Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in liver paraffin - fixated sections 24h after sepsis , measured with TUNEL depicted as % of PI positive / µm² tissue . P - values were calculated with 2 - way ANOVA . n = 6 - 7 / group , combined data of 2 independent experiments .

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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in liver paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Apoptosis in kidney paraffin - fixated sections 24h after sepsis , measured with TUNEL depicted as % of PI positive / µm² tissue . P - values were calculated with 2 - way ANOVA . n = 6 - 7 / group , combined data of 2 independent experiments .

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Apoptosis in kidney paraffin-fixated sections 24h after sepsis, measured with TUNEL staining and depicted as % of PI positive cells/µm² tissue area. P-values were calculated with 2-way ANOVA tests. n = 6-7/group, combined data of 2 independent experiments.


Mice were pre - treated with pemafibrate ( 1mg / kg ) or vehicle ( 0 , 9 % NaCl ) for 1 week before being subjected to CLP . Systemic load ( CFU / ml blood ) 24h post - sepsis in vehicle or pemafibrate treated mice . P - values were calculated with 2 - way Student ' s t - .

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

Mice were pre-treated with pemafibrate (1mg/kg) or vehicle (0,9% NaCl) for 1 week before being subjected to CLP. Systemic bacterial load (CFU/ml blood) 24h post-sepsis in vehicle or pemafibrate treated mice. P-values were calculated with 2-way Student's t-test.


Pemafibrate ( 1 mg / kg ) or vehicle ( 0 , 9 % NaCl ) was administered at different timepoints before after induction of sepsis survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via Log rank

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

Pemafibrate (1 mg/kg) or vehicle (0,9% NaCl) was administered at different timepoints before and after the induction of sepsis and survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via Log rank tests


Mice were injected with PPARα antagonist GW6471 ( 10µg / g ) or vehicle ( DMSO ) 3h pre - 3h post - CLP . Survival was monitored during 9 days , after which no further deaths occurred . Survival curve was analyzed via a Log rank . Combined data of 2 independent experiments , n = 16 / group .

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Mice were injected with the PPARα antagonist GW6471 (10µg/g) or vehicle (DMSO) 3h pre- and 3h post-CLP. Survival was monitored during 9 days, after which no further deaths occurred. Survival curve was analyzed via a Log rank test. Combined data of 2 independent experiments, n = 16/group.


Blood samples were collected from healthy volunteers septic ( n = 13 septic , = 15 healthy controls ) . Plasma was prepared ( A ) FFA ( B ) glycerol concentrations were determined as described in methods . P - values were calculated with two - way Student t - .

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

Blood samples were collected from healthy volunteers and septic patients (n= 13 septic patients, = 15 healthy controls). Plasma was prepared and (A) FFA and (B) glycerol concentrations were determined as described in the methods. P-values were calculated with two-way Student t-tests.


C . cGAMP - VLPs induce an IFN - I response in target . HEK293 were infected with decreasing amounts of cGAMP - VLPs Empty - VLPs ( 1 / 5 serial dilutions starting at 2μL of VLP stocks per well ) infection was monitored 24 hours later by quantifying GFP + by flow cytometry . Supernatants from same infected were then transferred to a reporter expressing firefly luciferase under a promoter induced by IFN - I ( ISRE ) . Luciferase activity measured 24 hours later indicated presence of IFN - I in supernatants .

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C. cGAMP-VLPs induce an IFN-I response in target cells. HEK293 cells were infected with decreasing amounts of cGAMP-VLPs and Empty-VLPs (1/5 serial dilutions starting at 2μL of VLP stocks per well) and the infection was monitored 24 hours later by quantifying GFP+ cells by flow cytometry. Supernatants from the same infected cells were then transferred to a reporter cell line expressing firefly luciferase under a promoter induced by IFN-I (ISRE). Luciferase activity measured 24 hours later indicated the presence of IFN-I in the supernatants.


