PMN were treated with 1 µM PPP ( blue ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Graph shows average velocity of HcT - containing organelles , after PPP treatment compared to controls ( DMSO : 138 endosomes , 24 axons , 6 , 433 movements ; PPP : 130 endosomes , 23 axons , 5 , 222 movements ) ( * * P = 0 . 0061 , Student ' s t - , N = 4 independent experiments , boxplot shows median , first third quartiles . Upper / lower whiskers extend to 1 . 5 * interquartile range ) . of time HcT - containing organelles pausing per axon . There was no difference in pausing between PPP - treated neurons controls ( P = 0 . 45 , Student ' s T - , N = 4 independent experiments data shown are mean ± ) .

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

PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles, after PPP treatment compared to controls (DMSO: 138 endosomes, 24 axons, 6,433 movements; PPP: 130 endosomes, 23 axons, 5,222 movements) (** P= 0.0061, Student's t-test, N=4 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. There was no difference in pausing between PPP-treated neurons and controls (P=0.45, Student's T-test, N=4 independent experiments data shown are mean ± SEM).


PMN were treated with 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Graph shows average velocity of HcT - containing organelles after IGF1 treatment compared to control ( water : 138 endosomes , 22 axons , 5 , 119 movements ; IGF1 : 106 endosomes , 20 axons , 5 , 202 movements ; * * * P = 2 . 83x10 - 5 , Student ' s T - , N = 3 independent experiments , boxplot shows median , first third quartiles . Upper / lower whiskers extend to 1 . 5 * interquartile range ) . of time HcT - containing organelles spent pausing per axon . IGF1 significantly increased pausing time of HcT - containing organelles ( * P = 0 . 011 , Student ' s T - , N = 3 independent experiments , data shown are mean ± ) .

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

PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles after IGF1 treatment compared to control (water: 138 endosomes, 22 axons, 5,119 movements; IGF1: 106 endosomes, 20 axons, 5,202 movements; *** P=2.83x10-5, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles spent pausing per axon. IGF1 significantly increased the pausing time of HcT-containing organelles (* P=0.011, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).


PMN were treated with 1 µM PPP ( blue ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Speed distribution profile of PPP - treated neurons controls . PPP caused an increase in instantaneous velocities of HcT - containing organelles ( N = 3 independent experiments , data shown are mean ± ) .

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 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

PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of PPP-treated neurons and controls. PPP caused an increase in instantaneous velocities of HcT-containing organelles (N=3 independent experiments, data shown are mean ± SEM).


PMN were treated with 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Speed distribution profile of IGF1 - treated neurons controls . IGF1 caused a decrease in instantaneous velocities of HcT - containing organelles compared to controls ( N = 3 independent experiments , data shown are mean ± ) .

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

PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of IGF1-treated neurons and controls. IGF1 caused a decrease in instantaneous velocities of HcT-containing organelles compared to controls (N=3 independent experiments , data shown are mean ± SEM).


Western blot showing of pAKT ( Ser473 ) , total Akt , pErk1 / 2 ( T202 / Y204 ) total Erk1 / 2 after treatment with 1 µM PPP or 50 ng / ml IGF1 for 60 min . was normalised to GAPDH . Western blot . PMN treated with PPP showed a significant decrease in Akt activation ( * P = 0 . 049 , N = 5 independent experiments , Student ' s T - ) , but no significant difference was seen on Erk1 / 2 activation ( P = 0 . 07 , Student ' s T - , N = 5 independent experiments ) . 50 ng / ml IGF1 treated neurons showed a significant increase in Akt activation ( * * P = 0 . 008 , Student ' s T - , N = 3 independent experiments ) . In contrast , no change in Erk1 / 2 activation ( P = 0 . 88 , Student ' s T - , N = 3 independent experiments ) was observed ( data shown are mean ± ) .

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

Western blot showing the levels of pAKT (Ser473), total Akt, pErk1/2 (T202/Y204) and total Erk1/2 after treatment with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Protein was normalised to GAPDH. Western blot quantification. PMN treated with PPP showed a significant decrease in Akt activation (* P=0.049, N=5 independent experiments, Student's T-test), but no significant difference was seen on Erk1/2 activation (P=0.07, Student's T-test, N=5 independent experiments). 50 ng/ml IGF1 treated neurons showed a significant increase in Akt activation (** P=0.008, Student's T-test, N=3 independent experiments). In contrast, no change in Erk1/2 activation (P=0.88, Student's T-test, N=3 independent experiments) was observed (data shown are mean ± SEM).


Graph shows average velocity of HcT - containing organelles treated with Capivasertib or Ipatasertib ( 10 nM ) for 30 min compared to controls ( DMSO : 483 endosomes , 34 axons , 41 , 897 movements ; Capivasertib : 658 endosomes , 43 axons , 49 , 841 movements ; Ipatasertib : 605 endosomes , 32 axons , 41 , 485 movements ) ( * * P = 2 x10 - 16 , One - way ANOVA , Tukey ' s post - hoc : DMSO - Capivasertib DMSO - Ipatasertib * * * P < 0 . 0001 ; N = 6 independent experiments , boxplot shows median , first third quartiles . Upper / lower whiskers extend to 1 . 5 * interquartile range ) . of time HcT - containing organelles pausing per axon . Treatment with Ipatasertib Capivasertib reduced amount of time organelles spent pausing ( * P = 1 . 14 x10 - 7 , One - way ANOVA , Tukey ' s post - hoc : DMSO - Capivasertib P = 9 . 37x10 - 5 , DMSO - Ipatasertib P = 2 x10 - 7 ; data shown are mean ± , N = 6 independent experiments ) .

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

Graph shows the average velocity of HcT-containing organelles treated with Capivasertib or Ipatasertib (10 nM) for 30 min compared to controls (DMSO: 483 endosomes, 34 axons, 41,897 movements; Capivasertib: 658 endosomes, 43 axons, 49,841 movements; Ipatasertib: 605 endosomes, 32 axons, 41,485 movements) (** P=2 x10-16, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib and DMSO-Ipatasertib *** P <0.0001; N=6 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing (* P=1.14 x10-7, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib P= 9.37x10-5, DMSO-Ipatasertib P= 2 x10-7; data shown are mean ± SEM, N=6 independent experiments).


PMN were treated with either an shRNA targeting IGF1R ( 1 µl ) or a scrambled control 6 h after plating . At DIV 6 - 7 , retrograde axonal transport was analysed using 30 nM AlexaFluor555 - HcT . Knockdown of IGF1R caused a significant increase in average velocity of signalling endosomes compared to controls ( IGF1R shRNA : 108 endosomes , 21 axons , 801 movements ; scrambled shRNA : 94 endosomes , 20 axons , 777 movements , * P = 0 . 011 , Student ' s T - , N = 3 independent experiments , boxplot shows median , first third quartiles . Upper / lower whiskers extend to 1 . 5 * interquartile range ) . Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control . IGF1R knockdown caused an increase in instantaneous velocities of HcT - containing organelles decreased amount of pausing ( N = 3 independent experiments , data shown are mean ± ) .

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

PMN were treated with either an shRNA targeting IGF1R (1 µl) or a scrambled control 6 h after plating. At DIV 6-7, retrograde axonal transport was analysed using 30 nM AlexaFluor555-HcT. Knockdown of IGF1R caused a significant increase in the average velocity of signalling endosomes compared to controls (IGF1R shRNA: 108 endosomes, 21 axons, 801 movements; scrambled shRNA: 94 endosomes, 20 axons, 777 movements, * P=0.011, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control. IGF1R knockdown caused an increase in instantaneous velocities of HcT-containing organelles and decreased the amount of pausing (N=3 independent experiments, data shown are mean ± SEM).


