A ) Low magnification merged split channels confocal of striatal sections showing immunolabelling for ChAT ( cyano ) , DRD2 ( red ) , stain DAPI ( ) . Bar = 200 μm . B1 , B2 ) Top . Representative ChAT - positive ( cyano ) cholinergic interneurons , labelled for DRD2 ( red ) , from a Tor1a + / + ( B1 ) or Tor1a + / - ( B2 ) striatal section . colocalization mask of two signals is shown in ( right ) . Bar = 10 μm . Bottom . Higher magnification ( bar = 5 μm ) of representative Tor1a + / + ( B1 ) Tor1a + / - ( B2 ) ChIs showing lack of colocalization of DRD2 with ER marker PDI ( green ) . A detail at higher magnification is shown in

O O O O O O O B-EXP_ASSAY O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O

A) Low magnification merged and split channels confocal images of striatal sections showing immunolabelling for ChAT (cyano), DRD2 (red), and the nuclear stain DAPI (white). Bar = 200 μm. B1,B2) Top. Representative ChAT-positive (cyano) cholinergic interneurons, labelled for DRD2 (red), from a Tor1a+/+ (B1) or Tor1a+/- (B2) striatal section. The colocalization mask of the two signals is shown in white (right). Bar = 10 μm. Bottom. Higher magnification images (bar = 5 μm) of representative Tor1a+/+ (B1) and Tor1a+/- (B2) ChIs showing the lack of colocalization of DRD2 with the ER marker PDI (green). A detail at higher magnification is shown in the insets


A ) Middle . Graphic reconstruction of an anteroposterior of coronal sections of mouse striatum , reporting coordinates relative to Bregma . HSV - LacZ ( left , grey ) HSV - RGS9 - 2 ( right , red ) were injected into left right striatum , respectively , mice sacrificed after 3 - 5 days . Left . Serial confocal showing extent of viral infection , as indicated by robust β - gal . Right . RGS9 - 2 immunolabelling ( red ) is particularly intense near injection site , indicating enhanced expression with respect to endogenous level . Bar = 200

O O O O O O O O O O B-TISSUE O O B-ORGANISM B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

A) Middle. Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum, reporting the coordinates relative to Bregma. HSV-LacZ (left, grey) and HSV-RGS9-2 (right, red) were injected into the left and right striatum, respectively, and mice sacrificed after 3-5 days. Left. Serial confocal images showing the extent of viral infection, as indicated by the robust dark β-gal staining. Right. The RGS9-2 immunolabelling (red) is particularly intense near the injection site, indicating enhanced protein expression with respect to the endogenous level. Bar = 200 µm


B ) A representative WB shows correlated viral - mediated increases in RGS9 - 2 DRD2 in Tor1a + / - dorsal striata ( lanes 4 5 ) . while in a sample where RGS9 - 2 level was not increased by viral infection ( lane 6 ) DRD2 level remained low as in sham - injected striatum ( lane 7 ) . C , D ) summary plots report mean ± values of of vs . loading control level measured from injected ( HSV / LV ) or sham Tor1a + / - ( + / - ) striata , normalized to Tor1a + / + ( + / + ) samples of same experiment . C ) RGS9 - 2 : Tor1a + / + ( N = 10 ) ; Tor1a + / - injected ( N = 7 ) vs . ( N = 7 ) : paired t * * P = 0 . 0011 ; D ) DRD2 : Tor1a + / + ( N = 9 ) ; Tor1a + / - injected ( N = 6 ) vs . ( N = 6 ) : paired t * P = 0 .

O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O 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-GENEPROD O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD 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) A representative WB shows correlated viral-mediated increases in RGS9-2 and DRD2 protein levels in Tor1a+/- dorsal striata (lanes 4 and 5) . while in a sample where RGS9-2 level was not increased by viral infection (lane 6) DRD2 level remained low as in the sham-injected striatum (lane 7). C,D) The summary plots report mean ± SEM values of the ratio of protein vs. loading control intensity level measured from the injected (HSV/LV) or sham contralateral Tor1a+/- (+/-) striata, normalized to the Tor1a+/+ (+/+) samples of the same experiment. C) RGS9-2: Tor1a+/+ (N=10); Tor1a+/- injected (N=7) vs. contralateral (N=7): paired t test **P=0.0011; D) DRD2: Tor1a+/+ (N=9); Tor1a+/- injected (N=6) vs. contralateral (N=6): paired t test *P=0.0380


A ) As depicted in cartoon shown by representative confocal , patch - clamp recordings were performed in dorsal striatum , in of HSV - LacZ ( blue , left ) HSV - RGS9 - 2 ( red , right ) viral infection , latter showing an increased RGS9 - 2 immunolabelling with respect to sham striatum of same mouse . Representative confocal at higher magnification show colocalization of RGS9 - 2 immunolabeling with Lucifer Yellow ( LY ) in two ChIs loaded intracellularly with LY during whole - recordings in infected

O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O O B-ORGANISM O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O

A) As depicted in the cartoon and shown by representative confocal images, patch-clamp recordings were performed in the dorsal striatum, in the area of HSV-LacZ (blue, left) and HSV-RGS9-2 (red, right) viral infection, the latter showing an increased RGS9-2 immunolabelling with respect to the sham contralateral striatum of the same mouse. Representative confocal images at higher magnification show colocalization of RGS9-2 immunolabeling with Lucifer Yellow (LY) fluorescence in two ChIs loaded intracellularly with LY during whole-cell recordings in the infected area


B ) Representative patch - clamp recordings of ChIs from hMT ( top , perforated configuration ) Tor1aΔgag / + ( bottom , - attached configuration ) mice , showing that viral - mediated delivery of RGS9 - 2 rescues physiological response to DRD2 agonist quinpirole ( 10 µM , 2 min ) . dot plot reports firing frequency changes induced by quinpirole in hMT striata ( N = 6 , HSV - RGS9 - 2 n = 9 , HSV - LacZ n = 14 , t P < 0 . 0001 )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O

B) Representative patch-clamp recordings of ChIs from hMT (top, perforated configuration) and Tor1aΔgag/+ (bottom, cell-attached configuration) mice, showing that viral-mediated delivery of RGS9-2 rescues the physiological response to the DRD2 agonist quinpirole (10 µM, 2 min). The dot plot reports the firing frequency changes induced by quinpirole in hMT striata (N=6, HSV-RGS9-2 n=9, HSV-LacZ n=14, t test P<0.0001)


C ) Representative traces show a typical response of Tor1a + / + ChIs to quinpirole , characterized by a reduction in frequency of ongoing firing activity of , as opposed to an aberrant increase in firing frequency in Tor1a + / - ChIs , quantified in plot ( right , Tor1a + / + N = 6 , n = 14 ; Tor1a + / - N = 10 , n = 27 ; Tor1a + / + vs . Tor1a + / - in quinpirole t * * * P = 0 . 0005 ; Tor1a + / - basal vs . quinpirole paired t * * * P = 0 . 0009 )

O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

C) Representative traces show a typical response of Tor1a+/+ ChIs to quinpirole, characterized by a reduction in the frequency of the ongoing firing activity of the cell, as opposed to an aberrant increase in firing frequency in Tor1a+/- ChIs, quantified in the plot (right, Tor1a+/+ N=6, n=14; Tor1a+/- N=10, n=27; Tor1a+/+ vs. Tor1a+/- in quinpirole t test ***P=0.0005; Tor1a+/- basal vs. quinpirole paired t test ***P=0.0009)


D ) LV - mediated delivery of RGS9 - 2 rescues physiological response to quinpirole in Tor1a + / - slices . Dot plot reporting firing frequency changes induced by quinpirole in Tor1a + / + ( N = 3 ) Tor1a + / - ( N = 6 ) striatal ChIs infected with LV / HSV - RGS9 - 2 or control LV - GFP HSV - LacZ ChIs ( Tor1a + / + RGS9 - 2 n = 7 ; Tor1a + / - RGS9 - 2 n = 9 ; Tor1a + / - LacZ / GFP n = 8 ; Tor1a + / - : LacZ / GFP vs . RGS9 - 2 t * P = 0 . 0136 )

