( D ) Time - dependent changes of H2O2 levels in the ER of PDI + / + and PDI - / - hMSCs at P8 . Left , the fluorescence intensity ratio of HyPer - ER at 535 nm with excitation at 488 and 405 nm were firstly monitored at resting state for 5 min , followed by addition of 0 . 5 mM DTT ( yellow bar ) for another 15 min . Right , statistical analysis of the fluorescence intensity ratio at 488 / 405 nm excitation of HyPer - ER probe at 15 min post DTT addition . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

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

(D) Time-dependent changes of H2O2 levels in the ER of PDI+/+ and PDI-/- hMSCs at P8. Left, the fluorescence intensity ratio of HyPer-ER at 535 nm with excitation at 488 and 405 nm were firstly monitored at resting state for 5 min, followed by addition of 0.5 mM DTT (yellow bar) for another 15 min. Right, statistical analysis of the fluorescence intensity ratio at 488/405 nm excitation of HyPer-ER probe at 15 min post DTT addition. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( E ) Analysis of the H2O2 levels in PDI + / + and PDI - / - hMSCs at P8 using Amplex Red assays . n = 3 biological repeats . ( F ) Analysis of the cellular total ROS levels in PDI + / + and PDI - / - hMSCs at P8 using H2DCFDA probe . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

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

(E) Analysis of the H2O2 levels in PDI+/+ and PDI-/- hMSCs at P8 using Amplex Red assays. n = 3 biological repeats. (F) Analysis of the cellular total ROS levels in PDI+/+ and PDI-/- hMSCs at P8 using H2DCFDA probe. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( M ) Confocal imaging of PDI + / + and PDI - / - hMSCs expressing Hyper7 - nucleus at steady state and upon addition of 2 mM DTT . The fluorescence intensity ratio at 488 / 405 nm obtained from individual cells was calculated at indicated times with a representative false color image . Scale bar , 10 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 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

(M) Confocal imaging of PDI+/+ and PDI-/- hMSCs expressing Hyper7-nucleus at steady state and upon addition of 2 mM DTT. The fluorescence intensity ratio at 488/405 nm obtained from individual cells was calculated at indicated times with a representative false color image. Scale bar, 10 μm.


( C , D ) Density plot showing that upregulated ( C ) or downregulated ( D ) genes in the LP hMSCs ( PDI + / + , LP vs . PDI + / + , EP ) that were restored after PDI deletion ( PDI - / - , LP vs . PDI + / + , LP ) . Comparisons were performed with two - sided Wilcoxon signed - rank test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Density plot showing that upregulated (C) or downregulated (D) genes in the LP hMSCs (PDI+/+, LP vs. PDI+/+, EP) that were restored after PDI deletion (PDI-/-, LP vs. PDI+/+, LP). Comparisons were performed with two-sided Wilcoxon signed-rank test.


( K ) Conjoint analysis showing the secretion levels of PDI and those in ( I ) and various SASPs from multiple senescence inducers and in plasma proteome from 3 , 087 healthy individuals RS , replicative senescence ( PDI + / + , LP vs . PDI + / + , EP ) . ATV , atazanavir treatment ; RAS , inducible RAS overexpression ; IR , X - irradiation . The color keys that change from dark blue to dark purple represent the fold change of gene and protein , change from white to red represent the change in protein levels per unit increase in the covariate in plasma proteomic .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

(K) Conjoint analysis showing the secretion levels of PDI and those in (I) and various SASPs from multiple senescence inducers and in plasma proteome from 3,087 healthy individuals RS, replicative senescence (PDI+/+, LP vs. PDI+/+, EP). ATV, atazanavir treatment; RAS, inducible RAS overexpression; IR, X-irradiation. The color keys that change from dark blue to dark purple represent the fold change of gene and protein, change from white to red represent the change in protein levels per unit increase in the covariate in plasma proteomic.


( A ) qPCR analysis of SERPINE1 expression in PDI + / + and PDI - / - hMSCs at EP and LP . n = 3 independent experiments . Data information : In ( A , data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of SERPINE1 expression in PDI+/+ and PDI-/- hMSCs at EP and LP. n = 3 independent experiments. Data information: In (A, data are presented as mean ± SEM, two-tailed Student's t-test.


( B ) Western blotting analysis of SERPINE1 protein in PDI + / + and PDI - / - hMSCs at EP and LP . GAPDH was used as the loading control of cell lysates . Ponceau was used as the loading control of conditional medium .

O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting analysis of SERPINE1 protein in PDI+/+ and PDI-/- hMSCs at EP and LP. GAPDH was used as the loading control of cell lysates. Ponceau was used as the loading control of conditional medium.


( F ) Left , Ki67 staining in PDI - / - hMSCs transduced with sgNTC or SERPINE1 - targeting activation sgRNAs . Scale bar , 20 μm . Right , statistical analysis of Ki67 - positive cells . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left, Ki67 staining in PDI-/- hMSCs transduced with sgNTC or SERPINE1-targeting activation sgRNAs. Scale bar, 20 μm. Right, statistical analysis of Ki67-positive cells. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( G ) Left , SA - β - gal staining in PDI - / - hMSCs transduced with sgNTC or SERPINE1 - targeting activation sgRNAs . Scale bar , 200 μm . Right , statistical analysis of SA - β - gal - positive cells . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

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

(G) Left, SA-β-gal staining in PDI-/- hMSCs transduced with sgNTC or SERPINE1-targeting activation sgRNAs. Scale bar, 200 μm. Right, statistical analysis of SA-β-gal-positive cells. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( H ) qPCR analysis of the SERPINE1 mRNA expression in PDI + / + and PDI - / - hMSCs treated with the indicated concentration H2O2 for 4 days . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

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

(H) qPCR analysis of the SERPINE1 mRNA expression in PDI+/+ and PDI-/- hMSCs treated with the indicated concentration H2O2 for 4 days. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( J ) H2O2 levels in PDI - / - hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer - ER at 488 / 405 nm excitation after the addition of 0 . 5 mM DTT for 15 min . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

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

(J) H2O2 levels in PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer-ER at 488/405 nm excitation after the addition of 0.5 mM DTT for 15 min. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( K ) H2O2 levels in PDI - / - hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer7 - nucleus at 500 / 400 nm excitation after the addition of 2 mM DTT for 20 min . n = 3 independent experiments . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

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

(K) H2O2 levels in PDI-/- hMSCs transduced with lentiviruses expressing Luc or PDI were determined by the fluorescence ratio of HyPer7-nucleus at 500/400 nm excitation after the addition of 2 mM DTT for 20 min. n = 3 independent experiments. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( A ) Western blotting of PDI expression in replicative senescent ( RS ) hMSCs , Hutchinson - Gilford Progeria Syndrome ( HGPS ) hMSCs , WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI - targeting sgRNA . GAPDH was used as the 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

(A) Western blotting of PDI expression in replicative senescent (RS) hMSCs, Hutchinson-Gilford Progeria Syndrome (HGPS) hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. GAPDH was used as the loading control.


