( 16 J / m2 )

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(C) Representative Western blots, stained with the indicated antibodies of HeLa cells pre-treated o/n with an S-phase inhibitor (PHA 767491 hydrochloride, 10 μM) and lysed at indicated time points after UV (16 J/m2)


A Treatment scheme of experimental timeline and Immunofluorescence staining of telogen backskin hair follicles ( HF ) for Integrin α6 ( Itgα6 , red ) , active Caspase 3 ( aCas3 , green ) and DAPI ( blue ) in control ( Ctr ) and Bcl - 2 inhibitor ( ABT - 199 ) treated mice . B : bulge , CH : club hair , DP : dermal papilla , HF : hair follicle , HG : secondary hair germ . Scale bar , 50 µm B Quantification of backskin HF with aCas3 + cells in bulge , hair germ and dermal papilla of Ctr and ABT - 199 treated mice ( n = 4 mice biological replicates ; mean ± standard deviation ( SD ) ; * * * p < 0 . 001 , ns = not significant , two - way ANOVA test ) . nd = not detected .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Treatment scheme of experimental timeline and Immunofluorescence staining of telogen backskin hair follicles (HF) for Integrin α6 (Itgα6, red), active Caspase 3 (aCas3, green) and DAPI (blue) in control (Ctr) and Bcl-2 inhibitor (ABT-199) treated mice. B: bulge, CH: club hair, DP: dermal papilla, HF: hair follicle, HG: secondary hair germ. Scale bar, 50 µm B Quantification of backskin HF with aCas3+ cells in bulge, hair germ and dermal papilla of Ctr and ABT-199 treated mice (n=4 mice biological replicates; mean ± standard deviation (SD); *** p<0.001, ns=not significant, two-way ANOVA test). nd=not detected.


C , D Treatment scheme , FACS plot ( C ) and quantification ( D ) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution ( Ctr ) or ABT - 199 ( n = 6 biological replicates ; mean ± standard deviation ( SD ) ; * * p < 0 . 01 , ns = not significant , two - tailed unpaired Student ' s t - test ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs and n - B ( non - bulge epidermal cells ) . * * p < 0 . 005 .

O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-TISSUE O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O

C,D Treatment scheme, FACS plot (C) and quantification (D) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution (Ctr) or ABT-199 (n=6 biological replicates; mean ± standard deviation (SD); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs and n-B (non-bulge epidermal cells). ** p<0.005.


B Treatment scheme of experimental timeline and histology of backskin sections of control ( Ctr ) and ABT - 199 treated mice . Scale bar , 200 µm .

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B Treatment scheme of experimental timeline and histology of backskin sections of control (Ctr) and ABT-199 treated mice. Scale bar, 200 µm.


D qRT - PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT - 199 treated and control ( Ctr ) mice at P77 ( n = 3 biological replicates ; mean ± standard error of mean ( SEM ) ; * * p < 0 . 01 , ns = not significant , two - tailed unpaired Student ' s t - test ) .

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D qRT-PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT-199 treated and control (Ctr) mice at P77 (n=3 biological replicates; mean ± standard error of mean (SEM); ** p<0.01, ns=not significant, two-tailed unpaired Student's t-test).


C qRT - PCR for mRNA expression of the anoikis - related markers Bim and Bmf in sorted n - B ( non - bulge ) , bBSC ( basal BSCs ) and sbBSC ( suprabasal BSCs ) ( n = 3 biological replicates ; mean ± standard error of mean ( SEM ) ; * p < 0 . 05 , ns = not significant , two - way ANOVA test ) .

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C qRT-PCR for mRNA expression of the anoikis-related markers Bim and Bmf in sorted n-B (non-bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs) (n=3 biological replicates; mean ± standard error of mean (SEM); * p<0.05, ns=not significant, two-way ANOVA test).


C FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8 week old control ( Ctr ) and Bcl - 2EOE mice ( n = 3 biological replicates ; mean ± standard deviation ( SD ) ; ns = not significant , two - tailed unpaired Student ' s t - test ) . suprabasal ( sbBSC , CD34 + / Itgα6low ) , basal ( bBSC , CD34 + / Itgα6high ) BSCs .

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C FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8 week old control (Ctr) and Bcl-2EOE mice (n=3 biological replicates; mean ± standard deviation (SD); ns=not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs.


D Immunofluorescence staining of telogen backskin HF for BrdU incorporation ( 1 h pulse , green ) and DAPI ( blue ) in 8 week old control ( Ctr ) and Bcl - 2EOE mice . Scale bar , 50 µm .