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

D) LV-mediated delivery of RGS9-2 rescues the physiological response to quinpirole in Tor1a+/- slices. Dot plot reporting firing frequency changes induced by quinpirole in Tor1a+/+ (N=3) and Tor1a+/- (N=6) striatal ChIs infected with LV/HSV-RGS9-2 or control LV-GFP and HSV-LacZ ChIs (Tor1a+/+ RGS9-2 n=7; Tor1a+/- RGS9-2 n=9; Tor1a+/- LacZ/GFP n=8; Tor1a+/-: LacZ/GFP vs. RGS9-2 t test *P=0.0136)


C . HEK293 were incubated with 5 µg of purified EVCx43 - or EVCx43 + for 24 h , in presence or absence of Bafilomycin A1 ( Baf ) in last 6 h . of LC3 were analyzed by WB are depicted on graph ( LC3 - II ; n = 4 biological replicates ) . Data information : p - values were derived by Mann - Whitney . Bars error bars indicate mean ± SD in graphs .

O O B-CELL_LINE O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O 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. HEK293 cells were incubated with 5 µg of purified EVCx43- or EVCx43+ for 24 h, in the presence or absence of Bafilomycin A1 (Baf) in the last 6 h. The levels of LC3 were analyzed by WB and are depicted on graph (LC3-II; n=4 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


E . HEK293 were incubated with EVCx43 - or EVCx43 + for 24 h before WB of ATP2A2 / SERCA2 , a predicted target of miRNAs enriched in Cx43 - containing EV . Graph depicts of ATP2A2 / SERCA2 ( n = 4 biological replicates ) . Data information : p - values were derived by Mann - Whitney . Bars error bars indicate mean ± SD in graphs .

O O B-CELL_LINE O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O O 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. HEK293 cells were incubated with EVCx43- or EVCx43+ for 24 h before WB analysis of ATP2A2/SERCA2, a predicted target of miRNAs enriched in Cx43-containing EV. Graph depicts quantification of ATP2A2/SERCA2 levels (n=4 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


A . Cx43 was immunoprecipitated ( IP ) from HEK293Cx43 + , after which interaction with hnRNPA2B1 hnRNPQ was analyzed by WB .

O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_LINE O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O

A. Cx43 was immunoprecipitated (IP) from HEK293Cx43+ cells, after which interaction with hnRNPA2B1 and hnRNPQ was analyzed by WB.


B . Co - localization between Cx43 ( green ) hnRNPA2B1 or hnRNPQ ( red ) in C33a was analyzed by confocal microscopy . Scale bars 10 μm . Arrowheads in inset highlight co - localization between Cx43 hnRNPs .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O

B. Co-localization between Cx43 (green) and hnRNPA2B1 or hnRNPQ (red) in C33a cells was analyzed by confocal microscopy. Scale bars 10 μm. Arrowheads in the inset images highlight co-localization between Cx43 and hnRNPs.


A . free EV biogenesis was assessed by relative protection of Cx43 - luciferase . Reactions were incubated at 4ºC or 30ºC in presence of membranes derived from HEK293Cx43 - transiently transfected with Cx43 - luciferase cytosol from HEK293Cx43 - , except in negative control . 1 % TX100 was used to disrupt membranes , where indicated ( n = 3 biological replicates ) . Data information : p - values were derived by Mann - Whitney . Bars error bars indicate mean ± SD in graphs .

O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O 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. Cell free EV biogenesis was assessed by relative protection of Cx43-luciferase. Reactions were incubated at 4ºC or 30ºC in the presence of membranes derived from HEK293Cx43- transiently transfected with Cx43-luciferase and cytosol from HEK293Cx43- cells, except in the negative control. 1% TX100 was used to disrupt membranes, where indicated (n=3 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


B . Crude membranes cytosol from broken HEK293Cx43 + or HEK293Cx43 - were mixed in an ATP regenerating system , supplemented with synthetic miR - 133b or miR - 509 - 3p , as indicated . Graph depicts indicated miRNAs , assessed by RT - qPCR represented as protected after RNAse I digestion of unincorporated miRNAs ( n = 7 - 8 biological replicates ) . Data information : p - values were derived by Mann - Whitney . Bars error bars indicate mean ± SD in graphs .

O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O 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. Crude membranes and cytosol from broken HEK293Cx43+ or HEK293Cx43- cells were mixed in an ATP regenerating system, supplemented with synthetic miR-133b or miR-509-3p, as indicated. Graph depicts the indicated miRNAs levels, assessed by RT-qPCR and represented as percent protected after RNAse I digestion of unincorporated miRNAs (n=7-8 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


C . Co - immunoprecipitation of endogenous miR - 133b miR - 509 - 3p following UV crosslinking immunoprecipitation ( CLIP ) of Cx43 ( n = 3 - 5 biological replicates ) . Data information : p - values were derived by Mann - Whitney . Bars error bars indicate mean ± SD in graphs .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O 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. Co-immunoprecipitation of endogenous miR-133b and miR-509-3p following UV crosslinking immunoprecipitation (CLIP) of Cx43 (n=3-5 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


D . WB for Cx43 in samples derived by miRNAs pulldowns performed with whole lysates of HEK293Cx43 + biotinylated miR - 133b miR - 509 - 3p ( n = 5 biological replicates ) . Data information : p - values were derived by Mann - Whitney . Bars error bars indicate mean ± SD in graphs .

O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-CELL_LINE O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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. WB analysis for Cx43 in samples derived by miRNAs pulldowns performed with whole lysates of HEK293Cx43+ cells and biotinylated miR-133b and miR-509-3p (n=5 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


EMSA was performed using either Cy3 - labeled miR - 133b ( E , Cx43 produced by in vitro translation inserted into lipid nanodiscs , For competition EMSA , an excess of unlabeled miR - 133b was used ( competitor ) . Empty nanodiscs were used to control unspecific binding .

B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O

EMSA was performed using either Cy3-labeled miR-133b (E , and Cx43 produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled miR-133b was used (competitor). Empty nanodiscs were used to control unspecific binding.


EMSA was performed using either Cy3 - labeled miR - 133b F ) Cx43 produced by in vitro translation inserted into lipid nanodiscs , in presence or absence of recombinant GST - hnRNPA2B1 or GST - hnRNPQ , as indicated .

B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

EMSA was performed using either Cy3-labeled miR-133b F) and Cx43 produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1 or GST-hnRNPQ, as indicated.


EMSA was performed using either Cy3 - labeled miR - 509 - 3p ( G Cx43 produced by in vitro translation inserted into lipid nanodiscs , For competition EMSA , an excess of unlabeled miR - 133b or miR - 509 - 3p was used ( competitor ) . Empty nanodiscs were used to control unspecific binding .

B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O

EMSA was performed using either Cy3-labeled miR-509-3p (G and Cx43 produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled miR-133b or miR-509-3p was used (competitor). Empty nanodiscs were used to control unspecific binding.


EMSA was performed using either Cy3 - labeled miR - 509 - 3p H ) Cx43 produced by in vitro translation inserted into lipid nanodiscs , in presence or absence of recombinant GST - hnRNPA2B1 or GST - hnRNPQ , as indicated .

B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

EMSA was performed using either Cy3-labeled miR-509-3p H) and Cx43 produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1 or GST-hnRNPQ, as indicated.


I . EMSA was performed using either Cy3 - labeled wild type or mutant miR - 199a - 3p , Cx43 - enriched membranes prepared from C33a parental ( Cx43 + ) . Cx43 - membranes were used to control unspecific binding .

