B . COS - 7 cells transfected with GFP - Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided . Arrows denoted by different colors indicate individual AL structures . Note that one of the AL structures ( marked by yellow arrow ) originated from the NE ( dotted line ) and fused with another AL structure ( marked by red arrow ) , which later fused with a different AL structure ( marked by blue arrow ) . N , nucleus ; C , cytoplasm . Scale bar , 5 μm .

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B. COS-7 cells transfected with GFP-Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided. Arrows denoted by different colors indicate individual AL structures. Note that one of the AL structures (marked by yellow arrow) originated from the NE (dotted line) and fused with another AL structure (marked by red arrow), which later fused with a different AL structure (marked by blue arrow). N, nucleus; C, cytoplasm. Scale bar, 5 μm.


C . Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 ( green ) , P bodies ( red , Dcp1a as a marker ) and SGs ( blue , eIF3η as a marker ) using specific antibodies . Scale bar , 10 μm . The histograms represent fluorescence intensity profile along the dotted arrows . Adjacent graph represents quantitative data showing percentage of P bodies ( top ) or SGs ( bottom ) associated with nuclear envelope ( NE ) , Nup358 positive AL , with each other or unassociated with any of the other mentioned structures ( others ) . Data are presented as mean SD ( n = 3 ) .

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C. Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker) and SGs (blue, eIF3η as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358 positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean SD (n = 3).


D . HeLa cells were transfected with Nup358 siRNA ( siNup358 ) or Nup214 siRNA ( siNup214 ) . Cells were fixed and stained for endogenous Nup358 ( green , upper panel ) or Nup214 ( green , lower panel ) and endogenous P body marker ( Dcp1a , red ) . Graph represents the quantitative data as mean ± SD . The data was obtained from three independent experiments , and in each experiment , 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Cells were fixed and stained for endogenous Nup358 (green, upper panel) or Nup214 (green, lower panel) and endogenous P body marker (Dcp1a, red). Graph represents the quantitative data as mean ± SD. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage.


D . HeLa cells were transfected with Nup358 siRNA ( siNup358 ) or Nup214 siRNA ( siNup214 ) . Western blot ( WB ) indicates the extent of Nup358 depletion and the level of Dcp1a .

O O O O O O O O O O O O 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. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Western blot (WB) indicates the extent of Nup358 depletion and the level of Dcp1a.


A . Western analysis of HeLa cells , treated with control ( siControl ) , Dicer ( siDicer ) , Nup214 ( siNup214 ) or Nup358 ( siNup358 ) siRNA , for assessing the extent of protein depletion using indicated antibodies . Vinculin was used as loading control .

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

A. Western analysis of HeLa cells, treated with control (siControl), Dicer (siDicer), Nup214 (siNup214) or Nup358 (siNup358) siRNA, for assessing the extent of protein depletion using indicated antibodies. Vinculin was used as loading control.


B . HeLa cells were initially transfected with indicated siRNAs , followed by renilla luciferase ( RL ) reporter constructs ; RL - control ( no let - 7a binding site in the 3 ' UTR ) or RL - 3xBulge ( 3 imperfect let - 7a binding sites in the 3 ' - UTR ) . Firefly luciferase ( FL ) was co - transfected to serve as internal control . RL / FL luminescence ratio was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

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B. HeLa cells were initially transfected with indicated siRNAs, followed by renilla luciferase (RL) reporter constructs; RL-control (no let-7a binding site in the 3'UTR) or RL-3xBulge (3 imperfect let-7a binding sites in the 3'-UTR). Firefly luciferase (FL) was co-transfected to serve as internal control. RL/FL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


C . HEK293T cells were transfected with indicated siRNAs , followed by pCMV - FL - miR30 ( P ) reporter with either pSUPER - control or pSUPER - miR30 constructs . RL was co - transfected as internal control . FL / RL luminescence ratio was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

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C. HEK293T cells were transfected with indicated siRNAs, followed by pCMV-FL-miR30 (P) reporter with either pSUPER-control or pSUPER-miR30 constructs. RL was co-transfected as internal control. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


D . HeLa cells were initially transfected with control ( siControl ) , Dicer ( siDicer ) or Nup358 ( siNup358 ) specific siRNAs , followed by FL constructs containing wild type ( HMGA2 - wt ) or mutant ( HMGA2 - mut ) HMGA2 3 ' - UTR , along with RL as internal control . Graph was plotted using data from three independent experiments . FL / RL luminescence ratio was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

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D. HeLa cells were initially transfected with control (siControl), Dicer (siDicer) or Nup358 (siNup358) specific siRNAs, followed by FL constructs containing wild type (HMGA2-wt) or mutant (HMGA2-mut) HMGA2 3'-UTR, along with RL as internal control. Graph was plotted using data from three independent experiments. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


A . HeLa cells were transfected with control , Dicer or Nup358 specific siRNAs , as indicated . Upper panel , total RNA was isolated and analyzed by northern blotting for let - 7a using radio - labeled probe . Ethidium bromide ( EtBr ) stained gel indicates equal loading of RNA samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HeLa cells were transfected with control, Dicer or Nup358 specific siRNAs, as indicated. Upper panel, total RNA was isolated and analyzed by northern blotting for let-7a using radio-labeled probe. Ethidium bromide (EtBr) stained gel indicates equal loading of RNA samples.


A . HeLa cells were transfected with control , Dicer or Nup358 specific siRNAs , as indicated . Bottom panel , total RNA was isolated from control , Dicer or Nup358 siRNA treated cells and was reverse - transcribed using TaqMan microRNA reverse transcription kit . The levels of let - 7a were quantified by qPCR following manufacturer ' s instructions . Graph represents the relative levels of miRNAs as compared to U6 RNA control . The values were further normalized to let - 7a levels in control siRNA knockdown condition . Data are presented as mean SD ( n = 3 ) .

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A. HeLa cells were transfected with control, Dicer or Nup358 specific siRNAs, as indicated. Bottom panel, total RNA was isolated from control, Dicer or Nup358 siRNA treated cells and was reverse-transcribed using TaqMan microRNA reverse transcription kit. The levels of let-7a were quantified by qPCR following manufacturer's instructions. Graph represents the relative levels of miRNAs as compared to U6 RNA control. The values were further normalized to let-7a levels in control siRNA knockdown condition. Data are presented as mean SD (n = 3).


B . HeLa cells were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these cells . Bottom right panel ; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody . Vinculin was used as loading control . Data are presented as mean SD ( 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 O O O 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. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom right panel; AGO2 immunoprecipitates were analyzed by western blotting using AGO2 specific antibody. Vinculin was used as loading control. Data are presented as mean SD (n = 3).


B . HeLa cells were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these cells . Bottom left panel ; RNA isolated from IgG control or AGO2 immunoprecipitate samples was reverse - transcribed using oligo ( dT ) primer . RL - 3xBulge mRNA association with AGO2 was quantified by q - PCR and normalized to the level of GAPDH mRNA associated with AGO2 .

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B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Bottom left panel; RNA isolated from IgG control or AGO2 immunoprecipitate samples was reverse-transcribed using oligo(dT) primer. RL-3xBulge mRNA association with AGO2 was quantified by q-PCR and normalized to the level of GAPDH mRNA associated with AGO2.


B . HeLa cells were transfected with indicated siRNAs , followed by RL - 3xBulge construct . Immunoprecipitation ( IP ) was performed with control mouse IgG ( IgG IP ) or anti - AGO2 antibody ( AGO2 IP ) using lysates prepared from these cells . Total RNA was isolated from control and AGO2 immunoprecipitates and the levels of AGO2 associated let - 7a ( top left panel ) and miR - 17 ( top right panel ) were quantified by q - PCR . Graph represents the extent of miRNAs associated with AGO2 as compared to U6 RNA ( as a negative control ) with AGO2 .