Immunisation with cGAMP - VLPs facilitates induction of HIV - 1 Gag - specific polyfunctional CD8 T responses . Alternatively , were analysed by ICS six hours after stimulation with peptide . CD8 T were gated as live , CD90 . 2 + , CD8 + . CD8 T expressing CD107a , IFNγ , TNFα or IL2 were analysed as shown in ( E ) .

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Immunisation with cGAMP-VLPs facilitates induction of HIV-1 Gag-specific polyfunctional CD8 T cell responses. Alternatively, cells were analysed by ICS six hours after stimulation with peptide. CD8 T cells were gated as live, CD90.2+, CD8+. CD8 T cells expressing CD107a, IFNγ, TNFα or IL2 were analysed as shown in (E).


A , B . cGAMP loading enhances IgG responses specific to VLP , including VSV - G . ELISA plates were coated with lysate from cGAMP - VLPs ( A ) or recombinant VSV - G ( B ) . Antibodies of different isotypes specific for these were measured in sera from immunised mice by ELISA . optical density at increasing serum dilutions is shown in first three graphs from left EC50 is on right .

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A, B. cGAMP loading enhances IgG responses specific to VLP proteins, including VSV-G. ELISA plates were coated with lysate from cGAMP-VLPs (A) or recombinant VSV-G protein (B). Antibodies of different isotypes specific for these proteins were measured in sera from immunised mice by ELISA. The optical density at increasing serum dilutions is shown in the first three graphs from the left and the EC50 is on the right.


C . Enhanced antibody production following immunisation with cGAMP - VLPs relies on STING signalling . WT or Sting - / - mice were immunised with PBS , cGAMP - VLPs , Empty - VLPs or Empty - VLPs + poly ( I : C ) . IgG2b antibodies recognising VLP were assessed by ELISA . optical density at increasing serum dilutions is shown in first two graphs from left EC50 is on right .

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

C. Enhanced antibody production following immunisation with cGAMP-VLPs relies on STING signalling. WT or Sting-/- mice were immunised with PBS, cGAMP-VLPs, Empty-VLPs or Empty-VLPs + poly(I:C). IgG2b antibodies recognising VLP proteins were assessed by ELISA. The optical density at increasing serum dilutions is shown in the first two graphs from the left and the EC50 is on the right.


Immunisation with cGAMP - VLPs enhances accumulation of Tfh in draining lymph node . T follicular ( Tf ) were identified by flow cytometry as CD4 + CD44 + CXCR5hiPD1hi were further subdivided into Tfr ( FoxP3 + ) Tfh ( FoxP3 - ) . percentages of Tf , Tfh Tfr within CD4 + are shown in ( B ) .

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Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B).


Immunisation with VLPs induces germinal centre formation . Germinal centre B were identified by flow cytometry as B220 + IgD - CD95 + GL7 + . of germinal centre B amongst B220 + is shown in ( E ) .

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Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. the percentage of germinal centre B cells amongst B220+ cells is shown in (E).


C , D . Low doses of cGAMP - HA - VLPs confer protection following IAV challenge . One month after immunisation with indicated doses of VLPs , were infected with 104 TCID50 of IAV PR8 . loss was monitored over following eleven days is shown as a of starting ( C , upper graph shows mean lower graphs show mice for each condition ) . approaching humane end - point of 20 % loss were culled survival to end - point curves are shown in ( D ) .

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C, D. Low doses of cGAMP-HA-VLPs confer protection following IAV challenge. One month after immunisation with the indicated doses of VLPs, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (C, upper graph shows mean and lower graphs show individual mice for each condition). Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (D).


B . cGAMP - HA - VLPs Empty - HA - VLPs + AddaVax protect against IAV challenge . One month after immunisation , were infected with 104 TCID50 of IAV PR8 . approaching humane end - point of 20 % loss were culled survival to end - point curves are shown in ( B ) .