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D Immunofluorescence staining of telogen backskin HF for BrdU incorporation (1 h pulse, green) and DAPI (blue) in 8 week old control (Ctr) and Bcl-2EOE mice. Scale bar, 50 µm.


A Histology of P19 catagen backskin HF of control ( Ctr ) and Bcl - 2EOE mice . RS : retracting strand , Scale bar , 50 µm . B Analysis of length and width of retracting strands of control ( Ctr ) and Bcl - 2EOE mice ( n = 4 ; multiple measurements / biological replicate ; whiskers : min and max ; boxes : median ± first and third quartile ; * * * * p < 0 . 0001 , two - tailed unpaired Student ' s t - test ) .

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A Histology of P19 catagen backskin HF of control (Ctr) and Bcl-2EOE mice. RS: retracting strand, Scale bar, 50 µm. B Analysis of length and width of retracting strands of control (Ctr) and Bcl-2EOE mice (n=4; multiple measurements/biological replicate; whiskers: min and max; boxes: median ± first and third quartile; **** p<0.0001, two-tailed unpaired Student's t-test).


C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control ( Ctr ) and Bcl - 2EOE mice ( n = 4 biological replicates ) . Dashed lines : retracting strands . Scale bar , 100 µm . D Quantification of aCas3 + cells in the RS of P19 Ctr and Bcl - 2EOE mice ( n = 4 biological replicates ; mean ± standard deviation ( SD ) ; * * * * p < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . HF : hair follicle .

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C Itgα6 and aCas3 Immunofluorescence staining of catagen tailskin HF of control (Ctr) and Bcl-2EOE mice (n=4 biological replicates). Dashed lines: retracting strands. Scale bar, 100 µm. D Quantification of aCas3+ cells in the RS of P19 Ctr and Bcl-2EOE mice (n=4 biological replicates; mean ± standard deviation (SD); **** p<0.0001, two-tailed unpaired Student's t-test). HF: hair follicle.


F , G qRT - PCR for mRNA expression of the quiescence - related markers Bmp6 ( F ) and Fgf18 ( G ) in epidermis of control ( Ctr ) and Bcl - 2EOE mice ( P27 ) , ( n = 4 biological replicates ; mean ± standard error of mean ( SEM ) ; * p < 0 . 05 , * * * p < 0 . 001 , two - tailed unpaired Student ' s t - test ) .

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F,G qRT-PCR for mRNA expression of the quiescence-related markers Bmp6 (F) and Fgf18 (G) in epidermis of control (Ctr) and Bcl-2EOE mice (P27), (n=4 biological replicates; mean ± standard error of mean (SEM); * p<0.05, *** p<0.001, two-tailed unpaired Student's t-test).


B Immunofluorescence staining of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , activated Caspase 3 ( aCas3 , red ) and DAPI ( blue ) . Note increased number of aCas + cells in the bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous gland . Scale bar , 100µm .

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O

B Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), activated Caspase 3 (aCas3, red) and DAPI (blue). Note increased number of aCas+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm.


D Immunofluorescence staining of epidermal tail whole mounts of control ( Ctr ) , K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice , stained for keratin 15 ( Krt15 , green ) , BrdU ( 1 h pulse , red ) and DAPI ( blue ) . Note increased number of BrdU + cells in the bulge of K15ΔNLef1 mice ( arrows ) . B : bulge , HG : secondary hair germ , SG : sebaceous gland . Scale bar ,

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O B-ORGANISM O O O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O

D Immunofluorescence staining of epidermal tail whole mounts of control (Ctr), K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice, stained for keratin 15 (Krt15, green), BrdU (1 h pulse, red) and DAPI (blue). Note increased number of BrdU+ cells in the bulge of K15ΔNLef1 mice (arrows). B: bulge, HG: secondary hair germ, SG: sebaceous gland. Scale bar, 100µm


B , C Tumour incidence ( B ) and frequency ( C ) in Ctr , Bcl - 2EOE , K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice ( n = 10 biological replicates ; mean ± standard error of mean ( SEM ) ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O

B,C Tumour incidence (B) and frequency (C) in Ctr, Bcl-2EOE, K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice (n=10 biological replicates; mean ± standard error of mean (SEM)).


D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1 ; Bcl - 2EOE mice . Scale bar , 100µm .

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D Histology of sebaceous tumours of K15ΔNLef1 and K15ΔNLef1;Bcl-2EOE mice. Scale bar, 100µm.


