of apoptotic corpse in meiotic compartment of CED - 1 : : GF relative to total number of meiotic , fed transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated wit 100µM cisplatin Data information : Data are presented as mean ± . For each panel at least N = 3 10 worms for replicate condition . * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit 100µM cisplatin Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Time course of apoptotic corpses relative to total number of meitic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 ( A left untreated or exposed to 1200 J / m2 UVB . Apoptotic were counted 12 , 24 36 hours after exposure Data information : Data are presented as mean ± , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 (A and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Time course of apoptotic corpses relative to total number of meitic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) o isp - 1 ( B ) , left untreated or exposed to 1200 J / m2 UVB . Apoptotic were counted 12 , 24 36 hours after exposure Data information : Data are presented as mean ± , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOV Tukey " s multiple

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

Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) o isp-1 (B), and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOV Tukey"s multiple comparisons


Real Time PCR gene expression of egl - genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated with gamma radiatio Data information : Data are presented a mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented a mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Real Time PCR gene expression of egl - genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated wit UV Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit UV Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Real Time PCR gene expression o ced - 1 genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated with gamma radiatio Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Real Time PCR gene expression o ced - 1 genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treate with UV Data information : Data are presented as mean ± SE N = F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s mu 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 O O O O O

Real Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treate with UV Data information: Data are presented as mean ± SE N= F). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s mu comparisons test


Representative pictures ( below pictures ) of dissected adult gonads relative to number of mitotic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 stained with DAPI ( blue ) either anti - PARP - 1 ( red ) ( A Scale bar : 20µ ( A Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate

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

Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue) and either anti-PARP-1 (red) (A Scale bar: 20µ (A Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


Representative pictures ( below pictures ) of dissected adult gonads relative to number of mitotic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 stained with DAPI ( blue eithe anti - RPA - 1 ( green ) anti - RAD - 51 ( red ) antibodies treated with gamma radiation ( 125 Gy ) ( B ) ( untreated displayed no of RPA - 1 RAD - 51 , not shown ) 15µm ( B Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate

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

Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue and eithe anti-RPA-1 (green) and anti-RAD-51 (red) antibodies and treated with gamma radiation (125 Gy) (B) (untreated animals displayed no staining of RPA-1 and RAD-51, not shown) 15µm (B Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


of mitotic in wild - type fed as ( A ) left untreated or treated with gamma radiation 125 G Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate

O O O O O O O O O O O O O O O O O O O O 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 mitotic cells in wild-type animals fed as (A) and left untreated or treated with gamma radiation 125 G Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


of mitotic in wild - type fed as ( A ) left untreated or treate with 1200 J / m2 UV Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate

O O O O O O O O O O O O O O O O O O O O O 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 mitotic cells in wild-type animals fed as (A) and left untreated or treate with 1200 J/m2 UV Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


A , B to number of mitotic of dissected gonads from wild - type stained with either anti - phosphohistone H3 ( PH3 ) ( A ) , anti - CDK - 1 ( CDK - 1 ) ( B ) transformed with either empty vector ( con ) or vector - expressing dsRNA against frh - 1 ( frh - 1 ) or isp - 1 ( isp - 1 ) treated with gamma radiation 125 Gy . is expressed as fold changes of positive stained treated over positive untreated Data information : Data are presented as mean ± SD ( A , B N = 2 ( A , B * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . One - way ANOVA Tukey " s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B Quantification to the number of mitotic cells of dissected gonads from wild-type animals stained with either anti-phosphohistone H3 (PH3) (A), anti-CDK-1 (CDK-1) (B) transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) and treated with gamma radiation 125 Gy. Quantification is expressed as fold changes of positive stained cells treated over the positive untreated cells Data information: Data are presented as mean ± SD (A, B N=2 (A, B * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. One-way ANOVA Tukey"s multiple comparisons test


Fertilit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated with gamma radiatio at indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified 2 - wa 2 - way ANOVA Tukey " s multiple compa ( C - H )

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

Fertilit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated with gamma radiatio at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified 2-wa 2-way ANOVA Tukey"s multiple compa (C-H)


fecundit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated wit UV at indicated doses Data information : Data are presented a mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

fecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit UV at the indicated doses Data information: Data are presented a mean ± SE ), N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compa


Fertilit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated wit Hydroxyure at indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

Fertilit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple comparisons


fecundit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated with gamma radiatio at indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

fecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated with gamma radiatio at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compa


fecundit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated wit UV at indicated doses Data information : Data are presente as mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

fecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit UV at the indicated doses Data information: Data are presente as mean ± SE ), N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compa


fecundit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , an left untreated or treated wit Hydroxyure at indicated doses Data information : Data are presente as mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

fecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, an left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presente as mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compa


A of BRD - 1 representative western blot from wild - type worms fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 Bar graphs are mean ± Data information : Data are presented as mean ± SD N = 8 - 1 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of BRD-1 protein and representative western blot from wild-type worms fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 Bar graphs are mean ± SEM Data information: Data are presented as mean ± SD N= 8-1 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified


B of mitotic nuclei in single gonad arm of wild - type ( WT ) , brc - 1 ( tm1145 ) brd - 1 ( gk297 ) strains fed as in ( A ) Data information : Data are presented as mean ± SD N = ) , 8 - 1 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is

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

B Quantification of mitotic nuclei in single gonad arm of wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) Data information: Data are presented as mean ± SD N= ), 8-1 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified


of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) brd - 1 ( gk297 ) strains fed as in ( A ) left untreated ( C Bar graphs are mean ± . Data information Data are presented a mean ± N = 8 - 10 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 apoptotic corpse in wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) and left untreated (C Bar graphs are mean ± SEM. Data information Data are presented a mean ± SEM N= 8-10 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparison


of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) brd - 1 ( gk297 ) strains fed as in ( A ) an treated with ionizing radiation 125 Gy ( IR ) ( D ) . Bar graphs are mean ± . Data information : Data are presented a mean ± N = 8 - 10 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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Quantification of apoptotic corpse in wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) an treated with ionizing radiation 125 Gy (IR) (D). Bar graphs are mean ± SEM. Data information: Data are presented a mean ± SEM N= 8-10 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparison


Kaplan - Meier survival curves of wild - type ( WT ) , ced - 9 ( n1653ts strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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Kaplan-Meier survival curves of wild-type (WT) and, ced-9(n1653ts strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1


Kaplan - Meier survival curves of wild - type ( WT ) an ) , brc - 1 ( tm1145 ) ( B strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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Kaplan-Meier survival curves of wild-type (WT) an ), brc-1(tm1145) (B strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1


Kaplan - Meier survival curves of wild - type ( WT ) an brd - 1 ( gk297 ) ( C strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

Kaplan-Meier survival curves of wild-type (WT) an brd-1(gk297) (C strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1


Kaplan - Meier survival curves of wild - type ( WT ) an glp - 1 ( e2141 ) ( D strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

Kaplan-Meier survival curves of wild-type (WT) an glp-1(e2141) (D strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1


Kaplan - Meier survival curves of wild - type ( WT ) an glp4 ( bn2 ) ( E ) , strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

Kaplan-Meier survival curves of wild-type (WT) an glp4(bn2) (E), strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1


Kaplan - Meier survival curves of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp - 1 ( F left untreated or treated with 600 , 1200 or 2400 J / m2

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Kaplan-Meier survival curves of wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1 (F and left untreated or treated with 600, 1200 or 2400 J/m2 UVB


Kaplan - Meier survival curves of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA agains isp - 1 ( G ) left untreated or treated with 600 , 1200 or 2400 J / m2

Kaplan-Meier survival curves of wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA agains isp-1 (G) and left untreated or treated with 600, 1200 or 2400 J/m2 UVB


A Kaplan - Meier survival curves of wild - type ( WT ) , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp -

A Kaplan-Meier survival curves of wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1


B of apoptotic corpses in wild - type ( WT ) , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed as ( A ) left untreated or treated with 600 J / m2 UVB . Data information : Data are presented as mean ± , N = 3 , 9 - 10 worms per replicate condition * * P < 0 . 01 vs con ; # # P < 0 . 01 vs WT ; ooo P < 0 . 00 oooo P < 0 . 0001 vs untreated . Not significant if nothing is specified . 2 - way ANOVA Tukey ' s multiple comparisons . C

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B Quantification of apoptotic corpses in wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed as (A) and left untreated or treated with 600 J/m2 UVB. Data information: Data are presented as mean ± SEM, N=3, 9-10 worms per replicate and condition ** P < 0.01 vs con; ## P < 0.01 vs WT; ooo P < 0.00 and oooo P < 0.0001 vs untreated . Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test. C


Western blot of indicated in T - inducible PSCs stimulated as indicated for 24 hr . GAPDH was used as a loading control .

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Western blot of the indicated proteins in T-inducible PSCs stimulated as indicated for 24 hr. GAPDH was used as a loading control.


derived from mean - centered expression values ( log2 scale ) of selected key pluripotency genes . See also Fig EV2C , Dataset EV1 for clustered DEGs .

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Heatmap derived from mean-centered expression values (log2 scale) of selected key pluripotency genes. See also Fig EV2C, Dataset EV1 for all the clustered DEGs.


ChIP - seq tracks of T ( in red ; input is in blue ) around Oct4 ( 7 . 5 kb ) . DE , distal enhancer ; PE , proximal enhancer . T - binding peak around Oct4 - DE is indicated ( arrowhead ) . Right : ChIP - qPCR for enrichment of T on DE regions indicated in Fig EV3E . NC , negative control ( locus without T binding ) . enrichment is indicated by input with SDs ( three independent experiments with two technical replicates ) .

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ChIP-seq tracks of T (in red; input is in blue) around the Oct4 (7.5 kb). DE, distal enhancer; PE, proximal enhancer. The T-binding peak around the Oct4-DE is indicated (arrowhead). Right: ChIP-qPCR for the enrichment of T on the DE and the regions indicated in Fig EV3E. NC, negative control (the locus without T binding). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).


