F , G Staining for EdU ( red ) and Ki67 ( green ) in control ( F ) and Gpr124KO ( G ) cortices at E13 . 5 , 24 hours after EdU injection . H , Quantification of Ki67 + NPCs in the VZ ( blue ) , SVZ / CP ( grey ) and of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control and Gpr124KO embryos ( mean ± SEM ; N = 4 ( control ) and N = 3 ( Gpr124KO ) ; * p < 0 . 05 , * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F,G Staining for EdU (red) and Ki67 (green) in control (F) and Gpr124KO (G) cortices at E13.5, 24 hours after EdU injection. H, Quantification of Ki67+ NPCs in the VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124KO embryos (mean±SEM; N=4 (control) and N=3 (Gpr124KO); * p<0.05, ** 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


I , J , Immunostaining for Tbr2 ( red ) and Pax6 ( green ) in control ( I ) and Gpr124KO ( J ) cortices . K , Quantification of newborn BPs in the VZ of control and Gpr124KO cortices ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,J, Immunostaining for Tbr2 (red) and Pax6 (green ) in control (I) and Gpr124KO (J) cortices. K, Quantification of newborn BPs in the VZ of control and Gpr124KO cortices (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


L , M , Immunostaining for Tbr2 ( red ) and Ngn1 ( green ) in control ( L ) and Gpr124KO ( M ) cortices . N , O Quantification of neurogenic ( Ngn1 + ) RGs and BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control and Gpr124KO cortices ( mean ± SEM ; N = 4 ; * p < 0 . 05 , * * * p < 0 . 001 ) . Note that the fraction of total BPs is significantly reduced ( Fig 1K ) while the fraction of Ngn1 + BPs is insignificantly reduced ( Fig 1N ) , because the proportion of Ngn1 + Tbr2 + cells within all BPs is slightly , though statisically insignificantly higher in the Gpr124KObrains ( see Fig S2M ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L,M, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (L) and Gpr124KO (M) cortices. N,O Quantification of neurogenic (Ngn1+) RGs and BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05, *** p<0.001). Note that the fraction of total BPs is significantly reduced (Fig 1K) while the fraction of Ngn1+BPs is insignificantly reduced (Fig 1N), because the proportion of Ngn1+Tbr2+ cells within all BPs is slightly, though statisically insignificantly higher in the Gpr124KObrains (see Fig S2M). 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


P , Q Stainings for Tbr2 ( red ) and for Tis21 - GFP expression ( green ) in control ( P ) and Gpr124KO ( Q ) cortices . R , S Quantification of neurogenic ( Tis21 - GFP + ) RGs and BPs ( R ) or expanding ( Tbr2 - Tis21 - GFP - ) RGs ( S ) in control and Gpr124KO cortices ( mean ± SEM ; N = 4 ; * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

P,Q Stainings for Tbr2 (red) and for Tis21-GFP expression (green) in control (P) and Gpr124KO (Q) cortices. R,S Quantification of neurogenic (Tis21-GFP+) RGs and BPs (R) or expanding (Tbr2- Tis21-GFP-) RGs (S) in control and Gpr124KO cortices (mean±SEM; N=4; *** 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 , B , Staining for Isolectin B4 + blood vessels in control ( A ) and Gpr124ECcKO ( B ) forebrains . Asterisks mark the lateral ventricles .

O O O O O 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,B, Staining for Isolectin B4+ blood vessels in control (A) and Gpr124ECcKO (B) forebrains. Asterisks mark the lateral ventricles.


C , D , Staining for pimonidazole ( PIMO , green ) and Isolectin B4 + blood vessels ( red ) in ctrl ( C ) and Gpr124ECcKO ( D ) cortices .

O O O O O O O O O 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,D, Staining for pimonidazole (PIMO, green) and Isolectin B4+ blood vessels (red) in ctrl (C) and Gpr124ECcKO (D) cortices.


E , F , Immunostaining for Pax6 in control ( E ) and Gpr124ECcKO ( F ) forebrain sections to reveal the lateral extension of the neocortex ( dashed line ) between the cortical hem ( arrowhead ) and the lateral ganglionic eminence ( arrow ) . G , Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains ( mean ± SEM ; N = 6 for ctrl and N = 8 for Gpr124ECcKO ; * p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,F, Immunostaining for Pax6 in control (E) and Gpr124ECcKO (F) forebrain sections to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). G, Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains (mean±SEM; N=6 for ctrl and N=8 for Gpr124ECcKO; * p<0.05).


H , I , Staining for EdU ( red ) and Ki67 ( green ) in control ( H ) and Gpr124ECcKO ( I ) cortices at E13 . 5 , 24 hours after EdU injection . J , 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 Gpr124ECcKO embryos ( mean ± SEM ; N = 4 ; * p < 0 . 05 , * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,I, Staining for EdU (red) and Ki67 (green) in control (H) and Gpr124ECcKO (I) cortices at E13.5, 24 hours after EdU injection. J, 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 Gpr124ECcKO embryos (mean±SEM; N=4; * p<0.05, ** 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


K , L , Immunostaining for Tbr2 ( red ) and Ngn1 ( green ) in control ( K ) and Gpr124KO ( L ) cortices . M - O , Quantification of neurogenic ( Ngn1 + ) RGs and BPs ( M ) , newborn BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control and Gpr124ECcKO cortices ( mean ± SEM ; N = 4 ; * p < 0 . 05 , * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K,L, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (K) and Gpr124KO (L) cortices. M-O, Quantification of neurogenic (Ngn1+) RGs and BPs (M), newborn BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124ECcKO cortices (mean±SEM; N=4; * p<0.05, ** 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 NSC - specific markers ( Fabp7 , Hes5 ) and neuronal markers ( Dcx , Tubb3 ) in the Prom1 + fraction , presented as fold change compared to the Prom1 - fraction ) from WT cortices at E14 . 5 ( mean ± SEM ; N = 3 for Dcx ; N = 6 for all other genes ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 NSC-specific markers (Fabp7, Hes5) and neuronal markers (Dcx, Tubb3) in the Prom1+ fraction, presented as fold change compared to the Prom1- fraction) from WT cortices at E14.5 (mean±SEM; N=3 for Dcx; N=6 for all other genes).


