HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 , ASC or pro - casapase - 1 ( Pro - Casp - 1 ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3, ASC or pro-casapase-1 (Pro-Casp-1). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


BMDMs were treated with indicated stimuli . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

BMDMs were treated with indicated stimuli. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Flag - ABRO1 and Myc - tagged NLRP3 or NLRP3 deletion mutants as indicated in ( F ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - c - Myc agarose .

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 O O O O O O O O O O

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Flag-ABRO1 and Myc-tagged NLRP3 or NLRP3 deletion mutants as indicated in (F). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 and Flag - tagged ABRO1 or ABRO1 deletion mutants as indicated in ( H ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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 O O O O O O O O O

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3 and Flag-tagged ABRO1 or ABRO1 deletion mutants as indicated in (H). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - tagged NLRP3 ( WT ) , NLRP3 ( S194D ) , or NLRP3 ( S194A ) . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-tagged NLRP3 (WT), NLRP3 (S194D), or NLRP3 (S194A). Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT BMDMs were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT BMDMs were pretreated with or without 100 nM SP600125 for 1 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194A ) were treated with or without 5 μM Anisomycin for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194A) were treated with or without 5 μM Anisomycin for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 and Flag - NLRP3 ( WT ) or Flag - NLRP3 ( S194D ) were treated with or without 100 nM SP600125 for 2 h . Immunoblot analysis of Myc - and Flag - tagged proteins in cell lysates immunoprecipitated with anti - Flag M2 beads .

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1 and Flag-NLRP3 (WT) or Flag-NLRP3 (S194D) were treated with or without 100 nM SP600125 for 2 h. Immunoblot analysis of Myc- and Flag-tagged proteins in cell lysates immunoprecipitated with anti-Flag M2 beads.


WT and Nlrp3S194A / S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng / ml LPS for 1 h . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti - ABRO1 antibody .

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

WT and Nlrp3S194A/S194A BMDMs were pretreated with or without 5 μM Anisomycin for 2 h and then left untreated or treated with 100 ng/ml LPS for 1 h. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with control IgG or anti-ABRO1 antibody.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - NLRP3 , Flag - ABRO1 and HA - ubiquitin ( HA - Ub ) , HA - Ub K48 only or K63 only . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-NLRP3, Flag-ABRO1 and HA-ubiquitin (HA-Ub), HA-Ub K48 only or K63 only. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding HA - Ub , Flag - ABRO1 , and NLRP3 truncations as indicated . Before collection , cells were treated with MG132 ( 10 μM ) for 6 h . Immunoblot analysis of ubiquitination of NLRP3 truncations ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - c - Myc agarose .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding HA-Ub, Flag-ABRO1, and NLRP3 truncations as indicated. Before collection, cells were treated with MG132 (10 μM) for 6 h. Immunoblot analysis of ubiquitination of NLRP3 truncations (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-c-Myc agarose.


LPS - primed Abro1 + / + and Abro1 − / − BMDMs were left untreated or treated with ATP ( left ) or nigericin ( right ) . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP (left) or nigericin (right). Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were left unstimulated or stimulated with LPS or LPS plus ATP . Immunoblot analysis of NLRP3 and NLRP3 ( A350V ) ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were left unstimulated or stimulated with LPS or LPS plus ATP. Immunoblot analysis of NLRP3 and NLRP3 (A350V) ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


HEK - 293T cells were transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 ( A350V ) and HA - ubiquitin ( HA - Ub ) . Immunoblot analysis of NLRP3 ( A350V ) ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3 (A350V) and HA-ubiquitin (HA-Ub). Immunoblot analysis of NLRP3 (A350V) ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


Nlrp3 - / - BMDMs transduced with lentiviruses expressing Flag - NLRP3 ( WT ) or Flag - NLRP3 ( A350V ) were stimulated with or without 100ng / ml LPS for 1 h . Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

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

Nlrp3-/- BMDMs transduced with lentiviruses expressing Flag-NLRP3 (WT) or Flag-NLRP3 (A350V) were stimulated with or without 100ng/ml LPS for 1 h. Immunoblot analysis of ABRO1 and NLRP3 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


HEK - 293T cells transfected with various combinations ( above lanes ) of plasmids encoding Myc - ABRO1 , Flag - NLRP3 , and HA - ubiquitin ( HA - Ub ) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h . Immunoblot analysis of NLRP3 ubiquitination ( detected by anti - HA antibody ) in cell lysates immunoprecipitated with anti - Flag M2 beads .

