I Altered localization of the GFP - labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV - infected Rpt3 CKO mice at 5 days after injury . J Fluorescent intensity scans of GFP and AnkG corresponding to ( I ) . K Graph represents the percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of Atf3 : BAC2 Tg mouse treated with AnkG shRNA ( n = 5 mice per group ) . Data information : For data are shown as the mean ± s . e . m . , * * P = 0 . 0064 in ( K ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . In I ) , closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in I )

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I Altered localization of the GFP-labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV-infected Rpt3 CKO mice at 5 days after injury. J Fluorescent intensity scans of GFP and AnkG corresponding to (I). K Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Atf3:BAC2 Tg mouse treated with AnkG shRNA (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0064 in (K) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In I), closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in I)


L Thionine - stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury . Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs . Dashed lines outline hypoglossal nucleus . XII , hypoglossal nucleus ; cc , central canal . M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs ( n = 4 mice per group ) . Data information : For M ) , data are shown as the mean ± s . e . m . , * P = 0 . 0405 , * * P = 0 . 003 in ( M ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 100 μm in ( L ) .

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L Thionine-stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury. Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc, central canal. M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs (n= 4 mice per group). Data information: For M), data are shown as the mean ± s.e.m. , * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 100 μm in (L).


B Whole - mount visualization of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in sciatic nerve . Dashed lines indicate the boundary of axon . Scale bar , 5 μm . C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1 - Mito Tg and mtGFP in injured sciatic nerves of Atf3 ; BAC2 and Rpt3 CKO mice .

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B Whole-mount visualization of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in sciatic nerve. Dashed lines indicate the boundary of axon. Scale bar, 5 μm. C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1-Mito Tg and mtGFP in injured sciatic nerves of Atf3;BAC2 and Rpt3 CKO mice.


D - F The number of mitochondria ( D ) , percentage of motile mitochondria ( E ) and stationary mitochondrial site ( F ) in axons . Data are shown as the mean ± s . e . m . * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( D ) , * * P = 0 . 0006 , * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( E ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected , n = 5 mice for each group .

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D-F The number of mitochondria (D), percentage of motile mitochondria (E) and stationary mitochondrial site (F) in axons. Data are shown as the mean ± s.e.m. *** P = 0.0001, **** P < 0.0001 in (D), ** P = 0.0006, *** P = 0.0001, **** P < 0.0001 in (E), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected, n = 5 mice for each group.


C Immunostaining of spinal cord in Atf3 : BAC Tg and Atf3 ; SOD1 mice at P70 . The boxed regions are magnified in lower panels . Arrows indicate GFP - and ATF3 - positive motor neurons ( MNs ) . Dashed lines outline the edge of the gray matter in spinal cord . D The percentage ( % ) of GFP - positive MNs ( black bar ) in ChAT - positive MNs of Atf3 : BAC and Atf3 ; SOD1 mice at P70 ( n = 5 mice per each group ) . E The percentage ( % ) of ATF3 , MMP - 9 or osteopontin ( OPN ) - positive MNs ( black bar ) in GFP - positive MNs of Atf3 ; SOD1 mice at P65 - 70 ( n = 5 - 7 mice per group ) . Data information : For ( D , E , data are shown as the mean ± s . e . m . , Scale bars , 50 μm in ( C ; upper panel ) , 20 μm in ( C ; lower )

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C Immunostaining of spinal cord in Atf3:BAC Tg and Atf3;SOD1 mice at P70. The boxed regions are magnified in lower panels. Arrows indicate GFP- and ATF3- positive motor neurons (MNs). Dashed lines outline the edge of the gray matter in spinal cord. D The percentage (%) of GFP-positive MNs (black bar) in ChAT-positive MNs of Atf3:BAC and Atf3;SOD1 mice at P70 (n = 5 mice per each group). E The percentage (%) of ATF3, MMP-9 or osteopontin (OPN)-positive MNs (black bar) in GFP-positive MNs of Atf3;SOD1 mice at P65-70 (n = 5-7 mice per group). Data information: For (D, E, data are shown as the mean ± s.e.m., Scale bars , 50 μm in (C; upper panel), 20 μm in (C; lower)


F Immunostaining of AnkG in ChAT - positive spinal MN of Atf3 : BAC and SOD1 and in GFP - positive spinal MN of Atf3 ; SOD1 mouse at P70 . G Quantification of the AIS length ( n = 5 mice per group ) . Data information : For G , data are shown as the mean ± s . e . m . , no significant difference in ( G ) determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 10 μm in (

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F Immunostaining of AnkG in ChAT-positive spinal MN of Atf3:BAC and SOD1 and in GFP-positive spinal MN of Atf3;SOD1 mouse at P70. G Quantification of the AIS length (n = 5 mice per group). Data information: For G, data are shown as the mean ± s.e.m., no significant difference in (G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in (F


H The localization of AnkG and GFP - labeled mitochondria in uninjured spinal MN of Thy1 - Mito ( without ATF3 induction ) , traumatically sciatic nerve - injured MN of Atf3 : BAC ( with ATF3 induction ) and pathologically damaged MN of Atf3 ; SOD1 mice ( with ATF3 induction ) . Closed arrowheads denote the presence of the AIS , while open arrowheads show disappeared AIS . I Fluorescent intensity scan of GFP and AnkG in corresponding to ( H ) . J The percentage ( % ) of GFP intensity in the AIS , compared with that in the AIS of uninjured MNs in Thy1 - Mito mice ( n = 5 mice per group ) . Data information : For data are shown as the mean ± s . e . m . , * * P = 0 . 0008 , * * * P = 0 . 0007 in ( J ) , * * determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 5 μm in ( H ) .

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H The localization of AnkG and GFP-labeled mitochondria in uninjured spinal MN of Thy1-Mito (without ATF3 induction), traumatically sciatic nerve-injured MN of Atf3:BAC (with ATF3 induction) and pathologically damaged MN of Atf3;SOD1 mice (with ATF3 induction). Closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. I Fluorescent intensity scan of GFP and AnkG in corresponding to (H). J The percentage (%) of GFP intensity in the AIS, compared with that in the AIS of uninjured MNs in Thy1-Mito mice (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0008, *** P = 0.0007 in (J), ** determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in (H).


K Whole - mount observation of mtGFP or mtCFP in sciatic nerve . Dashed lines outline the axon . L The number of mtGFP or mtCFP in sciatic nerve ( n = 5 mice ) Data information : For L ) , data are shown as the mean ± s . e . m . , , * * P = 0 . 0036 , * * * * P < 0 . 0001 in ( L ) , determined by one - way ANOVA followed by Tukey post - hoc test , n . d . , not detected . Scale bars , 10 μm in K )

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K Whole-mount observation of mtGFP or mtCFP in sciatic nerve. Dashed lines outline the axon. L The number of mtGFP or mtCFP in sciatic nerve (n = 5 mice) Data information: For L), data are shown as the mean ± s.e.m., , ** P = 0.0036, **** P <0.0001 in (L), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 10 μm in K)


( A ) Inducible GFP ‐ S ‐ Ubqln1 293 cell line was treated with 1 μg / ml of doxycycline for 24 h . GFP ‐ S ‐ Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis . Red sequences indicate Ubqln4 peptides identified by mass spectrometry .

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(A) Inducible GFP‐S‐Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP‐S‐Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry.


( B ) 293 cells were co ‐ transfected with GFP ‐ S or GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies . Lower molecular weight GFP ‐ tag ‐ containing bands that appear in the GFP ‐ S ‐ Ubqln1 lane might represent degradation products .

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(B) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. Lower molecular weight GFP‐tag‐containing bands that appear in the GFP‐S‐Ubqln1 lane might represent degradation products.


( C ) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG . The immunoprecipitates were analysed by western blotting using anti ‐ Ubqln1 and anti ‐ Ubqln4 antibodies .

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(C) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG. The immunoprecipitates were analysed by western blotting using anti‐Ubqln1 and anti‐Ubqln4 antibodies.


( D ) Confocal images of Hela cells transfected with GFP ‐ S ‐ Ubqln1 and RFP ‐ Ubqln4 . Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross ‐ section . GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate .

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(D) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and RFP‐Ubqln4. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; Ubqln, Ubiquilin; WCL, whole‐cell lysate.


Ubqln4 interacts with LC3 . Confocal images of ( A ) Hela cells transfected with GFP ‐ LC3 and RFP ‐ Ubqln4 and starved for 1 h

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Ubqln4 interacts with LC3. Confocal images of (A) Hela cells transfected with GFP‐LC3 and RFP‐Ubqln4 and starved for 1 h or


( B ) Hela cells starved for 1 h and stained for endogenous LC3 and Ubqln4 . The squares show colocalization . The insets show the enlargement of the indicated areas . Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross ‐ section .

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(B) Hela cells starved for 1 h and stained for endogenous LC3 and Ubqln4. The squares show colocalization. The insets show the enlargement of the indicated areas. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section.


( C ) 293 cells were transfected with GFP ‐ S , GFP ‐ S ‐ Ubqln1 or GFP ‐ S ‐ Ubqln4 and treated with 50 μM chloroquine for 14 h . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ LC3 antibodies .

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(C) 293 cells were transfected with GFP‐S, GFP‐S‐Ubqln1 or GFP‐S‐Ubqln4 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies.


( D ) Recombinant His ‐ Ubqln1 , His ‐ Ubqln4 , His ‐ S5a or His ‐ p62 was mixed with LC3 covalently coupled to agarose beads . The beads were washed , eluted and detected by western blot using anti ‐ His antibody . The extent of Ubqln1 , Ubqln4 , s5a or p62 co ‐ immunoprecipitation with LC3 was measured . Bars for Ubqln1 , Ubqln4 and s5a represents the average and s . e . m . of four independent experiments . * P ‐ value = 0 . 0094 .

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(D) Recombinant His‐Ubqln1, His‐Ubqln4, His‐S5a or His‐p62 was mixed with LC3 covalently coupled to agarose beads. The beads were washed, eluted and detected by western blot using anti‐His antibody. The extent of Ubqln1, Ubqln4, s5a or p62 co‐immunoprecipitation with LC3 was measured. Bars for Ubqln1, Ubqln4 and s5a represents the average and s.e.m. of four independent experiments. *P‐value=0.0094.


( E ) 293 cells were transfected with GFP ‐ S ‐ Ubqln4 WT , GFP ‐ S ‐ Ubqln4 ΔSTI1 12 , GFP ‐ S ‐ Ubqln4 Δ between STI1 or GFP ‐ S ‐ Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ LC3 antibodies . GFP , green fluorescent protein ; IP , immunoprecipitation ; LC3 , microtubule ‐ associated protein light chain 3 ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate ; WT , wild ‐ type .

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(E) 293 cells were transfected with GFP‐S‐Ubqln4 WT, GFP‐S‐Ubqln4 ΔSTI1 12, GFP‐S‐Ubqln4 Δ between STI1 or GFP‐S‐Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


( A ) Confocal images of Hela cells transfected with GFP ‐ S ‐ Ubqln1 and also transfected with either control or Ubqln4 siRNA . The cells were either untreated or were transferred to starvation medium for 1 h .

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(A) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 1 h.


( B ) Quantification of the number of GFP ‐ S ‐ Ubqln1 WT puncta per cell in ( A ) . Each bar represents the average and s . e . m . of 30 cells per condition in three independent experiments . * P ‐ value = 0 . 011 , * * P ‐ value = 0 . 0005 and * * * P ‐ value = 0 . 0001 .

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(B) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.011, **P‐value=0.0005 and ***P‐value=0.0001.


( C ) Depletion of Ubqln4 with siRNA in ( A ) was verified by western blot using anti ‐ Ubqln1 , anti ‐ Ubqln4 and anti ‐ tubulin antibodies .

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(C) Depletion of Ubqln4 with siRNA in (A) was verified by western blot using anti‐Ubqln1, anti‐Ubqln4 and anti‐tubulin antibodies.


( D ) Hela cells transfected with GFP ‐ S ‐ Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ LC3 antibodies .

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(D) Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies.


( E ) Confocal images of GFP ‐ S ‐ Ubqln1 WT in Hela cells transfected with RFP , RFP ‐ Ubqln4 or RFP ‐ Ubqln4 ΔUBL .

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(E) Confocal images of GFP‐S‐Ubqln1 WT in Hela cells transfected with RFP, RFP‐Ubqln4 or RFP‐Ubqln4 ΔUBL.


( F ) Quantification of the number of GFP ‐ S ‐ Ubqln1 WT puncta per cell in ( E ) . Each bar represents the average and s . e . m . of 30 cells per condition in three independent experiments . * P ‐ value = 0 . 0001 and * * P ‐ value = 0 . 0003 . GFP , green fluorescent protein ; IP , immunoprecipitation ; LC3 , microtubule ‐ associated protein light chain 3 ; UBL , ubiquitin ‐ like ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate ; WT , wild ‐ type .

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(F) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (E). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.0001 and **P‐value=0.0003. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


( A ) 293 cells were co ‐ transfected with GFP ‐ S ‐ Ubqln1 , GFP ‐ S ‐ Ubqln1 ΔUBL ( deletion of UBL domain ) or GFP ‐ S ‐ Ubqln1 ΔUBA ( deletion of UBA domain ) and HA ‐ Ubqln4 . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies .

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(A) 293 cells were co‐transfected with GFP‐S‐Ubqln1, GFP‐S‐Ubqln1 ΔUBL (deletion of UBL domain) or GFP‐S‐Ubqln1 ΔUBA (deletion of UBA domain) and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.


( B ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1 , Ubqln1ΔUBL and Ubqln1ΔUBA . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average and s . e . m . of four independent experiments . * P ‐ value = 0 . 0041 .

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(B) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1, Ubqln1ΔUBL and Ubqln1ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0041.


( C ) 293 cells were co ‐ transfected with GFP ‐ S or GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 , HA ‐ Ubqln4 ΔUBL or HA ‐ Ubqln4 ΔUBA . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies .

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(C) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBL or HA‐Ubqln4 ΔUBA. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.


( D ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4 , Ubqln4ΔUBL and Ubqln4ΔUBA . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average and s . e . m . of four independent experiments . * P ‐ value = 0 . 0003 and * * P ‐ value = 0 . 0001 .

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(D) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBL and Ubqln4ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0003 and **P‐value=0.0001.


( E ) 293 cells were co ‐ transfected with GFP ‐ S ‐ Ubqln1 and HA ‐ Ubqln4 , HA ‐ Ubqln4 ΔUBA or HA ‐ Ubqln4 ΔUBA I55A ( I55 in the UBL domain of Ubqln4 was mutated to A ) . GFP was immunoprecipitated and analysed by western blot using anti ‐ GFP and anti ‐ HA antibodies .

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(E) 293 cells were co‐transfected with GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBA or HA‐Ubqln4 ΔUBA I55A (I55 in the UBL domain of Ubqln4 was mutated to A). GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies.


( F ) The extent of Ubqln4 co ‐ immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4 , Ubqln4ΔUBA and Ubqln4ΔUBA I55A . The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT . Each bar represents the average of two independent experiments . GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; UBA , ubiquitin ‐ associated ; UBL , ubiquitin ‐ like ; Ubqln , Ubiquilin ; WCL , whole ‐ cell lysate ; WT , wild ‐ type .

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(F) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln4 was calculated for WT Ubqln4, Ubqln4ΔUBA and Ubqln4ΔUBA I55A. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average of two independent experiments. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; UBA, ubiquitin‐associated; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.


( A ) Confocal images of Hela cells transfected with mCherry ‐ GFP ‐ LC3 and also transfected with control , Ubqln1 or Ubqln4 siRNA . The cells were either untreated or were transferred to starvation medium for 8 h .

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(A) Confocal images of Hela cells transfected with mCherry‐GFP‐LC3 and also transfected with control, Ubqln1 or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 8 h.