Western blot of PMN treated with 1 µM PPP or 50 ng / ml IGF1 for 60 min . were lysed extracts assessed for expression of dynein adaptor , were assessed from same blot . Hook3 expression didn ' t change after treatment with PPP or IGF1 compared to controls ( DMSO , 100 % ; PPP , 102 . 16 ± 4 . 3 % , P = 0 . 67 , Student ' s T - , N = 3 independent experiments ; water , 100 % ; IGF1 , 93 . 48 ± 4 . 2 % , P = 0 . 26 , N = 3 independent experiments , data shown are mean ± ) . However , BICD1 expression was significantly increased in PMN treated with PPP compared to control ( DMSO , 100 % ; PPP , 137 . 1 ± 7 . 3 % , * * P = 0 . 002 , Student ' s T - , N = 7 independent experiments ) . In contrast , IGF1R stimulation by IGF1 had no effect on BICD1 expression ( water , 100 % ; IGF1 , 88 . 1 ± 14 . 0 % , P = 0 . 49 , Student ' s T - , N = 3 independent experiments , data shown are mean ± ) . BICD2 expression didn ' t change after treatment with PPP or IGF1 compared to controls ( DMSO , 100 % ; PPP , 104 . 44 ± 6 . 5 % , P = 0 . 51 , N = 3 independent experiments : water , 100 % ; IGF1 , 106 ± 7 . 5 % , P = 0 . 51 , Student ' s T - , N = 3 independent experiments , data shown are mean ± ) .

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

Western blot of PMN treated with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins, all proteins were assessed from the same blot. Hook3 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 102.16 ± 4.3%, P=0.67, Student's T-test, N=3 independent experiments; water, 100%; IGF1, 93.48 ± 4.2%, P=0.26, N=3 independent experiments, data shown are mean ± SEM). However, BICD1 expression was significantly increased in PMN treated with PPP compared to control (DMSO, 100%; PPP, 137.1 ± 7.3%, **P=0.002, Student's T-test, N=7 independent experiments). In contrast, IGF1R stimulation by IGF1 had no effect on BICD1 expression (water, 100%; IGF1, 88.1 ± 14.0%, P=0.49, Student's T-test, N=3 independent experiments, data shown are mean ± SEM). BICD2 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 104.44 ± 6.5%, P=0.51, N=3 independent experiments: water, 100%; IGF1, 106 ± 7.5%, P=0.51, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).


Western blot of PMN pre - treatment with cycloheximide ( 50 µg / ml ) or MG - 132 ( 10 µM ) for 30 min before incubation with 1 µM PPP for 60 min . Cycloheximide causes a significant decrease in BICD1 compared to PPP treated . ( DMSO , grey : 100 % , N = 7 independent experiments ; PPP , blue : 137 . 1 ± 7 . 3 % , N = 7 independent experiments ; CHX , pink : 89 . 5 ± 12 . 7 % , N = 5 independent experiments ; MG - 132 , teal : 124 ± 28 . 7 , N = 3 independent experiments ; P = 0 . 009 , one - way ANOVA ; Tukey ' s post - hoc : DMSO - PPP * P = 0 . 04 , PPP - PPP + CHX * P = 0 . 01 ; data shown are mean ± ) .

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

Western blot of PMN pre-treatment with cycloheximide (50 µg/ml) or MG-132 (10 µM) for 30 min before incubation with 1 µM PPP for 60 min. Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells. (DMSO, grey: 100%, N=7 independent experiments; PPP, blue: 137.1 ± 7.3%, N=7 independent experiments; CHX, pink: 89.5 ± 12.7%, N=5 independent experiments; MG-132, teal: 124 ± 28.7, N=3 independent experiments; P=0.009, one-way ANOVA; Tukey's post-hoc test: DMSO-PPP * P=0.04, PPP-PPP+CHX * P=0.01; data shown are mean ± SEM).


Puromycin - PLA signal in PMN grown in microfluidics devices . are magnifications from boxes in Appendix Fig S6 taken from axonal compartment . scale bars are 10 µm . arrows point to puromycin - PLA within β3 - tubulin mask ; empty arrow shows a PLA not associated with neurons .

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

Puromycin-PLA signal in PMN grown in microfluidics devices. Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment. The scale bars are 10 µm. White arrows point to puromycin-PLA puncta within the β3-tubulin mask; the empty arrow shows a PLA puncta not associated with neurons.


of puromycin - PLA signal . PPP causes an increase in PLA in axon of PMN , which is abolished by treatment with 40 µM anisomycin ( DMSO , grey : 100 % ; PPP , blue : 139 . 3 ± 7 . 7 % ; PPP + anisomycin , purple : 86 . 3 ± 10 . 0 ; P = 0 . 005 , one - way ANOVA , Tukey ' s post - hoc ; DMSO vs PPP , * P = 0 . 02 ; PPP vs PPP + anisomycin , * * 0 . 005 , N = 3 independent experiments , 27 - 57 , data shown are mean ± ) .

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

Quantification of puromycin-PLA signal. PPP causes an increase in PLA puncta in the axon of PMN, which is abolished by treatment with 40 µM anisomycin (DMSO, grey: 100%; PPP, blue: 139.3 ± 7.7%; PPP + anisomycin, purple: 86.3 ± 10.0; P=0.005, one-way ANOVA, Tukey's post-hoc test; DMSO vs PPP, * P=0.02; PPP vs PPP + anisomycin, ** 0.005, N=3 independent experiments, 27-57 images, data shown are mean ± SEM).


graph shows average velocity of HcT - containing organelles in vivo in D72 / 73 SOD1G93A mice wild type littermate controls . PPP ( blue ) treatment increased transport rate of signalling endosomes in wild type SOD1G93A mice compared to treated with vehicle control ( grey ) ( WT control : cargo 371 , movements 15 , 272 , N = 4 independent experiments ; WT + PPP : cargo 388 , movements 14 , 512 , N = 4 independent experiments ; SOD1G93A control : cargo 322 , movements 15 , 425 , N = 4 independent experiments ; SOD1G93A + PPP : cargo 404 , movements 17 , 643 , N = 4 independent experiments ; P = 2 . 24x10 - 12 ( treatment ) , P = 2 . 22 x10 - 16 ( genotype ) , two - way ANOVA , Tukey ' s post - hoc : conditions * * * P < 0 . 005 , boxplot shows median , first third quartiles . Upper / lower whiskers extend to 1 . 5 * interquartile range ) . PPP treatment does not alter pausing of signalling endosomes ( WT : 16 . 1 ± 1 . 5 % , WT + PPP : 13 . 1 ± 1 . 5 % ; SOD1G93A : 16 . 6 ± 1 . 9 % , SOD1G93A + PPP : 16 . 2 ± 2 . 1 % ; P = 0 . 36 ( treatment ) , P = 0 . 32 ( genotype ) , N = 4 independent experiments for each condition , data shown are mean ± .

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 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 O O O O 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 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 O O O O O O O O O O O O O O O O O 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 graph shows the average velocity of HcT-containing organelles in vivo in D72/73 SOD1G93A mice and wild type littermate controls. PPP (blue) treatment increased the transport rate of signalling endosomes in wild type and SOD1G93A mice compared to animals treated with vehicle control (grey) (WT control: cargo 371, movements 15,272, N=4 independent experiments; WT + PPP: cargo 388, movements 14,512, N=4 independent experiments; SOD1G93A control: cargo 322, movements 15,425, N=4 independent experiments; SOD1G93A + PPP: cargo 404, movements 17,643, N=4 independent experiments; P=2.24x10-12 (treatment), P=2.22 x10-16 (genotype), two-way ANOVA, Tukey's post-hoc test: all conditions *** P<0.005, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). PPP treatment does not alter the pausing of signalling endosomes (WT: 16.1 ± 1.5%, WT + PPP: 13.1 ± 1.5%; SOD1G93A: 16.6 ± 1.9%, SOD1G93A + PPP: 16.2 ± 2.1%; P=0.36 (treatment), P=0.32 (genotype), N=4 independent experiments for each condition, data shown are mean ± SEM.