Luciferase with T - binding elements ( TBEs ) around indicated loci as shown in ( A ) Fig EV3E . Relative luciferase activities of sorted fractions are shown with SDs . ( three independent experiments with two technical replicates ) .

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Luciferase assay with T-binding elements (TBEs) around the indicated loci as shown in (A) and Fig EV3E. Relative luciferase activities of sorted cell fractions are shown with SDs. (three independent experiments with two technical replicates).


Left : A representative confocal image of a mouse at E7 . 0 bearing T - GFP Blimp1 - RFP transgenic reporters . Scale bar : 50um . Arrowheads indicate Blimp1 - positive nascent germ in posterior proximal epiblast . Right : FACS of dissociated mouse without visceral endoderm .

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Left: A representative confocal image of a mouse embryo at E7.0 bearing T-GFP and Blimp1-RFP transgenic reporters. Scale bar: 50um. Arrowheads indicate Blimp1-positive nascent germ cells in the posterior proximal epiblast. Right: FACS analysis of dissociated mouse embryos without visceral endoderm.


Expression of indicated genes measured by qPCR in FACS - sorted ( E ) derived from at different developmental stages ( E7 . 0 - 7 . 5 ) . Ten FACS - sorted in different fractions ( E ) were used for each qPCR with two technical replicates . ∆ Ct values from average Ct values of Arbp Ppia are shown ( n ≥ 3 , log2 scale ) . P values were calculated by two - unpaired t - .

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Expression of the indicated genes measured by qPCR in FACS-sorted cells (E) derived from embryos at different developmental stages (E7.0-7.5). Ten FACS-sorted cells in different cell fractions (E) were used for each individual qPCR analysis with two technical replicates. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (n≥3, log2 scale). P values were calculated by two-tailed unpaired t-test.


ChIP - seq tracks of T ( in red ; input is in blue ) pluripotency factors ( in green ) around Blimp1 ( 100 kb ) Prdm14 ( 100 kb ) . co - bound locus ( L # 1 - 4 ) locus exclusively bound by pluripotency factors but not T ( L # 5 ) are indicated by arrow . NC , negative control ( locus without T binding ) .

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ChIP-seq tracks of T (in red; input is in blue) and core pluripotency factors (in green) around the Blimp1 (100 kb) and Prdm14 (100 kb). The co-bound locus (L#1-4) and the locus exclusively bound by core pluripotency factors but not T (L#5) are indicated by arrow. NC, negative control (the locus without T binding).


ChIP - qPCR for enrichment of indicated factors on loci ( L # 1 - 5 ) indicated in ( A ) . enrichment is indicated by input with SDs ( three independent experiments with two technical 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

ChIP-qPCR for the enrichment of the indicated factors on the loci (L#1-5) indicated in (A). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).


Luciferase with specific of locus bound by T around L # 1 L # 3 as shown in ( A ) . Relative luciferase activities of low T - inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs . ( three independent experiments with two technical replicates ) .

Luciferase assay with the specific sequence of the locus bound by T around L#1 and L#3 as shown in (A). Relative luciferase activities of low T-inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs. (three independent experiments with two technical replicates).


Expression of Blimp1 Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines . ∆ Ct values from average Ct values of Arbp Ppia are shown ( log2 scale , mean value of two independent experiments with two technical replicates ) .

Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (log2 scale, the mean value of two independent experiments with two technical replicates).


Top : Oct4 - GFP ( green ) reporter expression ( left ) , Blimp1 - RFP ( red ) Oct4 - GFP ( BROG ) reporter expression ( right ) in gonads of E13 . 5 generated by 4N complementation with indicated ESC lines . Scale bar : 100um . Bottom : of Oct4 - GFP reporter - positive PGCs ( left ) BROG reporter - positive PGCs ( right ) measured by FACS . Blue circles , red squares , green triangles represent of gonadal PGCs in first , second , third independent mutant , respectively , of examined locus ( L # 1 - 5 ) .

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Top: Oct4-GFP (green) reporter expression (left), and Blimp1-RFP (red) and Oct4-GFP (BROG) reporter expression (right) in the gonads of E13.5 embryos generated by 4N complementation with the indicated ESC lines. Scale bar: 100um. Bottom: percentage of Oct4-GFP reporter-positive PGCs (left) and BROG reporter-positive PGCs (right) measured by FACS analysis. Blue circles, red squares, and green triangles represent the percentage of gonadal PGCs in the first, second, and third independent mutant line, respectively, of the examined locus (L#1-5).


High constitutive expression of CXCR4 in of SW1417 CRC as measured by flow cytometry .

High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry.