( B ) Quantification of the number of mCherry / GFP double ‐ positive and mCherry single ‐ positive puncta per cell in ( A ) . Each bar represents the average and s . e . m . of 30 cells per condition in three independent experiments . The number of mCherry single ‐ positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA . * P ‐ value = 0 . 0475 and * * P ‐ value = 0 . 0373 . GFP , green fluorescent protein ; LC3 , microtubule ‐ associated protein light chain 3 ; Ubqln , Ubiquilin .

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(B) Quantification of the number of mCherry/GFP double‐positive and mCherry single‐positive puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. The number of mCherry single‐positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA. *P‐value=0.0475 and **P‐value=0.0373. GFP, green fluorescent protein; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin.


( A ) IP of GFP - Atg8 ( α ‐ GFP ) , Atg30 - Flag ( α ‐ Flag ) and Pex3 ( α ‐ Pex3 ) under pexophagy conditions . The abundant peroxisome matrix protein , AOX , was used as a negative control . Input : total lysate ; ϕ : IP without antibody .

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(A) IP of GFP-Atg8 (α‐GFP), Atg30-Flag (α‐Flag) and Pex3 (α‐Pex3) under pexophagy conditions. The abundant peroxisome matrix protein, AOX, was used as a negative control. Input: total lysate; ϕ: IP without antibody.


( B ) Two multiple sequence alignments , including 11 Atg30 homologues and 11 Atg32 homologues ( identical residues are indicated with black boxes , and similar residues with grey boxes ) , and a sequence logo of the combined multiple sequence alignments from Atg30 and Atg32 homologues listed currently in GenBank .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Two multiple sequence alignments, including 11 Atg30 homologues and 11 Atg32 homologues (identical residues are indicated with black boxes, and similar residues with grey boxes), and a sequence logo of the combined multiple sequence alignments from Atg30 and Atg32 homologues listed currently in GenBank.


( C , D ) Pexophagy experiments of Δatg30 ( ϕ ) , wild ‐ type ( Atg30 ) and Atg30 AIM mutant ( Atg30W73A F76A ) cells were done by fluorescence microscopy , following the degradation of peroxisomes labelled with BFP fused at its C ‐ terminus to the Ser - Lys - Leu peroxisomal targeting signal 1 ( BFP - SKL ) and biochemically by monitoring peroxisomal thiolase degradation . Vacuoles were labelled with FM4 ‐ 64 . Scale bar , 5 μm .

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(C,D) Pexophagy experiments of Δatg30 (ϕ), wild‐type (Atg30) and Atg30 AIM mutant (Atg30W73A F76A) cells were done by fluorescence microscopy, following the degradation of peroxisomes labelled with BFP fused at its C‐terminus to the Ser-Lys-Leu peroxisomal targeting signal 1 (BFP-SKL) and biochemically by monitoring peroxisomal thiolase degradation. Vacuoles were labelled with FM4‐64. Scale bar, 5 μm.


( E ) AH109 cells were transformed with two yeast two ‐ hybrid assay plasmids , AD and BD , which encode the indicated domains fused with Atg30 , Atg32 and Atg8 or an empty vector , as negative controls and grown on + His and − His + 40 mM 3 ‐ AT plates .

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(E) AH109 cells were transformed with two yeast two‐hybrid assay plasmids, AD and BD, which encode the indicated domains fused with Atg30, Atg32 and Atg8 or an empty vector, as negative controls and grown on +His and −His+40 mM 3‐AT plates.


( F ) . Δatg30 ( ϕ ) and Δatg30 cells complemented with Atg30 - HA ( Atg30 ) and several Atg30 - HA mutants were immunoprecipitated ( α ‐ HA IP ) under pexophagy conditions . In addition , α ‐ HA IP of Δatg30 cells ( ϕ ) and Δatg30 cells complemented with Atg30 - HA ( Atg30 ) were incubated with ( + ) and without ( − ) λPP . Input : total lysate .

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(F) . Δatg30 (ϕ) and Δatg30 cells complemented with Atg30-HA (Atg30) and several Atg30-HA mutants were immunoprecipitated (α‐HA IP) under pexophagy conditions. In addition, α‐HA IP of Δatg30 cells (ϕ) and Δatg30 cells complemented with Atg30-HA (Atg30) were incubated with (+) and without (−) λPP. Input: total lysate.


( G ) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate ‐ induced peroxisomes after shifting cells to SD ‐ N . aa , amino acid ; AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; AOX , alcohol oxidase ; BD , binding domain ; GFP , green fluorescent protein ; HA , haemagglutinin ; IP , immunoprecipitation ; λPP , λ protein phosphatase .

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(G) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate‐induced peroxisomes after shifting cells to SD‐N. aa, amino acid; AD, activation domain; AIM, Atg8‐family‐interacting motif; AOX, alcohol oxidase; BD, binding domain; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; λPP, λ protein phosphatase.


( A1 ) Atg30 sequences from aa 68 - 122 of wild ‐ type ( Atg30 ) , the S71A mutant ( Atg30S71A ) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 ( Atg30WDILSSS and Atg30WSILSSS ) .

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(A1) Atg30 sequences from aa 68-122 of wild‐type (Atg30), the S71A mutant (Atg30S71A) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 (Atg30WDILSSS and Atg30WSILSSS).


( A2 ) Pexophagy experiments of Δatg30 cells complemented with appropriate wild ‐ type or mutant Atg30 proteins described in A1 .

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(A2) Pexophagy experiments of Δatg30 cells complemented with appropriate wild‐type or mutant Atg30 proteins described in A1.


( A3 ) Two ‐ hybrid assays between Atg30 wild ‐ type or mutants ( described in A1 ) and Atg8 . Phosphomimic S71E was included to detect the interaction with Atg8 .

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(A3) Two‐hybrid assays between Atg30 wild‐type or mutants (described in A1) and Atg8. Phosphomimic S71E was included to detect the interaction with Atg8.


( B2 ) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B1 .

(B2) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B1.


( C2 ) Pexophagy assays of Δatg30 cells complemented with Atg30 wild ‐ type and mutants . aa , amino acids ; AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; BD , binding domain ; HA , haemagglutinin .

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(C2) Pexophagy assays of Δatg30 cells complemented with Atg30 wild‐type and mutants. aa, amino acids; AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; HA, haemagglutinin.


( A ) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP - Atg8 during pexophagy conditions .

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(A) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP-Atg8 during pexophagy conditions.


( B ) Localization of GFP - Atg11 during pexophagy of methanol ‐ induced peroxisomes in cells expressing WT or mutant Atg30 proteins . White arrows indicate correct localization and yellow arrows indicate mislocalization , or in case of Atg8 localization , indicate absence of phagophore membrane elongation . Peroxisomes were labelled with BFP - SKL and vacuoles with FM4 ‐ 64 . Scale bar , 5 μm . GFP , green fluorescent protein ; WT , wild type .

(B) Localization of GFP-Atg11 during pexophagy of methanol‐induced peroxisomes in cells expressing WT or mutant Atg30 proteins. White arrows indicate correct localization and yellow arrows indicate mislocalization, or in case of Atg8 localization, indicate absence of phagophore membrane elongation. Peroxisomes were labelled with BFP-SKL and vacuoles with FM4‐64. Scale bar, 5 μm. GFP, green fluorescent protein; WT, wild type.


( A ) Mitophagy experiments of WT ( PPY12 ) , Δatg5 , Δatg32 ( ϕ ) and Δatg32 complemented with WT Atg32 ( Atg32 ) , Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding ( Atg32WQVLSSS ) , Atg32 AIM mutant ( Atg32W121A V124A ) , mutants of the Thr upstream of the Atg32 AIM ( Atg32T119A ) and mutants altered in the Ser required for Atg11 binding ( Atg32S159A ) . The cells were grown in YPL medium and shifted to SD ‐ N . Mitophagy was followed by the transport of Tom20 - mCherry to the vacuole by fluorescence microscopy . Mitophagy was classified as − no mitophagy , + few cells show mitophagy , + + + most cells show mitophagy and + + + + almost all the cells show mitophagy ( the intensity and numbers of cells containing Tom20 - mCherry in the vacuole was considered ) . Scale bar , 5 μm .