O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-EXP_ASSAY O

I. EMSA was performed using either Cy3-labeled wild type or mutant miR-199a-3p, and Cx43-enriched membranes prepared from C33a parental cells (Cx43+). Cx43- membranes were used to control unspecific binding.


Representative maximum projection of microscopy - miRNA - interaction using beads coated with GST - tagged NT / CT - Cx43 , hnRNPA2B1 or hnRNPQ , as indicated , following addition of Cy3 - labelled miR - 133b or miR - 509 - 3p . Graph depicts of total / bead , as a measure of - miRNAs binding . Scale bars 50 μm ( n = 2 biological replicates ; average of 23 beads analyzed / condition ) . Data information : p - values were derived by Kruskal - Wallis ( Dunn ' s post - hoc ) . Bars error bars indicate mean ± SD in graphs .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative maximum projection images of microscopy-based miRNA-protein interaction using beads coated with GST-tagged NT/CT-Cx43, hnRNPA2B1 or hnRNPQ, as indicated, following the addition of Cy3-labelled miR-133b or miR-509-3p. Graph depicts quantification of total fluorescence/bead, as a measure of protein-miRNAs binding. Scale bars 50 μm (n=2 biological replicates; average of 23 beads analyzed/condition). Data information: p-values were derived by Kruskal-Wallis (Dunn's post-hoc) test. Bars and error bars indicate mean ± SD in all graphs.


A . Equal amounts of EVCx43 + EVCx43 - labelled with SYTO ® RNASelect ™ green stain were incubated with HEK293Cx43 + for 30 min , at 37ºC . Scale bars 10 μm ( n = 5 biological replicates ) . p - values were derived by Mann - Whitney .

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A. Equal amounts of EVCx43+ and EVCx43- labelled with SYTO® RNASelect™ green fluorescent stain were incubated with HEK293Cx43+ cells for 30 min, at 37ºC. Scale bars 10 μm (n=5 biological replicates). p-values were derived by Mann-Whitney test.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intravenously infected with VSV - GFP ( 2 × 108 PFU / mouse ) , serum samples were collected at 12 hpi ( hours post - infection ) . ( A ) titer was analyzed in a plaque .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (A) Virus titer was analyzed in a plaque assay.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intravenously infected with VSV - GFP ( 2 × 108 PFU / mouse ) , serum samples were collected at 12 hpi ( hours post - infection ) . ( B ) Secretion of IFN - β , IL - 6 , IL - 12 , TNFα was measured in specific ELISAs .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (B) Secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.


C MARCH2 + / + ( n = 6 ) or MARCH2 - / - ( n = 6 ) mice were intravenously injected with poly ( I : C ) ( 200 μg / mouse ) . Serum samples were collected at 2 hpi secretion of IFN - β , IL - 6 , IL - 12 , TNFα was measured in specific ELISAs .

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C MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were intravenously injected with poly(I:C) (200 μg/mouse). Serum samples were collected at 2 hpi and secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( D ) of surviving mice ( D , log - rank , * * P < 0 . 01 )

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (D) The percentage of surviving mice (D, log-rank test, **P < 0.01)


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . changes for each group ( E ) are shown .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. body weight changes for each group (E) are shown.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( F , G ) At 3 dpc , load in spleen ( F ) liver ( G ) was examined .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (F, G) At 3 dpc, the bacterial load in the spleen (F) and liver (G) was examined.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes . ( H - J ) Amount of IL - 6 , IL - 12 , CCL5 , CXCL10 in serum ( H ) , spleen ( I ) , liver ( J ) of mice in each group at 24 hpc .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (H-J) Amount of IL-6, IL-12, CCL5, and CXCL10 in serum (H), spleen (I), and liver (J) of mice in each group at 24 hpc.


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . of surviving mice ( A , log - rank , * * P < 0 . 01 )

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). Percentage of surviving mice (A, log-rank test, **P < 0.01)


MARCH2 + / + ( n = 8 ) or MARCH2 - / - ( n = 8 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . changes ( B ) in each groups .

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MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). body weight changes (B) in each groups.


C MARCH2 + / + ( n = 4 ) or MARCH2 - / - ( n = 4 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . Representing slides of H & E of spleen sections from each group . Scale bar , 6mm .

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C MARCH2+/+ (n = 4) or MARCH2-/- (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6mm.


D MARCH2 + / + ( n = 6 ) or MARCH2 - / - ( n = 6 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . of IL - 6 , TNF - α , CXCL - 10 , CCL - 5 in serum from mice in each groups were measured at12 hpc by ELISA .

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D MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, CCL-5 in serum from mice in each groups were measured at12 hpc by ELISA.


E , F MARCH2 + / + ( n = 5 ) or MARCH2 - / - ( n = 5 ) mice were challenged intraperitoneally with LPS ( 24 mg / kg ) . cDNA was prepared from total extracted from spleen liver of mice . Expression of mRNA encoding IL - 6 , TNF - α , CXCL10 , IL - 1β in spleen ( E ) liver ( F ) from mice in each group was examined at 6 hpc by qPCR .

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E, F MARCH2+/+ (n = 5) or MARCH2-/- (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR.


A - C BMDMs isolated from MARCH2 + / + or MARCH2 - / - mice were infected with PR8 - GFP ( A , MOI = 3 ) , VSV - GFP ( B , MOI = 5 ) , or HSV - GFP ( C , MOI = 3 ) , titer was measured in a plaque .

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A-C BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with PR8-GFP (A, MOI=3), VSV-GFP (B, MOI = 5), or HSV-GFP (C, MOI = 3), and virus titer was measured in a plaque assay.


D , E BMDMs isolated from MARCH2 + / + or MARCH2 - / - mice were infected with or treated with poly ( I : C ) ( 80 µg / ml ) or poly ( dA : dT ) ( 1 µg / ml ) . of secreted IFN - β ( D ) IL - 6 ( E ) in supernatants was measured in an ELISA .

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D, E BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with viruses or treated with poly(I:C) (80 µg/ml) or poly (dA:dT) (1 µg/ml). The concentration of secreted IFN-β (D) and IL-6 (E) in the supernatants was measured in an ELISA.


PBMCs isolated from whole peripheral blood from MARCH2 + / + or MARCH2 - / - mice were infected with VSV - GFP ( MOI = 1 ) . ( F ) titer was measured in a plaque .

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PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (F) The virus titer was measured in a plaque assay.


PBMCs isolated from whole peripheral blood from MARCH2 + / + or MARCH2 - / - mice were infected with VSV - GFP ( MOI = 1 ) . ( G ) of IFN - β IL - 6 in supernatant was measured in an ELISA .

B-CELL_TYPE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O

PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (G) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.


PMs isolated from MARCH2 + / + or MARCH2 - / - mice infected with CVB - GFP ( MOI = 3 ) . ( H ) titer was measured in a plaque .

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PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (H) Virus titer was measured in a plaque assay.


PMs isolated from MARCH2 + / + or MARCH2 - / - mice infected with CVB - GFP ( MOI = 3 ) . ( I ) of IFN - β IL - 6 in supernatant was measured in an ELISA .

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PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (I) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.


J , K BMDMs ( J ) or PMs ( K ) isolated from MARCH2 + / + or MARCH2 - / - mice were infected with Salmonella typhimurium or L . monocytogenes , or treated with LPS or zymosan . of IL - 6 IL - 12 in supernatant was analyzed in an ELISA .

O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O

J, K BMDMs (J) or PMs (K) isolated from MARCH2+/+ or MARCH2-/- mice were infected with Salmonella typhimurium or L. monocytogenes, or treated with LPS or zymosan. The concentration of IL-6 and IL-12 in supernatant was analyzed in an ELISA.


