D , E Knockdown of RNF123 enhances IFN - β RANTES transcription induced by SeV EMCV , but not that by cGAS STING . HEK293T were transfected with cGAS ( 1 μg ) STING ( 1 μg ) or empty vector for 12 h , then infected with SeV or EMCV for 12 h or left uninfected . Total was isolated RT - PCR ( D ) were performed using indicated primers .

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D, E Knockdown of RNF123 enhances the IFN-β and RANTES transcription induced by SeV and EMCV, but not that by cGAS and the STING. HEK293T cells were transfected with cGAS (1 μg) and STING (1 μg) or empty vector for 12 h, and then infected with SeV or EMCV for 12 h or left uninfected. Total RNA was isolated and RT-PCR (D) were performed using the indicated primers.


D , E Knockdown of RNF123 enhances IFN - β RANTES transcription induced by SeV EMCV , but not that by cGAS STING . HEK293T were transfected with cGAS ( 1 μg ) STING ( 1 μg ) or empty vector for 12 h , then infected with SeV or EMCV for 12 h or left uninfected . Total was isolated Q - PCR ( E ) were performed using indicated primers .

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D, E Knockdown of RNF123 enhances the IFN-β and RANTES transcription induced by SeV and EMCV, but not that by cGAS and the STING. HEK293T cells were transfected with cGAS (1 μg) and STING (1 μg) or empty vector for 12 h, and then infected with SeV or EMCV for 12 h or left uninfected. Total RNA was isolated and Q-PCR (E) were performed using the indicated primers.


F , G Time - course of potentiation of SeV - induced IFN - β transcription ( F ) by knockdown of RNF123 . HEK293T were infected with SeV for 6 , 12 , 18 , or 24 h , total was isolated , RT - PCR was performed using indicated primers ( F ) .

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F, G Time-course of potentiation of SeV-induced IFN-β transcription (F) by knockdown of RNF123. HEK293T cells were infected with SeV for 6, 12, 18, or 24 h, total RNA was isolated, and RT-PCR was performed using the indicated primers (F).


F , G Time - course of potentiation of SeV - induced IFN - β secretion ( G ) by knockdown of RNF123 . HEK293T were infected with SeV for 6 , 12 , 18 , or 24 h , total was isolated . Bioassays of type - I interferon in supernatants of HEK293T were performed , results are presented relative to untreated control ( G ) .

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F, G Time-course of potentiation of SeV-induced IFN-β secretion (G) by knockdown of RNF123. HEK293T cells were infected with SeV for 6, 12, 18, or 24 h, total RNA was isolated. Bioassays of type-I interferon in the supernatants of HEK293T cells were performed, and the results are presented relative to untreated control cells (G).


H HeLa with stable knockdown of RNF123 were generated by shRNA . Western blot was performed as described in Fig 2A .

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H HeLa cells with stable knockdown of RNF123 were generated by shRNA. Western blot was performed as described in Fig 2A.


I , J Knockdown of RNF123 enhances SeV - EMCV - induced IFN - β expression in HeLa . HeLa were infected with SeV or EMCV for 12 h , total was isolated , RT - PCR ( I ) was performed using indicated primers .

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I, J Knockdown of RNF123 enhances SeV- and EMCV- induced IFN-β expression in HeLa cells. HeLa cells were infected with SeV or EMCV for 12 h, total RNA was isolated, and RT-PCR (I) was performed using the indicated primers.


I , J Knockdown of RNF123 enhances SeV - EMCV - induced IFN - β expression in HeLa . HeLa were infected with SeV or EMCV for 12 h , total was isolated , Q - PCR ( J ) was performed using indicated primers .

I, J Knockdown of RNF123 enhances SeV- and EMCV- induced IFN-β expression in HeLa cells. HeLa cells were infected with SeV or EMCV for 12 h, total RNA was isolated, and Q-PCR (J) was performed using the indicated primers.


K Knockdown of RNF123 suppresses NDV - eGFP replication . HEK293T ( 2 × 105 ) were infected with NDV - eGFP . At 30 h after infection , viral replication was determined by GFP expression visualized by microscopy analyzed by western blot with GFP antibody .

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K Knockdown of RNF123 suppresses NDV-eGFP replication. HEK293T cells (2×105) were infected with NDV-eGFP. At 30 h after infection, viral replication was determined by GFP expression visualized by fluorescence microscopy and analyzed by western blot with GFP antibody.


L Knockdown of RNF123 inhibits EMCV replication . HeLa ( 2 × 105 ) were infected with EMCV for 0 , 12 , or 18 h .

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L Knockdown of RNF123 inhibits EMCV replication. HeLa cells (2×105) were infected with EMCV for 0, 12, or 18 h.


A , B RNF123 inhibits activation of IFN - β ( A ) promoters induced by overexpression of RIG - I , RIG - I CARD , MDA5 CARD , VISA , but has no effect on TBK1 - IRF3 - 5D - induced activation of IFN - β promoter ( A ) . HEK293T ( 2 × 105 ) were transfected with indicated plasmids ( 100 ng ) , IFN - β promoter luciferase plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , together with an empty vector RNF123 construct ( 100 ng ) . Luciferase assays were performed 24 h after transfection .

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A, B RNF123 inhibits the activation of the IFN-β (A) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TBK1- and IRF3-5D-induced activation of the IFN-β promoter (A) . HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), IFN-β promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection.


A , B RNF123 inhibits activation of NF - κB ( B ) promoters induced by overexpression of RIG - I , RIG - I CARD , MDA5 CARD , VISA , but has no effect on TAB2 - IKKβ - induced activation of NF - κB promoter ( B ) . HEK293T ( 2 × 105 ) were transfected with indicated plasmids ( 100 ng ) , NF - κB promoter luciferase plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , together with an empty vector RNF123 construct ( 100 ng ) . Luciferase assays were performed 24 h after transfection .

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A, B RNF123 inhibits the activation of the NF-κB (B) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TAB2- and IKKβ-induced activation of the NF-κB promoter (B). HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), NF-κB promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection.


C Knockdown of RNF123 potentiates activation of IFN - β promoter induced by RIG - I , RIG - I CARD , MDA5 , MDA5 CARD , but not that by VISA , TBK1 , cGAS / STING . 293T - GFPsh , shRNF123 # 1 , # 3 , # 4 stable pools ( 2 × 105 ) were transfected with indicated plasmids . Luciferase assays were performed 24 h after transfection .

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C Knockdown of RNF123 potentiates the activation of the IFN-β promoter induced by RIG-I, RIG-I CARD, MDA5, and MDA5 CARD, but not that by VISA, TBK1, and cGAS/STING. 293T-GFPsh, shRNF123 #1, #3, and #4 stable cell pools (2×105) were transfected with the indicated plasmids. Luciferase assays were performed 24 h after transfection.


D RNF123 interacted with RIG - I MDA5 , but not VISA , TBK1 , cGAS . HEK293T ( 1 × 106 ) were transfected with Flag - RNF123 ( F - RNF123 ) together with HA - RIG - I , HA - MDA5 , HA - VISA , HA - TBK1 , or HA - cGAS plasmids ( 8 μg each ) . lysates were immunoprecipitated ( IP ) with anti - HA ( αHA ) . immunoprecipitates were analyzed by western blotting ( immunoblotting [ IB ] ) with anti - Flag ( αF ) or anti - HA antibody ( αHA ) . Whole - lysates ( WCL ) were analyzed by western blotting with anti - HA anti - Flag antibodies to determine expression of transfected plasmids .

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D RNF123 interacted with RIG-I and MDA5, but not VISA, TBK1, and cGAS. HEK293T cells (1×106) were transfected with Flag-RNF123 (F-RNF123) together with HA-RIG-I, HA-MDA5, HA-VISA, HA-TBK1, or HA-cGAS plasmids (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-HA (αHA). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-Flag (αF) or anti-HA antibody (αHA). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies to determine the expression levels of transfected plasmids.


E Endogenous interaction of RNF123 with RIG - I upon SeV infection . HEK293T ( 3 × 106 ) were treated with SeV for 12 h or left untreated . lysates were immunoprecipitated with rabbit anti - RNF123 antibody or control IgG . immunoprecipitates were analyzed by western blotting with rabbit anti - RIG - I antibody . expression of endogenous were analyzed by western blotting with anti - RIG - I anti - RNF123 antibodies .

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E Endogenous interaction of RNF123 with RIG-I upon SeV infection. HEK293T cells (3×106) were treated with SeV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-RIG-I antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-RIG-I and anti-RNF123 antibodies.