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(A) Mitophagy experiments of WT (PPY12), Δatg5, Δatg32 (ϕ) and Δatg32 complemented with WT Atg32 (Atg32), Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding (Atg32WQVLSSS), Atg32 AIM mutant (Atg32W121A V124A), mutants of the Thr upstream of the Atg32 AIM (Atg32T119A) and mutants altered in the Ser required for Atg11 binding (Atg32S159A). The cells were grown in YPL medium and shifted to SD‐N. Mitophagy was followed by the transport of Tom20-mCherry to the vacuole by fluorescence microscopy. Mitophagy was classified as −no mitophagy,+few cells show mitophagy, +++ most cells show mitophagy and ++++ almost all the cells show mitophagy (the intensity and numbers of cells containing Tom20-mCherry in the vacuole was considered). Scale bar, 5 μm.


( B ) P . pastoris Δatg32 cells ( ΔPpatg32 ) expressing Tom20 - GFP and expressing the indicated Atg32 mutants were cultured in YPL medium for 12 , 18 and 36 h . Mitophagy was monitored by GFP appearance by immunoblotting with α ‐ GFP antibodies .

(B) P. pastoris Δatg32 cells (ΔPpatg32) expressing Tom20-GFP and expressing the indicated Atg32 mutants were cultured in YPL medium for 12, 18 and 36 h. Mitophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies.


( C ) N ‐ terminal sequence of Atg36 manually aligned against the several sequence alignments of Atg30 , Atg32 and ScAtg32 . Identical residues are indicated with black boxes , and similar residues with grey boxes .

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(C) N‐terminal sequence of Atg36 manually aligned against the several sequence alignments of Atg30, Atg32 and ScAtg32. Identical residues are indicated with black boxes, and similar residues with grey boxes.


( D ) Two ‐ hybrid protein - protein interaction analysis of ScAtg36 , ScAtg8 and ScAtg11 . The receptors were mutated at the AIM ( Atg36F33A L36A ) , at serine ( s ) upstream of the AIM ( Atg36S31A ) and at the Atg11 ‐ binding site ( Atg36S97A ) .

(D) Two‐hybrid protein-protein interaction analysis of ScAtg36, ScAtg8 and ScAtg11. The receptors were mutated at the AIM (Atg36F33A L36A), at serine(s) upstream of the AIM (Atg36S31A) and at the Atg11‐binding site (Atg36S97A).


( E ) S . cerevisiae Δatg36 cells ( ΔScatg36 ) expressing thiolase - GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid ‐ log growth and then shifted to SD ‐ N . Pexophagy was monitored by GFP appearance by immunoblotting with α ‐ GFP antibodies .

(E) S. cerevisiae Δatg36 cells (ΔScatg36) expressing thiolase-GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid‐log growth and then shifted to SD‐N. Pexophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies.


( F ) Y3H analysis of ScAtg36 with ScAtg8 and ScAtg11 . The Y3H technology is on the basis of the yeast two ‐ hybrid system but with the co ‐ expression of third protein as a competitor and indicated in the figure ( NLS - ScAtg11 or NLS - ScAtg8 ) . The positive control was the ScAtg36 mutant affected in Atg11 binding ( ScAtg36S97A ) or in Atg8 binding ( ScAtg36S31A ) , which should be unaffected by the competition of NLS - ScAtg11 or NLS - ScAtg8 , respectively . Appropriate auto ‐ activation and interaction controls were also included . AD , activation domain ; AIM , Atg8 ‐ family ‐ interacting motif ; BD , binding domain ; GFP , green fluorescent protein ; WT , wild type ; Y3H , yeast three ‐ hybrid .

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(F) Y3H analysis of ScAtg36 with ScAtg8 and ScAtg11. The Y3H technology is on the basis of the yeast two‐hybrid system but with the co‐expression of third protein as a competitor and indicated in the figure (NLS-ScAtg11 or NLS-ScAtg8). The positive control was the ScAtg36 mutant affected in Atg11 binding (ScAtg36S97A) or in Atg8 binding (ScAtg36S31A), which should be unaffected by the competition of NLS-ScAtg11 or NLS-ScAtg8, respectively. Appropriate auto‐activation and interaction controls were also included. AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; GFP, green fluorescent protein; WT, wild type; Y3H, yeast three‐hybrid.


Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti - TAPBPL or isotype Ab .

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Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti-TAPBPL or isotype Ab.


Splenocytes from C57BL / 6 mice were freshly harvested . Resting and activated T cells , monocytes , macrophages , DCs and B cells were obtained as in Figure 2 . The resting and activated immune cells were stained with biotinylated TAPBPL - Ig or control Ig ， followed by streptavidin PE , as well as anti - CD4 , CD8 , CD11b , F4 / 80 , CD11c , B220 or CD19 antibody to identify immune cells . statistical analysis showing the binding TAPBPL - Ig or control Ig to freshly harvest and activated immune cells ( n = 3 ) . Significance was calculated by two ‐ way ANOVA with Tukey test . * P < 0 . 05 compared with control Ig . * * P < 0 . 05 compared with resting cells .

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Splenocytes from C57BL/6 mice were freshly harvested. Resting and activated T cells, monocytes, macrophages, DCs and B cells were obtained as in Figure 2. The resting and activated immune cells were stained with biotinylated TAPBPL-Ig or control Ig，followed by streptavidin PE, as well as anti-CD4, CD8, CD11b, F4/80, CD11c, B220 or CD19 antibody to identify immune cells. statistical analysis showing the binding TAPBPL-Ig or control Ig to freshly harvest and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with control Ig. ** P<0.05 compared with resting cells.


Splenic cells from C57BL / 6 mice were cultured with anti - CD3 antibody and anti - CD28 ( 0 . 5 µg / ml ) antibodies in the presence of graded doses of hTAPBPL - Ig or equimolar amounts of control Ig ( 3 . 75 and 5 . 63 µg / ml ) for 1 day The cells were analyzed for CD69 + , cells in CD4 and CD8 T cells . Representative flow cytometric and statistical analyses of the percentages of CD69 + , and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells . The data are expressed as mean + SD ( n = 3 ) . Significance was calculated by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 compared with control Ig .

Splenic cells from C57BL/6 mice were cultured with anti-CD3 antibody and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 1 day The cells were analyzed for CD69+, cells in CD4 and CD8 T cells. Representative flow cytometric and statistical analyses of the percentages of CD69+, and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.


Splenic cells were labelled with CFSE and cultured in 96 - well plates that were precoated with anti - CD3 antibody and hTAPBPL - Ig or control Ig for 3 days as in ( A ) . The cells were analyzed for CFSE levels by CD4 + and CD8 + T cells . statistical analysis of ( D ) CD4 and ( E ) CD8 T cell proliferation .

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Splenic cells were labelled with CFSE and cultured in 96-well plates that were precoated with anti-CD3 antibody and hTAPBPL-Ig or control Ig for 3 days as in (A). The cells were analyzed for CFSE levels by CD4+ and CD8+ T cells. statistical analysis of (D) CD4 and (E) CD8 T cell proliferation.


Purified human T cells were cultured with plate - bound anti - human CD3 antibody ( 1 µg / ml ) in the presence of graded doses of hTAPBPL - Ig ( 1 . 5 and 3 µg / ml ) or control Ig protein ( 1 . 5 and 3 µg / ml ) for 3 days . Cell proliferation was measured by [ 3H ] thymidine incorporation . The data are expressed as mean + SD ( n = 3 ) . Significance was calculated by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 compared with control Ig .

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Purified human T cells were cultured with plate-bound anti-human CD3 antibody (1 µg/ml) in the presence of graded doses of hTAPBPL-Ig (1.5 and 3 µg/ml) or control Ig protein (1.5 and 3 µg/ml) for 3 days. Cell proliferation was measured by [3H] thymidine incorporation. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.


Anti - hTAPBPL mAb ( clone 54 ) neutralized the inhibitory activity of hTAPBPL - Ig on T cell proliferation Significance in was calculated by one ‐ way ANOVA with Dunnett test and others by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 , anti - hTAPBPL Ab group was compared with isotype Ab group .