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B. HeLa cells were transfected with indicated siRNAs, followed by RL-3xBulge construct. Immunoprecipitation (IP) was performed with control mouse IgG (IgG IP) or anti-AGO2 antibody (AGO2 IP) using lysates prepared from these cells. Total RNA was isolated from control and AGO2 immunoprecipitates and the levels of AGO2 associated let-7a (top left panel) and miR-17 (top right panel) were quantified by q-PCR. Graph represents the extent of miRNAs associated with AGO2 as compared to U6 RNA (as a negative control) with AGO2.


C . HeLa cells were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . Western blots indicate the extent of Nup358 depletion and AGO2 immunoprecipitation in siControl and siNup358 samples . Vinculin was used as loading control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s t ‐ test .

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C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Western blots indicate the extent of Nup358 depletion and AGO2 immunoprecipitation in siControl and siNup358 samples. Vinculin was used as loading control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


C . HeLa cells were transfected with control ( siControl ) or Nup358 specific siRNA ( siNup358 ) for 96 h . Cells were lysed and subjected to immunoprecipitation with control IgG ( IgG IP ) or anti - AGO2 ( AGO2 IP ) . Total RNA was extracted from initial lysate and IP samples and analyzed by q - PCR for validated miRNA targets , Serbp1 and Dnajb11 . GAPDH was considered as negative control . Data expressed as the relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA .

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C. HeLa cells were transfected with control (siControl) or Nup358 specific siRNA (siNup358) for 96 h. Cells were lysed and subjected to immunoprecipitation with control IgG (IgG IP) or anti-AGO2 (AGO2 IP). Total RNA was extracted from initial lysate and IP samples and analyzed by q-PCR for validated miRNA targets, Serbp1 and Dnajb11. GAPDH was considered as negative control. Data expressed as the relative amount of target mRNA associated with AGO2 as compared to GAPDH mRNA.


D . HeLa cells were treated with indicated siRNAs for 96 h . Cells were lysed and analyzed for extent of Nup358 depletion and for the levels of validated miRNA targets , Ras and c - Myc by western analysis . Vinculin was used as loading control .

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

D. HeLa cells were treated with indicated siRNAs for 96 h. Cells were lysed and analyzed for extent of Nup358 depletion and for the levels of validated miRNA targets, Ras and c-Myc by western analysis. Vinculin was used as loading control.


E . HeLa cells were lysed and immunoprecipitation was performed with control ( IgG - IP ) or Nup358 ( Nup358 - IP , left panel ) or AGO2 ( AGO2 - IP , right panel ) using specific antibodies . The immunoprecipitates were washed with a buffer containing ( + ) or not containing ( - ) RNAse A and probed for the presence of indicated proteins by western blotting .

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E. HeLa cells were lysed and immunoprecipitation was performed with control (IgG-IP) or Nup358 (Nup358-IP, left panel) or AGO2 (AGO2-IP, right panel) using specific antibodies. The immunoprecipitates were washed with a buffer containing (+) or not containing (-) RNAse A and probed for the presence of indicated proteins by western blotting.


F . HeLa cells were transfected with RL - 3xBulge construct and immunoprecipitation was performed using control ( IgG - IP ) or Nup358 ( Nup358 - IP ) antibodies . Total RNA was isolated from the immunoprecipitates and analyzed for let - 7a miRNA ( left panel ) , RL - 3xBulge mRNA and the endogenous miRNA target Dnajb11 using q - PCR ( right panel ) . Fold enrichment in Nup358 - IP as compared to IgG - control was calculated and the data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s t ‐ test .

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F. HeLa cells were transfected with RL-3xBulge construct and immunoprecipitation was performed using control (IgG-IP) or Nup358 (Nup358-IP) antibodies. Total RNA was isolated from the immunoprecipitates and analyzed for let-7a miRNA (left panel), RL-3xBulge mRNA and the endogenous miRNA target Dnajb11 using q-PCR (right panel). Fold enrichment in Nup358-IP as compared to IgG-control was calculated and the data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.


B . HEK293T cells were co - transfected with GFP - maltose binding protein ( MBP ) - control or GFP - tagged version of indicated Nup358 fragments along with HA - AGO2 . Immunoprecipitation ( IP ) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting ( WB ) using HA antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HEK293T cells were co-transfected with GFP-maltose binding protein (MBP)-control or GFP-tagged version of indicated Nup358 fragments along with HA-AGO2. Immunoprecipitation (IP) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting (WB) using HA antibodies.


C . Cells were transfected with indicated constructs and IP and WB were performed as described for B .

C. Cells were transfected with indicated constructs and IP and WB were performed as described for B.


D . HEK293T cells transfected with GFP - MBP ( control ) , GFP - Nup358 - C or GFP - Nup358 - CΔIR ( a mutant devoid of IR region ) and HA - AGO2 . IP and WB analyses were performed as indicated .

O O O O O O O O O O O O O O O O O O O O O O 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. HEK293T cells transfected with GFP-MBP (control), GFP-Nup358-C or GFP-Nup358-CΔIR (a mutant devoid of IR region) and HA-AGO2. IP and WB analyses were performed as indicated.


E . Cells were transfected with HA - tagged version of indicated AGO subfamily member and GFP - Nup358 - IR . IP and WB analyses were performed using indicated antibodies .

O O O O O O O O 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. Cells were transfected with HA-tagged version of indicated AGO subfamily member and GFP-Nup358-IR. IP and WB analyses were performed using indicated antibodies.


F . Lysates from cells expressing GFP - control , GFP - human ( h ) Nup358 - IR or GFP - zebrafish ( z ) Nup358 - IR and HA - AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA - AGO2 by western blotting .

F. Lysates from cells expressing GFP-control, GFP-human (h) Nup358-IR or GFP-zebrafish (z) Nup358-IR and HA-AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA-AGO2 by western blotting.


B . HEK293T cells were transfected with indicated constructs and immunoprecipitation ( IP ) was performed using GFP specific antibodies and probed for HA - AGO2 by western blotting ( WB ) using HA antibodies . Endogenous RanGAP and Ubc9 were probed with specific antibodies .

B. HEK293T cells were transfected with indicated constructs and immunoprecipitation (IP) was performed using GFP specific antibodies and probed for HA-AGO2 by western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies.


C . Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies . Presence of endogenous RanGAP and Ubc9 was determined by WB .

O O O O O O O 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. Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies. Presence of endogenous RanGAP and Ubc9 was determined by WB.


D . GFP - control , GFP - IR1 + 2 wild type or mutants were co - expressed with HA - AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies .

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D. GFP-control, GFP-IR1+2 wild type or mutants were co-expressed with HA-AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies.


E . Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies .

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E. Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies.


A . Top panel ; depicts the amino acid sequence corresponding to the Nup358 - SIM1 region and substitutions introduced in the SIM1 mutant . Bottom panel : HEK293T cells were co - transfected with GFP - control , GFP - SIM1 or GFP - SIM1 - mut along with HA - AGO2 and immunoprecipitation ( IP ) was performed with GFP specific antibodies and western analysis ( WB ) of the input lysate and immunoprecipitates were carried out using indicated antibodies .

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A. Top panel; depicts the amino acid sequence corresponding to the Nup358-SIM1 region and substitutions introduced in the SIM1 mutant. Bottom panel: HEK293T cells were co-transfected with GFP-control, GFP-SIM1 or GFP-SIM1-mut along with HA-AGO2 and immunoprecipitation (IP) was performed with GFP specific antibodies and western analysis (WB) of the input lysate and immunoprecipitates were carried out using indicated antibodies.


B . Top panel ; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study . Hydrophobic residues involved in SUMO binding are indicated in red . Bottom panel ; constructs expressing GFP - control or GFP - fused SIMs of indicated proteins were co - transfected with HA - AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction .

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B. Top panel; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study. Hydrophobic residues involved in SUMO binding are indicated in red. Bottom panel; constructs expressing GFP-control or GFP-fused SIMs of indicated proteins were co-transfected with HA-AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction.


