( e ) Hearts from fed and starved ROCK1 WT and KO mice were sectioned , fixed in acetone and immunofluorescence assay was performed against endogenous LC3 . Data are representative of two independent experiments from each genotype . Scale bar , 50 μm .

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

(e) Hearts from fed and starved ROCK1 WT and KO mice were sectioned, fixed in acetone and immunofluorescence assay was performed against endogenous LC3. Data are representative of two independent experiments from each genotype. Scale bar, 50 μm.


( E ) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay . Interaction between bls - CRBN and FLAG - GST - protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology .

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

(E) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology.


( F ) In vitro binding assay for thalidomide , pomalidomide and lenalidomide . Interaction between bls - CRBN and FLAG - GST - PLZF in the presence of DMSO ( 3 . 125 , 6 . 25 , 12 . 5 , 25 , 50 , 100 or 200 µM ) thalidomide , pomalidomide or lenalidomide was analysed using the AlphaScreen technology . Data information : All relative AlphaScreen ( AS ) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) .

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

(F) In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars denote ± standard deviation (n=3).


( E ) In vitro binding and ubiquitination assay of AGIA - PLZF . Empty vector , AGIA - PLZF or FLAG - CRBN expressing HEK293T cells were lysed and the lysates were mixed . The first immunoprecipitation with anti - AGIA or anti - FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide . The purified AGIA - PLZF or CRL4FLAG - CRBN complex , including AGIA - PLZF and FLAG - CRBN , was incubated with recombinant E1 , E2 and HA - ubiquitin in the presence of DMSO or 200 µM lenalidomide . The second immunoprecipitation was performed using anti - AGIA antibody . Ubiquitination of PLZF was analysed by immunoblot .

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

(E) In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide. The purified AGIA-PLZF or CRL4FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2 and HA-ubiquitin in the presence of DMSO or 200 µM lenalidomide. The second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.


( B ) In vitro binding assay using truncated PLZF . Thalidomide - dependent interaction between bls - CRBN and FLAG - GST - PLZF - full length ( FL ) or truncated FLAG - GST - PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology . Data information : All relative AlphaScreen ( AS ) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * * * * P < 0 .

O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 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) In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001


( D ) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . ( E ) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP - 1 cells treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . Data information : Data in bar graphs in ( D ) and ( E ) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100 . Error bars donated ± standard deviation ( n = 3 ) and P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ) .

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

(D) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. (E) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. Data information: Data in bar graphs in (D) and (E) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100. Error bars donated ± standard deviation (n = 3) and P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01 and ***P < 0.001).


( A , B ) Immunoblot analysis of AGIA - MmPLZF ( A ) or - MmSALL4 ( B ) in FLAG - MmCRBN - WT , - MmCRBN - I391V or - MmCRBN - V380E / I391V expressing CRBN - / - HEK293T cells treated with DMSO or thalidomide ( Tha ) for 16 h . Data information : Data in bar graphs were calculated as relative Mm ) PLZF or SALL4 band intensity with Mm ) PLZF or SALL4 , respectively . The band intensity of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * 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 B-CONTROLLED_VAR 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) Immunoblot analysis of AGIA-MmPLZF (A) or -MmSALL4 (B) in FLAG-MmCRBN-WT, -MmCRBN-I391V or -MmCRBN-V380E/I391V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. Data information: Data in bar graphs were calculated as relative Mm) PLZF or SALL4 band intensity with Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


( C , D ) Immunoblot analysis of AGIA - GgPLZF ( C ) or - GgSALL4 ( D ) in FLAG - GgCRBN - WT or - GgCRBN - I390V expressing CRBN - / - HEK293T cells treated with DMSO or thalidomide ( Tha ) for 16 h . ( E , F ) Immunoblot analysis of AGIA - Mm PLZF ( E ) or - MmSALL4 ( F ) in FLAG - MmCRBN - WT expressing CRBN - / - HEK293T cells treated with indicated concentration of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . ( G , H ) Immunoblot analysis of AGIA - GgPLZF ( G ) or - GgSALL4 ( H ) in FLAG - GgCRBN - WT expressing CRBN - / - HEK293T cells treated with indicated concentration of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . Data information : Data in bar graphs were calculated as relative ( Gg or Mm ) PLZF or SALL4 band intensity with ( Gg or Mm ) PLZF or SALL4 , respectively . The band intensity of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of AGIA-GgPLZF (C) or -GgSALL4 (D) in FLAG-GgCRBN-WT or -GgCRBN-I390V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. (E, F) Immunoblot analysis of AGIA-Mm PLZF (E) or -MmSALL4 (F) in FLAG-MmCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. (G, H) Immunoblot analysis of AGIA-GgPLZF (G) or -GgSALL4 (H) in FLAG-GgCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. Data information: Data in bar graphs were calculated as relative (Gg or Mm) PLZF or SALL4 band intensity with (Gg or Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


( E ) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl ( n = 10 ) or shPLZF ( n = 9 ) were analysed by Victoria blue staining . h ; humerus , r ; radius , u ; ulna , fe ; femur , fi ; fibula , t ; tibia . Data in bar graphs were calculated as relative bone length with bone length of shControl as 100 . Error bars denote ± standard deviation ( forelimb and hindlimb shControl ( n = 3 ) , forelimb shPLZF ( n = 5 ) or hindlimb shPLZF ( n = 6 ) ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * * P < 0 . 01 and * * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl (n = 3), forelimb shPLZF (n = 5) or hindlimb shPLZF (n = 6)). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01 and ****P < 0.0001).


( F ) Photographs show E4 chicken embryos treated with HBC ( control , n = 10 , C1 , C3 , C5 and C6 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 , T1 , T2 , T6 and T9 ) or 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 , H1 , H2 , H8 and H9 ) corresponding to immunoblot analysis in Fig 7G . Red arrows show treated regions . Data information Data in bar graphs in ( F ) were calculated as relative limb bud length with left limb bud length as one . Error bars denote ± standard deviation ( F ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * * P < 0 . 01 , and * * * P < 0 . 001 ) .