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Anti-hTAPBPL mAb (clone 54) neutralized the inhibitory activity of hTAPBPL-Ig on T cell proliferation Significance in was calculated by one‐way ANOVA with Dunnett test and others by two‐tailed Student's t‐test. * P<0.05, anti-hTAPBPL Ab group was compared with isotype Ab group.


DBA / 2J mice were injected s . c . with 1 × 105 P388 murine leukemia cells , followed by injection of the anti - hTAPBPL mAb ( 25 , 50 , or 100µg ) or isotype Ab ( 25 , 50 , or 100 µg ) 3 times per week . Tumors size was measured over time . The mean tumor diameter ( mm3 ) + S . D . at the indicated time points are shown .

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DBA/2J mice were injected s.c. with 1× 105 P388 murine leukemia cells, followed by injection of the anti-hTAPBPL mAb (25, 50, or 100µg) or isotype Ab (25, 50, or 100 µg) 3 times per week. Tumors size was measured over time. The mean tumor diameter (mm3) + S.D. at the indicated time points are shown.


Tumor infiltrating CD4 + and CD8 + T cells were examined by immunofluorescence .

Tumor infiltrating CD4+ and CD8+ T cells were examined by immunofluorescence.


B Western blot analyses after induced expression of FLAG - tagged UPF3A in WT HEK293 and HeLa cells ( n = 1 ) . Tubulin serves as control . The asterisk indicates unspecific bands .

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B Western blot analyses after induced expression of FLAG-tagged UPF3A in WT HEK293 and HeLa cells (n=1). Tubulin serves as control. The asterisk indicates unspecific bands.


C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR ( ISAR ) using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR (ISAR) using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D Volcano plot showing the differential gene expression analyses from the RNA - Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . OE = overexpression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plot showing the differential gene expression analyses from the RNA-Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A. The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. OE = overexpression.


E Read coverage of SMG5 from WT HEK293 and HeLa RNA - seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer ( IGV ) snapshot . Differential gene expression ( from DESeq2 ) is indicated as log2 fold change ( log2FC ) on the right . Schematic representation of the protein coding transcript below .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Read coverage of SMG5 from WT HEK293 and HeLa RNA-seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer (IGV) snapshot. Differential gene expression (from DESeq2) is indicated as log2 fold change (log2FC) on the right. Schematic representation of the protein coding transcript below.


A Western blot analysis of WT and UPF3A KO cells ( clones 4 , 14 and 20 ) with the indicated siRNA treatments ( n = 1 ) . UPF3A and UPF3B protein levels were detected , Tubulin serves as control . The asterisk indicates unspecific bands .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC ) ( n = 3 for RSRC2 and SRSF2 , n = 4 for ZFAS1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitative RT-PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC) (n=3 for RSRC2 and SRSF2, n=4 for ZFAS1).


C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in the indicated conditions using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D , E Volcano plots showing the differential gene expression analyses from the indicated RNA - Seq datasets ( D UPF3A KO clone 14 , E UPF3A KO clone 20 ) . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,E Volcano plots showing the differential gene expression analyses from the indicated RNA-Seq datasets (D UPF3A KO clone 14, E UPF3A KO clone 20). The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method


A Western blot analysis of WT and UPF3B KO cells ( clones 90 and 91 ) combined with the indicated knockdowns ( n = 1 ) . UPF3A and UPF3B ( AK - 141 ) protein levels were detected , Tubulin serves as control . Protein levels of UPF3A were quantified , normalized to tubulin expression , and shown as datapoints and mean . Fold - changes of relevant conditions are shown . The asterisk indicates unspecific bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of WT and UPF3B KO cells (clones 90 and 91) combined with the indicated knockdowns (n=1). UPF3A and UPF3B (AK-141) protein levels were detected, Tubulin serves as control. Protein levels of UPF3A were quantified, normalized to tubulin expression, and shown as datapoints and mean. Fold-changes of relevant conditions are shown. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of the indicated cell lines with the indicated knockdowns . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

B Quantitative RT-PCR of the indicated cell lines with the indicated knockdowns. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC, n=3).


D , E Volcano plots showing the differential transcript usage ( via IsoformSwitchAnalyzeR ) in various RNA - Seq data . Isoforms containing GENCODE ( release 33 ) annotated PTC ( red , TRUE ) , regular stop codons ( blue , FALSE ) or having no annotated open reading frame ( grey , NA ) are indicated . The change in isoform fraction ( dIF ) is plotted against the - log10 adjusted p - value ( adj . p - value ) . Density plots show the distribution of filtered isoforms in respect to the dIF , cutoffs were | dIF | > 0 . 1 and adj . p - value < 0 . 05 . P - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,E Volcano plots showing the differential transcript usage (via IsoformSwitchAnalyzeR) in various RNA-Seq data. Isoforms containing GENCODE (release 33) annotated PTC (red, TRUE), regular stop codons (blue, FALSE) or having no annotated open reading frame (grey, NA) are indicated. The change in isoform fraction (dIF) is plotted against the -log10 adjusted p-value (adj. p-value). Density plots show the distribution of filtered isoforms in respect to the dIF, cutoffs were |dIF| > 0.1 and adj. p-value < 0.05. P-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method.


A Western blot analysis of WT , UPF3B KO and UPF3A - UPF3B double KO cells ( clones 1 and 2 ) ( n = 1 ) . UPF3A and UPF3B protein levels were detected , Tubulin serves as control . The asterisk indicates unspecific bands .

O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of WT, UPF3B KO and UPF3A-UPF3B double KO cells (clones 1 and 2) (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


C Quantitative RT - PCR of the indicated samples with the indicated KDs . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitative RT-PCR of the indicated samples with the indicated KDs. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


E Northern blot analysis of globin reporter and xrFrag . Ethidium bromide stained 28S and 18S rRNAs are shown as controls . Quantification results are shown as data points and mean ( n = 3 ) .

O O O O O O O O O O O O O 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 Northern blot analysis of globin reporter and xrFrag. Ethidium bromide stained 28S and 18S rRNAs are shown as controls. Quantification results are shown as data points and mean (n=3).


A - C Volcano plots of label free mass spectrometry - based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B ( n = 4 biologically independent samples ) . ( A ) FLAG - UPF2 in WT against FLAG - GST control in WT cells , ( B ) UPF2 in 3B KO cells against FLAG control in WT cells , ( C ) UPF2 in dKO cells against FLAG control in WT cells . Points labeled in purple indicate NMD factors ; points labeled in turquoise indicate EJC components . Cut offs : Log2 fold change ≥

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

A-C Volcano plots of label free mass spectrometry-based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B (n = 4 biologically independent samples). (A) FLAG-UPF2 in WT against FLAG-GST control in WT cells, (B) UPF2 in 3B KO cells against FLAG control in WT cells, (C) UPF2 in dKO cells against FLAG control in WT cells. Points labeled in purple indicate NMD factors; points labeled in turquoise indicate EJC components. Cut offs: Log2 fold change ≥ 0


E Representative Western blot after FLAG co - immunoprecipitation ( IP ) of FLAG - tagged GST ( control ) or UPF3B WT and single mutant constructs in UPF3B KO cells ( n = 3 ) .

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

E Representative Western blot after FLAG co-immunoprecipitation (IP) of FLAG-tagged GST (control) or UPF3B WT and single mutant constructs in UPF3B KO cells (n = 3).


F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively . Monitored expression of the FLAG - tagged UPF3B rescue construct shown in ( D ) . Rescue construct protein levels were detected with anti - FLAG and anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) .

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

F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3B rescue construct shown in (D). Rescue construct protein levels were detected with anti-FLAG and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1).


G Quantitative RT - PCR of the samples from ( F ) . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitative RT-PCR of the samples from (F). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


B Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively . Monitored expression of the FLAG - tagged UPF3A and UPF3B rescue construct shown in ( A ) . Rescue construct protein levels were detected with anti - FLAG , anti - UPF3A and anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) . The asterisk indicates unspecific bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3A and UPF3B rescue construct shown in (A). Rescue construct protein levels were detected with anti-FLAG, anti-UPF3A and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1). The asterisk indicates unspecific bands.


C Quantitative RT - PCR of the samples from ( B ) . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . Data points and means are plotted as log2 fold change ( log2FC ) ( n = 3 ) .