F , Western blot for HIF - 1 ( top ) , HIF - 2 ( middle ) and a - tubulin as loading control ( bottom ) using forebrain lysates from E13 . 5 control ( left ) or Gpr124KOembryos ( middle ) . HIF - 1 / 2 - transfected HEK293 cells served as positive control ( right ) . G , Quantification of HIF - 1 band intensities shown in panel F ( mean ± SEM ; N = 3 ; * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Western blot for HIF-1 (top), HIF-2 (middle) and a-tubulin as loading control (bottom) using forebrain lysates from E13.5 control (left) or Gpr124KOembryos (middle). HIF-1/2-transfected HEK293 cells served as positive control (right). G, Quantification of HIF-1 band intensities shown in panel F (mean±SEM; N=3; ** p<0.01).


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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O 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, 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-MEASURED_VAR O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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


( D ) RNF145 and ADIPOR2 respond to a range of FAs . HEK - 293T cells were treated for 6h with saturated FAs ( SFAs ; 400 µM ( C12 : 0 - C18 : 0 ) ; 200 µM ( C20 : 0 ) ) or unsaturated FAs ( UFAs ; 400 µM ( C18 : 1 - n9 ) ; 200 µM ( C20 : 4 - n6 , C20 : 5 - n3 ) ) . * non - specific bands .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

(D) RNF145 and ADIPOR2 respond to a range of FAs. HEK-293T cells were treated for 6h with saturated FAs (SFAs; 400 µM (C12:0 - C18:0); 200 µM (C20:0)) or unsaturated FAs (UFAs; 400 µM (C18:1-n9); 200 µM (C20:4-n6, C20:5-n3)). * non-specific bands.


( E ) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels . HEK - 293T cells were treated with the SCD1 inhibitor MF - 438 ( SCDi , 100 nM , 18h ) and RNF145 / ADIPOR2 expression analysed by immunoblotting . * non - specific bands .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(E) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels. HEK-293T cells were treated with the SCD1 inhibitor MF-438 (SCDi, 100 nM, 18h) and RNF145/ADIPOR2 expression analysed by immunoblotting. * non-specific bands.


( F ) SCD1 overexpression affects RNF145 / ADIPOR2 . HEK - 293T cells containing an empty control vector or expressing SCD1 - HA were cultured in FCS - free DMEM for 24h and whole - cell lysates analysed . * non - specific bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) SCD1 overexpression affects RNF145/ADIPOR2. HEK-293T cells containing an empty control vector or expressing SCD1-HA were cultured in FCS-free DMEM for 24h and whole-cell lysates analysed. * non-specific bands.


( A ) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells . Representative 35S pulse - chase analysis in HeLa WT , RNF145 knockout ( ∆ RNF145 ) or ADIPOR2 knockout ( ∆ AR2 ) cells . Quantification of 35S - labelled ADIPOR2 normalised to t = 0 h is shown below .

(A) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells. Representative 35S pulse-chase analysis in HeLa WT, RNF145 knockout (∆RNF145) or ADIPOR2 knockout (∆AR2) cells. Quantification of 35S-labelled ADIPOR2 normalised to t = 0 h is shown below.


( B ) ADIPOR2 regulation requires the E3 ligase activity of RNF145 . HEK - 293T RNF145 knockout cells ( ∆ RNF145 ) were stably complemented with WT RNF145 ( RNF145 - V5 ) or an inactive RNF145C552A , H554A RING domain mutant ( mRNF145 - V5 ) . * non - specific bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ADIPOR2 regulation requires the E3 ligase activity of RNF145. HEK-293T RNF145 knockout cells (∆RNF145) were stably complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). * non-specific bands.


( C ) RNF145 binds ADIPOR2 . RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 ( RNF145 - V5 ) or an inactive RNF145C552A , H554A RING domain mutant ( mRNF145 - V5 ) . Cells were treated with VCP inhibitor ( NMS - 873 , 10 µM , 90 min ) prior to immunoprecipitation with an anti - V5 antibody .

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(C) RNF145 binds ADIPOR2. RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). Cells were treated with VCP inhibitor (NMS-873, 10 µM, 90 min) prior to immunoprecipitation with an anti-V5 antibody.


( D ) The E2 ubiquitin - conjugating enzyme UBE2G2 is involved in RNF145 - mediated ADIPOR2 degradation . Immunoblot analysis of HEK - 293T cells depleted of RNF145 ( gRNF145 ) , UBE2G2 ( gUBE2G2 ) or B2M ( control ; gCTR ) .

O O O O O O O O O O O O O O O O O O O O O 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) The E2 ubiquitin-conjugating enzyme UBE2G2 is involved in RNF145-mediated ADIPOR2 degradation. Immunoblot analysis of HEK-293T cells depleted of RNF145 (gRNF145), UBE2G2 (gUBE2G2) or B2M (control; gCTR).


( E ) ADIPOR2 degradation depends on UBXD8 . HEK - 293T cells were depleted of B2M ( gCTR ) or UBXD8 ( gUBXD8 ) and analysed by immunoblotting .

O O O O O O 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) ADIPOR2 degradation depends on UBXD8. HEK-293T cells were depleted of B2M (gCTR) or UBXD8 (gUBXD8) and analysed by immunoblotting.


( A ) FA - mediated regulation of RNF145 is proteasome - dependent . HEK - 293T cells were treated with BSA , PA ( 400 µM , 6h ) ± MG132 ( 10 µg / ml , 6h ) and analysed by immunoblotting . * non - specific bands .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(A) FA-mediated regulation of RNF145 is proteasome-dependent. HEK-293T cells were treated with BSA, PA (400 µM, 6h) ± MG132 (10 µg/ml, 6h) and analysed by immunoblotting. *non-specific bands.


( B ) UBXD8 participates in PA - modulated RNF145 degradation . CRISPR / Cas9 - mediated depletion of UBXD8 ( gUBXD8 ) or B2M ( gB2M , control ) in HEK - 293T cells , followed by exposure to PA ( 400 µM , 6h ) or BSA ( 6h , vehicle control ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O

(B) UBXD8 participates in PA-modulated RNF145 degradation. CRISPR/Cas9-mediated depletion of UBXD8 (gUBXD8) or B2M (gB2M, control) in HEK-293T cells, followed by exposure to PA (400 µM, 6h) or BSA (6h, vehicle control).