( B ) Single clonal formation assay in RS hMSCs , HGPS hMSCs , WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI - targeting sgRNA . n = 3 biological repeats . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Single clonal formation assay in RS hMSCs, HGPS hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. n = 3 biological repeats. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


( C ) SA - β - gal staining in RS hMSCs , HGPS hMSCs , WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI - targeting sgRNA . n = 3 biological repeats . Scale bar , 200 µm . Data information : data are presented as mean ± SEM , two - tailed Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SA-β-gal staining in RS hMSCs, HGPS hMSCs, WS hMSCs and human primary hMSCs from an old individual transduced with sgNTC or PDI-targeting sgRNA. n = 3 biological repeats. Scale bar, 200 µm. Data information: data are presented as mean ± SEM, two-tailed Student's t-test.


A1 ) Representative WBs showing DRD2 and RGS9 - 2 downregulation in adult ( P60 - P90 ) Tor1a + / - ( + / - ) striata , characterized by a reduced torsinA protein level with respect to Tor1a + / + ( + / + ) littermates . The striatal level of RGS7 is unchanged . The dot plot shows Tor1a + / - data , normalized to Tor1a + / + controls of the same experiment . TorsinA : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t test * * * P = 0 . 0003 ; DRD2 : Tor1a + / + N = 18 , Tor1a + / - N = 16 , t test * * P = 0 . 0029 ; RGS9 - 2 : Tor1a + / + N = 32 , Tor1a + / - N = 33 , t test * P = 0 . 0115 ) . RGS7 : Tor1a + / + N = 7 , Tor1a + / - N = 7 , t test P = 0 . 8721 ) . WB quantification data , expressed as the ratio of protein vs . loading control intensity level , are normalized to the wild - type samples of the same experiment . A2 , A3 , A4 ) Time - course of changes in torsinA , DRD2 and RGS9 - 2 striatal levels along postnatal development . Summary plot data are normalized to the Tor1a + / + P60 sample of each independent experiment . A2 ) TorsinA level is significantly reduced in mutants throughout the developmental period considered ( P7 N = 3 ; P14 - P60 N = 4 ; one - way ANOVA P < 0 . 0001 and Bonferroni ' s Multiple Comparison Test * * P < 0 . 01 at P7 , P14 and P60 ) . A3 , A4 ) DRD2 and RGS9 - 2 levels show parallel courses , with a similar increase from P7 to P21 in Tor1a + / + as well as Tor1a + / - striatal lysates ( P7 : DRD2 N = 3 , RGS9 - 2 N = 4 ; P14 - P21 N = 4 ; one - way ANOVA with Bonferroni ' s Multiple Comparison Test P > 0 . 05 ) . At P60 , a simultaneous reduction of DRD2 and RGS9 - 2 levels is observed in Tor1a + / - striatal lysates ( N = 4 ; Tor1a + / - one sample t test DRD2 : * P = 0 . 0250 , RGS9 - 2 : * P = 0 . 0175 ) . WB quantification data , expressed as the ratio of protein vs . loading control intensity level , are normalized to the wild - type P60 sample of the same experiment

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

A1) Representative WBs showing DRD2 and RGS9-2 downregulation in adult (P60-P90) Tor1a+/- (+/-) striata, characterized by a reduced torsinA protein level with respect to Tor1a+/+ (+/+) littermates. The striatal level of RGS7 is unchanged. The dot plot shows Tor1a+/- data, normalized to Tor1a+/+ controls of the same experiment. TorsinA: Tor1a+/+ N=7, Tor1a+/- N=6, t test ***P=0.0003; DRD2: Tor1a+/+ N=18, Tor1a+/- N=16, t test **P=0.0029; RGS9-2: Tor1a+/+ N=32, Tor1a+/- N=33, t test *P=0.0115). RGS7: Tor1a+/+ N=7, Tor1a+/- N=7, t test P=0.8721). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. A2,A3,A4) Time-course of changes in torsinA, DRD2 and RGS9-2 striatal levels along postnatal development. Summary plot data are normalized to the Tor1a+/+ P60 sample of each independent experiment. A2) TorsinA level is significantly reduced in mutants throughout the developmental period considered (P7 N=3; P14-P60 N=4; one-way ANOVA P<0.0001 and Bonferroni's Multiple Comparison Test **P<0.01 at P7, P14 and P60). A3,A4) DRD2 and RGS9-2 levels show parallel courses, with a similar increase from P7 to P21 in Tor1a+/+ as well as Tor1a+/- striatal lysates (P7: DRD2 N=3, RGS9-2 N=4; P14-P21 N=4; one-way ANOVA with Bonferroni's Multiple Comparison Test P>0.05). At P60, a simultaneous reduction of DRD2 and RGS9-2 levels is observed in Tor1a+/- striatal lysates (N=4; Tor1a+/- one sample t test DRD2: *P=0.0250, RGS9-2: *P= 0.0175). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type P60 sample of the same experiment


B ) Representative WB images and the dot plot show downregulation of torsinA , DRD2 and RGS9 - 2 striatal levels also in adult P60 - P90 Tor1a ∆ gag / + ( ∆ gag / + ) mice . TorsinA : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 12 , t test * * * P = 0 . 0006 ; DRD2 : Tor1a + / + N = 7 , Tor1a ∆ gag / + N = 9 , t test * P = 0 . 0183 ; RGS9 - 2 : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 9 , t test * P = 0 . 0192 . WB quantification data , expressed as the ratio of protein vs . loading control intensity level , are normalized to the wild - type samples of the same experiment . Mean ± SEM is represented in the

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative WB images and the dot plot show downregulation of torsinA, DRD2 and RGS9-2 striatal levels also in adult P60-P90 Tor1a∆gag/+ (∆gag/+) mice. TorsinA: Tor1a+/+ N=10, Tor1a∆gag/+ N=12, t test ***P= 0.0006; DRD2: Tor1a+/+ N=7, Tor1a∆gag/+ N=9, t test *P= 0.0183; RGS9-2: Tor1a+/+ N=10, Tor1a∆gag/+ N=9, t test *P= 0.0192. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. Mean ± SEM is represented in the graphs


C ) Left . Representative image of coronal striatal slices of fresh frozen brains of wild - type and Tor1a + / - mice probed with the DRD2 radioligand 3H - spiperone . Scale bar 1 . 5 mm . ( Right ) Graph showing DRD2 binding density in Tor1a + / - striatal sections , obtained from the densitometric quantification analysis , expressed as percentage variation compared to control

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

C) Left. Representative image of coronal striatal slices of fresh frozen brains of wild-type and Tor1a+/- mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. (Right) Graph showing DRD2 binding density in Tor1a+/- striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals


A1 ) Representative WB of RGS9 - 2 , R7BP , and Gβ5 proteins on four Tor1a + / + and four Tor1a + / - striatal lysates . The bar histogram on the right shows the quantification of the three proteins in each sample . A2 ) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a + / - mice ( Tor1a + / + N = 11 , Tor1a + / - N = 10 , Mann Whitney test P = 0 . 2453 ) . A3 ) Conversely , the level of the R7 binding protein R7BP is increased in Tor1a + / - striatum ( Tor1a + / + N = 12 , Tor1a + / - N = 12 , t test * * P = 0 . 0014 ) . Values are represented as ratio of protein vs . loading control intensity level , normalized to the mean of Tor1a + / + control values of the same

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

A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/- striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/- mice (Tor1a+/+ N=11, Tor1a+/- N=10, Mann Whitney test P=0.2453). A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/- striatum (Tor1a+/+ N=12, Tor1a+/- N=12, t test **P=0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experiment


B1 ) Representative WB of lysates of Tor1a + / + and Tor1a + / - dorsal striatum slices incubated for 5 hours in the presence ( w / leu ) or absence ( w / o leu ; contralateral striatum ) of the protease inhibitor leupeptin . Samples showing enhanced degradation of DRD2 and / or RGS9 - 2 proteins show additional bands at lower molecular weight . B2 ) Summary plot reporting mean ± SEM of RGS9 - 2 and DRD2 protein level values , expressed as the ratio of protein vs . loading control intensity level , normalized to the value of the Tor1a + / + w / leu sample measured in the same experiment . DRD2 : Tor1a + / + N = 4 , t test P = 0 . 4376 ; Tor1a + / - N = 4 , t test * P = 0 . 0427 ; RGS9 - 2 : Tor1a + / + N = 5 , t test * P = 0 . 0464 ; Tor1a + / - N = 5 , t test * P = 0 .