C . HA - MBP - control , HA - AGO1 or HA - AGO2 was co - expressed with GFP - SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates .

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C. HA-MBP-control, HA-AGO1 or HA-AGO2 was co-expressed with GFP-SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates.


D . SIM1 directly interacts with AGO2 . Bacterial lysates expressing MBP control or MBP - AGO2 and GST or GST - SIM1 were mixed and GST pull down assay was performed . The presence of proteins in the pull down samples was analyzed by WB using specific antibodies . The extent of GST pull down was monitored by coomassie staining of the membrane ( coomassie ) .

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D. SIM1 directly interacts with AGO2. Bacterial lysates expressing MBP control or MBP-AGO2 and GST or GST-SIM1 were mixed and GST pull down assay was performed. The presence of proteins in the pull down samples was analyzed by WB using specific antibodies. The extent of GST pull down was monitored by coomassie staining of the membrane (coomassie).


E . SIM1 interacts with PIWI subfamily of proteins . HEK293T cells were co - transfected with indicated constructs expressing HA - tagged version of PIWI proteins and GFP - SIM1 . IP was performed using HA antibodies . The immunoprecipitates were probed for the presence of GFP - SIM1 using GFP specific antibodies by WB .

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E. SIM1 interacts with PIWI subfamily of proteins. HEK293T cells were co-transfected with indicated constructs expressing HA-tagged version of PIWI proteins and GFP-SIM1. IP was performed using HA antibodies. The immunoprecipitates were probed for the presence of GFP-SIM1 using GFP specific antibodies by WB.


F . Putative SIM - binding regions in AGO2 . Three distinct superimpositions of AGO2 ( grey ) and SUMO ( blue ) , in complex with Nup358 - SIM1 ( red ) , are shown in cartoon representation rendered with Chimera [ 90 ] . The residue numbers on the AGO2 that could be the putative SIM - binding regions are labelled . To identify possible Nup358 binding sites on AGO2 , the 3D structure of AGO2 ( PDB ID : 4W5O ) [ 89 ] was compared with that of SUMO ( PDB ID : 1Z5S ) [ 47 ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Putative SIM-binding regions in AGO2. Three distinct superimpositions of AGO2 (grey) and SUMO (blue), in complex with Nup358-SIM1(red), are shown in cartoon representation rendered with Chimera [90]. The residue numbers on the AGO2 that could be the putative SIM-binding regions are labelled. To identify possible Nup358 binding sites on AGO2, the 3D structure of AGO2 (PDB ID: 4W5O) [89] was compared with that of SUMO (PDB ID: 1Z5S) [47].


A . Right panel ; Western blots showing the expression of transfected constructs using indicated antibodies . a - tubulin was used as loading control .

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A. Right panel; Western blots showing the expression of transfected constructs using indicated antibodies. a-tubulin was used as loading control.


A . Left panel ; HeLa cells were co - transfected with GFP - MBP ( control ) , GFP - SIM1 or GFP - SIM1 - mut ( AGO interaction defective ) along with RL - control or RL - 3xbulge . Firefly luciferase was used as transfection control . Dual luciferase assay was performed . RL / FL luminescence ration was plotted after normalization with GFP - control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O 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 panel; HeLa cells were co-transfected with GFP-MBP (control), GFP-SIM1 or GFP-SIM1-mut (AGO interaction defective) along with RL-control or RL-3xbulge. Firefly luciferase was used as transfection control. Dual luciferase assay was performed. RL/FL luminescence ration was plotted after normalization with GFP-control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


B . Bottom right panel ; Western blot for monitoring the expression levels of indicated proteins using antibodies . a - tubulin was used as loading control .

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

B. Bottom right panel; Western blot for monitoring the expression levels of indicated proteins using antibodies. a-tubulin was used as loading control.


B . Bottom left panel ; HEK293T cells were co - transfected with indicated expression constructs and the RL reporter . Firefly luciferase ( FL ) was used as transfection control . Dual luciferase assay was performed . The ratio of RL / FL luminescence was plotted after normalization with N - HA - MBP control . Data are presented as mean SD ( n = 3 ) , P ‐ value was calculated using Student ' s 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 O O O O O O O O O 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. Bottom left panel; HEK293T cells were co-transfected with indicated expression constructs and the RL reporter. Firefly luciferase (FL) was used as transfection control. Dual luciferase assay was performed. The ratio of RL/FL luminescence was plotted after normalization with N-HA-MBP control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.


C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Left panel : Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions . The data was obtained from three independent experiments , and in each experiment , 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Left panel: Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage.


C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Middle panel : HeLa cells were transfected with indicated siRNAs , followed by expression constructs and miRNA RL reporters . FL was used as transfection control . The ratio of RL / FL luminescence was calculated . Data are presented as mean SD ( n = 3 ) , P ‐ value was obtained using Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Middle panel: HeLa cells were transfected with indicated siRNAs, followed by expression constructs and miRNA RL reporters. FL was used as transfection control. The ratio of RL/FL luminescence was calculated. Data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test.


C . Ectopic expression of AGO2 restores P body formation , but fails to rescue the impairment in miRNA function in Nup358 depleted cells . HeLa cells were transfected with control ( siContriol ) or Nup358 ( siNup358 ) specific siRNA , followed by indicated constructs . Right panel : Western blot analysis for the relative expression of indicated constructs in HEK293T cells . a - tubulin is used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Right panel: Western blot analysis for the relative expression of indicated constructs in HEK293T cells. a-tubulin is used as loading control.


D . HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay . Fractions were collected and analyzed for the presence of indicated proteins by western blotting .

O O O O O O O O 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. HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay. Fractions were collected and analyzed for the presence of indicated proteins by western blotting.


( A , B ) Localization of oskar mRNA in wild - type ( A ) and in Tm1eg9 / Tm1eg9 ( B ) egg - chambers .

(A, B) Localization of oskar mRNA in wild-type (A) and in Tm1eg9/Tm1eg9 (B) egg-chambers.


( C ) Kymograph of oskMS2 - GFP mRNPs ( cyan ) travelling along polarity marked MTs ( red , EB1 protein ) in an ex vivo ooplasmic preparation . White dashed arrow shows the growing plus tip of the MT . Scale bars represent 1 s and 1 µm , respectively . ( D ) Distribution of oskMS2 - GFP mRNP runs towards plus ( white ) and minus ends ( grey ) . Numbers within the bars indicate number of runs . P value of Chi2 test against wild - type is indicated above each bar .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kymograph of oskMS2-GFP mRNPs (cyan) travelling along polarity marked MTs (red, EB1 protein) in an ex vivo ooplasmic preparation. White dashed arrow shows the growing plus tip of the MT. Scale bars represent 1 s and 1 µm, respectively.(D) Distribution of oskMS2-GFP mRNP runs towards plus (white) and minus ends (grey). Numbers within the bars indicate number of runs. P value of Chi2 test against wild-type is indicated above each bar.


( E , F ) oskar mRNA ( green ) and Oskar protein ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers expressing oskarMS2 ( 6x ) ( E ) or oskarMS2 ( 6x ) and Khc401 - MCP ( 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

(E, F) oskar mRNA (green) and Oskar protein (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers expressing oskarMS2(6x) (E) or oskarMS2(6x) and Khc401-MCP (F).


( G ) oskar mRNA ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers rescued with EmGFP - Tm1 - I ( green ) . Scale bars represent 50 µm .

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

(G) oskar mRNA (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers rescued with EmGFP-Tm1-I (green). Scale bars represent 50 µm.


