CSF extract was supplemented with Flag - Cdc20 wt IVT . Flag - Cdc20 was re - isolated by α - Flag immunoprecipitation , supplemented with calcium treated with recombinant hs _ CaN calmodulin as indicated . Samples were taken at indicated time points immunoblotted for Flag - tag pThr68 Cdc20 . pThr68 Cdc20 was stripped reprobed for catalytic calcineurin subunit CnA . Asterisk indicates 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 B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-SUBCELLULAR 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 , were fixed stained for anti - pTyr - 416 - Src ( green ) with DAPI ( blue ) . Solid arrows indicate with active Src in adhesions ; dashed arrows show with active Src in . Scale bars , 20 μm . Right , of with active Src localizing to intracellular structures was quantified . Data are presented as mean ± s . d . significance is P0 . 001 ( n = 3 ) .

O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD 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) 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 ) were fixed stained for anti - FAK ( red ) anti - pTyr - 416 - Src ( green ) . Merged higher - magnification of outlined areas are also shown . Solid dashed arrows show Src localization to adhesions or to , respectively . Scale bars , 20 μm .

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 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

(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 were immunoblotted with anti - FAK , anti - Src , anti - Yes , anti - Fyn , anti - pTyr - 416 - Src anti - actin antibodies . Uncropped of blots are shown in Supplementary Fig . S9 .

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

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 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 B-SUBCELLULAR I-SUBCELLULAR 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 + / + FAK − / − then immunoblotted with anti - Src , anti - pTyr - 416 - Src anti - LC3B antibodies .

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

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD 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 B-GENEPROD 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 B-GENEPROD 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 , were transiently transfected with an RFP - GFP tandem - tagged LC3 ( RFP - GFP - LC3 ) . Scale bars , 20 μm . Bottom , number of yellow number of RFP LC3 - positive in merged were counted total number of per was calculated . Data are presented as mean ± s . d . ( n = 3 ) .

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

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

O O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD 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 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 ) were treated with 200 nM epoxomicin for 4 h then immunoblotted probed with anti - pTyr - 416 - Src , anti - Src , anti - p53 anti - actin antibodies . Uncropped of blots are shown in Supplementary Fig . S9 .

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

O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD 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 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 − / − were transiently transfected with SrcY527F - GFP or with Src - 251 - GFP ( green ) stained for anti - LC3B ( red ) . Scale bars , 20 μm . Bottom , of with co - localization between Src LC3B . Data are presented as mean ± s . d . significance is P0 . 001 ( n = 3 ) .

O O O O O B-GENEPROD 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 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

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

O O O O O B-GENEPROD 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 B-GENEPROD 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 B-GENEPROD O B-SUBCELLULAR 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 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 were transfected with RFP - GFP - LC3 . Solid arrows indicate co - localization 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 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

(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 actin antibodies in absence presence of chloroquine . Densitometry was carried out to calculate increase in amount of LC3B - II relative to actin for each type after treatment with chloroquine is presented as a increase for immunoblot shown . Uncropped of blots are shown in Supplementary Fig . S9 .

O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY 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 were suspended in 1 . 4 % methylcellulose solution in growth media plated on agarose for 3 days . Top bottom right , were recovered cytospins prepared that were stained for anti - pTyr - 416 - Src ( red ) , LC3B ( green ) with DAPI ( blue ) or for paxillin ( red ) , LC3B ( green ) with DAPI ( blue ) . Higher - magnification of outlined areas are also shown . Solid arrows indicate localization of pTyr - 416 - Src in LC3B - positive . Scale bars , 20 μm . Bottom left , immunoblotting was carried out on lysates from adherent from recovered from methylcellulose using anti - pTyr - 397 FAK , anti - FAK , anti - pTyr - 416 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 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 B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O O O B-GENEPROD 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 + / + were suspended for 1 h in PBS cytospins were prepared that were stained for anti - pTyr - 416 - Src ( green ) with DAPI ( blue ) . A higher - magnification image of outlined is also shown . Scale bar , 20 μm .