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

B1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190


C1 , C2 , C3 , C4 ) Dot plots showing DRD2 , RGS9 - 2 , R7BP and Gβ5 protein levels measured in striatal detergent - resistant - membrane ( DRM ) preparations from Tor1a + / - ( + / - ) and WT ( + / + ) mice ( DRD2 : Tor1a + / + N = 16 , Tor1a + / - N = 13 , t test P = 0 . 0129 ; RGS9 - 2 : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t test * P = 0 . 0396 ; R7BP : Tor1a + / + N = 6 , Tor1a + / - N = 6 , t test * * * P = 0 . 0002 ; Gβ5 : Tor1a + / + N = 9 , Tor1a + / - N = 9 , t test P = 0 . 8461 )

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

C1, C2, C3, C4) Dot plots showing DRD2, RGS9-2, R7BP and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/- (+/-) and WT (+/+) mice (DRD2: Tor1a+/+ N=16, Tor1a+/- N=13, t test P=0.0129; RGS9-2: Tor1a+/+ N=7, Tor1a+/- N=6, t test *P=0.0396; R7BP: Tor1a+/+ N=6, Tor1a+/- N=6, t test ***P=0.0002; Gβ5: Tor1a+/+ N=9, Tor1a+/- N=9, t test P=0.8461)


WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a + / - mice . A ) The level of β - arrestin 2 ( β - Arr2 ) is not altered ( Tor1a + / + N = 11 , Tor1a + / - N = 12 , t test P = 0 . 6796 ) , whereas the level of spinophilin ( B ) is decreased ( Tor1a + / + N = 5 , Tor1a + / - N = 8 , t test * P = 0 . 0151 )

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

WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a+/- mice. A) The level of β-arrestin 2 (β-Arr2) is not altered (Tor1a+/+ N=11, Tor1a+/- N=12, t test P=0.6796), whereas the level of spinophilin (B) is decreased (Tor1a+/+ N=5, Tor1a+/- N=8, t test *P=0.0151)


C ) A Sh - RGS9 - 2 - GFP viral preparation injected into the right striatum ( + ) of C57Bl / 6 mice effectively reduces both RGS9 - 2 and DRD2 protein levels , with respect to the contralateral striatum ( - ) . The summary plot reports mean ± SEM values ( RGS9 - 2 : N = 4 , paired t test * P = 0 . 0100 ; DRD2 : N = 4 , paired t test * P = 0 . 0432 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A Sh-RGS9-2-GFP viral preparation injected into the right striatum (+) of C57Bl/6 mice effectively reduces both RGS9-2 and DRD2 protein levels, with respect to the contralateral striatum (-).The summary plot reports mean ± SEM values (RGS9-2: N=4, paired t test *P=0.0100; DRD2: N=4, paired t test *P=0.0432)


D ) The endosomal marker Rab4 is upregulated in the Tor1a + / - striatum ( Tor1a + / + N = 13 , Tor1a + / - N = 14 , t test * * P = 0 . 0087 )

O O O O O O O O O O O O O O O O O O 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) The endosomal marker Rab4 is upregulated in the Tor1a+/- striatum (Tor1a+/+ N=13, Tor1a+/- N=14, t test **P=0.0087)


E ) Basal levels of markers of the degradative pathway , p62 and LC3 - II , show a trend towards upregulation in Tor1a + / - striatal lysates ( p62 : Tor1a + / + N = 11 , Tor1a + / - N = 11 , t test P = 0 . 2292 ; LC3 - II : Tor1a + / - N = 9 , Tor1a + / - N = 11 , t test P = 0 . 1574 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Basal levels of markers of the degradative pathway, p62 and LC3-II, show a trend towards upregulation in Tor1a+/- striatal lysates (p62: Tor1a+/+ N=11, Tor1a+/- N=11, t test P=0.2292; LC3-II: Tor1a+/- N=9, Tor1a+/- N=11, t test P=0.1574)


F ) Treatment with bafilomycin A1 ( BafA1 ) induces a significantly more pronounced increase of LC3 - II in Tor1a + / - than in wild - type dorsal striatum slices . BafA1 - induced increase in LC3 - II level measured in Tor1a + / - slices was normalized to the increase observed in the Tor1a + / + samples of the same experiment ( N = 4 , one sample t test * * P = 0 . 0012 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Treatment with bafilomycin A1 (BafA1) induces a significantly more pronounced increase of LC3-II in Tor1a+/- than in wild-type dorsal striatum slices. BafA1-induced increase in LC3-II level measured in Tor1a+/- slices was normalized to the increase observed in the Tor1a+/+ samples of the same experiment (N=4, one sample t test **P=0.0012)


G ) LAMP - 2 , a lysosomal marker , shows a trend for upregulation ( Tor1a + / + N = 8 ; Tor1a + / - N = 6 , t test P = 0 . 2232 )

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

G) LAMP-2, a lysosomal marker, shows a trend for upregulation (Tor1a+/+ N=8; Tor1a+/- N=6, t test P= 0.2232)


Representative double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling ( red ) in choline acetyltransferase ( ChAT ) - positive ( cyano ) ChIs of Tor1a + / + and Tor1a + / - mice . A1 ) Two representative ChIs identified by the white box in the low magnification images ( bar = 20 µm ) are shown at higher magnification in ( A2 ) ( bar = 5 µm ) . A3 ) The graph reporting mean ± SEM values of p62 / ChAT signal overlap in ChIs , normalized to Tor1a + / + values of the same experiment , shows a significant increase of p62 in Tor1a + / - neurons ( Tor1a + / + n = 20 cells , N = 6 mice ; Tor1a + / - n = 20 cells , N = 6 mice , t test * * * P = 0 . 00008 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling (red) in choline acetyltransferase (ChAT)-positive (cyano) ChIs of Tor1a+/+ and Tor1a+/- mice. A1) Two representative ChIs identified by the white box in the low magnification images (bar = 20 µm) are shown at higher magnification in (A2) (bar = 5 µm). A3) The graph reporting mean ± SEM values of p62/ChAT signal overlap in ChIs, normalized to Tor1a+/+ values of the same experiment, shows a significant increase of p62 in Tor1a+/- neurons (Tor1a+/+ n=20 cells, N=6 mice; Tor1a+/- n=20 cells, N=6 mice, t test ***P=0.00008)