Lack of human SDF - 1α release from cultured SW1417 CRC , as measured by ELISA , whereas human control 1BR3 . G fibroblasts express high SDF - 1α , after 48h or 72h of growth in culture . ( N = 2 experiment in duplicate )

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Lack of human SDF-1α release from cultured SW1417 CRC cells, as measured by ELISA, whereas human control 1BR3.G fibroblasts express high SDF-1α levels, after 48h or 72h of growth in culture. (N=2 experiment in duplicate)


Nanoconjugate internalization in CXCR4 overexpressing ( CXCR4 + ) SW1417 CRC after 1 hour exposure at 1µM , as measured by emission using flow cytometry . ( N = 3 experiments in duplicate ) Significant difference at p = 0 . 002 between T22 - GFP - H6 - FdU AMD3100 + T22 - GFP - H6 - FdU

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

Nanoconjugate internalization in CXCR4 overexpressing (CXCR4+) SW1417 CRC cells after 1 hour exposure at 1µM, as measured by fluorescence emission using flow cytometry. (N=3 experiments in duplicate) Significant difference at p= 0.002 between T22-GFP-H6-FdU and AMD3100+T22-GFP-H6-FdU groups


Intracellular trafficking of T22 - GFP - H6 - FdU in CXCR4 + SW1417 by confocal microscopy after exposure at 1µM for 24 h . green corresponds to GFP containing nanoconjugates red corresponds to plasma membranes stained with a red dye ( CellMask ™ ) , whereas nucleus was stained in blue with hoescht . insets show detail of intracellular localization of nanostructured , entities , in an isosurface representation within a three - dimensional volumetric x - y - z data

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

Intracellular trafficking of T22-GFP-H6-FdU in CXCR4+ SW1417 cells by confocal microscopy after exposure at 1µM for 24 h. The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye (CellMask™), whereas cell nucleus was stained in blue with hoescht. The insets show detail of the intracellular localization of nanostructured, fluorescent entities, in an isosurface representation within a three-dimensional volumetric x-y-z data field


Linearized T22 - GFP - H6 - FdU dose - response trend representation compared with unconjugated free oligo - FdU exposure . Antitumor effect was measured as CXCR4 + SW1417 viability by MTT after 72 h exposure as described concentrations . ( N = 3 experiments in duplicate )

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Linearized T22-GFP-H6-FdU dose-response trend line representation compared with unconjugated free oligo-FdU exposure. Antitumor effect was measured as CXCR4+ SW1417 cell viability by MTT after 72 h exposure as the described concentrations. (N=3 experiments in duplicate)


Reduction of viability determined by optical microscope of SW1417 exposed to 1µM T22 - GFP - H6 - FdU for 48 h , as compared to T22 - GFP - H6 or free oligo - FdU . ( N = 3 experiments in duplicate ; Scale bar , 100µm ) .

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Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22-GFP-H6-FdU for 48 h, as compared to T22-GFP-H6 or free oligo-FdU. (N=3 experiments in duplicate; Scale bar, 100µm).


Selective T22 - GFP - H6 - FdU nanoconjugate biodistribution in subcutaneous CXCR4 + SW1417 CRC tissue 5h after a 100µg single intravenous dose , as measured by emission using IVIS Spectrum 200 ( N = 5 / group ) . Biodistribution is similar to that achieved by T22 - GFP - H6 targeting vector undetectable after Buffer or free oligo - FdU treatment . ( N = 5 mice / group )

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Selective T22-GFP-H6-FdU nanoconjugate biodistribution in subcutaneous CXCR4+ SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose, as measured by fluorescence emission using IVIS Spectrum 200 (N=5/group). Biodistribution is similar to that achieved by the T22-GFP-H6 targeting vector and undetectable after Buffer or free oligo-FdU treatment. (N=5 mice/group)


Co - localization ( yellow merged ) of T22 - GFP - H6 - FdU ( green ) CXCR4 receptor ( red ) release of T22 - GFP - H6 - FdU into cytosol in CXCR4 + 5h after a 100ug dose of nanoconjugate , as measured by dual anti - GFP / anti - CXCR4 immunofluorescence ( IF ) . DAPI ( blue ) . emission was measured in green red channels using Image J software expressed as mean ( A ) ± s . e . m ( µm2 ) ( N = 10 , 2 fields x 5 mice ; 200X ) . Note significant ( P = 0 . 003 ) increase in occupied by green dots ( nanoconjugate realeased to cytosol ) in T22 - GFP - H6 - FdU - treated tumors , compared to free oligo - FdU - treated control tissues . Scale bar , 50 µm .

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

Co-localization (yellow merged) of the T22-GFP-H6-FdU (green) and the CXCR4 receptor (red) and release of T22-GFP-H6-FdU into the cytosol in CXCR4+ tumor cells 5h after a 100ug dose of nanoconjugate, as measured by dual anti-GFP/anti-CXCR4 immunofluorescence (IF). DAPI (blue nuclear staining). Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area (A)±s.e.m (µm2) (N=10, 2 tumor fields x 5 mice; 200X). Note the significant (P=0.003) increase in the area occupied by the green dots (nanoconjugate realeased to the cell cytosol) in T22-GFP-H6-FdU-treated tumors, compared to free oligo-FdU-treated control tissues. Scale bar, 50 µm.


Administration of CXCR4 antagonist AMD3100 completely blocks T22 - GFP - H6 - FdU biodistribution , as measured by emission . is not detected in Buffer or free oligo - FdU controls ( N = 5 fields / group ) .