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B. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (B).


A . cGAMP - S - VLPs Empty - S - VLPs incorporate SARS - CoV - 2 S . Lysates from VLP producer VLP preparations were analysed by western blot for presence of S2 subunit of SARS - CoV - 2 S , Gag - GFP actin 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 O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

A. cGAMP-S-VLPs and Empty-S-VLPs incorporate SARS-CoV-2 S. Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS-CoV-2 S, Gag-GFP and actin using the indicated antibodies.


B , C . Immunisation with cGAMP - S - VLPs augments anti - Spike anti - RBD antibody titres . Mice were immunised via intra - muscular route with 5x105 IU of cGAMP - S - VLPs , Empty - S - VLPs , or PBS as a control . Three after immunisation , sera were collected , heat - inactivated , titres of antibodies capable of binding to SARS - CoV - 2 S ( B ) or its RBD ( C ) were determined by ELISA . antibody response was expressed as Endpoint titre defined as reciprocal of highest serum dilution that gives a positive signal ( blank + 10SD ) . dotted shows limit of detection ( LOD ) .

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B, C. Immunisation with cGAMP-S-VLPs augments anti-Spike and anti-RBD antibody titres. Mice were immunised via the intra-muscular route with 5x105 IU of cGAMP-S-VLPs, Empty-S-VLPs, or PBS as a control. Three weeks after immunisation, sera were collected, heat-inactivated, and titres of antibodies capable of binding to SARS-CoV-2 S (B) or its RBD (C) were determined by ELISA. The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10SD). The dotted line shows the limit of detection (LOD).


D . Immunisation with cGAMP - S - VLPs induces neutralising antibodies . Using serum samples from ( B ) , antibody titres capable of neutralising SARS - CoV - 2 were determined by microneutralisation ( MN ) . Calculated IC50 doses from multiple serum dilutions are shown . dotted shows LOD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunisation with cGAMP-S-VLPs induces neutralising antibodies. Using serum samples from (B), antibody titres capable of neutralising SARS-CoV-2 were determined by microneutralisation (MN) assay. Calculated IC50 doses from multiple serum dilutions are shown. The dotted line shows the LOD.


A1 ) Representative WBs showing DRD2 RGS9 - 2 downregulation in adult ( P60 - P90 ) Tor1a + / - ( + / - ) striata , characterized by a reduced torsinA level with respect to Tor1a + / + ( + / + ) . striatal level of RGS7 is unchanged . dot plot shows Tor1a + / - data , normalized to Tor1a + / + controls of same experiment . TorsinA : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t * * * P = 0 . 0003 ; DRD2 : Tor1a + / + N = 18 , Tor1a + / - N = 16 , t * * P = 0 . 0029 ; RGS9 - 2 : Tor1a + / + N = 32 , Tor1a + / - N = 33 , t * P = 0 . 0115 ) . RGS7 : Tor1a + / + N = 7 , Tor1a + / - N = 7 , t P = 0 . 8721 ) . WB data , expressed as of vs . loading control level , are normalized to wild - type samples of same experiment . A2 , A3 , A4 ) Time - course of changes in torsinA , DRD2 RGS9 - 2 striatal along postnatal development . Summary plot data are normalized to Tor1a + / + P60 sample of each independent experiment . A2 ) TorsinA level is significantly reduced in mutants throughout developmental period considered ( P7 N = 3 ; P14 - P60 N = 4 ; one - way ANOVA P < 0 . 0001 Bonferroni ' s Multiple Comparison * * P < 0 . 01 at P7 , P14 P60 ) . A3 , A4 ) DRD2 RGS9 - 2 show parallel courses , with a similar increase from P7 to P21 in Tor1a + / + as well as Tor1a + / - striatal lysates ( P7 : DRD2 N = 3 , RGS9 - 2 N = 4 ; P14 - P21 N = 4 ; one - way ANOVA with Bonferroni ' s Multiple Comparison P > 0 . 05 ) . At P60 , a simultaneous reduction of DRD2 RGS9 - 2 is observed in Tor1a + / - striatal lysates ( N = 4 ; Tor1a + / - one sample t DRD2 : * P = 0 . 0250 , RGS9 - 2 : * P = 0 . 0175 ) . WB data , expressed as of vs . loading control level , are normalized to wild - type P60 sample of same experiment