F Endogenous interaction of RNF123 with MDA5 upon EMCV infection . HeLa ( 3 × 106 ) were treated with EMCV for 12 h or left untreated . lysates were immunoprecipitated with rabbit anti - RNF123 antibody or control IgG . immunoprecipitates were analyzed by western blotting with rabbit anti - MDA5 antibody . expression of endogenous were analyzed by western blotting with anti - MDA5 anti - RNF123 antibodies . Each histogram shows mean ± SD of three independent experiments done in triplicate . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 1 ; ns , P > 0 . 05 .

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F Endogenous interaction of RNF123 with MDA5 upon EMCV infection. HeLa cells (3×106) were treated with EMCV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-MDA5 antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-MDA5 and anti-RNF123 antibodies. Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; *P <0.1; ns, P >0.05.


B SPRY coiled - coil domains are both indispensable for inhibiting IFN - β promoter activation . HEK293T ( 3 × 105 ) were transfected with IFN - β reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , expression plasmid for RNF123 its mutants ( 100 ng ) , empty vector or RIG - I CARD / MDA5 CARD ( 100 ng ) . Reporter assays were performed 24 h after transfection .

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B The SPRY and coiled-coil domains are both indispensable for inhibiting IFN-β promoter activation. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmid for RNF123 and its mutants (100 ng), and the empty vector or RIG-I CARD/MDA5 CARD (100 ng). Reporter assays were performed 24 h after transfection.


C SPRY domain two coiled - coil domains of RNF123 are both required for interaction with RIG - I CARD . HEK293T ( 1 × 106 ) were transfected with HA - RIG - I CARD together with F - RNF123 or its mutants ( 8 μg each ) . lysates were immunoprecipitated ( IP ) with anti - Flag ( α - F ) . immunoprecipitates were analyzed by western blotting ( immunoblotting [ IB ] ) with anti - HA ( α - HA ) or anti - Flag ( α - F ) . Whole - lysates ( WCL ) were analyzed by western blotting with anti - HA anti - Flag antibodies .

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C The SPRY domain and two coiled-coil domains of RNF123 are both required for interaction with RIG-I CARD. HEK293T cells (1×106) were transfected with HA-RIG-I CARD together with F-RNF123 or its mutants (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-Flag (α-F). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-HA (α-HA) or anti-Flag (α-F). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies.


D KPC2 has no effect on activation of IFN - β reporter induced by RIG - I CARD MDA5 CARD . HEK293T ( 3 × 105 ) were transfected with IFN - β reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , indicated expression plasmids . Reporter assays were performed 24 h after transfection .

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D KPC2 has no effect on the activation of the IFN-β reporter induced by RIG-I CARD and MDA5 CARD. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), and the indicated expression plasmids. Reporter assays were performed 24 h after transfection.


E Effect of shKPC2 plasmids on endogenous KPC2 . RT - PCR was performed with indicated primers after HEK293T were infected with six lentiviruses encoding an shRNA control ( GFPsh ) five shRNAs targeting kpc2 ( Kpc2 # 1 - # 5 ) .

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E Effect of shKPC2 plasmids on endogenous KPC2. RT-PCR was performed with the indicated primers after HEK293T cells were infected with six lentiviruses encoding an shRNA control (GFPsh) and five shRNAs targeting kpc2 (Kpc2 #1-#5).


F KPC2 is not required for inhibitory action of RNF123 . HEK293T ( 2 × 105 ) with GFPsh or KPC2 sh ( # 1 , # 2 , or # 4 ) were transfected with IFN - β reporter plasmid ( 100 ng ) pRL - TK Renilla luciferase plasmid ( 100 ng ) with empty vector or RNF123 ( 100 ng ) . Twenty - four hours after transfection , were infected with SeV or left uninfected . Reporter assays were performed 18 h after infection .

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F KPC2 is not required for the inhibitory action of RNF123. HEK293T cells (2×105) with GFPsh or KPC2 sh (#1, #2, or #4) were transfected with the IFN-β reporter plasmid (100 ng) and the pRL-TK Renilla luciferase plasmid (100 ng) with the empty vector or RNF123 (100 ng). Twenty-four hours after transfection, cells were infected with SeV or left uninfected. Reporter assays were performed 18 h after infection.


G Knockdown of KPC2 had no effect on SeV - induced IFN - β expression . HEK293T with GFPsh or KPC2 sh # 4 were infected with SeV for indicated times , total was isolated , RT - PCR experiments were performed .

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G Knockdown of KPC2 had no effect on SeV-induced IFN-β expression. HEK293T cells with GFPsh or KPC2 sh #4 were infected with SeV for the indicated times, total RNA was isolated, and RT-PCR experiments were performed.


A , B RNF123 interacts with RIG - I / MDA5 CARD . HEK293T ( 1 × 106 ) were transfected with expression plasmids for Flag - RNF123 together with HA - RIG - I or its truncations ( 8 μg each ) . Co - immunoprecipitation whole - lysates were analyzed as described in legend of Fig . 3D .

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A, B RNF123 interacts with RIG-I/MDA5 CARD. HEK293T cells (1×106) were transfected with expression plasmids for Flag-RNF123 together with HA-RIG-I or its truncations (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in the legend of Fig. 3D.


C , D RNF123 competes with VISA for RIG - I / MDA5 CARD binding . HEK293T ( 1 × 106 ) were transfected with expression plasmids for HA - RIG - I CARD / MDA5 CARD alone or together with Flag - VISA or with Flag - VISA Flag - RNF123 ( 8 μg each ) . Co - immunoprecipitation whole - lysates were analyzed as described in Fig . 3D .

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C, D RNF123 competes with VISA for RIG-I/MDA5 CARD binding. HEK293T cells (1×106) were transfected with expression plasmids for HA-RIG-I CARD/MDA5 CARD alone or together with Flag-VISA or with Flag-VISA and Flag-RNF123 (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in Fig. 3D.


E RNF123 does not compete with REUL for RIG - I CARD binding . methods of transfection co - immunoprecipitation were as described in Fig . 5C , D .

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E RNF123 does not compete with REUL for RIG-I CARD binding. The methods of transfection and co-immunoprecipitation were as described in Fig. 5C,D.


F Knockdown of RNF123 facilitates endogenous interaction between RIG - I VISA in 293T . 293T stably infected with SeV were treated for indicated times . lysates were immunoprecipitated with anti - VISA antibody . immunoprecipitates lysates were analyzed by western blotting with indicated antibody .

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F Knockdown of RNF123 facilitates the endogenous interaction between RIG-I and VISA in 293T cells. 293T cells stably infected with SeV were treated for the indicated times. Cell lysates were immunoprecipitated with anti-VISA antibody. The immunoprecipitates and lysates were analyzed by western blotting with the indicated antibody.


G Knockdown of RNF123 facilitates endogenous interaction between MDA5 VISA in HeLa . HeLa were stably infected with EMCV for indicated times . Co - immunoprecipitation whole - lysates were analyzed as described in ( F ) .

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G Knockdown of RNF123 facilitates the endogenous interaction between MDA5 and VISA in HeLa cells. HeLa cells were stably infected with EMCV for the indicated times. Co-immunoprecipitation and whole-cell lysates were analyzed as described in (F).


ELISA of fecal IgA1 ( A ) , IgA2 ( B ) , total IgA ( C ) , from non - IBD ( n = 7 ) , CD ( n = 18 ) , UC ( n = 12 ) ' stool . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( A ) * * : p = 0 , 0098 ; ( B ) * : p = 0 , 0406 ; ( C ) Non - IBD v . UC * : p = 0 , 0479 , CD vs . UC * : p = 0 , 0166 ;

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ELISA assay of fecal IgA1 (A), IgA2 (B), total IgA (C), levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (A)** : p= 0,0098; (B) *: p=0,0406; (C) Non-IBD v. UC *: p=0,0479, CD vs. UC *: p=0,0166;


ELISA of secretory component ( D ) from non - IBD ( n = 7 ) , CD ( n = 18 ) , UC ( n = 12 ) ' stool .

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ELISA assay of secretory component (D) levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool.


