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 ) .

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

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 )

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

The relative mRNA levels of genes 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 )

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 ) .

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


of PGC1α in these adipocytes , graph was shown on 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)


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

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

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


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

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 )

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

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

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


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 )

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

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 )

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


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 )

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 ) .

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


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 )

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 ) .

O O O O O O O O O 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 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 )

O O O O O O O O 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 )

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 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 )

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

The protein levels of 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 )

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

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

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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 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 .

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


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 .

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

B 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 .

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

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 .

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 .

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 .

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

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

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 .

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 - )

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

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

(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 .

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 .

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 .

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 .

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 .

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 .

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 .

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 .

(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 .

(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 .

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


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

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

(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 ‐ Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . were fixed subjected to immunofluorescence microscopy using anti ‐ Atg16L1 antibody . Note that full ‐ Atg16L1 Atg16L1 ( 1 - 230 ) , but not Atg16L1Δ ( 230 - 300 ) , showed punctate structures under starvation conditions ( inset ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 . were fixed analysed by immunofluorescence microscopy using anti ‐ GFP antibody . Arrowheads indicate perinuclear localization of Atg16L1 ( 1 - 230 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 , 2 μm in inset .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) A20OC - KO . Each dot represents an mouse ( A20WT , n = 6 ; A20OC - KO , n = 9 ) .

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

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

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

(d) Measurement of 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 of whole lysates from BMDMs cultures isolated from A20WT A20myel - KO mice stimulated with RANKL ( 100ng / ml ) for 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

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

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

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

(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 of whole lysates from BMDMs differentiated from A20WT A20ZnF4ZnF7 mice , stimulated with RANKL ( 100ng / ml ) for indicated time periods . Actin is shown as a loading control .

(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 A20DUB mice were stimulated with GST - RANKL ( 1 μg / ml ) for indicated time periods . RANK signaling was immunoprecipitated using glutathione - sepharose beads 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

(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 stimulated with GST - RANKL ( 1 μg / ml ) for indicated time periods . RANK signaling was immunoprecipitated using glutathione - sepharose beads immunoblotted for A20 . Actin was used as a loading control .

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

(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 transiently transfected with WT Golga7b or a palmitoylation - deficient mutant form of with cysteines replaced with alanine residues . hydroxylamine sensitivity of Golga7b signal in + HA condition of WT pull - down not in either - HA condition or + HA condition of mutant shows that Golga7b is a palmitoylated .

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

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 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 after transfection of WT DHHC5 but not by DHHC5 catalytic mutant .

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

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

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 Golga7b were expressed in in HeLa depleted of endogenous DHHC5 . Confocal of HeLa co - expressing WT Golga7b ( 1st column ) DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal of HeLa co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal of HeLa co - expressing WT Golga7b ( 1st column ) catalytic mutant DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal of HeLa co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal of HeLa co - expressing WT Golga7b ( 1st column ) DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Confocal of HeLa co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . of plasma signal of DHHC5 in each of ( A - F ) , graph displays mean + / - s . e . m . * * * * = p < 0 . 001 , n . s . = not significant , unpaired t - . 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 plasma . majority of DHHC5 C - terminal palmitoylation - deficient mutant is localised to plasma in a Golga7b independent manner . N numbers refer to number of imaged quantified , experiments repeated 4 times . Data information : show ectopic expression only . scale bars 10µm .

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

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 of endogenous DHHC5 in HeLa expressing FLAG - tagged WT Golga7b . Confocal of endogenous DHHC5 in HeLa 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 of endogenous Golga7b DHHC5 in HeLa treated with negative control non - targeting siRNA . Confocal of endogenous Golga7b DHHC5 in HeLa treated with Golga7b siRNA .

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.


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

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.


of plasma signal of DHHC5 in ( C D ) , * * * * = p < 0 . 001 t - , 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 co - expressing either wild - type ( WT ) or palmitoylation deficient mutant ( m ) Golga7b . interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting is significantly reduced in presence of palmitoylation deficient mutant Golga7b , normalised to input , * = p < 0 . 05 , paired t - , n = 3 .