A ) Hek293T cells expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) and schematic of BRET assay , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm . B ) Lamplight - driven response to increasing intensity 1s 405nm light C ) 405nm irradiance response curve D ) Response to 1s 405nm ( blue line ) and 525nm light ( green line ) E ) Response to 405nm ( blue line ) followed by 525nm light of different intensities ( green line )

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A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm. B) Lamplight-driven response to increasing intensity 1s 405nm light C) 405nm irradiance response curve D) Response to 1s 405nm (blue line) and 525nm light (green line) E) Response to 405nm (blue line) followed by 525nm light of different intensities (green line)


A ) Hek293T cells expressing Lamplight - 1D4 ( anti - 1D4 = green , DAPI = blue , Top ) and schematic of BRET assay , measuring interaction of liberated Gβγ - Venus with GRK3 fragment - Nanoluciferase ( Bottom ) . Scale bar = 20µm .

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A) Hek293T cells expressing Lamplight-1D4 (anti-1D4 = green, DAPI = blue, Top) and schematic of BRET assay, measuring interaction of liberated Gβγ-Venus with GRK3 fragment-Nanoluciferase (Bottom). Scale bar = 20µm.


G ) Response to 405nm alone or with 525nm

G) Response to 405nm alone or with 525nm light


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) dark or ( I ) after 8s 405nm light ( blue bar ) J ) Sustained Lamplight - driven activity ( mean BRET ratio 20 - 80s after stimulus ) after exposure to mixed stimuli from dark ( ON ) or 405nm light - adapted state ( OFF ) . Lamplight data fit with linear trendline . Best fitting parameters for ON , slope = 0 . 311 , Y - intercept = 0 . 04 , R2 = 0 . 85 . For OFF , slope = 0 . 30 , Y - intercept = 0 . 07 , R2 = 0 . 69 ) .

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H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar) J) Sustained Lamplight-driven activity (mean BRET ratio 20-80s after stimulus) after exposure to mixed stimuli from dark (ON) or 405nm light-adapted state (OFF). Lamplight data fit with linear trendline. Best fitting parameters for ON, slope = 0.311, Y-intercept = 0.04, R2 = 0.85. For OFF, slope = 0.30, Y-intercept = 0.07, R2 = 0.69).


H - I ) Representative time courses of Lamplight - driven responses to 8s mixed stimuli ( grey bar ) with different 405nm : 525nm ratios from 1 ( 100 % 405nm ) to 0 ( 100 % 525nm ) from ( H ) dark or ( I ) after 8s 405nm light ( blue bar )

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H-I) Representative time courses of Lamplight-driven responses to 8s mixed stimuli (grey bar) with different 405nm: 525nm ratios from 1 (100% 405nm) to 0 (100% 525nm) from (H) dark or (I) after 8s 405nm light (blue bar)


B ) Representative trace of hyperpolarisation to 405nm light in a Lamplight - expressing SCN neuron C ) Hyperpolarisation to 405nm was observed over a range of intensities . Recordings from 3 different cells across 2 SCN slices

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B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron C) Hyperpolarisation to 405nm was observed over a range of intensities. Recordings from 3 different cells across 2 SCN slices


B ) Representative trace of hyperpolarisation to 405nm light in a Lamplight - expressing SCN

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B) Representative trace of hyperpolarisation to 405nm light in a Lamplight-expressing SCN neuron


D ) Representative trace showing hyperpolarisation was sustained during dark after 405nm light ( 5s ) and reversed by 525nm light ( 15s )

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D) Representative trace showing hyperpolarisation was sustained during dark after 405nm light (5s) and reversed by 525nm light (15s)


E ) Hyperpolarisation and depolarisation of a Lamplight - expressing neuron by 405nm and 525nm light , respectively , across multiple stimulus presentations

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E) Hyperpolarisation and depolarisation of a Lamplight-expressing neuron by 405nm and 525nm light, respectively, across multiple stimulus presentations


E ) Hyperpolarisation and depolarisation of a Lamplight - expressing neuron by 405nm and 525nm light , respectively , across multiple stimulus

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F ) Three example traces from a single cell exposed to 405nm followed by 525nm light . Individual traces were fit with exponential decay ( 405nm , blue ) or association curves ( 525nm , green ) for 6 trials across 2 cells

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F) Three example traces from a single cell exposed to 405nm followed by 525nm light. Individual traces were fit with exponential decay (405nm, blue) or association curves (525nm, green) for 6 trials across 2 cells


F ) Three example traces from a single cell exposed to 405nm followed by 525nm light . Individual traces were fit with exponential decay ( 405nm , blue ) or association curves ( 525nm , green ) for 6 trials across 2

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G , H ) Parameters from best - fit curves used to calculate ( G ) latency ( ms ) and ( H ) half - life ( ms ) .

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G, H) Parameters from best-fit curves used to calculate (G) latency (ms) and (H) half-life (ms).