C Quantitative RT-PCR of the samples from (B). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC) (n=3).


B SDS - PAGE analysis of soluble ( Supernatant ) and insoluble proteins ( Pellet ) from human neurons after treatment for one hour with a mixture of RNase A and RNase T1 ( A / T1 ) , or vehicle ( Ve - ) .

O O O O O O O O O O O O O O O O O O O O O O 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 SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment for one hour with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-).


C Protein aggregation ( Pellet ) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg2 + . Ribonucleases used were RNase A ( A ) , RNase T1 ( T1 ) , a mixture of RNase A and RNase T1 ( A / T1 ) , RNase 1f ( 1f ) , and RNase V1 ( V1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg2+. Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1).


Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from human neurons ( A )

Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from human neurons (A)


Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from HEK293 cells expressing GFP - Aβ or GFP ( 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

Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from HEK293 cells expressing GFP-Aβ or GFP (B).


C Effect of an RNase A inhibitor ( RNasin ) on RNase A mediated protein aggregation in neuronal cell lysate .

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

C Effect of an RNase A inhibitor (RNasin) on RNase A mediated protein aggregation in neuronal cell lysate.


B Effect of RNA / protein ratio on protein aggregation after renaturing .

B Effect of RNA/protein ratio on protein aggregation after renaturing.


C Coomassie - stained gel of soluble ( Sup 1 / 2 ) and aggregated ( Pel 1 / 2 ) proteins after removal of GuHCl in the presence ( + ) or absence ( - ) of total RNA . Asterix ( * ) denotes added RNase A .

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

C Coomassie-stained gel of soluble (Sup 1/2) and aggregated (Pel 1/2) proteins after removal of GuHCl in the presence (+) or absence (-) of total RNA. Asterix (*) denotes added RNase A.


soluble ( Sup 1 / 2 ) and aggregated ( Pel 1 / 2 ) proteins after removal of GuHCl in the presence ( + ) or absence ( - ) of total RNA . D As in ( c ) but analysed by Western blot .

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

soluble (Sup 1/2) and aggregated (Pel 1/2) proteins after removal of GuHCl in the presence (+) or absence (-) of total RNA. D As in (c) but analysed by Western blot.


E ATP - hydrolysing activity of proteins renatured with total RNA or buffer ( Ve - ) after removal of GuHCl .

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

E ATP-hydrolysing activity of proteins renatured with total RNA or buffer (Ve-) after removal of GuHCl.


A Genomic attributes of RNA associated with renatured NF - H , Tau , or Aβ . Inset shows which fractions of the peaks that derive from exons or introns have their origin in coding or non - coding transcripts , respectively .

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

A Genomic attributes of RNA associated with renatured NF-H, Tau, or Aβ. Inset shows which fractions of the peaks that derive from exons or introns have their origin in coding or non-coding transcripts, respectively.


B Characterization of DNA associated with soluble , renatured Aβ or all proteins ( Total ) , captured either by immunoprecipitation ( Aβ ) or absorption to membranes ( Total ) .

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

B Characterization of DNA associated with soluble, renatured Aβ or all proteins (Total), captured either by immunoprecipitation (Aβ ) or absorption to membranes (Total).


A Proportion of aggregated proteins after renaturation with either single - stranded ( Forward or Reverse ) or complementary strands ( For / Rev ) of the selected M1x4 or the control oligonucleotide , M2x4 .

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

A Proportion of aggregated proteins after renaturation with either single-stranded (Forward or Reverse) or complementary strands (For/Rev) of the selected M1x4 or the control oligonucleotide, M2x4.


B Western blot analysis of proteins renatured with either single - stranded or double - stranded M1x4 or M2x4 DNA oligonucleotides .

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

B Western blot analysis of proteins renatured with either single-stranded or double-stranded M1x4 or M2x4 DNA oligonucleotides.


C The effect on protein re - aggregation from varying the number of DNA motif repeats when complementary oligonucleotides are given together .

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

C The effect on protein re-aggregation from varying the number of DNA motif repeats when complementary oligonucleotides are given together.


D Protein aggregation after renaturation with the M1x4 DNA oligonucleotides pre - annealed with 100 mM NaCl or vehicle ( None ) .

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

D Protein aggregation after renaturation with the M1x4 DNA oligonucleotides pre-annealed with 100 mM NaCl or vehicle (None).


B Proportion of protein aggregation following renaturing with the ds / ss / ds oligonucleotides shown in ( A ) .

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

B Proportion of protein aggregation following renaturing with the ds/ss/ds oligonucleotides shown in (A).


C Renaturing capacity of structurally different oligonucleotides . The diagrams on the right show a theoretical structure of each oligonucleotide . All oligonucleotides , except the 3x - loop and 3x - bulges , contain a stretch of 30 Ts in the single - stranded region and the same sequences ( 15 nucleotides each ) in the double stranded regions . The 3x - loops and bulges oligonucleotides have 3 stretches of 9 Ts and the same sequence in the ds - regions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Renaturing capacity of structurally different oligonucleotides. The diagrams on the right show a theoretical structure of each oligonucleotide. All oligonucleotides, except the 3x-loop and 3x-bulges, contain a stretch of 30 Ts in the single-stranded region and the same sequences (15 nucleotides each) in the double stranded regions. The 3x-loops and bulges oligonucleotides have 3 stretches of 9 Ts and the same sequence in the ds-regions.


D Proportion of protein aggregation in Jurkat cell lysate supplemented with various amounts and configurations of the M1x4 or M2x4 DNA oligonucleotides .

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

D Proportion of protein aggregation in Jurkat cell lysate supplemented with various amounts and configurations of the M1x4 or M2x4 DNA oligonucleotides.


e Insoluble proteins from two ALS brain tissues were chemically denatured in guanidine hydrochloride and treated with either buffer ( Vehicle ) , total RNA from Jurkat cells or the complementary strands of the M1x4 DNA oligonucleotides ( M1 F / R ) ( Pellet 1 , top panel ) . After removal of GuHCl , the soluble fraction from these samples was treated with RNase and any aggregated proteins ( Pellet 2 ) , analysed by western blot ( middle panel ) . Remaining supernatants were then treated with Benzonase to degrade any remaining nucleic acids and aggregated proteins collected by centrifugation and analysed by western blot ( bottom panel ) . Proteins aggregated by enzymatic RNA degradation in Jurkat cell lysates , which do not contain NF - H , were used as a control .

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

e Insoluble proteins from two ALS brain tissues were chemically denatured in guanidine hydrochloride and treated with either buffer (Vehicle), total RNA from Jurkat cells or the complementary strands of the M1x4 DNA oligonucleotides (M1 F/R) (Pellet 1, top panel). After removal of GuHCl, the soluble fraction from these samples was treated with RNase and any aggregated proteins (Pellet 2), analysed by western blot (middle panel). Remaining supernatants were then treated with Benzonase to degrade any remaining nucleic acids and aggregated proteins collected by centrifugation and analysed by western blot (bottom panel). Proteins aggregated by enzymatic RNA degradation in Jurkat cell lysates, which do not contain NF-H, were used as a control.


B . RNAseq data are plotted in log2 fold change ( FC ) levels of protrusions over cell bodies against adjusted - log10 false discovery rate ( FDR ) ( n = 2 replicates , average FC values are represented ) . The horizontal dashed line marks the FDR ( q ) = 0 . 05 threshold ; vertical dashed lines mark the FC = 0 . 625 ( left ) or FC = 1 . 6 ( right ) thresholds .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RNAseq data are plotted in log2 fold change (FC) levels of protrusions over cell bodies against adjusted -log10 false discovery rate (FDR) (n=2 replicates, average FC values are represented). The horizontal dashed line marks the FDR (q) = 0.05 threshold; vertical dashed lines mark the FC = 0.625 (left) or FC = 1.6 (right) thresholds.


C . Heat map represents the k - means clustering of transcript log2 FC levels ( protrusions over cell bodies ) extracted from RNAseq datasets published elsewhere . The corresponding HUVEC FC levels are shown in parallel .

