Embryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY an the membrane marker GAP43 : : mCherry ( D - I ) were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation ( D ) Cross section ( z - projection ) showing the plasma membrane and ( E ) top view showing the apical plasma membrane at the initial time point before photo - activation . At this stage , the central mesodermal tissue has started to internalize and cells were constricted at the apical surface . In an alternating fashion , the base of the cells in the anterior half of the embryo ( left ) was photo - activated for 100 sec and the GAP43 : : mCherry signal in the entire field of view was recorded . The furrow further ingressed and closed completely 3 min ( F ) and 6 min ( G ) after initial activation , respectively . In the activated region , the tissue failed to internalize and to form a closed furrow both at 3 min ( H ) and 6 min ( I ) after initial photo - activation . The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm ( D ) . In the non - activated region 6 min after initial activation the width of the tissue shrank to about 25 µm ( G ) , whereas it remained constant at 40 µm in the activated region ( I ) . Data information : Scale bars , 20

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

Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY an the membrane marker GAP43::mCherry (D-I) were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation (D) Cross section (z-projection) showing the plasma membrane and (E) top view showing the apical plasma membrane at the initial time point before photo-activation. At this stage, the central mesodermal tissue has started to internalize and cells were constricted at the apical surface. In an alternating fashion, the base of the cells in the anterior half of the embryo (left) was photo-activated for 100 sec and the GAP43::mCherry signal in the entire field of view was recorded. The furrow further ingressed and closed completely 3 min (F) and 6 min (G) after initial activation, respectively. In the activated region, the tissue failed to internalize and to form a closed furrow both at 3 min (H) and 6 min (I) after initial photo-activation. The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm (D). In the non-activated region 6 min after initial activation the width of the tissue shrank to about 25 µm (G), whereas it remained constant at 40 µm in the activated region (I). Data information: Scale bars, 20 µm


A , B Quantification of mitotic ( A ) and meiotic ( B ) cells in the distal germline in wild - type animals fed bacteria transformed with either empty vector ( con ) or vector - expressing dsRNA against frh - 1 ( frh - 1 ) or isp - 1 ( isp - 1 ) Data information : Data are presented as mean ± SE ( A ) N = 3 at least 5 worms per replicate and condition , ( B ) N = 3 at least 10 worms for conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of mitotic (A) and meiotic (B) cells in the distal germline in wild-type animals fed bacteria transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) Data information: Data are presented as mean ± SE (A) N=3 at least 5 worms per replicate and condition, (B) N=3 at least 10 worms for conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Quantification of dissected distal germline from wild - type animal stained either anti - phosphohistone H3 ( anti - PH3 ) ( C relative to the total number of mitotic cells of wild - type animal Data informatio Data are presented a as mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

Quantification of dissected distal germline from wild-type animal stained either anti-phosphohistone H3 (anti-PH3) (C relative to the total number of mitotic cells of wild-type animal Data informatio Data are presented a as mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Quantification of dissected distal germline from wild - type animal stained eithe anti - CDK - 1 ( anti - CDK - 1 ( D relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

Quantification of dissected distal germline from wild-type animal stained eithe anti-CDK-1 (anti-CDK-1 (D relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Quantification of dissected distal germline from wild - type animal stained eithe 5 - ethynyl - 2 ′ - deoxyuridine ( EdU - green ) ( E ) relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - 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

Quantification of dissected distal germline from wild-type animal stained eithe 5-ethynyl-2′-deoxyuridine (EdU -green) (E) relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


F Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild - type worms fed as in ( A ) Data information : Data are presented as mean ± SE ( F ) N = 5 at least 10 worms per replicate and condition . * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - 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

F Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild-type worms fed as in (A) Data information: Data are presented as mean ± SE (F) N=5 at least 10 worms per replicate and condition. * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


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 with gamma radiation 125 Gy ( A 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

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 with gamma radiation 125 Gy (A 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


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 ) , UVC 400J / m2 ( B 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

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ), UVC 400J/m2 (B 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


Quantification of apoptotic corpse in the meiotic compartment o wild - type strain 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 different doses of UVB ( C 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

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

Quantification of apoptotic corpse in the meiotic compartment o wild-type strain 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 different doses of UVB (C 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


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

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

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

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

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

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 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

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

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 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

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

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 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

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

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 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

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

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 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

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

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


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

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


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

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

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

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

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

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

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

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 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

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 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

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

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 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

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

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


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

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

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


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

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) and , ced - 9 ( n1653ts strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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

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 bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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

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 bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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

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 bacteria 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 bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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

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 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

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

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 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

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

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 ) and , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp -

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

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 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

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

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 the indicated proteins 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.


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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 ) .

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 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 ) .

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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 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 ) .

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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 ) , 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 ) .

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

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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 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 )

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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 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

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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 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

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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 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 )

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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 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 ) .

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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 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 )

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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 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 .

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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 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 ) .

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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).


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 ) .

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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 images of T22 - GFP - H6 - FdU uptake and 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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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

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 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 .

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

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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 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 .

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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 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 .

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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 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 ) .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O 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 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 ] .

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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 the holoElongator complex . Particles in side and tilted views are highlighted . Scale bar , 50 nm .

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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 and back - projections of the holoElongator model . Scale bar , 20 nm .

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


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 .

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

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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 the Elp123 sub - complex . Particles in side and top views are highlighted . Scale bar , 50 nm .

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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 and back - projections of the 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 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 .

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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 .

A. The model of the Elp123 sub-complex.


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 .

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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 the orientation of the 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 ) 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 .

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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 and variants of Elp1 , Elp2 , and Elp456 .

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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 length Elp1 and truncations and deletions of Elp456 .

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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 .

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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 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 .

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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 the localization of Elp3 and the orientation of its KAT and SAM domain in relation to Elp1 and Elp2 .

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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 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 .

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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 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

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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 . 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

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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 . 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 )

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


D . Miro shRNAs mitigate 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

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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 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

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


F . Miro shRNAs inhibit basal mitophagy in 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 )

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


G . Miro shRNAs decrease 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 ) .

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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).