Data were further separated according to disease activity for IgA1 ( E ) IgA2 ( F ) . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( F ) * : p = 0 ,

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Data were further separated according to disease activity for IgA1 (E) and IgA2 (F). Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (F) *: p=0,0167


In vitro of purified IgA1 ( G ) IgA2 ( H ) reverse - transcytosis abilities on an inverted model of FAE from Caco2 Raji co - culture . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( G ) * : p = 0 , 0441 ; ( H ) * : p = 0 , 423 ; For some , antibody purification did not yield a high enough , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-DISEASE O O O O O B-DISEASE O O O O O B-DISEASE O O O O O B-GENEPROD O O O B-DISEASE O O O O O B-DISEASE O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O

In vitro assay of purified IgA1 (G) and IgA2 (H) reverse-transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co-culture. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (G) *: p=0,0441; (H) *: p=0,423 ; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


( I ) ELISA of IgA1 - Dectin - 1 binding , at a rate of one receptor per 10 IgA ; ( J ) ELISA of IgA2 - Dectin - 1 binding , at a rate of one receptor per 10 IgA . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( J ) Non - IBD vs . UC * : p = 0 , 0199 , CD vs . UC * : p = 0 , 0243 ; For some , antibody purification did not yield a high enough , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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(I) ELISA assay of IgA1-Dectin-1 binding, at a rate of one receptor per 10 IgA; (J) ELISA assay of IgA2-Dectin-1 binding, at a rate of one receptor per 10 IgA. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (J) Non-IBD vs. UC *: p=0,0199 , CD vs. UC *: p= 0,0243; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


of IgA1 ( K , L ) IgA2 ( M , N ) reverse - transcytosis for weak ( K , M ) strong ( L , N ) Dectin - 1 binding . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( K ) * : p = 0 , 0221 ; ( L ) * : p = 0 , 0486 . For some , antibody purification did not yield a high enough , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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Percent of IgA1 (K, L) and IgA2 (M, N) reverse-transcytosis for weak (K, M) and strong (L, N) Dectin-1 binding. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (K) *: p=0,0221 ; (L) *: p=0,0486 . For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


Heatmaps displaying standardized log2 - transformed signal intensities per glycan for IgA1 ( A ) IgA2 ( B ) in non - IBD , CD UC groups , plotted by hierarchical clustering of Euclidean .

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Heatmaps displaying standardized log2-transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non-IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance.


Flow cytometry of total IgA1 - bound ( A ) IgA2 - bound ( B ) fecal microbiota . ( C ) Flow cytometry of stool IgA1 + IgA2 - ( grey dots ) , IgA1 - IgA2 + ( black dots ) IgA1 + IgA2 + ( empty dots ) . ( A - C ) Data were analysed with a Kruskall - Wallis multiple comparison . In ( C ) * : p = 0 , 0226 . Non - IBD : n = 7 , CD : n = 18 , UC : n = 12 .

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Flow cytometry analysis of total IgA1-bound (A) and IgA2-bound (B) fecal microbiota. (C) Flow cytometry analysis of stool IgA1+ IgA2- bacteria (grey dots), IgA1- IgA2+ bacteria (black dots) and IgA1+ IgA2+ bacteria (empty dots). (A-C) Data were analysed with a Kruskall-Wallis multiple comparison test. In (C) *: p= 0,0226. Non-IBD: n= 7, CD: n=18, and UC: n=12.


( A - E ) Optical density ( 600nm ) variations during in vitro growth of Salmonella enterica Typhimurium ( SL1344 ) co - cultured with stool - purified IgA1 ( A ) or IgA2 ( B ) from non - IBD ( n = 4 ) , CD ( n = 6 ) UC ( n = 3 ) . Comparison between IgA1 IgA2 co - culture in non - IBD ( C ) , CD ( D ) , UC ( E ) . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality , p - values are as follows : ( A ) UC curve : 2h - p = 0 , 0024 , 3h - p = 0 , 003 ; CD curve : 2h 3h p < 0 , 0001 ( B ) UC curve : 4h - p = 0 , 0186 , 5h - p = 0 , 0393 , 6h - p = 0 , 039 , 8h - p < 0 , 0001 ; CD curve : 4h - p = 0 , 0001 , 5h - p = 0 , 0028 , 6h - p = 0 , 0024 , 8h - p < 0 , 0001 . ( C ) 3h - p = 0 , 0422 , 4h - p = 0 , 0422 , 6h - p = 0 , 0012 , 8h - p < 0 , 0001 .

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(A-E) Optical density (600nm) variations during in vitro growth assay of Salmonella enterica Typhimurium (SL1344) co-cultured with stool-purified IgA1 (A) or IgA2 (B) from non-IBD (n=4), CD (n=6) and UC (n=3). Comparison between IgA1 and IgA2 co-culture in non-IBD (C), CD (D), and UC (E). Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (A) UC curve: 2h - p=0,0024, 3h - p=0,003; CD curve: 2h and 3h p<0,0001 (B) UC curve: 4h - p=0,0186, 5h - p=0,0393, 6h - p=0,039, 8h - p<0,0001; CD curve: 4h - p=0,0001, 5h - p=0,0028, 6h - p=0,0024, 8h - p<0,0001. (C) 3h - p=0,0422, 4h - p=0,0422, 6h - p=0,0012, 8h - p<0,0001.


( F - I ) Optical density ( 600nm ) during in vitro growth of Escherichia coli ( 25922 from ATCC ) co - cultured with stool - purified IgA1 ( F ) or IgA2 ( G ) from non - IBD ( n = 2 ) CD ( n = 2 ) . samples ( biological replicates ) have been tested in technical duplicates . Comparison between IgA1 IgA2 co - culture in non - IBD ( H ) , CD ( I ) . These IgA samples were from same throughout each experiment . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality , p - values are as follows : ( F ) Grey curve : 4h - p = 0 , 0004 , 5h - p = 0 , 0014 ; Black curve : 4h 5h - p < 0 , 0001 . ( G ) De - glycosylated CD vs . De - glycosylated non - IBD : 4h - p = 0 , 0003 , 5h - p < 0 , 0001 . ( H ) Native vs . de - glycosylated : 4h 5h - p < 0 , 0001 ; De - glycosylated IgA1 vs . De - glycosylated IgA2 : 5h - p = 0 , 0012 . ( H ) Native vs . de - glycosylated : 4h - p = 0 , 001 5h - p = 0 , 006 ; De - glycosylated IgA1 vs . De - glycosylated IgA2 : 5h - p < 0 , 0001 .

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(F-I) Optical density (600nm) during in vitro growth assay of Escherichia coli (25922 strain from ATCC) co-cultured with stool-purified IgA1 (F) or IgA2 (G) from non-IBD (n=2) and CD (n=2). All patient samples (biological replicates) have been tested in technical duplicates. Comparison between IgA1 and IgA2 co-culture in non-IBD (H), and CD (I). These IgA samples were from the same patients throughout each experiment. Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (F) Grey curve: 4h - p=0,0004, 5h - p=0,0014; Black curve: 4h and 5h - p<0,0001. (G) De-glycosylated CD vs. De-glycosylated non-IBD: 4h - p=0,0003, 5h - p<0,0001. (H) Native vs. de-glycosylated: 4h and 5h - p<0,0001; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p=0,0012. (H) Native vs. de-glycosylated: 4h - p=0,001 and 5h - p= 0,006; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p<0,0001.


( A ) Combined FISH - IF for each probe . ( B ) Total detected per probe per biopsy . Erec482 - * : p = 0 , 00233 ; Ato291 - * : p = 0 , 0268 ; Bac303 - * : p = 0 , 0196 . ( C ) of co - occurred IgA2 FISH - bound particles . Erec482 - * : p = 0 , 0073 . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality . Non - IBD : n = 3 ; Active CD : n = 6 ; Inactive CD : n = 4 . samples ( biological replicates ) have been tested in technical duplicates .

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(A) Combined FISH-IF staining for each probe. (B) Total fluorescence detected per probe and per biopsy. Erec482 - *: p=0,00233; Ato291 - *: p=0,0268; Bac303 - *: p=0,0196. (C) Percent of co-occurred IgA2 and FISH-bound particles. Erec482 - *: p=0, 0073.Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test. Non-IBD: n = 3; Active CD: n= 6; Inactive CD: n=4. All patient samples (biological replicates) have been tested in technical duplicates.


( B ) Immunoblots from ( cyt ) ( nuc ) fractionations of RPE - 1 in early - G1 showing presence of endogenous ACTN4 in fractions . were detected with antibodies against ( α ) ACTN4 , α - Tubulin or Histone 3 as

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(B) Immunoblots from cytosolic (cyt) and nuclear (nuc) fractionations of RPE-1 cells in early-G1 showing the presence of endogenous ACTN4 in nuclear fractions. Proteins were detected with antibodies against (α) ACTN4, α-Tubulin or Histone 3 as indicated


( C ) Upper middle panels : immunoblots showing precipitation of ACTN4 β - actin from RPE - 1 lysates in early - G1 in presence or absence of biotin - phalloidin , as indicated . Lower panel : Immunoblot of ( cyt ) ( nuc ) fractions . were detected with antibodies against ( α ) ACTN4 , β - Actin , α - Tubulin or Histone 3 as indicated .