O O O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Photographs show E4 chicken embryos treated with HBC (control, n = 10, C1, C3, C5 and C6), 1 µg/µl (3.9 mM) thalidomide (n = 11, T1, T2, T6 and T9) or 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n = 10, H1, H2, H8 and H9) corresponding to immunoblot analysis in Fig 7G. Red arrows show treated regions. Data information Data in bar graphs in (F) were calculated as relative limb bud length with left limb bud length as one. Error bars denote ± standard deviation (F), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, **P < 0.01, and ***P < 0.001).


( G ) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud . Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC ( n = 10 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 ) and 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 ) . Data information : Data in bar graphs in ( G ) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4 , respectively . The band intensity of HBC was 100 . Error bars denote ± standard deviation ( ( E ) , n = 3 or ( G ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , and * * P < 0 . 01 ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud. Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC (n =10), 1 µg/µl (3.9 mM) thalidomide (n =11) and 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n =10). Data information: Data in bar graphs in (G) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4, respectively. The band intensity of HBC was 100. Error bars denote ± standard deviation ((E), n = 3 or (G), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, and **P < 0.01).


E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS - GgPLZF was treated with HBC or 1 µg / µl ( 3 . 9 mM ) thalidomide , and the chicken forelimb bud phenotypes were observed in bright field ( BF ) or fluorescence of enhanced GFP ( EGFP ) . Fgf10 / Fgf8 expression was analysed by section by in situ hybridisation . ( Q ) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in ( Q ) were calculated as relative limb bud length with limb bud length of control embryo as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * * P < 0 . 01 ) .

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

E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS-GgPLZF was treated with HBC or 1 µg/µl (3.9 mM) thalidomide, and the chicken forelimb bud phenotypes were observed in bright field (BF) or fluorescence of enhanced GFP (EGFP). Fgf10/Fgf8 expression was analysed by section by in situ hybridisation. (Q) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in (Q) were calculated as relative limb bud length with limb bud length of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01).


( R ) Quantitative analysis of Fgf10 / Fgf8 expression Data in bar graphs in ( R ) were calculated as relative Fgf10 / Fgf8 mRNA expressions with Fgf10 / Fgf8 mRNA expressions of control embryo as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * P < 0 . 05 and * * P < 0 . 01 ) .

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

(R) Quantitative analysis of Fgf10/Fgf8 expression Data in bar graphs in (R) were calculated as relative Fgf10/Fgf8 mRNA expressions with Fgf10/Fgf8 mRNA expressions of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (*P < 0.05 and **P < 0.01).


Decreased Drp1S616 phosphorylation in PINK1 - null HEK293 cells Lysates of PINK1 - null ( PINK1KO ) and parkin - null ( parkinKO ) HEK293 cells ( a ) and their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Two independent PINK1KO and parkinKO HEK293 cell lines ( 1 , 2 ) were shown . Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

Decreased Drp1S616 phosphorylation in PINK1-null HEK293 cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) HEK293 cells (a) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO HEK293 cell lines (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Decreased Drp1S616 phosphorylation in PINK1 - null MEF cells Lysates of PINK1 - null ( PINK1KO ) and parkin - null ( parkinKO ) MEF cells ( c ) and their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

Decreased Drp1S616 phosphorylation in PINK1-null MEF cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) MEF cells (c) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Decreased Drp1S616 phosphorylation in PINK1 - null mouse substantial nigra tissues . Lysates of PINK1 - null ( PINK1KO ) and parkin - null ( parkinKO ) mouse substantial nigra tissues ( e ) , and their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Two independent PINK1KO and parkinKO substantial nigra from two separate PINK1KO and parkinKO mice ( 1 , 2 ) were shown . Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

Decreased Drp1S616 phosphorylation in PINK1-null mouse substantial nigra tissues. Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) mouse substantial nigra tissues (e), and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO substantial nigra from two separate PINK1KO and parkinKO mice (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( g , h ) . Overexpression of PINK1 , not PINK1 kinase mutants , reverses Drp1S616 phosphorylation in PINK1KO HEK293 cells . Lysates of PINK1KO and PINK1WT control ( WT ) HEK293 cells expressing PINK1 variants were immuno - detected with an anti - phospho ( Ser616 ) - Drp1 antibody ( pS616 ) , an anti - Drp1 antibody ( Drp1 ) and an anti - PINK1 antibody ( PINK1 ) . Cells with mock transfection ( Blank ) and transfected with an empty plasmid ( 3 . 1 ) were included as controls ( g ) . Quantitation of pS616 / Drp1 is shown ( h ) . PINK1 : wildtype PINK1 ; D384N : PINK1 kinase - dead mutant ; G309D , pathogenic PINK1 mutant ; ∆ 110 : PINK1 mito - target deficient mutant . Student ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(g, h). Overexpression of PINK1, not PINK1 kinase mutants, reverses Drp1S616 phosphorylation in PINK1KO HEK293 cells. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells expressing PINK1 variants were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1) and an anti-PINK1 antibody (PINK1). Cells with mock transfection (Blank) and transfected with an empty plasmid (3.1) were included as controls (g). Quantitation of pS616/Drp1 is shown (h). PINK1: wildtype PINK1; D384N: PINK1 kinase-dead mutant; G309D, pathogenic PINK1 mutant; ∆110: PINK1 mito-target deficient mutant. Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( i , j ) . PINK1 deficiency does not affect expression and kinase activity of CDK1 and ERK1 / 2 . Lysates of PINK1KO and PINK1WT control ( WT ) HEK293 cells were immuno - detected with an anti - phospho ( Ser616 ) - Drp1 antibody ( pS616 ) , an anti - Drp1 antibody ( Drp1 ) , an anti - PINK1 antibody ( PINK1 ) , an anti - CDK1 antibody ( CDK1 ) , an anti - p44 / p42 MAPK antibody ( ERK1 / 2 ) , an anti - phospho ( Thr161 ) - CDK1 antibody ( pThr161 ) , and an anti - phospho ( Thr202 / 204 ) - p44 / p42 MAPK antibody ( pThr202 / 204 ) . β - actin was detected as a loading control ( i ) . Quantitation of CDK1Thr161 phosphorylation ( pThr161 ) and ERK1 / 2Thr202 / 204 phosphorylation ( pThr202 / 204 ) was shown ( j ) . Student ' s test . ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(i, j). PINK1 deficiency does not affect expression and kinase activity of CDK1 and ERK1/2. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1), an anti-PINK1 antibody (PINK1), an anti-CDK1 antibody(CDK1), an anti-p44/p42 MAPK antibody (ERK1/2), an anti-phospho(Thr161)-CDK1 antibody (pThr161), and an anti-phospho(Thr202/204)-p44/p42 MAPK antibody (pThr202/204). β-actin was detected as a loading control (i). Quantitation of CDK1Thr161 phosphorylation (pThr161) and ERK1/2Thr202/204 phosphorylation (pThr202/204) was shown (j). Student's test. ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( l , m ) . PINK1 phosphorylates Drp1S616 in vitro . Recombinant TcPINK1 was incubated with either GST - Drp1518 - 736 ( WT ) or GST - Drp1518 - 736 , S616A ( S616A ) in the presence ( + ) / absence ( - ) of ATP . After heat inactivation , reactions were treated with ( + ) / without ( - ) shrimp alkaline phosphatase ( SAP ) . Phosphorylation of Drp1S616 ( pS616 ) was immuno - detected ( l , upper panel ) . Total Drp1 ( l , middle panel ) and protein loading stained with Coomassie blue staining ( l , lower panel ) were shown . Quantitation of pS616 / Drp1 ratio was shown ( m ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments .

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

(l, m). PINK1 phosphorylates Drp1S616 in vitro. Recombinant TcPINK1 was incubated with either GST-Drp1518-736 (WT) or GST-Drp1518-736, S616A (S616A) in the presence (+)/absence (-) of ATP. After heat inactivation, reactions were treated with (+)/without (-) shrimp alkaline phosphatase (SAP). Phosphorylation of Drp1S616 (pS616) was immuno-detected (l, upper panel). Total Drp1 (l, middle panel) and protein loading stained with Coomassie blue staining (l, lower panel) were shown. Quantitation of pS616/Drp1 ratio was shown (m). One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( n , o ) . Label - free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation . Representative spectra for the Drp1 phospho - peptide ( S616 ) SKPIPMPA ( p ) SPQK and non - phospho - peptide SKPIPMPA ( p ) SPQK . Spectra obtained for peptides from PINK1WT HEK293 cells are shown ( n ) . The relative ratios of the phospho - peptides containing phosphor - Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO HEK293 samples were calculated and plotted ( 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 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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

(n, o). Label-free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation. Representative spectra for the Drp1 phospho-peptide (S616) SKPIPMPA(p)SPQK and non-phospho-peptide SKPIPMPA(p)SPQK. Spectra obtained for peptides from PINK1WT HEK293 cells are shown (n). The relative ratios of the phospho-peptides containing phosphor-Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO HEK293 samples were calculated and plotted (o).


( p , q ) . Km and Kcat for PINK1 - mediated phosphorylation of ubiquitin and Drp1 . In vitro kinase assays were performed with different concentrations of ubiquitin ( p ) or Drp1 ( q ) with TcPINK1 at fixed ATP concentration ( 500 μM ) . Results were analyzed on phos ‐ tag gels and modeled to the Michaelis - Menten equation . The given graphs represent global fits to data collected from 3 sets of reactions for both Ub and Drp1 performed independently . Km : michaelis constant . Kcat : catalytic constant . Bars represent the mean ± SEM .

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

(p, q). Km and Kcat for PINK1-mediated phosphorylation of ubiquitin and Drp1. In vitro kinase assays were performed with different concentrations of ubiquitin (p) or Drp1 (q) with TcPINK1 at fixed ATP concentration (500 μM). Results were analyzed on phos‐tag gels and modeled to the Michaelis-Menten equation. The given graphs represent global fits to data collected from 3 sets of reactions for both Ub and Drp1 performed independently. Km: michaelis constant. Kcat: catalytic constant. Bars represent the mean ± SEM.


( a , b ) . Decreased Drp1S616 phosphorylation in PINK1 - null primary neurons . Lysates of DIV6 neurons generated from PINK1KO and PINK1WT control mice were immune - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) or Drp1 . Tuj1 was detected as a loading control ( a ) . Quantitation of pS616 / Drp1 for each experiment is shown ( b ) . Student ' s test . * * p < 0 . 01 . Data was presented as mean ± SEM of three independent experiments .

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

(a, b). Decreased Drp1S616 phosphorylation in PINK1-null primary neurons. Lysates of DIV6 neurons generated from PINK1KO and PINK1WT control mice were immune-detected with antibodies against either phospho (Ser616)-Drp1 (pS616) or Drp1. Tuj1 was detected as a loading control (a). Quantitation of pS616/Drp1 for each experiment is shown (b). Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.


( c , d ) . TEM analysis of mitochondria in mouse substantial nigra . Sections of substantial nigra from 18 - month - old PINK1KO and PINK1WT control mice were analyzed under TEM ( c , left panels , scale bar = 2 µm ) . Amplified images of mitochondria from dot line frames gated region are shown ( c , right panels , scale bar = 1 µm ) . Asterisks indicate abnormal elongated mitochondria . A quantitative analysis of mitochondria with perimeter > 2µm is shown ( d ) . Over 300 identifiable mitochondria ( cristae and / or double membrane ) per mice were randomly and blindly selected for perimeter measurement . Student ' s test . * p < 0 . 05 . Data was presented as mean ± SEM of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). TEM analysis of mitochondria in mouse substantial nigra. Sections of substantial nigra from 18-month-old PINK1KO and PINK1WT control mice were analyzed under TEM (c, left panels, scale bar=2 µm). Amplified images of mitochondria from dot line frames gated region are shown (c, right panels, scale bar=1 µm). Asterisks indicate abnormal elongated mitochondria. A quantitative analysis of mitochondria with perimeter >2µm is shown (d). Over 300 identifiable mitochondria (cristae and/or double membrane) per mice were randomly and blindly selected for perimeter measurement. Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.


( e , f ) . Overexpression of Drp1WT , but not phosphorylation mutant Drp1S616A , suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons . ( e ) . DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co - transfected mito - GFP with either pcDNA3 . 1 ( 3 . 1 ) or Drp1WT or Drp1S616A . Neurons were immuno - detected with an antibody against myc - tag ( Myc ) ( red , to detect exogenous Drp1 ) . Mito - GFP : green , to detect mitochondria . Representative images were shown , scale bar = 25 μm . Magnified dendritic shafts gated by a dot line frame are shown ( Zoom , right panels , scale bar = 5 μm ) . ( f ) . Quantification of Mito - index in dendrites of neurons from experiments of ( e ) . 10 neurons were randomly selected and analyzed in each experiment . One - way ANOVA followed with Tukey ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

O O O O O O O O B-CONTROLLED_VAR 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 O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Overexpression of Drp1WT, but not phosphorylation mutant Drp1S616A, suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons. (e). DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co-transfected mito-GFP with either pcDNA3.1 (3.1) or Drp1WT or Drp1S616A. Neurons were immuno-detected with an antibody against myc-tag (Myc) (red, to detect exogenous Drp1). Mito-GFP: green, to detect mitochondria. Representative images were shown, scale bar=25 μm. Magnified dendritic shafts gated by a dot line frame are shown (Zoom, right panels, scale bar=5 μm).(f). Quantification of Mito-index in dendrites of neurons from experiments of (e). 10 neurons were randomly selected and analyzed in each experiment. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - c ) . Drp1 is required for mitochondrial fission induced by PINK1 . Drp1 - null HeLa cells ( Drp1KO ) and their wildtype controls ( Drp1WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP with FRB - MTS . Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) and Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( Δ110 - PINK1 , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown ( a , upper panel , scale bar = 25 μm ) . Higher magnification images are also included ( a , lower panel , scale bar = 10 μm ) . Mitochondrial morphology in different transfections was quantified . ( b , c ) . Student ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(a-c). Drp1 is required for mitochondrial fission induced by PINK1. Drp1-null HeLa cells (Drp1KO) and their wildtype controls (Drp1WT) were co-transfected Δ110-PINK1-GFP-FKBP with FRB-MTS. Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (Δ110-PINK1, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown (a, upper panel, scale bar=25 μm). Higher magnification images are also included (a, lower panel, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified. (b, c). Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( d - f ) . Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1 . Drp1KO HeLa cells were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A . Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , blue ) , exogenous Drp1 ( myc , red ) , Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm ( d ) . Higher magnification images are also included ( d , Zoom , scale bar = 5 μm ) . Mitochondrial morphology in different transfections was quantified ( e , f ) . Student ' s test . * * p < 0 . 01 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f). Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1. Drp1KO HeLa cells were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A. Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, blue), exogenous Drp1 (myc, red), Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm (d). Higher magnification images are also included (d, Zoom, scale bar=5 μm). Mitochondrial morphology in different transfections was quantified (e, f). Student's test. **p<0.01, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( g , h ) . Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616 . HEK293 cells co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP were treated with 250 nM rapalog ( Rapalog ) . After 2hours treatment , cytosol protion ( cyto ) and mitochondria ( Mito ) were fractionated and immunobloted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , Drp1 ( Drp1 ) , or GFP ( PINK1 ) . Tom20 and β - actin were detected as mitochondrial loading control and cytosol loading control , respectively ( g ) . Quantitation of pS616 / Drp1 in various conditions was shown ( h ) . Student ' s test . * * p < 0 . 01 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(g, h). Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616. HEK293 cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP were treated with 250 nM rapalog (Rapalog). After 2hours treatment, cytosol protion (cyto) and mitochondria (Mito) were fractionated and immunobloted with antibodies against either phospho (Ser616)-Drp1 (pS616), Drp1 (Drp1), or GFP (PINK1). Tom20 and β-actin were detected as mitochondrial loading control and cytosol loading control, respectively (g). Quantitation of pS616/Drp1 in various conditions was shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( i , j ) . Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1 . HeLa cells co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP ( FKBP - PINK1 ) were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . Cells treated with solvent ( DMSO ) were included as a control . Cells were immunostained with antibodies against either Tom20 , Drp1 and GFP ( PINK1 - FKBP ) . Representative images were shown , scale bar = 2 μm ( i ) . Higher magnification images from gated box are shown ( bottom panels ) . Arrows indicate Drp1 puncta . Mitochondria - associated ( yellow ) and non - associated Drp1 ( Red ) are shown , scale bar = 1 μm . Mitochondria - associated Drp1 puncta were analyzed by using an ImageJ plugin - Coloc 2 and expressed as the Manders ' Colocalization Coefficients ( MCC ) ( j ) . Three different regions from each cell and > 15 cells for each experimental group were analyzed . Student ' s test . * * p < 0 . 01 . Data was presented as mean ± SEM of three independent experiments .

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

(i, j). Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1. HeLa cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP (FKBP-PINK1) were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cells were immunostained with antibodies against either Tom20, Drp1 and GFP (PINK1-FKBP). Representative images were shown, scale bar=2 μm (i). Higher magnification images from gated box are shown (bottom panels). Arrows indicate Drp1 puncta. Mitochondria-associated (yellow) and non-associated Drp1(Red) are shown, scale bar=1 μm. Mitochondria-associated Drp1 puncta were analyzed by using an ImageJ plugin-Coloc 2 and expressed as the Manders' Colocalization Coefficients (MCC) (j). Three different regions from each cell and >15 cells for each experimental group were analyzed. Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.


( k , l ) . PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms . DRP1KO HEK293 cells co - transfected FRB - MTS / Δ110 - PINK1 - GFP - FKBP ( PINK1WT ) with either myc - Drp1WT or myc - Drp1S616A were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . Cells treated with solvent ( DMSO ) were included as a control . Cell lysates were immunoblotting with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho - ( Ser637 ) - Drp1 ( pS637 ) , Drp1 and GFP ( to detect PINK1 - FKBP ) ( k ) . β - actin was detected as a loading control . Quantitation of pS637 / Drp1 in various conditions was shown ( l ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(k, l). PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms. DRP1KO HEK293 cells co-transfected FRB-MTS/Δ110-PINK1-GFP-FKBP (PINK1WT) with either myc-Drp1WT or myc-Drp1S616A were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cell lysates were immunoblotting with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho-(Ser637)-Drp1 (pS637), Drp1 and GFP (to detect PINK1-FKBP) (k). β-actin was detected as a loading control. Quantitation of pS637/Drp1 in various conditions was shown (l). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - e ) . ATG5 is dispensable for PINK1 - mediated mitochondrial fission . MEF cells derived from ATG5 - null ( ATG5KO ) and their wildtype control mice ( ATG5WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either PINK1WT or a kinase - dead mutant PINK1D384N . Cells were induced with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) and Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images are shown , scale bar = 25 μm ( a ) . Higher magnification images are also included ( panels beneath the large cell images , scale bar = 10 μm ) . Mitochondrial morphology in different transfections was quantified ( b - e ) . Student ' s test . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-e). ATG5 is dispensable for PINK1-mediated mitochondrial fission. MEF cells derived from ATG5-null (ATG5KO) and their wildtype control mice (ATG5WT) were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either PINK1WT or a kinase-dead mutant PINK1D384N. Cells were induced with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images are shown, scale bar=25 μm (a). Higher magnification images are also included (panels beneath the large cell images, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified (b-e). Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT and Drp1KO HeLa cells were transfected with GFP - parkin . Cells were treated with 20 μM CCCP for 2 hours ( f ) followed by immunodetecting mitochondria ( TOM20 , red ) and parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm . Cell with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 20 μM CCCP for 2 hours (f) followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT and Drp1KO HeLa cells were transfected with GFP - parkin . Cells were treated with 150 μM actinonin for 6 hours ( h ) , followed by immunodetecting mitochondria ( TOM20 , red ) and parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . Cells treated with solvent ( DMSO ) were used as a treatment control . Representative images were shown , scale bar = 25 μm . Cell with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments . For each condition , > 100 cells were analyzed .

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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 150 μM actinonin for 6 hours (h), followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( a , b ) . Drp1WT and Drp1S616D , but not Drp1S616A , rescue PINK1 - null induced crushed thorax in Drosophila . Representative thorax images of PINK1WT and PINK1 - deficiency ( PINK1KO ) Drosophila expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Images were taken either under light microscopy ( a , upper panels , yellow arrowhead indicates the collapsed throax ) or SEM ( b , lower panels ) . Quantitation of crushed thorax is shown ( b ) . > 200 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments . For each condition , > 200 flies were quantified .

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

(a, b). Drp1WT and Drp1S616D, but not Drp1S616A, rescue PINK1-null induced crushed thorax in Drosophila. Representative thorax images of PINK1WT and PINK1-deficiency (PINK1KO) Drosophila expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Images were taken either under light microscopy (a, upper panels, yellow arrowhead indicates the collapsed throax) or SEM (b, lower panels). Quantitation of crushed thorax is shown (b). > 200 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments. For each condition, >200 flies were quantified.


( c ) Drp1WT and Drp1S616D , but not Drp1S616A , restore abnormal ATP production in PINK1 - null flies . ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-MEASURED_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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

(c) Drp1WT and Drp1S616D, but not Drp1S616A, restore abnormal ATP production in PINK1-null flies. ATP production was measured using muscle lysates generated from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Drp1WT and Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative images of Mito - GFP ( green ) labeled mitochondria ( d ) from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 10 μm ( d ) Average mitochondrial size was quantified , > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s test . * p < 0 . 05 , * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments ( e ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of Mito-GFP (green) labeled mitochondria (d) from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar=10 μm (d) Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001. Data was presented as mean ± SEM of three independent experiments (e).


Drp1WT and Drp1S616D , but not Drp1S616A , rescue mitochondrial structures in PINK1KO flies . Representative images of TEM analysis ( f ) of IFMs from PINK1WT flies expressing mhc - gal4 ( Mhc / + ) ( as a control ) and PINK1KO flies expressing either mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1wt ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Scale bar = 1 μm ( f ) . Average mitochondrial size was quantified , > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Normal and abnormal mitochondrial number per mm2 were quantified . > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent the mean ± SEM of three independent experiments ( g ) .

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

Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of TEM analysis (f) of IFMs from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar= 1 μm (f). Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Normal and abnormal mitochondrial number per mm2 were quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (g).


( h , i ) . Drp1WT and Drp1S616D , but not Drp1S616A , suppress cell death in PINK1KO flies . Representative TUNEL staining ( Red ) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc - gal4 ( Mhc / + ) , Mhc - gal4 driven human Drp1wt ( Mhc > hDrp1WT ) or Mhc - gal4 driven human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven human Drp1S616D ( Mhc > hDrp1S616D ) are shown . Nuclei were counter - stained with DAPI ( blue ) , scale bar = 10 μm . Apoptotic cells ( % ) in different genotype flies were quantified ( i ) . > 5 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments .

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

(h, i). Drp1WT and Drp1S616D, but not Drp1S616A, suppress cell death in PINK1KO flies. Representative TUNEL staining (Red) images of IFM sections from PINK1WT and PINK1KO flies expressing either Mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1WT) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Nuclei were counter-stained with DAPI (blue), scale bar=10 μm. Apoptotic cells (%) in different genotype flies were quantified (i). >5 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat.One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( j ) . Expression and phosphorylation of hDrp1 in Drosophila . Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc - gal4 driven hDrp1WT ( WT ) and hDrp1S616A ( S616A ) were immunoblotted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) ( to detect phosphorylated hDrp1S616 ) , Drp1 ( to detect both endogenous and exogenous Drp1 ) , myc - tag ( to detect exogenous Drp1 ) . α - tubulin was detected as a loading control . Flies expressing mhc - gal4 ( - ) were included as an expression control .

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

(j). Expression and phosphorylation of hDrp1 in Drosophila. Fly muscle lysates generated from PINK1WT or PINK1KO flies expressing mhc-gal4 driven hDrp1WT (WT) and hDrp1S616A (S616A) were immunoblotted with antibodies against either phospho(Ser616)-Drp1(pS616) (to detect phosphorylated hDrp1S616), Drp1 (to detect both endogenous and exogenous Drp1), myc-tag (to detect exogenous Drp1). α-tubulin was detected as a loading control. Flies expressing mhc-gal4 (-) were included as an expression control.


( a , b ) . Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7 - deficient Drosophila . Representative SEM images of thorax from PINK1WT , ( upper panels ) and PINK1KO ( lower panels ) flies combined with dATP7 deficiency ( dATG7KO ) or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( a ) . Quantitation of crushed thorax in flies with indicated genotypes is presented ( b ) . > 100 flies for each experiment are analyzed . Scale bar = 200µm . One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 . Data was presented as mean ± SEM of three independent experiments .

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

(a, b). Drp1 rescues PINK1KO induced collapsed thorax phenotype in dATG7-deficient Drosophila. Representative SEM images of thorax from PINK1WT, (upper panels) and PINK1KO (lower panels) flies combined with dATP7 deficiency (dATG7KO) or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1 (Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D (Mhc>hDrp1S616D) are shown (a). Quantitation of crushed thorax in flies with indicated genotypes is presented (b). > 100 flies for each experiment are analyzed. Scale bar=200µm. One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( c , d ) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila . Representative TEM images of indirect flight muscle from PINK1WT ( upper panels ) and PINK1KO ( lower panels ) flies combined with dATG7KO or their controls ( dATG7WT ) , followed by expressing either mhc - gal4 ( Mhc / + ) alone , or Mhc - gal4 driven expression of human Drp1 ( Mhc > hDrp1WT ) , mhc - gal4 driven expression of human Drp1S616A ( Mhc > hDrp1S616A ) or mhc - gal4 driven expression of human Drp1S616D ( Mhc > hDrp1S616D ) are shown ( c ) . Scale bar = 1 μm . Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified . > 3 flies / group and 3 - 6 pictures of different microscopic fields from each fly were analyzed per repeat . Bars represent the mean ± SEM of three independent experiments ( d ) .

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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

(c, d) Drp1 rescues PINK1KO induced mitochondrial defects in dATG7KO Drosophila. Representative TEM images of indirect flight muscle from PINK1WT (upper panels) and PINK1KO (lower panels) flies combined with dATG7KO or their controls (dATG7WT), followed by expressing either mhc-gal4 (Mhc/+) alone, or Mhc-gal4 driven expression of human Drp1(Mhc>hDrp1WT), mhc-gal4 driven expression of human Drp1S616A(Mhc>hDrp1S616A) or mhc-gal4 driven expression of human Drp1S616D(Mhc>hDrp1S616D) are shown (c). Scale bar=1 μm. Normal and abnormal mitochondrial number per mm2 from each experimental group were and quantified. >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. Bars represent the mean ± SEM of three independent experiments (d).


( e ) . Drp1 rescues PINK1KO induced ATP reduction in Drosophila . ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One - way ANOVA followed with Tukey ' s test . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

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

(e). Drp1 rescues PINK1KO induced ATP reduction in Drosophila. ATP contents of thorax muscle tissues from the indicated genotypes were measured and normalized against the protein levels One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( g , h ) . Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD patients with PINK1 mutations . Lysates of cells derived from three unrelated normal control individuals ( Control , C ) and four familial PD patients with PINK1 mutations ( PINK1mutant ) were immunoblotted with antibodies recognizing either phosphor - Drp1S616 ( pS616 ) or Drp1 protein . β - actin was detected as a loading control ( g ) . A quantitative analysis of phospho - Drp1S616 ( pS616 / Drp1 ) is shown ( h ) . Student ' s test . * * p < 0 . 01 , ns : no significance . Data was presented as mean ± SEM of three independent experiments .

O O O O O O O O B-MEASURED_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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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

(g, h). Reduction of Drp1S616 phosphorylation in dermal fibroblasts derived from PD patients with PINK1 mutations. Lysates of cells derived from three unrelated normal control individuals (Control, C) and four familial PD patients with PINK1 mutations (PINK1mutant) were immunoblotted with antibodies recognizing either phosphor-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (g). A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) is shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients . Lysates of cells derived from five unrelated normal control individuals ( Control , C ) and seven unrelated sporadic PD patients ( Sporadic PD , SP ) were immunoblotted with antibodies recognizing either phospho - Drp1S616 ( pS616 ) or Drp1 protein . β - actin was detected as a loading control ( i ) .

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

Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. Lysates of cells derived from five unrelated normal control individuals (Control, C) and seven unrelated sporadic PD patients (Sporadic PD, SP) were immunoblotted with antibodies recognizing either phospho-Drp1S616 (pS616) or Drp1 protein. β-actin was detected as a loading control (i).


Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients . A quantitative analysis of phospho - Drp1S616 ( pS616 / Drp1 ) in sporadic cases is shown ( j ) . Student ' s test . * p < 0 . 05 . Data was presented as mean ± SEM of three independent experiments .

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

Drp1S616 phosphorylation in dermal fibroblasts derived from sporadic PD patients. A quantitative analysis of phospho-Drp1S616 (pS616/Drp1) in sporadic cases is shown (j). Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.


B . Volcano plot showing the magnitude of differential gene up - regulation , as measured by the respective log 2 - fold change in expression level , at day ( positive ) or night ( negative ) versus statistical significance , as quantified by a negative binomial exact test ( red dots , dashed line p < 0 . 05 adjusted for multiple testing using Benjamin - Hochberg ) . Known circadian - regulated genes are labelled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot showing the magnitude of differential gene up-regulation, as measured by the respective log 2-fold change in expression level, at day (positive) or night (negative) versus statistical significance, as quantified by a negative binomial exact test (red dots, dashed line p<0.05 adjusted for multiple testing using Benjamin-Hochberg). Known circadian-regulated genes are labelled.


B . Violin plots depicting the distribution of the quantified average cellular expression of Prok2 , ProkR2 , Vip , Vipr2 , Avp , Avpr1a , Grp and Grpr in each sub - cluster in CT7 . 5 ( left ) and CT15 . 5 ( right ) datasets .

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

B. Violin plots depicting the distribution of the quantified average cellular expression of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr in each sub-cluster in CT7.5 (left) and CT15.5 (right) datasets.


C . The period of Cry1 - luc bioluminescence rhythms in SCN slices transfected with either pProk2 . Cre . T2A . mCherry or pProkR2 . Cre . T2A . Venus does not differ between treatments ( p = 0 . 71 , n = 10 , 9 SCN slices per group , respectively ) . D . Similarly , the amplitude of Cry1 - luc bioluminescence rhythms from Prok2 + SCN cells and ProkR2 + SCN cells , does not differ between treatments ( p = 0 . 67 ) .

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

C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively). D. Similarly, the amplitude of Cry1-luc bioluminescence rhythms from Prok2+ SCN cells and ProkR2+ SCN cells, does not differ between treatments (p=0.67).


C . The period of Cry1 - luc bioluminescence rhythms in SCN slices transfected with either pProk2 . Cre . T2A . mCherry or pProkR2 . Cre . T2A . Venus does not differ between treatments ( p = 0 . 71 , n = 10 , 9 SCN slices per group , respectively ) .

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

C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively).


H . Left : confocal images of representative SCN slices dual - transfected with pProk2 . Cre . T2A . mCherry and Syn . GCaMP6f ( top ) , or pProkR2 . Cre . T2A . Venus and Syn . jRCaMP1a ( below ) ( scale bar = 500µm ) . Right , above : representative detrended plots of signal from GCaMP6f ( green ) traces and pCry1 - DIO - Luc , emitted from Prok2 + SCN cells ( red ) . Right , below : representative detrended plots of jRCaMP1a ( purple ) and pCry1 - DIO - Luc emitted from ProkR2 + cells ( light green ) . Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN .

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

H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN.


B . Comparison of the Per2 : : Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle ( applied CT23 - 20 dark grey n = 12 SCN slices , CT - 20 - 23 light grey n = 6 SCN slices ) or Prok2RA ( applied CT23 - 20 purple n = 13 SCN slices , CT20 - 23 pink n = 11 SCN slices ) treatment ( interaction , p < 0 . 0001 ) . A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20 - 23 ( * * * * p < 0 . 0001 ) .

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

B. Comparison of the Per2::Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle (applied CT23-20 dark grey n=12 SCN slices, CT-20-23 light grey n=6 SCN slices) or Prok2RA (applied CT23-20 purple n=13 SCN slices, CT20-23 pink n=11 SCN slices) treatment (interaction, p<0.0001). A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20-23 (****p<0.0001).


H . Period lengthening occurred at the same rate when Prok2 . Cre or ProkR2 . Cre was the first AAV to be applied ( p = 0 . 1 ; Prok2 . Cre - baseline vs . 1 AAV , * p = 0 . 01 ; baseline vs . both AAVs , * p = 0 . 04 ; ProkR2 . Cre - baseline vs . 1 AAV , * p = 0 . 03 ; baseline vs . both AAVs , * * p = 0 . 01 .

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

H. Period lengthening occurred at the same rate when Prok2.Cre or ProkR2.Cre was the first AAV to be applied (p=0.1; Prok2.Cre - baseline vs. 1 AAV, *p=0.01; baseline vs. both AAVs, *p=0.04; ProkR2.Cre - baseline vs. 1 AAV, *p=0.03; baseline vs. both AAVs, **p=0.01.


A . Representative Per2 : : Luciferase traces of Cry1 , 2 - / - SCN slices at baseline ( black , arrhythmic ) , after Cry1 complementation in Prok2 + cells ( red ) and further complementation in ProkR2 + cells ( yellow , rhythmic ) ( cohort n = 8 ) . B . as in A but order of complementation reversed ( green = Cry1 complementation in ProkR2 + cells ) ( cohort n = 8 ) .

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

A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8). B. as in A but order of complementation reversed (green =Cry1 complementation in ProkR2+ cells) (cohort n=8).


A . Representative Per2 : : Luciferase traces of Cry1 , 2 - / - SCN slices at baseline ( black , arrhythmic ) , after Cry1 complementation in Prok2 + cells ( red ) and further complementation in ProkR2 + cells ( yellow , rhythmic ) ( cohort n = 8 ) .

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

A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8).


( A , Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + and Nlrp3A350V / wt CreT - mice were incubated with LPS ( 3 hr ) before culture media were analysed for secreted IL - 1β ( A ) Cytokine values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA and P - values were corrected for multiple comparisons ( Bonferroni ) . * * , p ≤ 0 . 01 .

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

(A, Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS (3 hr) before culture media were analysed for secreted IL-1β (A) Cytokine values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA and P-values were corrected for multiple comparisons (Bonferroni). **, p ≤ 0.01.


B ) . Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + and Nlrp3A350V / wt CreT - mice were incubated with LPS and cell lysates and culture media were immunoblotted for β - actin , caspase - 1 and IL - 1β ( B ) .

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

B). Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS and cell lysates and culture media were immunoblotted for β-actin, caspase-1 and IL-1β (B).


( C ) Representative pictures of Nlrp3A350V / wt MRP8 - CreTg and Nlrp3A350V / wt MRP8 - Cre - mice on days 4 and 8 after birth . Left side in each picture : Nlrp3A350V / wt MRP8 - Cre - mice ; right side in each picture : Nlrp3A350V / wt MRP8 - CreTg . Abscesses ( day 4 ) and scaling erythema ( day 8 ) are observed in Nlrp3A350V / wt MRP8 - CreTg mice .

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

(C) Representative pictures of Nlrp3A350V/wt MRP8-CreTg and Nlrp3A350V/wt MRP8-Cre- mice on days 4 and 8 after birth. Left side in each picture: Nlrp3A350V/wt MRP8-Cre- mice; right side in each picture: Nlrp3A350V/wt MRP8-CreTg. Abscesses (day 4) and scaling erythema (day 8) are observed in Nlrp3A350V/wt MRP8-CreTg mice.


( D ) Survival curves of Nlrp3A350V / wt MRP8 - CreTg ( n = 22 ) and littermate Nlrp3A350V / wt MRP8 - Cre - ( n = 21 ) mice . Statistical significance was analysed by the log - rank Mantel - Cox test . * * * * , p ≤ 0 . 0001 .

O O O O O O B-CONTROLLED_VAR 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

(D) Survival curves of Nlrp3A350V/wt MRP8-CreTg (n=22) and littermate Nlrp3A350V/wt MRP8-Cre- (n=21) mice. Statistical significance was analysed by the log-rank Mantel-Cox test. ****, p ≤0.0001.


