( 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 ) .

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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 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 .

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

(c) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O 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)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 )

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

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 .

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 .

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

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 )

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

H&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 O O O 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 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 )

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

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 )

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

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 )

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

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 )

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

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O 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 .

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 )

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 )

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 )

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

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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 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 )

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 )

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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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 )

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 .

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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 - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O 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 )

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 )

O O O O O O O O O O O O O O O O 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 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 ) .

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

H&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 )

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 ) .

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

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 ) .

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

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 )

O O O O O O O O O O O O O O O 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)


The mRNA levels of genes related to energy consumption ( PGC1α , UCP1 and 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)


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 .

, 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

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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 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 .

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 .

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 .

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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 .

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 .

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(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 ) .

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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 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 .

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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 .

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 .

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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 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 .

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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 ‐ 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 .

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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 ‐ cell ERK activity trajectories . Cell ‐ averaged ER trajectories are color ‐ coded ( n = 78 cells ) , population average ( bottom ) . Vertical dotted line 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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) , and data within 1 . 5 IQR range of the lower and upper quartiles ( whiskers ) are shown ( n = 20 cells 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 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 ) .

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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 ‐ 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 .

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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 ‐ 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 .

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 ) , and 1 . 5 IQR ( whiskers ) range are shown for at least n = 30 cells .

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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 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 .

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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 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 .

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

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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 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 .

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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 DNA methylation levels at TAS1a , TAS1c , and 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 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 .

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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 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 .

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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 The largest subunit of Pol IV , NRPD1 ( red ) , was visualized by its specific antibody in cells 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 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 ) .

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 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 ) .

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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 levels of Pol V ‐ dependent transcripts . RNA levels 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 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 .

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 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 ( ) .

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

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


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 .

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 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 .

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

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


( 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 .

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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 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 .

(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 analysis of Atg proteins in the indicated MEFs .

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


( 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 .

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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 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 .

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

(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 ‐ 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 .

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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 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 .

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(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.


( D , E ) Cells stably expressing GFP - LC3 were cultured in regular DMEM , starvation medium or starvation medium in the presence of 0 . 2 μM WM or 100 nM BafA1 for 6 h . Total cellular GFP - LC3 signals were analysed by flow cytometry . Representative FACS data were shown ( D ) . The geometric mean of fluorescence intensity was determined . Values are expressed as a percentage of the mean of control cells cultured in regular DMEM . Data represent mean ± s . e . ( * P0 . 05 ) ( E ) . BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; KO , knockout ; MEFs , mouse embryonic fibroblasts ; NS , not significant ; WM , wortmannin ; WT , wild ‐ type .

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(D, E) Cells stably expressing GFP-LC3 were cultured in regular DMEM, starvation medium or starvation medium in the presence of 0.2 μM WM or 100 nM BafA1 for 6 h. Total cellular GFP-LC3 signals were analysed by flow cytometry. Representative FACS data were shown (D). The geometric mean of fluorescence intensity was determined. Values are expressed as a percentage of the mean of control cells cultured in regular DMEM. Data represent mean±s.e. (*P0.05) (E). BafA1, bafilomycin A1; GFP, green fluorescent protein; KO, knockout; MEFs, mouse embryonic fibroblasts; NS, not significant; WM, wortmannin; WT, wild‐type.


( A ) 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 for 1 h . Cells were fixed and subjected to immunofluorescence microscopy using anti ‐ Atg16L1 antibody . Note that full ‐ length Atg16L1 and Atg16L1 ( 1 - 230 ) , but not Atg16L1Δ ( 230 - 300 ) , showed punctate structures under starvation conditions ( inset ) .

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(A) 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 for 1 h. Cells were fixed and subjected to immunofluorescence microscopy using anti‐Atg16L1 antibody. Note that full‐length Atg16L1 and Atg16L1(1-230), but not Atg16L1Δ(230-300), showed punctate structures under starvation conditions (inset).