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(C) Upper and middle panels: immunoblots showing precipitation of ACTN4 and β-actin from RPE-1 nuclear lysates in early-G1 in the presence or absence of biotin-phalloidin, as indicated. Lower panel: Immunoblot of cytosolic (cyt) and nuclear (nuc) fractions. Proteins were detected with antibodies against (α) ACTN4, β-Actin, α-Tubulin or Histone 3 as indicated.


( D ) Immmunofluorescence of endogenous ACTN4 in non - dividing post - mitotic NIH3T3 RPE - 1 in as indicated . Shown are maximum projections . were fixed in 4 % PFA stained for ACTN4 nuclei ( DAPI ) as indicated . Scale bars 10 μm .

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(D) Immmunofluorescence images of endogenous ACTN4 in non-dividing and post-mitotic NIH3T3 and RPE-1 cells in as indicated. Shown are maximum intensity projections. Cells were fixed in 4% PFA and stained for ACTN4 and nuclei (DAPI) as indicated. Scale bars 10 μm.


( A ) in early G1 stably expressing nAC - mCherry doxycycline - inducible ACTN4 - SNAP were analyzed by time - lapse microscopy . ACTN4 - SNAP was labeled by SNAP - 647SiR dye ( green ) . Different arrows mark dynamic ACTN4 clusters . square 1 is shown as a timeseries on right . square 2 is shown as a magnification below . square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B . Scale bar overview ( nAC ) represents 10 µm . Scale bar time series magnification 5 µm .

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(A) Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a timeseries on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. Scale bar overview (nAC) represents 10 µm. Scale bar time series and magnification 5 µm.


in early G1 stably expressing nAC - mCherry doxycycline - inducible ACTN4 - SNAP were analyzed by time - lapse microscopy . ACTN4 - SNAP was labeled by SNAP - 647SiR dye ( green ) . square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B . B ) Linescan of an actin filament with associated ACTN4 from square 3 in A .

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Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. B) Linescan of an actin filament with associated ACTN4 from square 3 in A.


( C ) Automated tracking ( autoregressive motion ) of actin ACTN4 in nuclei by Imaris software . were treated like in A . Tracks are visualized by red ( actin ) or green ( ACTN4 ) lines in a representative image ( see corresponding Movie EV2 ) . Scale bar is 2 µm . track , track displacement ( between starting - end point ) , average speed maximum speed were quantified from 3 independent experiments . black shows median grey dashed lines show quartiles . Quantifications show similar motion characteristics for actin ACTN4 .

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(C) Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in A. Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2). Scale bar is 2 µm. The track length, track displacement (distance between starting- and end point), average speed and maximum speed were quantified from 3 independent experiments. The black line shows the median and grey dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.


( A ) NIH3T3 expressing nAC - mCherry were imaged for 120 min . Image frames represent maximum projections of 30 Z planes . Lower panel is a magnification of indicated ( orange box ) ; scale bars 10 µm .

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

(A) NIH3T3 cells expressing nAC-mCherry were imaged for 120 min. Image frames represent maximum projections of 30 Z planes. Lower panel is a magnification of the indicated area (orange box); scale bars 10 µm.


( B ) NIH3T3 stably expressing H2B - mCherry were transfected with siCtrl or siACTN1 + siACTN4 . After mitotic exit z - stacks ( 30 planes ) were acquired every 5 min . Nuclei were 3D - reconstructed volume was measured by Imaris . Data are mean ± from 3 biological replicates with 22 - 27 ( siCtrl ) 32 - 42 ( siACTN1 + 4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon * * * * p < 0 . 0001 by t - at 90 min .

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(B) NIH3T3 cells stably expressing H2B-mCherry were transfected with siCtrl or siACTN1 + siACTN4. After mitotic exit z-stacks (30 planes) were acquired every 5 min. Nuclei were 3D-reconstructed and volume was measured by Imaris. Data are mean ± SEM from 3 biological replicates with 22-27 (siCtrl) and 32-42 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by t-test at 90 min.


( C ) RPE - 1 were analyzed as in B . Data are mean ± from 5 biological replicates with 32 - 37 ( siCtrl ) 31 - 39 ( siACTN1 + 4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon * * * * p < 0 . 0001 by Mann Whitney at 90 min .

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(C) RPE-1 cells were analyzed as in B. Data are mean ± SEM from 5 biological replicates with 32-37 (siCtrl) and 31-39 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by Mann Whitney test at 90 min.


( E ) NIH3T3 stably expressing H2B - mCherry siRNA - resistant ACTN4 - NES were transfected with indicated siRNAs analyzed as described in B . Data are mean ± from 5 biological replicates with 63 - 71 ( siCtrl ) , 37 - 41 ( siACTN1 + 4 ) , 52 - 58 ( siACTN1 ) 52 - 55 ( siACTN4 ) analyzed nuclei ; * * * * p ( siACTN4 ) < 0 . 0001 ; * * * * p ( siACTN1 + 4 ) < 0 . 0001 by One Way ANOVA at 90 min .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD 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 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 O O

(E) NIH3T3 cells stably expressing H2B-mCherry and siRNA-resistant ACTN4-NES were transfected with the indicated siRNAs and analyzed as described in B. Data are mean ± SEM from 5 biological replicates with 63-71 (siCtrl), 37-41 (siACTN1+4), 52-58 (siACTN1) and 52-55 (siACTN4) analyzed nuclei; ****p(siACTN4) < 0.0001; ****p(siACTN1+4) < 0.0001 by One Way ANOVA at 90 min.


( F ) NIH3T3 stably expressing H2B - mCherry indicated ACTN4 mutants were transfected with indicated siRNAs analyzed as in B . Data are mean ± from 3 biological replicates with 63 - 69 ( ACTN4 wt + siACTN4 ) , 63 - 71 ( ACTN4 - NES + siCtrl ) 54 - 58 ( ACTN4 - NES + siACTN4 ) analyzed nuclei ; * * * * p ( ACTN4 - NES + siACTN4 ) < 0 . 0001 by One Way ANOVA at 90 min .

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(F) NIH3T3 cells stably expressing H2B-mCherry and the indicated ACTN4 mutants were transfected with the indicated siRNAs and analyzed as in B. Data are mean ± SEM from 3 biological replicates with 63-69 (ACTN4 wt + siACTN4), 63-71 (ACTN4-NES + siCtrl) and 54-58 (ACTN4-NES + siACTN4) analyzed nuclei; ****p(ACTN4-NES + siACTN4) < 0.0001 by One Way ANOVA at 90 min.


( G ) NIH3T3 expressing siRNA - resistant ACTN4 - NES were transfected with siCtrl or siACTN4 ; density was calculated from H2B - mCherry intensities by total volumes ( / μm3 ) normalized to t = 0 ( mitotic exit ) ; Data are mean ± from 5 biological replicates with 44 - 48 ( siCtrl ) 44 - 47 ( siACTN4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon * p < 0 . 05 by t - at 90 min .

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

(G) NIH3T3 cells expressing siRNA-resistant ACTN4-NES were transfected with siCtrl or siACTN4; chromatin density was calculated from H2B-mCherry fluorescence intensities by total nuclear volumes (intensity/μm3) and normalized to t=0 (mitotic exit); Data are mean ± SEM from 5 biological replicates with 44-48 (siCtrl) and 44-47 (siACTN4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and *p < 0.05 by t-test at 90 min.


( B ) Upper panel shows expression of ACTN4 constructs ( green , confocal image ) phalloidin - AF647 in same ( orange , dSTORM image ) scale bar 10 µm ; lower panel represents zoom of indicated areas ( boxes ) ; scale bars 5 µm .

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

(B) Upper panel shows expression of ACTN4 constructs (green, confocal image) and phalloidin-AF647 staining in the same cells (orange, dSTORM image) scale bar 10 µm; lower panel represents zoom of the indicated areas (white boxes); scale bars 5 µm.


( C ) Phalloidin - AF647 localizations per post - mitotic nuclei ; data shown as mean ± 2 . 5 - 97 . 5 percentile : 52 ACTN4 - wt nuclei , 55 ACTN4ΔCH1 - NLS nuclei 40 nuclei of non - dividing ( ACTN4 - wt ) from 5 biological replicates . * * * p ( ACTN4ΔCH1 - NLS post - mitotic ) < 0 . 001 , * * * * p ( ACTN4 - wt non - dividing ) < 0 . 0001 , * p ( ACTN4ΔCH1 - NLS post - mitotic vs . ACTN4 - wt non - dividing ) < 0 . 05 by One Way ANOVA .