Treatment with PPP had no effect on Akt in sciatic nerve ( WT : Control - 100 ± 8 . 6 , PPP - 91 . 5 ± 14 . 6 % ; SOD1 : Control - 101 . 4 ± 13 . 5 % , PPP - 117 . 6 ± 32 . 9 % , P = 0 . 81 ( treatment ) , P = 0 . 53 ( genotype ) , two - way ANOVA , N = 5 independent experiments , data shown are mean ± , Appendix Fig S7D ) . Erk1 / 2 activation was down - regulated after treatment with PPP in sciatic nerve of wild type SOD1G93A mice ( WT : control - 100 ± 15 . 4 % , PPP - 64 . 4 ± 3 . 2 % ; SOD1 : Control - 78 . 0 ± 11 . 9 % , PPP - 51 . 2 ± 8 . 2 % , * P = 0 . 013 ( treatment ) , P = 0 . 13 ( genotype ) , two - way ANOVA , N = 5 independent experiments , data shown are mean ± , Appendix Fig S7D ) . was normalised to GAPDH .

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

Treatment with PPP had no effect on Akt levels in the sciatic nerve (WT: Control-100 ± 8.6, PPP-91.5 ± 14.6%; SOD1: Control-101.4 ± 13.5%, PPP-117.6 ± 32.9%, P=0.81 (treatment), P=0.53 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D). Erk1/2 activation was down-regulated after treatment with PPP in the sciatic nerve of wild type and SOD1G93A mice (WT: control-100 ± 15.4%, PPP-64.4 ± 3.2%; SOD1: Control-78.0 ± 11.9 %, PPP-51.2 ± 8.2%, * P=0.013 (treatment), P=0.13 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D). Protein was normalised to GAPDH protein levels.


( A ) Electron of MEFs . Scale bar ; 1 μm . High - magnification depicting representative electron - dense materials are shown . Scale bar ; 0 . 2 μm .

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

(A) Electron micrographs of MEFs . Scale bar; 1 μm. High-magnification images depicting representative electron-dense materials are shown. Scale bar; 0.2 μm.


( B ) Citrate synthase activity ( absorbance / min ) was measured in MEFs . Error bars represent ± SD from eight independent experiments . Data information : For graph , P values was determined by a Mann - Whitney U . ns = not significant , * 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 O O O O O O O O O O O O O O O O O O O O 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) Citrate synthase activity (absorbance/min) was measured in MEFs . Error bars represent ±SD from eight independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( C , D ) Oxygen consumption rates ( OCRs ) of MEFs were measured on an XF24 Analyzer . ( C ) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin ( 2 μg / mL ) ( Oligo ) , FCCP ( 2 μM ) , rotenone ( 1 μM ) plus actinomycin ( 2 μM ) ( R & A ) . ( D ) OCR ( pmol O2 / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * 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 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 B-CONTROLLED_VAR 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 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

(C, D) Oxygen consumption rates (OCRs) of MEFs were measured on an XF24 Analyzer. (C) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin (2 μg/mL) (Oligo), FCCP (2 μM), and rotenone (1 μM) plus actinomycin (2 μM) (R & A). (D) OCR (pmol O2/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( E ) ATP production ( pmol / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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

(E) ATP production (pmol/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( F ) Representative immunoblot of LC3 p62 in MEFs of indicated genotype . MEFs were treated with tunicamycin ( 5 μg / ml ) or vehicle control in presence or absence of bafilomycin A1 ( 400 nM ) , endogenous LC3 p62 were measured by immunoblotting . Data represent ratios of LC3 - II to LC3 - I p62 to actin in absence of bafilomycin , which were normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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 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 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

(F) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype. MEFs were treated with tunicamycin (5 μg/ml) or vehicle control in the presence or absence of bafilomycin A1 (400 nM), and endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin in the absence of bafilomycin, which were normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( G ) Survival rate of MEFs treated with tunicamycin ( 5 μg / ml ) , thapsigargin ( 1 μM ) , or hydrogen peroxide ( 100 μM ) . Error bars represent ± SD from four independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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 B-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

(G) Survival rate of MEFs treated with tunicamycin (5 μg/ml), thapsigargin (1 μM), or hydrogen peroxide (100 μM). Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( A ) Absolute [ Ca2 + ] m changes in MEFs of indicated genotype in response to bradykinin ( 2 . 5 μM ) . ( B ) Basal peak values of Ca2 + transients ( μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (A) Absolute [Ca2+]m changes in MEFs of indicated genotype in response to bradykinin (2.5 μM). (B) Basal and peak values of Ca2+ transients (μM). Error bars represent ±SD from six independent experiments. Data information: For graph P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( C ) Peak values of Ca2 + transients in MEFs transfected with IP3R or shRNA against IP3R , or treated with 2 - AP ( 20 mM ) . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (C) Peak values of Ca2+ transients in MEFs transfected with IP3R or shRNA against IP3R, or treated with 2-AP (20 mM). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( D ) Peak values of Ca2 + transients in MEFs transfected with VDAC1 or shRNA against VDAC1 . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (D) Peak values of Ca2+ transients in MEFs transfected with VDAC1 or shRNA against VDAC1. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( E ) Representative electron of MEFs . Full arrowheads designate limits of zone of intimate contact between ER mitochondria 20 nm ) . Scale bar : 100 nm . Data represent of ERmitochondrial contacts per mitochondria perimeter . Error bars represent ± SD from 40 MEFs Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

(E) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm). Scale bar: 100 nm. Data represent percentage of ERmitochondrial contacts per mitochondria perimeter. Error bars represent ±SD from 40 images MEFs Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( F ) In situ PLA using anti - IP3R anti - VDAC1 antibodies . Scale bar : 20 μm . Data represent in MEFs normalized LRRK2 + / + MEFs . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

(F) In situ PLA images using anti-IP3R and anti-VDAC1 antibodies. Scale bar: 20 μm. Data represent fluorescent in MEFs normalized LRRK2+/+ MEFs. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( G ) Peak values of Ca2 + transients in MEFs transfected with synthetic tethering ( TOM - mRFP - ER ) to induce artificial tethering of ER mitochondria . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O 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 O O O O O O O O O O O O O O O O O O O O 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) Peak values of Ca2+ transients in MEFs transfected with synthetic tethering protein (TOM-mRFP-ER) to induce artificial tethering of the ER and mitochondria. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Representative immunoblots of MAM components in MEFs of indicated genotype . MAM fraction was extracted from MEFs by Percoll gradient method immunoblotted with antibodies indicated at right .

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

(A) Representative immunoblots of MAM components in MEFs of indicated genotype. MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right.


( C ) Peak values of Ca2 + transients in MEFs transfected with deletion constructs of LRRK2 ( G2019S ) . Error bars represent ± SD from six independent experiments . Data information : For graph , P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 .

O O O O O 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 O O O O 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) Peak values of Ca2+ transients in MEFs transfected with deletion constructs of LRRK2(G2019S). Error bars represent ±SD from six independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( D ) Binding assays to detect interactions between deletion constructs of LRRK2 ( G2019S ) E3 ubiquitin ligases . HEK293 were transfected with deletion constructs of LRRK2 ( G2019S ) E3 ubiquitin ligases , lysates were immunoprecipitated with antibody against V5 or HA epitope . Precipitated were subjected to SDS / PAGE , blots were stained with antibody against V5 or HA epitope , as indicated right of each panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding assays to detect interactions between deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases. HEK293 cells were transfected with deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases, and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the V5 or HA epitope, as indicated the right of each panel.