A ) Top . Low magnification merged confocal images of coronal striatal sections showing ChAT - labelled ( cyano ) ChIs in the area of mCherry fluorescence ( red ) indicating viral infection . Bar = 100 µm . Bottom . Higher magnification images of the ChIs identified by the white box in the upper images show viral - induced expression of mCherry and GFP signal . Bar = 20 µm . B ) The mCherry / ChAT overlap is increased in ChIs from Tor1a + / - mice ( Tor1a + / + n = 24 cells , Tor1a + / - n = 22 cells , N = 8 mice / genotype , t test * * * P = 0 . 0005 ) , but the mCherry / GFP fluorescence ratio indicated that the LC3 flux was unchanged ( Tor1a + / + n = 19 cells , Tor1a + / - n = 21 cells , N = 8 mice / genotype , Mann Whitney test P = 0 . 2334 )

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

A) Top. Low magnification merged confocal images of coronal striatal sections showing ChAT-labelled (cyano) ChIs in the area of mCherry fluorescence (red) indicating viral infection. Bar = 100 µm. Bottom. Higher magnification images of the ChIs identified by the white box in the upper images show viral-induced expression of mCherry and GFP signal. Bar = 20 µm. B) The mCherry/ChAT overlap is increased in ChIs from Tor1a+/- mice (Tor1a+/+ n=24 cells, Tor1a+/- n=22 cells, N=8 mice/genotype, t test ***P=0.0005), but the mCherry/GFP fluorescence ratio indicated that the LC3 flux was unchanged (Tor1a+/+ n=19 cells, Tor1a+/- n=21 cells, N=8 mice/genotype, Mann Whitney test P=0.2334)


C ) Merged and split channels confocal images ( bar = 10 µm ) of representative Tor1a + / + and Tor1a + / - ChIs showing red dots and yellow puncta ( indicated by white circles ) , quantified in ( D ) : red dots : Tor1a + / + n = 13 cells , Tor1a + / - n = 14 cells , t test * * * P = 0 . 0003 ; yellow dots : Tor1a + / + n = 13 cells , Tor1a + / - n = 14 cells , t test * * P = 0 . 0048 , N = 5 mice / genotype ) . Measurement of the mCherry / yellow dots ratio indicated that the autophagic flux was not altered ( Tor1a + / + n = 13 cells ; Tor1a + / - n = 14 cells , Mann Whitney test P = 0 . 8842 ) . Data in B , D ) are represented 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Merged and split channels confocal images (bar = 10 µm) of representative Tor1a+/+ and Tor1a+/- ChIs showing red dots and yellow puncta (indicated by white circles), quantified in (D): red dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test ***P=0.0003; yellow dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test **P=0.0048, N=5 mice/genotype). Measurement of the mCherry/yellow dots ratio indicated that the autophagic flux was not altered (Tor1a+/+ n=13 cells; Tor1a+/- n=14 cells, Mann Whitney test P=0.8842). Data in B,D) are represented as mean ± SEM


A ) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT ( cyano ) , DRD2 ( red ) , and the nuclear stain DAPI ( white ) . Bar = 200 μm . B1 , B2 ) Top . Representative ChAT - positive ( cyano ) cholinergic interneurons , labelled for DRD2 ( red ) , from a Tor1a + / + ( B1 ) or Tor1a + / - ( B2 ) striatal section . The colocalization mask of the two signals is shown in white ( right ) . Bar = 10 μm . Bottom . Higher magnification images ( bar = 5 μm ) of representative Tor1a + / + ( B1 ) and Tor1a + / - ( B2 ) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI ( green ) . A detail at higher magnification is shown in the

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT (cyano), DRD2 (red), and the nuclear stain DAPI (white). Bar = 200 μm. B1,B2) Top. Representative ChAT-positive (cyano) cholinergic interneurons, labelled for DRD2 (red), from a Tor1a+/+ (B1) or Tor1a+/- (B2) striatal section. The colocalization mask of the two signals is shown in white (right). Bar = 10 μm. Bottom. Higher magnification images (bar = 5 μm) of representative Tor1a+/+ (B1) and Tor1a+/- (B2) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI (green). A detail at higher magnification is shown in the insets


A ) Middle . Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum , reporting the coordinates relative to Bregma . HSV - LacZ ( left , grey ) and HSV - RGS9 - 2 ( right , red ) were injected into the left and right striatum , respectively , and mice sacrificed after 3 - 5 days . Left . Serial confocal images showing the extent of viral infection , as indicated by the robust dark β - gal staining . Right . The RGS9 - 2 immunolabelling ( red ) is particularly intense near the injection site , indicating enhanced protein expression with respect to the endogenous level . Bar = 200

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Middle. Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum, reporting the coordinates relative to Bregma. HSV-LacZ (left, grey) and HSV-RGS9-2 (right, red) were injected into the left and right striatum, respectively, and mice sacrificed after 3-5 days. Left. Serial confocal images showing the extent of viral infection, as indicated by the robust dark β-gal staining. Right. The RGS9-2 immunolabelling (red) is particularly intense near the injection site, indicating enhanced protein expression with respect to the endogenous level. Bar = 200 µm


B ) A representative WB shows correlated viral - mediated increases in RGS9 - 2 and DRD2 protein levels in Tor1a + / - dorsal striata ( lanes 4 and 5 ) . while in a sample where RGS9 - 2 level was not increased by viral infection ( lane 6 ) DRD2 level remained low as in the sham - injected striatum ( lane 7 ) . C , D ) The summary plots report mean ± SEM values of the ratio of protein vs . loading control intensity level measured from the injected ( HSV / LV ) or sham contralateral Tor1a + / - ( + / - ) striata , normalized to the Tor1a + / + ( + / + ) samples of the same experiment . C ) RGS9 - 2 : Tor1a + / + ( N = 10 ) ; Tor1a + / - injected ( N = 7 ) vs . contralateral ( N = 7 ) : paired t test * * P = 0 . 0011 ; D ) DRD2 : Tor1a + / + ( N = 9 ) ; Tor1a + / - injected ( N = 6 ) vs . contralateral ( N = 6 ) : paired t test * P = 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A representative WB shows correlated viral-mediated increases in RGS9-2 and DRD2 protein levels in Tor1a+/- dorsal striata (lanes 4 and 5) . while in a sample where RGS9-2 level was not increased by viral infection (lane 6) DRD2 level remained low as in the sham-injected striatum (lane 7). C,D) The summary plots report mean ± SEM values of the ratio of protein vs. loading control intensity level measured from the injected (HSV/LV) or sham contralateral Tor1a+/- (+/-) striata, normalized to the Tor1a+/+ (+/+) samples of the same experiment. C) RGS9-2: Tor1a+/+ (N=10); Tor1a+/- injected (N=7) vs. contralateral (N=7): paired t test **P=0.0011; D) DRD2: Tor1a+/+ (N=9); Tor1a+/- injected (N=6) vs. contralateral (N=6): paired t test *P=0.0380


A ) As depicted in the cartoon and shown by representative confocal images , patch - clamp recordings were performed in the dorsal striatum , in the area of HSV - LacZ ( blue , left ) and HSV - RGS9 - 2 ( red , right ) viral infection , the latter showing an increased RGS9 - 2 immunolabelling with respect to the sham contralateral striatum of the same mouse . Representative confocal images at higher magnification show colocalization of RGS9 - 2 immunolabeling with Lucifer Yellow ( LY ) fluorescence in two ChIs loaded intracellularly with LY during whole - cell recordings in the infected