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

(C) Phalloidin-AF647 localizations per post-mitotic nuclei; data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei and 40 nuclei of non-dividing cells (ACTN4-wt) from 5 biological replicates. ***p(ACTN4ΔCH1-NLS post-mitotic) < 0.001, ****p(ACTN4-wt non-dividing) < 0.0001, *p (ACTN4ΔCH1-NLS post-mitotic vs. ACTN4-wt non-dividing) < 0.05 by One Way ANOVA.


( D ) Number of filaments in post mitotic nuclei ; as in ( C ) ; data shown as mean ± 2 . 5 - 97 . 5 percentile : 52 ACTN4 - wt nuclei , 55 ACTN4ΔCH1 - NLS nuclei from 5 biological replicates , * * * p < 0 . 001 by t - .

O O O B-EXP_ASSAY O B-SUBCELLULAR 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 B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(D) Number of filaments in the post mitotic nuclei; as in (C); data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei from 5 biological replicates, ***p < 0.001 by t-test.


( E ) Actin filament widths for ACTN4 - wt ACTN4ΔCH1 - NLS in early G1 nuclei ; ) ; data shown as mean ± 2 . 5 - 97 . 5 percentile with 96 filaments ( ACTN4 - wt ) 36 filaments ( ACTN4ΔCH1 - NLS ) from 5 biological replicates , * * p < 0 . 01 by t - .

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

(E) Actin filament widths for ACTN4-wt and ACTN4ΔCH1-NLS in early G1 nuclei; ); data shown as mean ± 2.5-97.5 percentile with 96 filaments (ACTN4-wt) and 36 filaments (ACTN4ΔCH1-NLS) from 5 biological replicates, **p < 0.01 by t-test.


( a ) siRNA screening . MDA - MB - 231 reverse transfected with siRNA pools against MT1 - MMP , TKS5 , or each human RAB GTPase were plated on fluorescein - labeled - gelatin coated optical microplates . Degradation activity was measured by counting number of degrading gelatin identified as black foci ( similar results were obtained by measuring of degradation / number of ) . inset shows representative of degraded Oregon green gelatin ( upper panels ) merged with TRITC - phalloidin ( red ) DAPI ( blue ) in scrambled siRNA ( NC ) , or siRNA against RAB28 or RAB42 transfected ( lower panel ) . Data are means ± of 3 independent experiments . Bar , 50 µm .

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

(a) Primary siRNA screening analysis. MDA-MB-231 cells reverse transfected with siRNA pools against MT1-MMP, TKS5, or each human RAB GTPase were plated on fluorescein-labeled-gelatin coated optical microplates. Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci (similar results were obtained by measuring the area of degradation/number of cells). The inset shows representative images of degraded Oregon green gelatin (upper panels) and merged images with TRITC-phalloidin (red) and DAPI (blue) in scrambled siRNA (NC), or siRNA against RAB28 or RAB42 transfected cells (lower panel). Data are means ± SEM of 3 independent experiments. Bar, 50 µm.


( b ) Secondary siRNA screening was performed on candidate RAB GTPases selected from screen using 3 deconvoluted siRNAs . Data are mean ± SD ( n > 5 different fields / experiment ) .

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(b) Secondary siRNA screening analysis was performed on candidate RAB GTPases selected from the primary screen using 3 individual deconvoluted siRNAs. Data are mean ± SD (n> 5 different fields/experiment).


( c - e ) Tertiary siRNA screening . Pooled siRNAs against candidate RAB GTPases identified from secondary screen or TKS5 , used as control , were transfected into MCF10 . DCIS . com . plated onto fluorescently conjugated gelatin ( red ) were stimulated with 100 ng / ml HGF overnight in presence or absence of GM6001 , followed by with FITC - phalloidin ( green ) DAPI ( blue ) ( c ) . Data are mean ± ( d , error bars ; n > 100 / experiment in at least 4 independent ones ) .

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-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O 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 B-SMALL_MOLECULE O B-EXP_ASSAY O O O 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 O O O O O O O O

(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Cells plated onto fluorescently conjugated gelatin (red) were stimulated with 100 ng/ml HGF overnight in the presence or absence of GM6001, followed by staining with FITC-phalloidin (green) and DAPI (blue) (c). Data are the mean ± SEM (d, error bars; n > 100 cells/experiment in at least 4 independent ones).


( c - e ) Tertiary siRNA screening . Pooled siRNAs against candidate RAB GTPases identified from secondary screen or TKS5 , used as control , were transfected into MCF10 . DCIS . com . Efficacy of targeted gene silencing was verified by qPCR ( e ) . Bar , 50 µm .

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

(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Efficacy of targeted gene silencing was verified by qPCR (e). Bar, 50 µm.


( f - h ) Inverse invasion . An equal number of MDA - MB - 231 was reverse transfected with a mixture of three siRNA oligonucleotides targeting interest genes . Invasive activity is shown as average number of / field that migrate into fibronectin - supplemented ( 25 µg / ml ) Matrigel further than 20 µm ( f , g ) . Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed .

O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Invasive activity is shown as the average number of cells/field that migrate into fibronectin-supplemented (25 µg/ml) Matrigel further than 20 µm (f, g). Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed.


( f - h ) Inverse invasion . An equal number of MDA - MB - 231 was reverse transfected with a mixture of three siRNA oligonucleotides targeting interest genes . Efficacy of targeted gene silencing was verified by qPCR ( h ) . Bar , 250 µm . * P < 0 . 05 . * * * , P < 0 . 001 .

O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O 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 O O O O

(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Efficacy of targeted gene silencing was verified by qPCR (h). Bar, 250 µm. *P < 0.05. ***, P < 0.001.


( a ) Doxycycline - inducible empty vector ( EV ) or RAB2A - shRNA MDA - MB - 231 were cultured in absence ( - ) or presence ( + ) of doxycycline for three days , immunoblotted with anti - RAB2A anti - actin antibodies . of RAB2A relative to actin is shown on bottom . Relative density in uninduced EV was set to 1 .

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

(a) Doxycycline-inducible empty vector (EV) or RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (-) or presence (+) of doxycycline for three days, and immunoblotted with anti-RAB2A and anti-actin antibodies. The intensity of RAB2A relative to actin is shown on the bottom. Relative density in uninduced EV cells was set to 1.


( b ) Doxycycline - inducible RAB2A - shRNA MDA - MB - 231 were cultured in absence ( CTR ) or presence ( shRAB2A ) of doxycycline for three days . Invasion of MDA - MB - 231 through Matrigel - coated transwells toward HGF was assessed after 24 hours . Representative are shown . Data are mean ± SD ( n = 10 field / condition in at least 3 experiments ) . Bar , 200 µm .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE 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 O O O O O

(b) Doxycycline-inducible RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days. Invasion of MDA-MB-231 cells through Matrigel-coated transwells toward HGF was assessed after 24 hours. Representative images are shown. Data are the mean ± SD (n=10 field/condition in at least 3 experiments). Bar, 200 µm.


( c - e ) EV shRAB2AMDA - MB - 231 were embedded in Spheroid Formation ECMR in absence ( - ) or presence ( + ) of doxycycline / or GM6001 . After 3 days , were transferred into Invasive MatrixR . Phase contrast microscopy of representative spheroids are shown after 9 days ( c ) . Dashed circle indicates of spheroids at day 0 . Bars , 500 µm . Invasive activity into ECM was expressed as total spheroid ( relative to control at day 0 ) quantified by ImageJ ( d ) . number of at indicated days was quantified by CyQUANTR Proliferation Kit ( e ) . Data are mean ± SD ( n = 3 independent experiments ) .

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

(c-e) EV and shRAB2AMDA-MB-231 cells were embedded in Spheroid Formation ECMR in the absence (-) or presence (+) of doxycycline and/or GM6001. After 3 days, the cells were transferred into Invasive MatrixR. Phase contrast light microscopy images of representative spheroids are shown after 9 days (c). Dashed circle indicates the size of spheroids at day 0. Bars, 500 µm. Invasive activity into ECM was expressed as total spheroid area (relative to control cells at day 0) quantified by ImageJ (d). The number of cells at the indicated days was quantified by CyQUANTR Cell Proliferation Assay Kit (e). Data are the mean ± SD (n=3 independent experiments).