L , M RAW 264 . 7 transfected with control siRNA ( si - control ) or MARCH2 - specific siRNA ( si - MARCH2 ) were infected with PR8 - GFP ( MOI = 1 ) , VSV - GFP ( MOI = 0 . 5 ) , poly ( I : C ) ( 20 µg / ml ) or HSV - GFP ( MOI = 1 ) poly ( dA : dT ) ( 1 µg / ml ) IFN - β ( L ) , IL - 6 ( M ) in supernatant were measured in an ELISA .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-ORGANISM O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O

L, M RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with PR8-GFP (MOI = 1), VSV-GFP (MOI = 0.5), poly (I:C) (20 µg/ml) or HSV-GFP (MOI = 1) poly (dA:dT) (1 µg/ml) and IFN-β (L), and IL-6 (M) levels in the supernatant were measured in an ELISA.


N RAW 264 . 7 transfected with control siRNA ( si - control ) or MARCH2 - specific siRNA ( si - MARCH2 ) were infected with S . typhimurium , or L . monocytogenes , or treated with LPS ( 100 ng / ml ) or zymosan ( 100 µg / ml ) IL - 6 secretion into supernatant was measured in an

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N RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with S. typhimurium, or L. monocytogenes, or treated with LPS (100 ng/ml) or zymosan (100 µg/ml) and IL-6 secretion into the cell supernatant was measured in an ELISA


HEK293T were transfected with a firefly luciferase reporter plasmid encoding IFN - β promoter , plus a TK renilla plasmid an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for RIG - I 2CARD , RIG - I , MDA - 5 , MAVS or stimulated with poly ( I : C ) , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

B-CELL_LINE O O O O B-ORGANISM B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM B-GENEPROD O O O O O O

HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter , plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for RIG-I 2CARD, RIG-I, MDA-5, MAVS or stimulated with poly (I:C), for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T were transfected with a firefly luciferase reporter plasmid encoding IFN - β promoter plus a TK renilla plasmid an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TBK1 TRIF , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the IFN-β promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T were transfected with a firefly luciferase reporter plasmid encoding NF - κB promoter plus a TK renilla plasmid an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TRAF 6 , TRAF 2 , NEMO for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

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HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TRAF 6, TRAF 2, NEMO for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


HEK293T were transfected with a firefly luciferase reporter plasmid encoding NF - κB promoter plus a TK renilla plasmid an increasing dose of flag - tagged MARCH2 plasmid ( 50 , 100 , 200 , 400 ng ) plus expression plasmids for TBK1 TRIF , for 24 h . Results are expressed relative to those of renilla luciferase alone ( Internal control ) .

B-CELL_LINE O O O O B-ORGANISM B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM B-GENEPROD O O O O O O

HEK293T cells were transfected with a firefly luciferase reporter plasmid encoding the NF-κB promoter plus a TK renilla plasmid and an increasing dose of flag-tagged MARCH2 plasmid (50, 100, 200, 400 ng) plus expression plasmids for TBK1 TRIF , for 24 h. Results are expressed relative to those of renilla luciferase alone (Internal control).


Interaction between MARCH2 NEMO in response to viral infection . HEK293T ( E ) were infected with NDV - GFP in a time - dependent manner in presence of MG132 ( proteasome inhibitor , 10 μM ) . lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

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Interaction between MARCH2 and NEMO in response to viral infection. HEK293T cells (E) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


Interaction between MARCH2 NEMO in response to viral infection . Raw264 . 7 ( F ) were infected with NDV - GFP in a time - dependent manner in presence of MG132 ( proteasome inhibitor , 10 μM ) . lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

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Interaction between MARCH2 and NEMO in response to viral infection. Raw264.7 cells (F) were infected with NDV-GFP in a time-dependent manner in the presence of MG132 (proteasome inhibitor, 10 μM). Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


G Confocal microscopy was conducted to examine time - dependent colocalization of MARCH2 NEMO in HeLa upon NDV infection ( MOI = 1 ) in presence of MG132 ( 10 μM ) . Arrow indicates co - localized NEMO MARCH2 .

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G Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.


Interaction between MARCH2 NEMO in response to infection . Raw264 . 7 were infected with L . monocytogenes ( H ) or S . typhimurium ( I ) in a time - dependent manner in presence of MG132 . lysates were subjected to immunoprecipitation with an anti - NEMO antibody , followed by immunoblotting with an anti - MARCH2 antibody .

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Interaction between MARCH2 and NEMO in response to bacterial infection. Raw264.7 cells were infected with L. monocytogenes (H) or S. typhimurium (I) in a time-dependent manner in the presence of MG132. Cell lysates were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-MARCH2 antibody.


J Confocal microscopy was conducted to examine time - dependent colocalization of MARCH2 NEMO in HeLa expressing TLR2 upon zymosan treatment ( 100 μg / ml ) in presence of MG132 ( 10 μM ) . Arrow indicates co - localized NEMO MARCH2 .

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J Confocal microscopy was conducted to examine time-dependent colocalization of MARCH2 and NEMO in HeLa cells expressing TLR2 upon zymosan treatment (100 μg/ml) in the presence of MG132 (10 μM). Arrow indicates the co-localized NEMO and MARCH2 protein.


NEMO degradation . ( A ) HEK293T were transfected with different doses of Strep - tagged MARCH2 in presence HA - tagged NEMO plasmid .

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NEMO degradation assay. (A) HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence HA-tagged NEMO plasmid.


NEMO degradation . ( B ) MARCH2 + / + MARCH2 - / - HEK293T were infected with PR8 - GFP ( MOI = 1 ) in time - dependent manner . were harvested following infection , NEMO expression level , total phosphorylated IRF - 3 , TBK - 1 were measured by immunoblotting . β - actin was used to confirm equal loading of .

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NEMO degradation assay. (B) MARCH2+/+ and MARCH2-/- HEK293T cells were infected with PR8-GFP (MOI=1) in time-dependent manner. Cells were harvested following infection, NEMO expression level, total and phosphorylated IRF-3, and TBK-1 were measured by immunoblotting. β-actin was used to confirm equal loading of protein.


C HEK293T transfected with a Strep - tagged empty vector or MARCH2 together with GST - tagged NEMO were treated with MG132 ( 10 μM ) , chloroquine , NH4Cl , or Z - VAD for 6 h before harvest . Whole lysates were subjected to immunoblotting with indicated antibodies .

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C HEK293T cells transfected with a Strep-tagged empty vector or MARCH2 together with GST-tagged NEMO were treated with MG132 (10 μM), chloroquine, NH4Cl, or Z-VAD for 6 h before harvest. Whole cell lysates were subjected to immunoblotting with indicated antibodies.


D HEK293T were transfected with different doses of Strep - tagged MARCH2 together with GST - tagged NEMO plasmid . MG132 ( 10 μM ) was added to for 6 h prior to harvest . Whole lysates were subjected to immunoblotting with indicated antibodies .

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D HEK293T cells were transfected with different doses of Strep-tagged MARCH2 together with and GST-tagged NEMO plasmid. MG132 (10 μM) was added to cells for 6 h prior to harvest. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.


E Confocal microscopy was used to examine colocalization of MARCH2 NEMO in HeLa upon NDV infection ( MOI = 1 ) in presence or absence of MG132 ( 10 μM ) . Scale bar , 10μm . Arrow indicates co - localized NEMO MARCH2 .

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E Confocal microscopy was used to examine colocalization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence or absence of MG132 (10 μM). Scale bar, 10μm. Arrow indicates the co-localized NEMO and MARCH2 protein.


F HEK293T transfected with Strep - tagged empty vector or MARCH2 with GST - tagged NEMO each different HA - tagged ubiquitin mutants ( indicated lysine ( K ) only , other lysines ( K ) mutated to arginines ( R ) were treated with MG132 ( 10 μM ) for 6 h before harvest . Lysates were subjected to pull down with GST beads , followed by immunoblotting with an anti - HA antibody . Whole lysates were determined by immunoblotting with indicated antibodies .