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A1) Representative WBs showing DRD2 and RGS9-2 downregulation in adult (P60-P90) Tor1a+/- (+/-) striata, characterized by a reduced torsinA protein level with respect to Tor1a+/+ (+/+) littermates. The striatal level of RGS7 is unchanged. The dot plot shows Tor1a+/- data, normalized to Tor1a+/+ controls of the same experiment. TorsinA: Tor1a+/+ N=7, Tor1a+/- N=6, t test ***P=0.0003; DRD2: Tor1a+/+ N=18, Tor1a+/- N=16, t test **P=0.0029; RGS9-2: Tor1a+/+ N=32, Tor1a+/- N=33, t test *P=0.0115). RGS7: Tor1a+/+ N=7, Tor1a+/- N=7, t test P=0.8721). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. A2,A3,A4) Time-course of changes in torsinA, DRD2 and RGS9-2 striatal levels along postnatal development. Summary plot data are normalized to the Tor1a+/+ P60 sample of each independent experiment. A2) TorsinA level is significantly reduced in mutants throughout the developmental period considered (P7 N=3; P14-P60 N=4; one-way ANOVA P<0.0001 and Bonferroni's Multiple Comparison Test **P<0.01 at P7, P14 and P60). A3,A4) DRD2 and RGS9-2 levels show parallel courses, with a similar increase from P7 to P21 in Tor1a+/+ as well as Tor1a+/- striatal lysates (P7: DRD2 N=3, RGS9-2 N=4; P14-P21 N=4; one-way ANOVA with Bonferroni's Multiple Comparison Test P>0.05). At P60, a simultaneous reduction of DRD2 and RGS9-2 levels is observed in Tor1a+/- striatal lysates (N=4; Tor1a+/- one sample t test DRD2: *P=0.0250, RGS9-2: *P= 0.0175). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type P60 sample of the same experiment


B ) Representative WB dot plot show downregulation of torsinA , DRD2 RGS9 - 2 striatal also in adult P60 - P90 Tor1a ∆ gag / + ( ∆ gag / + ) mice . TorsinA : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 12 , t * * * P = 0 . 0006 ; DRD2 : Tor1a + / + N = 7 , Tor1a ∆ gag / + N = 9 , t * P = 0 . 0183 ; RGS9 - 2 : Tor1a + / + N = 10 , Tor1a ∆ gag / + N = 9 , t * P = 0 . 0192 . WB data , expressed as of vs . loading control level , are normalized to wild - type samples of same experiment . Mean ± is represented in

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B) Representative WB images and the dot plot show downregulation of torsinA, DRD2 and RGS9-2 striatal levels also in adult P60-P90 Tor1a∆gag/+ (∆gag/+) mice. TorsinA: Tor1a+/+ N=10, Tor1a∆gag/+ N=12, t test ***P= 0.0006; DRD2: Tor1a+/+ N=7, Tor1a∆gag/+ N=9, t test *P= 0.0183; RGS9-2: Tor1a+/+ N=10, Tor1a∆gag/+ N=9, t test *P= 0.0192. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. Mean ± SEM is represented in the graphs


C ) Left . Representative image of coronal striatal slices of fresh frozen brains of wild - type Tor1a + / - mice probed with DRD2 radioligand 3H - spiperone . Scale bar 1 . 5 mm . ( Right ) Graph showing DRD2 binding density in Tor1a + / - striatal sections , obtained from densitometric , expressed as variation compared to control