Peak values of Ca2 + transients in MEFs transfected with ligase - active MARCH5 ( WT ) , MULAN ( WT ) , Parkin ( W403A ) [ Parkin ( WA ) ] or dominant - negative forms of MARCH5 ( H43W ) [ MARCH ( HW ) ] , MULAN ( C339A ) [ MULAN ( CA ) ] , Parkin ( C431A ) [ Parkin ( CA ) ] ( E ) Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 .

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

Peak values of Ca2+ transients in MEFs transfected with ligase-active MARCH5(WT), MULAN(WT), and Parkin(W403A) [Parkin(WA)] or dominant-negative forms of MARCH5(H43W) [MARCH(HW)], MULAN(C339A) [MULAN(CA)], Parkin(C431A) [Parkin(CA)] (E) Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


Peak values of Ca2 + transients in MEFs transfected with active USP30 or inactive USP30 ( C77S ) [ USP ( CS ) ] ( F ) . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 .

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

Peak values of Ca2+ transients in MEFs transfected with active USP30 or inactive USP30(C77S) [USP(CS)] (F). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( A ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2 + / + LRRK2 ( G2019S ) - expressing MEFs treated with vehicle ( Control ) or tunicamycin ( 1 μg / ml ) . Endogenous PERK , MARCH5 , MULAN Parkin were immunoprecipitated with antibody , precipitates were immunoblotted with antibody indicated at right . Data represent of phosphorylated to total . Error bars represent ± SD from independent experiments . Data information : For graph A P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

(A) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2+/+ and LRRK2(G2019S)-expressing MEFs treated with vehicle (Control) or tunicamycin (1 μg/ml). Endogenous PERK, MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Data represent the ratio of phosphorylated to total protein levels. Error bars represent ±SD from independent experiments. Data information: For graph A the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) Immunoprecipitation / immunoblot of phosphorylated E3 ubiquitin ligases mitofusin 2 in MEFs in of tunicamycin ( 5 μg / ml ) . LRRK2 ( G2019S ) MEFs were also pretreated with LRRK2 - IN - 1 ( 1 μM ) . MEFs were transfected with each E3 ubiquitin ligase biquitin lysates were immunoprecipitated with antibody against Myc or HA epitope . Precipitated were subjected to SDS / PAGE , blots were stained with antibody against Myc epitope , phosphoserine or mitofin2 as indicated to right of each panel . Mfn2 : mitofusin 2 , Ub : ubiquitin . Data represent ratios of phosphorylated to total E3 ubiquitin ligase mitofusin2 to actin . Error bars represent ± SD from four independent experiments . Data information : For graph B , P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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 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 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

(B) Immunoprecipitation/immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin (5 μg/ml). LRRK2(G2019S) MEFs were also pretreated with LRRK2-IN-1 (1 μM). MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the Myc epitope, phosphoserine or mitofin2 as indicated to the right of each panel. Mfn2: mitofusin 2, Ub: ubiquitin. Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin. Error bars represent ±SD from four independent experiments. Data information: For graph B, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Peak values of Ca2 + transients in MEFs treated with LRRK2 - IN - 1 ( 1 μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph A , P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

(A) Peak values of Ca2+ transients in MEFs treated with LRRK2-IN-1 (1 μM). Error bars represent ±SD from six independent experiments. Data information: For graph A , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) ( Upper panel ) Diagram showing full - PERK Myc at N - terminus FLAG at C - terminus ( M - PERK - F ) . S1P recognition R33SLL is mutated to A33SLL ( M - PERK ( R33A ) - F ) . ( Lower panel ) Immunoblot of PERK PERK ( R33A ) from transfected MEFs under tunicamycin . MAM fraction cytosol were extracted from transfected MEFs by Percoll gradient method . Lysates were immunoprecipitated with antibody against FLAG . Precipitated were subjected to SDS / PAGE , blots were stained with antibody as indicated to right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)(Upper panel) Diagram showing full-length PERK Myc at the N-terminus and FLAG at the C-terminus (M-PERK-F). S1P recognition sequence R33SLL is mutated to A33SLL (M-PERK(R33A)-F). (Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against FLAG. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right.


( C ) In vitro kinase using isolated PERK , isolated E3 ubiquitin ligase [ γ - 32P ] ATP . Reaction mixture was subjected to SDS / PAGE . Blots were with antibody against Myc , FLAG or phosphoserine . [ γ - 32P ] ATP - incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , right blot for 36 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase and [γ-32P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were with antibody against Myc, FLAG or phosphoserine. [γ-32P]ATP-incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ).


( D ) In vitro ubiquitination using phosphorylated E3 ubiquitin ligases , HA - tagged Ubl , His - tagged mitofusin2 in presence of E1 enzyme , UbcH7 ATP . E3 ubiquitin ligases were initially phosphorylated PERK or PERK ( K618R ) in vitro . Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination . itofusin 2 with Ni - NTA subjected to SDS / PAGE . Blots were with antibody against HA or mitofusin2 . Mfn2 : mitofusin 2 , Ub : ubiquitin .

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

(D) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA-tagged Ubl, and His-tagged mitofusin2 in the presence of E1 enzyme, UbcH7 and ATP. E3 ubiquitin ligases were initially phosphorylated PERK or PERK(K618R) in vitro. Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination. itofusin 2 with Ni-NTA subjected to SDS/PAGE. Blots were with antibody against HA or mitofusin2. Mfn2: mitofusin 2, Ub: ubiquitin.


( E ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases mitofusin 2 in LRRK2 ( G2019S ) - expressing MEFs transfected with increasing amounts of LRRK2 - d1 - V5 ( 1 , 5 , 25 μg / 106 ) treated with tunicamycin ( 1 μg / ml ) . Endogenous MARCH5 , MULAN Parkin were immunoprecipitated with antibody , precipitates were immunoblotted with antibody indicated at right . Endogenous mitofusin 2 was immunoblotted with anti - mitofusin 2 antibody . Data represent of phosphorylated to total of MARCH5 , MULAN Parkin , of mitofusin 2 to actin . Error bars represent ± SD from independent experiments . Data information : For graph E , P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)-expressing MEFs transfected with the increasing amounts of LRRK2-d1-V5 (1, 5, 25 μg/106 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti-mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ±SD from independent experiments. Data information: For graph E, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK binding to mutant LRRK2 . HEK293 were transfected with PERK either MARCH5 , MULAN or Parkin , LRRK2 , LRRK2 ( D1994A ) or LRRK2 ( S1292D ) . lysates were immunoprecipitated with antibody against Myc or V5 , precipitates were immunoblotted with antibody indicated right .

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

(A) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2. HEK293 cells were transfected with PERK and either MARCH5, MULAN or Parkin, LRRK2, LRRK2(D1994A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc or V5, and precipitates were immunoblotted with antibody indicated the right.


( B ) In vitro kinase for auto - phosphorylated LRRK2 using isolated LRRK2 mutants [ γ - 32P ] ATP with or without LRRK2 - IN - 1 ( 1 μM ) . Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS / PAGE . Blots were with antibody against V5 or phosphoserine . [ γ - 32P ] ATP - incorporated LRRK2 visualized by autoradiography .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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 kinase assay for auto-phosphorylated LRRK2 using isolated LRRK2 mutants and [γ-32P]ATP with or without LRRK2-IN-1 (1 μM). Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS/PAGE. Blots were with antibody against V5 or phosphoserine. [γ-32P]ATP-incorporated LRRK2 visualized by autoradiography.


( C ) Binding assays to detect interactions between phosphomimetic LRRK2 ( S1292D ) or phosphorylation - defective LRRK2 ( S1292A ) E3 ubiquitin ligases . HEK293 were transfected with LRRK2 mutants E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope . Precipitated were subjected to SDS / PAGE , for immunoblotwith antibody against V5 or Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding assays to detect interactions between phosphomimetic LRRK2(S1292D) or phosphorylation-defective LRRK2(S1292A) and E3 ubiquitin ligases. HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope. Precipitated proteins were subjected to SDS/PAGE, for immunoblotwith antibody against V5 or Myc.


