( A and B ) Cross - sectional electron micrographs of the proximal ( A ) and distal ( B ) regions of centrioles containing eight or nine triplets in bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 cells . The arrangement of triplets in images of axonemes were circularized using CentrioleJ program The colored circles represent the circles inscribed to the innermost positions of the A - tubules in the centriole . Scale bar , 100 nm . ( C and D ) Distances between adjacent A - tubules at the proximal ( C ) and distal ( D ) regions of centrioles containing seven , eight , nine , and ten triplets in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 cells . The distances between the contact points of A - tubules with the inscribed circle were measured . These distances were ~ 10 % shorter in cells expressing truncated Bld10ps than in wild - type and bld12 cells . * * * , P < 0 . 0001 . n , number of centrioles measured . Data information : Data were analyzed by unpaired t - test . The graphs show mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Cross-sectional electron micrographs of the proximal (A) and distal (B) regions of centrioles containing eight or nine triplets in bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The arrangement of triplets in images of axonemes were circularized using CentrioleJ program The colored circles represent the circles inscribed to the innermost positions of the A-tubules in the centriole. Scale bar, 100 nm. (C and D) Distances between adjacent A-tubules at the proximal (C) and distal (D) regions of centrioles containing seven, eight, nine, and ten triplets in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The distances between the contact points of A-tubules with the inscribed circle were measured. These distances were ~10% shorter in cells expressing truncated Bld10ps than in wild-type and bld12 cells. ***, P<0.0001. n, number of centrioles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.


( E ) Lengths of A - C linkers in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 centrioles . Centrioles containing nine triplets and eight triplets were separately analyzed . n , number of A - Clinkers measured . ( F ) Angles between adjacent triplets in centrioles analyzed in panel € . The angle ( θ ) measured was defined as indicated in the schematic diagram ; the angle between the line connecting the centers of B - tubules of the two adjacent triplets ( black line ) and the line connecting the centers of A - and C - tubules of the triplet of the two ( brown line ) . * , P < 0 . 01 . n , number of triplet angles measured . Data information : Data were analyzed by unpaired t - test . The graphs show mean ± SEM .

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

(E) Lengths of A-C linkers in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 centrioles. Centrioles containing nine triplets and eight triplets were separately analyzed. n, number of A-Clinkers measured. (F) Angles between adjacent triplets in centrioles analyzed in panel €. The angle (θ) measured was defined as indicated in the schematic diagram; the angle between the line connecting the centers of B-tubules of the two adjacent triplets (black line) and the line connecting the centers of A- and C-tubules of the triplet of the two (brown line). *, P<0.01. n, number of triplet angles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.


( A , B ) Flies form well - organized compound eyes when they carry GMR - GAL4 without a target gene ( A ) but develop rough eyes when GMR - GAL4 drives expression of UAS - Foxn1 ( B ) . Panel B shows the typical severity of the rough - eye phenotype . Data information : Representative phenotypes are shown in all panels . Scale bars , 100 μm .

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

(A, B) Flies form well-organized compound eyes when they carry GMR-GAL4 without a target gene (A) but develop rough eyes when GMR-GAL4 drives expression of UAS-Foxn1 (B). Panel B shows the typical severity of the rough-eye phenotype. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.


( C , D ) Bristles are pigmented and properly shaped when flies carry hs - GAL4 without a target gene ( C ) but are often white , bent , and shortened when hs - GAL4 drives expression of UAS - Foxn1 ( D ) . Flies in panels C and D were raised at 25 ° C . Arrows denote examples of white bristles . Data information : Representative phenotypes are shown in all panels . Scale bars , 100 μm .

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

(C, D) Bristles are pigmented and properly shaped when flies carry hs-GAL4 without a target gene (C) but are often white, bent, and shortened when hs-GAL4 drives expression of UAS-Foxn1 (D). Flies in panels C and D were raised at 25°C. Arrows denote examples of white bristles. Data information: Representative phenotypes are shown in all panels. Scale bars, 100 μm.


AFF4 ( green ) and FOXN1 ( red ) are shown by immunofluorescence in the following wild - type tissue samples : ( B ) murine hair follicles , P9 ; DNA is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 and FOXN1 generates yellow color , visible in cell nuclei of all samples . Arrowheads mark the CH , cortex of the hair ; Scale bars , 20 µm .

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

AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (B) murine hair follicles, P9; DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. Arrowheads mark the CH, cortex of the hair; Scale bars, 20 µm.


AFF4 ( green ) and FOXN1 ( red ) are shown by immunofluorescence in the following wild - type tissue samples : ( C ) developing murine epidermis , E16 . 5 ; ( D ) human epidermis , adult ; ( E ) murine thymus , P0 , outer region . DNA is stained by Hoechst dye 33258 ( blue ) . Co - localization of AFF4 and FOXN1 generates yellow color , visible in cell nuclei of all samples . In panel C , the fluorescent signal in the periderm ( P ) is nonspecific , as it is present when primary antibodies are omitted from the staining . Inner regions of the murine thymus ( not shown ) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E . Arrowheads mark the dermal / epidermal border ( C , D ) . CT , cortex of the thymus . Scale bars , 20 µm .

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

AFF4 (green) and FOXN1 (red) are shown by immunofluorescence in the following wild-type tissue samples: (C) developing murine epidermis, E16.5; (D) human epidermis, adult; (E) murine thymus, P0, outer region. DNA is stained by Hoechst dye 33258 (blue). Co-localization of AFF4 and FOXN1 generates yellow color, visible in cell nuclei of all samples. In panel C, the fluorescent signal in the periderm (P) is nonspecific, as it is present when primary antibodies are omitted from the staining. Inner regions of the murine thymus (not shown) yielded the same extensive overlap of FOXN1 and AFF4 staining as that shown in panel E. Arrowheads mark the dermal/epidermal border (C, D). CT, cortex of the thymus. Scale bars, 20 µm.


Representative skin phenotypes of wild - type ( WT ) mice shown macroscopically The phenotypes compared are as follows : hair coats , 4 - month - old

O O O O 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 skin phenotypes of wild-type (WT) mice shown macroscopically The phenotypes compared are as follows: hair coats, 4-month-old adults


Representative skin phenotypes of Foxn1nu / nu ( Nude ) , or KRT14 - cre ; Aff4flox / flox ( Aff4 cko ) mice are shown macroscopically or histologically after hematoxylin and eosin staining ( H - N ) . The phenotypes compared are as follows : hair coats , 4 - month - old adults ; ( E - G ) vibrissae , P7 ; ( H ) hair follicles with growing hairs , juveniles ; ( I - K ) regressing hair follicles , juveniles ; ( L - N ) epidermis , P9 . In panel H , micrographs from left to right are WT , Nude , and Aff4 cko . In panels I - K , HF marks examples of hair follicles . In panels L - M , arrowheads indicate the dermal / epidermal border ; arrows mark examples of bent hair shafts . Scale bars : H , L - N , 20 µm ; I - K , 40 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 skin phenotypes of Foxn1nu/nu (Nude), or KRT14-cre; Aff4flox/flox (Aff4 cko) mice are shown macroscopically or histologically after hematoxylin and eosin staining (H-N). The phenotypes compared are as follows: hair coats, 4-month-old adults; (E-G) vibrissae, P7; (H) hair follicles with growing hairs, juveniles; (I-K) regressing hair follicles, juveniles; (L-N) epidermis, P9. In panel H, micrographs from left to right are WT, Nude, and Aff4 cko. In panels I-K, HF marks examples of hair follicles. In panels L-M, arrowheads indicate the dermal/epidermal border; arrows mark examples of bent hair shafts. Scale bars: H, L-N, 20 µm; I-K, 40 µm.