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F HEK293T cells transfected with Strep-tagged empty vector or MARCH2 with GST-tagged NEMO and each different HA-tagged ubiquitin mutants (indicated lysine (K) only, other lysines (K) mutated to arginines (R) were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to pull down with GST beads, followed by immunoblotting with an anti-HA antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.


G NEMO ubiquitination . HEK293T were transfected with different doses of Strep - tagged MARCH2 in presence of a HA - tagged NEMO plasmid . Lysates were immunoprecipitated with anti - HA antibody followed by immunoblotting with anti - K48 antibody .

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G NEMO ubiquitination assay. HEK293T cells were transfected with different doses of Strep-tagged MARCH2 in the presence of a HA-tagged NEMO plasmid. Lysates were immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-K48 antibody.


MARCH2 + / + HEK293T harboring empty vector or MARCH2 - / - HEK293T harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within catalytic domain : C64S , C67S , H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . Viral replication was assessed under a microscope ( H )

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MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). Viral replication was assessed under a fluorescence microscope (H)


MARCH2 + / + HEK293T harboring empty vector or MARCH2 - / - HEK293T harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within catalytic domain : C64S , C67S , H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . by measuring GFP expression using a

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MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). by measuring GFP expression using a fluorescence modulator


MARCH2 + / + HEK293T harboring empty vector or MARCH2 - / - HEK293T harboring empty vector , Flag - tagged MARCH2 , or MARCH2CCH ( a mutant harboring mutations in residues within catalytic domain : C64S , C67S , H90Q ) were infected with VSV - GFP ( MOI = 0 . 05 ) . ( J ) of IFN - β ( top ) IL - 6 ( bottom ) in supernatants were measured in an ELISA .

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MARCH2+/+ HEK293T cells harboring empty vector or MARCH2-/- HEK293T cells harboring empty vector, Flag-tagged MARCH2, or MARCH2CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV-GFP (MOI = 0.05). (J) Levels of IFN-β (top) and IL-6 (bottom) in cell supernatants were measured in an ELISA.


K In vitro ubiquitination for NEMO . HEK293T were transfected with GST - tagged NEMO , Strep - tagged MARCH2 or MARCH2CCH individually . Immunoprecipitates were incubated with a reaction mixture containing ubiquitin , E1 , UbcH6 ( E2 ) , followed by immunoblotting with an anti - K48 - linkage specific polyubiquitin antibody .

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K In vitro ubiquitination assay for NEMO. HEK293T cells were transfected with GST-tagged NEMO, Strep-tagged MARCH2 or MARCH2CCH individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.


( B ) HEK293T were transfected with Strep - tagged MARCH2 domain constructs together with GST - tagged NEMO . Whole lysates were subjected to a Strep - PD , followed by immunoblotting with an anti - GST antibody .

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(B) HEK293T cells were transfected with Strep-tagged MARCH2 domain constructs together with GST-tagged NEMO. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.


( D ) HEK293T were transfected with GST - tagged NEMO domain constructs together with Strep - tagged MARCH2 - TM . Whole lysates were subjected to a Strep - PD , followed by immunoblotting with an anti - GST antibody .

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(D) HEK293T cells were transfected with GST-tagged NEMO domain constructs together with Strep-tagged MARCH2-TM. Whole cell lysates were subjected to a Strep-PD assay, followed by immunoblotting with an anti-GST antibody.


( F ) HEK293T were transfected with HA - tagged NEMO domain constructs together with GST - tagged MARCH2 . Whole lysates were subjected to a GST - PD , followed by immunoblotting with an anti - HA antibody .

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(F) HEK293T cells were transfected with HA-tagged NEMO domain constructs together with GST-tagged MARCH2. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-HA antibody.


G HEK293T transfected with His - tagged ubiquitin HA - tagged NEMO WT or NEMO lacking CC1 domain together with Strep - tagged empty vector or MARCH2 were treated with MG132 ( 10 μM ) for 6 h before harvest . Lysates were subjected to immunoprecipitation with an anti - HA antibody , followed by immunoblotting with an anti - His antibody . WCL were subjected to immunoblotting with anti - HA , anti - Strep , or anti - β - actin antibody .

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G HEK293T cells transfected with His-tagged ubiquitin and HA-tagged NEMO WT or NEMO lacking the CC1 domain together with Strep-tagged empty vector or MARCH2 were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-His antibody. WCL were subjected to immunoblotting with anti-HA, anti-Strep, or anti-β-actin antibody.


( I ) HEK293T transfected with GST - tagged NEMO mutants together with Strep - tagged empty vector or MARCH2 were subjected to immunoblotting with anti - GST , anti - Strep , or anti - β - actin antibody .

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(I) HEK293T cells transfected with GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to immunoblotting with anti-GST, anti-Strep, or anti-β-actin antibody.


J HEK293T transfected with HA - tagged ubiquitin GST - tagged NEMO mutants together with Strep - tagged empty vector or MARCH2 were subjected to pulldown with GST beads , followed by immunoblotting with anti - Lys48 ( K48 ) - linkage specific polyubiquitin ( K48 - ub ) , anti - Lys63 ( K63 ) - linkage specific polyubiquitin ( K63 - ub ) , or anti - Strep antibody . Whole lysates were determined by immunoblotting with indicated antibodies .

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J HEK293T cells transfected with HA-tagged ubiquitin and GST-tagged NEMO mutants together with Strep-tagged empty vector or MARCH2 were subjected to pulldown with GST beads, followed by immunoblotting with anti-Lys48 (K48)-linkage specific polyubiquitin (K48-ub), anti-Lys63 (K63)-linkage specific polyubiquitin (K63-ub), or anti-Strep antibody. Whole cell lysates were determined by immunoblotting with the indicated antibodies.


A HEK293T were transfected with GST - tagged NEMO - WT its point mutants with or without Strep - tagged MARCH2 . Whole lysates were subjected to immunoblotting with indicated antibodies .

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A HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants with or without Strep-tagged MARCH2. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.


B HEK293T were transfected with GST - tagged NEMO - WT its point mutants together with Strep - tagged MARCH2 in presence of MG132 . Whole lysates were subjected to a GST - PD , followed by immunoblotting with an anti - K48 linkage specific polyubiquitin antibody .

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B HEK293T cells were transfected with GST-tagged NEMO-WT and its point mutants together with Strep-tagged MARCH2 in the presence of MG132. Whole cell lysates were subjected to a GST-PD assay, followed by immunoblotting with an anti-K48 linkage specific polyubiquitin antibody.


C In vitro ubiquitination for NEMO - K326R . HEK293T were transfected with GST - tagged NEMO , GST - tagged NEMO - K326R or Strep - tagged MARCH2 individually . Immunoprecipitates were incubated with a reaction mixture containing ubiquitin , E1 , UbcH6 ( E2 ) , followed by immunoblotting with an anti - K48 - linkage specific polyubiquitin antibody .

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C In vitro ubiquitination assay for NEMO-K326R. HEK293T cells were transfected with GST-tagged NEMO, GST-tagged NEMO-K326R or Strep-tagged MARCH2 individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti-K48-linkage specific polyubiquitin antibody.


HEK293T transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . ( D - F ) Viral replication was determined by microscopy ( D )

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HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (D-F) Viral replication was determined by fluorescence microscopy (D)


HEK293T transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . , absorbance ( E )

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HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. , fluorescence absorbance (E)


HEK293T transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . plaque ( F ) .

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HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. plaque assay (F).


HEK293T transfected with GST - tagged empty vector , NEMO - WT , or NEMO - K326R , with or without Strep - tagged MARCH2 were infected with VSV - GFP . ( G ) IFN - β IL - 6 in supernatants were measured in an ELISA .