( D ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK - ΔN in presence of LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . HEK293 were transfected with MARCH5 , MULAN , Parkin , PERK - ΔN LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . lysates were immunoprecipitated with antibody against Myc , precipitates were immunoblotted with antibody indicated right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK-ΔN in the presence of LRRK2(S1292A) or LRRK2(S1292D). HEK293 cells were transfected with MARCH5, MULAN, Parkin, and PERK-ΔN LRRK2(S1292A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc, and precipitates were immunoblotted with antibody indicated the right.


( A ) Immunoblot of LC3 p62 . MEFs were transfected with PERK - ΔN or PERK shRNA . Endogenous LC3 p62 were measured by immunoblotting . Data represent ratios of LC3 - II to LC3 - I p62 to actin normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph A , P values was determined by a Mann - Whitney U . ns = not significant , * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of LC3 and p62. MEFs were transfected with PERK-ΔN or PERK shRNA. Endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph A, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05.


( B ) Peak values of Ca2 + transients in MEFs transfected with PERK - ΔN or PERK shRNA . Error bars represent ± SD from six independent experiments .

O O O O O 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) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments.


Left panel , a scheme of chromosome 18 copy number alterations depicting distal long arm loss across TCGA Pan - Cancer dataset . Vertical red indicates localization of VPS4B . Right panel , enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples . Deletions are marked in blue , amplified regions are marked in red . of VPS4B copy number alterations in TCGA Pan - Cancer dataset . Cancer types were sorted according to mean VPS4B copy number after removing germline values .

Left panel, a scheme of chromosome 18 copy number alterations depicting the distal long arm loss across TCGA Pan-Cancer dataset. Vertical red line indicates the localization of VPS4B. Right panel, enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples. Deletions are marked in blue, amplified regions are marked in red. Analysis of VPS4B copy number alterations in TCGA Pan-Cancer dataset. Cancer types were sorted according to the mean VPS4B copy number after removing germline values.


Scatter plot , of VPS4B mRNA expression ( number of transcripts per million ) plotted against VPS4B copy number from TCGA CRC samples ( n = 376 ) ; plot generated using cBioPortal Gao et al , 2013 ( ) . Pie chart , summary of types of VPS4B copy number alterations on of data from 615 CRC samples deposited in Colorectal Adenocarcinoma ( TCGA , Provisional )

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

Scatter plot, analysis of VPS4B mRNA expression (number of transcripts per million) plotted against VPS4B copy number from TCGA CRC patient samples (n=376); plot generated using cBioPortal Gao et al, 2013(). Pie chart, summary of all types of VPS4B copy number alterations based on the analysis of data from 615 CRC samples deposited in the Colorectal Adenocarcinoma (TCGA, Provisional)


qPCR of VPS4B mRNA abundance in normal colon , adenoma CRC samples . Adenocarcinoma ( n = 26 ) ; adenoma ( n = 42 ) ; normal colon ( n = 24 ) . Green horizontal bars indicate means red whiskers indicate SD .

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

qPCR analysis of VPS4B mRNA abundance in normal colon, adenoma and CRC samples. Adenocarcinoma (n=26); adenoma (n=42); normal colon (n=24). Green horizontal bars indicate means and red whiskers indicate SD.


Examples of immunohistochemical of VPS4B in normal colon matched CRC samples as an illustration of scoring system used for evaluation presented in ( F ) . 3 + - very intensive , 2 + - medium - intensive , 1 + - weak , 0 - no . Scale bar , 50 µm .

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

Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in (F). 3+ - very intensive staining, 2+ - medium-intensive staining, 1+ - weak staining, 0 - no staining. Scale bar, 50 µm.


of viability of HCT116 assessed 96 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 1 or # 2 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± . values were normalized , averaged ( avrg ) viability of siCTRL # 1 # 2 - transfected was set as 100 % , Kruskal - Wallis followed by Dunn ' s multiple comparison post hoc ; ns - non - significant ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #1 or #2), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%, the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test; ns - non-significant (p≥0.05), **p<0.01.


Left panel , of clonogenic growth of HCT116 assessed 15 days after transfection with non - targeting ( siCTRL # 1 ) , VPS4A - or / VPS4B - targeting siRNAs used in various duplex combinations as indicated . Right panel , of HCT116 clones taken at day of clonogenic growth assessment . Data are means of 3 independent experiments ± . NT - non - transfected . values were normalized , clonogenic growth of siCTRL # 1 - transfected was set as 1 . One - sample t - ; ns - non - significant ( p ≥ 0 . 05 ) , * * * * 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

Left panel, analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non-targeting (siCTRL#1), VPS4A- or/and VPS4B-targeting siRNAs used in various duplex combinations as indicated. Right panel, images of HCT116 clones taken at the day of clonogenic growth assessment. Data are means of 3 independent experiments ±SEM. NT - non-transfected. All values were normalized, clonogenic growth of siCTRL#1-transfected cells was set as 1. One-sample t-test; ns - non-significant (p≥0.05), ****p<0.0001.


Viability of RKO SW480 assessed as in ( A ) , growth of DLD - 1 assessed in BrdU incorporation 96 h after siRNA transfection . Various independent non - targeting ( siCTRL # 1 or # 2 ) VPS4A - or B - targeting siRNA duplexes were used ( # 1 , # 2 , # 5 or # 1 , # 2 , respectively ) . values were normalized , averaged ( avrg ) viability of siCTRL # 1 # 2 - transfected was set as 100 % . Data are means of 3 independent experiments ± . Kruskal - Wallis followed by Dunn ' s multiple comparison post hoc ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Viability of RKO and SW480 cells assessed as in (A), growth of DLD-1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection. Various independent non-targeting (siCTRL#1 or #2) and VPS4A- or B-targeting siRNA duplexes were used (#1, #2, #5 or #1, #2, respectively). All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%. Data are means of 3 independent experiments ±SEM. The Kruskal-Wallis test followed by Dunn's multiple comparison post hoc; ns - non-significant (p≥0.05), **p<0.01, ***p<0.001.


VPS4B copy number dependency scores of selected cancer lines obtained in CRISPR / Cas9 RNAi screens deposited in DepMap portal ( https : / / depmap . org / portal / ) .

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

VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR/Cas9 and RNAi screens deposited in the DepMap portal (https://depmap.org/portal/).


VPS4B copy number status estimated across different lines using TaqMan . whiskers represent minimal maximal copy number in a given triplicate readout . BCL2 gene localized on 18q in close vicinity to VPS4B was analyzed as a control .

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

VPS4B copy number status estimated across different cell lines using TaqMan assay. The whiskers represent the minimal and maximal copy number in a given triplicate readout. The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control.


VPS4B VPS4A abundance in selected normal cancer lines analyzed by immunoblotting . Vinculin GAPDH were used as loading controls .

VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting. Vinculin and GAPDH were used as loading controls.


of viability of HOP62 lung cancer assessed 144 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 2 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 4 independent experiments ± . values were normalized , viability of averaged ( avrg ) siCTRL # 1 - # 2 - transfected was set as 100 % . Two - unpaired t - ; * * * * 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

Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #2 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 4 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1- and #2-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.