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C) Left. Representative image of coronal striatal slices of fresh frozen brains of wild-type and Tor1a+/- mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. (Right) Graph showing DRD2 binding density in Tor1a+/- striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals


A1 ) Representative WB of RGS9 - 2 , R7BP , Gβ5 on four Tor1a + / + four Tor1a + / - striatal lysates . bar histogram on right shows of three in each sample . A2 ) Dot plot showing that Gβ5 striatal are unchanged in Tor1a + / - mice ( Tor1a + / + N = 11 , Tor1a + / - N = 10 , Mann Whitney P = 0 . 2453 ) . A3 ) Conversely , level of R7 binding R7BP is increased in Tor1a + / - striatum ( Tor1a + / + N = 12 , Tor1a + / - N = 12 , t * * P = 0 . 0014 ) . Values are represented as of vs . loading control level , normalized to mean of Tor1a + / + control values of same

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A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/- striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/- mice (Tor1a+/+ N=11, Tor1a+/- N=10, Mann Whitney test P=0.2453). A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/- striatum (Tor1a+/+ N=12, Tor1a+/- N=12, t test **P=0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experiment


B1 ) Representative WB of lysates of Tor1a + / + Tor1a + / - dorsal striatum slices incubated for 5 hours in presence ( w / leu ) or absence ( w / o leu ; striatum ) of protease inhibitor leupeptin . Samples showing enhanced degradation of DRD2 / or RGS9 - 2 show additional bands at lower molecular . B2 ) Summary plot reporting mean ± of RGS9 - 2 DRD2 level values , expressed as of vs . loading control level , normalized to value of Tor1a + / + w / leu sample measured in same experiment . DRD2 : Tor1a + / + N = 4 , t P = 0 . 4376 ; Tor1a + / - N = 4 , t * P = 0 . 0427 ; RGS9 - 2 : Tor1a + / + N = 5 , t * P = 0 . 0464 ; Tor1a + / - N = 5 , t * P = 0 .

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B1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190


C1 , C2 , C3 , C4 ) Dot plots showing DRD2 , RGS9 - 2 , R7BP Gβ5 measured in striatal detergent - resistant - ( DRM ) preparations from Tor1a + / - ( + / - ) WT ( + / + ) mice ( DRD2 : Tor1a + / + N = 16 , Tor1a + / - N = 13 , t P = 0 . 0129 ; RGS9 - 2 : Tor1a + / + N = 7 , Tor1a + / - N = 6 , t * P = 0 . 0396 ; R7BP : Tor1a + / + N = 6 , Tor1a + / - N = 6 , t * * * P = 0 . 0002 ; Gβ5 : Tor1a + / + N = 9 , Tor1a + / - N = 9 , t P = 0 . 8461 )

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C1, C2, C3, C4) Dot plots showing DRD2, RGS9-2, R7BP and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/- (+/-) and WT (+/+) mice (DRD2: Tor1a+/+ N=16, Tor1a+/- N=13, t test P=0.0129; RGS9-2: Tor1a+/+ N=7, Tor1a+/- N=6, t test *P=0.0396; R7BP: Tor1a+/+ N=6, Tor1a+/- N=6, t test ***P=0.0002; Gβ5: Tor1a+/+ N=9, Tor1a+/- N=9, t test P=0.8461)


WB of markers of endocytic trafficking degradative pathway in dorsal striatum of Tor1a + / - mice . A ) level of β - arrestin 2 ( β - Arr2 ) is not altered ( Tor1a + / + N = 11 , Tor1a + / - N = 12 , t P = 0 . 6796 ) , whereas level of spinophilin ( B ) is decreased ( Tor1a + / + N = 5 , Tor1a + / - N = 8 , t * P = 0 . 0151 )

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WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a+/- mice. A) The level of β-arrestin 2 (β-Arr2) is not altered (Tor1a+/+ N=11, Tor1a+/- N=12, t test P=0.6796), whereas the level of spinophilin (B) is decreased (Tor1a+/+ N=5, Tor1a+/- N=8, t test *P=0.0151)