( C ) PA and OA treatment alter RNF145 ubiquitination . Immunoprecipitation of RNF145 from cells treated with BSA ( control ) , PA or OA ( each 200 µM , 3h ) . During FA treatments , VCP inhibitor ( NMS - 873 , 10 µM ) was present to stabilise ubiquitinated proteins . RNF145 ubiquitination ( ubi ) was detected using an anti - ubiquitin antibody ( VU - 1 ) .

O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O 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-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O

(C) PA and OA treatment alter RNF145 ubiquitination. Immunoprecipitation of RNF145 from cells treated with BSA (control), PA or OA (each 200 µM, 3h). During FA treatments, VCP inhibitor (NMS-873, 10 µM) was present to stabilise ubiquitinated proteins. RNF145 ubiquitination (ubi) was detected using an anti-ubiquitin antibody (VU-1).


( D ) HEK - 293T cells were treated with cycloheximide ( CHX , 1 µg / ml ) and OA ( 400 µM , 6h ) ± MG132 ( 10 µg / ml ) for the indicated times . LE , long exposure ; * non - specific bands .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(D) HEK-293T cells were treated with cycloheximide (CHX, 1 µg/ml) and OA (400 µM, 6h) ± MG132 (10 µg/ml) for the indicated times. LE, long exposure; * non-specific bands.


( E ) Lysine - less RNF145 is stabilised under PA treatment . RNF145 knockout HEK - 293T cells ( ∆ RNF145 ) were complemented with RNF145 - V5 or a lysine - less variant of RNF145 ( RNF145 - V5 K > R ) and treated BSA or PA ( 400 µM , 6h ) . Dotted lines demarcate the different cell lines .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O

(E) Lysine-less RNF145 is stabilised under PA treatment. RNF145 knockout HEK-293T cells (∆RNF145) were complemented with RNF145-V5 or a lysine-less variant of RNF145 (RNF145-V5 K > R) and treated BSA or PA (400 µM, 6h). Dotted lines demarcate the different cell lines.


( F ) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity . Immunoblot of whole - cell lysates from RNF145 knockout HEK - 293T ( ∆ RNF145 ) cells stably complemented with RNF145 - V5 or RNF145C552A , H554A - V5 ( mRNF145 - V5 ) and treated with BSA or PA ( 400 µM , 6h ) . LE , long exposure .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O

(F) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity. Immunoblot of whole-cell lysates from RNF145 knockout HEK-293T (∆RNF145) cells stably complemented with RNF145-V5 or RNF145C552A, H554A-V5 (mRNF145-V5) and treated with BSA or PA (400 µM, 6h). LE, long exposure.


( A ) ADIPOR2 regulation by FAs is completely dependent on RNF145 . WT or RNF145 knockout HEK - 293T cells ( ∆ RNF145 ) were treated with BSA , PA or OA ( 400 µM , 6h ) and analysed by immunoblotting . Non - specific bands are indicated ( * ) .

O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(A) ADIPOR2 regulation by FAs is completely dependent on RNF145. WT or RNF145 knockout HEK-293T cells (∆RNF145) were treated with BSA, PA or OA (400 µM, 6h) and analysed by immunoblotting. Non-specific bands are indicated (*).


( B ) RNF145 - ADIPOR2 interaction is FA - modulated . Immunoprecipitation of endogenous RNF145 HEK - 293T cells depleted of UBE2G2 ( gUBE2G2 ) and / or RNF145 ( gRNF145 ) and treated with 400 µM of the indicated PA , OA or BSA ( control ) for 3h . LE , long exposure . * non - specific bands .

O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(B) RNF145 - ADIPOR2 interaction is FA-modulated. Immunoprecipitation of endogenous RNF145 HEK-293T cells depleted of UBE2G2 (gUBE2G2) and/or RNF145 (gRNF145) and treated with 400 µM of the indicated PA, OA or BSA (control) for 3h. LE, long exposure. * non-specific bands.


( C ) Immunoprecipitation ( IP ) of endogenous ADIPOR2 from WT or ∆ RNF145 cells ( see ( C ) ) treated with MG132 ( 5 µg / ml , 6h ) ± OA ( 200 µM , 6h ) . Ubiquitinated ADIPOR2 was detected using a specific anti - ubiquitin antibody ( clone VU - 1 ) . Note that ADIPOR2 levels in RNF145 knockout HEK - 293T cells are increased .

O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(C) Immunoprecipitation (IP) of endogenous ADIPOR2 from WT or ∆RNF145 cells (see (C)) treated with MG132 (5 µg/ml, 6h) ± OA (200 µM, 6h). Ubiquitinated ADIPOR2 was detected using a specific anti-ubiquitin antibody (clone VU-1). Note that ADIPOR2 levels in RNF145 knockout HEK-293T cells are increased.


( D ) ADIPOR2 knockout HEK - 293T cells were stably complemented with ADIPOR2 - HA , an ADIPOR2 mutant in which all lysines were mutated to arginines ( K - less ADIPOR2 - HA ) , or an empty vector control and exposed to OA ( 200 µM , 6h ) or BSA ( control ) . ADIPOR2 variants were visualised by immunoblotting using a specific anti - HA antibody . LE , long exposure .

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(D) ADIPOR2 knockout HEK-293T cells were stably complemented with ADIPOR2-HA, an ADIPOR2 mutant in which all lysines were mutated to arginines (K-less ADIPOR2-HA), or an empty vector control and exposed to OA (200 µM, 6h) or BSA (control). ADIPOR2 variants were visualised by immunoblotting using a specific anti-HA antibody. LE, long exposure.