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HEK293T cells transfected with GST-tagged empty vector, NEMO-WT, or NEMO-K326R, with or without Strep-tagged MARCH2 were infected with VSV-GFP. (G) IFN-β and IL-6 in the cell supernatants were measured in an ELISA.


H , I IFN - β ( H ) NF - κB ( I ) luciferase reporter .

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H, I IFN-β (H) and NF-κB (I) luciferase reporter assay.


( E ) ( left ) Western blot of NANOG , OCT4 , SOX2 . ( right ) Relative NANOG as estimated by densitometry ( n = 8 ) . NANOG was nearly 7 - fold more in PD , which is twice that of 2iL / SL2i . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns = not significant ( paired two - student ' s t - ) .

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(E) (left) Western blot of NANOG, OCT4, and SOX2. (right) Relative NANOG levels as estimated by densitometry (n=8). NANOG was nearly 7-fold more in PD, which is twice that of 2iL/SL2i. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).


( F ) Western blot of pERK ERK at 0 , 1 , 4 , 8 , 12 , 16 , 24 hrs after media change in indicated treatments ( n = 3 ) .

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(F) Western blot of pERK and ERK at 0, 1, 4, 8, 12, 16, and 24 hrs after media change in indicated treatments (n=3).


( G ) Western blot of pERK ERK in SLPD , SLCHIR , 2iL , SL2i after 8 , 12 , 16 , 24 hrs of culture relative to SL , where in SL were harvested 24 hrs after media change ( n > 3 ) .

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(G) Western blot of pERK and ERK in SLPD, SLCHIR, 2iL, and SL2i after 8, 12, 16, and 24 hrs of culture relative to SL, where the cells in SL were harvested 24 hrs after the media change (n>3).


( H ) ( left ) Western blot of pERK ERK in 1μM PD increasing concentrations of CHIR in serum - free N2B27 media . ( right ) Relative pERK ( n = 3 ) . ( I ) ( left ) Western blot of pERK ERK in 3μM CHIR increasing concentrations of PD in serum - free N2B27 media . ( right ) Relative pERK ( n = 6 ) . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± in H , I . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ns = not significant ( paired two - student ' s t - ) .

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(H) (left) Western blot of pERK and ERK in 1μM PD and increasing concentrations of CHIR in serum-free N2B27 media. (right) Relative pERK levels (n=3). (I) (left) Western blot of pERK and ERK in 3μM CHIR and increasing concentrations of PD in serum-free N2B27 media. (right) Relative pERK levels (n=6). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in H, and I. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).


( G ) ( left ) Western blot of pERK in TNERT , TNERTFgfr1 - / - , TNERTFgfr2 - / - , TNERTFgf4 - / - , TNERTFgfbp1 - / - with or no OHT . ( Right ) relative pERK estimated by densitometry ( n = 4 ) . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± in G , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant ( paired two - student ' s t - ) .

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(G) (left) Western blot of pERK in TNERT, TNERTFgfr1-/-, TNERTFgfr2-/-, TNERTFgf4-/-, and TNERTFgfbp1-/- with or no OHT. (Right) relative pERK levels estimated by densitometry (n=4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in G, *P < 0.05, **P < 0.01, ***P < 0.001, and ns = not significant (paired two-tailed student's t-test).


( E ) Western blot of FGFR1 , FGFR2 , pERK in TNERT at one - hour intervals at indicated time points after OHT treatment ( n > 3 ) .

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(E) Western blot of FGFR1, FGFR2, and pERK in TNERT at one-hour intervals at indicated time points after OHT treatment (n>3).


( C ) Western blot of pERK ERK in TNERT TNERTNono - / - treated with or no OHT ( n = 3 ) .

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(C) Western blot of pERK and ERK in TNERT and TNERTNono-/- cells treated with or no OHT (n=3).


( D ) Immunoprecipitation showing interactions between ERK NONO in presence or absence of Nanog induction by Doxycycline in TDiN .

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(D) Immunoprecipitation analysis showing interactions between ERK and NONO in the presence or absence of Nanog induction by Doxycycline in TDiN cells.


( E ) ( left ) Western blot of pERK ERK in E14Tg2a treated with PD or FGF4 . ( Middle right ) Relative of pERK NONO in E14Tg2a treated with PD or FGF4 ( n > 4 ) . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± in E * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ns = not significant ( paired two - student ' s t - ) .

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(E) (left) Western blot of pERK and ERK in E14Tg2a cells treated with PD or FGF4. (Middle and right) Relative levels of pERK and NONO in E14Tg2a cells treated with PD or FGF4 (n>4). Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in E *P < 0.05, **P < 0.01, ****P < 0.0001 and ns = not significant (paired two-tailed student's t-test).


( L ) Cycloheximide chase of NANOG in SL , SLPD , SLFGF4 in E14Tg2a .

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(L) Cycloheximide chase assay of NANOG in SL, SLPD, and SLFGF4 in E14Tg2a cells.


( A ) Immunofluorescence of pERK ( red ) NANOG ( green ) in indicated ESCs . Scale bars 25 μM . ( B ) normalized of pERK was plotted against normalized of NANOG .

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(A) Immunofluorescence of pERK (red) and NANOG (green) in the indicated ESCs. Scale bars 25 μM. (B)The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG.


( C ) ( left ) Immunofluorescence of pERK NANOG in 10 % NANOG - high NiRFP2A at indicated time cultured in same media collected from before FACS . ( Right ) normalized of pERK was plotted against normalized of NANOG at different time points . Scale bars 25 μM .

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(C) (left) Immunofluorescence of pERK and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FACS. (Right) The normalized fluorescence intensity of pERK was plotted against the normalized fluorescent intensity of NANOG at different time points. Scale bars 25 μM.


( D ) ( left ) Western blot of pERK , NANOG in 10 % NANOG - high NiRFP2A at indicated time cultured in same media collected from before FAC sorting . ( Right ) Expression of NANOG pERK in 10 % NANOG - high NiRFP2A relative to 0 hrs during time course of culture . NANOG pERK expression oscillate between high low in during culture . Data information : n ≥ 3 biological replicates ( each dot represents a biological replicate ) . Data are presented as mean ± in D . * P < 0 . 05 , * * P < 0 . 01 ns = not significant ( paired two - student ' s t - ) .

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(D) (left) Western blot analysis of pERK, and NANOG in 10% NANOG-high NiRFP2A cells at indicated time cultured in the same media collected from cells before FAC sorting. (Right) Expression levels of NANOG and pERK in 10% NANOG-high NiRFP2A cells relative to 0 hrs during the time course of culture. The NANOG and pERK expression oscillate between high and low levels in the cells during culture. Data information: n ≥ 3 biological replicates (each dot represents a biological replicate). Data are presented as mean ± SEM in D. *P < 0.05, **P < 0.01 and ns = not significant (paired two-tailed student's t-test).


Analysis of DNA content by flow cytometry.


of BrdU incorporation by BrdU - IP - chip 45 minutes after release into S phase in treated with 0 . 1 % MMS as described in ( A ) . A representative region on chromosome VII is shown . position of replication origins is indicated above map . Asterisks : late origins firing in mrc1 ∆ .

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Analysis of BrdU incorporation by BrdU-IP-chip 45 minutes after release into S phase in cells treated with 0.1% MMS as described in (A). A representative region on chromosome VII is shown. The position of replication origins is indicated above the map. Asterisks: late origins firing in mrc1∆ cells.


of BrdU incorporation ( signal log ) at 384 early late replication origins in wild type , rad9 ∆ mrc1 ∆ strains exposed to 0 . 1 or 0 . 033 % MMS . Red lines indicate median values . Median replication times ( Trep ) are indicated for bins of 96 origins .

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Quantification of the BrdU incorporation (signal log ratio) at 384 early and late replication origins in wild type, rad9∆ and mrc1∆ strains exposed to 0.1 or 0.033% MMS. Red lines indicate median values. Median replication times (Trep) are indicated for bins of 96 origins.


Wild type , mrc1 ∆ rad9 ∆ were released into S phase in presence of BrdU 0 . 033 % MMS , as described in Fig . 1A . combing was performed for samples collected 45 60 min after release into S phase . Representative are shown . Red : , green : BrdU . Bar is 20 kb .

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Wild type, mrc1∆ and rad9∆ cells were released into S phase in the presence of BrdU and 0.033% MMS, as described in Fig. 1A. DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase. Representative DNA fibers are shown. Red: DNA, green: BrdU. Bar is 20 kb.


Distribution of center - to - center distances in released for 45 minutes into S phase .

Distribution of center-to-center distances in cells released for 45 minutes into S phase.


Distribution of BrdU track lengths in released for 45 or 60 minutes into S phase , respectively .

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Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase, respectively.


Wild type , mrc1 ∆ rad9 ∆ were synchronized in G1 with α - factor prior to release with pronase in presence of 0 . 033 % MMS . were collected at indicated times Rad53 phosphorylation was monitored by western blot asterisk indicates a non - specific band detected by anti - Rad53 antibody .

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Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.


Wild type , mrc1 ∆ rad9 ∆ were synchronized in G1 with α - factor prior to release with pronase in presence of 0 . 033 % MMS . were collected at indicated times Rad53 phosphorylation was monitored with an in situ phosphorylation - ISA ( F ) .

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Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored with an in situ phosphorylation assay - ISA (F).


of Rad53 phosphorylation by Western blot .

Analysis of Rad53 phosphorylation by Western blot.


of replication by combing in released for 45 60 minutes into S phase in presence of 100 μg / ml Zeocin . IdU tracks ( red ) correspond to initiation events occurring during HU treatment . Elongation events after HU removal appear as CldU tracks ( yellow ) flanking IdU tracks . New initiation events as detected as isolated CldU tracks ( asterisks ) . Bar is 20 kb .

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Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg/ml Zeocin. IdU tracks (red) correspond to initiation events occurring during the HU treatment. Elongation events after HU removal appear as CldU tracks (yellow) flanking IdU tracks. New initiation events as detected as isolated CldU tracks (asterisks). Bar is 20 kb.


Number of new initiation events per detected at 60 minutes in experiment shown in ( D ) . For each sample , at least 65 fiber larger than 200 kb were analyzed , for a total of > 17 Mb .

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Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in (D). For each sample, at least 65 individual DNA fiber larger than 200 kb were analyzed, for a total of >17 Mb.


2D gel of initiation of replication at early origin ARS305 late origin ARS1212 . experiment was performed as in ( A ) for indicated times . Arrows indicate late origin firing .

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2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212. The experiment was performed as in (A) for the indicated times. Arrows indicate late origin firing.


distribution of CldU tracks 45 minutes after release into S phase in combing experiment shown in ( D ) . Median values are indicated in red . For each sample , at least 250 tracks were analyzed in two biological replicates .

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Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates.


2D gel of initiation elongation in wild type rad9 ∆ at early origin ARS719 ( probe 1 ) at a locus 7 kb away of origin ( probe 2 ) . Time in Zeocin after HU wash is indicated . Arrows point to Y arcs ( fork signals ) with strongest .

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2D gel analysis of initiation and elongation in wild type and rad9∆ cells at the early origin ARS719 (probe 1) and at a locus 7 kb away of the origin (probe 2). Time in Zeocin after the HU wash is indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.


2D gel of fork progression in wild type rad9 ∆ through a 70 - kb region of chromosome VI for which active origins downstream of ARS607 have been deleted . position of probes time after release from HU are indicated . Arrows point to Y arcs ( fork signals ) with strongest .

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2D gel analysis of fork progression in wild type and rad9∆ cells through a 70-kb region of chromosome VI for which all active origins downstream of ARS607 have been deleted. The position of probes and the time after release from HU are indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.


of Rad53 phosphorylation in G1 prior to Zeocin addition ( G1 ) at indicated times . asterisk indicates a non - specific band detected by anti - Rad53 antibody .

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Analysis of Rad53 phosphorylation in G1 prior to Zeocin addition (G1) and at indicated times. The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.


combing of replication profiles 60 minutes after release into S phase in presence of Zeocin . Representative are shown . Red : , green : BrdU . green channel is also shown separately . Bar : 20 kb .

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DNA combing analysis of replication profiles 60 minutes after release into S phase in the presence of Zeocin. Representative DNA fibers are shown. Red: DNA, green: BrdU. The green channel is also shown separately. Bar: 20 kb.


Distribution of BrdU tracks in wild type , mrc1 ∆ mrc1AQ . are fully labeled in case of rad9 ∆ . Median values are indicated in red correspond to of at least 150 tracks from two biological replicates . ns : non - significant Mann - Whitney rank sum .

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Distribution of BrdU tracks length in wild type, mrc1∆ and mrc1AQ cells. DNA fibers are fully labeled in the case of rad9∆ cells. Median values are indicated in red and correspond to the analysis of at least 150 tracks from two biological replicates. ns: non-significant Mann-Whitney rank sum test.


from indicated strains were released synchronously into S phase in presence of Zeocin as described in Fig . 5A content was analyzed by flow cytometry .

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Cells from the indicated strains were released synchronously into S phase in the presence of Zeocin as described in Fig. 5A and DNA content was analyzed by flow cytometry.


of chromosome mobility by Field Gel Electrophoresis ( PFGE ) after of with ethidium bromide . In G1 , chromosomes are linear are sorted according to their . Upon entry into S phase ( 30 min ) , they cannot enter gel due to presence of replication intermediates . Quantitation of chromosomes re - entering gel shows completion of replication in 40 % of rad9 ∆ , rad53 - 11 rad9 ∆ rad53 - 11 mutants , but not in wild type mrc1 mutants as determined for a representative experiment . Error bars indicate SD between signal determined for three larger chromosomes ( arrowheads ) .

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Analysis of chromosome mobility by Pulse Field Gel Electrophoresis (PFGE) after staining of DNA with ethidium bromide. In G1, the chromosomes are linear and are sorted according to their length. Upon entry into S phase (30 min), they cannot enter the gel due to the presence of replication intermediates. Quantitation of chromosomes re-entering the gel shows completion of DNA replication in 40% of rad9∆, rad53-11 and rad9∆ rad53-11 mutants, but not in wild type cells and mrc1 mutants as determined for a representative experiment. Error bars indicate SD between the signal intensity determined for the three larger chromosomes (arrowheads).


combing of replication profiles 30 minutes after release into S phase in presence of Zeocin . Representative are shown . Red : , green : BrdU . Bar is 20 kb .

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DNA combing analysis of replication profiles 30 minutes after release into S phase in the presence of Zeocin. Representative images are shown. Red: DNA, green: BrdU. Bar is 20 kb.


distribution of BrdU tracks measured at that time for wild type , rad9 ∆ , rad53 - 11 rad53 - 11 rad9 ∆ . Median lengths are indicated in red . For each sample , at least 200 tracks were measured from two biological replicates .

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Length distribution of BrdU tracks measured at that time for wild type, rad9∆, rad53-11 and rad53-11 rad9∆ cells. Median lengths are indicated in red. For each sample, at least 200 tracks were measured from two biological replicates.


C - E . Full GFP : Mud shows wild type pattern of localization in mud3 / mud4 mitotic follicle ( C ) rescues spindle orientation ( D E ) . GFP : Mud lacking Pins binding domain localizes only at spindle poles ( C ' ) in mud3 / mud4 mitotic follicle fails to rescue spindle orientation ( D E ) . Statistical significance in spindle orientation was determined using Mann - Whitney .