Top panel , of clonogenic growth of HOP62 assessed 14 days after transfection with various siRNA duplexes as indicated . Bottom panel , of HOP62 clones taken at day of clonogenic growth assessment . Data are means of 4 independent experiments ± . values were normalized , colony of averaged ( avrg ) siCTRL # 1 # 2 - transfected was set as 1 . Mann - Whitney U ; * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Top panel, analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated. Bottom panel, images of HOP62 clones taken at the day of clonogenic growth assessment. Data are means of 4 independent experiments ±SEM. All values were normalized, colony area of averaged (avrg) siCTRL#1 and #2-transfected cells was set as 1. The Mann-Whitney U test; **p<0.01.


of viability of SNU410 pancreatic cancer assessed 168 h after transfection with independent non - targeting siRNA ( three different duplexes used , siCTRL # 1 , # 2 or # 3 ) or targeting VPS4A ( duplexes # 2 , # 4 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± . values were normalized , viability of averaged ( avrg ) siCTRL # 1 - , # 2 - # 3 - transfected was set as 100 % . Two - unpaired t - ; * * * * 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

Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non-targeting siRNA (three different duplexes used, siCTRL#1, #2 or #3) or targeting VPS4A (duplexes #2, #4 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1-, #2- and #3-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.


Left panel , growth of HCT116 VPS4B - / - shVPS4A as xenografts in mice in presence or absence of doxycycline . Day 1 indicates first day of doxycycline administration . n = 9 for each group , each mouse bearing one , ± . Two - unpaired t - ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 . Right panel , scatter plot representing end - point volumes of single xenografts . Bars represent means ± .

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

Left panel, growth of HCT116 VPS4B-/- shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n=9 for each group, each mouse bearing one tumor, ±SEM. Two-tailed unpaired t-test; ns - non-significant (p≥0.05), **p<0.01. Right panel, scatter plot representing end-point volumes of single xenografts. Bars represent means ±SEM.


Immunoblotting of VPS4A abundance in xenograft samples from untreated doxycycline - treated mice ( 6 separate xenograft samples for each group ) . Lysates of HCT116 VPS4B - / - transfected with control or VPS4A - targeting siRNA marked VPS4A detection . GAPDH served as a loading control .

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

Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline-treated mice (6 separate xenograft samples for each group). Lysates of HCT116 VPS4B-/- cells transfected with control or VPS4A-targeting siRNA marked VPS4A protein detection. GAPDH served as a loading control.


Growth of HCT116 VPS4B - / - shCTRL # 1 as xenografts in mice in presence or absence of doxycycline . arrow indicates first day of doxycycline ( Dox ) administration . n = 2 mice for each group , each mouse bearing two tumors , ± .

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

Growth of HCT116 VPS4B-/- shCTRL#1 cells as xenografts in mice in the presence or absence of doxycycline. The arrow indicates the first day of doxycycline (Dox) administration. n=2 mice for each group, each mouse bearing two tumors, ±SEM.


visualizing expression of genes related to inflammatory response ( left panel ) positive regulation of ( right panel ) generated from GO of biological processes across different transfection conditions with at least 3 biological replicates .

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

Heatmap visualizing expression of genes related to inflammatory response (left panel) and positive regulation of cell death (right panel) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates.


Immunoblotting of canonical noncanonical branches of NF - κB pathway mediators of caspase - dependent . Lysates of HCT116 VPS4B - / - were collected 66 h after transfection with siRNA ( siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . Lysates of HCT116 VPS4B + / + non - transfected HCT116 VPS4B - / - were used to monitor basal pathway activity . Representative blot from 10 experiments is shown . NT - non - transfected ; p - RelA - phospho - RelA ; p - IκBα - phospho - IκBα ; cl - caspases or PARP - 1 . GAPDH or vinculin served as loading controls .

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

Immunoblotting analysis of the canonical and noncanonical branches of the NF-κB pathway and mediators of caspase-dependent cell death. Lysates of HCT116 VPS4B-/- cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Lysates of HCT116 VPS4B+/+ and non-transfected HCT116 VPS4B-/- cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT- non-transfected; p-RelA - phospho-RelA; p-IκBα - phospho-IκBα; cl - cleaved caspases or PARP-1. GAPDH or vinculin served as loading controls.


Densitometry of abundance of indicated on immunoblot as shown in ( A ) . Data are means of 10 ( phospho - Rel caspase 7 ) , 9 ( p52 , caspase 8 caspase 9 ) , 7 ( phosphorylated total IκB ) or 5 ( caspase 3 ) independent experiments . Error bars are . Statistical significance was assessed using following : one - sample t - ( caspase 8 , caspases 9 , 7 3 , total phospho - IκBα ) or Wilcoxon signed rank ( phospho - RelA p52 ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho-Rel and cleaved caspase 7), 9 (p52, caspase 8 and cleaved caspase 9), 7 (phosphorylated and total IκB) or 5 (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one-sample t-test (caspase 8, cleaved caspases 9, 7 and 3, total and phospho-IκBα) or Wilcoxon signed rank test (phospho-RelA and p52); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Immunoblot showing induction of NF - κB pathway ( p52 ) apoptosis ( caspase 7 ) in xenograft samples described in Fig 3C . cl - caspase . Vinculin was used as a loading control .

Immunoblot showing induction of the NF-κB pathway (p52) and apoptosis (cleaved caspase 7) in xenograft samples described in Fig 3C. cl - cleaved caspase. Vinculin was used as a loading control.


of impact of RIPK1 inhibitor ( necrostatin - 1 ) or pan - caspase inhibitor ( Q - VD - Oph ) on viability of HCT116 VPS4B - / - transfected with siRNA ( non - targeting siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . viability was assessed 72 h after siRNA transfection . Necrostatin - 1 ( 50 µM ) , Q - VP - Oph ( 20 µM ) or vehicle were added to medium 48 h before viability assessment . Data are means of 5 independent experiments ± . values were normalized , viability of siCTRL - transfected vehicle - treated was set as 100 % . Two - unpaired t - ; * * p < 0 . 01 .

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

Analysis of the impact of RIPK1 inhibitor (necrostatin-1) or pan-caspase inhibitor (Q-VD-Oph) on cell viability of HCT116 VPS4B-/- cells transfected with siRNA (non-targeting siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Cell viability was assessed 72 h after siRNA transfection. Necrostatin-1 (50 µM), Q-VP-Oph (20 µM) or vehicle were added to the medium 48 h before viability assessment. Data are means of 5 independent experiments ±SEM. All values were normalized, viability of siCTRL-transfected and vehicle-treated cells was set as 100%. Two-tailed unpaired t-test; **p<0.01.


Measurement of ATP released to medium by HCT116 VPS4B - / - non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . culture media were exchanged 16 h after transfection , fresh media were conditioned for next 52 - 58 h . For non - transfected ( NT ) , same treatment protocol was used but without transfection mixture .

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

Measurement of ATP released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.


Measurement of HMGB1 released to medium by HCT116 VPS4B - / - non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . culture media were exchanged 16 h after transfection , fresh media were conditioned for next 52 - 58 h . For non - transfected ( NT ) , same treatment protocol was used but without transfection mixture .

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

Measurement of HMGB1 released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.


Microscopy presenting surface calreticulin ( green ) in HCT116 VPS4B - / - 48 h after transfection with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A # 2 ) . As a positive control for detection of surface calreticulin , non - transfected ( NT ) were treated with 2 µM mitoxantrone for 24 h . In blue , DAPI . Scale bar , 15 µm .

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

Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm.


Flow cytometric of calreticulin exposed on surface of VPS4A - depleted HCT116 VPS4B - / - 66 h after siRNA transfection ( siVPS4A duplexes : # 2 , # 4 or # 5 were used ) . Non - transfected ( NT ) or siCTRL # 1 - transfected served as negative controls . Left panel , of positive for calreticulin in population of live ( DAPI - negative ) , data are means of 4 independent experiments ± . Mann - Whitney U ; * * p < 0 . 01 . Right panel , representative dot plot diagrams of flow cytometric of surface - exposed calreticulin . rabbit anti - calreticulin control isotype IgG antibodies were used for , followed by secondary AlexaFluor 647 - conjugated antibody .