( B ) FIP200 KO MEFs stably expressing either CFP - Atg16L1 ( 1 - 588 ) , CFP - Atg16L1 ( 1 - 230 ) or CFP - Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . Cells were fixed and analysed by immunofluorescence microscopy using anti ‐ GFP antibody . Arrowheads indicate the perinuclear localization of Atg16L1 ( 1 - 230 ) .

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(B) FIP200 KO MEFs stably expressing either CFP-Atg16L1(1-588), CFP-Atg16L1(1-230) or CFP-Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP antibody. Arrowheads indicate the perinuclear localization of Atg16L1(1-230).


( C , D ) FIP200 KO MEFs stably expressing CFP - Atg16L1 ( 1 - 230 ) were cultured in regular DMEM . Scale bar , 10 μm , and 2 μm in inset .

(C, D) FIP200 KO MEFs stably expressing CFP-Atg16L1(1-230) were cultured in regular DMEM. Scale bar, 10 μm, and 2 μm in inset.


( a ) Representative micro - CT pictures of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Note the severe osteoporosis in A20myel - KO mice . ( b ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Each dot represents an individual mouse ( A20WT , n = 10 ; A20myel - KO , n = 8 ) .

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(a) Representative micro-CT pictures of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Note the severe osteoporosis in A20myel-KO mice. (b) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Each dot represents an individual mouse (A20WT, n=10; A20myel-KO, n=8).


( a ) Levels of TNF and IL - 6 in serum of A20WT , A20OC - KO and A20myel - KO mice . Each dot represents an individual mouse ( A20WT , n = 11 ; A20OC - KO , n = 11 and A20myel - KO , n = 14 ) . Data are expressed as mean ± SEM . *

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(a) Levels of TNF and IL-6 in serum of A20WT, A20OC-KO and A20myel-KO mice. Each dot represents an individual mouse (A20WT, n=11; A20OC-KO, n=11 and A20myel-KO, n=14). Data are expressed as mean ± SEM. *


( c ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20OC - KO littermates . Each dot represents an individual mouse ( A20WT , n = 6 ; A20OC - KO , n = 9 ) .

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(c) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20OC-KO littermates. Each dot represents an individual mouse (A20WT, n=6; A20OC-KO, n=9).


( e ) Representative pictures of TRAP - stained sections from tibia of 20 week - old A20WT and A20OC - KO mice . Red arrows indicate osteoclasts .

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(e) Representative pictures of TRAP-stained sections from tibia of 20 week-old A20WT and A20OC-KO mice. Red arrows indicate osteoclasts.


( f ) Quantification of the number of TRAP - positive cells on sections from tibia of 20 week - old A20WT and A20OC - KO mice . Each dot represents an individual mouse ( A20WT , n = 5 ; A20OC - KO , n = 6 ) .

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(f) Quantification of the number of TRAP-positive cells on sections from tibia of 20 week-old A20WT and A20OC-KO mice. Each dot represents an individual mouse (A20WT, n=5; A20OC-KO, n=6).


( d ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF - κB reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and with either an empty vector or a vector expressing wild - type A20 . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency .

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(d) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF-κB reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and with either an empty vector or a vector expressing wild-type A20. Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency.


( e ) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel - KO mice and stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control .

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(e) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel-KO mice and stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.


( f ) Wild - type and A20 deficient BMDMs were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for RANK , A20 , TRAF6 , HOIP and Sharpin .

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

(f) Wild-type and A20 deficient BMDMs were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for RANK, A20, TRAF6, HOIP and Sharpin.


( g ) BMDMs from A20WT and A20myel - KO mice were stimulated with RANKL ( 100ng / ml ) for the indicated time periods and immunoblotted to visualize p100 processing to p52 . Actin is shown as a loading control .

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

(g) BMDMs from A20WT and A20myel-KO mice were stimulated with RANKL (100ng/ml) for the indicated time periods and immunoblotted to visualize p100 processing to p52. Actin is shown as a loading control.


( a ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transfected with an NF - κB luciferase reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and plasmids encoding different A20 variants : A20WT , A20DUB ( C103A mutation ) , A20ZnF4 ( C624A / C627A ) , A20ZnF7 ( C775A / C779A ) , and A20ZnF4ZnF7 ( C624A / C627A / C775A / C779A ) . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency . Data are expressed as the mean of technical triplicates ± SD .

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

(a) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transfected with an NF-κB luciferase reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and plasmids encoding different A20 variants: A20WT, A20DUB (C103A mutation), A20ZnF4 (C624A/C627A), A20ZnF7 (C775A/C779A), and A20ZnF4ZnF7 (C624A/C627A/C775A/C779A). Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency. Data are expressed as the mean of technical triplicates ± SD.


( c - d ) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice , stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control .

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

(c-d) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice, stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.


( e ) BMDMs isolated from control ( A20WT ) , A20ZnF7 , A20ZnF4ZnF7 and A20DUB mice were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control .

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

(e) BMDMs isolated from control (A20WT), A20ZnF7, A20ZnF4ZnF7 and A20DUB mice were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.


( f ) Wild - type BMDMs were pretreated for 1h with HOIPIN - 8 ( 50µM ) or left untreated and stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control .

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(f) Wild-type BMDMs were pretreated for 1h with HOIPIN-8 (50µM) or left untreated and stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.


Immunoblots of acyl - biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation - deficient mutant form of the protein with cysteines replaced with alanine residues . The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull - down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein .

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Immunoblots of acyl-biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation-deficient mutant form of the protein with cysteines replaced with alanine residues. The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull-down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein.


Immunoblots of acyl - biotin exchange purifications from Hela cells treated with either DHHC5 siRNA or control non - targeting siRNA .

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Immunoblots of acyl-biotin exchange purifications from Hela cells treated with either DHHC5 siRNA or control non-targeting siRNA.


Immunoblots showing rescue of Golga7b palmitoylation in DHHC5 depleted cells after transfection of WT DHHC5 but not by the DHHC5 catalytic mutant .

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Immunoblots showing rescue of Golga7b palmitoylation in DHHC5 depleted cells after transfection of WT DHHC5 but not by the DHHC5 catalytic mutant.


A significant reduction in the Golga7b signal is observed in the + HA condition of the pull - down when DHHC5 is depleted with siRNA . The graph displays mean + / - s . e . m . N = 4 , * = p < 0 . 05 , paired t - test .

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A significant reduction in the Golga7b signal is observed in the + HA condition of the pull-down when DHHC5 is depleted with siRNA. The graph displays mean +/- s.e.m. N=4, *=p<0.05, paired t-test.


Immunoblots of WT and non - palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants ( DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine , C - terminal mutant = mutant where the 3 C - terminal palmitoylation sites of DHHC5 have been mutated to alanine residues . ) . WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C - terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C - terminus of DHHC5 is essential for the stability of this interaction .

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

Immunoblots of WT and non-palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants (DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine, C-terminal mutant = mutant where the 3 C-terminal palmitoylation sites of DHHC5 have been mutated to alanine residues.). WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C-terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C-terminus of DHHC5 is essential for the stability of this interaction.


Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5 . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and the catalytic mutant DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Quantification of the plasma membrane signal of DHHC5 in each of the images ( A - F ) , the graph displays mean + / - s . e . m . * * * * = p < 0 . 001 , n . s . = not significant , unpaired t - test . N = 35 , DHHC5 + WT Golga7b . N = 41 , DHHC5 + mutant Golga7b . N = 35 , DHHS5 + WT Golga7b . N = 31 , DHHS5 + Golga7b mutant . N = 29 , C - terminal mutant + WT Golga7b . N = 30 , C - terminal mutant + mutant Golga7b . Co expression of palmitoylation - deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane . The majority of the DHHC5 C - terminal palmitoylation - deficient mutant is localised to the plasma membrane in a Golga7b independent manner . All N numbers refer to the number of cells imaged and quantified , all experiments repeated 4 times . Data information : All images show ectopic protein expression only . All scale bars 10µm .