( f - h ) Doxycycline - inducible murine RAB2A ( mRAB2A ) - expression MCF10 . DCIS . com were transfected with scrambled siRNA or siRNA against human RAB2A ( siRAB2A ) in absence ( - ) or presence ( + ) of doxycycline . Serum - starved were plated onto fluorescently conjugated gelatin ( red ) , stimulated with HGF ( 100 ng / ml ) overnight , stained with phalloidin ( green ) DAPI ( blue ) ( f ) . Bar , 50 μm . Data are mean ± ( error bars ; n > 100 / experiment in 4 independent ones ) ( g ) . Silencing of endogenous RAB2A ( lower band ) ectopic RAB2A expression ( upper band ) was verified by immunoblotting ( h ) . * P < 0 . 05 , * * , P < 0 . 01 , * * * , P < 0 . 001 , NS , not significant .

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

(f-h) Doxycycline-inducible murine RAB2A (mRAB2A)-expression MCF10.DCIS.com cells were transfected with scrambled siRNA or siRNA against human RAB2A (siRAB2A) in the absence (-) or presence (+) of doxycycline. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue) (f). Bar, 50 μm. Data are the mean ± SEM (error bars; n > 100 cells/experiment in 4 independent ones) (g). Silencing of endogenous RAB2A protein (lower band) and ectopic RAB2A expression (upper band) was verified by immunoblotting (h). * P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.


RAB2A expression was performed on Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in European Institute of Oncology , Milano , Italy , in year 2000 . Expression was measured by on TMA . ( a ) are representative of RAB2A expression scoring according to in TMA . In tissues signals were associated with component not with adjacent or infiltrating stroma .

B-GENEPROD 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 B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(a) Images are representative of RAB2A expression scoring according to intensity staining in TMA. In tumor tissues the IHC signals were associated with the tumor cell component and not with the adjacent or infiltrating stroma.


RAB2A expression was performed on Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in European Institute of Oncology , Milano , Italy , in year 2000 . Expression was measured by on TMA . ( b ) Correlation of RAB2A expression with clinicopathological parameters . Only for 520 of 622 samples expression data for RAB2A was available . Note that number of scored cases is lower than total number of cases since : i ) in some cases , cores detached from slides during manipulations ; ii ) clinical information was not available for . In tissues signal was associated with component not with adjacent or infiltrating stroma .

B-GENEPROD 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 B-EXP_ASSAY O O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(b) Correlation of RAB2A expression with clinicopathological parameters. Only for 520 of 622 samples expression data for RAB2A was available. Note that the number of scored cases is lower than the total number of cases since: i) in some cases, individual cores detached from the slides during the manipulations; ii) clinical information was not available for all patients. In tumor tissues the IHC signal was associated with the tumor cell component and not with the adjacent or infiltrating stroma.


RAB2A expression was performed on Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in European Institute of Oncology , Milano , Italy , in year 2000 . Expression was measured by on TMA . ( c ) Kaplan - Meier Distant Recurrence Free Interval survival ( DRFI [ 70 ] ) ( d ) Univariate Multivariate Cox - regression , shows that in BC ' high of RAB2A predict a poor prognosis , that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence .

B-GENEPROD 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 B-EXP_ASSAY O O O O O O O O O O O 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 B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(c) Kaplan-Meier Distant Recurrence Free Interval survival (DRFI [70]) and (d) Univariate and Multivariate Cox-regression analysis, shows that in BC patients' high levels of RAB2A protein predict a poor prognosis, and that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence.


( a , b ) MCF10 . DCIS . com were transfected with FLAG - empty vector ( EV ) , constitutively active RAB2A - Q65L , or dominant negative RAB2A - S20N mutant . were immunostained with anti - FLAG ( red ) RAB7 ( a , green ) or anti - EEA1 ( b ) antibodies . Merged with DAPI ( blue ) are shown on right . Enlarged endosomes in boxed region are highly magnified in insets . Diameters of RAB7 positive vesicles are also shown . n , number of scored for each population . RGB profiler plugin from ImageJ was used to plot colocalization in lines drawn in inset . Bars , 10 μm , ( inset 2 μm ) .

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

(a, b) MCF10.DCIS.com cells were transfected with FLAG-empty vector (EV), constitutively active RAB2A-Q65L, or dominant negative RAB2A-S20N mutant. Cells were immunostained with anti-FLAG (red) and RAB7 (a, green) or anti-EEA1 (b) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. The RGB profiler plugin from ImageJ was used to plot colocalization in the lines drawn in the inset images. Bars, 10 μm, (inset 2 μm).


( c ) MDA - MB - 231 were transfected with EGFP - fused EV , RAB2A - Q65L , or RAB2A - S20N . are immunostained with anti - RAB7 ( blue ) anti - MT1 - MMP ( red ) antibodies , their merged are shown . Merged are shown on right . Enlarged endosomes containing MT1 - MMP in boxed region are highly magnified in insets . Note that RAB2A - Q65L RAB7 - positive vesicles contain MT1 - MMP ( arrows ) . Bars , 10 μm , ( inset 2 μm ) .

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

(c) MDA-MB-231 cells were transfected with EGFP-fused EV, RAB2A-Q65L, or RAB2A-S20N. Cells are immunostained with anti-RAB7 (blue) and anti-MT1-MMP (red) antibodies, and their merged images are shown. Merged images are shown on the right. Enlarged endosomes containing MT1-MMP in the boxed region are highly magnified in the insets. Note that RAB2A-Q65L and RAB7-positive vesicles contain MT1-MMP (arrows). Bars, 10 μm, (inset 2 μm).


( d - f ) MDA - MB - 231 expressing MT1 - MMP - pHluorin transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A were seeded on type I collagen , were acquired for 20 min period by TIRF microscopy . Frequency of MT1 - MMP - pHluorin exocytic events was quantified ( e ) . corresponding time - lapse series are shown in Movie 1 , while an entire shown in Movie 2 . Efficacy of RAB2A gene silencing was verified by qPCR ( f ) . Bars , 2 μm . * * , P < 0 . 01 .

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

(d-f) MDA-MB-231 cells expressing MT1-MMP-pHluorin transfected with scrambled siRNA (siNC) or siRNA against RAB2A were seeded on type I collagen, and images were acquired for 20 min period by TIRF microscopy. Frequency of MT1-MMP-pHluorin exocytic events was quantified (e). The corresponding time-lapse series are shown in Movie 1, while an entire cell shown in Movie 2. Efficacy of RAB2A gene silencing was verified by qPCR (f). Bars, 2 μm. **, P < 0.01.


( a ) Total lysates from HEK293T transfected with myc - VPS39 or VPS41 were incubated with GST - empty vector ( EV ) , RAB2A - S20N ( SN ) , or RAB2A - Q65L ( QL ) bound to glutathione sepharose beads . Immunoblotting was performed with anti - myc antibody . Input lysates are also shown . GST loaded in gels were stained with CBB .

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(a) Total cellular lysates from HEK293T cells transfected with myc-VPS39 or VPS41 were incubated with GST-empty vector (EV), RAB2A-S20N (SN), or RAB2A-Q65L (QL) proteins bound to glutathione sepharose beads. Immunoblotting was performed with anti-myc antibody. Input lysates are also shown. GST proteins loaded in gels were stained with CBB.


( b ) FLAG - EV or RAB2A were transiently co - transfected with EGFP - VPS39 or VPS41 into HEK293T . Immunoprecipitated ( IP ) fraction from lysates with anti - FLAG antibody total lysates were immunoblotted with antibodies indicated on right .

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(b) FLAG-EV or RAB2A were transiently co-transfected with EGFP-VPS39 or VPS41 into HEK293T cells. Immunoprecipitated (IP) fraction from cell lysates with anti-FLAG antibody and total lysates were immunoblotted with the antibodies indicated on the right.


( c , d ) MDA - MB - 231 were transfected with EGFP - fused empty EV , VPS39 , or VPS41 . were immunostained with RAB7 ( c , red ) or anti - MT1 - MMP ( d ) antibodies . Merged with DAPI ( blue ) are shown on right . Enlarged endosomes in boxed region are highly magnified in insets . Diameters of RAB7 positive vesicles are also shown . n , number of scored for each population . Bars , 10 μm , ( inset 5 μm ) .

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

(c, d) MDA-MB-231 cells were transfected with EGFP-fused empty EV, VPS39, or VPS41. Cells were immunostained with RAB7 (c, red) or anti-MT1-MMP (d) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. Bars, 10 μm, (inset 5 μm).