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) As depicted in the cartoon and shown by representative confocal images, patch-clamp recordings were performed in the dorsal striatum, in the area of HSV-LacZ (blue, left) and HSV-RGS9-2 (red, right) viral infection, the latter showing an increased RGS9-2 immunolabelling with respect to the sham contralateral striatum of the same mouse. Representative confocal images at higher magnification show colocalization of RGS9-2 immunolabeling with Lucifer Yellow (LY) fluorescence in two ChIs loaded intracellularly with LY during whole-cell recordings in the infected area


B ) Representative patch - clamp recordings of ChIs from hMT ( top , perforated configuration ) and Tor1aΔgag / + ( bottom , cell - attached configuration ) mice , showing that viral - mediated delivery of RGS9 - 2 rescues the physiological response to the DRD2 agonist quinpirole ( 10 µM , 2 min ) . The dot plot reports the firing frequency changes induced by quinpirole in hMT striata ( N = 6 , HSV - RGS9 - 2 n = 9 , HSV - LacZ n = 14 , t test P < 0 . 0001 )

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

B) Representative patch-clamp recordings of ChIs from hMT (top, perforated configuration) and Tor1aΔgag/+ (bottom, cell-attached configuration) mice, showing that viral-mediated delivery of RGS9-2 rescues the physiological response to the DRD2 agonist quinpirole (10 µM, 2 min). The dot plot reports the firing frequency changes induced by quinpirole in hMT striata (N=6, HSV-RGS9-2 n=9, HSV-LacZ n=14, t test P<0.0001)


C ) Representative traces show a typical response of Tor1a + / + ChIs to quinpirole , characterized by a reduction in the frequency of the ongoing firing activity of the cell , as opposed to an aberrant increase in firing frequency in Tor1a + / - ChIs , quantified in the plot ( right , Tor1a + / + N = 6 , n = 14 ; Tor1a + / - N = 10 , n = 27 ; Tor1a + / + vs . Tor1a + / - in quinpirole t test * * * P = 0 . 0005 ; Tor1a + / - basal vs . quinpirole paired t test * * * P = 0 . 0009 )

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

C) Representative traces show a typical response of Tor1a+/+ ChIs to quinpirole, characterized by a reduction in the frequency of the ongoing firing activity of the cell, as opposed to an aberrant increase in firing frequency in Tor1a+/- ChIs, quantified in the plot (right, Tor1a+/+ N=6, n=14; Tor1a+/- N=10, n=27; Tor1a+/+ vs. Tor1a+/- in quinpirole t test ***P=0.0005; Tor1a+/- basal vs. quinpirole paired t test ***P=0.0009)


D ) LV - mediated delivery of RGS9 - 2 rescues the physiological response to quinpirole in Tor1a + / - slices . Dot plot reporting firing frequency changes induced by quinpirole in Tor1a + / + ( N = 3 ) and Tor1a + / - ( N = 6 ) striatal ChIs infected with LV / HSV - RGS9 - 2 or control LV - GFP and HSV - LacZ ChIs ( Tor1a + / + RGS9 - 2 n = 7 ; Tor1a + / - RGS9 - 2 n = 9 ; Tor1a + / - LacZ / GFP n = 8 ; Tor1a + / - : LacZ / GFP vs . RGS9 - 2 t test * P = 0 . 0136 )

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LV-mediated delivery of RGS9-2 rescues the physiological response to quinpirole in Tor1a+/- slices. Dot plot reporting firing frequency changes induced by quinpirole in Tor1a+/+ (N=3) and Tor1a+/- (N=6) striatal ChIs infected with LV/HSV-RGS9-2 or control LV-GFP and HSV-LacZ ChIs (Tor1a+/+ RGS9-2 n=7; Tor1a+/- RGS9-2 n=9; Tor1a+/- LacZ/GFP n=8; Tor1a+/-: LacZ/GFP vs. RGS9-2 t test *P=0.0136)


A Effects of myeloid - specific Lamtor1 deficiency on peritoneal IL - 1β production , CD11b + Ly6C + monocyte recruitment , and CD11b + Ly6G + neutrophil recruitment after an intraperitoneal injection of 600 μL alum solution ( 20 mg / mL , 6 h ) , Data are shown as means ± SEM . * indicates P < 0 . 05 and * * * indicates P < 0 . 001 by Student ' s t - test , n = 6 mice ( peritoneal IL - 1β production ) and 4 mice ( FACS analysis ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effects of myeloid-specific Lamtor1 deficiency on peritoneal IL-1β production, CD11b+ Ly6C+ monocyte recruitment, and CD11b+ Ly6G+ neutrophil recruitment after an intraperitoneal injection of 600 μL alum solution (20 mg/mL, 6 h), Data are shown as means ± SEM. * indicates P < 0.05 and *** indicates P < 0.001 by Student's t-test, n = 6 mice (peritoneal IL-1β production) and 4 mice (FACS analysis).


B Effects of myeloid - specific Lamtor1 deficiency in an acute gouty arthritis model . MSU crystals ( 0 . 5 mg ) or the PBS control were injected intra - articularly into the tibia - tarsal joint of Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre mice . Mice were assessed for joint swelling using electronic calipers . Images show photomicrographs ( upper panel ) and hematoxylin and eosin - stained sections ( middle and lower panels ) of ankle joints obtained at 24 h . Data are shown as the means ± SEM . * * * indicates P < 0 . 001 by Student ' s t - test , n = 7 mice each .

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

B Effects of myeloid-specific Lamtor1 deficiency in an acute gouty arthritis model. MSU crystals (0.5 mg) or the PBS control were injected intra-articularly into the tibia-tarsal joint of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre mice. Mice were assessed for joint swelling using electronic calipers. Images show photomicrographs (upper panel) and hematoxylin and eosin-stained sections (middle and lower panels) of ankle joints obtained at 24 h. Data are shown as the means ± SEM. *** indicates P < 0.001 by Student's t-test, n = 7 mice each.


C Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation . ELISA assay shows IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with LPS ( 200 ng / mL , 4 h ) , followed by nigericin ( 15 μM , 1 h ) , ATP ( 10 mM , 1 h ) , or GPN ( 100 nM , 6 h ) .

C Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation. ELISA assay shows IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h), followed by nigericin (15 μM, 1 h), ATP (10 mM, 1 h), or GPN (100 nM, 6 h).


D Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO different single clones , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO different single clones, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


E , F Effects of Lamtor1 deficiency on IL - 1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous ROS ( hydrogen peroxide ) . ELISA assay shows IL - 1β secretion in supernatants after treatment with LPS ( 200 ng / mL ) , Rotenone ( 10 μM ) , or Antimycin ( 10 μg / mL ) for 6 h or followed by hydrogen peroxide ( 3 . 86 mM ) .

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E, F Effects of Lamtor1 deficiency on IL-1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous ROS (hydrogen peroxide). ELISA assay shows IL-1β secretion in supernatants after treatment with LPS (200 ng/mL), Rotenone (10 μM), or Antimycin (10 μg/mL) for 6 h or followed by hydrogen peroxide (3.86 mM).