( A - C ) Thymi from E18 . 5 embryos are shown : ( A ) intact with genotypes indicated , or ( B , C ) after sectioning , immunofluorescent staining for DLL4 ( red ) , and counterstaining of DNA ( blue ) . Sectioned samples are wild - type ( B ) or Aff4ko / ko ; Foxn1 + / - ( C ) . Scale bars : A , 1 mm ; B - C , 50 µm .

(A-C) Thymi from E18.5 embryos are shown: (A) intact with genotypes indicated, or (B, C) after sectioning, immunofluorescent staining for DLL4 (red), and counterstaining of DNA (blue). Sectioned samples are wild-type (B) or Aff4ko/ko; Foxn1+/- (C). Scale bars: A, 1 mm; B-C, 50 µm.


( C - E ) KRT86 ( red ) is shown by immunofluorescence in skin sections from wild - type ( WT ) , Foxn1 null , or Aff4 cko mice at P9 . DNA is stained blue . HS marks examples of hair shafts . Scale bar , 40 µm .

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

(C-E) KRT86 (red) is shown by immunofluorescence in skin sections from wild-type (WT), Foxn1 null, or Aff4 cko mice at P9. DNA is stained blue. HS marks examples of hair shafts. Scale bar, 40 µm.


Graphs present pre - mRNA ( B , C ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P6 ( B , C ) All values are derived from 3 independent experiments and presented as mean ± S . D . Pre - mRNA measurements were analyzed statistically using two - way ANOVA with post - hoc Sidak ' s multiple comparison test at a 95 % confidence interval .

Graphs present pre-mRNA (B, C) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P6 (B, C) All values are derived from 3 independent experiments and presented as mean ± S.D. Pre-mRNA measurements were analyzed statistically using two-way ANOVA with post-hoc Sidak's multiple comparison test at a 95% confidence interval.


Graphs present ChIP ( D - G ) assays of Krt86 using skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 ( D - G ) . ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated . All values are derived from 3 independent experiments and presented as mean ± S . D . ChIP assays were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison test at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

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

Graphs present ChIP (D-G) assays of Krt86 using skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7 (D-G). ChIP assays were performed with antibodies to FOXN1 or AFF4 as indicated. All values are derived from 3 independent experiments and presented as mean ± S.D. ChIP assays were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.


( B - G ) Graphs present ChIP assays of Krt86 using antibodies to total ( B , C ) , P - Ser5 ( D , E ) , or P - Ser2 ( F , G ) Pol II and skin from mutant mice ( Foxn1 null , Aff4 cko ) or corresponding wild - type controls at P7 . All values are derived from at least 3 independent experiments and presented as mean ± S . D . Assay results were analyzed statistically using one - way ANOVA with post - hoc Tukey ' s multiple comparison test at a 95 % confidence interval . * * * * , p ≤ 0 . 0001 .

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

(B-G) Graphs present ChIP assays of Krt86 using antibodies to total (B, C), P-Ser5 (D, E), or P-Ser2 (F, G) Pol II and skin from mutant mice (Foxn1 null, Aff4 cko) or corresponding wild-type controls at P7. All values are derived from at least 3 independent experiments and presented as mean ± S.D. Assay results were analyzed statistically using one-way ANOVA with post-hoc Tukey's multiple comparison test at a 95% confidence interval. ****, p ≤ 0.0001.


( B ) Immunoblotting of whole cell extracts obtained from parental and RAP80 - / - clones in the RPE - 1 hTERT p53 - / - Cas9 , RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 , U2OS , U2OS - 2 - 6 - 3 and U2OS Flp - In / TREx cell lines with RAP80 antibodies . Tubulin is used as loading control . Representative of at least two independent immunoblots . See Figure EV1 for further details on the gene editing strategy used to knockout RAP80 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblotting of whole cell extracts obtained from parental and RAP80-/- clones in the RPE-1 hTERT p53-/- Cas9, RPE-1 hTERT p53-/- BRCA1-/- Cas9, U2OS, U2OS-2-6-3 and U2OS Flp-In/TREx cell lines with RAP80 antibodies. Tubulin is used as loading control. Representative of at least two independent immunoblots. See Figure EV1 for further details on the gene editing strategy used to knockout RAP80.


( C , D ) The indicated parental and RAP80 - / - cell lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) and stained with antibodies against ABRAXAS1 and γH2AX . Shown in ( C ) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs of RPE1 - hTERT p53 - / - Cas9 wild type ( WT ) and RAP90 - / - cells are shown in ( D ) .

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

(C, D) The indicated parental and RAP80-/- cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against ABRAXAS1 and γH2AX. Shown in (C) is the percentage of cells with > 5 ABRAXAS1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs of RPE1-hTERT p53-/- Cas9 wild type (WT) and RAP90-/- cells are shown in (D).


( E , The indicated cell lines were processed for immunofluorescence 1 h post - irradiation ( 10 Gy ) and stained with antibodies against BRCA1 and γH2AX . Shown in ( E ) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates for all conditions except n = 5 for U2OS WT , n = 2 for U2OS 2 - 6 - 3 cells , n = 7 , 5 , 4 for U2OS Flp - In / T - REx cells ) .

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(E, The indicated cell lines were processed for immunofluorescence 1 h post-irradiation (10 Gy) and stained with antibodies against BRCA1 and γH2AX. Shown in (E) is the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates for all conditions except n=5 for U2OS WT, n=2 for U2OS 2-6-3 cells, n=7, 5, 4 for U2OS Flp-In/T-REx cells).


( G ) RPE - 1 hTERT p53 - / - Cas9 parental ( WT ) and RAP80 - / - cells were untreated ( t = 0 h ) or irradiated with a 10 Gy dose . Samples were collected at the indicated time points , processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX . Shown is the percentage of cells with > 5 BRCA1 foci . A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S . D . ( n = 3 biological replicates ) . All scale bars are 5 μm .

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

(G) RPE-1 hTERT p53-/- Cas9 parental (WT) and RAP80-/- cells were untreated (t=0 h) or irradiated with a 10 Gy dose. Samples were collected at the indicated time points, processed for immunofluorescence and stained with antibodies against BRCA1 and γH2AX. Shown is the percentage of cells with > 5 BRCA1 foci. A minimum of 100 cells per replicate were analyzed and the datapoints represent the mean ± S.D. (n=3 biological replicates). All scale bars are 5 μm.


( A , B ) U2OS Flp - In / T - Rex parental ( WT ) and RAP80 - / - cells were transfected with siRNA pools targeting BRCA1 , RNF8 , RNF168 or BARD1 or with a non - targeting control siRNA ( CTRL ) . 48 h post - transfection cells were irradiated ( 2 Gy ) and processed for immunofluorescence 1 h post - IR treatment using antibodies against BRCA1 and γH2AX . Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in ( A ) . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs are shown in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS Flp-In/T-Rex parental (WT) and RAP80-/- cells were transfected with siRNA pools targeting BRCA1, RNF8, RNF168 or BARD1 or with a non-targeting control siRNA (CTRL). 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 5 BRCA1 foci that colocalize with γH2AX is shown in (A). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (B).


( C , D ) Parental ( WT ) RPE1 - hTERT p53 - / - Cas9 , RNF8 - / - , and RNF168 - / - cells were treated with either a non - targeting siRNA pool ( CTRL ) or a pool targeting RAP80 . 48 h post - transfection cells were irradiated ( 2 Gy ) and processed for immunofluorescence 1 h post - IR treatment using antibodies against BRCA1 and γH2AX . Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in ( C ) . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D . ( n = 3 biological replicates ) . Representative micrographs are shown in ( D ) . All scale bars are 5 μm .

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

(C, D) Parental (WT) RPE1-hTERT p53-/- Cas9, RNF8-/-, and RNF168-/- cells were treated with either a non-targeting siRNA pool (CTRL) or a pool targeting RAP80. 48 h post-transfection cells were irradiated (2 Gy) and processed for immunofluorescence 1 h post-IR treatment using antibodies against BRCA1 and γH2AX. Quantitation of the percentage of cells with > 10 BRCA1 foci that colocalize with γH2AX is shown in (C). A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D. (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.