( A - B " ) Colocalization of oskMS2 - mCherry ( red , A , B ) ( A ' , B ' ) with Khc - EGFP ( green , A ) ( A ' ) or with EmGFP - Tm1 - I ( green B ) ( B ' ) in ex vivo ooplasmic preparations . ( C ) Fraction of oskMS2 - MCP - mCherry mRNPs located non - randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations . MCP indicates MCP - EGFP which , like MCP - mCherry , can bind to MS2 loops . Staufen ( Stau ) is a dsRNA binding protein and bona fide partner of oskar mRNA ( St Johnston et al . , 1991 ; St Johnston et al . , 1992 ) . All values are significantly different from zero ( p < 10 - 3 , one sample t - test ) . ( D ) Fraction of oskMS2 - mCherry mRNPs co - localizing ( max . 200 nm ) non - randomly with Khc - EGFP particles in wild - type and in Tm1eg1 / Tm1eg9 ooplasms in presence of two ( white ) or one ( grey ) copy of endogenous Khc . ( C , D ) P values of two sample t - tests are indicated above the relevant bar pairs . Numbers indicate the number of particle clusters ( 160 mRNPs in each ) and the number of preparations ( in brackets ) analysed . Error bars represent 95 % confidence intervals .

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

(A-B") Colocalization of oskMS2-mCherry (red, A, B) (A', B') with Khc-EGFP (green, A) (A') or with EmGFP-Tm1-I (green B) (B') in ex vivo ooplasmic preparations.(C) Fraction of oskMS2-MCP-mCherry mRNPs located non-randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations. MCP indicates MCP-EGFP which, like MCP-mCherry, can bind to MS2 loops. Staufen (Stau) is a dsRNA binding protein and bona fide partner of oskar mRNA (St Johnston et al., 1991; St Johnston et al., 1992). All values are significantly different from zero (p<10-3, one sample t-test).(D) Fraction of oskMS2-mCherry mRNPs co-localizing (max. 200 nm) non-randomly with Khc-EGFP particles in wild-type and in Tm1eg1/Tm1eg9 ooplasms in presence of two (white) or one (grey) copy of endogenous Khc.(C, D) P values of two sample t-tests are indicated above the relevant bar pairs. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed. Error bars represent 95% confidence intervals.


( A , B ) Kymographs of oskMS2 - GFP mRNPs ( green ) associated with Khc - mKate2 ( A , red ) and mCherry - Tm1 - I / C ( B , red ) ex vivo . Arrows indicate motile RNPs in stable complex with Khc ( A ) or Tm1 - I / C ( B ) , the arrowheads point to non - motile oskMS2 - RNPs showing no obvious accumulation of the tagged protein . Note that mCherry - Tm1 - I was exposed twice as long as Khc - mKate to obtain comparable red fluorescence signals . Scale bars represent 1 µm and 1 second , respectively . ( C - E ) Relative Khc - mKate2 ( C ) , mCherry - Tm1 - I / C ( D ) coverage of motile ( white ) and non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within the boxes indicate the number of trajectories and the number of ooplasms ( in brackets ) analysed . Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein ( for at least half of the duration of the trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U tests are indicated above the boxplots .

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

(A, B) Kymographs of oskMS2-GFP mRNPs (green) associated with Khc-mKate2 (A, red) and mCherry-Tm1-I/C (B, red) ex vivo. Arrows indicate motile RNPs in stable complex with Khc (A) or Tm1-I/C (B), the arrowheads point to non-motile oskMS2-RNPs showing no obvious accumulation of the tagged protein. Note that mCherry-Tm1-I was exposed twice as long as Khc-mKate to obtain comparable red fluorescence signals. Scale bars represent 1 µm and 1 second, respectively.(C-E) Relative Khc-mKate2 (C), mCherry-Tm1-I/C (D) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.


( C - E ) Relative GFP - Staufen ( E ) coverage of motile ( white ) and non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within the boxes indicate the number of trajectories and the number of ooplasms ( in brackets ) analysed . Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein ( for at least half of the duration of the trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U tests are indicated above the boxplots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Relative GFP-Staufen (E) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.


( F ) Example kymograph of Khc molecules ( red ) associating with an oskMS2 - GFP mRNP ( green ) before and during its run . White arrowheads indicate two association events , yellow arrow indicates the onset of motility . Scale bars represent 5 µm and 1 second , respectively . ( G ) Frequency of Khc - mKate2 appearance on motile ( white ) , before ( motility - primed , checked ) and after ( dotted ) the onset of motility , and non - motile ( grey ) oskMS2 - GFP trajectories . Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed . Indicated P values show results of pairwise Fisher ' s exact test . The Khc association frequency observed on RNPs before ( checked ) and during ( dotted ) their motility is not significantly different from wild - type motile RNP controls ( p > 0 . 01 ) .

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

(F) Example kymograph of Khc molecules (red) associating with an oskMS2-GFP mRNP (green) before and during its run. White arrowheads indicate two association events, yellow arrow indicates the onset of motility. Scale bars represent 5 µm and 1 second, respectively.(G) Frequency of Khc-mKate2 appearance on motile (white), before (motility-primed, checked) and after (dotted) the onset of motility, and non-motile (grey) oskMS2-GFP trajectories. Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed. Indicated P values show results of pairwise Fisher's exact test. The Khc association frequency observed on RNPs before (checked) and during (dotted) their motility is not significantly different from wild-type motile RNP controls (p>0.01).


( A ) Western blots of oskar mRNP components ( Staufen , Bruno and Y14 ) and motor associated proteins ( Khc , BicD , Dic ) co - immunoprecipitated with EmGFP - Tm1 - I from ovarian lysates . Protein marker bands and their molecular weight in kDa are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 blots of oskar mRNP components (Staufen, Bruno and Y14) and motor associated proteins (Khc, BicD, Dic) co-immunoprecipitated with EmGFP-Tm1-I from ovarian lysates. Protein marker bands and their molecular weight in kDa are indicated.


( B ) Signal developed with oligo ( dT ) 25 - TexasRed probes on GFP - Trap _ M beads binding EmGFP - Tm1 - I or FlagMycGFP after denaturing washes . 594 nm reflection marks bead boundaries . ( B ' ) Quantification of mean oligo ( dT ) signal measured on beads .

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

(B) Signal developed with oligo(dT)25-TexasRed probes on GFP-Trap_M beads binding EmGFP-Tm1-I or FlagMycGFP after denaturing washes. 594 nm reflection marks bead boundaries.(B') Quantification of mean oligo(dT) signal measured on beads.


( C ) qRT - PCR of EmGFP - Tm1 - I and control Flag - Myc - GFP and bead alone ( w1118 ) bound oskar and gapdh RNAs after stringent washes ( mean ± s . e . m . , 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

(C) qRT-PCR of EmGFP-Tm1-I and control Flag-Myc-GFP and bead alone (w1118) bound oskar and gapdh RNAs after stringent washes (mean ± s.e.m., N = 3).


( D ) Images of beads binding to EmGFP - Tm1 - I ( green ) and DIG labelled in vitro transcribed RNA fragments ( magenta ) . ( E , F ) Mean DIG - Cy5 fluorescence measured on beads capturing the RNA fragment , with or without UV cross - linking , indicated below the charts . P values of pairwise Mann - Whitney U tests are indicated . In panel F , none of the non - cross - linked samples differ significantly from the no RNA control ( p > 0 . 05 ) . Numbers below the plots indicate the number of beads analysed .

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

(D) Images of beads binding to EmGFP-Tm1-I (green) and DIG labelled in vitro transcribed RNA fragments (magenta).(E, F) Mean DIG-Cy5 fluorescence measured on beads capturing the RNA fragment, with or without UV cross-linking, indicated below the charts. P values of pairwise Mann-Whitney U tests are indicated. In panel F, none of the non-cross-linked samples differ significantly from the no RNA control (p>0.05). Numbers below the plots indicate the number of beads analysed.