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

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

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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 from HeLa co - expressing FLAG - DHHC5 WT Golga7b highlighting interacting involved in clathrin - mediated endocytosis , : adhesion known substrates of DHHC5 .

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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 co - expressing DHHC5 either Wild - type ( WT ) or mutant ( m ) Golga7b normalised to input , * = p < 0 . 05 , paired t - , n = 3 . Co - IP immunoblots of clathrin heavy chain from Hela co - expressing DHHC5 either wild - type ( WT ) or mutant ( m ) Golga7b normalised to input , * = p < 0 . 05 , paired t - , n = 3 . Co - IP immunoblots of ZO - 1 from Hela co - expressing DHHC5 either wild - type ( WT ) or mutant ( m ) Golga7b normalised to input , * * = p < 0 . 01 paired t - , 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 co - expressing WT DHHC5 ( FLAG tagged ) mutant Golga7b ( HA tagged ) . 30 minute Dynasore treatment ( 25µM ) of HeLa co - expressing WT DHHC5 ( FLAG tagged ) 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).


Non - targeting control siRNA treatment ( 10nM ) of HeLa expressing mutant Golga7b ( HA tagged ) . are detecting endogenous DHHC5 HA tagged Golga7b AP2µ siRNA treatment ( 10nM ) of HeLa that were subsequently transfected with mutant Golga7b ( HA tagged ) . are detecting endogenous DHHC5 HA tagged Golga7b .

Non-targeting control siRNA treatment (10nM) of HeLa cells expressing mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b AP2µ siRNA treatment (10nM) of HeLa cells that were subsequently transfected with mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b.


of plasma signal of DHHC5 in ( A B ) . * * * * = p < 0 . 001 , unpaired t - . N = 37 , + Dynasore . N = 28 , + DMSO .

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Quantification of plasma membrane signal of DHHC5 in (A and B). ****=p<0.001, unpaired t-test. N=37, +Dynasore. N=28, +DMSO.


of plasma signal of DHHC5 in ( C D ) . * * * * = p < 0 . 001 , unpaired t - . scale bars 10μm .

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Quantification of plasma membrane signal of DHHC5 in (C and D). ****=p<0.001, unpaired t-test. All scale bars 10μm.


Biotin endocytosis assays of HeLa with endogenous DHHC5 , over - expression of DHHC5 alone , co - expression of DHHC5 WT Golga7b , co - expression of DHHC5 mutant Golga7b or expression of DHHC5 C - terminal mutant . Biotinylated surface endocytosed within 15 minutes were purified using streptavidin resin blots probed with an antibody to DHHC5 . lack of signal in pull - down of DHHC5 C - terminal mutant or when DHHC5 WT Golga7b are co - expressed indicates that DHHC5 is stabilised at plasma in these conditions . robust signal in pull - down of endocytosed from co - expressing DHHC5 mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition . N = 3 , * = p < 0 . 05 , paired t "

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Biotin endocytosis assays of HeLa cells with endogenous DHHC5, over-expression of DHHC5 alone, co-expression of DHHC5 and WT Golga7b, co-expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C-terminal mutant. Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5. The lack of signal in the pull-down of the DHHC5 C-terminal mutant or when DHHC5 and WT Golga7b are co-expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions. The robust signal in the pull-down of endocytosed proteins from cells co-expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition. N=3, *=p<0.05, paired t test"


ABE purifications from HeLa treated with 10nM non - targeting siRNA ( nt siRNA ) or DHHC5 siRNA were immunoblotted for Dsg2 . Loss of band in + HA pull - down in DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5 .

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ABE purifications from HeLa cells treated with 10nM non-targeting siRNA (nt siRNA) or DHHC5 siRNA were immunoblotted for Dsg2. Loss of the band in the +HA pull-down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5.


Confocal of DHHC5 Dsg2 in nt siRNA treated A431 three hours after calcium was reintroduced to media in calcium switch experiments . Confocal of DHHC5 Dsg2 in DHHC5 depleted A431 three hours after calcium was reintroduced to media in calcium switch experiments .