( A , B ) U2OS Flp - In / T - REx cells with integrated transgenes encoding GFP - BRCA1BRCT or - BRCA1BRCT S1655A were transfected with non - targeting siRNA ( CTRL ) or siRNA targeting RAP80 or BRCA1 . Following doxycycline treatment to induce transgene expression ( 5 μg / mL , 24 h ) , cells were irradiated ( 10 Gy ) and processed for immunofluorescence 1 h post - IR for GFP and antibodies against RAP80 and γH2AX . Shown in ( A ) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S . D ( n = 4 ) . Representative micrographs are shown in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS Flp-In/T-REx cells with integrated transgenes encoding GFP-BRCA1BRCT or -BRCA1BRCT S1655A were transfected with non-targeting siRNA (CTRL) or siRNA targeting RAP80 or BRCA1. Following doxycycline treatment to induce transgene expression (5 μg/mL, 24 h), cells were irradiated (10 Gy) and processed for immunofluorescence 1 h post-IR for GFP and antibodies against RAP80 and γH2AX. Shown in (A) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=4). Representative micrographs are shown in (B).


( C , D ) U2OS Flp - In / T - REx cells stably integrated with the indicated transgenes were treated with doxycycline ( 5 μg / mL , 36 h ) to induce protein expression and transfected with an siRNA targeting BRCA1 and also either non - targeting siRNA ( CTRL ) or siRNAs targeting RAP80 . 1 h post - irradiation ( 10 Gy ) cells were processed for immunofluorescence using GFP ( to detect the BRCA1 fusions ) and antibodies against γH2AX . Shown in ( C ) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in ( D ) . All scale bars are 5 μm .

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

(C, D) U2OS Flp-In/T-REx cells stably integrated with the indicated transgenes were treated with doxycycline (5 μg/mL, 36 h) to induce protein expression and transfected with an siRNA targeting BRCA1 and also either non-targeting siRNA (CTRL) or siRNAs targeting RAP80. 1 h post-irradiation (10 Gy) cells were processed for immunofluorescence using GFP (to detect the BRCA1 fusions) and antibodies against γH2AX. Shown in (C) is the quantitation of a minimum of 100 cells per replicate where the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in (D). All scale bars are 5 μm.


( A , B ) U2OS 2 - 6 - 3 parental ( WT ) or RAP80 - / - cell lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of the indicated GFP fusion proteins . 48 h post - nucleofection , mCherry - LacR - FokI expression was induced for 5 h prior to being processed for fluorescence microscopy for GFP and mCherry . Shown in ( A ) is the quantitation of GFP fluorescence at the mCherry focus where the bars represent the mean ± S . D ( n = 50 , 50 , 40 , 40 , 40 , 40 , 20 , 20 , 30 , 30 cells analyzed from 3 biological replicates ) . Representative micrographs for the BRCA1 and BRCA1 - I26A conditions are shown in ( B ) . Additional micrographs for the other conditions are in Figure EV2C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) U2OS 2-6-3 parental (WT) or RAP80-/- cell lines were transfected with siRNAs targeting BRCA1 followed by nucleofection of the indicated GFP fusion proteins. 48 h post-nucleofection, mCherry-LacR-FokI expression was induced for 5 h prior to being processed for fluorescence microscopy for GFP and mCherry. Shown in (A) is the quantitation of GFP fluorescence at the mCherry focus where the bars represent the mean ± S.D (n= 50, 50, 40, 40, 40, 40, 20, 20, 30, 30 cells analyzed from 3 biological replicates). Representative micrographs for the BRCA1 and BRCA1-I26A conditions are shown in (B). Additional micrographs for the other conditions are in Figure EV2C.


( C ) RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 cells ( WT ) or their isogenic RAP80 - / - counterparts expressing the indicated GFP fusion proteins were processed 1 h post - irradiation ( 10 Gy ) for immunofluorescence using GFP and antibodies against BRCA1 and γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in Figure EV3B . ( D ) RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 cells or their isogenic RAP80 - / - counterparts expressing the indicated GFP fusion proteins were processed 1 h post - irradiation ( 10 Gy ) for immunofluorescence using GFP and antibodies against RAD51 and γH2AX . A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in Figure EV3C . Scale bar is 5 μm in ( B ) .

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

(C) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells (WT) or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against BRCA1 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3B. (D) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against RAD51 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3C. Scale bar is 5 μm in (B).


( A , B ) MDA - MB - 436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated ( 10 Gy ) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation . Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies . Shown in ( A ) is the quantitation of a minimum of 100 EdU + cells per replicate and the bars represent the mean ± S . D ( n = 3 biological replicates ) . Representative micrographs are shown in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies. Shown in (A) is the quantitation of a minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). Representative micrographs are shown in (B).


MDA - MB - 436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated ( 10 Gy ) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation . Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies . Representative micrographs are shown in ( D ) .

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

MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies. Representative micrographs are shown in (D).


( E ) Clonogenic survival assays using the indicated dose of olaparib and MDA - MB - 436 cells transduced with lentivirus expressing the indicated HA fusions . Data is shown as the mean ± S . D ( n = 3 biological replicates ) . Representative clonogenic images are shown in Figure EV5D .

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

(E) Clonogenic survival assays using the indicated dose of olaparib and MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions. Data is shown as the mean ± S.D (n=3 biological replicates). Representative clonogenic images are shown in Figure EV5D.


SDS - PAGE of SecYEG - ND sample after size - exclusion chromatography . Asterisks indicate translocon - enriched fractions used for forming the RNC FtsQ : SecYEG - ND complex . Lipid - loaded " empty " nanodiscs elute at larger volumes and so can be separated .

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

SDS-PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated.


Representative cryo - EM micrograph of RNC FtsQ : SecYEG - ND . Exemplary individual ribosomes are encircled . Examples of two - dimensional classes of imaged particles . RNC : nanodisc assemblies can be seen at different view angles .

O O O O O O O O O O O O O O O O O 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 cryo-EM micrograph of RNC FtsQ:SecYEG-ND. Exemplary individual ribosomes are encircled. Examples of two-dimensional classes of imaged particles. RNC:nanodisc assemblies can be seen at different view angles.


Western - blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross - linking product of ~ 80 kDa in presence of nanodisc - reconstituted SecYC282EG and SecYC283EG , presumably assigned to FtsQxSecY adduct . The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa - histidine tag by 3C protease .

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

Western-blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross-linking product of ~80 kDa in presence of nanodisc-reconstituted SecYC282EG and SecYC283EG, presumably assigned to FtsQxSecY adduct. The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa-histidine tag by 3C protease.


In - gel fluorescence imaging of SecYCF488A reveals a cross - linking product of ~ 80 kDa assigned to the FtsQC40xSecYC283 adduct ( " SecYxFtsQ " ) . Bands for covalently cross - linked SecY dimer ( " SecYxSecY " ) , and adducts of SecY and ribosomal proteins ( " SecYxRiboP " ) are indicated . The nascent chain - specific adduct " SecYxFtsQ " does not depend on the presence of PE lipids .

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

In-gel fluorescence imaging of SecYCF488A reveals a cross-linking product of ~80 kDa assigned to the FtsQC40xSecYC283 adduct ("SecYxFtsQ"). Bands for covalently cross-linked SecY dimer ("SecYxSecY"), and adducts of SecY and ribosomal proteins ("SecYxRiboP") are indicated. The nascent chain-specific adduct "SecYxFtsQ" does not depend on the presence of PE lipids.


FtsQ : SecY cross - linking is position specific . FtsQ TMH C - terminal residues 40 - 42 could be cross - linked to the lateral gate ( SecY residue 283 ) , while no adduct could be detected at the upstream position 35 .