( A , B , C ) Confocal image of a Tm1eg9 homozygous egg - chamber expressing EmGFP - Tm1 - I ( green ) and Khc - mKate2 ( magenta ) . oskar mRNA labelled with osk1 - 5 FIT probes ( Hovelmann et al . , 2014 ) is in yellow . ( B ' , C ' ) oskar mRNPs co - localizing with both EmGFP - Tm1 - I and Khc - mKate2 . Colors indicate the maximal co - localization distance ( C ' ) . Panels B - C ' represent the boxed regions in panel 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

(A, B, C) Confocal image of a Tm1eg9 homozygous egg-chamber expressing EmGFP-Tm1-I (green) and Khc-mKate2 (magenta). oskar mRNA labelled with osk1-5 FIT probes(Hovelmann et al., 2014) is in yellow.(B', C') oskar mRNPs co-localizing with both EmGFP-Tm1-I and Khc-mKate2. Colors indicate the maximal co-localization distance (C'). Panels B-C' represent the boxed regions in panel A.


( D , E ) Localization of Khc - mKate2 ( green ) and oskar mRNA ( magenta ) in wild - type and Tm1gs mutant nurse cells . ( F , G ) Fraction of oskar mRNPs co - localizing with Khc - mKate ( dark grey ) , EmGFP - Tm1 - I ( white ) , or both of these proteins ( light grey ) in the oocyte ( F ) or in the nurse cells ( G ) ( max . colocalization distance is 250 nm ) . None of the values are significantly different from each other ( one - way ANOVA , p > 10 - 3 ) . Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent ( see Fig EV3C and D ) . Significance of the observed co - localization values versus the expected values are shown . ( H ) Fraction of oskar mRNPs co - localizing with Khc - mKate in wild - type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled ( as indicated above the graph ) . P values of pair - wise t - tests are indicated . ( I ) Fraction of oskar mRNPs co - localizing with free mCherry in wild - type nurse cells used as negative control . The measured fraction ( ~ 1 . 6 % ) is significantly different from zero ( one sample t - test ) . All other measured co - localization values are significantly different form this negative control ( p < 0 . 001 , one - way ANOVA ) . ( F - I ) Numbers indicate the number of particle clusters ( 100 oskar mRNPs in each ) and the number of egg - chambers ( in brackets ) analysed . Error bars represent 95 % confidence intervals . All values ( F - I ) are significantly different from zero ( p < 10 - 3 , one sample 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization of Khc-mKate2 (green) and oskar mRNA (magenta) in wild-type and Tm1gs mutant nurse cells.(F, G) Fraction of oskar mRNPs co-localizing with Khc-mKate (dark grey), EmGFP-Tm1-I (white), or both of these proteins (light grey) in the oocyte (F) or in the nurse cells (G) (max. colocalization distance is 250 nm). None of the values are significantly different from each other (one-way ANOVA, p>10-3). Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent (see Fig EV3C and D). Significance of the observed co-localization values versus the expected values are shown.(H) Fraction of oskar mRNPs co-localizing with Khc-mKate in wild-type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled (as indicated above the graph). P values of pair-wise t-tests are indicated.(I) Fraction of oskar mRNPs co-localizing with free mCherry in wild-type nurse cells used as negative control. The measured fraction (~1.6%) is significantly different from zero (one sample t-test). All other measured co-localization values are significantly different form this negative control (p<0.001, one-way ANOVA).(F-I) Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg-chambers (in brackets) analysed. Error bars represent 95% confidence intervals. All values (F-I) are significantly different from zero (p<10-3, one sample t-test).


( A - C ) Localization of EmGFP - Tm1 - I ( A ) oskar mRNA ( B ) and Khc ( C ) around the nurse cell nuclear envelope ( magenta , WGA staining ) . ( D - F ) Mean distribution profile of Khc ( D ) mKate2 - Tm1 - I , EmGFP - Tm1 - I ( E ) and oskar mRNA ( F ) around the NE of nurse cells ( marked with a dotted vertical line ) . Genotypes are indicated as follows : wild - type control with solid black line ( D - F ) , oskar RNA null with solid grey line ( D , E ) , Tm1eg9 / Tm1eg9 with dotted black line ( D , F ) Lines indicate mean and 95 % confidence intervals . We analysed the accumulation of mKate2 - Tm1 - I instead of EmGFP - Tm1 - I due to unexpected GFP expression from the oskar0 allele .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization of EmGFP-Tm1-I (A) oskar mRNA (B) and Khc (C) around the nurse cell nuclear envelope (magenta, WGA staining).(D-F) Mean distribution profile of Khc (D) mKate2-Tm1-I, EmGFP-Tm1-I (E) and oskar mRNA (F) around the NE of nurse cells (marked with a dotted vertical line). Genotypes are indicated as follows: wild-type control with solid black line (D-F), oskar RNA null with solid grey line (D, E), Tm1eg9/Tm1eg9 with dotted black line (D, F) Lines indicate mean and 95% confidence intervals. We analysed the accumulation of mKate2-Tm1-I instead of EmGFP-Tm1-I due to unexpected GFP expression from the oskar0 allele.


( G ) Khc accumulation around the NE . To calculate accumulation the signal intensity measured at the position of the peak observed in the wild - type control ( D , arrowhead , 356 ± 17 . 6 nm away from NE ) was divided by the signal intensity 2 s . d . away ( d , arrow , at 356 + 2 * 410 nm ) . P values of pairwise Mann - Whitney U tests against wild - type control or Tm1gs rescued with EmGFP - Tm1 - I are indicated above the boxplots . Numbers indicate the number of nuclei and the number of egg - chambers ( in brackets ) analysed . ( H - K ) Khc accumulation around the NE of nurse cells over - expressing EmGFP - Tm1 - I within Tm1eg9 / Tm1eg9 ( H ) , and oskar RNA null egg - chambers ( I ) expressing either the oskar 3 ' UTR ( J ) or oskar Δi ( 1 , 2 , 3 ) ( K ) .

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(G) Khc accumulation around the NE. To calculate accumulation the signal intensity measured at the position of the peak observed in the wild-type control (D, arrowhead, 356±17.6 nm away from NE) was divided by the signal intensity 2 s.d. away (d, arrow, at 356+2*410 nm). P values of pairwise Mann-Whitney U tests against wild-type control or Tm1gs rescued with EmGFP-Tm1-I are indicated above the boxplots. Numbers indicate the number of nuclei and the number of egg-chambers (in brackets) analysed.(H-K) Khc accumulation around the NE of nurse cells over-expressing EmGFP-Tm1-I within Tm1eg9/Tm1eg9 (H), and oskar RNA null egg-chambers (I) expressing either the oskar 3' UTR (J) or oskar Δi(1,2,3) (K).


( A - E ) Mean oskar mRNA distribution ( green , detected by conventional FISH ) within oocytes in which oskar mRNA is substituted by oskar Δi ( 1 , 2 , 3 ) mRNA ( A , B ) , oskar 3 ' UTR mRNA ( D ) and that ( in addition ) over - express EmGFP - Tm1 - I ( B , D and E ) . Wild - type control ( C ) and oocytes expressing Khc RNAi and EmGFP - Tm1 - I ( E ) . Magenta indicates standard deviation of the distribution of oskar mRNA . Scale bar is 10 % of total oocyte length ( C ) . ( F ) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte ( dotted horizontal line ) along the anteroposterior ( AP ) axis . Posterior pole is the top of the chart . P values of pairwise Mann - Whitney U tests are indicated . Numbers indicate number of oocytes analysed .

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(A-E) Mean oskar mRNA distribution (green, detected by conventional FISH) within oocytes in which oskar mRNA is substituted by oskar Δi(1,2,3) mRNA (A, B), oskar 3'UTR mRNA (D) and that (in addition) over-express EmGFP-Tm1-I (B, D and E). Wild-type control (C) and oocytes expressing Khc RNAi and EmGFP-Tm1-I (E). Magenta indicates standard deviation of the distribution of oskar mRNA. Scale bar is 10% of total oocyte length (C).(F) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte (dotted horizontal line) along the anteroposterior (AP) axis. Posterior pole is the top of the chart. P values of pairwise Mann-Whitney U tests are indicated. Numbers indicate number of oocytes analysed.


C . HDo - TRaMs were engineered with vectors relevant to treatment of Fabry , Gaucher , Farber and Pompe diseases . Intracellular lysosomal enzyme specific activities were measured in transduced T - Rapa and controls . Transductions varied across vectors / donors , as indicated . D , E . Enzyme activity was assessed in transduced T - Rapa supernatants compared to controls in activated / dividing ( D ) and quiescent / resting ( E ) states .

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C. HDo-TRaMs were engineered with vectors relevant to treatment of Fabry, Gaucher, Farber and Pompe diseases. Intracellular lysosomal enzyme specific activities were measured in transduced T-Rapa and controls. Transductions varied across vectors/donors, as indicated. D, E. Enzyme activity was assessed in transduced T-Rapa supernatants compared to controls in activated/dividing (D) and quiescent/resting (E) states.


F . Cells from all FDo were stably transduced , as indicated . Intracellular α - galactosidase A ( α - gal A ) specific activities were determined . G , H . Secreted α - gal A activity was measured from FDo - derived TRaMs in activated ( G ) , and quiescent / resting ( H ) states .

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F. Cells from all FDo were stably transduced, as indicated. Intracellular α-galactosidase A (α-gal A) specific activities were determined. G, H. Secreted α-gal A activity was measured from FDo-derived TRaMs in activated (G), and quiescent/resting (H) states. I


I . α - Gal A specific activity in Fabry patient - derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P .

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I. α-Gal A specific activity in Fabry patient-derived skin fibroblasts after 6 hours of exposure to conditioned media from TRaMs in the presence or absence of 1mM M6P.


B . Engraftment was evaluated by flow cytometry for human CD3 expression .

B. Engraftment was evaluated by flow cytometry for human CD3 expression.


C , D . α - Gal A activity in plasma , and specific activities in liver , spleen , heart and kidneys were measured after transplant of transduced healthy donor ( HDo ) - derived ( C ) or Fabry donor ( FDo ) - derived ( D ) TRaMs , non - transduced ( NT ) cells , or in sham - treated NOD / SCID ( NS ) and NSF mice .

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C, D. α-Gal A activity in plasma, and specific activities in liver, spleen, heart and kidneys were measured after transplant of transduced healthy donor (HDo)-derived (C) or Fabry donor (FDo)-derived (D) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.


A , B . Levels of globotriaosylceramide ( Gb3 ) were measured by LC / MS in plasma and extracts of liver , spleen , heart , and kidneys of mice engrafted with healthy donor ( HDo ) - derived ( A ) or Fabry donor ( FDo ) - derived ( B ) TRaMs , non - transduced ( NT ) cells , or in sham - treated NOD / SCID ( NS ) and NSF mice .

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A, B. Levels of globotriaosylceramide (Gb3) were measured by LC/MS in plasma and extracts of liver, spleen, heart, and kidneys of mice engrafted with healthy donor (HDo)-derived (A) or Fabry donor (FDo)-derived (B) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.


C , D . Levels of lyso - Gb3 were measured by LC / MS in plasma and extracts of tissues of mice engrafted with HDo - derived ( C ) or FDo - derived ( D ) TRaMs .

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C, D. Levels of lyso-Gb3 were measured by LC/MS in plasma and extracts of tissues of mice engrafted with HDo-derived (C) or FDo-derived (D) TRaMs.


RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h , Ca WT or Ca YL , and Lm WT or Lm Δhly for 60 min . ( A ) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry . Data represent the mean ± SEM of three independent biological experiments . One - way ANOVA followed by Dunnett ' s test compared to WT strains . ns = non - significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 for 24 h, Ca WT or Ca YL, and Lm WT or Lm Δhly for 60 min. (A) Levels of Rab8A and Rab8A pT72 phosphorylation were analysed by Western blot and quantified by densitometry. Data represent the mean ± SEM of three independent biological experiments. One-way ANOVA followed by Dunnett's test compared to WT strains. ns= non-significant; *p ≤0.05; **p ≤0.01.


( B ) Recruitment of endogenous LRRK2 and EGFP - Rab8A to M . tuberculosis ( Mtb ) was visualised by confocal microscopy . Scale bar = 5 μm .

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(B) Recruitment of endogenous LRRK2 and EGFP-Rab8A to M. tuberculosis (Mtb) was visualised by confocal microscopy. Scale bar = 5 μm.


( C ) C . albicans ( Ca ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative analysis of marker association to the respective pathogen . Data represents the mean ± SEM of two to four independent biological replicates .

(C) C. albicans (Ca) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.


( D ) L . monocytogenes ( Lm ) was analysed by confocal microscopy . Scale bar = 5 μm . Bottom panels show quantitative analysis of marker association to the respective pathogen . Data represents the mean ± SEM of two to four independent biological replicates .

(D) L. monocytogenes (Lm) was analysed by confocal microscopy. Scale bar = 5 μm. Bottom panels show quantitative analysis of marker association to the respective pathogen. Data represents the mean ± SEM of two to four independent biological replicates.


( A ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot .

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(A) RAW264.7 macrophages were treated with 1 mM LLOMe for the indicated time and Rab8A and Rab8A pT72 levels were analysed by Western blot.


( B ) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot .

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(B) WT or LRRK2 KO macrophages were treated with 1 mM LLOMe for 30 min and Rab8A and Rab8A pT72 levels were analysed by Western blot.


( C ) Macrophages were pre - treated with either 1 μM GSK inh or 0 . 1 μM MLi - 2 and then treated with 1 mM LLOMe for 30 min . Rab8A and Rab8A pT72 levels were analysed by Western blot .

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(C) Macrophages were pre-treated with either 1 μM GSK inh or 0.1 μM MLi-2 and then treated with 1 mM LLOMe for 30 min. Rab8A and Rab8A pT72 levels were analysed by Western blot.


( D - F ) RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ( D ) Endogenous LRRK2 and ( E ) Rab8A recruitment to LAMP - 1 positive compartments was visualised by immunofluorescence . Scale bar = 10 μm . ( F ) Quantification of D - E . Data represents the mean ± SEM of three independent biological experiments .

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(D-F) RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. (D) Endogenous LRRK2 and (E) Rab8A recruitment to LAMP-1 positive compartments was visualised by immunofluorescence. Scale bar = 10 μm. (F) Quantification of D-E. Data represents the mean ± SEM of three independent biological experiments.


LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min . The number of LRRK2 per cell were monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . Data represent the mean ± SEM of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s test against the untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 .

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LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of LRRK2 per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.


LRRK2 WT or LRRK2 KO macrophages were pre - treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min . The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . Data represent the mean ± SEM of three to four independent biological experiments . One - way ANOVA followed by Dunnett ' s test against the untreated WT control . ns = non - significant ; * * p ≤ 0 . 01 .

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LRRK2 WT or LRRK2 KO macrophages were pre-treated or not with 1 μM GSK2578215A and treated with 1 mM LLOMe for 30 min. The number of Rab8A positive vesicles per cell were monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. Data represent the mean ± SEM of three to four independent biological experiments. One-way ANOVA followed by Dunnett's test against the untreated WT control. ns=non-significant; **p≤0.01.


( A ) RAW264 . 7 macrophages were electroporated with EGFP - Rab8A and treated with 1 mM LLOMe for 30 min . Intracellular distribution of Galectin - 3 , CHMP4B or LC3B was visualised by immunofluorescence . Scale bar = 5 μm .

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(A) RAW264.7 macrophages were electroporated with EGFP-Rab8A and treated with 1 mM LLOMe for 30 min. Intracellular distribution of Galectin-3, CHMP4B or LC3B was visualised by immunofluorescence. Scale bar = 5 μm.


( B ) Macrophages were electroporated with EGFP - Rab8A and RFP - Galectin - 3 . Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging . Snapshots at the indicated time points after LLOMe addition are shown . Scale bar = 5 μm .

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(B) Macrophages were electroporated with EGFP-Rab8A and RFP-Galectin-3. Cells were then treated with 1 mM of LLOMe and monitored by live cell imaging. Snapshots at the indicated time points after LLOMe addition are shown. Scale bar = 5 μm.


( C ) RAW264 . 7 macrophages were treated with 1 mM of LLOMe and LRRK2 , Rab8A , CHMP4B , Galectin - 3 and LC3B foci over time was analysed by high - content imaging . Data shows the mean ± SEM of five biological replicates .

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(C) RAW264.7 macrophages were treated with 1 mM of LLOMe and LRRK2, Rab8A, CHMP4B, Galectin-3 and LC3B foci over time was analysed by high-content imaging. Data shows the mean ± SEM of five biological replicates.


RAW264 . 7 macrophages were pre - treated with 10 μM BAPTA - AM for 1 hr , and treated with 1 mM LLOMe for 30 min . ( D ) CHMP4B recruitment was monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm .

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RAW264.7 macrophages were pre-treated with 10 μM BAPTA-AM for 1 hr, and treated with 1 mM LLOMe for 30 min. (D) CHMP4B recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm.


( E ) Rab8A pT72 phosphorylation was analysed by Western blot .

(E) Rab8A pT72 phosphorylation was analysed by Western blot.


( F ) LRRK2 and ( G ) Rab8A recruitment was monitored by immunofluorescence and high - content imaging . Scale bar = 10 μm . ( H ) Quantification of E and F .

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(F) LRRK2 and (G) Rab8A recruitment was monitored by immunofluorescence and high-content imaging. Scale bar = 10 μm. (H) Quantification of E and F.


RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show the quantification of number of CHMP4B positive vesicles per cell . Mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

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RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. CHMP4B vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of CHMP4B positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


RAW264 . 7 WT , LRRK2 KO or Rab8A macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) were treated with 1 mM LLOMe for 30 min . Galectin - 3 vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show the quantification of number of Galectin - 3 positive vesicles per cell . Mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

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RAW264.7 WT, LRRK2 KO or Rab8A macrophages pre-treated with 1 μM GSK2578215A (GSK inh) were treated with 1 mM LLOMe for 30 min. Galectin-3 vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show the quantification of number of Galectin-3 positive vesicles per cell. Mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


Live cell imaging of LysoTracker positive spots in WT , LRRK2 KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in the LLOMe treatment window were estimated using linear regression .

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Live cell imaging of LysoTracker positive spots in WT, LRRK2 KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.


Live cell imaging of LysoTracker positive spots in WT , Rab8A KO macrophages treated with 1 mM LLOMe , followed by lysosomal recovery after LLOMe wash - out . One representative experiment out of three shown . Differences between slopes in the LLOMe treatment window were estimated using linear regression .

Live cell imaging of LysoTracker positive spots in WT, Rab8A KO macrophages treated with 1 mM LLOMe, followed by lysosomal recovery after LLOMe wash-out. One representative experiment out of three shown. Differences between slopes in the LLOMe treatment window were estimated using linear regression.


WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) and WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show quantification of number of positive vesicles per cell . Data show the mean ± SEM of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

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WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. LC3B positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT or LRRK2 KO RAW264 . 7 macrophages , RAW264 . 7 macrophages pre - treated with 1 μM GSK2578215A ( GSK inh ) and WT or Rab8A KO RAW264 . 7 macrophages were treated with 1 mM LLOMe for 30 min . ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging . Scale bar = 20 μm . Right panels show quantification of number of positive vesicles per cell . Data show the mean ± SEM of three to four biological replicates . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

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WT or LRRK2 KO RAW264.7 macrophages, RAW264.7 macrophages pre-treated with 1 μM GSK2578215A (GSK inh) and WT or Rab8A KO RAW264.7 macrophages were treated with 1 mM LLOMe for 30 min. ubiquitin K63 positive vesicle numbers were analysed by immunofluorescence and high content imaging. Scale bar = 20 μm. Right panels show quantification of number of positive vesicles per cell. Data show the mean ± SEM of three to four biological replicates. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


( C ) Representative images of LC3B / LAMP - 1 double positive endolysosomes in control and LLOMe treated macrophages . Scale bar = 10 μm . ( D ) Analysis of the % of LC3B / LAMP - 1 double positive endolysosomes in LLOMe treated macrophages . Data shows mean ± SEM of three independent biological experiments . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 by Student ' s t - test .

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(C) Representative images of LC3B/LAMP-1 double positive endolysosomes in control and LLOMe treated macrophages. Scale bar = 10 μm. (D) Analysis of the % of LC3B/LAMP-1 double positive endolysosomes in LLOMe treated macrophages. Data shows mean ± SEM of three independent biological experiments. *p≤0.05, ***p≤0.001 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Mtb for 24 h , Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb for 24 h, Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Ca for 60 min Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Ca for 60 min Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Lm for 60 min . Galectin - 3 and LC3B recruitment was analysed by immunofluorescence and high content imaging . Scale bar = 10 μm . Data show the mean ± SEM of two to three experiments . ns = non - significant , * p ≤ 0 . 05 by Student ' s t - test .

WT and LRRK2 KO RAW264.7 macrophages were infected with Lm for 60 min. Galectin-3 and LC3B recruitment was analysed by immunofluorescence and high content imaging. Scale bar = 10 μm. Data show the mean ± SEM of two to three experiments. ns=non-significant, *p≤0.05 by Student's t-test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

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WT and LRRK2 KO RAW264.7 macrophages were infected with Mtb WT or Mtb ΔRD1 Growth measured for 72 h Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h . Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

WT and LRRK2 KO RAW264.7 macrophages were infected with Ca WT or Ca YL Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


WT and LRRK2 KO RAW264 . 7 macrophages were infected with Lm WT or Lm Δhly . Growth measured for 2 h . Data show the mean ± SEM of three independent biological experiments . ns = non - significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 by One - way ANOVA followed by Sidak ' s multiple comparisons test .

WT and LRRK2 KO RAW264.7 macrophages were infected with Lm WT or Lm Δhly. Growth measured for 2 h. Data show the mean ± SEM of three independent biological experiments. ns=non-significant, *p≤0.05, **p≤0.01 by One-way ANOVA followed by Sidak's multiple comparisons test.


Monocyte - derived macrophages from healthy controls and PD patients carrying the G2019S ( Donor 2 - 3 ) or R1441C ( Donor 1 ) LRRK2 mutation were pre - treated with 0 . 1 μM MLi - 2 and treated with 1 mM LLOMe for 30 min . ( A ) Rab8A and Rab8A pT72 levels were analysed by Western blot .

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Monocyte-derived macrophages from healthy controls and PD patients carrying the G2019S (Donor 2-3) or R1441C (Donor 1) LRRK2 mutation were pre-treated with 0.1 μM MLi-2 and treated with 1 mM LLOMe for 30 min. (A) Rab8A and Rab8A pT72 levels were analysed by Western blot.


( B ) Rab8A and Galectin - 3 positive vesicle numbers were analysed by immunofluorescence . Data show the mean ± SEM of biological replicates . Scale bar = 20 μm . ns = non - significant , * * p ≤ 0 . 01 , by One - way ANOVA followed by Sidak ' s multiple comparisons test .

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(B) Rab8A and Galectin-3 positive vesicle numbers were analysed by immunofluorescence. Data show the mean ± SEM of biological replicates. Scale bar = 20 μm. ns=non-significant, **p≤0.01, by One-way ANOVA followed by Sidak's multiple comparisons test.


a , Survival curves of wild type ( n = 27 ) and Cdc14 DKO ( n = 51 ) mice during the first 6 weeks of life . ( * * * , p < 0 . 001 ; Log - rank , Mantel - Cox test ) . b , Representative images of postnatal day 9 Cdc14 DKO and wild - type littermates . c , Weight of Cdc14 DKO and wild - type littermates during the first 3 weeks of life ( n = 4 per genotype ) . Data information : Bars indicate mean ± SEM . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction .

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a, Survival curves of wild type (n=27) and Cdc14 DKO (n=51) mice during the first 6 weeks of life. (***, p<0.001; Log-rank, Mantel-Cox test). b, Representative images of postnatal day 9 Cdc14 DKO and wild-type littermates. c, Weight of Cdc14 DKO and wild-type littermates during the first 3 weeks of life (n=4 per genotype). Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.


d , Representative macroscopic pictures of brain dorsal view of wild type ( WT ) and Cdc14 KO mice at postnatal 0 , 5 and 9 days ( P0 , P5 and P9 ) . Scale bar , 2 mm . The bar plots show the quantification of brain width , length and area , respectively . Data information : Bars indicate mean ± SEM . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice .

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d, Representative macroscopic pictures of brain dorsal view of wild type (WT) and Cdc14 KO mice at postnatal 0, 5 and 9 days (P0, P5 and P9). Scale bar, 2 mm. The bar plots show the quantification of brain width, length and area, respectively. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.


e , Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0 , P5 and P9 . Fissures underdeveloped in mutant samples are labeled with arrowheads . Scale bar , 700 μm . The bar plots show the quantification of the cerebellar vermis area , the number of folia and the average length of the fissures , respectively . Data information : Bars indicate mean ± SEM . p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice .

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e, Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0, P5 and P9. Fissures underdeveloped in mutant samples are labeled with arrowheads. Scale bar, 700 μm. The bar plots show the quantification of the cerebellar vermis area, the number of folia and the average length of the fissures, respectively. Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.


f , Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples . The molecular layer ( ML ) , internal granule cell layer ( IGL ) and external granule cell layer ( EGL ) are indicated . The width of these layers is shown in the bar plots . Data information : Bars indicate mean ± SEM . * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction . Non - significant data ( p > 0 . 05 ) are not labeled . In data show the individual data , average and SEM from 3 WT and 3 DKO mice . In f , each point represents the average value of 9 different random measurements across distinctive folia for each mouse .

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f, Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples. The molecular layer (ML), internal granule cell layer (IGL) and external granule cell layer (EGL) are indicated. The width of these layers is shown in the bar plots. Data information: Bars indicate mean ± SEM. *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice. In f, each point represents the average value of 9 different random measurements across distinctive folia for each mouse.


g , Representative images of misdigital sections stained with DAPI ( blue ) , Calbindin ( green ) and GFAP ( red ) of cerebellar vermis of wild - type ad Cdc14 DKO mice at 9 days after birth . Scale bar , 50 μm . Plots show the quantification of Purkinje cell dendritic length ( average distance of 30 random measurements ) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot . Data information : Bars indicate mean ± SEM . , * , p > 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction .

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g, Representative images of misdigital sections stained with DAPI (blue), Calbindin (green) and GFAP (red) of cerebellar vermis of wild-type ad Cdc14 DKO mice at 9 days after birth. Scale bar, 50 μm. Plots show the quantification of Purkinje cell dendritic length (average distance of 30 random measurements) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.


a . Confocal microscopy analysis of wild - type and Cdc14 DKO embryonic stem cells ( ESCs ) subjected to neural differentiation . Neuronal marker TUJ1 ( green ) , glial marker GFAP ( red ) and nuclear marker DAPI ( blue ) are stained and quantified after 18 days in differentiation media . Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones . * * * * p - value < 0 . 0001 . Student ' s t - test . Scale bar 50 μm .

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a. Confocal microscopy analysis of wild-type and Cdc14 DKO embryonic stem cells (ESCs) subjected to neural differentiation. Neuronal marker TUJ1 (green), glial marker GFAP (red) and nuclear marker DAPI (blue) are stained and quantified after 18 days in differentiation media. Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones. **** p-value<0.0001. Student's t-test. Scale bar 50 μm.


c , Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation . Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media . Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells . * * * * , p < 0 . 0001 ; Students ' t - test .

c, Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation. Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media. Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells. ****, p<0.0001; Students' t-test.


d , Western blot analysis of CDC14B - GFP co - immunoprecipitations . HeLa cells were transfected with UTF1 and GFP or CDC14B - GFP . Cytoplasmic and nuclear soluble proteins were extracted with RIPA , and nucleolar and chromatin - bound proteins were extracted with high salt RIPA . Immunoprecipitations with anti - GFP antibody were performed . Total protein extracts and immunoprecipitates were loaded and UTF ( upper panel ) and GFP ( lower panel ) were blotted .

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d, Western blot analysis of CDC14B-GFP co-immunoprecipitations. HeLa cells were transfected with UTF1 and GFP or CDC14B-GFP. Cytoplasmic and nuclear soluble proteins were extracted with RIPA, and nucleolar and chromatin-bound proteins were extracted with high salt RIPA. Immunoprecipitations with anti-GFP antibody were performed. Total protein extracts and immunoprecipitates were loaded and UTF (upper panel) and GFP (lower panel) were blotted.


e , Immunoprecipitation of UTF1 upon co - transfection with CDC14B . Total protein was extracted with high - salt RIPA and immunoprecipitations with anti - UTF1 antibody were performed .

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e, Immunoprecipitation of UTF1 upon co-transfection with CDC14B. Total protein was extracted with high-salt RIPA and immunoprecipitations with anti-UTF1 antibody were performed.


f , Expression of active CDC14B , but not a phosphatase - dead ( PD ) isoform , reduces slow - mobility bands of UTF1 . Vinculin was used as a loading control .

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f, Expression of active CDC14B, but not a phosphatase-dead (PD) isoform, reduces slow-mobility bands of UTF1. Vinculin was used as a loading control.


h , In vitro kinase assay of recombinant human UTF1 with ERK2 , CDK1 - Cyclin B1 , CDK1 - Cyclin A2 or in absence of kinases ( C ) .

h, In vitro kinase assay of recombinant human UTF1 with ERK2, CDK1-Cyclin B1, CDK1-Cyclin A2 or in absence of kinases (C).


j , In vitro phosphatase assay of UTF1 . Three different fragments of UTF1 were phosphorylated by CDK1 - Cyclin A2 and subsequently exposed to wild - type or a catalytically inactive form ( PD ) of CDC14B .

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j, In vitro phosphatase assay of UTF1. Three different fragments of UTF1 were phosphorylated by CDK1-Cyclin A2 and subsequently exposed to wild-type or a catalytically inactive form (PD) of CDC14B.


a , Quantitative RT - PCR in wild - type ( WT ) or Cdc14 DKO ESCs of the indicated transcripts during neural differentiation . n = 3 independent experiments ( ± S . E . M . ) ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Student ' s t - test .

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a, Quantitative RT-PCR in wild-type (WT) or Cdc14 DKO ESCs of the indicated transcripts during neural differentiation. n=3 independent experiments (±S.E.M.); *, p<0.05; **, p<0.01; ***, p<0.001; Student's t-test.


b , Expression of a CDC14 - GFP fusion protein in ESCs before ( T0 ) and after three days ( T3 ) in differentiation conditions . The arrowheads indicate the concentration of CDC14 - GFP in the nucleolus before but not after the induction of differentiation . Scale bars , 5 μM . The plot shows the ratio between nucleolar vs . total nuclear URF1 intensity at T0 ( n = 26 cells ) or T3 ( n = 31 ) in cells from two separate experiments . * * * , p < 0 . 001 , Student ' s t - test with Welch ' s correction .

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b, Expression of a CDC14-GFP fusion protein in ESCs before (T0) and after three days (T3) in differentiation conditions. The arrowheads indicate the concentration of CDC14-GFP in the nucleolus before but not after the induction of differentiation. Scale bars, 5 μM. The plot shows the ratio between nucleolar vs. total nuclear URF1 intensity at T0 (n= 26 cells) or T3 (n=31) in cells from two separate experiments. ***, p<0.001, Student's t-test with Welch's correction.