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Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments.


Confocal of DHHC5 , Dsg2 Golga7b in Golga7b depleted A431 three hours after calcium was reintroduced to media in calcium switch experiments . See Appendix Figure S6 for of data in panels B - D .

Confocal images of DHHC5, Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. See Appendix Figure S6 for quantification of data in panels B-D.


Immunoblots of Dsg2 pulled - down in surface biotinylation assays in nt siRNA , DHHC5 siRNA or Golga7b siRNA treated conditions . of of DSG2 signal in pulldown is normalised to input . A significant reduction of surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA . * =

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Immunoblots and quantification of Dsg2 pulled-down in cell surface biotinylation assays in nt siRNA, DHHC5 siRNA or Golga7b siRNA treated conditions. Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input. A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA. *=


Immunoblot of an ABE purification for Dsg2 in with either normal media or low calcium ( 5µM calcium ) conditions , with of of DSG2 signal in pulldown normalised to input . There is a significant reduction in Dsg2 signal in + HA pull - down from low calcium showing that Dsg2 is significantly less palmitoylated in this condition . * = p < 0 . 05 , paired t - , N = 4 , error bars represent s . e . m . scale bars 10μm .

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Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium (5µM calcium) conditions, with quantification of the intensity of the DSG2 signal in the pulldown normalised to input. There is a significant reduction in the Dsg2 signal in the +HA pull-down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition. *=p<0.05, paired t-test, N=4, error bars represent s.e.m. All scale bars 10μm.


10x brightfield of A431 treated with non - targeting siRNA at time 0 ( left ) after 48 hours ( right ) of 5 ng / ml EGF treatment . 10x brightfield of A431 treated with DHHC5 siRNA at time 0 ( left ) after 48 hours ( right ) of 5ng / ml EGF treatment . of scattering of A431 , showing a significant increase in scatter after 48 hours of DHHC5 knockdown therefore a decrease in adhesion was observed .

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10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5 ng/ml EGF treatment. 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed.


10x brightfield of A431 treated with non - targeting siRNA at time 0 ( left ) after 48 hours ( right ) of 5ng / ml EGF treatment . 10x brightfield of A431 treated with Golga7b siRNA at time 0 ( left ) after 48 hours ( right ) of 5ng / ml EGF treatment . of scattering of A431 , showing a significant increase in scatter after 48 hours of Golga7b knockdown therefore a decrease in adhesion , was observed .

10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion, was observed.


Plot showing resistance of A431 from a trans - epithelial electrical resistance treated with either DHHC5 siRNA , Golga7b siRNA or non - targeting siRNA . A significant reduction in TEER demonstrates a decrease in adhesion after both DHHC5 Golga7b depletion .

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Plot showing the resistance of A431 cells from a trans-epithelial electrical resistance assay treated with either DHHC5 siRNA, Golga7b siRNA or non-targeting siRNA. A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion.


B . accessibility p300 binding at differential ATAC - seq peaks visualized using K - means clustering .

B. Chromatin accessibility and p300 binding at differential ATAC-seq peaks visualized using K-means clustering.


B . Boxplots showing differential gene expression ( AC versus DMZ ) associated ATAC - seq signals ( Two biological replicates ) . central band within boxplot represents median whiskers show 1 . 5 times range between first third quartile ( IQR ) .

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B. Boxplots showing differential gene expression (AC versus DMZ) and associated ATAC-seq signals (Two biological replicates). The central band within the boxplot represents the median and the whiskers show 1.5 times the range between the first and third quartile (IQR).


F . Genomic tracks showing aggregated accessibility of single - ATAC - seq clusters at t ( tbxt ) , tfap2a , gsc , ctcf , lhx1 sox11 loci .

F. Genomic tracks showing aggregated accessibility of single-cell ATAC-seq clusters at the t (tbxt), tfap2a, gsc, ctcf, lhx1 and sox11 loci.