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

(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 + / + then immunoblotted with anti - Src anti - LC3B antibodies . Lysates were also immunoblotted with anti - pTyr - 397 - FAK anti - actin antibodies . Uncropped 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 B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY 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 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 − / − were sparsely plated then treated with 3 - MA ( for 48 h ) , chloroquine or dasatinib ( both for 24 h ) . were then left at 37 ° C for 1 week to allow colony formation , fixed then stained with crystal violet . Bottom , number of colonies formed was then quantified normalized against untreated controls . Data are presented as mean ± s . d . significance is P0 . 001 for 3 - MA , P0 . 01 for chloroquine P > 0 . 5 for dasatinib ( n = 3 ) .

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

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

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

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

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

(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 − / − 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 then immunoblotted with an anti - PARP antibody . Uncropped of blots are shown in Supplementary Fig . S9 .

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

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

O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD 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 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 + / + FAK − / − then immunoblotted with anti - Src , anti - pTyr - 416 - Src anti - c - Cbl antibodies .

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

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

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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 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 B-GENEPROD O O O O O O O B-GENEPROD 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 − / − expressing either scrambled or c - Cbl siRNA then immunoblotted with anti - Src anti - LC3B antibodies . Uncropped 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 − / − 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 , were stained for anti - p - Tyr - 416 - Src ( green ) , anti - HA ( red ) with DAPI ( blue ) . Solid arrows show co - localization in intracellular . Scale bars , 20 μm . Right , of of that contained active Src in intracellular . Data are presented as mean ± s . d . significance is P > 0 . 5 ( n = 3 ) .

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(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 of HA - tagged wild - type c - Cbl , c - CblAA ( W802A , L805A ) c - CblAAAA ( W802A , L803A , S804A , L805A ) expressed in HEK293T 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 − / − expressing siRNA - resistant HA - tagged wild - type c - Cbl or c - CblAAAA . Left , were stained for anti - pTyr - 416 - Src ( green ) , anti - HA ( red ) with DAPI ( blue ) . Solid arrows indicate pTyr - 416 localization to adhesions dashed arrows indicate localization to intracellular . Scale bars , 20 μm . Right , of of that contained active Src in intracellular . Data are presented as mean ± s . d . significance is P0 . 01 ( n = 3 ) . Uncropped of blots are shown in Supplementary Fig . S9 .

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

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(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 QKI KO fl / fl : AP2 - Cre mice fed a NCD or HFD for 20 . arrow indicates time when high fat diet （ HFD ） starts , 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 ingWAT fat pads from WT fl / fl QKI KO fl / fl : AP2 - Cre mice after 20 on 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 of BAT , eWAT ingWAT from WT fl / fl QKI KO fl / fl : AP2 - Cre mice fed NCD or HFD for 20 . ( Scale bar represents 50 μm )

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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)


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

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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).


increase in relative 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 of liver , BAT 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 of WT fl / fl QKI KO fl / fl : AP2 - Cre mice on an NCD or HFD , showing whole - fat composition .

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

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

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

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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 ) . average O2 consumption rates are shown on right .

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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 QKI KO fl / fl : AP2 - Cre mice ( n = 8 , per genotype ) . average heat production rates are shown on right .

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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 oxidation . ( 3 samples , per group )

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


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

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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)


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

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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 QKI KO fl / fl : AP2 - Cre mice under basal conditions were measured using a Clark electrode . ( n = 3 biological replicates )

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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)


relative mRNA 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 QKI KO fl / fl : AP2 - Cre mice under basal conditions . ( n = 3 biological replicates )

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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 QKI KO fl / fl : AP2 - Cre mice on NCD ; Oxygen consumption was performed under basal conditions , following addition of 0 . 25 mg mitochondria ( Mit ) , 5 mM substrate glycerol - 3 - phosphate ( G3P ) , 2 mM GDP , 450 mM ADP , 2 μg / ml oligomycin , 1 . 5 μM FCCP . 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 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 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 B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE 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 , on 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)


of PGC1α QKI in siRNA - mediated QKI knockdown differentiated , graph was shown on right . ( n = 3 biological replicates )

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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 UCP1 in NC , Si - QKI 1 , 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).