( A ) Loss of ADIPOR2 sensitises cells to PA - induced cytotoxicity . HEK - 293T cells were depleted of RNF145 ( purple ) , ADIPOR2 ( blue ) , B2M ( CTR ; grey ) , or both ADIPOR2 and RNF145 ( blue + purple ) , exposed to PA ( 200 µM , 96h ) and stained with crystal violet . Quantification of crystal - violet stained cells is shown alongside a representative micrograph ( below ) . The response of each cell line to PA was normalised to its BSA - treated condition ( dotted line ) . Statistically significant differences to cells containing gCTR cells are indicated ( robust one - way ANOVA with Benjamini - Hochberg multiple testing correction ) . Mean ± SD are shown . Boxplots represent the median , first and third quantiles . Upper and lower whiskers extend to values up to 1 . 5 - fold interquartile range . N = 3 biological replicates , * * q ≤ 0 . 01 . Double knockdowns were generated by sequential constitutive CRISPR / Cas9 - mediated depletion of RNF145 or ADIPOR2 . Knockdown efficiencies are shown in Appendix Fig S1A .

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(A) Loss of ADIPOR2 sensitises cells to PA-induced cytotoxicity. HEK-293T cells were depleted of RNF145 (purple), ADIPOR2 (blue), B2M (CTR; grey), or both ADIPOR2 and RNF145 (blue + purple), exposed to PA (200 µM, 96h) and stained with crystal violet. Quantification of crystal-violet stained cells is shown alongside a representative micrograph (below). The response of each cell line to PA was normalised to its BSA-treated condition (dotted line). Statistically significant differences to cells containing gCTR cells are indicated (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction). Mean ± SD are shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. N = 3 biological replicates, ** q ≤ 0.01. Double knockdowns were generated by sequential constitutive CRISPR/Cas9-mediated depletion of RNF145 or ADIPOR2. Knockdown efficiencies are shown in Appendix Fig S1A.


( B ) Cells were treated as in ( A ) and lysates analysed by immunoblotting . LE , long exposure ; * non - specific bands .

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(B) Cells were treated as in (A) and lysates analysed by immunoblotting. LE, long exposure; *non-specific bands.


( C ) HEK - 293T cells depleted of B2M ( gCTR ) or ADIPOR2 ( gADIPOR2 ) were complemented with empty vector ( blue ) , full - length ADIPOR2 - HA ( green ) , or a catalytically inactive variant of ADIPOR2 - HA ( dead ADIPOR2 ; red ) and treated with PA ( 200 µM , 96h ) or BSA ( vehicle control , 96h ) . Measurements from PA - treated cells were normalised to their BSA - treated condition ( dotted line ) . Cell growth was quantified , and representative crystal violet staining is shown . Boxplots represent the median , first and third quantiles . Upper and lower whiskers extend to values up to 1 . 5 - fold interquartile range . * * q ≤ 0 . 01 ( robust one - way ANOVA with Benjamini - Hochberg multiple testing correction ) .

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(C) HEK-293T cells depleted of B2M (gCTR) or ADIPOR2 (gADIPOR2) were complemented with empty vector (blue), full-length ADIPOR2-HA (green), or a catalytically inactive variant of ADIPOR2-HA (dead ADIPOR2; red) and treated with PA (200 µM, 96h) or BSA (vehicle control, 96h). Measurements from PA-treated cells were normalised to their BSA-treated condition (dotted line). Cell growth was quantified, and representative crystal violet staining is shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. ** q ≤ 0.01 (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction).


MITOL - KO MEFs was vulnerable to ER stress . Control MEFs ( MITOLF / F ) and MITOL - KO MEFs ( MITOL - / - ) were treated with DMSO as control , 0 . 8 µM thapsigargin ( Tg ) , 0 . 7 µg / mL tunicamycin ( Tu ) or 1 . 2 µg / mL brefeldin A ( Br ) for 24 hours . All experiments using Tu below were performed at concentration of 0 . 7 µg / mL . The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers . Viable cells were detected by cell viability assay using cell counting kit - 8 .

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MITOL-KO MEFs was vulnerable to ER stress. Control MEFs (MITOLF/F) and MITOL-KO MEFs (MITOL-/-) were treated with DMSO as control, 0.8 µM thapsigargin (Tg), 0.7 µg/mL tunicamycin (Tu) or 1.2 µg/mL brefeldin A (Br) for 24 hours. All experiments using Tu below were performed at concentration of 0.7 µg/mL. The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers. Viable cells were detected by cell viability assay using cell counting kit-8.


MITOL KO increased ER stress - induced apoptosis . MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V - FITC for the detection of apoptosis using flow cytometry .

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MITOL KO increased ER stress-induced apoptosis. MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V-FITC for the detection of apoptosis using flow cytometry.


MITOL re - expression in MITOL - KO MEFs rescued ER stress vulnerability . MEFs were transfected with MITOL - coding vector ( WT ) , MITOL C65 / 68S - coding vector ( CS ) , or empty vector ( Vec ) 24 hours prior to Tu treatment for 18 hours , followed by immunoblotting with indicated antibodies . Cleaved caspase - 3 ( cC3 ) and cleaved PARP ( cPARP ) were used as apoptosis markers .

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MITOL re-expression in MITOL-KO MEFs rescued ER stress vulnerability. MEFs were transfected with MITOL-coding vector (WT), MITOL C65/68S-coding vector (CS), or empty vector (Vec) 24 hours prior to Tu treatment for 18 hours, followed by immunoblotting with indicated antibodies. Cleaved caspase-3 (cC3) and cleaved PARP (cPARP) were used as apoptosis markers.


MITOL KO enhanced ER stress - induced Bax translocation and cytochrome c release . Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours , followed by immunoblotting with indicated antibodies .

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MITOL KO enhanced ER stress-induced Bax translocation and cytochrome c release. Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours, followed by immunoblotting with indicated antibodies.


MITOL KO induced excessive mitochondrial depolarization by ER stress . MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM , followed by flow cytometric analysis .

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MITOL KO induced excessive mitochondrial depolarization by ER stress. MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM, followed by flow cytometric analysis.


IRE1α inhibition compensated MITOL - KO MEFs for vulnerability to ER stress . MEFs were transfected with scramble or siIRE1α ( # 1 , # 2 ) . After 24 hours , these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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IRE1α inhibition compensated MITOL-KO MEFs for vulnerability to ER stress. MEFs were transfected with scramble or siIRE1α (#1, #2). After 24 hours, these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MEFs were treated with 4µ8C ( 25 µM ) for 2 hours prior to Tu treatment for 24 hours ( B ) , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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MEFs were treated with 4µ8C (25 µM) for 2 hours prior to Tu treatment for 24 hours (B), followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MITOL KO did not affect the autophosphorylation of IRE1α . MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos - tag SDS - PAGE .