O O O O O O O O O O O O O O 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. Heat map represents the k-means clustering of transcript log2 FC levels (protrusions over cell bodies) extracted from RNAseq datasets published elsewhere. The corresponding HUVEC FC levels are shown in parallel.


D . smFISH co - detection of k 5 mRNAs and GAPDH in subconfluent motile HUVECs . arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 20

O O O O O O O O O O O O O O O O O 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. smFISH co-detection of k 5 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm


E . Polarisation Index ( PI ) of k 5 mRNAs and GAPDH in co - detected in HUVECs ( n ≥ 28 cells ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Wilcoxon test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Polarisation Index (PI) of k 5 mRNAs and GAPDH in co-detected in HUVECs (n≥28 cells; **P<0.01, ***P<0.001, ****P<0.0001; Wilcoxon test).


F . k 5 mRNAs PIs plotted against respective GAPDH PIs . The slope of the coloured lines represents the average k 5 mRNA / GAPDH PI ratio ; the dashed grey line represents a 1 : 1 ratio ( n ≥ 28 cells ) .

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

F. k 5 mRNAs PIs plotted against respective GAPDH PIs. The slope of the coloured lines represents the average k 5 mRNA/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n≥28 cells).


G . Top : distribution pattern of mRNAs clustered in k 2 , k 5 , k 7 and GAPDH . Bottom : smFISH co - detection of exemplar k7 / k 2 mRNAs and GAPDH in subconfluent motile HUVECs . arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Top: distribution pattern of mRNAs clustered in k 2, k 5, k 7 and GAPDH. Bottom: smFISH co-detection of exemplar k7 / k 2 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm


H . PIs of k 7 mRNAs and GAPDH co - detected in HUVECs ( n ≥ 25 cells ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; paired t test ) .

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

H. PIs of k 7 mRNAs and GAPDH co-detected in HUVECs (n≥25 cells; *P<0.05, **P<0.01, ***P<0.001; paired t test).


I . PIs of k 2 mRNAs and GAPDH co - detected in HUVECs ( n ≥ 19 cells ; * P < 0 . 05 ; Wilcoxon test ) .

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

I. PIs of k 2 mRNAs and GAPDH co-detected in HUVECs (n≥19 cells; *P<0.05; Wilcoxon test).


E . Left : representative subconfluent motile cells co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls and 24xMS2 - RAB13 3 ' UTR or 24xMS2 . Right : percentage of cells with MCP - GFP localised to protrusions when co - transfected with full length or deletion versions of RAB13 3 ' UTR ( n ≥ 3 experiments ) . white arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate the orientation of RNA localisation ; yellow dashed lines outline cell borders ; scale bars = 20 µm For each k 5 mRNA 3 ' UTR , a diagram of the full length 3 ' UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions ; black arrowheads indicate predicted polyadenylation

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left: representative subconfluent motile cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR or 24xMS2. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of RAB13 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sites


F . Left : representative cells co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls and 24xMS2 - k 5 3 ' UTRs . Right : percentage of cells with MCP - GFP localised to protrusions when co - transfected with full length or deletion versions of k 5 3 ' UTR ( n ≥ 3 experiments ) . white arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate the orientation of RNA localisation ; yellow dashed lines outline cell borders ; scale bars = 20 µm For each k 5 mRNA 3 ' UTR , a diagram of the full length 3 ' UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions ; black arrowheads indicate predicted polyadenylation

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

F. Left: representative cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-k 5 3'UTRs. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of k 5 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sites


B . Representative genotyping PCR demonstrates the band size shift in ∆ LE HUVECs .

B. Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs.


D . Wt and ∆ LE HUVEC RNAseq mapped reads depicting RAB13 exon usage .

D. Wt and ∆LE HUVEC RNAseq mapped reads depicting RAB13 exon usage.


E . Quantification of RAB13 mRNA smFISH spot number in Wt and ∆ LE HUVECs ( n = 3 experiments ; ns : not significant ; unpaired t test ) .

E. Quantification of RAB13 mRNA smFISH spot number in Wt and ∆LE HUVECs (n=3 experiments; ns: not significant; unpaired t test).


F . Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index ( PI ) ( n = 29 cells ; ns : not significant ; linear regression ) .

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

F. Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index (PI) (n=29 cells; ns: not significant; linear regression).


G . Left : representative Western blotting ( WB ) of Wt and ∆ LE HUVECs . Right : densitometry analysis of WB data ( n = 3 samples ; ns : not significant ; unpaired t test ) .

O O O O O O O O O O O O O O O O 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. Left: representative Western blotting (WB) of Wt and ∆LE HUVECs. Right: densitometry analysis of WB data (n=3 samples; ns: not significant; unpaired t test).


H . smFISH co - detection of RAB13 and control GAPDH in Wt and ∆ LE motile HUVECs cultured under subconfluent conditions . Arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 20 µm ( H ) . Bar charts are presented as means ± s . 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

H. smFISH co-detection of RAB13 and control GAPDH in Wt and ∆LE motile HUVECs cultured under subconfluent conditions. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm (H). Bar charts are presented as means±s.d.


I . PI of RAB13 and GAPDH co - detected in Wt and ∆ LE HUVECs ( n = 29 cells ; P * * * < 0 . 001 , ns : not significant ; Mann - Whitney test ) .

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

I. PI of RAB13 and GAPDH co-detected in Wt and ∆LE HUVECs (n=29 cells; P***<0.001, ns: not significant; Mann-Whitney test).


J . RAB13 PI plotted against respective GAPDH PI . The slope of the coloured lines represents the average RAB13 / GAPDH PI ratio ; the dashed grey line represents a 1 : 1 ratio ( n = 29 cells ) .

J. RAB13 PI plotted against respective GAPDH PI. The slope of the coloured lines represents the average RAB13/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n=29 cells).


A . Representative time - lapse microscopy of a bEnd . 3 cell co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls and 24xMS2 - RAB13 3 ' UTR . Arrowheads indicate

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

A. Representative time-lapse microscopy of a bEnd.3 cell co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR. Arrowheads indicate filopodia


B . Frequency of newly formed filopodia formed within 5 µm intervals relative to the nearest MCP - GFPnls particle or a randomised ( ctrl ) position ( n = 99 filopodia ; * * P < 0 . 01 , ns : not significant ; Pearson r correlation ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Frequency of newly formed filopodia formed within 5 µm intervals relative to the nearest MCP-GFPnls particle or a randomised (ctrl) position (n=99 filopodia; **P<0.01, ns: not significant; Pearson r correlation).


C . Distance of newly - formed filopodia to MCP - GFPnls or a ctrl position plotted against filopodia duration ( n = 99 filopodia ; * P < 0 . 05 , ns : not significant ; Spearman r correlation ) .

O O O O O O O O O O O O O O O O O O O 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. Distance of newly-formed filopodia to MCP-GFPnls or a ctrl position plotted against filopodia duration (n=99 filopodia; *P<0.05, ns: not significant; Spearman r correlation).


D . Wt and ∆ LE HUVECs co - cultured on fibroblast monolayers . Endothelial cells were identified either with an antibody against the endothelial cell marker PECAM - 1 ( left ) or through expression of a nucleofected plasmid encoding the cytoskeletal marker Lifeact - GFP ( right ) . Arrowheads indicate

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Wt and ∆LE HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified either with an antibody against the endothelial cell marker PECAM-1 (left) or through expression of a nucleofected plasmid encoding the cytoskeletal marker Lifeact-GFP (right). Arrowheads indicate filopodia


E . Number of filopodia detected in co - cultured HUVECs ( n = 30 cells ; * * P < 0 . 01 ; unpaired t test )

O O O O O 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. Number of filopodia detected in co-cultured HUVECs (n=30 cells; **P<0.01; unpaired t test)


F . Number of filopodia detected in co - cultured HUVECs within 12 µm intervals relative to cell distal tip ( n = 30 cells ; * * P < 0 . 01 , P * * * < 0 . 001 , ns : not significant ; one - way ANOVA with Bonferroni ' s correction )

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

F. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n=30 cells; **P<0.01, P***<0.001, ns: not significant; one-way ANOVA with Bonferroni's correction)


G . Number of filopodia detected in individual clones of co - cultured HUVECs within 12 µm intervals relative to cell distal tip .