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

FtsQ:SecY cross-linking is position specific. FtsQ TMH C-terminal residues 40-42 could be cross-linked to the lateral gate (SecY residue 283), while no adduct could be detected at the upstream position 35.


C . Proteins from whole cell extracts and culture supernatants of PAKΔretS , the derivative tssA1 mutant ( ΔA1 ) ( both carrying the pBBR plasmid ) and the complemented ΔA1 mutant carrying pBBR - tssA1 ( ΔA1 + A1 ) were analysed by Western blot . Polyclonal antibodies directed against TssA1 ( A1 ) , Hcp1 , VgrG1a and Tse3 were used . The anti - VgrG1a antibody detects VgrG1a , VgrG1b and VgrG1c , as indicated on the left . Cytoplasmic RNA polymerase ( RNAP ) was monitored using monoclonal antibody directed against its β - subunit .

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

C. Proteins from whole cell extracts and culture supernatants of PAKΔretS, the derivative tssA1 mutant (ΔA1) (both carrying the pBBR plasmid) and the complemented ΔA1 mutant carrying pBBR-tssA1 (ΔA1+A1) were analysed by Western blot. Polyclonal antibodies directed against TssA1 (A1), Hcp1, VgrG1a and Tse3 were used. The anti-VgrG1a antibody detects VgrG1a, VgrG1b and VgrG1c, as indicated on the left. Cytoplasmic RNA polymerase (RNAP) was monitored using monoclonal antibody directed against its β-subunit.


A . BTH experiments showing that TssA1 is able to self - interact in vivo . A graphical representation of β - galactosidase activity from co - transformants of E . coli DHM1 cells producing TssA1 ( A1 ) fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in quadruplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BTH experiments showing that TssA1 is able to self-interact in vivo. A graphical representation of β-galactosidase activity from co-transformants of E. coli DHM1 cells producing TssA1 (A1) fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in quadruplicate.


B . In vitro cross - linking experiments of purified His6 - TssA1 . About 30 μg of purified His6 - TssA1 was cross - linked ( 30 min , 1h and 2h ) at room temperature using increasing amount of ethylene glycol - bis ( succinimidylsuccinate ) EGS ( 2 mM and 5 mM ) where indicated . Western blot analysis of cross - linked products using an anti - His6 monoclonal antibody is shown . The cross - linked species are highlighted with stars with the corresponding oligomeric state indicated on the right ( 1x = monomer ; 2x = dimer ; 6x = hexamer ) . Molecular weight markers ( kDa ) are indicated on the left .

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

B. In vitro cross-linking experiments of purified His6-TssA1. About 30 μg of purified His6-TssA1 was cross-linked (30 min, 1h and 2h) at room temperature using increasing amount of ethylene glycol-bis(succinimidylsuccinate) EGS (2 mM and 5 mM) where indicated. Western blot analysis of cross-linked products using an anti-His6 monoclonal antibody is shown. The cross-linked species are highlighted with stars with the corresponding oligomeric state indicated on the right (1x = monomer; 2x = dimer; 6x = hexamer). Molecular weight markers (kDa) are indicated on the left.


C . Analysis of the oligomeric state of TssA1 by AUC . Sedimentation data of His6 - TssA1 ( 0 . 56 mg / ml ) recorded at a rotor speed of 20 , 000 rpm is shown . The left panel shows the sedimentation boundary fits and for clarity , only every third scan is shown in the fitted data plots . The experimental absorbance data are shown as black circles , whereas the boundary fits are shown as grey lines . The right panel shows the size - distribution analysis c ( s ) , obtained from fitting the scan boundaries using SEDFIT , revealing a dodecameric peak ( Mr ~ 479 kDa ) at S20 , w value of 16 . 6 S with a minor peak ( Mr ~ 1 MDa ) at S20 , w value of 29 . 01 S .

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

C. Analysis of the oligomeric state of TssA1 by AUC. Sedimentation data of His6-TssA1 (0.56 mg/ml) recorded at a rotor speed of 20,000 rpm is shown. The left panel shows the sedimentation boundary fits and for clarity, only every third scan is shown in the fitted data plots. The experimental absorbance data are shown as black circles, whereas the boundary fits are shown as grey lines. The right panel shows the size-distribution analysis c(s), obtained from fitting the scan boundaries using SEDFIT, revealing a dodecameric peak (Mr ~ 479 kDa) at S20,w value of 16.6 S with a minor peak (Mr ~1 MDa) at S20,w value of 29.01 S.


A . A representative micrograph of the TssA1 complex ( left panel ) showing discrete ring - shaped particles with a diameter of ~ 260 Å . The rotationally symmetrised average of the data set ( inset panel ) presents two distinct rings ( numbered ) , disclosing the layered nature of the TssA1 complex . Representative class averages of the TssA1 complex ( right panel ) demonstrating the wide range of conformations taken by the ring - shaped structures . Scale bars are 500 Å in the left panel , 130 Å in the inset and 100 Å in the right panels , respectively .

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

A. A representative micrograph of the TssA1 complex (left panel) showing discrete ring-shaped particles with a diameter of ~260 Å. The rotationally symmetrised average of the data set (inset panel) presents two distinct rings (numbered), disclosing the layered nature of the TssA1 complex. Representative class averages of the TssA1 complex (right panel) demonstrating the wide range of conformations taken by the ring-shaped structures. Scale bars are 500 Å in the left panel, 130 Å in the inset and 100 Å in the right panels, respectively.


B . Double MSA classification : ( i ) representative orientation class averages show TssA1 rings arranged into four - fold ( 4x ) , five - fold ( 5x ) and six - fold ( 6x ) symmetry - like complexes ( indicated in red , blue and green , respectively ) ; ( ii ) sub - class averages obtained from sub - classification of the corresponding orientation class ; and ( iii ) calculated difference images showing some white or black peaks ( coloured circles ) at the outer ring region . Scale bar is 100 Å .

B. Double MSA classification: (i) representative orientation class averages show TssA1 rings arranged into four-fold (4x), five-fold (5x) and six-fold (6x) symmetry-like complexes (indicated in red, blue and green, respectively); (ii) sub-class averages obtained from sub-classification of the corresponding orientation class; and (iii) calculated difference images showing some white or black peaks (coloured circles) at the outer ring region. Scale bar is 100 Å.


D . SEC - MALS analysis of the purified His6 - TssA11 - 245 . The TssA11 - 245 elution peak is shown and the corresponding mass is indicated . The molar mass and the differential refractive index ( RI ) are plotted versus the elution volume . Data were analysed using the ASTRA method ( Slotboom , Duurkens et al . , 2008 ) .

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

D. SEC-MALS analysis of the purified His6-TssA11-245. The TssA11-245 elution peak is shown and the corresponding mass is indicated. The molar mass and the differential refractive index (RI) are plotted versus the elution volume. Data were analysed using the ASTRA method (Slotboom, Duurkens et al., 2008).


A . Co - purification experiments using His6 - TssA1 and untagged TssB1C1 . Western blot analysis of the Ni - NTA outputs using antibodies directed against the His6 - tag ( upper panel ) and TssB1 ( lower panel ) . I , Input ; FT , Flow through ; W , Wash and E , Elution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-purification experiments using His6-TssA1 and untagged TssB1C1. Western blot analysis of the Ni-NTA outputs using antibodies directed against the His6-tag (upper panel) and TssB1 (lower panel). I, Input; FT, Flow through; W, Wash and E, Elution.


B . SEC elution fraction of the co - purified His6 - TssA1 and TssB1C1 . SDS - PAGE showing the presence of the three proteins His6 - TssA1 ( A1 ) , TssB1 ( B1 ) and TssC1 ( C1 ) . Molecular weight markers ( kDa ) are indicated on the left .

B. SEC elution fraction of the co-purified His6-TssA1 and TssB1C1. SDS-PAGE showing the presence of the three proteins His6-TssA1 (A1), TssB1 (B1) and TssC1 (C1). Molecular weight markers (kDa) are indicated on the left.