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Administration of the CXCR4 antagonist AMD3100 completely blocks T22-GFP-H6-FdU tumor biodistribution, as measured by fluorescence emission. Fluorescence is not detected in Buffer or free oligo-FdU controls (N=5 tumor fields/group).


uptake of T22 - GFP - H6 - FdU observed in CXCR4 + SW1417 tissues is almost completely blocked by prior AMD3100 administration , as quantified using anti - GFP H - score . ( N = 5 fields / group ) .

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The uptake of T22-GFP-H6-FdU observed in CXCR4+ SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration, as quantified using the anti-GFP IHC H-score. (N=5 tumor fields/group).


Representative of T22 - GFP - H6 - FdU uptake AMD3100 competition by anti - GFP inmunostaining , which quantitation is reported in panel E . Scale bar , 50

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Representative images of T22-GFP-H6-FdU uptake and AMD3100 competition by anti-GFP inmunostaining, which quantitation is reported in panel E. Scale bar, 50 µm


Representative of CXCR4 overexpression in subcutaneous tissue , showing similar CXCR4 among compared groups ( N = 5 / group ; Buffer , T22 - GFP - H6 - FdU , T22 - GFP - H6 free oligo - FdU ) before treatment ( upper panels ) . Representative of double - strand break induction caspase 3 activation ( measured with anti - ɣH2AX or anti - caspase 3 by ) 5h post - administration ( middle panels ) . Apoptotic induction ( Hoescht , 24h post - administration , lower panels ) . Note higher number of positive for DSBs , Caspase 3 activation apoptosis induction in T22 - GFP - H6 - FdU as compared to free oligo - FdU . Black or arrows indicate dead . Quantitation of number of containing DSBs or active Caspase - 3 in - stained sections 5h post - treatment number of condensated or disaggregated nuclei ( by Hoescht ) 24h post - treatment in sections of 10 high - powder fields ( 400x magnification ) using D software . ( N = 50 ; 10 fields / mice ; 5 mice / group ) . Data expressed as mean ± s . e . m .

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Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N=5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6 and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase 3 activation (measured with anti-ɣH2AX or anti-cleaved caspase 3 by IHC) 5h post-administration (middle panels). Apoptotic induction (Hoescht staining, 24h post-administration, lower panels). Note the higher number of cells positive for DSBs, Caspase 3 activation and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells. Quantitation of the number of cells containing DSBs or active Caspase-3 in IHC-stained tumor sections 5h post-treatment and the number of condensated or disaggregated nuclei (by Hoescht staining) 24h post-treatment in tumor sections of 10 high-powder fields (400x magnification) using the Cell D software. (N=50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean±s.e.m.


T22 - GFP - H6 - FdU depletes CXCR4 + cancer from SW1417 CRC tissue after a 100 µg single dose administration . Note reduction in CXCR4 + fraction in 24h after injection , their almost complete elimination at 48h re - emergence of CXCR4 + 72h post - administration , using anti - CXCR4 . In contrast , CXCR4 + cancer fraction ( CXCR4 + CCF ) in tissue remains constant along time after free oligo - FdU treatment . three day time - lapse for CXCR4 + re - appearance defines dosage interval used in a repeated dose schedule of nanoconjugate administration in experiments to evaluate its antimetastatic effect . ( N = 5 : 5 mice / group ; 1 samples / mouse ) . Scale bar , 50 µm . Data expressed as mean ± s . e .

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 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

T22-GFP-H6-FdU depletes CXCR4+ cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration. Note the reduction in CXCR4+ cell fraction in the tumor 24h after injection, their almost complete elimination at 48h and the re-emergence of CXCR4+ cells 72h post-administration, using anti-CXCR4 IHC. In contrast, the CXCR4+ cancer cell fraction (CXCR4+ CCF) in tumor tissue remains constant along time after free oligo-FdU treatment. The three day time-lapse for CXCR4+ tumor cell re-appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect. (N=5: 5 mice/group; 1 samples/mouse). Scale bar, 50 µm. Data expressed as mean±s.e.m


Significant reduction in number of spheroid formed ( C . optical microscope ) their bioluminescence emission ( D , IVIS Spectrum 200 ) , generated by 1x106 disaggregated ( cultured in conditioned media low - adhesion plates ) , obtained from CXCR4 + luciferase + SW1417 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . D . Quantitation of bioluminescent signal ( BLI ) expressed as Average Radiant , obtained using IVIS spectrum 200 equipment ( N = 2 plates / group ) .

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

Significant reduction in the number of spheroid formed (C. optical microscope) and their bioluminescence emission (D, IVIS Spectrum 200), generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates), obtained from CXCR4+ luciferase+ SW1417 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. D. Quantitation of the bioluminescent signal (BLI) expressed as Average Radiant Intensity, obtained using the IVIS spectrum 200 equipment (N=2 plates/group).


Reduction in number of formed spheroids ( optical microscope ) generated by 1x106 disaggregated ( cultured in conditioned media low - adhesion plates ) obtained from CXCR4 + M5 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . ( N = 8 ; 2 mice / group ; 4 plates / mouse ) . ( Scale bar , 100µm ) .