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

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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)


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

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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 in three adipocyte types : ShScramble + ovCon , QKI knockdown ( ShQKI + ovCon ) QKI knockdown with QKI - 5 re - expression ( ShQKI + ovQKI ) .

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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).


of PGC1α in these adipocytes , graph was shown on right . ( n = 3 biological replicates )

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


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

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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 - 3 ' UTR T , A , B , C fragments PGC1α 3 ' UTR QRE motif sites mutant B M1 , B M2 , C M1 , C M2 fragments ) . Luciferase activity assays to examine functional QRE motif sites in 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 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 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 - 3 ' UTR T fragment UCP1 3 ' UTR QRE motif sites mutant T M1 , T M2 , T M3 fragments ) . Luciferase activity assays to examine functional QRE motif sites in UCP1 3 ' UTR affected by QKI . ( n = 4 biological replicates )

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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)


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

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


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

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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 were transfected with PGC1α wild type or QRE sites mutant 3 ' UTR fragments . immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . results showed that endogenous QKI interacted with full - wild type QRE motif regions within 3 ' UTRs of

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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 were transfected with UCP1 wild type or QRE sites mutant 3 ' UTR fragments . immunoprecipitation ( RIP ) to identify these QRE motif sites region bound to endogenous QKI by using anti - QKI antibodies . results showed that endogenous QKI interacted with full - wild type QRE motif regions within 3 ' UTRs of UCP1 .

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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.


mRNA stability in adipocytes was measured by incubating with actinomycin D ( 5 μg / ml ) . Actinomycin D was added to stop transcription , RNAs were harvested at indicated times ( x - axis ) after transcription inhibition . Real - time PCR was used to determine remaining UCP1 , PGC1α Actin mRNA compared with 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 )

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


fractionation was valid by immunoblotting using markers ( GAPDH Lamin B1 ) to show purity of fractions .

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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 of mRNA transcript of QKI targets UCP1 PGC1α in fractions of differentiated MEF transfected with ShScramble or ShQKI . Values represent - to - 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE 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

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 fractionation were harvested after immunoprecipitation ( RIP ) of - complexes using either anti - QKI antibody or control IgG . RIP - RT - PCR showing PGC1α UCP1 transcripts majorly interacted with endogenous QKI in nucleus . ( n = 3 biological replicates )

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

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


rectal of WT fl / fl 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 UCP1 immunostaining of sections of ingWAT from WT fl / fl 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).


of UCP1 in ingWAT from WT fl / fl QKI KO fl / fl : AP2 - Cre mice at RT or following 72 h of cold exposure ( 4 ° C ) , quantified 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 in BAT injected with Control or QKI knockdown AAV at RT ( 22 ° C ) or following 6 h 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 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).


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

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

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)


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

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


of QKI in BAT ingWAT during cold stimulation ( 4 ° C ) quantified . ( 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)


relative luminescence of a QKI promoter reporter under cAMP ( Forskolin 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)


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

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

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


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

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

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.


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

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

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 of SVF isolated from iWAT of mice . Differentiation was induced with curcumin up to day 8 . C ORO of 3T3 - L1 . 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 isolated from iWAT of mice . D mRNA expression of PPARγ , C / EBPα FABP4 expression of 3T3 - L1 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.


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

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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 of Vector or OE - ALKBH5 transfected 3T3 - L1 . 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 of control , ALKBH5 - depleted ALKBH5 + TRAF4 depleted 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 of PPARγ C / EBPα in , of normalized to β - actin expression .