C . Representative micrographs of the immunogold - labelled complex His6 - TssA1 / TssB1C1 . The presence of gold particles is indicated with white arrows . Two close - up views of TssB1C1 sheath displaying gold particles at one extremity are shown ( inset panels ) . Scale bars are 2 , 000 Å and 500 Å for images 1 and 2 - 6 , respectively .

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

C. Representative micrographs of the immunogold-labelled complex His6-TssA1/TssB1C1. The presence of gold particles is indicated with white arrows. Two close-up views of TssB1C1 sheath displaying gold particles at one extremity are shown (inset panels). Scale bars are 2,000 Å and 500 Å for images 1 and 2-6, respectively.


D . BTH experiment showing interaction between TssA1 ( A1 ) and Hcp1 . A graphical representation of β - galactosidase activity from E . coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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

D. BTH experiment showing interaction between TssA1 (A1) and Hcp1. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


B . Cartoon representations of the dodecameric gp6 _ 334C structure ( PDB code 3H3W ) . Chains A and B of gp6 dimers are shown in magenta and green respectively , and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange . A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly , is shown on the right .

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

B. Cartoon representations of the dodecameric gp6_334C structure (PDB code 3H3W). Chains A and B of gp6 dimers are shown in magenta and green respectively, and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange. A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly, is shown on the right.


A Fluorescence microscopy images showing the presence / absence of TssBC sheaths ( seen as fluorescent foci ) in the ∆ B1 : : B1 - sfGFP ( top panel ) , ∆ B1 ∆ A1 : : B1 - sfGFP , ∆ B1 ∆ E1 : : B1 - sfGFP cells ( middle panels ) and in the complemented strains ∆ B1 ∆ A1 : : B1 - sfGFP + A1 and ∆ B1 ∆ E1 : : B1 - sfGFP + E1 ( bottom panels ) . Scale bars are 2 μm . B . Quantification of TssB1 - sfGFP foci and of diffuse fluorescence in various P . aeruginosa backgrounds imaged in panel A . Values are normalized to the total number of bacteria ( % ) . Data were obtained from three independent experiments , with images and values from representative replicates being shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence microscopy images showing the presence/absence of TssBC sheaths (seen as fluorescent foci) in the ∆B1::B1-sfGFP (top panel), ∆B1∆A1::B1-sfGFP, ∆B1∆E1::B1-sfGFP cells (middle panels) and in the complemented strains ∆B1∆A1::B1-sfGFP + A1 and ∆B1∆E1::B1-sfGFP + E1 (bottom panels). Scale bars are 2 μm. B. Quantification of TssB1-sfGFP foci and of diffuse fluorescence in various P. aeruginosa backgrounds imaged in panel A. Values are normalized to the total number of bacteria (%). Data were obtained from three independent experiments, with images and values from representative replicates being shown.


BTH screen for interactions between TssA1 and T6SS components of interest . A graphical representation ( bottom ) of β - galactosidase activity from E . coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits and images of corresponding spots on X - gal LB agar plates ( top ) are shown . The combination of T25 / T18 fusion proteins are indicated as 25 or 18 followed by the name of the fused T6SS protein . T6SS proteins are abbreviated as follows : A1 = TssA1 , E1 = TssE1 , F1 = TssF1 , G1 = TssG1 and K1 = TssK1 . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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

BTH screen for interactions between TssA1 and T6SS components of interest. A graphical representation (bottom) of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits and images of corresponding spots on X-gal LB agar plates (top) are shown. The combination of T25/T18 fusion proteins are indicated as 25 or 18 followed by the name of the fused T6SS protein. T6SS proteins are abbreviated as follows: A1=TssA1, E1=TssE1, F1=TssF1, G1=TssG1 and K1=TssK1. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


B . Cartoon representation of the gp6 / gp8 baseplate subcomplex of the T4 phage . The gp6 ring structure ( PDB code 3H3W ) is shown using the same colour code described in figure 5 . The chains A and B of the gp8 dimers ( PDB code 1PDM ) are shown in blue and yellow , respectively . The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange . A close up view of the gp8 structure ( bottom ) shows that the conserved blocks are involved in dimerization and interaction with gp6 .

B. Cartoon representation of the gp6/gp8 baseplate subcomplex of the T4 phage. The gp6 ring structure (PDB code 3H3W) is shown using the same colour code described in figure 5. The chains A and B of the gp8 dimers (PDB code 1PDM) are shown in blue and yellow, respectively. The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange. A close up view of the gp8 structure (bottom) shows that the conserved blocks are involved in dimerization and interaction with gp6.


A . Structure of the sheath / baseplate complex of the bacteriophage T4 , adapted from Kostyuchenko et al . ( Kostyuchenko , Leiman et al . , 2003 ) . A colour code with corresponding gp protein is provided . The proposed corresponding homolog in the T6SS is indicated with parentheses .

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A. Structure of the sheath/baseplate complex of the bacteriophage T4, adapted from Kostyuchenko et al. (Kostyuchenko, Leiman et al., 2003). A colour code with corresponding gp protein is provided. The proposed corresponding homolog in the T6SS is indicated with parentheses.


I Correlation between peptide - spectrum matches ( PSMs ) of identified proteins and their fold change calculated by SAINTexpress .

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I Correlation between peptide-spectrum matches (PSMs) of identified proteins and their fold change calculated by SAINTexpress.


A Pulldown and Western blot analysis validated the interaction between the viral protein ORF3a and its interactors , VPS11 and CLCC1 , and the binding of S protein to SPCS2 . HEK293T cells with SFB - tagged bait expression were collected and lysed . Cell lysates were subjected to pulldown assay using S - protein beads . Western blot analysis was conducted with the indicated antibodies . Cells transfected with vector or construct encoding control genes were included as controls in these experiments .

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A Pulldown and Western blot analysis validated the interaction between the viral protein ORF3a and its interactors, VPS11 and CLCC1, and the binding of S protein to SPCS2. HEK293T cells with SFB-tagged bait expression were collected and lysed. Cell lysates were subjected to pulldown assay using S-protein beads. Western blot analysis was conducted with the indicated antibodies. Cells transfected with vector or construct encoding control genes were included as controls in these experiments.


B Immunostaining analysis of protein localization . U2OS cells were transfected with construct encoding ORF3a . Cells were fixed and stained with the indicated antibodies . The green signal is CLCC1 or lysosome marker LAMP1 , the red signal is flag ( for SFB - ORF3a ) , and the blue signal indicates DAPI / nuclei . Scale bar : 10 µm .

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B Immunostaining analysis of protein localization. U2OS cells were transfected with construct encoding ORF3a. Cells were fixed and stained with the indicated antibodies. The green signal is CLCC1 or lysosome marker LAMP1, the red signal is flag (for SFB-ORF3a), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.


B Comparison of SARS - CoV - 2 virus - host interaction to the genetic screening with CRISPR technique . Two different MOIs ( MOI 0 . 01 and MOI 0 . 3 ) were used in these screens and we compared our results with both of these datasets . Blue dots are the identified genes from the genetic screening . Orange dots are the highlighted overlapped genes between our virus - host interaction study and the results from the genetic screening . Top five overlapped genes are indicated .

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B Comparison of SARS-CoV-2 virus-host interaction to the genetic screening with CRISPR technique. Two different MOIs (MOI 0.01 and MOI 0.3) were used in these screens and we compared our results with both of these datasets. Blue dots are the identified genes from the genetic screening. Orange dots are the highlighted overlapped genes between our virus-host interaction study and the results from the genetic screening. Top five overlapped genes are indicated.


C Analyzing the number of HCIPs with RNA expression changes identified by single - cell RNA sequencing using cells from COVID - 19 patients .

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C Analyzing the number of HCIPs with RNA expression changes identified by single-cell RNA sequencing using cells from COVID-19 patients.


D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1 / PYCR2 . Cells transfected with vector or construct encoding GFP were included as controls in these experiments .

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D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1/PYCR2. Cells transfected with vector or construct encoding GFP were included as controls in these experiments.


C Heat map of the N protein HCIPs identified by SFB - tandem affinity purification . N protein HCIPs were compared among seven human coronaviruses using the preys ' spectral counts . Three areas of the heat map are manually selected , enlarged and labeled as ( 1 ) , ( 2 ) , and ( 3 ) , which are enriched by all N proteins of the seven human coronaviruses . Functional characterization for each protein is shown with the red circles below the enlarged images .

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C Heat map of the N protein HCIPs identified by SFB-tandem affinity purification. N protein HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Three areas of the heat map are manually selected, enlarged and labeled as (1), (2), and (3), which are enriched by all N proteins of the seven human coronaviruses. Functional characterization for each protein is shown with the red circles below the enlarged images.


A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses . PSM , peptide - spectrum match .

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A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses. PSM, peptide-spectrum match.


B Pulldown and Western blot validation of the interaction between N protein and G3BP1 / G3BP2 . Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments .

B Pulldown and Western blot validation of the interaction between N protein and G3BP1/G3BP2. Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments.


C , D Effect of SARS - CoV - 2 N protein on stress granule formation . A549 ( C ) and MCF10A ( D ) cells were transfected with pinducer20 - N protein of SARS - CoV - 2 . All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression . Cells were fixed and stained with the indicated antibodies . The green signal is G3BP1 , the red signal is HA ( for N protein ) , and the blue signal indicates DAPI / nuclei . Scale bar : 10 µm .

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C,D Effect of SARS-CoV-2 N protein on stress granule formation. A549 (C) and MCF10A (D) cells were transfected with pinducer20-N protein of SARS-CoV-2. All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression. Cells were fixed and stained with the indicated antibodies. The green signal is G3BP1, the red signal is HA (for N protein), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.


E Quantification of the stress granule formation in A549 and MCF10A cells . Total of 30 cells were counted and a student ' s t test was used for the statistical analysis ( * * * , p < 0 . 001 ) . Box limits represent 25th percentile and 75th percentile ; horizontal line represents median . Whiskers display min . to max . values .

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E Quantification of the stress granule formation in A549 and MCF10A cells. Total of 30 cells were counted and a student's t test was used for the statistical analysis (***, p<0.001). Box limits represent 25th percentile and 75th percentile; horizontal line represents median. Whiskers display min. to max. values.


( A ) Electron micrograph of an area with abundant double - membrane spherules ( DMSs ) . DMVs ( asterisks ) are interspersed and surrounding the DMS cluster . ( B ) Slice through a tomogram ( left ) and corresponding surface - rendered model ( right ) of a representative area containing the different types of MERS - CoV - induced membrane modifications : DMSs ( orange ) , convoluted membranes ( CM , blue ) and DMVs ( yellow and lilac , outer and inner membranes ) . The model also highlights ER membranes ( green ) and a vesicle ( silver ) containing new virions ( pink ) . ( See also S1 Video ) . ( C ) Comparison of DMSs and virions ( arrowheads in left and right panels , respectively ) in enlarged views of tomographic slices from the regions boxed in ( B ) . The DMSs are similar in size but distinct in appearance from newly - formed MERS - CoV particles . ( D ) Whisker plots of the size distribution of DMSs ( n = 58 ) , virions ( n = 28 ) and DMVs ( n = 109 ) , as measured from the tomograms . DMSs and virions have a comparable size ( median diameter , 80 nm ) , while the median diameter of the DMVs is 247 nm . ( E ) Models and tomographic slices through an open ( left ) and closed ( right ) DMS . Both types of DMSs are connected with the CM . In open DMSs , both the inner and outer membranes ( dark blue and orange , respectively ) are continuous with CM . Two ~ 8 - nm apart slices through the reconstruction are shown . For closed DMSs only the outer membrane is connected to CM , while the inner membrane seems to define a closed compartment .

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(A) Electron micrograph of an area with abundant double-membrane spherules (DMSs). DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video). (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly-formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n=58), virions (n=28) and DMVs (n=109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), while the median diameter of the DMVs is 247 nm. (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two ~8-nm apart slices through the reconstruction are shown. For closed DMSs only the outer membrane is connected to CM, while the inner membrane seems to define a closed compartment.


( F ) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS - CoV RO , and of these with the ER . These include CM - ER ( black arrowheads ) , DMV - ER ( white arrowheads ) , CM - DMV ( dark grey arrowheads ) , and CM - DMS ( light grey arrowhead ) connections . Scale bars , 250 nm ( A , B ) , and 100 nm ( C - F ) .

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(F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO, and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (dark grey arrowheads), and CM-DMS (light grey arrowhead) connections. Scale bars, 250 nm (A, B), and 100 nm (C -F).


alpha - CoVs . 2D - EM images from 100 - nm thick sections of different mammal cells infected with ( A ) SARS - CoV ( MOI 10 , 9 hpi ) , ( B ) MHV ( MOI 10 , 8 hpi ) and ( C ) HCoV - 229E ( MOI 5 , 24 hpi ) . Both beta - CoVs ( A , B ) and the alpha - CoV ( C ) induce membrane modifications that include not only DMVs ( asterisks ) and CM , but also DMSs ( white arrowheads ) . Scale bars , 250 nm .

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alpha-CoVs.2D-EM images from 100-nm thick sections of different mammal cells infected with (A) SARS-CoV (MOI 10, 9 hpi), (B) MHV (MOI 10, 8 hpi) and (C) HCoV-229E (MOI 5, 24 hpi). Both beta-CoVs (A,B) and the alpha-CoV (C) induce membrane modifications that include not only DMVs (asterisks) and CM, but also DMSs (white arrowheads). Scale bars, 250 nm.


( A ) Overview of an infected cell in which regions with different virus - induced modifications are annotated in yellow ( DMVs ) , blue ( CM ) and orange ( DMSs ) . Several densely - labelled regions containing DMVs , but not the other virus - induced structures are apparent . A close - up of one of these regions ( boxed area ) is shown in ( B ) .

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(A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM) and orange (DMSs). Several densely-labelled regions containing DMVs, but not the other virus-induced structures are apparent. A close-up of one of these regions (boxed area) is shown in (B).


( C , D ) Distribution of the autoradiography signal around DMVs ( nDMVs = 36 , see Material and Methods for selection criteria and details ) . The data is plotted ( C ) as a histogram , or ( D ) normalized by the radius to the DMV centre to account for the increase in the screened area . Scale bars , ( A ) 5 μm , ( B ) 500 nm .

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(C, D) Distribution of the autoradiography signal around DMVs (nDMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the screened area. Scale bars, (A) 5 μm, (B) 500 nm.


( A ) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS - CoV - infected Vero E6 cells ( MOI 10 ) , as measured by scintillation counting on the RNA isolated from the cells . The label was provided simultaneously to all the samples at 6 hpi .

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(A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells. The label was provided simultaneously to all the samples at 6 hpi.


( B - D ) EM detection by autoradiography . ( B ) Overview of a SARS - CoV - infected Vero E6 cell ( MOI 10 ) were labelled for 20 min just before fixation at 7 hpi , and then processed for EM autoradiography . Autoradiography grains accumulate in the RO regions . N , nucleus ; m , mitochondria ; VCR , virion - containing regions ; LD , lipid droplets . Scale bar , 1 μm .

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(B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10) were labelled for 20 min just before fixation at 7 hpi, and then processed for EM autoradiography. Autoradiography grains accumulate in the RO regions. N, nucleus; m, mitochondria; VCR, virion-containing regions; LD, lipid droplets. Scale bar, 1 μm.