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

Reduction in the number of formed spheroids (optical microscope) generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates) obtained from CXCR4+ M5 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. (N=8; 2 mice/group; 4 plates/mouse). (Scale bar, 100µm).


Reduction in re - initiation capacity after subcutaneous inoculation of 5x106 in NSG mice ( N = 4 tumors / group ) derived from disaggregated obtained from SC tumors 10 days after administration of 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Free oligo - FdU - treated or Buffer - treated mice . Recording of number of positive tumors . ( N = 4 ; 2 mice / group , 2 injection points / mouse ) .

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

Reduction in tumor re-initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice (N=4 tumors/group) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days , as compared to Free oligo-FdU-treated or Buffer-treated mice. Recording of the number and size of positive tumors. (N=4; 2 mice/group, 2 injection points/mouse).


T22 - GFP - H6 - FdU - induced reduction in number of intravasated emboli in peri - tumoral vessels of M5 - orthotopic ( E . optical ; F . emboli number quantitation ) reduction of CXCR4 expression in these emboli ( G ) , treated 7 days after implantation with 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . emboli counting in 10 high powder field at 200x magnification in H & E stained sections from each . ( N = 5 / group ) .

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 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

T22-GFP-H6-FdU-induced reduction in the number of intravasated tumor emboli in peri-tumoral vessels of the M5-orthotopic primary tumor (E. optical images; F. emboli number quantitation) and reduction of CXCR4 expression in these emboli (G), treated 7 days after tumor cell implantation with 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. Tumor emboli counting in 10 high powder field at 200x magnification in H&E stained sections from each tumor. (N=5/group).


T22 - GFP - H6 - FdU prevents metastases in CXCR4 + - derived M5 model by potently reducing total mean number of liver , lung peritoneal mets , as recorded in H & E stained histology sections at end of treatment , in comparison to free oligo - FdU o Buffer - treatment . In contrast , number of LN mets is not reduced after T22 - GFP - H6 - FdU or free oligo - FdU administration ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e . m .

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

T22-GFP-H6-FdU prevents metastases in the CXCR4+ patient-derived M5 model by potently reducing the total and mean number of liver, lung and peritoneal mets, as recorded in H&E stained histology sections at the end of treatment, in comparison to free oligo-FdU o Buffer-treatment. In contrast, the number of LN mets is not reduced after T22-GFP-H6-FdU or free oligo-FdU administration (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m.


T22 - GFP - H6 - FdU induces a higher reduction of CXCR4 + cancer fraction ( CXCR4 + CCF ) in liver , lung peritoneal metastatic tissue , at end of treatment , than free oligo - FdU , as measured by anti - CXCR4 . In contrast , T22 - GFP - H6 - FdU or free oligo - FdU does not reduce CXCR4 + CCF in LN mets or tissue after therapy . ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e .

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

T22-GFP-H6-FdU induces a higher reduction of CXCR4+ cancer cell fraction (CXCR4+ CCF) in liver, lung and peritoneal metastatic tissue, at the end of treatment, than free oligo-FdU, as measured by anti-CXCR4 IHC. In contrast, T22-GFP-H6-FdU or free oligo-FdU does not reduce the CXCR4+ CCF in LN mets or primary tumor tissue after therapy. (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m


Representative CXCR4 of reduction in CXCR4 + CCF induced by T22 - GFP - H6 - FdU ( or its absence in free oligo - FdU mice ) at end of treatment , in M5 - derived CRC model , which quantitation is reported in panel B . In M5 model , highest reduction in foci number occurs in liver metastases , which show highest reduction in CXCR4 + CCF .

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

Representative CXCR4 IHC images of the reduction in CXCR4+ CCF induced by T22-GFP-H6-FdU (or its absence in free oligo-FdU mice) at the end of treatment, in the M5 patient-derived CRC model, which quantitation is reported in panel B. In the M5 model, the highest reduction in foci number and size occurs in liver metastases, which show the highest reduction in CXCR4+ CCF.


Undetectable T22 - GFP - H6 - FdU emitted in normal tissues , except for a transient accumulation 5h after a 100μg dose in liver , which disappears at 24 h . Liver emitted is transient significantly lower than one registered in tissue . / Liver = 7 . 5 ( see in Fig 2B , which was registered in same experiment ) ( N = 5 mice / group ) . Scale bar , 1 cm .

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

Undetectable T22-GFP-H6-FdU emitted fluorescence in normal tissues, except for a transient accumulation 5h after a 100μg dose in the liver, which disappears at 24 h. Liver emitted fluorescence is transient and significantly lower than the one registered in tumor tissue. Tumor/Liver ratio= 7.5 (see tumor intensity in Fig 2B, which was registered in the same experiment) (N=5 mice/group). Scale bar, 1 cm.


Representative depicting level of double strand break ( DSB ) induction in histologically normal bone marrow 5h after treatment , as measured by anti - ɣ - H2AX , which is higher in free oligo - FdU - treated mice than in T22 - GFP - H6 - FdU ( P = 0 . 047 ) . Low level of containing DSBs in histologically normal kidney after T22 - GFP - H6 - FdU or free oligo - FdU treatment , a finding occurring in normal tissues analysed . ( N = 50 , 5 mice / group ; 10 fields / mouse ) .