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

Flow cytometric analysis of calreticulin exposed on the cell surface of VPS4A-depleted HCT116 VPS4B-/- cells 66 h after siRNA transfection (siVPS4A duplexes: #2, #4 or #5 were used). Non-transfected (NT) or siCTRL#1-transfected cells served as negative controls. Left panel, percentage of cells positive for calreticulin in the population of live (DAPI-negative) cells, data are means of 4 independent experiments ±SEM. The Mann-Whitney U test; **p<0.01. Right panel, representative dot plot diagrams of flow cytometric analysis of cell surface-exposed calreticulin. Primary rabbit anti-calreticulin and control isotype IgG antibodies were used for staining, followed by secondary AlexaFluor 647-conjugated antibody.


qPCR of M1 ( pro - inflammatory ) M2 ( anti - inflammatory ) macrophage polarization markers in mouse bone marrow - derived macrophages ( BMDMs ) incubated for 24 h in conditioned media ( CM ) collected from control ( siCTRL # 1 ) , Vps4a - / or Vps4b - depleted CT - 26 . For double Vps4a + b depletion various combinations of siVps4a # 2 or # 5 siVps4B # 3 or # 4 duplexes were used . Data were normalized are presented as fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4 - depleted compared to its expression in BMDMs treated with CM from siCTRL # 1 - transfected ( set as 1 ) . Data are means of 4 independent experiments ± . One - sample t - ; ns - non - significant ( p ≥ 0 . 05 ) , * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analysis of M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage polarization markers in mouse bone marrow-derived macrophages (BMDMs) incubated for 24 h in conditioned media (CM) collected from control (siCTRL#1), Vps4a- and/or Vps4b-depleted CT-26 cells. For double Vps4a+b depletion various combinations of siVps4a#2 or #5 and siVps4B#3 or #4 duplexes were used. Data were normalized and are presented as the fold change of expression of a given M1 or M2 marker in BMDMs treated with CM from Vps4-depleted cells compared to its expression in BMDMs treated with CM from siCTRL#1-transfected cells (set as 1). Data are means of 4 independent experiments ±SEM. One-sample t-test; ns - non-significant (p≥0.05), *p<0.05, **p<0.01.


( A ) Stable RNAi screening of cancer - related genes was conducted to identify gene knockdowns that enhance reprogramming of MEFs into iPSCs . graph shows 10 shRNAs that were most strongly , positively selected in iPSCs compared to non - reprogrammed on day 14 of reprogramming ( n = 3 biological replicates ) .

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

(A) Stable RNAi screening of cancer-related genes was conducted to identify gene knockdowns that enhance reprogramming of MEFs into iPSCs. The graph shows the 10 shRNAs that were most strongly, positively selected in iPSCs compared to non-reprogrammed cells on day 14 of reprogramming (n=3 biological replicates).


( B - E ) Mouse embryonic fibroblasts ( MEFs ) were co - infected with shRNAs against Tnfaip2 or a scramble shRNA reprogramming factors . co - transduced were sorted were reprogrammed for 14 days : ( B ) Representative ( C ) of alkaline - phosphatase - positive ( AP + ) iPSC colonies on day 14 of reprogramming by transfection of 4 reprogramming factors ( = 4F : Oct4 , Sox2 , Klf4 , c - Myc = OSKM ) or 3 factors ( = 3F : OSK ) . [ n = 3 biological replicates per group ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk ) analyzed by one - sided t - analyzed by one - sided t - with Holm - Sidak correction for multiple testing ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (B) Representative images and (C) quantification of alkaline-phosphatase-positive (AP+) iPSC colonies on day 14 of reprogramming by transfection of 4 reprogramming factors (=4F: Oct4, Sox2, Klf4, c-Myc = OSKM) or 3 factors (=3F: OSK). [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test analyzed by one-sided t-test with Holm-Sidak correction for multiple testing].


Mouse embryonic fibroblasts ( MEFs ) were co - infected with shRNAs against Tnfaip2 or a scramble shRNA reprogramming factors . co - transduced were sorted were reprogrammed for 14 days : ( D ) Representative FACS

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

Mouse embryonic fibroblasts (MEFs) were co-infected with shRNAs against Tnfaip2 or a scramble shRNA and reprogramming factors. The co-transduced cells were sorted and were reprogrammed for 14 days: (D) Representative FACS profiles


( E ) of Oct4 - GFP + iPSCs obtained after 14 days of reprogramming of MEFs from Oct4 - eGFP mice using 4F - transduction on day zero that were infected with shRNAs targeting Tnfaip2 or a scrambled shRNA control [ n = 5 biological replicates ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk ) analyzed by one - sided t - with Holm - Sidak correction for multiple testing ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) percentage of Oct4-GFP+ iPSCs obtained after 14 days of reprogramming of MEFs from Oct4-eGFP mice using 4F-transduction on day zero that were infected with shRNAs targeting Tnfaip2 or a scrambled shRNA control [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm-Sidak correction for multiple testing].


] . ( F ) mRNA of Tnfaip2 was measured by RT - qPCR at indicated days of reprogramming by 4F - transduction on day zero [ n = 3 biological replicates ; log - transformed data were not normally distributed ( P > 0 . 05 as per Shapiro - Wilk ) , P value for upregulation of Tnfaip2 on consecutive days - 9 , - 12 - 14 was calculated starting with probability of maximum rank - difference , i . e . having two groups of three data points perfectly separating , p1 , b = 0 . 1 ( bidirectional ) p1 , u = 0 . 05 ( unidirectional ) ; probability of finding a triple series of max . difference starting at some timepoint is Ptriple , tp = p1 , b · p1 , u · p1 , u , finding such a triple somewhere across 5 - step time series is Ptriple = Ptriple , tp + 2 · ( 1 - p1 , u ) · Ptriple , tp = 0 . 0007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mRNA of Tnfaip2 was measured by RT-qPCR at the indicated days of reprogramming by 4F-transduction on day zero [n=3 biological replicates; log-transformed data were not normally distributed (P>0.05 as per Shapiro-Wilk test), P value for the upregulation of Tnfaip2 on consecutive test days-9,-12 and -14 was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and p1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 5-step time series is Ptriple = Ptriple,tp + 2 · (1- p1,u) · Ptriple,tp = 0.0007.


( C ) RT - qPCR of Smed - exoc3 mRNA expression of total ( whole - derived ) from exoc3 ( RNAi ) - treated planarians compared to control - injected planarians on day - 38 of injection scheme [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk ) ; data were analyzed by one - sided t - ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR analysis of Smed-exoc3 mRNA expression of total RNA (whole body-derived) from exoc3(RNAi)-treated planarians compared to control-injected planarians on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test); data were analyzed by one-sided t-test].


( D ) Representative of exoc3 ( RNAi ) - treated planarians vs . control planarians at day - 38 of injection protocol as shown in panel B ( n = 4 experimental replicates with 6 - 10 biological replicates per experiment , scale bar : 0 . 5 mm ) . Number ratios in depict total number of showing indicated phenotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative photographs of exoc3(RNAi)-treated planarians vs. control planarians at day-38 of the injection protocol as shown in panel B (n=4 experimental replicates with 6-10 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs depict the total number of animals showing the indicated phenotype.


Planarians were injected with Smed - exoc3 dsRNA = exoc3 ( RNAi ) or with control gfp ( RNAi ) injections . ( A ) representative show for phosphorylation of 10th serine residue of Histone 3 ( H3S10p , a marker in planarians ) at days - 38 of injection protocol ( n = 3 experimental replicates with 8 - 10 biological replicates per experiment , scale bar : 0 . 5 mm ) .

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

Planarians were injected with Smed-exoc3 dsRNA = exoc3(RNAi) or with control gfp(RNAi) injections. (A) The representative photographs show staining for phosphorylation of 10th serine residue of Histone 3 (H3S10p, a stem cell marker in planarians) at days-38 of the injection protocol (n=3 experimental replicates with 8-10 biological replicates per experiment, scale bar: 0.5 mm).


exoc3 ( RNAi ) - treated planarians versus gfp ( RNAi ) - treated controls were analyzed at indicated time - points of dsRNA injection protocol : ( B ) of H3S10p + per mm2 in planarians at day - 30 , - 34 - 38 of injection protocol [ n = 3 experimental replicates with 5 - 7 biological replicates per experiment ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk ) analyzed by multiple t - with Holm - Sidak correction for multiple comparisons ] .