( C , D ) Quantification of the autoradiography signal per subcellular structure ( see also S1 Table ) . Labelling densities and relative labelling indexes ( RLI ) in different subcellular regions of ( C ) Vero E6 cells infected with SARS - CoV ( MOI 10 ) or ( D ) Huh7 cells infected with MERS - CoV ( MOI 5 ) . Radioactively - labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi , respectively . Control mock - infected cells are excluded from the RLI plots , as RLI comparisons between conditions require the same number of classes ( subcellular regions ) and these cells lack ROs and virions .

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(C, D) Quantification of the autoradiography signal per subcellular structure (see also S1 Table). Labelling densities and relative labelling indexes (RLI) in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively-labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions.


( A ) Overview of a cluster of MERS - CoV - induced membrane modifications in Huh7 cells prepared as described in Fig . 4 . Some DMSs are boxed in orange while regions with CM are encircled in blue . In comparison with the densely - labelled surrounding DMVs , these regions are relatively devoid of autoradiography signal .

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(A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig. 4. Some DMSs are boxed in orange while regions with CM are encircled in blue. In comparison with the densely-labelled surrounding DMVs, these regions are relatively devoid of autoradiography signal.


( B ) The distribution of autoradiography grains on CM was not homogeneous ( nCM = 9 ) , and label was predominantly found close to the boundaries of the CM , as expected if the signal arises from the surrounding DMVs .

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(B) The distribution of autoradiography grains on CM was not homogeneous (nCM=9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs.


( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . ( C ) Enlargements of the DMS areas boxed in ( A ) .

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(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A).


( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . Most DMSs were devoid of signal and those who contained label were close to labelled DMVs ( D ) ( nDMS = 105 ) .

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(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). Most DMSs were devoid of signal and those who contained label were close to labelled DMVs (D) (nDMS = 105).


( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . ( E ) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre , as expected from a random distribution ( nDMSs = 54 ) . Scale bars , ( A ) 500 nm , ( C ) 100 nm .

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(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre, as expected from a random distribution (nDMSs = 54). Scale bars, (A) 500 nm, (C) 100 nm.


IEM detection of viral markers in MERS - CoV - infected cells . ( A - G ) Immunogold labeling of thawed cryo - sections of MERS - CoV - infected Huh7 cells ( 12 hpi ) for the detection of the indicated viral proteins . ( A - C ) Structural proteins were detected on virions ( black arrowheads ) and , for the M and S proteins , also on Golgi cisterna . While regions containing DMS ( white arrowheads ) and CM labelled for the N protein ( D ) and nsp3 ( G ) , the M and S protein were not detected in these areas . ( H - I ) Immunogold labeling of dsRNA in HPF - FS samples of MERS - CoV - infected Huh7 cells ( 13 hpi ) . The label accumulated on DMVs , which could be easily detected in this type of samples ( grey arrowheads ) , while the regions with CM and DMSs , which appeared as dark areas among the DMV clusters , where devoid of dsRNA signal . G , Golgi complex ; m , mitochondria . Scale bars , 250 nm .

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IEM detection of viral markers in MERS-CoV-infected cells.(A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.


( A ) Netrin - 1 ( NTN1 ) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas .

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(A) Netrin-1 (NTN1) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas.


( B ) Reporter gene expression in the midbrain ( substantia nigra ( SN ) and ventral tegmental area ( VTA ) ) of netrin - 1 + / lacZ + adult ( P60 ) mice incubated with S - Gal . ( Scale bar , 2000 µm ) . Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel .

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(B) Reporter gene expression in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA)) of netrin-1+/lacZ+ adult (P60) mice incubated with S-Gal. (Scale bar, 2000 µm). Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel.


( C ) Netrin - 1 protein localization in the SN of adult rat . ( Scale bar , 200 µm ) . Upper panel , confocal image of the SN . Higher magnification of the SNpc ( bottoms panels ) . Netrin - 1 ( red ) , Tyrosine hydroxylase ( TH ) ( green ) .

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(C) Netrin-1 protein localization in the SN of adult rat. (Scale bar, 200 µm). Upper panel, confocal image of the SN. Higher magnification of the SNpc (bottoms panels). Netrin-1 (red), Tyrosine hydroxylase (TH) (green).


( D ) Localization of netrin - 1 protein in the adult rat striatum . ( Scale bar , 600µm ) . Left panel , confocal image of the rat striatum . Netrin - 1 ( red ) , Dopamine transporter ( DAT ) ( green ) . Right panels , higher magnification .

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(D) Localization of netrin-1 protein in the adult rat striatum. (Scale bar, 600µm). Left panel, confocal image of the rat striatum. Netrin-1 (red), Dopamine transporter (DAT) (green). Right panels, higher magnification.


( A ) Representative images of dopamine neurons ( TH - positive ) in the substantia nigra ( SN ) ( left panel ) and in the SN and striatum ( right panel top , and bottom respectively ) by immunofluorescence , six weeks after the intranigral injection of control ( CTL ) ( AAV6 - GFP ) or Cre ( AAV6 - GFP Cre ) adenovirus vectors in Netrin - 1fl / fl mice ( left panel : green - GFP , red - TH ; right panel : Cy - 5 - TH ) ( Scale bar , 1000 μm ) . Filled arrows indicate SN TH dopamine neurons .

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(A) Representative images of dopamine neurons (TH-positive) in the substantia nigra (SN) (left panel) and in the SN and striatum (right panel top, and bottom respectively) by immunofluorescence, six weeks after the intranigral injection of control (CTL) (AAV6-GFP) or Cre (AAV6-GFP Cre) adenovirus vectors in Netrin-1fl/fl mice (left panel: green-GFP, red-TH; right panel: Cy-5-TH) (Scale bar, 1000 μm). Filled arrows indicate SN TH dopamine neurons.


( B ) Representative images of TH and netrin - 1 stainings by immunofluorescence ( scale bar , 2000 μm ) six weeks after the intranigral injection of CTL adenovirus vector ( left panel ) or Cre adenovirus vector ( right panel ) , in the midbrain ( substantia nigra ( SN ) and ventral tegmental area ( VTA ) of Netrin - 1fl / fl mice .

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(B) Representative images of TH and netrin-1 stainings by immunofluorescence (scale bar, 2000 μm) six weeks after the intranigral injection of CTL adenovirus vector (left panel) or Cre adenovirus vector (right panel), in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA) of Netrin-1fl/fl mice.


( C ) Quantification of fluorescence intensity of TH - positive neurons , in the substantia nigra ( SN ) ( left ) , and fibres , in the striatum ( right ) , six weeks after adenovirus vector injection , in Netrin - 1fl / fl mice . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; * * P < 0 . 01 . N = 3 each group .

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(C) Quantification of fluorescence intensity of TH-positive neurons, in the substantia nigra (SN) (left), and fibres, in the striatum (right), six weeks after adenovirus vector injection, in Netrin-1fl/fl mice. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; **P< 0.01. N=3 each group.


( D ) Terminal deoxynucleotidyl transferase ( TdT ) dUTP Nick - End Labelling ( TUNEL ) reactivity in the SN after Cre and control adenovirus vector injection , in Netrin - 1fl / fl mice ( Scale bar , 50μm ) . The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH - positive neurons . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * * P < 0 . 01 . N = 3 each group .

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(D) Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) reactivity in the SN after Cre and control adenovirus vector injection, in Netrin-1fl/fl mice (Scale bar, 50μm). The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. **P< 0.01. N=3 each group.


( E ) Motor behavior assays at six weeks after treatment . N = 8 animals / group . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 .

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(E) Motor behavior assays at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05


( F ) Immunoblot of SN lysates from netrin - 1 wild type ( WT ) and netrin - 1 conditional KO mice ( left upper panel ) . Densitometry band quantification of cleaved receptor over full length receptor ratio ( lower right panel ) . We randomly selected 2 mice from each group for immunoblot analysis . N = 3 independent experiments . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; P * * < 0 . 01 .