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C-E. Full length GFP:Mud shows the wild type pattern of localization in mud3/mud4 mitotic follicle cells (C) and rescues spindle orientation (D E). GFP:Mud lacking the Pins binding domain localizes only at spindle poles (C') in mud3/mud4 mitotic follicle cells and fails to rescue spindle orientation (D E). Statistical significance in spindle orientation was determined using the Mann-Whitney test.


C , D . Pins : Tom GFP : Mud have overlapping but not identical localization patterns in mitotic follicle . Representative pictures ( C ) from 9 ( D ) are shown . Scale bars = 5 microns .

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C, D. Pins:Tom and GFP:Mud have overlapping but not identical localization patterns in mitotic follicle cells. Representative pictures (C) and quantification from 9 cells (D) are shown. Scale bars = 5 microns.


A - C . Pins - myr - GFP myr - RFP show a similar localization pattern to Pins : Tom during mitosis . Representative pictures are shown ( A , B ) ; a lookup table ( A ' , B ' ) emphasizes relative enrichments of Pins - myr - GFP myr - RFP along cortex . ( Pins - myr : 4 , myr - RFP : 2 ) ( C ) .

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A-C. Pins-myr-GFP and myr-RFP show a similar localization pattern to Pins:Tom during mitosis. Representative pictures are shown (A, B); a heatmap lookup table (A', B') emphasizes the relative enrichments of Pins-myr-GFP and myr-RFP along the cortex. Quantification (Pins-myr: 4 cells, myr-RFP: 2 cells) (C).


D - F . Random spindle orientation in pinsp62 / pinsp62 null mutant follicle is rescued by expression of full - Pins or Pins - myr - GFP . Representative pictures ( D , E ) ( F ) are shown . Scale bars = 5 microns . Statistical significance in spindle orientation was determined using Mann - Whitney . * * * * p < 0 . 0001 .

O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

D-F. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full-length Pins or Pins-myr-GFP. Representative pictures (D, E) and quantification (F) are shown. Scale bars = 5 microns. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001.


A . Spindle orientation is randomized in Dlg1P20 / Dlg1P20 Dlg1P20 / Dlg2 mutant follicle . B . Random spindle orientation in pinsp62 / pinsp62 null mutant follicle is rescued by expression of full Pins or phosphomimetic ( S436D ) Pins . Unphosphorylatable ( S436A ) Pins fails to rescue . C . of spindle orientation . Statistical significance in spindle orientation was determined using Mann - Whitney . * * * * p < 0 . 0001 , * * * p < 0 . 001 . D .

O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O 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. Spindle orientation is randomized in Dlg1P20/Dlg1P20 and Dlg1P20/Dlg2 mutant follicle cells. B. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full length Pins or phosphomimetic (S436D) Pins. Unphosphorylatable (S436A) Pins fails to rescue. C. Quantification of spindle orientation phenotypes. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001, *** p<0.001. D.


H . of spindle orientation in Stage 9 / 10 embryonic ectoderm . n = 17 from 2 in w1118 n = 18 from 2 in Dlg1P20 / Dlg2 . Statistical significance in spindle orientation was determined using Mann - Whitney . * * p = 0 . 0015 .

O O O B-SUBCELLULAR O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

H. Quantification of spindle orientation phenotypes in the Stage 9/10 embryonic ectoderm. n = 17 cells from 2 embryos in w1118 and n = 18 cells from 2 embryos in Dlg1P20/Dlg2 embryos. Statistical significance in spindle orientation was determined using the Mann-Whitney test. ** p=0.0015.


A - C . Multiple genetic approaches were used to a role for either Khc73 or GUKHolder in metaphase spindle orientation . Representative pictures ( A - B ) ( C ) are shown . Scale bars = 5 microns . Spindle angle randomization in pinsp62 / pinsp62 null mutant follicle is included as a positive control for comparison .

O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O

A-C. Multiple genetic approaches were used to test a role for either Khc73 or GUKHolder in metaphase spindle orientation. Representative pictures (A-B) and quantification (C) are shown. Scale bars = 5 microns. Spindle angle randomization in pinsp62/pinsp62 null mutant follicle cells is included as a positive control for comparison.


A - D . Two distinct patterns of Inscuteable localization are observed in follicular epithelium when UAS - Inscuteable is driven by Traffic jam - GAL4 . ( A , C ) In first pattern ( InscA ) , Inscuteable is highly enriched at apical surface . ( B , D ) In second pattern ( InscB ) , Inscuteable is observed at both lateral apical cortex . Mud Pins localizations are also shown . Representative pictures ( A , B ) quantifications ( InscA : average of 5 , InscB : average of 7 ) ( C , D ) are shown .

O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR B-TISSUE O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O

A-D. Two distinct patterns of Inscuteable localization are observed in the follicular epithelium when UAS-Inscuteable is driven by Traffic jam-GAL4. (A, C) In the first pattern (InscA), Inscuteable is highly enriched at the apical cell surface. (B, D) In the second pattern (InscB), Inscuteable is observed at both the lateral and apical cortex. Mud and Pins localizations are also shown. Representative pictures (A, B) and quantifications (InscA: average of 5 cells, InscB: average of 7 cells) (C, D) are shown.


E . aPKC localization in InscA InscB follicle . F . of spindle orientation shows that mitotic spindles are reoriented in InscA , but not InscB . Reorientation relies on Mud but not on interaction between Pins Dlg .

O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-CELL_TYPE O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O

E. aPKC localization in InscA and InscB follicle cells. F. Quantification of spindle orientation phenotypes shows that mitotic spindles are reoriented in InscA cells, but not InscB cells. Reorientation relies on Mud but not on the interaction between Pins and Dlg. G


Domain architecture structure of Miro . ( A ) Bar diagram of Miro domain structure . Numbering corresponds to dMiro isoform D , domain names are described in text ( B ) Ribbon diagram of MiroS structure in ' side view ' . Note three distinct domains : ELM1 ( blue ) , ELM2 ( green ) , cGTPase ( grey / yellow ) , joined by two linkers : Lnk1 ( orange ) , Lnk2 ( red ) . structure shown is MgGDP ‐ MiroS , with Ca2 + ion from Ca ‐ MiroS structure overlaid for orientation ( Ca2 + / Mg2 + / GDP as yellow / purple / multicoloured spheres ) . cGTPase , C ‐ terminal GTPase ; dMiro , Drosophila Miro ; ELM , EF pair with ligand mimic .

O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O O

Domain architecture and structure of Miro. (A) Bar diagram of Miro domain structure. Numbering corresponds to dMiro isoform D, domain names are described in the text (B) Ribbon diagram of the MiroS structure in the 'side view'. Note three distinct domains: ELM1 (blue), ELM2 (green), cGTPase (grey/yellow), joined by two linkers: Lnk1 (orange), and Lnk2 (red). The structure shown is MgGDP‐MiroS, with the Ca2+ ion from the Ca‐MiroS structure overlaid for orientation ( Ca2+/Mg2+/GDP as yellow/purple/multicoloured spheres). cGTPase, C‐terminal GTPase; dMiro, Drosophila Miro; ELM, EF hand pair with ligand mimic.


( A ) Top , MiroS shown in ' top view ' . ELM1 ELM2 are related by pseudo twofold symmetry , represented by dyad between them . Bottom , overlay of two ELM domains ( rmsd of 1 . 8 Å over 84 Cα positions ) . ELM1 ( blue ) , ELM2 ( green ) identical or similar conserved residues in identical positions are shown as sticks .

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top, MiroS shown in the 'top view'. ELM1 and ELM2 are related by pseudo twofold symmetry, represented by the dyad between them. Bottom, overlay of the two ELM domains (rmsd of 1.8 Å over 84 Cα positions). ELM1 (blue), ELM2 (green) and identical or similar conserved residues in identical positions are shown as sticks.