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

Representative images depicting the level of DNA double strand break (DSB) induction in histologically normal bone marrow 5h after treatment, as measured by anti-ɣ-H2AX, which is higher in free oligo-FdU-treated mice than in T22-GFP-H6-FdU (P=0.047). Low level of cells containing DSBs in histologically normal kidney after T22-GFP-H6-FdU or free oligo-FdU treatment, a finding occurring in all normal tissues analysed. (N=50, 5 mice/group; 10 fields/mouse).


Representative showing lack of histopathological alterations in H & E stained tissue or apoptotic induction in H & E stained samples of CXCR4 + ( bone marrow ) CXCR4 - ( brain , kidney , liver , lung heart ) normal tissues 24h after administration of a 100 μg dose of T22 - GFP - H6 - FdU or an equimolecular dose of free oligo - FdU ( N = 5 / group ) . Note that transient nanoconjugate distribution to liver or damage induced in bone marrow do not lead to cytoxicity on these no - tissues . ( N = 50 , 5 mice / group ; 10 fields / mouse )

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

Representative images showing lack of histopathological alterations in H&E stained tissue or apoptotic induction in H&E stained samples of CXCR4+ (bone marrow) and CXCR4- (brain, kidney, liver, lung and heart) normal tissues 24h after the administration of a 100 μg dose of T22-GFP-H6-FdU or an equimolecular dose of free oligo-FdU (N=5/group). Note that the transient nanoconjugate distribution to liver or the DNA damage induced in bone marrow do not lead to cytoxicity on these no-tumor tissues. (N=50, 5 mice/group; 10 fields/mouse)


Lack of differences in among groups registered along time in SW1417 - derived CCR model regression of metastases protocol ( N = 10 mice / group ) . Lack of differences in mouse among groups registered along time in SW1417 - derived model prevention of metastasis protocol ( Buffer ( N = 11 ; Free oligo - FdU ( N = 12 ) , T22 - GFP - H6 - FdU ( N = 12 ) ) . Lack of differences in mouse among groups registered along time in M5 - derived model prevention of metastasis protocol ( Buffer ( N = 6 ) ; Free oligo - FdU ( N = 17 ) ; T22 - GFP - H6 - FdU ( N = 8 ) . Scale bar , 100

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

Lack of differences in body weight among groups registered along time in the SW1417-derived CCR model and the regression of metastases protocol (N=10 mice/group). Lack of differences in body mouse weight among groups registered along time in the SW1417 cell line-derived model and the prevention of metastasis protocol (Buffer (N=11; Free oligo-FdU (N=12), T22-GFP-H6-FdU (N=12)). Lack of differences in body mouse weight among groups registered along time in the M5 patient-derived model and the prevention of metastasis protocol (Buffer (N=6); Free oligo-FdU (N=17); T22-GFP-H6-FdU (N=8). Scale bar, 100 μm


A . SDS - PAGE gel showing purified holoElongator used as input for gel filtration . bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 that have been previously described [ 34 ] .

O O O O O O O O O O O O O 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. SDS-PAGE gel showing the purified holoElongator complex used as input for gel filtration. Protein bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 that have been previously described [34].


B . Representative negative stain - EM field of holoElongator . Particles in side tilted views are highlighted . Scale bar , 50 nm .

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

B. Representative negative stain-EM field of the holoElongator complex. Particles in side and tilted views are highlighted. Scale bar, 50 nm.


C . Reference - free class averages back - projections of holoElongator model . Scale bar , 20 nm .

C. Reference-free class averages and back-projections of the holoElongator model. Scale bar, 20 nm.


D . EM reconstruction of holoElongator at 31 Å resolution . E . Fitting of Elp456 crystal structure into ring density of holoElongator reconstruction . F . Fitting of Elp1 CTD into holoElongator reconstruction .

O O O O O O O O O O O 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. EM reconstruction of the holoElongator complex at 31 Å resolution.E. Fitting of the Elp456 crystal structure into the ring density of the holoElongator reconstruction.F. Fitting of the Elp1 CTD into the holoElongator reconstruction.


A . SDS - PAGE gel showing purified Elp123 sub - used as input for gel filtration . bands marked with asterisks presumably correspond to different phosphorylation states of Elp1 .

O O O O O O O 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. SDS-PAGE gel showing the purified Elp123 sub-complex used as input for gel filtration. Protein bands marked with asterisks presumably correspond to different phosphorylation states of Elp1.


B . Representative negative stain - EM field of Elp123 sub - . Particles in side top views are highlighted . Scale bar , 50 nm .

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

B. Representative negative stain-EM field of the Elp123 sub-complex. Particles in side and top views are highlighted. Scale bar, 50 nm.


C . Reference - free class averages back - projections of Elp123 model . Scale bar , 20 nm .