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

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 of YTHDF1 , ALKBH5 TRAF4 in control , ALKBH5 - depleted ALKBH5 + YTHDF1 depleted of normalized to β - actin expression .

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

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 of control , ALKBH5 - depleted ALKBH5 + YTHDF1 depleted 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 of PPARγ , C / EBPα FABP4 expression in β - Actin was used as an internal control .

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

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


C , D Western blot of PPARγ in control TRAF4 overexpressing in absence or presence of MG132 or 3MA of normalized to β - actin expression .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O 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 TRAF4 overexpressing were differentiated by treatment with DMI . were treated with MG132 for 6 h .

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

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 - unpaired t - )

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O 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 O O O O O O O O O O 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) 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 ( * : D21 , p = 0 . 0454 ; D28 , p = 0 . 0487 ; D35 , p = 0 . 0475 ; n = 5 ; ANOVA with post - hoc comparisons using two - unpaired t - ) .

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

(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 ) 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 case of response to thermal hyperalgesis , 4 - AP treated mice showed a significantly more rapid return to baseline . ( * : ( 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 - unpaired t - . ) In this other figures data is mean + 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 - unpaired t - ) .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of healthy , crushed , 4 - AP treated crushed sciatic nerve at 21 days post - injury . Scale bar at 3000x = 5m ; scale bar at 8000x = 2m ) ( B ) Comparison of axonal of randomly chosen axons of vehicle treated 4 - AP treated mice ( n = 4 for each experimental group ; 40 axons analyzed per mouse ) . 4 - AP treated sciatic nerve showed statistically greater axonal compared to 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 mean value for uninjured mice ( shown in green ) , with inset figure displaying 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 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 B-SUBCELLULAR 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 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 B-ORGANISM 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 close - to - normal Garea - compared to untreated group , as determined by of 40 randomly chosen myelinated axons from sections of 4 nerves for each group . ( * * : p < 0 . 0001 ; ANOVA , with post - hoc comparisons using two - unpaired t - ) . 4 - AP treated mice had a greater proportion of axons for which associated myelin was greater than mean value for uninjured mice ( shown in green ) , with inset figure displaying 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 of P0 as detected by western blot ( * * : p < 0 . 01 ; ANOVA , with post - hoc comparisons using two - unpaired t - ) .

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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 over time also were observed by immunofluorescence at different time points , with P0 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 number of myelinated axons ( * * : p = 0 . 002 , n = 4 ; ANOVA , with post - hoc comparisons using two - unpaired t - ) . Even though 4 - AP treated group also exhibited a greater number of total axons , this difference was not statistically significant . myelinated 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 ( * : 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 ) .

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

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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 .

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


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

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


Selected EKAR2G 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 .

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

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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 ‐ ERK activity trajectories . ‐ averaged ER trajectories are color ‐ coded ( n = 78 ) , population average ( bottom ) . Vertical dotted indicates GF application .

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

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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 .

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


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

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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.


A - DSingle GF experiments . ‐ averaged ERs , population average , StDev range for n = 10 . application indicated by black bars . 3 ′ ( A , C ) 10 ′ ( B , D ) . High ( A , B ) low ( C , D ) GF concentrations .

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A-DSingle GF pulse experiments. Cell‐averaged ERs, population average, and StDev range for n = 10 cells. Pulse application indicated by black bars. 3′ (A, C) and 10′ (B, D) pulse. High (A, B) and low (C, D) GF concentrations.


ERationale for measuring amplitude duration of 1st ERK activity peak in response to GF stimulation . Peak amplitude was measured as ER change from when GF stimulation starts until highest ERK activity before adaptation occurs . Peak duration was estimated as time between first point before reaching half ‐ maximum of peak in ascending phase , first point after half ‐ maximum in descending phase .