C ) A Sh - RGS9 - 2 - GFP viral preparation injected into right striatum ( + ) of C57Bl / 6 mice effectively reduces both RGS9 - 2 DRD2 , with respect to striatum ( - ) . summary plot reports mean ± values ( RGS9 - 2 : N = 4 , paired t * P = 0 . 0100 ; DRD2 : N = 4 , paired t * P = 0 . 0432 )

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C) A Sh-RGS9-2-GFP viral preparation injected into the right striatum (+) of C57Bl/6 mice effectively reduces both RGS9-2 and DRD2 protein levels, with respect to the contralateral striatum (-).The summary plot reports mean ± SEM values (RGS9-2: N=4, paired t test *P=0.0100; DRD2: N=4, paired t test *P=0.0432)


D ) endosomal marker Rab4 is upregulated in Tor1a + / - striatum ( Tor1a + / + N = 13 , Tor1a + / - N = 14 , t * * P = 0 . 0087 )

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D) The endosomal marker Rab4 is upregulated in the Tor1a+/- striatum (Tor1a+/+ N=13, Tor1a+/- N=14, t test **P=0.0087)


E ) Basal of markers of degradative pathway , p62 LC3 - II , show a trend towards upregulation in Tor1a + / - striatal lysates ( p62 : Tor1a + / + N = 11 , Tor1a + / - N = 11 , t P = 0 . 2292 ; LC3 - II : Tor1a + / - N = 9 , Tor1a + / - N = 11 , t P = 0 . 1574 )

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E) Basal levels of markers of the degradative pathway, p62 and LC3-II, show a trend towards upregulation in Tor1a+/- striatal lysates (p62: Tor1a+/+ N=11, Tor1a+/- N=11, t test P=0.2292; LC3-II: Tor1a+/- N=9, Tor1a+/- N=11, t test P=0.1574)


F ) Treatment with bafilomycin A1 ( BafA1 ) induces a significantly more pronounced increase of LC3 - II in Tor1a + / - than in wild - type dorsal striatum slices . BafA1 - induced increase in LC3 - II level measured in Tor1a + / - slices was normalized to increase observed in Tor1a + / + samples of same experiment ( N = 4 , one sample t * * P = 0 . 0012 )

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F) Treatment with bafilomycin A1 (BafA1) induces a significantly more pronounced increase of LC3-II in Tor1a+/- than in wild-type dorsal striatum slices. BafA1-induced increase in LC3-II level measured in Tor1a+/- slices was normalized to the increase observed in the Tor1a+/+ samples of the same experiment (N=4, one sample t test **P=0.0012)


G ) LAMP - 2 , a lysosomal marker , shows a trend for upregulation ( Tor1a + / + N = 8 ; Tor1a + / - N = 6 , t P = 0 . 2232 )

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G) LAMP-2, a lysosomal marker, shows a trend for upregulation (Tor1a+/+ N=8; Tor1a+/- N=6, t test P= 0.2232)


Representative double immunofluorescence confocal of coronal sections of dorsal striatum showing p62 immunolabelling ( red ) in choline acetyltransferase ( ChAT ) - positive ( cyano ) ChIs of Tor1a + / + Tor1a + / - mice . A1 ) Two representative ChIs identified by box in low magnification ( bar = 20 µm ) are shown at higher magnification in ( A2 ) ( bar = 5 µm ) . A3 ) graph reporting mean ± values of p62 / ChAT signal overlap in ChIs , normalized to Tor1a + / + values of same experiment , shows a significant increase of p62 in Tor1a + / - neurons ( Tor1a + / + n = 20 , N = 6 mice ; Tor1a + / - n = 20 , N = 6 mice , t * * * P = 0 . 00008 )

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Representative double immunofluorescence confocal images of coronal sections of the dorsal striatum showing p62 immunolabelling (red) in choline acetyltransferase (ChAT)-positive (cyano) ChIs of Tor1a+/+ and Tor1a+/- mice. A1) Two representative ChIs identified by the white box in the low magnification images (bar = 20 µm) are shown at higher magnification in (A2) (bar = 5 µm). A3) The graph reporting mean ± SEM values of p62/ChAT signal overlap in ChIs, normalized to Tor1a+/+ values of the same experiment, shows a significant increase of p62 in Tor1a+/- neurons (Tor1a+/+ n=20 cells, N=6 mice; Tor1a+/- n=20 cells, N=6 mice, t test ***P=0.00008)


A ) Top . Low magnification merged confocal of coronal striatal sections showing ChAT - labelled ( cyano ) ChIs in of mCherry ( red ) indicating viral infection . Bar = 100 µm . Bottom . Higher magnification of ChIs identified by box in upper show viral - induced expression of mCherry GFP signal . Bar = 20 µm . B ) mCherry / ChAT overlap is increased in ChIs from Tor1a + / - mice ( Tor1a + / + n = 24 , Tor1a + / - n = 22 , N = 8 mice / genotype , t * * * P = 0 . 0005 ) , but mCherry / GFP indicated that LC3 flux was unchanged ( Tor1a + / + n = 19 , Tor1a + / - n = 21 , N = 8 mice / genotype , Mann Whitney P = 0 . 2334 )

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A) Top. Low magnification merged confocal images of coronal striatal sections showing ChAT-labelled (cyano) ChIs in the area of mCherry fluorescence (red) indicating viral infection. Bar = 100 µm. Bottom. Higher magnification images of the ChIs identified by the white box in the upper images show viral-induced expression of mCherry and GFP signal. Bar = 20 µm. B) The mCherry/ChAT overlap is increased in ChIs from Tor1a+/- mice (Tor1a+/+ n=24 cells, Tor1a+/- n=22 cells, N=8 mice/genotype, t test ***P=0.0005), but the mCherry/GFP fluorescence ratio indicated that the LC3 flux was unchanged (Tor1a+/+ n=19 cells, Tor1a+/- n=21 cells, N=8 mice/genotype, Mann Whitney test P=0.2334)


C ) Merged split channels confocal ( bar = 10 µm ) of representative Tor1a + / + Tor1a + / - ChIs showing red dots yellow ( indicated by circles ) , quantified in ( D ) : red dots : Tor1a + / + n = 13 , Tor1a + / - n = 14 , t * * * P = 0 . 0003 ; yellow dots : Tor1a + / + n = 13 , Tor1a + / - n = 14 , t * * P = 0 . 0048 , N = 5 mice / genotype ) . Measurement of mCherry / yellow dots indicated that autophagic flux was not altered ( Tor1a + / + n = 13 ; Tor1a + / - n = 14 , Mann Whitney P = 0 . 8842 ) . Data in B , D ) are represented as mean ±

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C) Merged and split channels confocal images (bar = 10 µm) of representative Tor1a+/+ and Tor1a+/- ChIs showing red dots and yellow puncta (indicated by white circles), quantified in (D): red dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test ***P=0.0003; yellow dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test **P=0.0048, N=5 mice/genotype). Measurement of the mCherry/yellow dots ratio indicated that the autophagic flux was not altered (Tor1a+/+ n=13 cells; Tor1a+/- n=14 cells, Mann Whitney test P=0.8842). Data in B,D) are represented as mean ± SEM


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

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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

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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 .

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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

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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 )

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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 )

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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 )

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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 .

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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 .

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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 .

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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 .

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

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

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

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 O O B-MEASURED_VAR 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 I-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

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 .

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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 .

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

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

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

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

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 .

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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 .

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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 .

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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 ) .

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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 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 ) .

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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 .

(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 )

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

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 ) .

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 .

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

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).