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

exoc3(RNAi)-treated planarians versus gfp(RNAi)-treated controls were analyzed at the indicated time-points of the dsRNA injection protocol: (B) Quantification of H3S10p+ cells per mm2 in planarians at day-30, -34 and -38 of the injection protocol [n=3 experimental replicates with 5-7 biological replicates per experiment; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


FACS - time - course on changes in fraction of somatic ( X1 - population ) at indicated time points . were obtained from whole - trypsinized planarians of gfp ( RNAi ) - injected controls exoc3 ( RNAi ) - treated planarians stained with dye Calcein - AM ( ) dye Hoechst 33342 : ( C ) Representative FACS

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

FACS-based time-course analysis on changes in the fraction of somatic stem cells (X1-population) at indicated time points. Cells were obtained from whole body-trypsinized planarians of gfp(RNAi)-injected controls and exoc3(RNAi)-treated planarians and stained with the cytoplasmic dye Calcein-AM and the nuclear (DNA) dye Hoechst 33342: (C) Representative FACS profiles


( D ) of relative number of X1 [ n = 3 biological replicates ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk ) analyzed by multiple t - with Holm - Sidak correction for multiple comparisons ] .

O O O O O O O O O O O O O O O O O O O O O O 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) quantification of the relative number of X1 cells [n=3 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


RT - qPCR for makers of differentiating progenitor from epidermal lineage on total ( whole derived ) from exoc3 ( RNAi ) - treated planarians ( E ) Expression of differentiation marker prog - 2 on day - 38 of injection scheme [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk analyzed by one - sided t - ] ,

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

RT-qPCR analysis for makers of differentiating progenitor cells from the epidermal lineage on total (whole body derived) RNA from exoc3(RNAi)-treated planarians (E) Expression analysis of differentiation marker prog-2 on day-38 of the injection scheme [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test and analyzed by one-sided t-test],


( F ) Expression of marker genes of differentiated ( prog - 1 Smed - odc - 1 ; on day - 30 , - 34 - 38 of injection scheme [ n = 3 biological replicates per group time point except for prog - 1 expression at day 38 , which includes 6 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk ) analyzed by multiple t - with Holm - Sidak correction for multiple comparisons ] . For Smed - odc - 1 log - transformed mean centered group data were normally distributed analyzed by multiple t - with Holm - Sidak correction for multiple comparisons ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression analysis of marker genes of differentiated cells (prog-1 and Smed-odc-1; on day-30, -34 and -38 of the injection scheme [n= 3 biological replicates per group and time point except for prog-1 expression at day 38, which includes 6 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. For Smed-odc-1 log-transformed mean centered group data were normally distributed and and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].


Planarians were injected with exoc3 ( RNAi ) or with control gfp ( RNAi ) injections . Organ were analyzed on day - 38 of injection protocol . Organ homeostasis was analyzed by against specific markers : ( A ) structure of brain / central nervous system ( CNS ) was analyzed by against synapsin ( 3C11 ) . cartoon represents phenotypic change ( upper panel ) . Representative are shown in lower panel ( 3 experimental replicates with 2 - 3 biological replicates per experiment , scale bar : 0 . 5 mm ) . Number ratios in indicate total number of exhibiting represented phenotype . There was a significant increase in occurrence of atrophic cephalic ganglia sensory neuron loss in exoc3 ( RNAi ) - treated planarians vs . controls ( cg : cephalic ganglia ; sn : sensory neurons ) .

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

Planarians were injected with exoc3(RNAi) or with control gfp(RNAi) injections. Organ phenotypes were analyzed on day-38 of the injection protocol. Organ homeostasis was analyzed by staining against specific markers: (A) The structure of the brain/central nervous system (CNS) was analyzed by staining against synapsin (3C11). The cartoon represents the phenotypic change (upper panel). Representative photographs are shown in the lower panel (3 experimental replicates with 2-3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. There was a significant increase in the occurrence of atrophic cephalic ganglia and sensory neuron loss in exoc3(RNAi)-treated planarians vs. controls (cg: cephalic ganglia; sn: sensory neurons).


( B ) Planarian visual system was analyzed by against VC1 . Schematic cartoon ( upper panel ) representative ( lower panel ) are shown ( 3 experimental replicates with 3 biological replicates per experiment , scale bar : 0 . 5 mm ) . Number ratios in indicate total number of exhibiting represented phenotype . Arrows point to photosensitive ( ps ) ; pigmentary ( pc ) to optic chiasm ( oc ) .

(B) Planarian visual system was analyzed by staining against VC1. Schematic cartoon (upper panel) and representative images (lower panel) are shown (3 experimental replicates with 3 biological replicates per experiment, scale bar: 0.5 mm). Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype. Arrows point to photosensitive cells (ps); pigmentary cells (pc) and to the optic chiasm (oc).


- seq was conducted on freshly isolated X1 from exoc3 ( RNAi ) - treated compared to X1 from gfp ( RNAi ) - treated planarians on day - 38 of injection protocol . histograms depict overlap ( blue ) of DEGs in X1 of Smed - exoc3 - depleted planarians versus controls ( n = 3 biological replicates ; adjusted P < 0 . 05 ) with genes expressed in indicated sample sets of ( A ) neoblast

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

RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (A) neoblast stem cells


( B - E ) heat maps depict intersection of DEGs with strong expression signals ( log2 fold change > 0 . 5 or < - 0 . 5 ) discovered in X1 of exoc3 - depleted planarians versus controls ( n = 3 biological replicates ) with indicated , previously published data sets on gene expression profiles in ( B - D ) neoblast or ( E ) differentiating progenitor of epidermal lineage . colour scale represents gene - wise z - score calculated from normalized gene expression while purple green indicate upregulated downregulated genes in X1 of exoc3 - depleted planarians versus controls , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) The heat maps depict the intersection of DEGs with strong expression signals (log2 fold change > 0.5 or < -0.5) discovered in X1 cells of exoc3-depleted planarians versus controls (n= 3 biological replicates) with the indicated, previously published data sets on gene expression profiles in (B-D) neoblast cells or (E) differentiating progenitor cells of epidermal lineage. The colour scale represents the gene-wise z-score calculated from normalized gene expression levels while purple and green indicate upregulated and downregulated genes in X1 cells of exoc3-depleted planarians versus controls, respectively.


- seq was conducted on freshly isolated X1 from exoc3 ( RNAi ) - treated compared to X1 from gfp ( RNAi ) - treated planarians on day - 38 of injection protocol . histograms depict overlap ( blue ) of DEGs in X1 of Smed - exoc3 - depleted planarians versus controls ( n = 3 biological replicates ; adjusted P < 0 . 05 ) with genes expressed in indicated sample sets of ( F ) differentiating progenitor of epidermal lineage from indicated publications while sum of overlapping ( blue ) set - exclusive ( grey ) genes represents total number of genes of a cluster that were analyzed in this study .

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

RNA-seq was conducted on freshly isolated X1 cells from exoc3(RNAi)-treated compared to X1 cells from gfp(RNAi)-treated planarians on day-38 of the injection protocol. The histograms depict the overlap (blue) of DEGs in X1 cells of Smed-exoc3-depleted planarians versus controls (n=3 biological replicates; adjusted P< 0.05) with genes expressed in the indicated sample sets of (F) differentiating progenitor cells of epidermal lineage from the indicated publications while the sum of overlapping (blue) and set-exclusive (grey) genes represents the total number of genes of a cluster that were analyzed in this study.