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ERationale for measuring amplitude and duration of 1st ERK activity peak in response to GF stimulation. Peak amplitude was measured as the ER change from when the GF stimulation starts until the highest ERK activity before adaptation occurs. Peak duration was estimated as the time between the first point before reaching the half‐maximum of the peak in the ascending phase, and the first point after the half‐maximum in the descending phase.


FAmplitude of 1st ERK activity peak in response to different sustained or pulsed GF stimulation experiments . Notched boxplots of 1st ERK activity peak amplitude with median , interquartile range ( box ) , data within 1 . 5 IQR range of lower upper quartiles ( whiskers ) are shown ( n = 20 per experiment ) .

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FAmplitude of 1st ERK activity peak in response to different sustained or pulsed GF stimulation experiments. Notched boxplots of 1st ERK activity peak amplitude with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).


GDuration of 1st ERK activity peak in response to sustained pulsed GF stimulation . Boxplots of 1st ERK activity peak duration with median , interquartile range ( box ) , data within 1 . 5 IQR range of lower upper quartiles ( whiskers ) are shown ( n = 20 per experiment ) .

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GDuration of 1st ERK activity peak in response to sustained and pulsed GF stimulation. Boxplots of 1st ERK activity peak duration with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).


A - CDouble ‐ GF experiments . ( A ) 3 ′ GF / 3 ′ pause / 3 ′ GF . ( B ) 3 ′ GF / 10 ′ pause / 3 ′ GF . ( C ) 3 ′ GF / 20 ′ pause / 3 ′ GF . ‐ averaged ERs , population average , StDev range for n = 10 .

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A-CDouble‐pulse GF experiments. (A) 3′ GF/3′ pause/3′ GF. (B) 3′ GF/10′ pause/3′ GF. (C) 3′ GF/20′ pause/3′ GF. Cell‐averaged ERs, population average, and StDev range for n = 10 cells.


D - GMulti ‐ GF experiments . Population average ERs ( n = at least 30 ) for different EGF / NGF dosages . ( D ) 3 ′ GF / 3 ′ pause . ( E ) 3 ′ GF / 10 ′ pause . ( F ) 3 ′ GF / 20 ′ pause . ( G ) 3 ′ GF / 60 ′ pause .

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D-GMulti‐pulse GF experiments. Population average ERs (n= at least 30 cells) for different EGF/NGF dosages. (D) 3′ GF/3′pause. (E) 3′ GF/10′ pause. (F) 3′ GF/20′ pause. (G) 3′ GF/60′ pause.


HDecay kinetics of ERK activity maxima from ( D - G ) . Boxplots with median , interquartile ( box ) , 1 . 5 IQR ( whiskers ) range are shown for at least n = 30 .

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HDecay kinetics of ERK activity maxima from (D-G). Boxplots with median, interquartile (box), and 1.5 IQR (whiskers) range are shown for at least n = 30 cells.


ARepresentative of tubulin ‐ stained PC ‐ 12 NS ‐ 1 subclone stimulated with sustained multi ‐ GF regimes . Scale bar = 20 μm . B , CQuantification of neuronal differentiation fate . ( B ) Total neurite outgrowth per normalized to major axis of soma for sustained multi ‐ GF regimes . ( C ) Differentiation calculated as fraction of with normalized total neurite outgrowth per larger than 1 . At least 5 , 000 quantified in at least 2 independent experiments for each condition . Boxplots with median , interquartile ( Box ) , 1 . 5 IQR ( Whiskers ) are shown .

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ARepresentative micrographs of tubulin‐stained PC‐12 NS‐1 subclone cells stimulated with sustained and multi‐pulse GF regimes. Scale bar = 20 μm.B, CQuantification of neuronal differentiation fate. (B) Total neurite outgrowth per cell normalized to the major axis length of cell soma for sustained and multi‐pulse GF regimes. (C) Differentiation ratio calculated as the fraction of cells with normalized total neurite outgrowth per cell larger than 1. At least 5,000 cells quantified in at least 2 independent experiments for each condition. Boxplots with median, interquartile (Box), and 1.5 IQR (Whiskers) are shown.


A depiction of 3 , 731 hypomethylation loci in ago4 ‐ 6 . Each hypomethylated region corresponds to a colored horizontal bar , bars are clustered numerically into a column ( y ‐ axis ) . Cytosines were examined as CG , CHG , CHH . color ‐ scaled methylation indicate ratios of each type of methylated cytosines over total cytosines of same type within examined hypomethylated regions .

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A Heatmap depiction of the 3,731 DNA hypomethylation loci in ago4‐6. Each hypomethylated region corresponds to a colored horizontal bar, and the bars are clustered numerically into a column (y‐axis). Cytosines were examined as CG, CHG, and CHH. The color‐scaled methylation levels indicate the ratios of each type of methylated cytosines over total cytosines of the same type within the examined hypomethylated regions.


B depiction of 3 , 678 hypomethylation loci in ago6 ‐ 2 .

B Heatmap depiction of the 3,678 DNA hypomethylation loci in ago6‐2.


B Box plots showing methylation patterns of different groups of loci as categorized in ( A ) .

B Box plots showing methylation patterns of different groups of loci as categorized in (A).


B methylation in different mutants . 2 , 174 loci where AGO4 AGO6 are mutually dependent are numerically clustered ( y ‐ axis ) . Cytosines were examined as CG , CHG , CHH .

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B DNA methylation levels in different mutants. The 2,174 loci where AGO4 and AGO6 are mutually dependent are numerically clustered (y‐axis). Cytosines were examined as CG, CHG, and CHH.


C methylation at TAS1a , TAS1c , TAS3a loci . Snapshots from whole ‐ genome bisulfite sequencing results are shown .

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C DNA methylation levels at TAS1a, TAS1c, and TAS3a loci. Snapshots from whole‐genome bisulfite sequencing results are shown.


D RT - qPCR measurements of transposon . Double : ago4 ‐ 6 ago6 ‐ 2 double mutant . Actin 2 was used as an internal control . in mutants are relative to those in wild ‐ type ( Col ‐ 0 ) . Means ± SD are shown , n = 3 .

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D RT-qPCR measurements of transposon RNA levels. Double: ago4‐6 ago6‐2 double mutant. Actin 2 was used as an internal control. RNA levels in the mutants are relative to those in the wild‐type (Col‐0). Means ± SD are shown, n = 3.


A AGO4 AGO6 were visualized by immunofluorescence microscopy using their specific antibodies . Yellow signals would be expected in merged if two co ‐ localize , as a result of overlap of red green channels . ( blue ) was stained with DAPI .

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A AGO4 and AGO6 were visualized by immunofluorescence microscopy using their specific antibodies. Yellow signals would be expected in the merged images if the two proteins co‐localize, as a result of the overlap of red and green channels. DNA (blue) was stained with DAPI.


B largest subunit of Pol IV , NRPD1 ( red ) , was visualized by its specific antibody in expressing FLAG ‐ tagged AGO4 or AGO6 ( green ) .

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B The largest subunit of Pol IV, NRPD1 (red), was visualized by its specific antibody in cells expressing FLAG‐tagged AGO4 or AGO6 (green).


A largest subunit of Pol V , NRPE1 ( red ) , was visualized by immunofluorescence microscopy using its specific antibody in expressing FLAG ‐ tagged AGO4 ( green ) . AGO4 ‐ NRPE1 co ‐ localized to nucleolar dot ( yellow dot , marked with arrow , upper panel ) but not in nucleoplasmic foci ( lower panel ) .

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A The largest subunit of Pol V, NRPE1 (red), was visualized by immunofluorescence microscopy using its specific antibody in cells expressing FLAG‐tagged AGO4 (green). AGO4‐NRPE1 co‐localized to nucleolar dot (yellow dot, marked with white arrow, upper panel) but not in nucleoplasmic foci (lower panel).


B NRPE1 ( red ) was visualized by its specific antibody in expressing FLAG ‐ tagged AGO6 ( green ) . yellow signals due to overlap of red green channels in merged indicate co ‐ localization . ( blue ) was stained with DAPI . AGO6 ‐ NRPE1 co ‐ localized to nucleoplasmic foci ( yellow dots , marked with arrow ) .

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B NRPE1 (red) was visualized by its specific antibody in cells expressing FLAG‐tagged AGO6 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co‐localization. DNA (blue) was stained with DAPI. AGO6‐NRPE1 co‐localized to nucleoplasmic foci (yellow dots, marked with white arrow).


A AGO6 dysfunction reduces of Pol V ‐ dependent transcripts . were measured by RT - qPCR . Means ± SD are shown , n = 3 .

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A AGO6 dysfunction reduces levels of Pol V‐dependent transcripts. RNA levels were measured by RT-qPCR. Means ± SD are shown, n = 3.


B AGO6 dysfunction decreases Pol V occupancy at of loci where Pol V transcript are affected by AGO6 . Anti ‐ NRPE1 antibody was used for ChIP assays . Means ± SD are shown , n = 3 .

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B AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6. Anti‐NRPE1 antibody was used for ChIP assays. Means ± SD are shown, n = 3.


A depiction of dependence of AGO4 ‐ AGO6 ‐ bound siRNAs on Pol IV . Identities of siRNAs were retrieved from Havecker et al ( ) , siRNAs were grouped into 1 , 210 AGO4 ‐ bound clusters 1 , 486 AGO6 ‐ bound clusters ( see Materials Methods for details ) , each of which corresponds to a colored horizontal bar ; bars are stacked numerically into a column ( y ‐ axis ) . Information on siRNA in Pol IV mutant wild ‐ type was retrieved from Zhang et al ( ) .

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A Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol IV. Identities of siRNAs were retrieved from Havecker et al (), and siRNAs were grouped into 1,210 AGO4‐bound clusters and 1,486 AGO6‐bound clusters (see Materials and Methods for details), each of which corresponds to a colored horizontal bar; the bars are stacked numerically into a column (y‐axis). Information on siRNA levels in Pol IV mutant and wild‐type plants was retrieved from Zhang et al ().


B depiction of dependence of AGO4 ‐ AGO6 ‐ bound siRNAs on Pol V .

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B Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol V.


C of 24 ‐ nt siRNAs by TaqMan small assays . Double : ago4 ‐ 6 ago6 ‐ 2 double mutant . snoR101 was used as an internal control . in mutants were relative to those of wild ‐ type ( Col ‐ 0 ) . Error bars indicate SD , n ≥ 3 .

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C Quantification of individual 24‐nt siRNAs by TaqMan small RNA assays. Double: ago4‐6 ago6‐2 double mutant. snoR101 was used as an internal control. RNA levels in the mutants were relative to those of the wild‐type (Col‐0). Error bars indicate SD, n ≥ 3.


( B , C ) HEK293T were co ‐ transfected with indicated constructs . lysates were subjected to IP using anti ‐ FLAG antibodies . resulting precipitates were examined by immunoblot with anti ‐ FLAG anti ‐ HA antibodies .

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(B,C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were subjected to IP using anti‐FLAG antibodies. The resulting precipitates were examined by immunoblot analysis with anti‐FLAG and anti‐HA antibodies.


( D ) HEK293T were cultured in regular or starvation medium for 2 h . were harvested treated with DSP .

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(D) HEK293T cells were cultured in regular or starvation medium for 2 h. Cells were harvested and treated with DSP.


( E ) HEK293T were harvested treated with DSP .

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(E) HEK293T cells were harvested and treated with DSP.


( F ) HEK293T were cultured in regular DMEM or starvation medium in presence or absence of 200 nM wortmannin for 2 h . After treatment with DSP , lysates were subjected to IP .

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(F) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h. After treatment with DSP, cell lysates were subjected to IP analysis.


( G ) Atg14F / F ( undeleted ) or Atg14Δ / Δ ( deleted ) MEFs were harvested treated with DSP . * ( E ) * * ( G ) indicate positions of immunoglobulin heavy chains , respectively . aa , acid ; DSP , dithiobis ( succinimidyl propionate ) ; IP , immunoprecipitation ; MEFs , mouse embryonic fibroblasts .

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(G) Atg14F/F (undeleted) or Atg14Δ/Δ (deleted) MEFs were harvested and treated with DSP. * (E) and ** (G) indicate the positions of the immunoglobulin light and heavy chains, respectively. aa, amino acid; DSP, dithiobis(succinimidyl propionate); IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts.


( A ) Immunoblot of Atg in indicated MEFs .

(A) Immunoblot analysis of Atg proteins in the indicated MEFs.


( B ) m5 ‐ 7 were cultured in regular medium containing 10 ng / ml Dox for 8 days , then cultured in absence of Dox for a further 2 days .

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(B) m5‐7 cells were cultured in regular medium containing 10 ng/ml Dox for 8 days, and then cultured in the absence of Dox for a further 2 days.


( C , D ) m5 ‐ 7 were cultured in regular medium with or without 10 ng / ml Dox for 4 days , then cultured in presence of 50 μg / ml CHX for indicated time periods ( C ) . mRNA of indicated genes were measured by quantitative PCR following 4 ‐ day Dox treatment . Data represent mean ± s . e . ( * P0 . 05 ) ( D ) . CHX , cycloheximide ; Dox , doxycycline ; MEFs , mouse embryonic fibroblasts ; mRNA , messenger ; WT , wild ‐ type .

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

(C, D) m5‐7 cells were cultured in regular medium with or without 10 ng/ml Dox for 4 days, and then cultured in the presence of 50 μg/ml CHX for the indicated time periods (C). mRNA levels of the indicated genes were measured by quantitative PCR following the 4‐day Dox treatment. Data represent mean±s.e. (*P0.05) (D). CHX, cycloheximide; Dox, doxycycline; MEFs, mouse embryonic fibroblasts; mRNA, messenger RNA; WT, wild‐type.


( B , C ) HEK293T were co ‐ transfected with indicated constructs . lysates were analysed by IP . IP , immunoprecipitation ; ND , not determined .

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(B, C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were analysed by IP. IP, immunoprecipitation; ND, not determined.


( A ) WT MEFs , Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full ‐ Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium in presence or absence of 100 nM BafA1 for 2 h . * indicates nonspecific immunoreactive bands .

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(A) WT MEFs, Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h. * indicates nonspecific immunoreactive bands.


( B , C ) Atg16L1 KO MEFs stably expressing GFP - ULK1 either full ‐ Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM ( C ) or starvation medium ( B , C ) for 1 h . were fixed analysed by immunofluorescence microscopy using anti ‐ GFP , anti ‐ Atg16L1 anti ‐ LC3 antibodies . Arrowheads indicate perinuclear localization of Atg16L1 ( 1 - 230 ) . number of dots was quantified from more than 30 randomly selected from three independent samples as described in Methods . Data represent mean ± s . e . ( * P0 . 001 , of variance followed by Bonferroni / Dunn post hoc ) . Scale bar , 10 μm , 2 μm in inset .

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

(B, C) Atg16L1 KO MEFs stably expressing GFP-ULK1 and either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM (C) or starvation medium (B,C) for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP, anti‐Atg16L1 and anti‐LC3 antibodies. Arrowheads indicate the perinuclear localization of Atg16L1(1-230). The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods. Data represent mean±s.e. (*P0.001, analysis of variance followed by Bonferroni/Dunn post hoc test). Scale bar, 10 μm, and 2 μm in inset.