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(F) Immunoblot of SN lysates from netrin-1 wild type (WT) and netrin-1 conditional KO mice (left upper panel). Densitometry band quantification of cleaved receptor over full length receptor ratio (lower right panel). We randomly selected 2 mice from each group for immunoblot analysis. N=3 independent experiments. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.


( A ) Representative images of TH + dopamine neurons and fibers staining by immunofluorescence , in the SN and the striatum ( Str ) 6 weeks after CTL ( AAV6 - GFP ) ; Cre ( AAV6 - GFP Cre ) ; sh DCC ( AAV6 - GFP Cre + Sh - DCC ) and shUNC5B ( AAV6 GFP Cre + Sh - UNC5B ) injection into the SN of Netrin - 1fl / fl mice . ( Scale bar , 2000 µm ) . ( B ) Quantification of TH fluorescent intensity in the SN ( upper bar graph ) and striatum ( Str ) of Netrin - 1fl / fl mice ( lower bar graph ) . Data are shown as mean + SEM . Statistical significance was determined by an unpaired t - test . N = 3 each group . Tukey post hoc analysis after one - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , N . S . , not significant .

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(A) Representative images of TH+ dopamine neurons and fibers staining by immunofluorescence, in the SN and the striatum (Str) 6 weeks after CTL (AAV6-GFP); Cre (AAV6-GFP Cre); sh DCC (AAV6-GFP Cre + Sh-DCC) and shUNC5B (AAV6 GFP Cre + Sh-UNC5B) injection into the SN of Netrin-1fl/fl mice. (Scale bar, 2000 µm). (B) Quantification of TH fluorescent intensity in the SN (upper bar graph) and striatum (Str) of Netrin-1fl/fl mice (lower bar graph). Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 3 each group. Tukey post hoc analysis after one-way ANOVA *P < 0.05, **P <0.01, N.S., not significant.


( C ) DCC depletion mitigates netrin - 1 depletion - induced apoptosis in Netrin - 1fl / fl mice . TH - positive cell loss was assessed by TUNEL assay in the SN . Upper panel , TUNEL ( red ) and TH ( Cy5 - white ) ( Scale bar , 50 µm ) . Apoptotic index ( bar graph , bottom panel ) expressed as a percentage of TUNEL positive neurons out of the total number of TH - positive neurons . N = 3 each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . * P < 0 . 05 ; P * * < 0 . 01 .

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(C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01.


( D ) Immunoblot of SN lysates ( upper panel ) and corresponding quantification ( bottom panel ) . Quantification of band intensity ( bottom ) . Mouse N = 2 . N = 3 independent immunoblot analysis . Bars and error bars represent the mean ± SEM . Statistical significance was determined by an unpaired t - test . * P < 0 . 05 ; P * * < 0 . 01 .

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(D) Immunoblot of SN lysates (upper panel) and corresponding quantification (bottom panel). Quantification of band intensity (bottom). Mouse N=2. N=3 independent immunoblot analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.


( E ) Motor behaviour tests at six weeks after treatment . N = 8 animals / group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . * P < 0 . 05 ; N . S . , not significant .

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(E) Motor behaviour tests at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; N.S., not significant.


( B ) Amphetamine - induced rotations at two and six weeks after lesion . Individual values and Mean ± SD are shown , n = 11 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * P < 0 . 05 compared to WT .

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(B) Amphetamine-induced rotations at two and six weeks after lesion. Individual values and Mean ± SD are shown, n=11 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05 compared to WT.


( C ) Optical density of TH - positive fibres in the striatum . Individual values and mean ± SD , n = 10 animals , unpaired t - test , * P < 0 . 05 .

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(C) Optical density of TH-positive fibres in the striatum. Individual values and mean ± SD, n= 10 animals, unpaired t-test, *P < 0.05.


( D ) TH - positive cell count in the SN . Individual values and mean ± SD , n = 10 animals , unpaired t - test .

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(D) TH-positive cell count in the SN. Individual values and mean ± SD, n= 10 animals, unpaired t-test.


( F ) TH staining by immunohistochemistry ( Scale bar , 2000µm ) in the SN ( left ) and striatum ( right ) of WT and SNCA Tg mice , 6 days after treatment .

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(F) TH staining by immunohistochemistry (Scale bar, 2000µm) in the SN (left) and striatum (right) of WT and SNCA Tg mice, 6 days after treatment.


( G ) TH - positive cell count bar graph . All experiments were repeated 3 times . We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * * P < 0 . 01 ; N . S . , not significant .

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(G) TH-positive cell count bar graph. All experiments were repeated 3 times. We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. **P< 0.01; N.S., not significant.


Motor behavioural assays ( Rotarod rod test ( H ) N = 8 animals each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 .

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Motor behavioural assays (Rotarod rod test (H) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.


Motor behavioural assays Cylinder test ( I ) , Grid test ( J ) ) N = 8 animals each group . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni test for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 .

Motor behavioural assays Cylinder test (I), Grid test (J)) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.


( B ) Cumulative amphetamine - induced ipsilateral rotations . Progression of amphetamine - induced ipsilateral rotations over time ( right panel ) . Mean + SD are shown , n = 11 - 12 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * * * * P < 0 . 0001 compared to the initial rotation score at week 2 . Comparison of the treatment ' s effect between groups ( left panel ) . Mean + SD are shown , n = 11 - 12 animals in each group . Tukey post hoc analysis after two - way RM ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group .

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(B) Cumulative amphetamine-induced ipsilateral rotations. Progression of amphetamine-induced ipsilateral rotations over time (right panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, **** P <0.0001 compared to the initial rotation score at week 2. Comparison of the treatment's effect between groups (left panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group.


( C ) Dopamine fibres in the striatum of 6 - OHDA - lesionned rats , ten weeks after treatment . Representative images of coronal sections of the striatum showing TH IHC staining ( left panel ) , intact side on the left and 6 - OHDA lesioned side on the right . ( Scale bar , 2000 µm ) . Optical density measurement of TH - positive fibres in the lesioned side of the striatum ( bar graph , right panel ) . Individual values and mean ± SD are shown , N > 10 animals in each group . Tukey post hoc analysis after one - way ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group . Images are oriented with the dorsal part of the tissue at the top , and ventral part at the

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(C) Dopamine fibres in the striatum of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the striatum showing TH IHC staining (left panel), intact side on the left and 6-OHDA lesioned side on the right. (Scale bar, 2000 µm). Optical density measurement of TH-positive fibres in the lesioned side of the striatum (bar graph, right panel). Individual values and mean ± SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom


( D ) Dopamine cell bodies in the SN of 6 - OHDA - lesionned rats , ten weeks after treatment . Representative images of coronal sections of the SN ( lesioned side ) showing TH - positive cell bodies stained by IHC ( left panel ) ( Scale bar , 2000 µm ) . TH - positive cell count in the SN ( right panel ) . Individual values and mean + SD are shown , N > 10 animals in each group . Tukey post hoc analysis after one - way ANOVA , * * P < 0 . 01 compared to vehicle group . Images are oriented with the dorsal part of the tissue at the top , and ventral part at the bottom .

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(D) Dopamine cell bodies in the SN of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the SN (lesioned side) showing TH-positive cell bodies stained by IHC (left panel) (Scale bar, 2000 µm). TH-positive cell count in the SN (right panel). Individual values and mean + SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.


( A ) Netrin - 1 gene expression profiling by array of substantiae nigrae from PD and non - PD ( Control ) patients using the GEO dataset GSE7621 that has a total of 25 samples ( n = 9 control and n = 16 PD cases ) . Unpaired t - test , * * * P < 0 . 0005 , Mean ± SD are shown .

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(A) Netrin-1 gene expression profiling by array of substantiae nigrae from PD and non-PD (Control) patients using the GEO dataset GSE7621 that has a total of 25 samples (n=9 control and n=16 PD cases). Unpaired t-test, ***P < 0.0005, Mean ± SD are shown.