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MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE.


MITOL KO enhanced RIDD and xbp1 splicing . MEFs were incubated with Tu for 4 hours . Expression levels of various UPR associated genes were determined by qRT -

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MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR


MITOL KO enhanced RIDD and xbp1 splicing . MEFs were incubated with Tu for 4 hours . MEFs were transfected with either miR - 17 or miR - 34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours , followed by luciferase reporter assay ( F ) . Error bars represent SD ( n = 3 ) .

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MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. MEFs were transfected with either miR-17 or miR-34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours, followed by luciferase reporter assay (F). Error bars represent SD (n=3).


MEFs were incubated with Tu for 4 hours . Expression levels of various UPR associated genes were determined by qRT -

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MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR


IRE1α - mediated JNK phosphorylation was prolonged by MITOL KO . MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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IRE1α-mediated JNK phosphorylation was prolonged by MITOL KO. MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


JNK inhibition reduced ER stress - induced apoptosis of MITOL - KO MEFs . MEFs were treated with 20 µM JNK inhibitor - II ( JNK inh ) 2 hours prior to Tu treatment for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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JNK inhibition reduced ER stress-induced apoptosis of MITOL-KO MEFs. MEFs were treated with 20 µM JNK inhibitor-II (JNK inh) 2 hours prior to Tu treatment for 24 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MITOL KO facilitated IRE1α oligomerization . MEFs were transfected with IRE1α - GFP expression vector for 24 hours prior to Tu treatment for indicated periods ( A ) . The right panels show 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection in each independent experiment ( A ) . To evaluate the oligomerization level of endogenous IRE1α , cells without any transfection were solubilized and separated by sucrose density - gradient centrifugation , followed by immunoblotting with anti - IRE1α antibody ( B ) .

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MITOL KO facilitated IRE1α oligomerization. MEFs were transfected with IRE1α-GFP expression vector for 24 hours prior to Tu treatment for indicated periods (A). The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment (A). To evaluate the oligomerization level of endogenous IRE1α, cells without any transfection were solubilized and separated by sucrose density-gradient centrifugation, followed by immunoblotting with anti-IRE1α antibody (B).


IRE1α oligomers were stabilized by MITOL KO . MEFs transfected with IRE1α - GFP were treated with Tu . After 4 hours , these cells were washed with PBS and subsequently re - fed with fresh media for indicated periods . The right panels show 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment . Scale bar represents 10 µm .

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IRE1α oligomers were stabilized by MITOL KO. MEFs transfected with IRE1α-GFP were treated with Tu. After 4 hours, these cells were washed with PBS and subsequently re-fed with fresh media for indicated periods. The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment. Scale bar represents 10 µm.


Sustained IRE1α autophosphorylation after wash out of Tu in MITOL - KO MEFs . MEFs were treated with Tu for 4 hours and washed with PBS and re - fed with fresh media for indicated periods , following by immunoblotting anti - IRE1α antibody after Phos - tag SDS - PAGE . Error bars represent SD ( n = 3 ) .

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Sustained IRE1α autophosphorylation after wash out of Tu in MITOL-KO MEFs. MEFs were treated with Tu for 4 hours and washed with PBS and re-fed with fresh media for indicated periods, following by immunoblotting anti-IRE1α antibody after Phos-tag SDS-PAGE. Error bars represent SD (n=3).


MITOL deletion promoted the interaction between IRE1α and BIM under ER stress . MITOL - depleted HEK293 cells ( KO ) were generated using CRISPR - Cas9 system . Cells were co - transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours . Cells were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies .

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MITOL deletion promoted the interaction between IRE1α and BIM under ER stress. MITOL-depleted HEK293 cells (KO) were generated using CRISPR-Cas9 system. Cells were co-transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours. Cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.


Interaction between MITOL and IRE1α . Lysates of HEK293 cells were subjected to immunoprecipitation with anti - IRE1α antibody , followed by immunoblotting with indicated antibodies ( A ) .

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Interaction between MITOL and IRE1α. Lysates of HEK293 cells were subjected to immunoprecipitation with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies (A).


Interaction between MITOL and IRE1α . Lysates of HEK293 cells co - transfected with indicated vectors were subjected to immunoprecipitation , followed by immunoblotting with indicated antibodies ( B ) .

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Interaction between MITOL and IRE1α. Lysates of HEK293 cells co-transfected with indicated vectors were subjected to immunoprecipitation, followed by immunoblotting with indicated antibodies (B).


Interaction between MITOL and IRE1α . GST - pull - down assay of IRE1α or MITOL was performed on lysates of MEFs , followed by immunoblotting with indicated antibodies ( C , D ) . N - ter : 1 - 90 aa of MITOL , Loop : 158 - 210 aa of MITOL , C - ter : 241 - 278 aa of MITOL , 465 - 977 aa of IRE1α .

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Interaction between MITOL and IRE1α. GST-pull-down assay of IRE1α or MITOL was performed on lysates of MEFs, followed by immunoblotting with indicated antibodies (C, D). N-ter: 1-90 aa of MITOL, Loop: 158-210 aa of MITOL, C-ter: 241-278 aa of MITOL, 465-977 aa of IRE1α.


MITOL ubiquitylated IRE1α . Lysates of HEK293 cells immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

MITOL ubiquitylated IRE1α. Lysates of HEK293 cells immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL ubiquitylated IRE1α . Lysates of HEK293 cells transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

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MITOL ubiquitylated IRE1α. Lysates of HEK293 cells transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL ubiquitylated IRE1α . Lysates of MEFs ( G ) transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

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MITOL ubiquitylated IRE1α. Lysates of MEFs (G) transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL adds lysine 63 - linked polyubiquitin chains to IRE1α . Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody . IRE1α ubiquitylation was

MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected


MITOL added polyubiquitin chains to K481 of IRE1α . Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies . 481 : K481R , 545 : K545R , 568 : K568R .

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MITOL added polyubiquitin chains to K481 of IRE1α. Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. 481: K481R, 545: K545R, 568: K568R.


IRE1α K481R interacted with MITOL . Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies .

IRE1α K481R interacted with MITOL. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.


Overexpression of IRE1α K481R induced apoptosis . MEFs were transfected with indicated vectors . After 24 hours , these cells were stained with Annexin V -

Overexpression of IRE1α K481R induced apoptosis. MEFs were transfected with indicated vectors. After 24 hours, these cells were stained with Annexin V-FITC


Overexpression of IRE1α K481R induced apoptosis . or subjected by immunoblotting with indicated antibodies ( D ) .

Overexpression of IRE1α K481R induced apoptosis. or subjected by immunoblotting with indicated antibodies (D).


Enhanced RIDD activity in IRE1α K481R . MEFs were transfected with indicated vectors 24 hours prior to analysis ( E , F ) . The RIDD activity was measured by qRT - PCR .

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Enhanced RIDD activity in IRE1α K481R. MEFs were transfected with indicated vectors 24 hours prior to analysis (E, F). The RIDD activity was measured by qRT-PCR.


Hyper - oligomerization of IRE1α K481R . MEFs were transfected with IRE1α - GFP or IRE1α K481R - GFP . After 24 hours , these cells were exposed with Tu for indicated periods and GFP signals were observed . The right panels show 5 . 5 - fold magnification images of the boxed regions . Percentages of cells with IRE1α - foci were calculated from 100 cells by visual inspection in each independent experiment .

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Hyper-oligomerization of IRE1α K481R. MEFs were transfected with IRE1α-GFP or IRE1α K481R-GFP. After 24 hours, these cells were exposed with Tu for indicated periods and GFP signals were observed. The right panels show 5.5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment.


Increased interaction of IRE1α K481R with BIM . Cells transfected with indicated vectors 24 hours prior to immunoprecipitation , followed by immunoblotting with indicated antibodies .

Increased interaction of IRE1α K481R with BIM. Cells transfected with indicated vectors 24 hours prior to immunoprecipitation, followed by immunoblotting with indicated antibodies.


Accumulations of MITOL and IRE1α in the MAM fraction . Each organelle fraction was isolated from MEFs by percoll density - gradient centrifugation , followed by immunoblotting with indicated antibodies . mito : pure mitochondria , micro : microsome , cyto : pure cytosol .

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Accumulations of MITOL and IRE1α in the MAM fraction. Each organelle fraction was isolated from MEFs by percoll density-gradient centrifugation, followed by immunoblotting with indicated antibodies. mito: pure mitochondria, micro: microsome, cyto: pure cytosol.


High solubility of MITOL and IRE1α in the low dose digitonin . Raft - like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin . S : supernatant , P : pellet .

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High solubility of MITOL and IRE1α in the low dose digitonin. Raft-like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin. S: supernatant, P: pellet.


IRE1α regulation by MITOL occurred in the MAM - rich mitochondrial fraction . Crude mitochondrial fraction containing MAM ( Mito plus MAM ) and the cytosolic fraction containing ER ( Cyto plus ER ) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the interaction between MITOL and

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the interaction between MITOL and IRE1α


IRE1α regulation by MITOL occurred in the MAM - rich mitochondrial fraction . Crude mitochondrial fraction containing MAM ( Mito plus MAM ) and the cytosolic fraction containing ER ( Cyto plus ER ) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the level of IRE1α ubiquitylation ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the level of IRE1α ubiquitylation (D).


Knockdown of PACS2 impaired the interaction of MITOL with IRE1α . HEK293 cells were transfected with either scramble RNA or siPACS2 and indicated vectors for 24 hours prior to immunoprecipitation assay .

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Knockdown of PACS2 impaired the interaction of MITOL with IRE1α. HEK293 cells were transfected with either scramble RNA or siPACS2 and indicated vectors for 24 hours prior to immunoprecipitation assay.


Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL . HEK293 cells were transfected with either scramble RNA , siPACS2 and indicated vectors 24 hours prior to immunoprecipitation 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

Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siPACS2 and indicated vectors 24 hours prior to immunoprecipitation assay.


Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL . HEK293 cells were transfected with either scramble RNA , siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay .

Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay.


MITOL ubiquitylated IRE1α in the spinal cord of mice . Lysates of the spinal cord in mice were immunoprecipitated with anti - IRE1α antibody , followed by immunoblotting with indicated antibodies . WT : MITOLF / F mice , MITOLnestin : MITOLF / F , Nestin - Cre mice .

MITOL ubiquitylated IRE1α in the spinal cord of mice. Lysates of the spinal cord in mice were immunoprecipitated with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies. WT: MITOLF/F mice, MITOLnestin: MITOLF/F, Nestin-Cre mice.


Loss of MITOL led to hyper - oligomerization of IRE1α in the spinal cord under ER stress . 3 month - old WT or MITOLnestin mice were treated with 1 mg / kg Tu for 24 hours . Spinal cord was solubilized with NP - 40 lysis buffer and separated by sucrose - density gradient centrifugation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Loss of MITOL led to hyper-oligomerization of IRE1α in the spinal cord under ER stress. 3 month-old WT or MITOLnestin mice were treated with 1 mg/kg Tu for 24 hours. Spinal cord was solubilized with NP-40 lysis buffer and separated by sucrose-density gradient centrifugation.


MITOL deletion resulted in hyper - activation of IRE1α RNase in ER stress . WT or MITOLnestin mice were injected with Tu for 24 hours and spinal cord was analyzed by qRT - PCR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MITOL deletion resulted in hyper-activation of IRE1α RNase in ER stress. WT or MITOLnestin mice were injected with Tu for 24 hours and spinal cord was analyzed by qRT-PCR.


Increased ER stress - induced apoptosis in the MITOL - KO spinal cord . 3 month - old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using TUNEL and hoechst ( F ) Arrowheads indicate the representative neurons .

O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using TUNEL and hoechst (F) Arrowheads indicate the representative neurons.


Increased ER stress - induced apoptosis in the MITOL - KO spinal cord . 3 month - old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using cresyl violet ( G ) . Arrowheads indicate the representative neurons .

Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using cresyl violet (G). Arrowheads indicate the representative neurons.