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. Number of filopodia detected in individual clones of co-cultured HUVECs within 12 µm intervals relative to cell distal tip.


B . Representative Puro - PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes . Puro : puromycin ; Aniso : anisomycin ; 1ary Abs : primary antibodies . white arrowheads indicate Puro - PLA punctae ; yellow dashed lines outline protrusion borders ( B ) ;

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

B. Representative Puro-PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes. Puro: puromycin; Aniso: anisomycin; 1ary Abs: primary antibodies. white arrowheads indicate Puro-PLA punctae; yellow dashed lines outline protrusion borders (B);


C . Quantification of RAB13 Puro - PLA punctae normalised to protrusion area ( n ≥ 40 protrusions ; * P < 0 . 05 , * * * * P < 0 . 0001 ; Kruskal - Wallis test with Dunn ' s correction ) .

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

C. Quantification of RAB13 Puro-PLA punctae normalised to protrusion area (n≥40 protrusions; *P<0.05, ****P<0.0001; Kruskal-Wallis test with Dunn's correction).


D . Representative RAB13 IF assay on migrating HUVECs .

D. Representative RAB13 IF assay on migrating HUVECs.


E . Left : representative Western blotting ( WB ) of siRNA transfected HUVECs . Right : densitometry analysis of WB data ( n = 3 samples ; * P < 0 . 05 ; paired t test ) .

E. Left: representative Western blotting (WB) of siRNA transfected HUVECs. Right: densitometry analysis of WB data (n=3 samples; *P<0.05; paired t test).


F . Control ( ctrl ) and RAB13 siRNA - treated HUVECs co - cultured on fibroblast monolayers . Endothelial cells were identified with an antibody against the endothelial cell marker PECAM - 1 . black arrowheads indicate filopodia ( F )

F. Control (ctrl) and RAB13 siRNA-treated HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified with an antibody against the endothelial cell marker PECAM-1. black arrowheads indicate filopodia (F)


G . Number of filopodia detected in co - cultured HUVECs ( n ≥ 35 cells ; * * P < 0 . 01 ; unpaired t test ) .

O O O O O O 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. Number of filopodia detected in co-cultured HUVECs (n≥35 cells; **P<0.01; unpaired t test).


H . Number of filopodia detected in co - cultured HUVECs within 12 µm intervals relative to cell distal tip ( n ≥ 35 cells ; * P < 0 . 05 , ns : not significant ; Kruskal - Wallis test with Dunn ' s correction ) .

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

H. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n≥35 cells; *P<0.05, ns: not significant; Kruskal-Wallis test with Dunn's correction).


A . Left : Tg ( fli1ep : MCP - GFPnls ) zebrafish embryo at 26 hours post fertilisation ( hpf ) displaying vascular - specific expression of MCP - GFPnls . Inset shows the nuclear expression of MCP - GFPnls in the intersomitic vessels ( ISVs ) sprouting from the dorsal aorta ( DA ) . Middle : scheme depicts the in vivo MS2 system strategy with fli1 enhancer / promoter ( fli1ep ) - driven expression of reporter constructs , simultaneous translation of Lyn - mCherry reporter and binding of MCP - GFPnls to 24xMS2 - rab13 3 ' UTR . Right : scheme illustrates ISV cells expressing Lyn - mCherry imaged in panels B - D . TC : tip cell ; SC : stalk cell . Arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate direction of ISV sprouting ; yellow dashed lines outline ISV ( A )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left: Tg(fli1ep:MCP-GFPnls) zebrafish embryo at 26 hours post fertilisation (hpf) displaying vascular-specific expression of MCP-GFPnls. Inset shows the nuclear expression of MCP-GFPnls in the intersomitic vessels (ISVs) sprouting from the dorsal aorta (DA). Middle: scheme depicts the in vivo MS2 system strategy with fli1 enhancer/promoter (fli1ep)-driven expression of reporter constructs, simultaneous translation of Lyn-mCherry reporter and binding of MCP-GFPnls to 24xMS2-rab13 3'UTR. Right: scheme illustrates ISV cells expressing Lyn-mCherry imaged in panels B-D. TC: tip cell; SC: stalk cell. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV (A)


( B - D ) Time - lapse microscopy of Tg ( fli1ep : MCP - GFPnls ) tip ( C ) and stalk ( B , D ) cells displaying mosaic expression of Lyn - mCherry - 24xMS2 - rab13 3 ' UTR in ISV cells . Arrowheads indicate non - nuclear localisation of MCP - GFPnls ; arrows indicate direction of ISV sprouting ; yellow dashed lines outline ISV cell ( B - 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

(B-D) Time-lapse microscopy of Tg(fli1ep:MCP-GFPnls) tip (C) and stalk (B,D) cells displaying mosaic expression of Lyn-mCherry-24xMS2-rab13 3'UTR in ISV cells. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV cell (B-D) borders


B . Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆ 482 rab13 3 ' UTR . Asterisk marks a heteroduplex formed between Wt and ∆ 482 rab13 3 ' UTR PCR amplicons .

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

B. Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆482 rab13 3'UTR. Asterisk marks a heteroduplex formed between Wt and ∆482 rab13 3'UTR PCR amplicons.


D . RNAseq mapped reads depicting rab13 exon usage in Tg ( kdrl : EGFP ) rab13 + / + and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR zebrafish embryos . Coloured lines indicate SNPs .

O O O O O O O O O O O O O O O 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. RNAseq mapped reads depicting rab13 exon usage in Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Coloured lines indicate SNPs.


E . qPCR analysis of rab13 mRNA levels in individual 26 - 28 hpf clutch - matched sibling embryos ( n ≥ 9 embryos ; ns : not significant ; Kruskal - Wallis test with Dunn ' s correction ) . : + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qPCR analysis of rab13 mRNA levels in individual 26-28 hpf clutch-matched sibling embryos (n≥9 embryos; ns: not significant; Kruskal-Wallis test with Dunn's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively


F . smFISH detection of rab13 and kdr mRNA in cultured GFP - expressing endothelial cells extracted from 48 hpf Tg ( kdrl : EGFP ) rab13 + / + and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR zebrafish embryos . Arrows indicate orientation of RNA localisation ; yellow dashed lines outline cell borders ; red circles highlight smFISH spots ; scale bars = 10 µm ( F ) .

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

F. smFISH detection of rab13 and kdr mRNA in cultured GFP-expressing endothelial cells extracted from 48 hpf Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 10 µm (F).


G . Polarisation Index ( PI ) of rab13 and kdr detected by smFISH in individual zebrafish cells ( n ≥ 8 cells ; * * P < 0 . 01 , ns : not significant ; one - way ANOVA with Bonferroni ' s correction ) . : + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Polarisation Index (PI) of rab13 and kdr detected by smFISH in individual zebrafish cells (n≥8 cells; **P<0.01, ns: not significant; one-way ANOVA with Bonferroni's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively


H . rab13 PI plotted against respective kdr PI . The slope of the coloured lines represents the average rab13 / kdr PI ratio ; the dashed grey line represents a 1 : 1 ratio ( n ≥ 8 cells ) . : + / + , + / ∆ and ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos ,

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

H. rab13 PI plotted against respective kdr PI. The slope of the coloured lines represents the average rab13/kdr PI ratio; the dashed grey line represents a 1:1 ratio (n≥8 cells). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively


A . Time - lapse confocal microscopy of representative Tg ( kdrl : EGFP ) rab13 + / + and rab13 ∆ 3 ' UTR / ∆ 3 ' UTR embryos . DLAV : dorsal longitudinal anastomotic vessel ; HM : horizontal myoseptum . T0 = 25 hpf ; arrowheads indicate extra branches emerging from the main ISVs at the HM position ; scale bars = 50 µm ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time-lapse confocal microscopy of representative Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR embryos. DLAV: dorsal longitudinal anastomotic vessel; HM: horizontal myoseptum. T0= 25 hpf; arrowheads indicate extra branches emerging from the main ISVs at the HM position; scale bars = 50 µm (A).