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

HEK-293T cells transfected with various combinations (above lanes) of plasmids encoding Myc-ABRO1, Flag-NLRP3, and HA-ubiquitin (HA-Ub) were left untreated or treated with 5 μM Anisomycin for 2 h or 100 nM SP600125 for 2 h. Immunoblot analysis of NLRP3 ubiquitination (detected by anti-HA antibody) in cell lysates immunoprecipitated with anti-Flag M2 beads.


LPS - primed WT and Nlrp3S194A / S194A BMDMs were left untreated or treated with ATP . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed WT and Nlrp3S194A/S194A BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


WT BMDMs were stimulated with LPS for 1 h . Immunoblot analysis of NLRP3 , ABRO1 , and BRCC3 from the cell lysates immunoprecipitated with control IgG , anti - ABRO1 , anti - BRCC3 or anti - NLRP3 antibody .

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

WT BMDMs were stimulated with LPS for 1 h. Immunoblot analysis of NLRP3, ABRO1, and BRCC3 from the cell lysates immunoprecipitated with control IgG, anti-ABRO1, anti-BRCC3 or anti-NLRP3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Brcc3 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of ABRO1 . Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti - ABRO1 antibody .

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

Brcc3+/+ and Brcc3−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Brcc3−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of ABRO1. Immunoblot analysis of NLRP3 and ABRO1 proteins in cell lysates immunoprecipitated with anti-ABRO1 antibody.


Abro1 + / + and Abro1 − / − BMDMs were treated with or without LPS for 1 h . Before LPS treatment , Abro1 − / − BMDMs were pretreated with MG132 ( 10 μM ) for 6 h to rescue the expression of BRCC3 . Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti - BRCC3 antibody .

Abro1+/+ and Abro1−/− BMDMs were treated with or without LPS for 1 h. Before LPS treatment, Abro1−/− BMDMs were pretreated with MG132 (10 μM) for 6 h to rescue the expression of BRCC3. Immunoblot analysis of NLRP3 and BRCC3 proteins in cell lysates immunoprecipitated with anti-BRCC3 antibody.


A constant amount of Myc - NLRP3 plasmid together with an increasing amount of Myc - ABRO1 plasmid were co - transfected in HEK - 293T cells . Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti - BRCC3 antibody .

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

A constant amount of Myc-NLRP3 plasmid together with an increasing amount of Myc-ABRO1 plasmid were co-transfected in HEK-293T cells. Immunoblot analysis of NLRP3 and BRCC3 from the cell lysates immunoprecipitated with control IgG or anti-BRCC3 antibody.


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP were treated with LPS and nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - NLRP3 antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3

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

Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP were treated with LPS and nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-NLRP3 antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody


Brcc3 + / + and Brcc3 − / − BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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

Brcc3+/+ and Brcc3−/− BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Abro1 + / + , Abro1 − / − , BMDMs transduced with lentiviruses expressing GFP , ABRO1 - GFP or BRCC3 - GFP CBA analysis of IL - 1β in culture

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

Abro1+/+, Abro1−/−, BMDMs transduced with lentiviruses expressing GFP, ABRO1-GFP or BRCC3-GFP CBA analysis of IL-1β in culture supernatants


Brcc3 + / + and Brcc3 − / − BMDMs were left untreated ( Con ) , treated with LPS alone ( LPS ) or pretreated with LPS and then stimulated with ATP , nigericin , MSU , Alum , poly ( dA : dT ) or flagellin . CBA analysis of IL - 1β secretion in the culture supernatants .

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

Brcc3+/+ and Brcc3−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS and then stimulated with ATP, nigericin, MSU, Alum, poly (dA:dT) or flagellin. CBA analysis of IL-1β secretion in the culture supernatants.


Immunoblot analysis of cleaved caspase - 1 and IL - 1β in culture supernatants ( SN ) of LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs treated with ATP , nigericin , MSU , flagellin , and poly ( dA : dT ) .

Immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Brcc3+/+ and Brcc3−/− BMDMs treated with ATP, nigericin, MSU, flagellin, and poly (dA: dT).


Immunostaining of endogenous ASC specks in LPS - primed Brcc3 + / + and Brcc3 - / - BMDMs left untreated or treated with nigericin or poly ( dA : dT ) . ASC speck positive cells were counted and analyzed as described in Fig . 1I . UD , undetectable .

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

Immunostaining of endogenous ASC specks in LPS-primed Brcc3+/+ and Brcc3-/- BMDMs left untreated or treated with nigericin or poly (dA:dT). ASC speck positive cells were counted and analyzed as described in Fig. 1I. UD, undetectable.


LPS - primed Brcc3 + / + and Brcc3 − / − BMDMs were left unstimulated or stimulated with nigericin . Immunoblot analysis of NLRP3 ubiquitination ( detected by mouse anti - ubiquitin antibody ) in cell lysates immunoprecipitated with rabbit anti - NLRP3 antibody .

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

LPS-primed Brcc3+/+ and Brcc3−/− BMDMs were left unstimulated or stimulated with nigericin. Immunoblot analysis of NLRP3 ubiquitination (detected by mouse anti-ubiquitin antibody) in cell lysates immunoprecipitated with rabbit anti-NLRP3 antibody.


Survival of Brcc3 + / + and Brcc3 − / − mice subjected to LPS ( 15 mg / kg ) . n = 20 per group .

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

Survival of Brcc3+/+ and Brcc3−/− mice subjected to LPS (15 mg/kg). n=20 per group.


ELISA of serum IL - 1β from Brcc3 + / + and Brcc3 − / − mice 3 h after intraperitoneal injection of LPS ( 15mg / kg ) or vehicle ( PBS ) . n = 6 for PBS groups and n = 12 for LPS groups .

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

ELISA of serum IL-1β from Brcc3+/+ and Brcc3−/− mice 3 h after intraperitoneal injection of LPS (15mg/kg) or vehicle (PBS). n = 6 for PBS groups and n = 12 for LPS groups.


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . ELISA of IL - 1β and TNF - α in the peritoneal lavage fluid ( G )

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

Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. ELISA of IL-1β and TNF-α in the peritoneal lavage fluid (G)


Brcc3 + / + and Brcc3 − / − mice were intraperitoneally injected with MSU ( 1 mg per mouse ) or vehicle ( PBS ) for 6 h . flow cytometric analysis of peritoneal cell exudates ( H ) . n = 6 for PBS groups and n = 12 for MSU groups .

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Brcc3+/+ and Brcc3−/− mice were intraperitoneally injected with MSU (1 mg per mouse) or vehicle (PBS) for 6 h. flow cytometric analysis of peritoneal cell exudates (H). n = 6 for PBS groups and n = 12 for MSU groups.


A , B , Immunostaining for CD31 + blood vessels in control ( ctrl , A ) and Gpr124KO ( B ) forebrains . Note the normal vasculature in the cortical hem ( asterisk ) of Gpr124KO mice .

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A,B, Immunostaining for CD31+ blood vessels in control (ctrl, A) and Gpr124KO (B) forebrains. Note the normal vasculature in the cortical hem (asterisk) of Gpr124KO mice.


C , D , Immunostaining for Pax6 in control ( C ) and Gpr124KO ( D ) forebrains to reveal the lateral extension of the neocortex ( dashed line ) between the cortical hem ( arrowhead ) and the lateral ganglionic eminence ( arrow ) . E , Quantification of the lateral extension of the cortex in control and Gpr124KO brains ( mean ± SEM ; N = 10 ; * * * p < 0 . 001 ) .

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

C,D, Immunostaining for Pax6 in control (C) and Gpr124KO (D) forebrains to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). E, Quantification of the lateral extension of the cortex in control and Gpr124KO brains (mean±SEM; N=10; *** p<0.001).


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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 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 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

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 B-MEASURED_VAR O O O O B-MEASURED_VAR 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 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

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

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

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

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

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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-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 O 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 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

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 B-MEASURED_VAR I-MEASURED_VAR I-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 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 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

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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

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 B-MEASURED_VAR O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 B-MEASURED_VAR 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

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 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

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 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

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

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

(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 .

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

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

(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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 ( * ) .

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(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 .

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(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 .

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(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 .

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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

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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 .

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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 -

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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 ) .

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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.