G Effects of Lamtor1 reconstitution in Lamtor1 - deficient macrophages on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in BMDM supernatants from Lamtor1flox / flox cells , Lamtor1flox / flox LysM - Cre cells , and Lamtor1flox / flox LysM - Cre cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with LPS ( 200 ng / mL , 4 h ) .

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G Effects of Lamtor1 reconstitution in Lamtor1-deficient macrophages on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in BMDM supernatants from Lamtor1flox/flox cells, Lamtor1flox/flox LysM-Cre cells, and Lamtor1flox/flox LysM-Cre cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with LPS (200 ng/mL, 4 h).


H Effects of Lamtor1 deficiency on IL - 1β secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) .

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H Effects of Lamtor1 deficiency on IL-1β secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).


I Effects of Lamtor1 deficiency on IL - 18 secretion after NLRP3 inflammasome activation in human THP - 1 monocytes . ELISA assay shows IL - 18 secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with full - length Lamtor1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) .

I Effects of Lamtor1 deficiency on IL-18 secretion after NLRP3 inflammasome activation in human THP-1 monocytes. ELISA assay shows IL-18 secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full-length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).


J Effects of Lamtor1 deficiency on IL - 1β secretion after Pyrin inflammasome activation . ELISA assay showing IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with TcdB ( 0 . 5 μg / mL , 3 h ) after LPS ( 200 ng / mL , 4 h ) stimulation .

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J Effects of Lamtor1 deficiency on IL-1β secretion after Pyrin inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with TcdB (0.5 μg/mL, 3 h) after LPS (200 ng/mL, 4 h) stimulation.


K Effects of Lamtor1 deficiency on IL - 1β secretion after AIM2 inflammasome activation . ELISA assay showing IL - 1β secretion in supernatants from Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with Poly ( dA : dT ) ( 5 μg / mL , overnight ) and Lipofectamine 3000 after LPS ( 200 ng / mL , 4 h ) stimulation .

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K Effects of Lamtor1 deficiency on IL-1β secretion after AIM2 inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with Poly(dA:dT) (5 μg/mL, overnight) and Lipofectamine 3000 after LPS (200 ng/mL, 4 h) stimulation.


Effects of Lamtor1 deficiency on Caspase - 1 processing after NLRP3 inflammasome activation . Western blot shows caspase - 1 / p10 , pro - caspase - 1 , Lamtor1 , and β - actin . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs ( A ) ( B ) were treated with nigericin ( 15 μM , 1 h ) after LPS [ 200 ng / mL , for 4 h ( BMDM ) stimulation .

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Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs (A) (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL, for 4 h (BMDM) stimulation.


Effects of Lamtor1 deficiency on Caspase - 1 processing after NLRP3 inflammasome activation . Western blot shows caspase - 1 / p10 , pro - caspase - 1 , Lamtor1 , and β - actin . WT and Lamtor1 KO THP - 1 cells ( B ) were treated with nigericin ( 15 μM , 1 h ) after LPS [ 200 ng / mL 2 h ( THP - 1 ) ] stimulation .

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Effects of Lamtor1 deficiency on Caspase-1 processing after NLRP3 inflammasome activation. Western blot shows caspase-1/p10, pro-caspase-1, Lamtor1, and β-actin. WT and Lamtor1 KO THP-1 cells (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/mL 2 h (THP-1)] stimulation.


C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation . Western blot shows GSDMD , Lamtor1 , and β - actin . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with nigericin ( 15 μM , 1 h ) after LPS ( 200 ng / mL , 4 h ) stimulation .

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C Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation. Western blot shows GSDMD, Lamtor1, and β-actin. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation.


D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with nigericin ( 15 μM , 1 h ) after LPS ( 200 ng / mL , 4 h ) stimulation and the percentage of propidium iodide positive cells were counted by flow - cytometry .

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D Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/mL, 4 h) stimulation and the percentage of propidium iodide positive cells were counted by flow-cytometry.


E Effects of Lamtor1 deficiency on IL - 1β dynamics . Western blot shows IL - 1β and β - actin . WT cells and Lamtor1 KO cells were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) stimulation .

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E Effects of Lamtor1 deficiency on IL-1β dynamics. Western blot shows IL-1β and β-actin. WT cells and Lamtor1 KO cells were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) stimulation.


F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP - 1 cells . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with active form of GSDMD , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) .

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F Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with active form of GSDMD, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).


G Confirmation of the reconstitution of the active form Caspase - 1 ( p10 and p20 ) or GSDMD ( GSDMD - NT ) .

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G Confirmation of the reconstitution of the active form Caspase-1 (p10 and p20) or GSDMD (GSDMD-NT).


H Effects of Lamtor1 deficiency on the priming phase . Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs were treated with treated with LPS ( 200 ng / mL , 4 h ) .

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H Effects of Lamtor1 deficiency on the priming phase. Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs were treated with treated with LPS (200 ng/mL, 4 h).


I Effects of IL - 1β and active form of Caspase - 1 reconstitution on Lamtor1 KO THP - 1 cells . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with IL - 1β , active form of Caspase - 1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) .

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I Effects of IL-1β and active form of Caspase-1 reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with IL-1β, active form of Caspase-1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).


A Representative images of mutant Lamtor1 . PMA - primed Lamtor1 KO THP - 1 macrophages reconstituted with full - length or truncated Lamtor1 .

A Representative images of mutant Lamtor1. PMA-primed Lamtor1 KO THP-1 macrophages reconstituted with full-length or truncated Lamtor1.


B Effects of the Lamtor1 point mutation on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion in THP - 1 cell supernatants from WT cells , Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full - length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine ( G2A ) ; all cell types were then treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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B Effects of the Lamtor1 point mutation on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion in THP-1 cell supernatants from WT cells, Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full-length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine (G2A); all cell types were then treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


C Effects of Lamtor1 deficiency on mTORC1 activity . Immunoblot analysis of Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with LPS ( 200 ng / mL , 4 h ) .

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C Effects of Lamtor1 deficiency on mTORC1 activity. Immunoblot analysis of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h).


D Effect of the rapamycin treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from PMA ( 50 nM , overnight ) primed WT THP - 1 cells treated with rapamycin at indicated concentration for 2 h , then treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) .

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D Effect of the rapamycin treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from PMA (50 nM, overnight) primed WT THP-1 cells treated with rapamycin at indicated concentration for 2 h, then treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h).


E Effect of rapamycin treatment on Caspase - 1 processing . Western blot shows caspase - 1 / p20 . LPS ( 200 ng / mL , 4 h ) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin ( 15 μM , 1 h ) .

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E Effect of rapamycin treatment on Caspase-1 processing. Western blot shows caspase-1/p20. LPS (200 ng/mL, 4 h) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin (15 μM, 1 h).


F Effect of Torin - 1 treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from WT BMDMs pretreated with Torin - 1 ( 0 , 1 , 10 , 100 , or 250 nM ) for 2 h before LPS ( 200 ng / ml , 4 h ) and nigericin ( 15 μM , 1 h ) treatment ( left panel ) . Western blot shows phospho - p70 - S6K from BMDMs pretreated with Torin - 1 at the indicated concentrations for 2 h before LPS and nigericin treatment ( right panel ) .

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F Effect of Torin-1 treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from WT BMDMs pretreated with Torin-1 (0, 1, 10, 100, or 250 nM) for 2 h before LPS (200 ng/ml, 4 h) and nigericin (15 μM, 1 h) treatment (left panel). Western blot shows phospho-p70-S6K from BMDMs pretreated with Torin-1 at the indicated concentrations for 2 h before LPS and nigericin treatment (right panel).


A Effects of the HDAC6 inhibitor treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion and caspase - 1 processing ( p10 ) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin ( left to right : 10 and 20 mM ) or rocilinostat ( left to right : 30 and 60 mM ) .

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A Effects of the HDAC6 inhibitor treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion and caspase-1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM).


Co - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated

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Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids


Co - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Lamtor1 - Flag co - immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1 - Flag knocked - in THP - 1 cells . ( D )

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Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Lamtor1-Flag co-immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1-Flag knocked-in THP-1 cells. (D)


Endogenous Lamtor1 co - immunoprecipitated with endogenous HDAC6 from lysates of WT THP - 1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6 . Co - immunoprecipitation assay shows interactions between Lamtor1 and HDAC6 .

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Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.


G PLA to confirm endogenous binding between Lamtor1 and HDAC6 . Lamtor1 - HDAC6 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over nine images in each experiment .

G PLA to confirm endogenous binding between Lamtor1 and HDAC6. Lamtor1-HDAC6 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over nine images in each experiment.


H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells . HEK293T cells were transiently transfected with NanoLuc - fused Lamtor1 and Halo - tag - fused HDAC6 using Lipofectamine 2000 . Luminescence was measured 48 h after transfection following the addition of Nano - Glo Luciferase Assay Substrate ( Promega ) .

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H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc-fused Lamtor1 and Halo-tag-fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano-Glo Luciferase Assay Substrate (Promega).


I Immunoblot analysis of the truncated form of Flag - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - 144 ; ∆ 95 - 161 , Met1 - Val94 ) co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids .

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I Immunoblot analysis of the truncated form of Flag-tagged Lamtor1 mutant (∆145-161, Met1-144; ∆95-161, Met1-Val94) co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids.


Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP - 1 cells . ELISA assay showing IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , Lamtor1 KO cells reconstituted with full - length Lamtor1 , and Met1 - Val94 Lamtor1 cells ; all cell types were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP-1 cells. ELISA assay showing IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full-length Lamtor1, and Met1-Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


A Yeast two hybrid ( Y2H ) assays for Lamtor1 interactions with NLRP3 . Positive Y2H interaction between Lamtor1 as prey , and NLRP3 as bait . Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control .

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A Yeast two hybrid (Y2H) assays for Lamtor1 interactions with NLRP3. Positive Y2H interaction between Lamtor1 as prey, and NLRP3 as bait. Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control.


B PLA to confirm endogenous binding between Lamtor1 and NLRP3 . Lamtor1 - NLRP3 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over 10 images in each experiment .

B PLA to confirm endogenous binding between Lamtor1 and NLRP3. Lamtor1-NLRP3 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over 10 images in each experiment.


C , D Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids .

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C, D Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.


Immunoblot analysis of the truncated form of V5 - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - Ser144 ) co - immunoprecipitated with NLRP3 - flag from lysates of HEK293T cells transfected with the indicated plasmids .

Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.


G Effect of the HDAC6 knockdown on NLRP3 - Lamtor1 interactions . Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids .

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G Effect of the HDAC6 knockdown on NLRP3-Lamtor1 interactions. Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.


A , B Representative images of Lamtor1 and ASC . PMA - treated THP - 1 cells stably expressing GFP - tagged ASC and td - Tomato - tagged Lamtor1 were primed with LPS ( 200 ng / mL , 2 h ) and then stimulated with nigericin for the indicated duration . Time shows after nigericin stimulation ( min : s ) .

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A, B Representative images of Lamtor1 and ASC. PMA-treated THP-1 cells stably expressing GFP-tagged ASC and td-Tomato-tagged Lamtor1 were primed with LPS (200 ng/mL, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s).


C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation . Representative images of PMA - primed THP - 1 macrophages stably expressing GFP - tagged ASC treated with or without LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) . Scale bars = 10 μm .

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C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA-primed THP-1 macrophages stably expressing GFP-tagged ASC treated with or without LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm.


Effects of DL - all - rac - α - tocopherol on the interactions between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL - all - rac - α - tocopherol ( B )

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

Effects of DL-all-rac-α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL-all-rac-α-tocopherol (B)


IL - 1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL - all - rac - α - tocopherol ( C ) at the indicated concentrations . ELISA assay showing IL - 1β secretion in supernatants from WT THP - 1 cells treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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

IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL-all-rac-α-tocopherol (C) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


D ) IL - 6 secretion after overnight pretreatment with DL - all - rac - α - tocopherol . ELISA assay showing IL - 6 secretion in supernatants from WT BMDMs treated with LPS ( 200 ng / mL , overnight ) and DL - all - rac - α - tocopherol at the indicated concentrations ( overnight ) .

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

D) IL-6 secretion after overnight pretreatment with DL-all-rac-α-tocopherol. ELISA assay showing IL-6 secretion in supernatants from WT BMDMs treated with LPS (200 ng/mL, overnight) and DL-all-rac-α-tocopherol at the indicated concentrations (overnight).


Effects of α - tocopherol on the interactions between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D - α - tocopherol ( E )

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

Effects of α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D -α-tocopherol (E)


IL - 1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D - α - tocopherol ( F ) at the indicated concentrations . ELISA assay showing IL - 1β secretion in supernatants from WT THP - 1 cells treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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

IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D -α-tocopherol (F) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


G Effects of DL - all - rac - α - tocopherol in an acute gouty arthritis model . MSU crystals ( 0 . 5 mg ) suspended in 20 μL endotoxin - free PBS or PBS control were injected intra - articularly into the tibia - tarsal joint ( ankle ) of C57BL / 6 WT mice with or without DL - all - rac - α - tocopherol . Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin - stained sections of ankle joints .

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

G Effects of DL-all-rac-α-tocopherol in an acute gouty arthritis model. MSU crystals (0.5 mg) suspended in 20 μL endotoxin-free PBS or PBS control were injected intra-articularly into the tibia-tarsal joint (ankle) of C57BL/6 WT mice with or without DL-all-rac-α-tocopherol. Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin-stained sections of ankle joints.


H Effects of DL - all - rac - α - tocopherol in an MSU - induced peritonitis model . Twelve hours after DL - all - rac - α - tocopherol pretreatment , 100 μL MSU solution ( 10 mg / mL ) was intraperitoneally injected for 4 h . Peritoneal IL - 1β production was then measured by ELISA , and CD11b + Ly6G + neutrophil recruitment was measured by FACS .

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

H Effects of DL-all-rac-α-tocopherol in an MSU-induced peritonitis model. Twelve hours after DL-all-rac-α-tocopherol pretreatment, 100 μL MSU solution (10 mg/mL) was intraperitoneally injected for 4 h. Peritoneal IL-1β production was then measured by ELISA, and CD11b+ Ly6G+ neutrophil recruitment was measured by FACS.


( b ) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy . HeLa cells transfected with HA - Parkin were treated with CCCP for the indicated times . Mitochondria were stained by anti - TOM20 ( pseudo coloured ; blue ) and a ΔΨm dependent MitoTracker ( red ) . Parkin was stained with anti - HA ( green ) . Without treatment , mitochondria are intact and stained by both mitochondrial markers , whereas Parkin is equally distributed in the cytoplasm . After 2 h of CCCP treatment , mitochondria are depolarized as shown by the loss of MitoTracker staining . Parkin completely translocates to mitochondria clustering at perinuclear regions . After 24 h of CCCP treatment , massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker . Only Parkin - positive cells show mitochondrial clustering and clearance , in contrast to adjacent untransfected cells . Scale bars , 10 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

(b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA-Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a ΔΨm dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm.


( c ) Pathogenic Parkin mutations abrogate mitochondrial translocation and / or clearance . HeLa cells transfected with Flag - Parkin wild - type or pathogenic mutants were treated with CCCP and stained with anti - Flag ( green ) , anti - TOM20 ( red ) and Hoechst33342 ( blue ) . Representative merged images are shown . Scale bars , 10 μm . Immunostaining of cells transfected with non - functional cysteine mutants and functional Parkin variants is shown in Supplementary Information , Fig . S1a and S1b , respectively .

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

(c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag-Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively.


( d , e ) Based on the mitochondrial effects , Parkin mutants can be classified into functional and non - functional classes . ( d ) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment , as defined by complete colocalization of Parkin with the mitochondrial marker ( n = 3 ) . ( e ) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment , as shown by complete loss of mitochondrial staining ( n = 3 ) . Data represent the mean ± s . d . , * P ≤ 0 . 05 , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with wild - type . 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 O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type.


Stable Parkin wild - type or pathogenic mutants expressing SH - SY5Y cell lines were induced with doxycycline ( DOX ) for 72 h and treated with CCCP for the indicated times . Cells were stained with anti - Parkin ( green ) , anti - TOM20 ( red ) and Hoechst33342 ( blue ) . ( a - b ) CCCP treatment of stable SH - SY5Y cells for 0 h . Unstimulated control cells ( - DOX ) show relatively low levels of endogenous Parkin , compared to cells with induced expression of Parkin wild - type or K161N , R275W and G430D mutants ( + DOX ) . Immunostaining ( a ) and western blot analysis ( b ) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones . β - Actin served as a loading control .

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

Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control.


( c , d ) After 2 h of CCCP treatment , massive colocalization of wild - type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls . Similarly , Parkin K161N and R275W mutants translocate to mitochondria , whereas G430D does not .

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

(c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not.


( e , f ) After 24 h of CCCP treatment , induced wild - type Parkin - expressing cells show full clearance of damaged mitochondria . In contrast , the pathogenic mutants analysed fail to execute mitophagy and , therefore , damaged mitochondria remain uncleared . Representative images of three independent experiments are shown . Scale bars , 10 μm ( a , c and e ) . ( d , f ) Quantifications of the observed mitochondrial effects in Parkin - expressing SH - SY5Y cells ( n = 3 ) . Data represent the mean ± s . d . , * * * P ≤ 0 . 0005 compared with induced wild - type Parkin . WT , wild - type .

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

(e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.


( b ) Co - immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization . Endogenous Parkin and PINK1 were co - immunoprecipitated from neuronal SH - SY5Y cells after CCCP treatment with specific antibodies .

O O O O O 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

(b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies.


( c ) Western blot of PINK1 siRNA - transfected HeLa cells used in d - h demonstrates the knockdown efficiency and specificity of PINK1 silencing .

O O O 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) Western blot of PINK1 siRNA-transfected HeLa cells used in d-h demonstrates the knockdown efficiency and specificity of PINK1 silencing.


d - h ) PINK1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , and mitochondrial clearance after 24 h , of CCCP treatment . This can be rescued by re - transfection with wild - type PINK1 , but not with a kinase - dead mutant or a PINK1 variant lacking the MTS . Control siRNA - or PINK1 siRNA - silenced HeLa cells were transfected with EGFP - Parkin wild - type and PINK1 - V5 wild - type , 3 × KD or ΔMTS mutants . Use of appropriate empty vector controls is depicted . Cells were treated with CCCP and stained with anti - TOM20 ( pseudo coloured ; blue ) and anti - V5 ( red ) to visualize mitochondrial morphology and re - transfected PINK1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells are surrounded by a line . Representative images of four independent experiments are shown . Scale bars , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm.


( e ) Wild - type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed ( n = 4 ) . ( g ) Quantification of results from cells in e .

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

(e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (g) Quantification of results from cells in e.


( f ) Uncleared mitochondria were analysed after 24 h of CCCP treatment ( n = 4 ) . ( h ) Quantification of results from cells in f . Data represent the mean ± s . d . , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with control siRNA - transfected cells . Immunostaining of control siRNA - transfected cells at all analysed time points is shown in Supplementary Information , Fig . S2 . WT , wild - type .

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

(f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.


a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa cells were transfected with Flag - Parkin wild - type or pathogenic mutants . Cells were treated with CCCP before immunostaining of Parkin with anti - Flag ( green ) and endogenous poly - ubiquitin with a specific antibody FK1 ( red ) . ( a ) In contrast to pathogenic mutations , wild - type Parkin shows colocalization with poly - ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria . ( b ) After 24 h of CCCP treatment , K161N and R275W Parkin mutants colocalize with both poly - ubiquitin and mitochondria . In contrast , translocation - deficient Parkin mutants K211N , T240R and G430D lack the mitochondria - associated poly - ubiquitin signal . Representative images are shown . Scale bars , 10 μm . Complementary Parkin of untreated cells and quantification of colocalization of Parkin , mitochondria and poly - ubiquitin upon CCCP treatment are shown in Supplementary Information , Fig . S3a , b and c , respectively .

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

a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively.


( c , d ) Parkin - directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63 . HeLa cells were transfected with EGFP - Parkin wild - type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti - HtrA2 / Omi , as a mitochondrial marker ( pseudo coloured ; blue ) and anti - His ( red ) to detect ectopic ubiquitin . Parkin was visualized by its fusion to EGFP ( green ) . ( c ) Note that only wild - type , K27 and K63 6 × His - tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP - Parkin after 6 h of CCCP treatment . All other ubiquitin mutants interfere with Parkin translocation to mitochondria . ( d ) Only co - expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6 × His - tagged wild - type ubiquitin after 24 h of CCCP treatment . Representative images are shown . Scale bars , 10 μm . Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information , Fig . S4a and b , c , respectively . WT , wild - type ; PolyUb , poly - ubiquitin .

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

(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.


( a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa cells were transfected with Flag - Parkin wild - type or pathogenic mutants . Cells were treated with CCCP for the indicated times before immunostaining with anti - Flag ( green ) and anti - p62 ( red ) . ( a ) In contrast to the pathogenic mutants , wild - type Parkin colocalizes with p62 at the site of clustered mitochondria , after 6 h of CCCP treatment . ( b ) After 24 h of CCCP treatment , wild - type Parkin - transfected cells show complete loss of mitochondria , whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria . In contrast , translocation - compromised mutants K211N , T240R and G430D , lacking the mitochondria - associated poly - ubiquitin signal ( Fig . 4b ) , did not colocalize with p62 . Representative images of four independent experiments are shown . Data from untreated control cells and quantification of colocalization of Parkin , mitochondria and p62 upon CCCP treatment are shown in Supplementary Information , Fig . 5a and b , c , respectively .

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

(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively.