C . kinetics of EYFP - Parkin translocation is different in PC6 transfected with scrambled or Miro shRNAs . were plated in separate compartments of same dish , enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment ( A & O , both 10 µM ) . spatial heterogeneity of EYFP - Parkin ( coefficient of variation of of pixels ) was estimated for for each time point ( n = 39 - 47 ; P < 0 . 0001 difference between curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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C. The kinetics of EYFP-Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separate compartments of the same dish, enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment (A&O, both 10 µM). The spatial heterogeneity of EYFP-Parkin (coefficient of variation of the intensity of individual pixels) was estimated for individual cells for each time point (n = 39-47 cells; P < 0.0001 difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


D . Miro shRNAs mitigate effect of A & O on EYFP - Parkin translocation . PC6 transfected with EYFP - Parkin with without Miro shRNAs were treated with DMSO or A & O ( both 10 µM ) for 3h . figure demonstrates that of with Parkin translocated to mitochondria was lower in Miro shRNAs - expressing group when compared with scrambled shRNA - expressing group . * * * * P < 0 . 0001 , n = 4 dishes , 20 fields per dish , one - way

O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 µM) for 3h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVA


E . Miro shRNAs inhibit PINK1 - induced mitophagy in PC6 . figure shows changes in excitation spectra of mitochondrially targeted pH - sensitive Keima ( of 561 nm / 458nm intensities ) . * * * * P < 0 . 0001 , n = 80 from 4 dishes , Kruskal - Wallis

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E. Miro shRNAs inhibit PINK1-induced mitophagy in PC6 cells. The figure shows the changes in the excitation spectra of mitochondrially targeted pH-sensitive Keima (ratio of 561 nm/458nm fluorescence intensities). ****P < 0.0001, n = 80 cells from 4 dishes, Kruskal-Wallis test


F . Miro shRNAs inhibit basal mitophagy in cortical neurons expressing mitochondrially targeted Keima . Mitochondria in lysosomes ( acidic pH ) were counted manually , by a blinded observer ( * * P < 0 . 01 , n = 9 dishes , 9 fields per dish , from 2 independent experiments , one - way ANOVA )

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F. Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima. Mitochondria in lysosomes (acidic pH) were counted manually, by a blinded observer (**P < 0.01, n = 9 dishes, 9 fields per dish, from 2 independent experiments, one-way ANOVA)


G . Miro shRNAs decrease number of mitochondria co - localised with autophagosome marker EGFP - LC3B in cortical neurons ( * * P < 0 . 01 , n = 5 dishes , 9 fields per dish , one - way ANOVA ) .

O O B-GENEPROD O O B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O

G. Miro shRNAs decrease the number of mitochondria co-localised with the autophagosome marker EGFP-LC3B in primary cortical neurons (**P < 0.01, n = 5 dishes, 9 fields per dish, one-way ANOVA).


A . pattern of Parkin translocation caused by Miro1 overexpression is different from A & O - induced translocation . figure illustrates - like EYFP - Parkin on mitochondria in antimycin oligomycin - treated ( both 10 µM for 3h ) PC6 ( middle panels ) , whereas in Miro1 - overexpressing , EYFP - Parkin appears along rod - shaped mitochondria visualised with CFP - mito ( lower panels ) . merged panels present EYFP - Parkin ( green ) CFP - mito ( red )

O O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O

A. The pattern of Parkin translocation caused by Miro1 overexpression is different from A&O-induced translocation. The figure illustrates puncta-like EYFP-Parkin on mitochondria in antimycin and oligomycin-treated (both 10 µM for 3h) PC6 cells (middle panels), whereas in Miro1-overexpressing cells, EYFP-Parkin appears along rod-shaped mitochondria visualised with CFP-mito (lower panels). The merged panels present EYFP-Parkin (green) and CFP-mito (red)


B . Overexpression of myc - Miro1 untagged Miro2 , but not myc - Miro2 , induce EYFP - Parkin translocation to mitochondria . * * * * P < 0 . 0001 vs control group , n = 5 - 6 dishes , 20 fields per dish , one - way ANOVA or t -

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

B. Overexpression of myc-Miro1 and untagged Miro2, but not myc-Miro2, induce EYFP-Parkin translocation to mitochondria. ****P < 0.0001 vs control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA or t-test


C . of EYFP - Parkin signal heterogeneity in PC6 demonstrates that Miro1 overexpression induces Parkin translocation from cytosol to mitochondria , which is significantly weaker than PINK1 overexpression - or A & O ( 3 h ) - induced translocation . * * P < 0 . 01 * * * * P < 0 . 0001 vs control group , n = 18 - 32 from 3 dishes per group , Kruskal - Wallis

O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that Miro1 overexpression induces Parkin translocation from the cytosol to mitochondria, which is significantly weaker than PINK1 overexpression- or A&O (3 h)-induced translocation. **P < 0.01 and ****P < 0.0001 vs control group, n = 18-32 cells from 3 dishes per group, Kruskal-Wallis test


Miro1 overexpression does not cause mitochondrial depolarization ( D ) Representative image of Miro1 EYFP - Parkin - transfected stained with TMRE . merged panels present EYFP - Parkin ( green ) TMRE ( red )

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Miro1 overexpression does not cause mitochondrial depolarization (D) Representative image of Miro1 and EYFP-Parkin-transfected cells stained with TMRE. The merged panels present EYFP-Parkin (green) and TMRE (red)


Miro1 overexpression does not cause mitochondrial depolarization ( E ) Relative TMRE quantified in neurons transfected with Miro1 ( detected by CFP - mito co - transfection ) ; control group is non - transfected in same image . relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs - expressing neurons . Note that FCCP treatment ( 3 μM for 15 min ) almost completely abolished TMRE . * * * * P < 0 . 0001 vs control group , n = 94 - 186 from 4 dishes per group , Kruskal - Wallis

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Miro1 overexpression does not cause mitochondrial depolarization (E) Relative TMRE intensity quantified in neurons transfected with Miro1 (detected by CFP-mito co-transfection); the control group is non-transfected cells in the same image. The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs-expressing neurons. Note that FCCP treatment (3 μM for 15 min) almost completely abolished TMRE fluorescence. ****P < 0.0001 vs control group, n = 94-186 cells from 4 dishes per group, Kruskal-Wallis test


F . Co - expression of HA - Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons . Overexpression of Miro1 alone increased motion time in both directions as well as relative mitochondrial velocity . Co - transfection of Parkin did not affect significantly motility parameters . Data are presented as Tukey boxplot . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , n = 689 - 846 mitochondria from 39 - 44 axons per group pooled from at least from 10 dishes from 4 independent experiments , Kruskal - Wallis

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

F. Co-expression of HA-Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons. Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity. Co-transfection of Parkin did not affect significantly the motility parameters. Data are presented as Tukey boxplot. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, n = 689-846 individual mitochondria from 39-44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments, Kruskal-Wallis test


G . Parkin overexpression does not induce degradation of myc - Miro1 in control conditions . Representative Western blot image of myc - Miro1 expression in HEK . Treatment with A & O ( both 15 µM ) for 3h in absence of MG132 led to slight degradation of myc - Miro1 . Co - expression of EYFP - Parkin did not induce degradation of Miro1 although an additional band above Miro1 band was present . However , Parkin co - expression enhanced Miro1 degradation when were treated with A & O . * P < 0 . 05 * * * * P < 0 . 0001 , ns : not significant , n = 4 independent experiments , one way ANOVA . Note that only main band of myc - Miro1 at 80 kDa was analysed that YFP was used to compensate in groups not expressing EYFP -

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G. Parkin overexpression does not induce degradation of myc-Miro1 in control conditions. Representative Western blot image and analysis of myc-Miro1 expression in HEK cells. Treatment with A&O (both 15 µM) for 3h in the absence of MG132 led to slight degradation of myc-Miro1. Co-expression of EYFP-Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present. However, Parkin co-expression enhanced Miro1 degradation when cells were treated with A&O. *P < 0.05 and ****P < 0.0001, ns: not significant, n = 4 independent experiments, one way ANOVA. Note that only the main band of myc-Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP-Parkin


H . EYFP - Parkin co - immunoprecipitates with overexpressed myc - Miro1 , both in DMSO - or A & O ( both 15 µM for 3h ) treated HEK

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H. EYFP-Parkin co-immunoprecipitates with overexpressed myc-Miro1, both in DMSO- or A&O (both 15 µM for 3h) treated HEK cells


I . Endogenous Parkin co - immunoprecipitates with endogenous Miro1 . Miro1 was immunoprecipitated from non - transfected non - treated HEK using mouse monoclonal anti - Miro1 antibody immunoblotted for endogenous Parkin .

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

I. Endogenous Parkin co-immunoprecipitates with endogenous Miro1. Miro1 was immunoprecipitated from non-transfected and non-treated HEK cells using mouse monoclonal anti-Miro1 antibody and immunoblotted for endogenous Parkin.


A . Miro1 induces EYFP - Parkin translocation in presence of PINK1 shRNA . PC6 were transfected with EYFP - Parkin Miro1 or treated for 3h with A & O ( both 10 µM ) in presence of scrambled shRNA or PINK1 shRNA . Miro1 induced Parkin translocation also in presence of PINK1 shRNA while A & O treatment did not . * * * * P < 0 . 0001 compared with respective control group , n = 5 - 6 dishes , 20 fields per dish , one - way

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A. Miro1 induces EYFP-Parkin translocation in the presence of PINK1 shRNA. PC6 cells were transfected with EYFP-Parkin and Miro1 or treated for 3h with A&O (both 10 µM) in the presence of scrambled shRNA or PINK1 shRNA. Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A&O treatment did not. ****P < 0.0001 compared with the respective control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA


B . Miro1 induces EYFP - Parkin translocation in PINK1 - / - MEFs . MEFs obtained from PINK1 + / + - / - mice were transfected with EYFP - Parkin Miro1 or treated with A & O ( both 10 µM for 3h ) . Miro1 induced Parkin translocation in both MEF lines but A & O only in PINK1 + / + MEFs . * * * * P < 0 . 0001 compared with respective control group , n = 6 dishes , one - way

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

B. Miro1 induces EYFP-Parkin translocation in PINK1 -/- MEFs. MEFs obtained from PINK1 +/+ and -/- mice were transfected with EYFP-Parkin and Miro1 or treated with A&O (both 10 µM for 3h). Miro1 induced Parkin translocation in both MEF lines but A&O only in PINK1 +/+ MEFs. ****P < 0.0001 compared with respective control group, n = 6 dishes, one-way ANOVA


C . Mutated EYFP - Parkin is recruited to mitochondria in response to Miro1 overexpression . PC6 were transfected with wild type , T240R or C431N EYFP - Parkin Miro1 or treated for 3 hours with A & O ( both 10 µM ) . Miro1 induced translocation of EYFP - Parkin mutants whereas A & O treatment was not able to induce equivalent translocation of Parkin mutants . * * P < 0 . 01 * * * * P < 0 . 0001 compared with respective control group , n = 3 - 6 dishes , 20 fields per dish , one - way

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

C. Mutated EYFP-Parkin is recruited to mitochondria in response to Miro1 overexpression. PC6 cells were transfected with wild type, T240R or C431N EYFP-Parkin and Miro1 or treated for 3 hours with A&O (both 10 µM). Miro1 induced translocation of all EYFP-Parkin mutants whereas A&O treatment was not able to induce equivalent translocation of Parkin mutants. **P < 0.01 and ****P < 0.0001 compared with respective control group, n = 3-6 dishes, 20 fields per dish, one-way ANOVA


D . Parkin mutants T240R C431N interact with Miro1 both in DMSO - or A & O - treated ( both 15 µM for 3h ) HEK . Note that higher molecular bands of Miro1 were observed only in WT Parkin - expressing groups .

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

D. Parkin mutants T240R and C431N interact with Miro1 both in DMSO- or A&O-treated (both 15 µM for 3h) HEK cells. Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin-expressing groups.


A . Miro1 K572R mutant interacts with HA - Parkin similarly to Miro1 WT . HEK overexpressing myc - Miro1 WT or K572R mutant were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated probed for myc - Miro1 HA - Parkin . Note diminished degradation of Miro1 K572R in input additional band above Miro1 WT in IP , which is missing in case of K572R

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

A. Miro1 K572R mutant interacts with HA-Parkin similarly to Miro1 WT. HEK cells overexpressing myc-Miro1 WT or K572R mutant were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probed for myc-Miro1 and HA-Parkin. Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP, which is missing in the case of the K572R mutant


B . Miro1 K572R ubiquitination by Parkin is reduced . HEK overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 K572R EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated probes were immunoblotted for ubiquitin - HA myc -

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B. Miro1 K572R ubiquitination by Parkin is reduced. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1 K572R and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin-HA and myc-Miro1


C . Representative of PC6 overexpressing Miro1 K572R or Miro1 WT together with EYFP - Parkin . K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A & O ( both 7 . 5 µM ) for 3 hours . Note spatial heterogeneity values shown in

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

C. Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP-Parkin. K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A&O (both 7.5 µM) for 3 hours. Note the spatial heterogeneity values shown in the images


D . of EYFP - Parkin signal heterogeneity in PC6 demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A & O ( both 7 . 5 µM ) treatment for 3 hours , but K572R mutation of Miro1 abolishes this effect . * * * P < 0 . 001 * * * * P < 0 . 0001 , n = 152 - 172 from 3 dishes per group , Kruskal - Wallis . Data are presented as means ± .

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D. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A&O (both 7.5 µM) treatment for 3 hours, but K572R mutation of Miro1 abolishes this effect. ***P < 0.001 and ****P < 0.0001, n = 152-172 cells from 3 dishes per group, Kruskal-Wallis test. Data are presented as means ± SEM.


A . A Ca2 + - insensitive mutant of Miro1 ( Miro1 - EF ) interacts with Parkin . HEK overexpressing myc - Miro1 or myc - Miro1 - EF EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated probes were immunoblotted for Miro1 ( red ) Parkin ( green ) . high - contrast grayscale image from green channel of this blot shows that endogenous Parkin co - immunoprecipitated with Miro1 in conditions . bar chart shows of co - immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP - Parkin signal ( * P < 0 . 05 * * P < 0 . 01 , repeated measures

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

A. A Ca2+-insensitive mutant of Miro1 (Miro1-EF) interacts with Parkin. HEK cells overexpressing myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red) and Parkin (green). The high-contrast grayscale image from the green channel of this blot shows that endogenous Parkin co-immunoprecipitated with Miro1 in all conditions. The bar chart shows the quantification of co-immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP-Parkin signal (*P < 0.05 and **P < 0.01, repeated measures ANOVA


B . Miro1 - EF is less susceptible to ubiquitination by Parkin . HEK overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 - EF EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated probes were immunoblotted for ubiquitin ( red ) , Miro1 ( green ) Parkin ( green ; representative are from same experimental material on separate SDS - PAGE - WB membranes ) . high - contrast grayscale shows green channel of this blot , bar chart shows of co - immunoprecipitated Miro from 3 different blots ( * * P < 0 . 01 * * * P < 0 . 001 , repeated measures ANOVA ) . Note that EYFP - Parkin co - immunoprecipitated with ubiquitin only after A & O

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

B. Miro1-EF is less susceptible to ubiquitination by Parkin. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin (red), Miro1 (green) and Parkin (green; representative images are from the same experimental material on separate SDS-PAGE-WB membranes). The high-contrast grayscale shows the green channel of this blot, and the bar chart shows quantification of co-immunoprecipitated Miro from 3 different blots (**P < 0.01 and ***P < 0.001, repeated measures ANOVA). Note that EYFP-Parkin co-immunoprecipitated with ubiquitin only after A&O treatment


C . Mutation of Miro1 EF - hands diminishes Miro1 ubiquitination with S65 - phosphorylated ubiquitin . HEK were transfected with EYFP - Parkin with either myc - Miro1 - WT or myc - Miro1 - EF with or without PINK1 - V5 . were treated with either DMSO or A & O ( both 10 µM ) for 5 hrs with exception of PINK1 - V5 group . Myc - Miro1 was immunoprecipitated probes were immunoblotted for Miro1 ( red ) , S65 - Phosphoubiquitin PINK1 - V5 ( green ; weak bands marked with asterisk are nonspecific ) .

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C. Mutation of Miro1 EF-hands diminishes Miro1 ubiquitination with S65-phosphorylated ubiquitin. HEK cells were transfected with EYFP-Parkin with either myc-Miro1-WT or myc-Miro1-EF with or without PINK1-V5. Cells were treated with either DMSO or A&O (both 10 µM) for 5 hrs with the exception of the PINK1-V5 group. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red), S65-Phosphoubiquitin and PINK1-V5 (green; the weak bands marked with the asterisk are nonspecific).


A . Kinetics of EYFP - Parkin translocation in response to A & O is different in control Miro1 - EF - expressing PC6 . inset shows how different compartments of same dish were transfected with EYFP - Parkin control plasmid or Miro1 - EF followed over 3 hrs using a confocal microscope after addition of A & O ( both 10 µM ) . graph shows average spatial heterogeneity of EYFP - Parkin , reflecting of mitochondrial - to - Parkin over time ( n = 40 - 43 , difference between curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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A. Kinetics of EYFP-Parkin translocation in response to A&O is different in control and Miro1-EF-expressing PC6 cells. The inset shows how different compartments of the same dish were transfected with EYFP-Parkin and control plasmid or Miro1-EF and followed over 3 hrs using a confocal microscope after addition of A&O (both 10 µM). The graph shows the average spatial heterogeneity of EYFP-Parkin fluorescence, reflecting the ratio of mitochondrial-to-cytosolic Parkin over time (n = 40-43 cells, difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


of where EYFP - Parkin translocates in response to A & O ( both 10 µM , B is decreased in Miro1 - EF - expressing PC6 . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with indicated group ( n = 3 - 6 dishes , 20 fields per dish , one way ANOVA

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The percentage of cells where EYFP-Parkin translocates in response to A&O (both 10 µM, B is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dish, one way ANOVA for


of where EYFP - Parkin translocates in response t PINK1 overexpression ( C ) is decreased in Miro1 - EF - expressing PC6 . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with indicated group ( n = 3 - 6 dishes , 20 fields per dis Welch " s ANOVA

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

The percentage of cells where EYFP-Parkin translocates in response t PINK1 overexpression (C) is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dis Welch"s ANOVA f


D . Time course showing - induced EYFP - Parkin accumulation in irradiated of neuronal soma of rat cortical neurons . were transfected with CFP - mito EYFP - Parkin visualized using a confocal microscope . Mitochondria were irradiated in a 0 . 9 µm x 0 . 9 µm - frame using a 405 - nm ( 25 iterations using 100 % power , pixel time 164 µs )

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D. Time course showing laser-induced EYFP-Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons. Cells were transfected with CFP-mito and EYFP-Parkin and visualized using a confocal microscope. Mitochondria were irradiated in a 0.9 µm x 0.9 µm-frame using a 405-nm laser line (25 iterations using 100% laser power, pixel time 164 µs)


E , F Miro1 - EF delays reduces irradiation - induced EYFP - Parkin translocation . ( E ) time course of translocation strength in control Miro1 - EF - expressing neurons . Difference between curves P = 0 . 0016 , two - way ANOVA . ( F ) Comparison at 60 - min time point ( * P < 0 . 05 , n = 6 dishes , at least 9 neurons per dish , t - ) .

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E, F Miro1-EF delays and reduces irradiation-induced EYFP-Parkin translocation. (E) The time course of translocation strength in control and Miro1-EF-expressing neurons. Difference between the curves P = 0.0016, two-way ANOVA. (F) Comparison at the 60-min time point (*P < 0.05, n = 6 dishes, at least 9 neurons per dish, t-test).


A . EYFP - Parkin translocation requires Ca2 + . PC6 plated in separated compartments of same dish maintained during experiment in Krebs containing 1 mM Ca2 + or in Ca2 + free Krebs containing 5 μM BAPTA - AM , were treated with A & O ( both 10 μM ) . ( left panels ) demonstrate that EYFP - Parkin translocation was significantly weaker at zero Ca2 + at 195 min after A & O treatment ( compared with 15 min ; note spatial heterogeneity values shown in photos ) . Spatial heterogeneity ( right panel ) demonstrated that Parkin translocation was significantly decelerated in zero Ca2 + conditions ( n = 25 - 27 ; difference between curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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

A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


B . EYFP - Parkin translocation accelerates in presence of L - type Ca2 + channel agonist Bay K 8644 ( 5 μM ; n = 19 - 21 ; difference between curves p < 0 . 0001 , two - way ANOVA ; lines show mean , dashed shows )

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

B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into cytosol , visualised by increased GCaMP7b

O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-GENEPROD

Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescence


Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into cytosol Glutamate - induced Ca2 + entry was not affected by Miro1 WT or Miro1 EF overexpression ; n = 31 - 48 neurons per group ; ns , Kruskal - Wallis )

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Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test)


E , F Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) led to appearance of a few Parkin - positive strong co - localising with mitochondrial marker ( arrow ) , imaged 2 . 5 h after treatment ( E ) . Further demonstrated that expression of Miro1 - EF mutant decreased Miro1 WT increased number of neurons with EYFP - Parkin - positive mitochondria following glutamate treatment ( F ; n = 6 - 10 ; * * P < 0 . 01 * * * * P < 0 . 0001 , one - way ANOVA )

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

E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA)


G . Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) was associated with an increased number of mitochondrial co - localisation with autophagosome marker EGFP - LC3B in neuronal soma at 5 - 6 h later , which was not observed in Miro1 - EF - expressing neurons ( n = 8 dishes ; * P < 0 . 05 , one - way ANOVA )

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

G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA)


H . Glutamate treatment ( 30 μM glutamate 1 μM glycine for 10 min ) was also associated with a significant loss of mitochondrial density in axons at 6 h later , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 60 - 90 axons from 6 - 9 dishes ; * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , Kruskal - Wallis )

O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test)


I . Glutamate treatment ( 100 μM glutamate 10 μM glycine for 10 min ) led to a decline , at 6 - 8 h , in ATP measured using ATP / ADP sensor Perceval HR , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 59 - 60 neurons from 6 dishes ; * * P < 0 . 01 , Welch " s

O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O

I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch"s A


J . Glutamate ( 100 μM glutamate 10 μM glycine for 10 min ) induced excitotoxicity ( 24 h after treatment ) that was partially reversed by Miro1 - EF but not by Miro1 WT . Data is presented as a of control neuron ( n = 10 dishes ; * * * P < 0 . 001 , repeated measures ANOVA ) .

O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

J. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).


A microfluidic device for quantitative recovery time measurements . Each PDMS - fabricated chip consists of six independent channels , one of which is shown , from top view ( top row ) . Green circles indicate media inflow ; black circle indicates outflow . are manually loaded in inflow ports are trapped in culturing chambers ( gray circles ) , height of which ensures that are imaged in a monolayer ( bottom row ) . Growth conditions ( e . g . antibiotic dose profile ) are controlled via programmable syringe pumps . ( bottom row ) show representative growth of a monolayer of E . coli BW25113 constitutively expressing GFP over four hours .

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

A microfluidic device for quantitative recovery time measurements. Each PDMS-fabricated chip consists of six independent channels, one of which is shown, from top view (top row). Green circles indicate media inflow; black circle indicates outflow. Bacteria are manually loaded in inflow ports and are trapped in individual culturing chambers (gray circles), the height of which ensures that bacteria are imaged in a monolayer (bottom row). Growth conditions (e.g. antibiotic dose profile) are controlled via programmable syringe pumps. Fluorescent images (bottom row) show representative growth of a monolayer of E. coli BW25113 cells constitutively expressing GFP over four hours.


Representative time series data showing dose - dependent population recovery in response to transient streptomycin treatment . Here , time zero corresponds to end of treatment ( 120 minutes ) . Trajectories show mean standard deviation for five replicates ; colors indicate increasing antibiotic ( 2 , 4 , 6 , 8 , 10 , 12 μg / mL ) . values are normalized to those at time zero . Dotted indicates a 2 - fold increase , corresponding to recovery time for each population .

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

Representative time series fluorescence data showing dose-dependent population recovery in response to transient streptomycin treatment. Here, time zero corresponds to the end of treatment (120 minutes). Trajectories show mean and standard deviation for five replicates; colors indicate increasing antibiotic concentration (2, 4, 6, 8, 10, 12 μg/mL). Fluorescence values are normalized to those at time zero. Dotted line indicates a 2-fold increase, corresponding to the recovery time for each population.


Recovery time increases with total antibiotic exposure . Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations ( as in panel C ) . When plotted against total antibiotic exposure ( calculated as ) , recovery time values collapse onto a single exponential function ( black , R2 = 0 . 83 ) . Recovery time was measured in response to streptomycin concencentrations of 0 , 2 , 4 , 6 , 8 , 10 , 12 μg / mL , treatment durations 30 , 60 , 90 , 120 minutes .

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

Recovery time increases with total antibiotic exposure. Inset shows recovery time as a function of dose duration for increasing streptomycin concentrations (as in panel C). When plotted against total antibiotic exposure (calculated as ), all recovery time values collapse onto a single exponential function (black line, R2 = 0.83). Recovery time was measured in response to streptomycin concencentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL, and treatment durations 30, 60, 90, and 120 minutes.


Streptomycin treatment ( closed data points , R2 = 0 . 83 ) results in significantly longer recovery times than chloramphenicol treatment ( open data points ) . Here , difference between two responses is statistically significant ( p < 0 . 05 by ANOVA ) .

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

Streptomycin treatment (closed data points, R2 = 0.83) results in significantly longer recovery times than chloramphenicol treatment (open data points). Here, the difference between two responses is statistically significant (p<0.05 by ANOVA).


addition of efflux pump inhibitor ( CCCP ) ( closed data points , R2 = 0 . 91 ) increases population recovery time in response to chloramphenicol treatment ( open data points , R2 = 0 . 80 ) . Here , CCCP was added at subinhibitory concentrations ( 3μg / mL ) ; in absence of antibiotic treatment , CCCP alone did not inhibit population recovery . CCCP - mediated increase in recovery time is statistically significant ( p < 0 . 01 ) .

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

The addition of efflux pump inhibitor (CCCP) (closed data points, R2 = 0.91) increases population recovery time in response to chloramphenicol treatment (open data points, R2 = 0.80). Here, CCCP was added at subinhibitory concentrations (3μg/mL); in the absence of antibiotic treatment, CCCP alone did not inhibit population recovery. The CCCP-mediated increase in recovery time is statistically significant (p < 0.01).


ribosome synthesis rate was increased by increasing of casamino in media from 0 . 01 % w / v ( closed data points ) to 0 . 05 % w / v ( open data points ) . Faster synthesis resulted in lower recovery times ( p < 0 . 001 ) in response to streptomycin treatment . In each of these panels , color scheme indicates increasing antibiotic , as in Figure 1C .

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

The ribosome synthesis rate was increased by increasing the concentration of the casamino acids in the media from 0.01% w/v (closed data points) to 0.05% w/v (open data points). Faster synthesis resulted in lower recovery times (p<0.001) in response to streptomycin treatment. In each of these panels, the color scheme indicates increasing antibiotic concentration, as in Figure 1C.


Inhibiting antibiotic efflux with CCCP significantly increased recovery time for that induced rapid target degradation involved a positive feedback loop . are as follows : penicillin G ( PenG ) , spectinomycin ( Spec ) , gentamicin ( Gent ) , streptomycin ( Str ) , chloramphenicol ( Cm ) , tetracyclin ( Tet ) , carbenicillin ( Carb ) . Antibiotic concentrations used have been scaled by their respective IC50 values ( solid points open circles show response with no efflux inhibition 2μg / mL CCCP , respectively ) . Colors correspond to motifs of action shown in Figure 4A .

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

Inhibiting antibiotic efflux with CCCP significantly increased recovery time for antibiotics that induced rapid target degradation and involved a positive feedback loop. Antibiotics are as follows: penicillin G (PenG), spectinomycin (Spec), gentamicin (Gent), streptomycin (Str), chloramphenicol (Cm), tetracyclin (Tet), and carbenicillin (Carb). Antibiotic concentrations used have been scaled by their respective IC50 values (solid points and open circles show response with no efflux inhibition and 2μg/mL CCCP, respectively). Colors correspond to the motifs of action shown in Figure 4A.


a , Silver of LC3 complexes purified from U2OS expressing ZZ - Flag - LC3 in normal medium or Earle ' s balanced salt solution ( EBSS ) for 2 h . Asterisks mark centriolar satellite .

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

a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ-Flag-LC3 in normal medium or Earle's balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins.


b - d , Co - immunoprecipitation ( IP ) of OFD1 with LC3 , LC3 with PCM1 , or OFD1 with PCM1 in HEK293T . HA , haemagglutinin .

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b-d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin.


e , Western blotting of PCM1 OFD1 in control or PCM1 knockdown ( KD ) HEK293T .

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e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells.


f , Co - immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T . IP efficiency is calculated as of immunoprecipitated OFD1 / LC3 .

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f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3.


g , h , Western blotting of of indicated in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation ( SS ) . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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

g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


g , h , Western blotting of of indicated in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation ( SS ) , 50 nM bafilomycin A1 ( Baf ) , 20 µM chloroquine ( CQ ) or 1 µM MG132 . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O B-TISSUE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


a , Representative confocal of endogenous OFD1 with cilium marker acetylated tubulin ( Ac - tubulin ) , or centriole marker γ - tubulin in hTERT - RPE1 in normal medium ( Un ) or subjected to 24 hserum starvation ( SS ) .

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

a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker γ-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 hserum starvation (SS).


b , Representative confocal of endogenous OFD1 acetylated tubulin in Atg5 + / + Atg5 − / − MEFs subjected to 24 hserum starvation . a , b , Data shown represent mean ± s . d . of with centriolar satelliteOFD1 for 100 per well in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - . Similar results were observed in three independent experiments .

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

b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 hserum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments.


a , b , Representative confocal of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation . Data shown represent mean ± s . d . of with cilia or of cilia for 500 or 100 per well , respectively , in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - . Similar results were observed in three independent experiments . Un , untreated ; SS , serum - starved .

O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O

a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.


a , Western blotting of OFD1 in control or OFD1 knockdown MCF7 ( C - 19 ) , quantified OFD1 level was normalized with alpha - tubulin .

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

a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with alpha-tubulin.


b , of of with cilia in MCF7 or C19 subjected to 72 h serum starvation . Data shown represent mean ± s . d . of with cilia for 500 per well in triplicate samples . * * * P 0 . 001 , two - unpaired student ' s t - .

O O O O O B-SUBCELLULAR O B-CELL_LINE O B-CELL_LINE O O O O B-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

b, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test.


c , of of cilia in C19 subject to 48 h or 72 h serum starvation .

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c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation.


d , e , Representative confocal of cilia with variable formed in MCF7 or C19 subject to 72 h serum starvation . Data shown represent 100 per well in triplicate samples .

O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-CELL_LINE O B-CELL_LINE O O O O O B-TISSUE O O O O O O O O O O O O

d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples.


d , e , Representative confocal of cilia with variable formed in C19 subject to 72 hserum starvation . Data shown represent 100 per well in triplicate samples .

O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O

d, e, Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation. Data shown represent 100 cells per well in triplicate samples.


f , Scanning electron microscope of cilia ( marked by arrows ) formed in C - 19 subject to 72 h serum starvation cycling Atg5 + / + MEFs in normal medium for 24 h . Similar results were observed in three independent experiments .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-TISSUE O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O

f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.


( A ) PI3Kγ is expressed by human hepatocytes infiltrating immune in biopsies from with minimal to mild inflammatory activity . Triangles point to immune ( clusters ) , including some neutrophils known to express PI3Kγ highly . In negative control , antibody was replaced by an equal volume buffer . number of included , gender , diagnosis are summarized

O O O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O B-CELL_TYPE O O O O O O O B-DISEASE O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

(A) PI3Kγ is expressed by human hepatocytes and infiltrating immune cells in biopsies from patients with minimal to mild inflammatory activity. Triangles point to immune cells (clusters), including some neutrophils known to express PI3Kγ highly. In the negative control, the primary antibody was replaced by an equal volume buffer. The number of included patients, gender, and diagnosis are summarized


( A ) PI3Kγ is expressed by human hepatocytes infiltrating immune in biopsies from with minimal to mild inflammatory activity . Triangles point to immune ( clusters ) , including some neutrophils known to express PI3Kγ highly . In negative control , antibody was replaced by an equal volume buffer . number of included , gender , diagnosis are

O O O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O B-CELL_TYPE O O O O O O O B-DISEASE O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O


( B ) PI3Kγ expression in human hepatocytes from 20 ( ♂ ) or ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . characterization of donor pools is summarized

O O O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O

(B) PI3Kγ expression in human primary hepatocytes from 20 male (♂) or female (♀) donor pools (HEP, DP20), but not non-parenchymal cells (NPC). Isolated human leukocytes from healthy volunteers (LEU) served as positive controls. The characterization of the donor pools is summarized


( B ) PI3Kγ expression in human hepatocytes from 20 ( ♂ ) or ( ♀ ) donor pools ( HEP , DP20 ) , but not non - parenchymal ( NPC ) . Isolated human leukocytes from healthy volunteers ( LEU ) served as positive controls . characterization of donor pools is

O O O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O


( D ) Survival of WT , PI3Kγ null ( left ) liver - specific PI3Kγ knockout mice ( PI3Kγ floxtg / tg x AlbCre ( tg ) / tg , middle ) or systemic application of PI3Kγ inhibitor , AS605240 ( right ) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension . numbers : B6 : 86 ( % : 52 , % : 48 ) , B6 - Pi3kcgko / ko : 59 ( % : 51 , % 49 ) , B6 - Pi3kcgflox / flox x AlbCre ( tg ) / tg : 25 ( % : 68 , % 32 ) , FVB / N Vehicle : 25 ( % : 44 , % 56 ) , FVB / N AS : 43 ( % : 44 , % 56 )

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

(D) Survival of WT, PI3Kγ null (left) and liver-specific PI3Kγ knockout mice (PI3Kγ floxtg/tg x AlbCre(tg)/tg, middle) or systemic application of the PI3Kγ inhibitor, AS605240 (right) in a model of polymicrobial sepsis induced by peritoneal contamination with a human stool suspension. Animal numbers: B6: 86 (%male: 52, %female: 48), B6-Pi3kcgko/ko: 59 (%male: 51, %female 49), B6-Pi3kcgflox/flox x AlbCre(tg)/tg: 25 (%male: 68, %female 32), FVB/N Vehicle: 25 (%male: 44, %female 56), FVB/N AS: 43 (%male: 44, %female 56)


( C ) Uptake characteristics of T - LipoAS by human hepatocytes demonstrate competitive inhibition by cyclosporin A - energy dependence , confirming active uptake by organic anion transporters ( OATPs ) . data points are depicted mean ± SD from two batches of human hepatocytes in quadruples .

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(C) Uptake characteristics of T-LipoAS by primary human hepatocytes demonstrate competitive inhibition by cyclosporin A- and energy dependence, confirming active uptake by organic anion transporters (OATPs). The data points are depicted mean ± SD from two batches of primary human hepatocytes in quadruples.


( F ) T - LipoAS effectively inhibits fMLP - stimulated PI3Kγ , reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes . mean pAKT / AKT standard deviation ( SD ) were calculated for experiments separately .

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

(F) T-LipoAS effectively inhibits fMLP-stimulated PI3Kγ, reflected by inhibiting AKT S473 phosphorylation activity in hepatocytes from three different batchtes. The mean pAKT/AKT ratio and standard deviation (SD) were calculated for all experiments separately.


( G ) Episcopic imaging of mice treated with T - LipoAS for 15 or 60 min relative of targeting moiety DY - 635 in different compartments .

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

(G) Episcopic imaging of mice treated with T-LipoAS for 15 or 60 min and relative concentration of the targeting moiety DY-635 in the different compartments.


( B Pro - anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination infection ( PCI ) sham . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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(B Pro- anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


C ) anti - inflammatory cytokines were analyzed in EDTA - plasma from peritoneal contamination infection ( PCI ) sham . # p < 0 . 05 against sham , * p < 0 . 05 as indicated ; Kruskal - Wallis ANOVA with controlled false - discovery rates ( Benjamini - Hochberg procedure ) .

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C) anti-inflammatory cytokines were analyzed in EDTA-plasma from peritoneal contamination and infection (PCI) and sham animals. #p<0.05 against sham, *p<0.05 as indicated; Kruskal-Wallis ANOVA with controlled false-discovery rates (Benjamini-Hochberg procedure).


( F , G ) of ( F ) neutrophils ( G ) monocytes in 2 mL peritoneal lavage .

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(F,G) Concentration of (F) neutrophils and (G) monocytes in 2 mL peritoneal lavage.


( A ) Intravital microscopy of elimination of CDFDA , a substrate subject to excretory elimination by hepatocytes via Mrp - 2 , allows direct visualization of excretory function in sham or septic ( PCI ) treated with vehicle or a hepatocyte - directed inhibitor of PI3Kγ ( T - LipoAS ) .

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

(A) Intravital microscopy of elimination of CDFDA, a fluorescent substrate subject to excretory elimination by hepatocytes via Mrp-2, allows direct visualization of excretory function in sham or septic (PCI) animals treated with vehicle or a hepatocyte-directed inhibitor of PI3Kγ (T-LipoAS).


( A ) GFP - PAQR3 transfected HeLa were fixed for immunofluorescence with indicated antibodies before or after glucose starvation ( GS ) for 4 h . nuclei were stained with Hoechst 33342 . Scale bar : 10 μm .

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(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm.


( B ) Immunoblotting ( IB ) of wild type ( WT ) PAQR3 - deficient MEFs after GS for different time as indicated .

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(B) Immunoblotting (IB) analysis of wild type (WT) and PAQR3-deficient MEFs after GS for different time as indicated.


( C ) WT PAQR3 - deficient MEFs were treated with 80 mM chloroquine ( CQ ) for 2 h to induce LC3 - II accumulation . Whole - lysates were harvested for IB of LC3 - II .

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

(C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II.


( D ) Autophagosome - like structures in WT PAQR3 - deleted MEFs were detected after GS for 4 h by transmission electron microscopy . * indicates autophagosome - like structures . N , nucleus . ( E ) Quantitative of autophagic vacuoles in transmission electron microscopy . Thirty were quantified from each independent experiment ， which was repeated for three times with similar results . Values are presented as mean ± SD , * * * p < 0 . 001 .

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(D) Autophagosome-like structures in WT and PAQR3-deleted MEFs were detected after GS for 4 h by transmission electron microscopy. * indicates autophagosome-like structures. N, nucleus.(E) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images. Thirty cells were quantified from each independent experiment，which was repeated for three times with similar results. Values are presented as mean ± SD, ***p < 0.001.


( F ) Immunofluorescence of LC3 in WT or PAQR3 - deficient MEFs after GS for 4 h . arrowhead indicates representative LC3 - positive . Scale bar : 10 μm .

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(F) Immunofluorescence staining of LC3 in WT or PAQR3-deficient MEFs after GS for 4 h. The arrowhead indicates representative LC3-positive puncta. Scale bar: 10 μm.


( G , H ) HeLa were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control . After GS for 1 h , lysates were harvested for IB .

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(G, H) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus. After GS for 1 h, the cell lysates were harvested for IB.


( A ~ C ) HeLa stably expressing GFP - DFCP1 were transfected with or without PAQR3 - specific shRNA . GFP signals were observed in these before or after glucose starvation ( GS , shown in A ) . knockdown efficiency of PAQR3 is shown in B statistics of GFP - positive per is illustrated in C . Values are presented as mean ± SD , * * * P < 0 . 001 . Scale bar : 10 μm .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm.


( D ) GFP - WIPI1 was transfected into WT or PAQR3 - deficient MEFs . 24 h later , were incubated under GS for 4 h , followed by observation . Scale bar : 10 μm .

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(D) GFP-WIPI1 was transfected into WT or PAQR3-deficient MEFs. 24 h later, the cells were incubated under GS for 4 h, followed by fluorescence observation. Scale bar: 10 μm.


( E ) Different VPS34 complexes of WT or PAQR3 knockout HeLa were immunoprecipitated by ATG14L UVRAG antibodies respectively in normal medium ( NM ) or GS . Then PI ( 3 ) P level was determined by a quantitative ELISA . PI ( 3 ) P level was normalized to amount of ATG14L or UVRAG used in ( n = 5 ; * * * p < 0 . 001 ; ns , not significant ) .

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

(E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant).


( F ) HeLa infected with control or PAQR3 - overexpressed lentivirus were treated with GS for 4 h . PI ( 3 ) P were determined as in Figure 2E ( n = 5 ; * * p < 0 . 01 ; ns , not significant ) .

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(F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant).


( G , H ) PAQR3 knockout HeLa were transfected with plasmids of multiple PAQR3 deletion mutants . At 24 h after transfection , lysates were used to immunoprecipitate VPS34 by ATG14L antibody , followed by immunoblotting . activity of ATG14L - linked VPS34 activity was detected by a quantitative ELISA . PI ( 3 ) P level was normalized to amount of ATG14L ( n = 5 ; * * p < 0 . 01 ) .

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

(G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).


( A ) Interaction between endogenous PAQR3 components of VPS34 in HEK293T . lysates were used for immunoprecipitation ( IP ) immunoblotting ( IB ) with antibodies as indicated .

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(A) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells. The cell lysates were used for immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) Cytosol Golgi fractionations were isolated from livers of PAQR3 - deleted mice their littermate controls . Equal amounts of cytosol Golgi fractions were subjected to IB .

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(B) Cytosol and Golgi fractionations were isolated from the livers of PAQR3-deleted mice and their littermate controls. Equal protein amounts of cytosol and Golgi fractions were subjected to IB.


( C ) HEK293T were transiently transfected with Myc - tagged PAQR3 . At 24 h after transfection , were treated with normal medium ( NM ) ， acid starvation ( AS ) or glucose starvation ( GS ) , HBSS solution , or rapamycin ( Rapa , 50 nM ) for 4 h respectively . lysates were then used in IP IB .

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(C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB.


( D ) Four VPS34 complexes of MEFs were immunopurified using indicated antibodies under NM , AS or GS conditions . relative abundance of VPS34 - binding partners was determined by IB . Each IP was normalized to amount of VPS34 .

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(D) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS or GS conditions. The relative abundance of VPS34-binding partners was determined by IB. Each IP was normalized to the amount of VPS34.


( E ) HEK293T were transfected with plasmids as indicated lysates were used in IP IB .

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(E) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB.


( F ) lysates of WT or PAQR3 knockout MEFs were used in IP IB .

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(F) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB.


( G ) MEFs were infected with control or PAQR3 - expressing lentivirus . Then a quantitative immunodepletion was performed with increasing amounts of indicated antibodies . Supernatants of MEF lysates after immunodepletion ( Post - IP ) were examined to determine level of VPS34 .

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(G) MEFs were infected with control or PAQR3-expressing lentivirus. Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies. Supernatants of MEF lysates after immunodepletion (Post-IP) were examined to determine the level of VPS34 complex proteins.


( A ) A two - step co - IP in HEK293T to determine ternary containing PAQR3 , Beclin1 ATG14L . procedures of two - step co - IP are outlined in top . samples were subjected to immunoprecipitation ( IP ) immunoblotting ( IB ) with antibodies as indicated .

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(A) A two-step co-IP assay in HEK293T cells to determine the ternary complex containing PAQR3, Beclin1 and ATG14L. The procedures of the two-step co-IP are outlined in the top. The samples were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) HeLa were transiently transfected with plasmids as indicated . 24 h after transfection , were treated with or without glucose starvation ( GS ) for 4 h . lysates were used in IP IB .

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(B) HeLa cells were transiently transfected with the plasmids as indicated. 24 h after transfection, the cells were treated with or without glucose starvation (GS) for 4 h. The cell lysates were used in IP and IB.


( C , D ) Different truncations of ATG14L ( C ) were co - transfected with Myc - tagged PAQR3 into HEK293T as indicated , followed by IP IB .

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(C, D) Different truncations of ATG14L (C) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( C , D ) Different truncations of Beclin1 ( D ) were co - transfected with Myc - tagged PAQR3 into HEK293T as indicated , followed by IP IB .

(C, D) Different truncations of Beclin1 (D) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( E ) Purified GST or GST - fused PAQR3 ( 1 - 71 ) ( GST fused NH2 - terminal 71aa of PAQR3 ) on glutathione agarose beads was mixed with purified His tagged as indicated . After incubation at 4 ° C for 3 h , samples were washed extensively subjected to IB .

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(E) Purified GST or GST-fused PAQR3 (1-71) (GST fused NH2-terminal 71aa of PAQR3) on glutathione agarose beads was mixed with purified His tagged proteins as indicated. After incubation at 4 °C for 3 h, the samples were washed extensively and subjected to IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with Flag - tagged Beclin1 ( F ) into HEK293T . were then subjected to IP IB .

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(F, G) Different PAQR3 deletion constructs were co-transfected with Flag-tagged Beclin1 (F) into HEK293T cells. The cells were then subjected to IP and IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with GFP - fused ATG14L ( G ) into HEK293T . were then subjected to IP IB .

(F, G) Different PAQR3 deletion constructs were co-transfected with GFP-fused ATG14L (G) into HEK293T cells. The cells were then subjected to IP and IB.


( A ) HeLa were cultured in normal medium ( NM ) or glucose starvation ( GS ) for 4 h . starved lysates were treated with calf intestinal phosphatase ( CIP ) to remove phosphate groups analyzed by immunoblotting ( IB ) with Phos - tag gel or regular SDS - PAGE .

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(A) HeLa cells were cultured in normal medium (NM) or glucose starvation (GS) for 4 h. The starved lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate groups and analyzed by immunoblotting (IB) with Phos-tag gel or regular SDS-PAGE.


( B ) lysates from HeLa were collected analyzed after GS for indicated time ( lane 1 ~ 5 ) , or GS for 2 h followed by 2 h chase in NM ( lane 6 ) . lysates were subjected to Phos - tag gel regular SDS PAGE respectively .

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(B) The cell lysates from HeLa cells were collected and analyzed after GS for indicated time (lane 1~5), or GS for 2 h followed by 2 h chase in NM (lane 6). The cell lysates were subjected to Phos-tag gel and regular SDS PAGE respectively.


( C ) WT or AMPKa1 / 2 double knockout MEFs were incubated under NM or GS for 4 h , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(C) WT or AMPKa1/2 double knockout MEFs were incubated under NM or GS for 4 h, followed by IB with Phos-tag gel or regular SDS-PAGE.


( D ) HeLa were incubated with compound C ( 20 mM , 30 min ) with or without GS , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(D) HeLa cells were incubated with compound C (20 mM, 30 min) with or without GS, followed by IB with Phos-tag gel or regular SDS-PAGE.


( E ) purified His - tagged NH2 - terminal 71 acid of PAQR3 was incubated with or without purified Flag - tagged ATG14L . Then complexes were treated with WT AMPK or DN - AMPK ( dominant negative AMPK ) for indicated time in vitro . PAQR3 phosphorylation was examined by Phos - tag gel .

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(E) Bacterial purified His-tagged NH2-terminal 71 amino acid of PAQR3 was incubated with or without purified Flag-tagged ATG14L. Then the complexes were treated with WT AMPK or DN-AMPK (dominant negative AMPK) for the indicated time in vitro. PAQR3 phosphorylation was examined by Phos-tag gel.


( F ) HeLa infected with control or ATG14L - expressing lentivirus were incubated under GS for different time as indicated . Whole lysates were analyzed by both Phos - tag gel regular SDS - PAGE .

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(F) HeLa cells infected with control or ATG14L-expressing lentivirus were incubated under GS for different time as indicated. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( G ) HeLa were infected by lentivirus expressing control shRNA or ATG14L - specific shRNA . Then ATG14L knockdown were stably re - expressed with shRNA - resistant ATG14L ( rATG14L ) or Beclin1 incubated under NM or GS for 4 h . Whole lysates were analyzed by both Phos - tag gel regular SDS - PAGE .