( e - i ) Scrambled siRNA ( siNC ) or siRNA against RAB2A ( siRAB2A ) , VPS39 ( siVPS39 ) , or VPS41 ( siVPS41 ) were transfected into MCF10 . DCIS . com . Serum - starved were then plated onto - conjugated gelatin ( red ) , stimulated with HGF ( 100 ng / ml ) overnight , stained with phalloidin ( green ) DAPI ( blue ) . of gelatin degradation ( f ) efficacy of targeted gene silencing verified by qPCR ( g - i ) . Data are mean ± ( error bars ; n > 100 / experiment in at least 4 independent ones ) . Bars , 50 µm .

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

(e-i) Scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A), VPS39 (siVPS39), or VPS41 (siVPS41) were transfected into MCF10.DCIS.com cells. Serum-starved cells were then plated onto fluorescent-conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue). Quantification of gelatin degradation (f) and efficacy of targeted gene silencing verified by qPCR (g-i). Data are the mean ± SEM (error bars; n > 100 cells/experiment in at least 4 independent ones). Bars, 50 µm.


( j - l ) Inverse invasion of MDA - MB - 231 transfected with siRNA oligonucleotides targeting interest genes . Invasive activity is numbers of / field that migrate through Matrigel further than 50 µm ( k ) . Efficacy of targeted gene silencing was verified by qPCR ( l ) . Data are mean ± SD from 3 different fields . Bar , 250 µm .

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(j-l) Inverse invasion assay of MDA-MB-231 cells transfected with siRNA oligonucleotides targeting interest genes. Invasive activity is the numbers of cells/field that migrate through Matrigel further than 50 µm (k). Efficacy of targeted gene silencing was verified by qPCR (l). Data are mean ± SD from 3 different fields. Bar, 250 µm.


( m - q ) Doxycycline - inducible murine RAB2A - expression MCF10 . DCIS . com were transfected with siNC , siRAB2A , or siVPS39 in absence ( CTR ) or presence ( RAB2A ) of doxycycline . Their ECM degradation activities were quantified ( n ) . Data are mean ± ( error bars ; n > 100 / experiment in 3 independent ones ) . Total lysates were immunoblotted by anti - RAB2A anti - tubulin antibodies ( o ) . Efficacy of targeted gene silencing was verified by qPCR ( p - q ) . Bar , 50 μm . * * , P < 0 . 01 , * * * , P < 0 . 001 .

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

(m-q) Doxycycline-inducible murine RAB2A-expression MCF10.DCIS.com cells were transfected with siNC, siRAB2A, or siVPS39 in the absence (CTR) or presence (RAB2A) of doxycycline. Their ECM degradation activities were quantified (n). Data are the mean ± SEM (error bars; n > 100 cells/experiment in 3 independent ones). Total cell lysates were immunoblotted by anti-RAB2A and anti-tubulin antibodies (o). Efficacy of targeted gene silencing was verified by qPCR (p-q). Bar, 50 μm. **, P < 0.01, ***, P < 0.001.


( a ) Doxycycline - inducible RAB2A - shRNA MCF10 . DCIS . com were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in absence ( CTR ) or presence ( shRAB2A ) of doxycycline . were stimulated with 5 ng / ml EGF 20 ng / ml HGF ( EGF + HGF ) or left starved ( STV ) for 9 days . Representative phase contrast microscopy are shown . Invasive activity of was expressed by circularity ( with a value of 1 representing perfect circularity = no invasion ) of each acini . Bar , 100 µm .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O 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 O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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 O O O B-CELL_TYPE O O O O O O

(a) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were incubated on a thick layer of Matrigel for 4 days to allow spheroid formation in the absence (CTR) or presence (shRAB2A) of doxycycline. Cells were stimulated with 5 ng/ml EGF and 20 ng/ml HGF (EGF+HGF) or left starved (STV) for 9 days. Representative phase contrast light microscopy images are shown. Invasive activity of the cells was expressed by circularity (with a value of 1 representing perfect circularity = no invasion) of each acini. Bar, 100 µm.


( b - d ) MCF10 . DCIS . com ( 104 ) transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A ( siRAB2A ) were plated onto 6 - well dish . Representative phase contrast microscopy ( b ) distribution of numbers per cluster of indicated number ( c ) are shown . Data are means ± SD of 10 fields ( n > 60 / field ) of independent 4 independent experiments . Bars , 100 μm . Efficacy of RAB2A gene silencing was verified by qPCR ( d ) . Bar , 200 µm .

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(b-d) MCF10.DCIS.com cells (104 cells) transfected with scrambled siRNA (siNC) or siRNA against RAB2A (siRAB2A) were plated onto 6-well dish. Representative phase contrast light microscopy images (b) and the distribution of cell numbers per cluster of the indicated cell number (c) are shown. Data are means ± SD of 10 fields (n > 60 cells/field) of independent 4 independent experiments. Bars, 100 μm. Efficacy of RAB2A gene silencing was verified by qPCR (d). Bar, 200 µm.


( e ) Doxycycline - inducible RAB2A - shRNA MCF10 . DCIS . com were cultured in absence ( CTR ) or presence ( shRAB2A ) of doxycycline for three days , immunostained with antibodies indicated on top ( green ) DAPI ( blue ) .

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(e) Doxycycline-inducible RAB2A-shRNA MCF10.DCIS.com cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days, and immunostained with antibodies indicated on the top (green) and DAPI (blue).


( f , g ) MCF - 10AneoT ( f ) were transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A , immunostained with antibodies indicated on top ( green ) DAPI ( blue ) . Bars , 10 μm .

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(f, g) MCF-10AneoT (f) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm.


( f , g ) MCF10 - CA1a ( g ) were transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A , immunostained with antibodies indicated on top ( green ) DAPI ( blue ) . Bars , 10 μm .

(f, g) MCF10-CA1a (g) cells were transfected with scrambled siRNA (siNC) or siRNA against RAB2A, and immunostained with antibodies indicated on the top (green) and DAPI (blue). Bars, 10 μm.


( h ) Total lysates from ( e ) , ( f ) , ( g ) were immunoblotted by anti - E - cadherin , RAB2A , anti - actin antibodies . * * * , P < 0 . 001 , NS , not significant .

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(h) Total cell lysates from (e), (f), and (g) were immunoblotted by anti-E-cadherin, RAB2A, and anti-actin antibodies. ***, P < 0.001, NS, not significant.


( a , b ) Doxycycline - inducible RAB2A - expression MCF10 . DCIS . com ( a ) MCF10A ( b ) were cultured in absence ( CTR ) or presence ( RAB2A ) of doxycycline , immunostained with anti - RAB2A ( blue ) , anti - E - cadherin ( green ) , anti - TGN46 ( red ) antibodies . Merged with DAPI ( magenta ) are shown on right . Bars , 10 μm .

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

(a, b) Doxycycline-inducible RAB2A-expression MCF10.DCIS.com (a) and MCF10A (b) cells were cultured in the absence (CTR) or presence (RAB2A) of doxycycline, and immunostained with anti-RAB2A (blue), anti-E-cadherin (green), and anti-TGN46 (red) antibodies. Merged images with DAPI (magenta) are shown on the right. Bars, 10 μm.


( c ) CTR RAB2AMCF10A ( 104 ) were plated onto 6 - well dish . Representative phase contrast microscopy ( left ) distribution of numbers per cluster composed of indicated number of are shown . Data are means ± SD of 10 fields ( n > 20 / field ) from 3 independent experiments . Bar , 100 μm .

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(c) CTR and RAB2AMCF10A cells (104 cells) were plated onto 6-well dish. Representative phase contrast light microscopy images (left) and the distribution of cell numbers per cluster composed of the indicated number of cells are shown. Data are means ± SD of 10 fields (n > 20 cells/field) from 3 independent experiments. Bar, 100 μm.


( d ) circularity of CTR RAB2A MCF10A of with protrusive structure were quantified . Data are means ± SD of ten independent fields ( n > 100 / field ) . n , number of scored for each population .

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(d) The circularity of CTR and RAB2A MCF10A cells and the percentage of cells with protrusive structure were quantified. Data are means ± SD of ten independent fields (n > 100 cells/field). n, number of cells scored for each cell population.


( e ) Total lysate from ( c ) were immunoblotted with antibodies anti - E - cadherin , which recognize either an epitope in domain ( IC ) , an epitope in extracellular domain of mature E - cadherin ( SHE78 - 7 ) , respectively , or anti - RAB2A , anti - actin . An arrow indicates unprocessed form of E - cadherin .

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(e) Total cell lysate from (c) were immunoblotted with antibodies anti-E-cadherin, which recognize either an epitope in the cytoplasmic domain (IC), an epitope in the extracellular domain of mature E-cadherin (SHE78-7), respectively, or anti-RAB2A, and anti-actin. An arrow indicates the unprocessed form of E-cadherin.


( f - h ) MCF10A were transfected with scrambled siRNA ( siNC ) or siRNA against Furin , immunostained with anti - E - cadherin ( green ) anti - TGN46 ( red ) antibodies . Merged with DAPI ( blue ) are shown on right ( f ) . Bar , 20 μm . Total lysate were immunoblotted with antibodies indicated on right ( g ) . Efficacy of Furin silencing was verified by qPCR ( h ) .

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(f-h) MCF10A cells were transfected with scrambled siRNA (siNC) or siRNA against Furin, and immunostained with anti-E-cadherin (green) and anti-TGN46 (red) antibodies. Merged images with DAPI (blue) are shown on the right (f). Bar, 20 μm. Total cell lysate were immunoblotted with antibodies indicated on the right (g). Efficacy of Furin silencing was verified by qPCR (h).


( i ) Representative electron microscopy of Golgi stacks ( arrows ) of CTR RAB2A MCF10 . DCIS . com . Bars , 1 μm .

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(i) Representative electron microscopy of Golgi stacks (arrows) of CTR and RAB2A MCF10.DCIS.com cells. Bars, 1 μm.


( j ) CTR RAB2A MCF10A were transfected with Str - KDEL _ SBP - EGFP - E - cadherin ( green ) . At indicated time points after addition of biotin , were fixed , stained without permeabilization with anti - GFP antibody , followed by Alexa568 - conjugated secondary antibody ( red ) . surface total of SBP - EGFP - E - cadherin at 60 min after addition of biotin were quantified using ImageJ software on non - saturated , surface / total was expressed as a surface GFP index ( relative to control ) . Bar , 20 µm . * * * , P < 0 . 001 .

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

(j) CTR and RAB2A MCF10A cells were transfected with Str-KDEL_SBP-EGFP-E-cadherin (green). At the indicated time points after the addition of biotin, cells were fixed, and stained without permeabilization with anti-GFP antibody, followed by Alexa568-conjugated secondary antibody (red). The cell surface and total levels of SBP-EGFP-E-cadherin at 60 min after the addition of biotin were quantified using ImageJ software on non-saturated images, and surface/total ratio was expressed as a surface GFP index (relative to control cells). Bar, 20 µm. ***, P < 0.001.


A , B . Coronal CT image of chest at baseline with pleural fluid intralobar septal thickening ( arrow ) ( A ) , 3 months after starting trametinib ( B ) .

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A, B. Coronal CT image of the chest at baseline with pleural fluid and intralobar septal thickening (arrow) (A), and 3 months after starting trametinib (B).


C , D . MR T2 weighted coronal slice of chest at baseline demonstrating bilateral pleural effusions ( arrow heads ) pulmonary interstitial edema ( arrows ) ( C ) , 3 months on trametinib therapy ( D ) .

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C, D. MR T2 weighted coronal slice of the chest at baseline demonstrating bilateral pleural effusions (arrow heads) and pulmonary interstitial edema (arrows) (C), and 3 months on trametinib therapy (D).


IN - DCMRL coronal MIP of chest at baseline demonstrating dilated tortuous TD ( arrow ) with dilated central lymphatic networks extensive bilateral pulmonary perfusion ( arrowheads ) ( E ) , 6 months after trametinib therapy demonstrating dilated tortuous TD ( arrow ) with reduction in extent of dilated central lymphatic networks resolution of bilateral pulmonary perfusion

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O B-TISSUE O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY

IN-DCMRL coronal MIP of the chest at baseline demonstrating dilated and tortuous TD (arrow) with dilated central lymphatic networks and extensive bilateral pulmonary perfusion (arrowheads) (E), and 6 months after trametinib therapy demonstrating dilated and tortuous TD (arrow) with reduction in the extent of the dilated central lymphatic networks and resolution of bilateral pulmonary perfusion


IN - DCMRL coronal MIP of chest at baseline demonstrating dilated tortuous TD ( arrow ) with dilated central lymphatic networks extensive bilateral pulmonary perfusion ( arrowheads ) ( E ) , 6 months after trametinib therapy demonstrating dilated tortuous TD ( arrow ) with reduction in extent of dilated central lymphatic networks resolution of bilateral pulmonary

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O B-TISSUE O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY


IN - DCMRL coronal slice of chest at level of carina demonstrating extensive mediastinal pulmonary interstitial perfusion ( arrows ) at baseline ( G ) , after 6 months of treatment with trametinib ( H ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

IN-DCMRL coronal slice of the chest at the level of the carina demonstrating extensive mediastinal and pulmonary interstitial perfusion (arrows) at baseline (G), and after 6 months of treatment with trametinib (H).


A . Deep coverage exome sequencing Integrative Genomics Viewer ( IGV ) view of CBL c . 2322T > G in sample that was derived from CD31 selected from pleural effusion fluid . alternate allele fraction was 4 % ( 20 / 493 ) .

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

A. Deep coverage exome sequencing Integrative Genomics Viewer (IGV) view of CBL c.2322T>G in DNA sample that was derived from CD31 selected cells from pleural effusion fluid. The alternate allele fraction was 4% (20/493).


B . Droplet digital polymerase chain reaction ( ddPCR ) result with 5 mutant positive droplets ( circle ) from a low left over sample which resulted in ~ 0 . 005 % fractional abundance .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 O O O O O

B. Droplet digital polymerase chain reaction (ddPCR) result with 5 mutant positive droplets (circle) from a low concentration left over sample which resulted in ~0.005% fractional abundance.


( B ) In vivo epifluorescence microscopy following de novo expressed UapA - GFP in a single growing germling , at 70 , 80 , 100 , 120 , 140 , 160 170 min after derepression of transcription via its native uapA promoter ( for details see text Materials methods ) . Notice that in growing germling tip UapA localizes in a membranous mesh some whereas in more tip - distal parts of germlings UapA is in PM - localized ( see arrows ) , which progressively become more abundant so that at more posterior areas PM is homogeneously labeled .

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

(B) In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a single growing germling, at 70, 80, 100, 120, 140, 160 and 170 min after derepression of transcription via its native uapA promoter (for details see text and Materials and methods). Notice that in the growing germling tip UapA localizes in a membranous mesh and some cytosolic puncta whereas in more tip-distal parts of the germlings UapA is in PM-localized puncta (see white arrows), which progressively become more abundant so that at more posterior areas the PM is homogeneously labeled.


( C ) In vivo epifluorescence microscopy following de novo expressed UapA - GFP in a germling maturing to young hypha at 180 , 240 , 300 , 360 400 min after derepression of transcription via its native uapA promoter . During this developmental transition , UapA apical localization is gradually diminished . As tip acquires a faster growth rate , UapA is completely retracted from hyphal apex ( see zoon - in panels on right arrows ) .

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

(C) In vivo epifluorescence microscopy following de novo expressed UapA-GFP in a germling maturing to young hypha at 180, 240, 300, 360 and 400 min after derepression of transcription via its native uapA promoter. During this developmental transition, UapA apical localization is gradually diminished. As the cell tip acquires a faster growth rate, UapA is completely retracted from the hyphal apex (see zoon-in panels on the right and white arrows).


( D ) In a mature hyphal , UapA is no longer found in PM of extreme apical compartment , but in a a network resembling ER . As from apex increases , UapA progressively populates PM as cortical ( see arrows ) .

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

(D) In a mature hyphal cell, UapA is no longer found in the PM of the extreme apical compartment, but in a cytoplasmic a membrane network resembling the ER. As the distance from the apex increases, UapA progressively populates the PM as cortical puncta (see white arrows).


( F ) In vivo epifluorescence microscopy following neosynthesized alcAp - UapA - GFP in a single hypha at 120 , 130 , 140 , 150 , 160 170 min , under overexpressing ( derepression / ethanol - induction ) conditions , as described in Materials methods . Notice that UapA labels perinuclear ER rings ( arrows ) .

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

(F) In vivo epifluorescence microscopy following neosynthesized alcAp-UapA-GFP in a single hypha at 120, 130, 140, 150, 160 and 170 min, under overexpressing (derepression/ethanol-induction) conditions, as described in Materials and methods. Notice that UapA labels the perinuclear ER rings (white arrows).