( A ) 293T cells were co - transfected with expression plasmids for Cherry - STING , the murine IFNβ - luciferase reporter ( IFNβ - Luc ) , a Renilla luciferase normalization control ( pRL - TK ) and the indicated expression plasmids or empty vector ( ev ) . Cells were additionally co - transfected with expression plasmids for cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) . 20 hours post transfection , cells were lysed and a dual - luciferase assay was performed . Data information : ( A - G ) Data is combined from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids for Cherry-STING, the murine IFNβ-luciferase reporter (IFNβ-Luc), a Renilla luciferase normalization control (pRL-TK) and the indicated expression plasmids or empty vector (ev). Cells were additionally co-transfected with expression plasmids for cGAS-GFP (stimulated) or IRES-GFP (unstimulated). 20 hours post transfection, cells were lysed and a dual-luciferase assay was performed. Data information: (A-G) Data is combined from three independent experiments.


( B ) An expression plasmid for RIG - I N ( stimulated ) or ev ( unstimulated ) was co - transfected with IFNβ - Luc , pRL - TK and the indicated expression plasmids in 293T cells and analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) An expression plasmid for RIG-I N (stimulated) or ev (unstimulated) was co-transfected with IFNβ-Luc, pRL-TK and the indicated expression plasmids in 293T cells and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.


( C ) An expression plasmid for TBK1 ( stimulated ) or ev ( unstimulated ) was co - transfected together with IFNβ - Luc , pRL - TK and the indicated expression plasmids and analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co-transfected together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.


( D ) 293T cells were co - transfected with a plasmid expressing constitutively active IRF3 ( IRF3 - 5D ; stimulated ) or IRES - GFP ( unstimulated ) together with IFNβ - Luc , pRL - TK and the indicated expression plasmids and analyzed as in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with a plasmid expressing constitutively active IRF3 (IRF3-5D; stimulated) or IRES-GFP (unstimulated) together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). Data information: (A-G) Data is combined from three independent experiments.


( E ) The ISG56 - luciferase reporter , pRL - TK and the indicated expression plasmids were co - transfected in 293T cells . 24 hours post transfection , cells were stimulated with 0 . 1 ng / µl human IFNβ or mock stimulated and analyzed 16 hours later as described in ( A ) . Data information : ( A - G ) Data is combined from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The ISG56-luciferase reporter, pRL-TK and the indicated expression plasmids were co-transfected in 293T cells. 24 hours post transfection, cells were stimulated with 0.1 ng/µl human IFNβ or mock stimulated and analyzed 16 hours later as described in (A). Data information: (A-G) Data is combined from three independent experiments.


( F - G ) iMEFgt / gt stably expressing Cherry - STING and either ev or V5 - tagged m152 were stimulated with 5 µg / ml ISD ( F ) , 10 µg / ml poly ( I : C ) ( G ) or mock stimulated with Lipofectamine . 4 hours post stimulation , RNA was extracted to determine IFNβ mRNA transcripts by qRT - PCR . Data information : ( A - G ) Data is combined from three independent experiments .

O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-G) iMEFgt/gt stably expressing Cherry-STING and either ev or V5-tagged m152 were stimulated with 5 µg/ml ISD (F), 10 µg/ml poly(I:C) (G) or mock stimulated with Lipofectamine. 4 hours post stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT-PCR. Data information: (A-G) Data is combined from three independent experiments.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with 10 µg / ml cGAMP ( H ) , 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 10 µg/ml cGAMP (H), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with 5 µg / ml ISD ( I ) , 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 5 µg/ml ISD (I), 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with Newcastle ' s Disease Virus ( NDV ) infection ( J ) 6 ( H ) or 16 ( I - K ) hours later , secreted IFNβ ( H - J ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with Newcastle's Disease Virus (NDV) infection (J) 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( H - K ) iBMDM stably expressing ev or m152 - V5 were stimulated in duplicates with µM CpG DNA ( K ) . 6 ( H ) or 16 ( I - K ) hours later , TNFα ( K ) levels were determined by ELISA . ( H - K ) Experiments were performed three ( H , I , K ) or two ( J ) times independently and one representative experiment is shown . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Data are shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with µM CpG DNA (K). 6 (H) or 16 (I-K) hours later, TNFα (K) levels were determined by ELISA. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are shown as mean ± SD.


( A ) HeLa cells were co - transfected with expression plasmids for Cherry - STING , V5 - tagged m152 and either ev ( unstimulated ) or cGAS - GFP ( stimulated ) .

(A) HeLa cells were co-transfected with expression plasmids for Cherry-STING, V5-tagged m152 and either ev (unstimulated) or cGAS-GFP (stimulated).


( B ) iMEFgt / gt were co - transfected with expression plasmids for V5 - tagged m152 together with either Cherry - STING or ev in combination with cGAS - GFP ( stimulated ) or ev ( unstimulated ) . 24 hours post transfection cells were fixed for immunolabeling with an anti - V5 antibody . White boxes indicate the region shown at a higher magnification . Scale bar represents 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) iMEFgt/gt were co-transfected with expression plasmids for V5-tagged m152 together with either Cherry-STING or ev in combination with cGAS-GFP (stimulated) or ev (unstimulated). 24 hours post transfection cells were fixed for immunolabeling with an anti-V5 antibody. White boxes indicate the region shown at a higher magnification. Scale bar represents 10 µm.


( C ) Lysates of Cherry - STING and either CD4 - V5 or m152 - V5 expressing 293T cells were subjected to immunoprecipitation ( IP ) with an anti - V5 antibody . Input and IP samples were analyzed by IB with the indicated antibodies . Data information : IB shown are representative of three ( C ) or two ( D ) independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysates of Cherry-STING and either CD4-V5 or m152-V5 expressing 293T cells were subjected to immunoprecipitation (IP) with an anti-V5 antibody. Input and IP samples were analyzed by IB with the indicated antibodies. Data information: IB shown are representative of three (C) or two (D) independent experiments.