C. Reference-free class averages and back-projections of the Elp123 model. Scale bar, 20 nm.


D . EM reconstruction of Elp123 sub - at 27 Å resolution . E . Fitting of Elp1 CTD into Elp123 reconstruction . F . Fitting of Elp2 crystal structure in difference density generated by subtracting partial Elp123 reconstruction from complete Elp123 reconstruction .

O O O O O O O O O O O O O O O O O O O O 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. EM reconstruction of the Elp123 sub-complex at 27 Å resolution.E. Fitting of the Elp1 CTD into the Elp123 reconstruction.F. Fitting of the Elp2 crystal structure in the difference density generated by subtracting the partial Elp123 reconstruction from the complete Elp123 reconstruction.


A. The model of the Elp123 sub-complex.


B . Crosslinks supporting orientation of Elp3 ( left ) Elp2 ( right ) . crosslinks are shown separately to highlight crosslinks linking from Elp3 Elp2 to other subunits , 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

B. Crosslinks supporting the orientation of Elp3 (left) and Elp2 (right). The crosslinks are shown separately to highlight crosslinks linking from Elp3 and Elp2 to other subunits, respectively.


D . Crosslinks supporting orientation of Elp456 hexamer .

D. Crosslinks supporting the orientation of the Elp456 hexamer.


A . GST - pull down assays of purified GST - tagged Elp3 SAM domain ( aa72 - 389 ) KAT domain ( aa348 - 557 ) with untagged full Elp1 Elp451 - 2706 . GSH resin were used as input controls . Lower panel shows 5 % of input upper panel shows bound fractions . samples were analyzed by SDS - PAGE stained by Coomassie . Identities of respective are indicated on 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 O O O O O O O O O

A. GST-pull down assays of purified GST-tagged Elp3 SAM domain (aa72-389) and KAT domain (aa348-557) with untagged full length Elp1 and Elp451-2706. GSH resin and proteins were used as input controls. Lower panel shows 5% of the input and upper panel shows bound fractions. All samples were analyzed by SDS-PAGE and stained by Coomassie. Identities of respective proteins are indicated on the right.


B . same as A , but using only GST - tagged Elp3 SAM domain with combinations variants of Elp1 , Elp2 , Elp456 .

B. same as A, but using only GST-tagged Elp3 SAM domain with combinations and variants of Elp1, Elp2, and Elp456.


C . same as A using GST - tagged full Elp1 truncations deletions of Elp456 .

C. same as A using GST-tagged full length Elp1 and truncations and deletions of Elp456.


D . same as A using GST - tagged C - terminally truncated Elp1 in combination with truncated versions of Elp4 .

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

D. same as A using GST-tagged C-terminally truncated Elp1 in combination with truncated versions of Elp4.


B . Same as A but tilted view to highlight active site cavity ( left ) . Close up view of active site in which known tRNA binding sites a region of Elp2 important for tRNA modification activity are highlighted . structures of subunits are shown in cartoon surface representation .

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

B. Same as A but tilted view to highlight the active site cavity (left). Close up view of the active site in which all known tRNA binding sites and a region of Elp2 important for tRNA modification activity are highlighted. Individual structures of the subunits are shown in cartoon and surface representation.


C . Close up view on localization of Elp3 orientation of its KAT SAM domain in relation to Elp1 Elp2 .

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

C. Close up view on the localization of Elp3 and the orientation of its KAT and SAM domain in relation to Elp1 and Elp2.


D . Close up view on relative spatial orientation of conserved basic patch in Elp2 potential interaction site between Elp456 a region of Elp2 important for tRNA modification activity .

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

D. Close up view on the relative spatial orientation of the conserved basic patch in Elp2 and the potential interaction site between Elp456 and a region of Elp2 important for tRNA modification activity.


A . Representative pseudocolor of PC6 transfected with EYFP - Parkin ( green ) mitochondrially targeted CFP ( red ) with without PINK1 Miro1 shRNA Miro2 shRNA . Zoomed show translocation of EYFP - Parkin to mitochondria in PINK1 - overexpressing PC6

O O O O O O O O O O O O O O O O O O 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 pseudocolor images of PC6 cells transfected with EYFP-Parkin (green) and mitochondrially targeted CFP (red) with and without PINK1 and Miro1 shRNA and Miro2 shRNA. Zoomed images show translocation of EYFP-Parkin to mitochondria in PINK1-overexpressing PC6 cells


B . of EYFP - Parkin translocation to mitochondria . of PC6 with EYFP - Parkin translocated to mitochondria in response to PINK1 overexpression is lower in Miro shRNAs - expressing groups when compared with scrambled shRNA - expressing group ( scr shRNA ) . * * * * P < 0 . 0001 , n = 6 dishes per group , 20 fields per dish , one - way

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

B. Quantification of EYFP-Parkin translocation to mitochondria. The percentage of PC6 cells with EYFP-Parkin translocated to mitochondria in response to PINK1 overexpression is lower in the Miro shRNAs-expressing groups when compared with the scrambled shRNA-expressing group (scr shRNA). ****P < 0.0001, n = 6 dishes per group, 20 fields per dish, one-way ANOVA


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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