( E ) Representative H & E - stained sections of spleen ( top , scale bar : 100 µM ) and liver ( bottom , scale bar : 200 µM ) of Nlrp3A350V / wt MRP8 - CreTg and littermate Nlrp3A350V / wt MRP8 - Cre - mice . Spleens of Nlrp3A350V / wt MRP8 - Cre - control mice ( top left ) display characteristic white pulp ( WP ) regions ( example shown surrounded by dashed line ) composed of lymphoid follicles and periarteriolar lymphoid sheaths ( PALS ) , which at this magnification are recognized by dark - purple staining , large aggregates of lymphocytes surrounding the central artery ( CA ) and their distinctive separation from the red - staining red pulp ( RP ) . WP was largely absent in spleens of Nlrp3A350V / wt MRP8 - CreTg ( top middle ) . Megakaryocytes ( MK ) are indicated for easier recognition of areas of extramedullary hematopoiesis ( EMH ) . Most of the spleen parenchyma of Nlrp3A350V / wt MRP8 - CreTg animals was replaced by EMH of mostly myeloid lineage , which can be recognized by the medium - sized cells with more cytoplasm , giving a lighter appearance ( inset ) . In both the overview picture ( top middle picture ) and inset ( top right ) of Nlrp3A350V / wt MRP8 - CreTg spleen , a small amount of EMH of the erythroid lineage is present that can be recognized by small aggregates of darker - stained , smaller cells ( asterix , * ) . Liver sections of both Nlrp3A350V / wtMRP8 - Cre - control mice and diseased Nlrp3A350V / wt MRP8 - CreTg pups displayed comparable EMH of the erythroid lineage ( bottom left picture , indicated with yellow arrows ) . Liver sections of Nlrp3A350V / wt MRP8 - CreTg pups also showed a modest increase in EMH of the myeloid lineage ( bottom middle picture and inset ( scale bar : 100 µM ) in bottom right , indicated with yellow arrowheads ) , as well as hepatocyte enlargement . ( F - I ) H & E - stained histological sections of spleen ( F , G ) or liver ( H , I ) of Nlrp3A350V / wt MRP8 - Cre - ( Cre - , n = 8 ) and Nlrp3A350V / wt MRP8 - CreTg ( CreTg , n = 6 ) mice were assigned a score of 0 - 5 for lymphoid depletion ( F ) or EMH ( G , H ) or hepatocyte enlargement ( I ) by a board - certified pathologist . Values represent mean ± SEM of n = 6 - 8 biological repeats . Statistical significance was analysed by the Mann - Whitney U - test . * * * , p ≤ 0 . 001 .

O O O O O O O O O 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 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 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 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 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-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 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 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

(E) Representative H&E-stained sections of spleen (top, scale bar: 100 µM) and liver (bottom, scale bar: 200 µM) of Nlrp3A350V/wt MRP8-CreTg and littermate Nlrp3A350V/wt MRP8-Cre- mice. Spleens of Nlrp3A350V/wt MRP8-Cre- control mice (top left) display characteristic white pulp (WP) regions (example shown surrounded by dashed line) composed of lymphoid follicles and periarteriolar lymphoid sheaths (PALS), which at this magnification are recognized by dark-purple staining, large aggregates of lymphocytes surrounding the central artery (CA) and their distinctive separation from the red-staining red pulp (RP). WP was largely absent in spleens of Nlrp3A350V/wt MRP8-CreTg (top middle). Megakaryocytes (MK) are indicated for easier recognition of areas of extramedullary hematopoiesis (EMH). Most of the spleen parenchyma of Nlrp3A350V/wt MRP8-CreTg animals was replaced by EMH of mostly myeloid lineage, which can be recognized by the medium-sized cells with more cytoplasm, giving a lighter appearance (inset). In both the overview picture (top middle picture) and inset (top right) of Nlrp3A350V/wt MRP8-CreTg spleen, a small amount of EMH of the erythroid lineage is present that can be recognized by small aggregates of darker-stained, smaller cells (asterix,*). Liver sections of both Nlrp3A350V/wtMRP8-Cre- control mice and diseased Nlrp3A350V/wt MRP8-CreTg pups displayed comparable EMH of the erythroid lineage (bottom left picture, indicated with yellow arrows). Liver sections of Nlrp3A350V/wt MRP8-CreTg pups also showed a modest increase in EMH of the myeloid lineage (bottom middle picture and inset (scale bar: 100 µM) in bottom right, indicated with yellow arrowheads), as well as hepatocyte enlargement. (F-I) H&E-stained histological sections of spleen (F, G) or liver (H, I) of Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) mice were assigned a score of 0-5 for lymphoid depletion (F) or EMH (G, H) or hepatocyte enlargement (I) by a board-certified pathologist. Values represent mean ± SEM of n=6-8 biological repeats. Statistical significance was analysed by the Mann-Whitney U-test. ***, p ≤0.001.


( J - L ) Cytokine secretion analysis ( Luminex ) of serum samples obtained at day 7 from Nlrp3A350V / wt MRP8 - Cre - ( Cre - , n = 8 ) and Nlrp3A350V / wt MRP8 - CreTg ( CreTg , n = 6 ) pups . Values represent mean ± SEM of n = 6 - 8 biological repeats . Statistical analysis of serum cytokine levels was performed by the Welch ' s t - test . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

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

(J-L) Cytokine secretion analysis (Luminex) of serum samples obtained at day 7 from Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) pups. Values represent mean ± SEM of n=6-8 biological repeats. Statistical analysis of serum cytokine levels was performed by the Welch's t-test. *, p ≤0.05; **, p ≤0.01.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with LeTx ( 3 hr ) ( A , B ) IL - 1β secretion levels were determined in culture media ( A , and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) ( B , Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with LeTx (3 hr) (A, B) IL-1β secretion levels were determined in culture media (A, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) (B, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) ( C , D ) IL - 1β secretion levels were determined in culture media C , and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) D , Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (C, D) IL-1β secretion levels were determined in culture media C, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) D, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or infected with F . tularensis ( 18 hr ) ( E , F ) IL - 1β secretion levels were determined in culture media E , and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) F , Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or infected with F. tularensis (18 hr) (E, F) IL-1β secretion levels were determined in culture media E, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) F, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).