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Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5. Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and the catalytic mutant DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Quantification of the plasma membrane signal of DHHC5 in each of the images (A-F), the graph displays mean +/- s.e.m. ****=p<0.001, n.s. = not significant, unpaired t-test. N=35, DHHC5 + WT Golga7b. N=41, DHHC5 + mutant Golga7b. N=35, DHHS5 + WT Golga7b. N=31, DHHS5 + Golga7b mutant. N=29, C-terminal mutant + WT Golga7b. N=30, C-terminal mutant + mutant Golga7b. Co expression of palmitoylation-deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane. The majority of the DHHC5 C-terminal palmitoylation-deficient mutant is localised to the plasma membrane in a Golga7b independent manner. All N numbers refer to the number of cells imaged and quantified, all experiments repeated 4 times. Data information: All images show ectopic protein expression only. All scale bars 10µm.


Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged WT Golga7b . Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged mutant Golga7b .

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Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged WT Golga7b. Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged mutant Golga7b.


Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non - targeting siRNA . Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA .

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Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non-targeting siRNA. Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA.


Quantification of plasma membrane signal of endogenous DHHC5 when co - expressed with WT or mutant Golga7b or without any transfected Golga7b . * * * * = P < 0 . 001 , t - test , endogenous + WT Golga7b n = 37 , endogenous + mutant Golga7b n = 23 , endogenous n = 42 . Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell , while WT Golga7b stabilises DHHC5 at the plasma membrane .

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

Quantification of plasma membrane signal of endogenous DHHC5 when co-expressed with WT or mutant Golga7b or without any transfected Golga7b. ****=P<0.001, t-test, endogenous + WT Golga7b n=37, endogenous + mutant Golga7b n=23, endogenous n=42. Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell, while WT Golga7b stabilises DHHC5 at the plasma membrane.


Quantification of the plasma membrane signal of DHHC5 in ( C and D ) , * * * * = p < 0 . 001 t - test , control siRNA n = 23 , Golga7b siRNA n = 23 .

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Quantification of the plasma membrane signal of DHHC5 in (C and D), ****=p<0.001 t-test, control siRNA n=23, Golga7b siRNA n=23.


FLAG - DHHC5 immunoprecipitations were performed using lysates from Hela cells co - expressing either wild - type ( WT ) or palmitoylation deficient mutant ( m ) Golga7b . The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b , quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 .

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FLAG-DHHC5 immunoprecipitations were performed using lysates from Hela cells co-expressing either wild-type (WT) or palmitoylation deficient mutant (m) Golga7b. The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b, quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.


Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG - DHHC5 is expressed alone or co - expressed with either WT or mutant Golga7b . IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation .

Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG-DHHC5 is expressed alone or co-expressed with either WT or mutant Golga7b. IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation.


Volcano plot of DHHC5 interactors from AP - MS analysis from HeLa cells co - expressing FLAG - DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin - mediated endocytosis , cell : cell adhesion and known substrates of DHHC5 .

Volcano plot of DHHC5 interactors from AP-MS analysis from HeLa cells co-expressing FLAG-DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin-mediated endocytosis, cell:cell adhesion and known substrates of DHHC5.


Validation of novel DHHC5 interactors by co - IP : immunoblots of Dsg2from Hela cells co - expressing DHHC5 and either Wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of clathrin heavy chain from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of ZO - 1 protein from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * * = p < 0 . 01 ratio paired t - test , n = 3 .

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Validation of novel DHHC5 interactors by co-IP: immunoblots of Dsg2from Hela cells co-expressing DHHC5 and either Wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of clathrin heavy chain from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of ZO-1 protein from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, **=p<0.01 ratio paired t-test, n=3.


30 minute DMSO treatment of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) . 30 minute Dynasore treatment ( 25µM ) of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) .

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30 minute DMSO treatment of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged). 30 minute Dynasore treatment (25µM) of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged).