b . The K63 - linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K ( h . sapiens ) were employed in single turnover ubiquitination experiments in presence of K63 - Ub2 with Ub ( K48R ) at the proximal or distal position or in presence of K48 ‑ Ub2 . Initial rate determination c . Experiment as in ( B ) but with the Ubc1 homologue ubc - 20 ( c . elegans ) .

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

b. The K63-linked chain building enzyme Ubc13 with its cofactor Uev1a or the Ubc1 homologue Ube2K (h. sapiens) were employed in single turnover ubiquitination experiments in presence of K63-Ub2 with Ub(K48R) at the proximal or distal position or in presence of K48‑Ub2. Initial rate determination c. Experiment as in (B) but with the Ubc1 homologue ubc-20 (c. elegans).


D . Alexa488 - labeled monoubiquitin and C - terminally capped K63 - Ub4 were used in a 4 : 1 molar ratio for in vitro ubiquitination reactions with full ‑ length Ubc1 or Ubc1 - ΔUBA . Where indicated , Alexa488 - labeled monoubiquitin was methylated prior to the reaction ( met - Ub ) to block de novo chain assembly . Reactions were incubated overnight , quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH ( lanes 4 - 6 ) . Samples were analyzed by SDS - PAGE and fluorescence scan .

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

D. Alexa488-labeled monoubiquitin and C-terminally capped K63-Ub4 were used in a 4:1 molar ratio for in vitro ubiquitination reactions with full‑length Ubc1 or Ubc1-ΔUBA. Where indicated, Alexa488-labeled monoubiquitin was methylated prior to the reaction (met-Ub) to block de novo chain assembly. Reactions were incubated overnight, quenched and half the reaction mix from each condition was treated with the K63 specific deubiquitinating enzyme AMSH (lanes 4-6). Samples were analyzed by SDS-PAGE and fluorescence scan.


F . Ubc1 - EA was genomically integrated into various yeast strains . Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10 ‑ fold dilutions . Where noted cells were incubated at elevated temperature ( 37 ° C ) or in presence of hydroxyurea ( HU ) to induce DNA replication stress .

O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

F. Ubc1-EA was genomically integrated into various yeast strains. Exponentially growing cells of the indicated genotype were spotted onto YPD plates in serial 10‑fold dilutions. Where noted cells were incubated at elevated temperature (37 °C) or in presence of hydroxyurea (HU) to induce DNA replication stress.


CSF extract was treated with DMSO , CsA , buffer or His - CnA420 - 508 at the indicated concentrations . Meiotic exit was induced by calcium addition and samples were taken at the indicated time points . Samples were immunoblotted for cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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

CSF extract was treated with DMSO, CsA, buffer or His-CnA420-508 at the indicated concentrations. Meiotic exit was induced by calcium addition and samples were taken at the indicated time points. Samples were immunoblotted for cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was treated with DMSO or CsA . Both reactions were divided and supplemented with the indicated amounts of calcium or H2O . After 20min , samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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

CSF extract was treated with DMSO or CsA. Both reactions were divided and supplemented with the indicated amounts of calcium or H2O. After 20min, samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was supplemented with Myc - XErp1 wt IVT at the indicated dilutions . An empty IVT reaction not expressing Myc - XErp1 served as control . All reactions were divided and treated with DMSO or CsA . Calcium was added to all reactions and samples were taken at the indicated time points , treated with λ - phosphatase and immunoblotted for XErp1 , the Myc - tag , cyclin B2 and α - tubulin . Asterisks indicate unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 wt IVT at the indicated dilutions. An empty IVT reaction not expressing Myc-XErp1 served as control. All reactions were divided and treated with DMSO or CsA. Calcium was added to all reactions and samples were taken at the indicated time points, treated with λ-phosphatase and immunoblotted for XErp1, the Myc-tag, cyclin B2 and α-tubulin. Asterisks indicate unspecific bands.


CSF extract was supplemented with Myc - XErp1 wt IVT . The reaction was divided and treated with DMSO or CsA . Both reactions were treated with calcium , samples were taken at the indicated time points and as indicated incubated with λ - phosphatase . Samples were immunoblotted for XErp1 and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 wt IVT. The reaction was divided and treated with DMSO or CsA. Both reactions were treated with calcium, samples were taken at the indicated time points and as indicated incubated with λ-phosphatase. Samples were immunoblotted for XErp1 and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT and as indicated treated with DMSO or CsA . Both reactions were treated with calcium and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag , cyclin B2 and α - tubulin . A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A . Arrow marks the meiotic phosphorylation state of Myc - XErp1 . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands.


CSF extract was supplemented with Myc - XErp1 DSG - DSA - ZBR - ( S33N S38N S284N S288N C583A ) IVT that was either wild - type or mutated to alanine at the p90RSK - target sites S335 , T336 and S342 ( Rsk3A ) . Both reactions were divided and treated with DMSO or CsA . Calcium was added and samples were taken at the indicated time points . Samples were immunoblotted for the Myc - tag and cyclin B2 . The cyclin B2 membrane was stripped and reprobed for α - tubulin . Asterisk indicates unspecific bands .

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

CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.


CSF extract was treated with Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT . An empty IVT reaction not expressing XErp1 was used as control . The extracts were treated with DMSO or CsA as indicated . Calcium was added , samples were taken at the indicated time points and immunoblotted for cyclin B2 , pSer335 XErp1 and α - tubulin . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisks indicate unspecific bands .

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

CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands.


Myc - XErp1 CaMKII - ZBR - ( T195A C583A ) IVT was in vitro phosphorylated by recombinant PKA and isolated by α - Myc immunoprecipitation . The immunoprecipitate was supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1 . The pSer335 XErp1 membrane was stripped and reprobed for the Myc - tag . Asterisk indicates the IgG heavy chain .

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

Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.


CSF extract was supplemented with mRNA encoding Flag - Cdc20 that was either wild - type or mutated to serine at Thr64 , Thr68 and Thr79 ( 3TS ) . After expression , calcium was added , samples were taken at the indicated time points and resolved by either conventional or Phos - tagTM SDS - PAGE . Samples were immunoblotted for the Flag - tag , Cdc27 and cyclin B2 .

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

CSF extract was supplemented with mRNA encoding Flag-Cdc20 that was either wild-type or mutated to serine at Thr64, Thr68 and Thr79 (3TS). After expression, calcium was added, samples were taken at the indicated time points and resolved by either conventional or Phos-tagTM SDS-PAGE. Samples were immunoblotted for the Flag-tag, Cdc27 and cyclin B2.


CSF extract was supplemented with Myc - XErp1 wt IVT and treated with DMSO or CsA . Calcium was added , samples were taken at the indicated time points , resolved by either conventional or Phos - tagTM SDS - PAGE and immunoblotted for Cdc20 , Cdc27 and cyclin B2 . A control sample was treated with λ - phosphatase . A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig . EV4A . The cyclin B2 membrane was stripped and reprobed for α - tubulin .

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

CSF extract was supplemented with Myc-XErp1 wt IVT and treated with DMSO or CsA. Calcium was added, samples were taken at the indicated time points, resolved by either conventional or Phos-tagTM SDS-PAGE and immunoblotted for Cdc20, Cdc27 and cyclin B2. A control sample was treated with λ-phosphatase. A pseudocoloured representation of the Cdc20 and the Cdc27 immunoblots is shown in Fig. EV4A. The cyclin B2 membrane was stripped and reprobed for α-tubulin.


CSF extract was supplemented with Flag - Cdc20 wt IVT coupled to α - Flag beads . Calcium was added and Flag - Cdc20 was isolated by α - Flag immunoprecipitation at the indicated time points . Input and pellet ( IP : Flag ) samples were immunoblotted for the Flag - tag , pThr68 Cdc20 and p150 ( Glued ) . The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2 . Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain . Asterisks in other immunoblots indicate unspecific bands .

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

CSF extract was supplemented with Flag-Cdc20 wt IVT coupled to α-Flag beads. Calcium was added and Flag-Cdc20 was isolated by α-Flag immunoprecipitation at the indicated time points. Input and pellet (IP: Flag) samples were immunoblotted for the Flag-tag, pThr68 Cdc20 and p150(Glued). The pThr68 Cdc20 membrane was stripped and reprobed for cyclin B2. Asterisk in the cyclin B2 immunoblot indicates the IgG heavy chain. Asterisks in other immunoblots indicate unspecific bands.


CSF extract was supplemented with Flag - Cdc20 wt IVT . Flag - Cdc20 was re - isolated by α - Flag immunoprecipitation , supplemented with calcium and treated with recombinant hs _ CaN and calmodulin as indicated . Samples were taken at the indicated time points and immunoblotted for the Flag - tag and pThr68 Cdc20 . The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA . Asterisk indicates the IgG heavy chain .

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

CSF extract was supplemented with Flag-Cdc20 wt IVT. Flag-Cdc20 was re-isolated by α-Flag immunoprecipitation, supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the Flag-tag and pThr68 Cdc20. The pThr68 Cdc20 membrane was stripped and reprobed for the catalytic calcineurin subunit CnA. Asterisk indicates the IgG heavy chain.


( a ) Left , cells were fixed and stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . Solid arrows indicate cells with active Src in adhesions ; dashed arrows show cells with active Src in puncta . Scale bars , 20 μm . Right , percentage of cells with active Src localizing to intracellular structures was quantified . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) .

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

(a) Left, cells were fixed and stained for anti-pTyr-416-Src (green) and with DAPI (blue). Solid arrows indicate cells with active Src in adhesions; dashed arrows show cells with active Src in puncta. Scale bars, 20 μm. Right, percentage of cells with active Src localizing to intracellular structures was quantified. Data are presented as mean±s.d. and significance is P0.001 (n=3).


( b ) Cells were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Merged and higher - magnification images of the outlined areas are also shown . Solid and dashed arrows show Src localization to adhesions or to puncta , respectively . Scale bars , 20 μm .

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

(b) Cells were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Merged and higher-magnification images of the outlined areas are also shown. Solid and dashed arrows show Src localization to adhesions or to puncta, respectively. Scale bars, 20 μm.


( c ) Lysates from cells were immunoblotted with anti - FAK , anti - Src , anti - Yes , anti - Fyn , anti - pTyr - 416 - Src and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(c) Lysates from cells were immunoblotted with anti-FAK, anti-Src, anti-Yes, anti-Fyn, anti-pTyr-416-Src and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Cells were fixed and stained for anti - pTyr - 416 - Src ( red ) and co - stained for anti - LC3B , anti - Atg7 or anti - Atg12 ( all green ) . Scale bars , 20 μm . ( b ) Higher - magnification images of the areas outlined in a . Solid arrows indicate co - localization in focal adhesions and dashed arrows show puncta in the cytoplasm .

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

(a) Cells were fixed and stained for anti-pTyr-416-Src (red) and co-stained for anti-LC3B, anti-Atg7 or anti-Atg12 (all green). Scale bars, 20 μm. (b) Higher-magnification images of the areas outlined in a. Solid arrows indicate co-localization in focal adhesions and dashed arrows show puncta in the cytoplasm.


( c ) LC3B was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - LC3B antibodies .

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

(c) LC3B was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-LC3B antibodies.


( d ) Cells were transfected with 40 nM scrambled , Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti - Atg5 , anti - Atg12 and anti - actin antibodies ( lower panels ) or fixed and stained for anti - pTyr - 416 - Src ( green ) , anti - paxillin ( red ) and with DAPI ( blue ) . Dashed arrows point to intracellular phospho - Src - containing puncta ; solid arrows point to peripheral phospho - Src . Scale bars , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(d) Cells were transfected with 40 nM scrambled, Atg5 or Atg12 siRNA for 72 h and immunoblotted with anti-Atg5, anti-Atg12 and anti-actin antibodies (lower panels) or fixed and stained for anti-pTyr-416-Src (green), anti-paxillin (red) and with DAPI (blue). Dashed arrows point to intracellular phospho-Src-containing puncta; solid arrows point to peripheral phospho-Src. Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , cells were transiently transfected with an RFP - GFP tandem fluorescent - tagged LC3 ( RFP - GFP - LC3 ) . Scale bars , 20 μm . Bottom , the number of yellow puncta and the number of RFP LC3 - positive puncta in the merged images were counted and the total number of puncta per cell was calculated . Data are presented as mean ± s . d . ( n = 3 ) .

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

(a) Top, cells were transiently transfected with an RFP-GFP tandem fluorescent-tagged LC3 (RFP-GFP-LC3). Scale bars, 20 μm. Bottom, the number of yellow puncta and the number of RFP LC3-positive puncta in the merged images were counted and the total number of puncta per cell was calculated. Data are presented as mean±s.d. (n=3).


b ) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti - pTyr - 416 - Src , anti - Src , anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown .

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

b) Cells were treated with 10 μM chloroquine for 24 h and then immunoblotted using anti-pTyr-416-Src, anti-Src, anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type following treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.


( c ) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti - pTyr - 416 - Src , anti - Src , anti - p53 and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(c) Cells were treated with 200 nM epoxomicin for 4 h and then immunoblotted and probed with anti-pTyr-416-Src, anti-Src, anti-p53 and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , FAK − / − cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti - Src antibody ( green ) . Solid arrows indicate Src at the cell periphery ; dashed arrows indicate Src in autophagosomes . Scale bars , 20 μm . Bottom , quantification of dasatinib - treated cells with Src in puncta . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) . Middle , lysates from cells treated with dasatinib were immunoblotted with anti - Src and anti - pTyr - 416 - Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti - LC3B and anti - actin antibodies . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown .

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

(a) Top, FAK−/− cells were treated with 200 nM dasatinib for 24 h and then fixed and stained for anti-Src antibody (green). Solid arrows indicate Src at the cell periphery; dashed arrows indicate Src in autophagosomes. Scale bars, 20 μm. Bottom, quantification of dasatinib-treated cells with Src in puncta. Data are presented as mean±s.d. and significance is P0.001 (n=3). Middle, lysates from cells treated with dasatinib were immunoblotted with anti-Src and anti-pTyr-416-Src and lysates from cells treated with dasatinib and chloroquine were immunoblotted with anti-LC3B and anti-actin antibodies. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each condition after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown.


( b ) Top , FAK − / − cells were transiently transfected with SrcY527F - GFP or with Src - 251 - GFP ( green ) and stained for anti - LC3B ( red ) . Scale bars , 20 μm . Bottom , quantification of cells with co - localization between Src and LC3B . Data are presented as mean ± s . d . and significance is P0 . 001 ( n = 3 ) .

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

(b) Top, FAK−/− cells were transiently transfected with SrcY527F-GFP or with Src-251-GFP (green) and stained for anti-LC3B (red). Scale bars, 20 μm. Bottom, quantification of cells with co-localization between Src and LC3B. Data are presented as mean±s.d. and significance is P0.001 (n=3).


( c ) Left , FAK − / − cells stably re - expressing wild - type FAK , FAKY397F or FAKY4F - Y9F were fixed and stained for anti - FAK ( red ) and anti - pTyr - 416 - Src ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate active Src in autophagosomes . Scale bars , 20 μm . Right , lysates from these cells were also immunoblotted with anti - FAK and anti - actin antibodies .

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

(c) Left, FAK−/− cells stably re-expressing wild-type FAK, FAKY397F or FAKY4F-Y9F were fixed and stained for anti-FAK (red) and anti-pTyr-416-Src (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate active Src in autophagosomes. Scale bars, 20 μm. Right, lysates from these cells were also immunoblotted with anti-FAK and anti-actin antibodies.