C . Feature maps , showing expression of selected genes in single ( cf . Appendix Fig . S2 ) . Color scale represents gene expression value ( log1p - transformed per 10 , 000 unique reads ) for each for a given gene , from low ( grey ) to high ( orange ) .

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C. Feature maps, showing the expression of selected genes in single cells (cf. Appendix Fig. S2). Color scale represents the gene expression value (log1p-transformed counts per 10,000 unique reads) for each cell for a given gene, from low (grey) to high (orange).


A . Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1 , Foxb1 - Eomes , Eomes , Irx3 , Irx3 - Otx2 , Otx2 Lhx8 .

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A. Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1, Foxb1-Eomes, Eomes, Irx3, Irx3-Otx2, Otx2 and Lhx8.


C . Fold enrichment of genes with zygotically defined ( ZyD ) H3K4me3 at their promoter in AC with transcription factor overexpression . Asterisk indicates hypergeometric p - value < = 0 . 01 .

C. Fold enrichment of genes with zygotically defined (ZyD) H3K4me3 at their promoter in AC with transcription factor overexpression. Asterisk indicates hypergeometric p-value <= 0.01.


( A , B ) Actively dividing labelled by BrdU Ki67 are reduced in SGZ of DSCR1 mutants compared to that of wild type mice . Brain sections were prepared 1 day after BrdU injection . box is magnified in lower panels : DAPI ( blue ) , Ki67 ( green ) , BrdU ( red ) . Arrow heads indicate BrdU Ki - 67 double positive in SGZ .

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(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ.


( C , D ) Differentiating are identified by with BrdU DCX . Brain sections were prepared 10 days after BrdU injection .

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(C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection.


( E , F ) Maturation of progenitor are assessed by with BrdU NeuN . Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness , a total of 24 sections were obtained from one hippocampus . Scale bars : 100 μm in large panel , 10 μm in magnified . Values are shown as mean ± tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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(E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.


( A ) miR - 124 expression is altered in DSCR1 mutants . Values are shown as mean ± tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 01 .

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(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01.


( B ) Promoter activity of miR - 124 in Neuro2 A with DSCR1 reduction or overexpression . Firefly luciferase reporter under control of miR - 124 promoter is measured . Values are shown as mean ± tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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(B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01.


( C ) Bisulfite sequencing analyses of miR - 124 promoter . Each CpG site is indicated , methylation status of two different regions are shown . Open filled circles represent unmethylated methylated CpG sites , respectively . of methylated CpGs among total number of CpGs is also shown .

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(C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown.


( D ) Neuro2A containing DSCR1shRNA show increased activity of miR - 124 promoter measured . miR - 124 promoter was inserted in front of luciferase reporter . Site - directed mutation of two methylation sites ( 31 58 ) in promoter shows luciferase activity similar to that of DSCR1 reduction . Values are shown as mean ± , tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 05 .

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(D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( E ) Overexpression of DSCR1 reduced luciferase activity , while removing methylation sites in miR - 124 promoter increases it . Values are shown as mean ± , tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 05 .

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(E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


( A ) DSCR1 binds to TET1 introns . binding affinity of DSCR1 to intron 8 of TET1 decreased with increased dosage of U1 snRNA U2 snRNA .

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(A) DSCR1 binds to TET1 introns. The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA.


( B ) DSCR1 missing recognition motif ( △ RRM ) does not bind to TET1 intron 8 .

(B) DSCR1 missing RNA recognition motif (△RRM) does not bind to the TET1 intron 8.


( C , D ) Schematic diagram of luciferase reporter separated by TET1 intron 8 9 or GAPDH intron 3 4 . DSCR1 reduction increases activity of this luciferase construct , while DSCR1 overexpression decreases its activity . In contrast , altering DSCR1 did not affect luciferase activity of construct containing GAPDH introns . Values are shown as mean ± , tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 . * * * P < 0 . 001 .

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(C, D) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4. DSCR1 reduction increases the activity of this luciferase construct, while DSCR1 overexpression decreases its activity. In contrast, altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. ***P< 0.001.