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

(G) HeLa cells were infected by lentivirus expressing control shRNA or ATG14L-specific shRNA. Then the ATG14L knockdown cells were stably re-expressed with shRNA-resistant ATG14L (rATG14L) or Beclin1 and incubated under NM or GS for 4 h. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( A ) purified His - tagged WT or T32A NH2 - terminal 71aa of PAQR3 was incubated with or without Flag - tagged ATG14L purified from HEK293T . Then complexes were incubated with AMPK for indicated time subjected to Phos - tag gel or regular SDS - PAGE .

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

(A) Bacterial purified His-tagged WT or T32A NH2-terminal 71aa of PAQR3 was incubated with or without Flag-tagged ATG14L purified from HEK293T cells. Then the complexes were incubated with AMPK for the indicated time and subjected to Phos-tag gel or regular SDS-PAGE.


( B ) PAQR3 - deficient HeLa were transfected with WT or T32A mutants of PAQR3 plasmids . 24 h later , were incubated with normal medium ( NM ) or glucose starvation ( GS ) for 4 h , followed by immunoblotting ( IB ) in Phos - tag gel or regular SDS - PAGE .

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(B) PAQR3-deficient HeLa cells were transfected with WT or T32A mutants of PAQR3 plasmids. 24 h later, the cells were incubated with normal medium (NM) or glucose starvation (GS) for 4 h, followed by immunoblotting (IB) in Phos-tag gel or regular SDS-PAGE.


( C ) HeLa were incubated with NM or GS for 1 h or treated with compound C ( C . C , 20 mM , 30 min ) prior to GS for 1 h . In parallel , 25 mM 2 - DG or 1 mM AICAR was added in NM for 1 h . lysates were analyzed by IB .

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(C) HeLa Cells were incubated with NM or GS for 1 h or treated with compound C (C.C, 20 mM, 30 min) prior to GS for 1 h. In parallel, 25 mM 2-DG or 1 mM AICAR was added in NM for 1 h. Cell lysates were analyzed by IB.


( D ) HEK293T were transfected with Myc - tagged PAQR3 . 24 h after transfection , were treated with GS for 4 h . Then lysates were used in IB immunoprecipitation ( IP ) with antibodies as indicated . immunoprecipitates were treated with CIP to remove phosphate groups analyzed by IB in Phos - tag gel or regular SDS - PAGE .

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

(D) HEK293T cells were transfected with Myc-tagged PAQR3. 24 h after transfection, the cells were treated with GS for 4 h. Then the cell lysates were used in IB and immunoprecipitation (IP) with the antibodies as indicated. The immunoprecipitates were treated with CIP to remove phosphate groups and analyzed by IB in Phos-tag gel or regular SDS-PAGE.


( E ) WT , phosphorylation defective ( T32A ) , or phospho - mimetic ( T32E ) PAQR3 were transfected in presence or absence of constitutively active AMPK ( CA - AMPK ) into PAQR3 - deficient HeLa as indicated . 24 h later , without AMPK transfection were treated with GS for 4 h . 10 % of lysates were subjected to IB to detect PAQR3 expression level ( shown in Appendix Fig S4A ) , remaining lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody , followed by VPS34 activity measurement by a quantitative PI ( 3 ) P ELISA . PI ( 3 ) P level was normalized to amount of ATG14L ( n = 5 ; * p < 0 . 05 ; * * p < 0 . 01 as compared to first group ) .

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

(E) WT, phosphorylation defective (T32A), or phospho-mimetic (T32E) PAQR3 were transfected in the presence or absence of constitutively active AMPK (CA-AMPK) into PAQR3-deficient HeLa cells as indicated. 24 h later, the cells without AMPK transfection were treated with GS for 4 h. 10% of the cell lysates were subjected to IB to detect PAQR3 expression level (shown in Appendix Fig S4A), the remaining cell lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody, followed by VPS34 activity measurement by a quantitative PI(3)P ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; *p < 0.05; **p < 0.01 as compared to the first group).


( F ) PAQR3 - deficient HeLa were infected with lentivirus expressing WT or T32A PAQR3 . Then PAQR3 expression were examined by IB .

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(F) PAQR3-deficient HeLa cells were infected with lentivirus expressing WT or T32A PAQR3. Then PAQR3 expression levels were examined by IB.


( G ) Both WT HeLa PAQR3 knockout HeLa infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively . lysates were analyzed by IB .

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(G) Both WT HeLa cells and PAQR3 knockout HeLa cells infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively. Cell lysates were analyzed by IB.


( A , B ) Representative immunohistochemical of LC3 in skeletal muscle ( A ) liver ( B ) after 80 min exercise in WT PAQR3 - deleted mice . Scale bar in ( A ) : 20 μm . Scale bar in ( B ) : 50 μm .

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(A, B) Representative immunohistochemical images of LC3 staining in skeletal muscle (A) and liver (B) after 80 min exercise in WT and PAQR3-deleted mice. Scale bar in (A): 20 μm. Scale bar in (B): 50 μm.


( C , D ) Immunoblotting of skeletal muscle ( C ) liver ( D ) from WT or PAQR3 knockout mice at or after 80 min exercise .

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(C, D) Immunoblotting analysis of skeletal muscle (C) and liver (D) from WT or PAQR3 knockout mice at rest or after 80 min exercise.


( E ) Abnormal limb - clasping of PAQR3 knockout mice when suspended by its tail . scoring of limb clasping phenotype is shown on right ( n = 7 for each group ; * * p < 0 . 01 ) .

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

(E) Abnormal limb-clasping of PAQR3 knockout mice when suspended by its tail. Quantification scoring of the limb clasping phenotype is shown on the right (n = 7 for each group; **p < 0.01).


( F ) Motor performance was measured using an accelerating rotarod apparatus , time of each mouse spent on rotating rod until it fell off was recorded . Average time on rod for each group is shown ( n = 7 for each group ; * * p < 0 . 01 ) .

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(F) Motor performance was measured using an accelerating rotarod apparatus, and the time of each mouse spent on the rotating rod until it fell off was recorded. Average time on the rod for each group is shown (n = 7 for each group; **p < 0.01).


( G ) Co - immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype . Immunoprecipitation ( IP ) immunoblotting ( IB ) were performed using antibodies as indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O B-ORGANISM O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O

(G) Co-immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype. Immunoprecipitation (IP) and immunoblotting (IB) were performed using the antibodies as indicated.


( H ) supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration , followed by IB .

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(H) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis, followed by IB.


( I ) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI ( 3 ) P ELISA ( n = 5 ; * * p < 0 . 01 ) .

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

(I) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI(3)P ELISA assay (n = 5; **p < 0.01).


A . Labeling of non - permeabilized ( top ) permeabilized ( bottom ) demonstrate that surface labeling does not label internal . GluA2 labeling ( green ) is visible both on surface internally , while DARPP32 labeling ( red ) is only visible internally .

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

A. Labeling of non-permeabilized (top) and permeabilized (bottom) cells demonstrate that surface labeling does not label internal proteins. GluA2 labeling (green) is visible both on the surface and internally, while DARPP32 labeling (red) is only visible internally.


B . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no treatment or 20 min 2APB dantrolene . Bottom . Quantified expression of GluA2 under both conditions , n = 9 replicates of 10 each , Student ' s t .

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

B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student's t test.