( d ) Left , wild - type FAK , FAKY397F or FAKY4F - Y9F cells were transfected with RFP - GFP - LC3 . Solid arrows indicate co - localization and dotted arrows indicate its absence . Scale bars , 20 μm .

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

(d) Left, wild-type FAK, FAKY397F or FAKY4F-Y9F cells were transfected with RFP-GFP-LC3. Solid arrows indicate co-localization and dotted arrows indicate its absence. Scale bars, 20 μm.


( e ) Lysates were immunoblotted with anti - LC3B and actin antibodies in the absence and presence of chloroquine . Densitometry was carried out to calculate the increase in the amount of LC3B - II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(e) Lysates were immunoblotted with anti-LC3B and actin antibodies in the absence and presence of chloroquine. Densitometry was carried out to calculate the increase in the amount of LC3B-II relative to actin for each cell type after treatment with chloroquine and is presented as a percentage increase for the immunoblot shown. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Wild - type FAK cells were suspended in 1 . 4 % methylcellulose solution in growth media and plated on agarose for 3 days . Top and bottom right , cells were recovered and cytospins prepared that were stained for anti - pTyr - 416 - Src ( red ) , LC3B ( green ) and with DAPI ( blue ) or for paxillin ( red ) , LC3B ( green ) and with DAPI ( blue ) . Higher - magnification images of the outlined areas are also shown . Solid arrows indicate localization of pTyr - 416 - Src in LC3B - positive puncta . Scale bars , 20 μm . Bottom left , immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti - pTyr - 397 FAK , anti - FAK , anti - pTyr - 416 and anti - Src antibodies .

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

(a) Wild-type FAK cells were suspended in 1.4% methylcellulose solution in growth media and plated on agarose for 3 days. Top and bottom right, cells were recovered and cytospins prepared that were stained for anti-pTyr-416-Src (red), LC3B (green) and with DAPI (blue) or for paxillin (red), LC3B (green) and with DAPI (blue). Higher-magnification images of the outlined areas are also shown. Solid arrows indicate localization of pTyr-416-Src in LC3B-positive puncta. Scale bars, 20 μm. Bottom left, immunoblotting was carried out on lysates from adherent cells and from cells recovered from methylcellulose using anti-pTyr-397 FAK, anti-FAK, anti-pTyr-416 and anti-Src antibodies.


( b ) FAK + / + cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti - pTyr - 416 - Src ( green ) and with DAPI ( blue ) . A higher - magnification image of the area outlined is also shown . Scale bar , 20 μm .

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

(b) FAK+/+ cells were suspended for 1 h in PBS and cytospins were prepared that were stained for anti-pTyr-416-Src (green) and with DAPI (blue). A higher-magnification image of the area outlined is also shown. Scale bar, 20 μm.


( c ) LC3B was immunoprecipitated ( IP ) from adherent or suspended FAK + / + and then immunoblotted with anti - Src and anti - LC3B antibodies . Lysates were also immunoblotted with anti - pTyr - 397 - FAK and anti - actin antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(c) LC3B was immunoprecipitated (IP) from adherent or suspended FAK+/+ and then immunoblotted with anti-Src and anti-LC3B antibodies. Lysates were also immunoblotted with anti-pTyr-397-FAK and anti-actin antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Top , FAK − / − cells were sparsely plated and then treated with 3 - MA ( for 48 h ) , chloroquine or dasatinib ( both for 24 h ) . Cells were then left at 37 ° C for 1 week to allow colony formation , fixed and then stained with crystal violet . Bottom , the number of colonies formed was then quantified and normalized against untreated controls . Data are presented as mean ± s . d . and significance is P0 . 001 for 3 - MA , P0 . 01 for chloroquine and P > 0 . 5 for dasatinib ( n = 3 ) .

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

(a) Top, FAK−/− cells were sparsely plated and then treated with 3-MA (for 48 h), chloroquine or dasatinib (both for 24 h). Cells were then left at 37 °C for 1 week to allow colony formation, fixed and then stained with crystal violet. Bottom, the number of colonies formed was then quantified and normalized against untreated controls. Data are presented as mean±s.d. and significance is P0.001 for 3-MA, P0.01 for chloroquine and P>0.5 for dasatinib (n=3).


( b ) Top , wild - type FAK and FAK − / − cells were transiently transfected with two individual Atg5 shRNAs ( 1 and 2 ) , selected in puromycin and then the clonogenic assay was carried out as described above . Bottom , quantification ; data are presented as mean ± s . d . and significance is P > 0 . 1 for Atg5 A and B in wild - type FAK cells and P0 . 01 for Atg5 A and P0 . 05 for Atg5 B in FAK − / − cells ( n = 3 ) .

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(b) Top, wild-type FAK and FAK−/− cells were transiently transfected with two individual Atg5 shRNAs (1 and 2), selected in puromycin and then the clonogenic assay was carried out as described above. Bottom, quantification; data are presented as mean±s.d. and significance is P>0.1 for Atg5 A and B in wild-type FAK cells and P0.01 for Atg5 A and P0.05 for Atg5 B in FAK−/− cells (n=3).


( c ) Cells were also stained with TUNEL . The positive control was DNase1 - treated cells and the negative control lacked TUNEL reaction mix . Quantification is shown and data are presented as mean ± s . d . and significance is P0 . 005 for 3 - MA and P0 . 001 for Atg5 siRNA ( n = 3 ) .

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(c) Cells were also stained with TUNEL. The positive control was DNase1-treated cells and the negative control lacked TUNEL reaction mix. Quantification is shown and data are presented as mean±s.d. and significance is P0.005 for 3-MA and P0.001 for Atg5 siRNA (n=3).


( d ) FAK − / − cells were untreated or treated with dasatinib , 3 - MA , chloroquine or bafilomycin A ( BFA ) for 24 h , or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti - PARP antibody . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(d) FAK−/− cells were untreated or treated with dasatinib, 3-MA, chloroquine or bafilomycin A (BFA) for 24 h, or were transfected with scrambled or Atg5 siRNA for 72 h and then immunoblotted with an anti-PARP antibody. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) Cells untreated , or treated with chloroquine for 24 h , were fixed and stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ) . Solid arrows indicate co - localization at adhesions and dashed arrows indicate co - localization in autophagosomes . Scale bars , 20 μm .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O

(a) Cells untreated, or treated with chloroquine for 24 h, were fixed and stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green). Solid arrows indicate co-localization at adhesions and dashed arrows indicate co-localization in autophagosomes. Scale bars, 20 μm.


( b ) c - Cbl was immunoprecipitated ( IP ) from FAK + / + and FAK − / − cells and then immunoblotted with anti - Src , anti - pTyr - 416 - Src and anti - c - Cbl antibodies .

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(b) c-Cbl was immunoprecipitated (IP) from FAK+/+ and FAK−/− cells and then immunoblotted with anti-Src, anti-pTyr-416-Src and anti-c-Cbl antibodies.


( c , d ) Cells were transfected with scrambled siRNA or with c - Cbl siRNA for 72 h and then immunoblotted with anti - c - Cbl and actin antibodies ( c ) or stained for anti - pTyr - 416 - Src ( red ) and anti - c - Cbl ( green ; d , left ) . Dashed arrows indicate co - localization and solid arrows indicate Src at focal adhesions . Scale bars , 20 μm . Quantification is shown ( d , right ) . Data are presented as mean ± s . d . and significance is P > 0 . 5 for FAK + / + cells and P0 . 001 for FAK − / − cells ( n = 3 ) .

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

(c,d) Cells were transfected with scrambled siRNA or with c-Cbl siRNA for 72 h and then immunoblotted with anti-c-Cbl and actin antibodies (c) or stained for anti-pTyr-416-Src (red) and anti-c-Cbl (green; d, left). Dashed arrows indicate co-localization and solid arrows indicate Src at focal adhesions. Scale bars, 20 μm. Quantification is shown (d, right). Data are presented as mean±s.d. and significance is P>0.5 for FAK+/+ cells and P0.001 for FAK−/− cells (n=3).


( e ) LC3B was immunoprecipitated from FAK − / − cells expressing either scrambled or c - Cbl siRNA and then immunoblotted with anti - Src and anti - LC3B antibodies . Uncropped images of blots are shown in Supplementary Fig . S9 .

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(e) LC3B was immunoprecipitated from FAK−/− cells expressing either scrambled or c-Cbl siRNA and then immunoblotted with anti-Src and anti-LC3B antibodies. Uncropped images of blots are shown in Supplementary Fig. S9.


( a ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA resistant , HA - tagged wild - type c - Cbl or c - Cbl mutants with defective E3 ligase activity ( c - CblC381A or c - Cbl - 70Z ) . Left , cells were stained for anti - p - Tyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows show co - localization in intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P > 0 . 5 ( n = 3 ) .

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

(a) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA resistant, HA-tagged wild-type c-Cbl or c-Cbl mutants with defective E3 ligase activity (c-CblC381A or c-Cbl-70Z). Left, cells were stained for anti-p-Tyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows show co-localization in intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P>0.5 (n=3).


( c ) GST - pulldown assay of HA - tagged wild - type c - Cbl , c - CblAA ( W802A , L805A ) and c - CblAAAA ( W802A , L803A , S804A , L805A ) expressed in HEK293T cells was carried out using GST - LC3 .

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(c) GST-pulldown assay of HA-tagged wild-type c-Cbl, c-CblAA (W802A, L805A) and c-CblAAAA (W802A, L803A, S804A, L805A) expressed in HEK293T cells was carried out using GST-LC3.


( d ) c - Cbl siRNA was transfected into FAK − / − cells expressing siRNA - resistant HA - tagged wild - type c - Cbl or c - CblAAAA . Left , cells were stained for anti - pTyr - 416 - Src ( green ) , anti - HA ( red ) and with DAPI ( blue ) . Solid arrows indicate pTyr - 416 localization to adhesions and dashed arrows indicate localization to intracellular puncta . Scale bars , 20 μm . Right , quantification of percentage of cells that contained active Src in intracellular puncta . Data are presented as mean ± s . d . and significance is P0 . 01 ( n = 3 ) . Uncropped images of blots are shown in Supplementary Fig . S9 .

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

(d) c-Cbl siRNA was transfected into FAK−/− cells expressing siRNA-resistant HA-tagged wild-type c-Cbl or c-CblAAAA. Left, cells were stained for anti-pTyr-416-Src (green), anti-HA (red) and with DAPI (blue). Solid arrows indicate pTyr-416 localization to adhesions and dashed arrows indicate localization to intracellular puncta. Scale bars, 20 μm. Right, quantification of percentage of cells that contained active Src in intracellular puncta. Data are presented as mean±s.d. and significance is P0.01 (n=3). Uncropped images of blots are shown in Supplementary Fig. S9.


( A ) The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice ( n = 8 ) and lean controls ( n = 8 ) .

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

(A)The mRNA levels of QKI relative to those of 36B4 in BAT and ingWAT from HFD mice (n = 8) and lean controls (n = 8).


WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed a NCD or HFD for 20 weeks . The arrow indicates the time when the high fat diet （ HFD ） starts , and body weight was monitored weekly . ( n = 10 , per genotype )

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WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed a NCD or HFD for 20 weeks. The arrow indicates the time when the high fat diet（HFD）starts, and body weight was monitored weekly. (n = 10, per genotype)


Representative pictures of adipocyte - specific QKI - deficient mice under NCD or HFD conditions .

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Representative pictures of adipocyte-specific QKI-deficient mice under NCD or HFD conditions.


Gross appearance of tissues from BAT , eWAT and ingWAT fat pads from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice after 20 weeks on the NCD or HFD .

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Gross appearance of tissues from BAT, eWAT and ingWAT fat pads from WT fl/fl and QKI KO fl/fl:AP2-Cre mice after 20 weeks on the NCD or HFD.


H & E staining of BAT , eWAT and ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm )

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

H&E staining of BAT, eWAT and ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)


The gross appearance of BAT , eWAT and ingWAT fat pads after administration of AAV - ShQKI or AAV - NC vectors in local adipose ( ingWAT and BAT ) .

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

The gross appearance of BAT, eWAT and ingWAT fat pads after administration of AAV-ShQKI or AAV-NC vectors in local adipose (ingWAT and BAT).


The increase in relative body weights following HFD feeding . ( n = 8 , per group )

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The increase in relative body weights following HFD feeding. (n = 8, per group)


H & E staining of the liver , BAT and ingWAT from AAV - NC or AAV - ShQKI mice . ( Scale bar represents 50 μm ) .

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H&E staining of the liver, BAT and ingWAT from AAV-NC or AAV-ShQKI mice. (Scale bar represents 50 μm).


Representative CT images of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on an NCD or HFD , showing the whole - body fat composition .

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

Representative CT images of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on an NCD or HFD, showing the whole-body fat composition.


Glucose tolerance tests ( GTT ) and Insulin tolerance tests ( ITT ) were performed on WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . Blood glucose concentrations during GTT and ITT were monitored . ( n = 10 , per genotype )

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

Glucose tolerance tests (GTT) and Insulin tolerance tests (ITT) were performed on WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. Blood glucose concentrations during GTT and ITT were monitored. (n= 10, per genotype)


Blood glucose and plasma insulin levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype )

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Blood glucose and plasma insulin levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)


Plasma Free Fatty Acid ( FFA ) and Glycerol levels in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( n = 8 , per genotype )

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

Plasma Free Fatty Acid (FFA) and Glycerol levels in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (n = 8, per genotype)


H & E and Oil Red O staining , and liver triglyceride ( TG ) content in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice fed the NCD or HFD for 20 weeks . ( Scale bar represents 50 μm )

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

H&E and Oil Red O staining, and liver triglyceride (TG) content in WT fl/fl and QKI KO fl/fl:AP2-Cre mice fed the NCD or HFD for 20 weeks. (Scale bar represents 50 μm)


O2 consumption rates were measured via indirect calorimetry using CLAMS under chow - fed conditions ( n = 8 , per genotype ) . And average light and dark O2 consumption rates are shown on the right .

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

O2 consumption rates were measured via indirect calorimetry using CLAMS under chow-fed conditions (n = 8, per genotype). And average light and dark O2 consumption rates are shown on the right.


Heat production rates in WT fl / fl and QKI KO fl / fl : AP2 - Cre mice ( n = 8 , per genotype ) . And average light and dark heat production rates are shown on the right .

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

Heat production rates in WT fl/fl and QKI KO fl/fl:AP2-Cre mice (n = 8, per genotype). And average light and dark heat production rates are shown on the right.


Heatmaps of log2 ( fold change QKI KO fl / fl : AP2 - Cre BAT vs . WT fl / fl BAT ) for genes involved in thermogenesis and oxidation . ( 3 samples , per group )

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

Heatmaps of log2 (fold change QKI KO fl/fl:AP2-Cre BAT vs. WT fl/fl BAT) for genes involved in thermogenesis and oxidation. (3 samples, per group)


Photograph of WT fl / fl and QKI KO fl / fl : AP2 - Cre classical BAT in 4 % paraformaldehyde solution , representative transmission electron micrographs , and UCP1 staining of BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice .

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Photograph of WT fl/fl and QKI KO fl/fl:AP2-Cre classical BAT in 4% paraformaldehyde solution, representative transmission electron micrographs, and UCP1 staining of BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice.


The average diameter of lipid droplets and the number of mitochondria per field from WT fl / fl and QKI KO fl / fl : AP2 - Cre BAT . ( n = 3 biological replicates )

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

The average diameter of lipid droplets and the number of mitochondria per field from WT fl/fl and QKI KO fl/fl:AP2-Cre BAT. (n = 3 biological replicates)


The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT - PCR . ( n = 4 biological replicates )

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

The relative mRNA levels of genes related to thermogenesis in BAT analyzed by RT-PCR. (n = 4 biological replicates)


Total oxygen consumption rates ( OCR ) per gram in BAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions were measured using a Clark electrode . ( n = 3 biological replicates )

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

Total oxygen consumption rates (OCR) per gram in BAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions were measured using a Clark electrode. (n = 3 biological replicates)


The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT - PCR . ( n = 4 biological replicates )

The relative mRNA levels of genes related to thermogenesis in ingWAT analyzed by RT-PCR. (n = 4 biological replicates)


Total oxygen consumption rates ( OCR ) per gram in ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice under basal conditions . ( n = 3 biological replicates )

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

Total oxygen consumption rates (OCR) per gram in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice under basal conditions. (n = 3 biological replicates)


Representative recording of Continuous measurement of oxygen consumption rate ( OCR ) in isolated mitochondria from BAT of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice on NCD ; Oxygen consumption was performed under basal conditions , following the addition of 0 . 25 mg mitochondria ( Mit ) , 5 mM substrate glycerol - 3 - phosphate ( G3P ) , 2 mM GDP , and 450 mM ADP , 2 μg / ml oligomycin , and 1 . 5 μM FCCP . Quantification of experimental data . ( n = 3 biological replicates , per group )

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

Representative recording of Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from BAT of WT fl/fl and QKI KO fl/fl:AP2-Cre mice on NCD; Oxygen consumption was performed under basal conditions, following the addition of 0. 25 mg mitochondria (Mit), 5 mM substrate glycerol-3-phosphate (G3P), 2 mM GDP, and 450 mM ADP, 2 μg/ml oligomycin, and 1.5 μM FCCP. Quantification of experimental data. (n=3 biological replicates, per group)


UCP1 - dependent oxygen consumption rates estimated as GDP - inhabitable rates , based on the data shown in ( K ) . ( n = 3 biological replicates )

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UCP1-dependent oxygen consumption rates estimated as GDP-inhabitable rates, based on the data shown in (K). (n=3 biological replicates)


The protein levels of PGC1α and QKI in siRNA - mediated QKI knockdown differentiated cell , and the quantification graph was shown on the right . ( n = 3 biological replicates )

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

The protein levels of PGC1α and QKI in siRNA-mediated QKI knockdown differentiated cell, and the quantification graph was shown on the right. (n=3 biological replicates)


Immunostaining of PGC1α and UCP1 in NC , Si - QKI 1 , and Si - QKI 2 adipocytes . ( Scale bar represents 20 μm ) .

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Immunostaining of PGC1α and UCP1 in NC, Si-QKI 1, and Si-QKI 2 adipocytes. (Scale bar represents 20 μm).


The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates )

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

The relative mRNA levels of genes related to thermogenesis in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)


The oxygen consumption rates in adipocytes treated with NC , QKI siRNA or both QKI and PGC1α siRNA . ( n = 3 biological replicates )

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

The oxygen consumption rates in adipocytes treated with NC, QKI siRNA or both QKI and PGC1α siRNA. (n=3 biological replicates)


Representative pictures of oil red O staining in three adipocyte types : ShScramble + ovCon cells , QKI knockdown cells ( ShQKI + ovCon ) and QKI knockdown with QKI - 5 re - expression cells ( ShQKI + ovQKI ) .

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

Representative pictures of oil red O staining in three adipocyte types: ShScramble+ovCon cells, QKI knockdown cells (ShQKI+ovCon) and QKI knockdown with QKI-5 re-expression cells (ShQKI+ovQKI).


The protein levels of PGC1α in these adipocytes , and the quantification graph was shown on the right . ( n = 3 biological replicates )

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

The protein levels of PGC1α in these adipocytes, and the quantification graph was shown on the right. (n=3 biological replicates)


The relative mRNA levels of genes associated with energy dissipation in ShScramble + ovCon , ShQKI + ovCon and ShQKI + ovQKI differentiated cells . The results represent 3 independent experiments .

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

The relative mRNA levels of genes associated with energy dissipation in ShScramble+ovCon, ShQKI+ovCon and ShQKI+ovQKI differentiated cells. The results represent 3 independent experiments.


Construction of a series of luciferase reporter vectors ( PGC1α full - length 3 ' UTR T , A , B , C fragments and PGC1α 3 ' UTR QRE motif sites mutant B M1 , B M2 , C M1 , C M2 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3 ' UTR affected by QKI . ( n = 4 biological replicates )

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

Construction of a series of luciferase reporter vectors (PGC1α full-length 3'UTR T, A, B, C fragments and PGC1α 3'UTR QRE motif sites mutant B M1, B M2, C M1, C M2 fragments). Luciferase activity assays to examine the functional QRE motif sites in the PGC1α 3'UTR affected by QKI. (n=4 biological replicates)


Construction of a series of luciferase reporter vectors ( UCP1 full - length 3 ' UTR T fragment and UCP1 3 ' UTR QRE motif sites mutant T M1 , T M2 , T M3 fragments ) . Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3 ' UTR affected by QKI . ( n = 4 biological replicates )

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

Construction of a series of luciferase reporter vectors (UCP1 full-length 3'UTR T fragment and UCP1 3'UTR QRE motif sites mutant T M1, T M2, T M3 fragments). Luciferase activity assays to examine the functional QRE motif sites in the UCP1 3'UTR affected by QKI. (n=4 biological replicates)


Based on RIP - sequencing data , the distribution of QKI - binding peaks within UCP1 and PGC1α transcript regions .

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

Based on RIP-sequencing data, the distribution of QKI-binding peaks within UCP1 and PGC1α transcript regions.


RNA immunoprecipitation followed by real - time PCR . Specific primer for PGC1α 3 ' UTR region 1 , region 2 and UCP1 3 ' UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue , and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP . ( n = 4 biological replicates )

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

RNA immunoprecipitation followed by real-time PCR. Specific primer for PGC1α 3'UTR region 1, region 2 and UCP1 3'UTR to detect these QRE motif sites region bound to endogenous QKI in brown fat tissue, and data showing the PGC1α and UCP1 transcripts abundance in QKI IP compared with IgG IP. (n=4 biological replicates)


293T cells were transfected with PGC1α wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of

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

293T cells were transfected with PGC1α wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of PGC1α


293T cells were transfected with UCP1 wild type or QRE sites mutant 3 ' UTR fragments . RNA immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . The results showed that endogenous QKI interacted with full - length wild type QRE motif regions within 3 ' UTRs of UCP1 .

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

293T cells were transfected with UCP1 wild type or QRE sites mutant 3'UTR fragments. RNA immunoprecipitation (RIP) to identify these QRE motif sites region bound to endogenous QKI by using anti-QKI antibodies. The results showed that endogenous QKI interacted with full-length wild type QRE motif regions within 3'UTRs of UCP1.


The mRNA stability in adipocytes was measured by incubating cells with actinomycin D ( 5 μg / ml ) . Actinomycin D was added to stop transcription , and RNAs were harvested at the indicated times ( x - axis ) after transcription inhibition . Real - time PCR was used to determine remaining UCP1 , PGC1α and Actin mRNA levels compared with the starting time . Increasing PGC1α mRNA half - life by depletion of QKI ( ShScramble T1 / 2 = 4 . 73 h , ShQKI T1 / 2 = 8 . 04 h ) . ( n = 4 biological replicates )

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

The mRNA stability in adipocytes was measured by incubating cells with actinomycin D (5 μg/ml). Actinomycin D was added to stop transcription, and RNAs were harvested at the indicated times (x-axis) after transcription inhibition. Real-time PCR was used to determine remaining UCP1, PGC1α and Actin mRNA levels compared with the starting time. Increasing PGC1α mRNA half-life by depletion of QKI (ShScramble T1/2 = 4.73 h, ShQKI T1/2 = 8.04 h). (n=4 biological replicates)


The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers ( GAPDH and Lamin B1 ) to show the purity of fractions .

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

The nuclear and cytoplasmic fractionation was valid by immunoblotting using cytoplasmic and nuclear protein markers (GAPDH and Lamin B1) to show the purity of fractions.


Real - time PCR analysis of mRNA transcript levels of the QKI targets UCP1 and PGC1α in the nuclear and cytoplasmic fractions of differentiated MEF cells transfected with ShScramble or ShQKI . Values represent the cytoplasmic - to - nuclear ratio of each transcript . Actin served as a negative control . ( n = 4 biological replicates )

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

Real-time PCR analysis of mRNA transcript levels of the QKI targets UCP1 and PGC1α in the nuclear and cytoplasmic fractions of differentiated MEF cells transfected with ShScramble or ShQKI. Values represent the cytoplasmic-to-nuclear ratio of each transcript. Actin served as a negative control. (n=4 biological replicates)


RNAs from isolated nuclear and cytoplasmic fractionation were harvested after RNA immunoprecipitation ( RIP ) of RNA - protein complexes using either anti - QKI antibody or control IgG . And RIP - RT - PCR showing the PGC1α and UCP1 transcripts majorly interacted with endogenous QKI in the nucleus . ( n = 3 biological replicates )

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

RNAs from isolated nuclear and cytoplasmic fractionation were harvested after RNA immunoprecipitation (RIP) of RNA-protein complexes using either anti-QKI antibody or control IgG. And RIP-RT-PCR showing the PGC1α and UCP1 transcripts majorly interacted with endogenous QKI in the nucleus. (n=3 biological replicates)


Control ( ShScramble ) and QKI knockdown ( ShQKI ) adipocyte lysates were subjected to polysome profiling , and a representative polysome profile is shown . Total RNA was extracted from each fraction , and the distribution of UCP1 and PGC1α mRNA was measured via real - time PCR ( K and L ) . The results are shown as the percentage of total mRNA ( % mRNA ) in polysome fractions and represent 3 independent experiments .

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

Control (ShScramble) and QKI knockdown (ShQKI) adipocyte lysates were subjected to polysome profiling, and a representative polysome profile is shown. Total RNA was extracted from each fraction, and the distribution of UCP1 and PGC1α mRNA was measured via real-time PCR (K and L). The results are shown as the percentage of total mRNA (% mRNA) in polysome fractions and represent 3 independent experiments.


The rectal temperature of WT fl / fl and QKI KO fl / fl : AP2 - Cre mice was monitored upon acute cold exposure ( 4 ° C ) . ( n = 10 mice , per group )

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The rectal temperature of WT fl/fl and QKI KO fl/fl:AP2-Cre mice was monitored upon acute cold exposure (4 °C). (n = 10 mice, per group)


H & E staining and UCP1 immunostaining of sections of ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice at RT ( 22 ° C ) or following 72 h of cold exposure ( 4 ° C ) . ( Scale bar : 50 μm ) .

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H&E staining and UCP1 immunostaining of sections of ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT (22 °C) or following 72 h of cold exposure (4 °C). (Scale bar: 50 μm).


The protein levels of UCP1 in ingWAT from WT fl / fl and QKI KO fl / fl : AP2 - Cre mice at RT or following 72 h of cold exposure ( 4 ° C ) , and quantified protein levels are shown in ( D ) . ( n = 3 biological replicates , per group )

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The protein levels of UCP1 in ingWAT from WT fl/fl and QKI KO fl/fl:AP2-Cre mice at RT or following 72 h of cold exposure (4 °C), and quantified protein levels are shown in (D). (n=3 biological replicates, per group)


Red Nile staining in BAT injected with Control or QKI knockdown AAV at RT ( 22 ° C ) or following 6 h and 72 h of cold exposure ( 4 ° C ) .

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Red Nile staining in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).


TG content in BAT injected with Control or QKI knockdown AAV at RT ( 22 ° C ) or following 6 h and 72 h of cold exposure ( 4 ° C ) .

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TG content in BAT injected with Control or QKI knockdown AAV at RT (22 °C) or following 6 h and 72 h of cold exposure (4 °C).


The UCP1 protein level in BAT at RT or following 6 h and 72 h of cold exposure ( 4 ° C ) and the quantified protein levels . ( n = 4 biological replicates , per group )

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The UCP1 protein level in BAT at RT or following 6 h and 72 h of cold exposure (4 °C) and the quantified protein levels. (n = 4 biological replicates, per group)


The mRNA levels of genes related to energy consumption ( PGC1α , UCP1 and Dio2 ) . ( n = 4 biological replicates , per group )

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

The mRNA levels of genes related to energy consumption (PGC1α, UCP1 and Dio2). (n=4 biological replicates, per group)


The protein levels of QKI in BAT and ingWAT during cold stimulation ( 4 ° C ) and the quantified protein levels . ( n = 3 biological replicates )

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The protein levels of QKI in BAT and ingWAT during cold stimulation (4 °C) and the quantified protein levels. (n=3 biological replicates)


The relative luminescence of a QKI promoter reporter under cAMP ( Forskolin and IBMX ) stimulus . ( n = 3 biological replicates )

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The relative luminescence of a QKI promoter reporter under cAMP (Forskolin and IBMX) stimulus. (n=3 biological replicates)


The effect of transcriptional factors on the QKI promoter , assessed by luciferase activity assays . ( n = 3 biological replicates )

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The effect of transcriptional factors on the QKI promoter, assessed by luciferase activity assays. (n=3 biological replicates)


The relative mRNA levels of genes ( UCP1 , PGC1α , Dio2 ) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus , following cAMP ( Forskolin and IBMX ) stimulation . ( n = 3 biological replicates )

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The relative mRNA levels of genes (UCP1, PGC1α, Dio2) in differentiated MEFs transfected with ShScramble or ShQKI lentivirus, following cAMP (Forskolin and IBMX) stimulation. (n=3 biological replicates)


, body weight trajectories of the mice .

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, body weight trajectories of the mice.


L , M , N , O The blood glucose level of mice after intraperitoneal injection of glucose or insulin for glucose ( GTT ) ( L ) and insulin tolerance tests ( ITT ) ( N ) , and AUC , area under the curve ( M , O ) .

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L, M, N, O The blood glucose level of mice after intraperitoneal injection of glucose or insulin for glucose (GTT) (L) and insulin tolerance tests (ITT) (N), and AUC, area under the curve (M, O).


A ORO staining of SVF cells isolated from iWAT of male mice . Differentiation was induced with curcumin up to day 8 . C ORO staining of 3T3 - L1 cells . Differentiation was induced with curcumin up to day 8 .

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A ORO staining of SVF cells isolated from iWAT of male mice. Differentiation was induced with curcumin up to day 8. C ORO staining of 3T3-L1 cells. Differentiation was induced with curcumin up to day 8.


B mRNA expression of adipogenesis - related factors of SVF cells isolated from iWAT of male mice . D mRNA expression of PPARγ , C / EBPα and FABP4 expression of 3T3 - L1 cells treated with curcumin .

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B mRNA expression of adipogenesis-related factors of SVF cells isolated from iWAT of male mice. D mRNA expression of PPARγ, C/EBPα and FABP4 expression of 3T3-L1 cells treated with curcumin.


protein expression levels of m6A regulator in iWAT from mice in HFD and HFD + CUR groups . protein expression levels of m6A regulator in 3T3 - L1 cells were

protein expression levels of m6A regulator in iWAT from mice in HFD and HFD+CUR groups. protein expression levels of m6A regulator in 3T3-L1 cells were analyzed


K Oil Red O staining of Vector or OE - ALKBH5 transfected 3T3 - L1 cells . Differentiation was induced with curcumin up to day 8 .

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K Oil Red O staining of Vector or OE-ALKBH5 transfected 3T3-L1 cells. Differentiation was induced with curcumin up to day 8.


E Oil Red O staining of control , ALKBH5 - depleted and ALKBH5 + TRAF4 depleted cells after induced for 8 days .

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E Oil Red O staining of control, ALKBH5-depleted and ALKBH5+TRAF4 depleted cells after induced for 8 days.


G Western blot analysis of PPARγ and C / EBPα protein levels in cells , and quantification of protein levels normalized to β - actin expression .

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G Western blot analysis of PPARγ and C/EBPα protein levels in cells , and quantification of protein levels normalized to β-actin expression.


Western blot analysis of YTHDF1 , ALKBH5 and TRAF4 in control , ALKBH5 - depleted and ALKBH5 + YTHDF1 depleted cells and quantification of protein levels normalized to β - actin expression .

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Western blot analysis of YTHDF1, ALKBH5 and TRAF4 in control, ALKBH5-depleted and ALKBH5 + YTHDF1 depleted cells and quantification of protein levels normalized to β-actin expression.


L Oil Red O staining of control , ALKBH5 - depleted and ALKBH5 + YTHDF1 depleted cells on day 8 of differentiation .

L Oil Red O staining of control, ALKBH5-depleted and ALKBH5+YTHDF1 depleted cells on day 8 of differentiation.


M qPCR analysis of PPARγ , C / EBPα and FABP4 expression in cell β - Actin was used as an internal control .

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M qPCR analysis of PPARγ, C/EBPα and FABP4 expression in cell β-Actin was used as an internal control.


C , D Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells in the absence or presence of MG132 or 3MA and quantification of protein levels normalized to β - actin expression .

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

C, D Western blot analysis of PPARγ protein levels in control and TRAF4 overexpressing cells in the absence or presence of MG132 or 3MA and quantification of protein levels normalized to β-actin expression.


E Ubiquitination of endogenous PPARγ in control and TRAF4 overexpressing cells were differentiated by treatment with DMI . Cells were treated with MG132 for 6 h .

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E Ubiquitination of endogenous PPARγ in control and TRAF4 overexpressing cells were differentiated by treatment with DMI. Cells were treated with MG132 for 6 h.


( A ) Daily 10 μg 4 - AP enhanced recovery of sciatic nerve motor function as compared with treatment with vehicle at 3 days post injury ( dpi ) through 8 dpi . ( * : Day3 ( D3 ) , p = 0 . 0131 ; D5 , p = 0 . 0475 ; D8 , p = 0 . 0472 ; n = 6 ; ANOVA with post - hoc comparisons using two - tailed unpaired t - test )

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(A) Daily 10 μg 4-AP enhanced recovery of sciatic nerve motor function as compared with treatment with vehicle at 3 days post injury (dpi) through 8 dpi. (*: Day3 (D3), p= 0.0131; D5, p = 0.0475; D8, p =0.0472; n=6; ANOVA with post-hoc comparisons using two-tailed unpaired t-test)


( B ) Daily 10 μg 4 - AP administration also enhanced recovery of nerve conduction velocity ( NCV ) as observed beginning at 21 days post - injury , eventually restoring NCV to near - normal values while NCV in vehicle - treated mice remained less than half that of uninjured animals ( * : D21 , p = 0 . 0454 ; D28 , p = 0 . 0487 ; D35 , p = 0 . 0475 ; n = 5 ; ANOVA with post - hoc comparisons using two - tailed unpaired t - test ) .

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(B) Daily 10 μg 4-AP administration also enhanced recovery of nerve conduction velocity (NCV) as observed beginning at 21 days post-injury, eventually restoring NCV to near-normal values while NCV in vehicle-treated mice remained less than half that of uninjured animals (*: D21, p= 0.0454; D28, p = 0.0487; D35, p =0.0475; n=5; ANOVA with post-hoc comparisons using two-tailed unpaired t-test).


( C , D ) Analysis of withdrawal latency in response to thermal ( C ) or mechanical ( D ) stimuli revealed that 4 - AP treatment did not worsen these diagnostics of neuropathic pain syndromes . In the case of response to thermal hyperalgesis , 4 - AP treated mice showed a significantly more rapid return to baseline levels . ( * : ( C ) D3 , D5 , D8 : p < 0 . 001 saline vs . baseline ; D3 p = 0 . 006 ; 4 - AP vs . baseline ; ( D ) D3 , p = 0 . 014 ; D5 , p = 0 . 008 ; saline versus baseline ; D3 , p = 0 . 006 4 - AP vs . baseline ; D8 : p = 0 . 0472 ; n = 10 ; ANOVA with post - hoc comparisons using two - tailed unpaired t - test . ) In this and all other figures data is mean + SEM and shows a representative experiment from 2 - 3 repetitions .

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(C, D) Analysis of withdrawal latency in response to thermal (C) or mechanical (D) stimuli revealed that 4-AP treatment did not worsen these diagnostics of neuropathic pain syndromes. In the case of response to thermal hyperalgesis, 4-AP treated mice showed a significantly more rapid return to baseline levels. (*: (C) D3, D5, D8 : p<0.001 saline vs. baseline; D3 p= 0.006; 4-AP vs. baseline; (D) D3, p= 0.014; D5, p = 0.008; saline versus baseline; D3, p = 0.006 4-AP vs. baseline; D8: p =0.0472; n=10 ; ANOVA with post-hoc comparisons using two-tailed unpaired t-test.) In this and all other figures data is mean + SEM and shows a representative experiment from 2-3 repetitions.


( A ) Local 4 - AP treated crushed sciatic nerve ( black : vehicle PLGA films ; red : ( 4 - AP ) - PLGA films ) regained partial walking ability as early as 3 days post - injury compared to vehicle treated group . ( * : D5 , p = 0 . 0004 ; D8 , p = 0 . 0012 ; D11 , p = 0 . 003 ; D14 , p = 0 . 0089 ; n = 6 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) .

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(A) Local 4-AP treated crushed sciatic nerve (black: vehicle PLGA films; red: (4-AP)-PLGA films) regained partial walking ability as early as 3 days post-injury compared to vehicle treated group. (*: D5, p= 0.0004; D8, p = 0.0012; D11, p =0.003; D14, p=0.0089; n=6; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).


( B ) Local 4 - AP treated crushed sciatic nerve ( black : vehicle PLGA films ; red : ( 4 - AP ) - PLGA films ) showed faster improvement in NCV restoration compared with vehicle - treated mice , beginning at 21 days post - injury .

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(B) Local 4-AP treated crushed sciatic nerve (black: vehicle PLGA films; red: (4-AP)-PLGA films) showed faster improvement in NCV restoration compared with vehicle-treated mice, beginning at 21 days post-injury.


( A ) Electron microscopy images of healthy , crushed , and 4 - AP treated crushed sciatic nerve at 21 days post - injury . Scale bar at 3000x = 5m ; scale bar at 8000x = 2m ) ( B ) Comparison of the axonal area of randomly chosen individual axons of vehicle treated and 4 - AP treated mice ( n = 4 for each experimental group ; 40 axons analyzed per mouse ) . 4 - AP treated sciatic nerve showed statistically greater axonal area compared to the vehicle - treated group ( p < 0 . 05 ; ANOVA ; restricted - maximum - likelihood ) . Moreover , 4 - AP treated mice had a greater proportion of axons with areas greater than the mean value for uninjured mice ( shown in the green line ) , with inset figure displaying all axons with values above this mean . This experiment represents a single group of mice of one of three replicates on NCV recovery .