( D ) iMEF stably expressing ev or m152 - V5 were left unstimulated or stimulated with 10 µg / ml ISD and lysed 90 minutes later . m152 was immunoprecipitated with an anti - V5 antibody and samples were analyzed by IB with V5 , STING and phospho - TBK1 ( pTBK1 ) specific antibodies . Data information : IB shown are representative of three ( C ) or two ( D ) independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) iMEF stably expressing ev or m152-V5 were left unstimulated or stimulated with 10 µg/ml ISD and lysed 90 minutes later. m152 was immunoprecipitated with an anti-V5 antibody and samples were analyzed by IB with V5, STING and phospho-TBK1 (pTBK1) specific antibodies. Data information: IB shown are representative of three (C) or two (D) independent experiments.


( B ) Cherry - STING , IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and indicated expression plasmids as shown in ( A ) were transiently expressed in 293T cells and a dual luciferase assay was performed . Data is combined from two out of three independent experiments and shown as mean ± SD . Data information : Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cherry-STING, IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids as shown in (A) were transiently expressed in 293T cells and a dual luciferase assay was performed. Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001


( C ) 293T cells were co - transfected with expression plasmids for Cherry - STING and indicated chimeras as shown in ( A ) . An anti - V5 IP was performed and samples were analyzed by IB with indicated antibodies . IB shown is representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids for Cherry-STING and indicated chimeras as shown in (A). An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. IB shown is representative of three independent experiments.


( D ) Cherry - STING , IFNβ - Luc , pRL - TK and either CD4 , m152 , m152 - Δstalk or m152 - GSstalk were transiently expressed in 293T cells . For stimulation , samples were co - transfected with cGAS - GFP , and unstimulated samples with IRES - GFP . Lysates were analyzed as described in ( B ) . Data is combined from two out of three independent experiments and shown as mean ± SD . Data information : Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cherry-STING, IFNβ-Luc, pRL-TK and either CD4, m152, m152-Δstalk or m152-GSstalk were transiently expressed in 293T cells. For stimulation, samples were co-transfected with cGAS-GFP, and unstimulated samples with IRES-GFP. Lysates were analyzed as described in (B). Data is combined from two out of three independent experiments and shown as mean ± SD. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **p<0.01, ***p<0.001, ****p<0.0001


( E ) 293T cells were co - transfected with expression plasmids for Cherry - STING and either CD4 , m152 , m152 - Δstalk or m152 - GSstalk . An anti - V5 IP was performed and samples were analyzed by IB with indicated antibodies . Immunoblot shown is representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids for Cherry-STING and either CD4, m152, m152-Δstalk or m152-GSstalk. An anti-V5 IP was performed and samples were analyzed by IB with indicated antibodies. Immunoblot shown is representative of three independent experiments.


( A ) 293T cells were co - transfected with expression plasmids for IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and indicated expression plasmids together with murine Cherry - STING ( left panel ) or human STING ( right panel ) . Lysates were analyzed by dual - luciferase assay . Data is combined from two out of three independent experiments and shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and indicated expression plasmids together with murine Cherry-STING (left panel) or human STING (right panel). Lysates were analyzed by dual-luciferase assay. Data is combined from two out of three independent experiments and shown as mean ± SD.


( C ) IFNβ - Luc , pRL - TK , cGAS - GFP ( stimulated ) or IRES - GFP ( unstimulated ) and either LacZ or m152 together with the indicated murine STING mutants described in ( B ) were transiently expressed in 293T cells . Luciferase activity was measured as described in ( A ) . Data information : Data in ( C ) is normalized to LacZ , combined from three independent experiments and shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated) or IRES-GFP (unstimulated) and either LacZ or m152 together with the indicated murine STING mutants described in (B) were transiently expressed in 293T cells. Luciferase activity was measured as described in (A). Data information: Data in (C) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD.


( D ) 293T cells were co - transfected with expression plasmids for either LacZ or m152 together with the indicated murine STING mutants described in ( B ) . An anti - V5 IP was performed and samples were analyzed by IB with the indicated antibodies . IB shown in ( D ) representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids for either LacZ or m152 together with the indicated murine STING mutants described in (B). An anti-V5 IP was performed and samples were analyzed by IB with the indicated antibodies. IB shown in (D) representative of three independent experiments.


( E - F ) Luciferase assay ( E ) were performed as described in ( C ) and ( D ) , respectively , using the indicated human STING mutants in place of the murine STING mutants described in ( B ) . Data information : Data in ( E ) is normalized to LacZ , combined from three independent experiments and shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Luciferase assay (E) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: Data in (E) is normalized to LacZ, combined from three independent experiments and shown as mean ± SD.


( E - F ) co - IP ( F ) were performed as described in ( C ) and ( D ) , respectively , using the indicated human STING mutants in place of the murine STING mutants described in ( B ) . Data information : IB shown in ( F ) are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) co-IP (F) were performed as described in (C) and (D), respectively, using the indicated human STING mutants in place of the murine STING mutants described in (B). Data information: IB shown in (F) are representative of three independent experiments.


( B ) 293T cells were co - transfected with expression plasmids for IFNβ - Luc , pRL - TK and either ev or m152 . Cells were further co - transfected with either Cherry - STING WT or the constitutively active Cherry - STING V154M ( stimulated ) or with IRES - GFP ( unstimulated ) . Data is combined from three independent experiments and shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK and either ev or m152. Cells were further co-transfected with either Cherry-STING WT or the constitutively active Cherry-STING V154M (stimulated) or with IRES-GFP (unstimulated). Data is combined from three independent experiments and shown as mean ± SD.


( C ) iMEF stably expressing ev or m152 - V5 were stimulated with 10 µg / ml ISD for the indicated times or left unstimulated ( mock ) . Cell lysates were analyzed by SDS - PAGE under non - reducing conditions and subjected to IB with the specified antibodies . Data information : IB shown in ( C ) are representative of three and two independent experiments , respectively . Student ' s t - test ( unpaired , two - tailed ) , n . s . not significant , * p < 0 . 05 , * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) iMEF stably expressing ev or m152-V5 were stimulated with 10 µg/ml ISD for the indicated times or left unstimulated (mock). Cell lysates were analyzed by SDS-PAGE under non-reducing conditions and subjected to IB with the specified antibodies. Data information: IB shown in (C) are representative of three and two independent experiments, respectively. Student's t-test (unpaired, two-tailed), n.s. not significant, *p<0.05, ****p<0.0001