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

(A) Electron microscopy images of healthy, crushed, and 4-AP treated crushed sciatic nerve at 21 days post-injury. Scale bar at 3000x = 5m; scale bar at 8000x = 2m) (B) Comparison of the axonal area of randomly chosen individual axons of vehicle treated and 4-AP treated mice (n=4 for each experimental group; 40 axons analyzed per mouse). 4-AP treated sciatic nerve showed statistically greater axonal area compared to the vehicle-treated group (p<0.05; ANOVA; restricted-maximum-likelihood). Moreover, 4-AP treated mice had a greater proportion of axons with areas greater than the mean value for uninjured mice (shown in the green line), with inset figure displaying all axons with values above this mean. This experiment represents a single group of mice of one of three replicates on NCV recovery.


( A , B ) Sustained local 4 - AP administration was associated with increased myelin area and close - to - normal Garea - ratio compared to untreated group , as determined by analysis of 40 randomly chosen myelinated axons from sections of 4 nerves for each group . ( * * : p < 0 . 0001 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) . 4 - AP treated mice had a greater proportion of axons for which the associated myelin area was greater than the mean value for uninjured mice ( shown in the green line ) , with inset figure displaying all axons with values above this mean .

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(A, B) Sustained local 4-AP administration was associated with increased myelin area and close-to-normal Garea-ratio compared to untreated group, as determined by analysis of 40 randomly chosen myelinated axons from sections of 4 nerves for each group. (**: p<0.0001; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). 4-AP treated mice had a greater proportion of axons for which the associated myelin area was greater than the mean value for uninjured mice (shown in the green line), with inset figure displaying all axons with values above this mean.


( C , D ) 4 - AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis ( * * : p < 0 . 01 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) .

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(C, D) 4-AP treated nerves also showed increases in the levels of P0 protein as detected by western blot analysis (**:p<0.01; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test).


( E ) Increases in P0 protein over time also were observed by immunofluorescence analysis at different time points , with P0 protein expression increasing to a greater extent in nerves of mice treated with 4 - AP . Scale bar = 200 m .

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(E) Increases in P0 protein over time also were observed by immunofluorescence analysis at different time points, with P0 protein expression increasing to a greater extent in nerves of mice treated with 4-AP. Scale bar = 200 m.


( F , G ) 4 - AP treatment increased the number of myelinated axons ( * * : p = 0 . 002 , n = 4 ; ANOVA , with post - hoc comparisons using two - tailed unpaired t - test ) . Even though the 4 - AP treated group also exhibited a greater number of total axons , this difference was not statistically significant . All myelinated and total axons were counted in 5 randomly chosen grids from each of 4 nerves for each group . This experiment represents a single group of mice of one of three replicates on NCV recovery .

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(F, G) 4-AP treatment increased the number of myelinated axons (**: p=0.002, n=4; ANOVA, with post-hoc comparisons using two-tailed unpaired t-test). Even though the 4-AP treated group also exhibited a greater number of total axons, this difference was not statistically significant. All myelinated and total axons were counted in 5 randomly chosen grids from each of 4 nerves for each group. This experiment represents a single group of mice of one of three replicates on NCV recovery.


( A ) At 1 day post - injury , a single dose of 10 μg 4 - AP significantly improved walking function as determined by SFI analysis ( * : p = 0 . 001 , n = 5 ) . ( B ) In contrast , even higher doses of 4 - AP administration ( 50 μg , ip ) had no effect on SFI in mice with transected nerves ( n = 10 ) . ( C ) In contrast with effects of 4 - AP , treatment with neostigmine did not cause improvements in SFI . ( p = 0 . 0024 for 4 - AP vs . saline , n = 8 , 1 - way ANOVA with Tukey ' s multiple comparison test ) .

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

(A) At 1 day post-injury, a single dose of 10 μg 4-AP significantly improved walking function as determined by SFI analysis (*: p=0.001, n=5). (B) In contrast, even higher doses of 4-AP administration (50 μg, ip) had no effect on SFI in mice with transected nerves (n=10). (C) In contrast with effects of 4-AP, treatment with neostigmine did not cause improvements in SFI. (p = 0.0024 for 4-AP vs. saline, n=8, 1-way ANOVA with Tukey's multiple comparison test).


Ratiometric and mTFP1 donor images of EKAR2G in an EGF ‐ stimulated PC ‐ 12 cell at the indicated time points , with t = 0 ' corresponding to EGF application . Upper panel : FRET ratio image is color ‐ coded for ERK activity . Lower panel : raw mTFP1 donor image in black / white contrast . Scale bar = 20 μm .

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

Ratiometric and mTFP1 donor images of EKAR2G in an EGF‐stimulated PC‐12 cell at the indicated time points, with t = 0' corresponding to EGF application. Upper panel: FRET ratio image is color‐coded for ERK activity. Lower panel: raw mTFP1 donor image in black/white contrast. Scale bar = 20 μm.


Population average of ERK activation dynamics measured by Western blot using a phosphoERK antibody .

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

Population average of ERK activation dynamics measured by Western blot using a phosphoERK antibody.


Population average of ERK activation dynamics cell ‐ averaged EKAR2G emission ratios ( ERs ) from n = at least 111 cells . StDev are shown .

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

Population average of ERK activation dynamics cell‐averaged EKAR2G emission ratios (ERs) from n= at least 111 cells. StDev are shown.


Selected EKAR2G ratio time series illustrating , from top to bottom , sustained , oscillatory , or transient ERK activity dynamics . ER is color ‐ coded as in bar . Scale bar = 20 μm .

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

Selected EKAR2G ratio time series illustrating, from top to bottom, sustained, oscillatory, or transient ERK activity dynamics. ER is color‐coded as in bar. Scale bar = 20 μm.


Single ‐ cell ERK activity trajectories . Cell ‐ averaged ERs for n = 10 cells , standard deviation range ( StDev ) , population average for the indicated GFs and dosages . Experimental time courses were normalized to the mean of 5 time points immediately preceding GF application .

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

Single‐cell ERK activity trajectories. Cell‐averaged ERs for n = 10 cells, standard deviation range (StDev), population average for the indicated GFs and dosages. Experimental time courses were normalized to the mean of 5 time points immediately preceding GF application.


Waterfall plots of single ‐ cell ERK activity trajectories . Cell ‐ averaged ER trajectories are color ‐ coded ( n = 78 cells ) , population average ( bottom ) . Vertical dotted line indicates GF application .

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

Waterfall plots of single‐cell ERK activity trajectories. Cell‐averaged ER trajectories are color‐coded (n = 78 cells), population average (bottom). Vertical dotted line indicates GF application.


Trajectories from all sustained GF stimulation experiments ( n = 307 cells , same ERK activation trajectories as in Fig 1 ) were pooled and five representative trajectories were identified using k ‐ means clustering with squared Euclidean distance . Raw ( color ‐ coded by cluster ) and cluster representative trajectories ( black ) .

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

Trajectories from all sustained GF stimulation experiments (n = 307 cells, same ERK activation trajectories as in Fig 1) were pooled and five representative trajectories were identified using k‐means clustering with squared Euclidean distance. Raw (color‐coded by cluster) and cluster representative trajectories (black).


Overlaid cluster representative trajectories .

Overlaid cluster representative trajectories.


Population distribution of representative ERK activity trajectories in response to different GF dosages . Data representative of n = 3 experiments .

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

Population distribution of representative ERK activity trajectories in response to different GF dosages. Data representative of n = 3 experiments.


Peak emission ratio intensity for each cluster . Boxplots with median , interquartile ( box ) and 1 . 5 IQR ( whiskers ) range , and raw datasets are shown . Boxplot notches extend 1 . 58 IQR / √ Nobs , which gives approximately 95 % confidence interval for comparing medians .

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

Peak emission ratio intensity for each cluster. Boxplots with median, interquartile (box) and 1.5 IQR (whiskers) range, and raw datasets are shown. Boxplot notches extend 1.58 IQR / √Nobs, which gives approximately 95% confidence interval for comparing medians.