( H ) WT and FAK − / − PEMs were pretreated with rapamycin ( 4 µm ) , Akt inhibitor AKTV / triciribine ( 10 µm ) or left untreated ( - Tx ) or before infection with S . typhimurium strain ΔinvG for a further 5 hours . WT macrophages were also pretreated with the FAK inhibitor PF228 ( 0 . 5 µm ) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours . Cells were then assessed for the percentage of ΔinvG Salmonella co - localizing with LC3 . At least 100 bacteria were counted per condition . Values are means ± SEM , N = 3 , * p < 0 . 05 . N . s . not significant .

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 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) WT and FAK−/− PEMs were pretreated with rapamycin (4 µm), Akt inhibitor AKTV/triciribine (10 µm) or left untreated (-Tx) or before infection with S. typhimurium strain ΔinvG for a further 5 hours. WT macrophages were also pretreated with the FAK inhibitor PF228 (0.5 µm) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours. Cells were then assessed for the percentage of ΔinvG Salmonella co-localizing with LC3. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05. N.s. not significant.


A ) WT and FAK − / − PEMs were incubated with LPS - coated beads ( 50 ∶ 1 ; beads ∶ cell ) for 1 hour before analysis with antibodies recognizing LC3 . DAPI was used to visualize nuclei . Bars represent 10 µm . The percentage of LPS - beads co - localizing with LC3 is displayed to the right . At least 100 beads were counted per condition . Values are means ± SEM , N = 3 .

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

A) WT and FAK−/− PEMs were incubated with LPS-coated beads (50∶1; beads∶cell) for 1 hour before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei. Bars represent 10 µm. The percentage of LPS-beads co-localizing with LC3 is displayed to the right. At least 100 beads were counted per condition. Values are means ± SEM, N = 3.


( B ) PEMs were treated with Atg5 , ULK1 or control siRNAs for 48 hours before immunoblotting with the 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

(B) PEMs were treated with Atg5, ULK1 or control siRNAs for 48 hours before immunoblotting with the indicated antibodies.


. ( C - D ) FAK − / − PEMs treated with Atg5 , ULK1 or control siRNA for 48 hours were incubated with LPS - coated beads or ΔinvG Salmonella for 1 or 5 hours before analysis with antibodies recognizing LC3 . The percentage of LPS - beads or Salmonella co - localizing with LC3 is displayed to the right . At least 100 beads or bacteria were counted per condition . Values are means ± SEM , N = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

. (C-D) FAK−/− PEMs treated with Atg5, ULK1 or control siRNA for 48 hours were incubated with LPS-coated beads or ΔinvG Salmonella for 1 or 5 hours before analysis with antibodies recognizing LC3. The percentage of LPS-beads or Salmonella co-localizing with LC3 is displayed to the right. At least 100 beads or bacteria were counted per condition. Values are means ± SEM, N = 3.


E ) WT and FAK − / − PEMs were infected with ΔinvG for 5 hours before preparation and examination by transmission electron microscopy ( TEM ) . Arrowheads indicate double membrane .

O O O O O O O O O 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) WT and FAK−/− PEMs were infected with ΔinvG for 5 hours before preparation and examination by transmission electron microscopy (TEM). Arrowheads indicate double membrane.


( A ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 1 . 5 , 3 and 5 hours . Bacterial survival was then assayed using a standard gentamycin resistance assay described in the Materials and Methods . Values are means ± SEM , N = 6 , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 1.5, 3 and 5 hours. Bacterial survival was then assayed using a standard gentamycin resistance assay described in the Materials and Methods. Values are means ± SEM, N = 6, *p<0.05.


( B ) WT and FAK − / − PEMs were pretreated with IFN - γ ( 50 ng / ml ) , rapamycin ( 4 µm ) , or left untreated before incubation with ΔinvG Salmonella for 3 hours . Bacterial survival was then assayed as described in ( A ) . Values are means ± SEM , N = 6 , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and FAK−/− PEMs were pretreated with IFN-γ (50 ng/ml), rapamycin (4 µm), or left untreated before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 6, *p<0.05.


( C ) PEMs were treated with control siRNA or depleted of Atg5 before incubation with ΔinvG Salmonella for 3 hours . Bacterial survival was then assayed as described in ( A ) . Values are means ± SEM , N = 3 , * p < 0 . 05 vs WT siControl .

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(C) PEMs were treated with control siRNA or depleted of Atg5 before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 3, *p<0.05 vs WT siControl.


. ( D ) Bacterial loads in WT ( black circles ) and FAKΔmyeloid mice ( white circles ) in the indicated tissues 5 days after oral infection with S . typhimurium strain SL1344 . Each point indicates data from an individual mouse . * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial loads in WT (black circles) and FAKΔmyeloid mice (white circles) in the indicated tissues 5 days after oral infection with S. typhimurium strain SL1344. Each point indicates data from an individual mouse. *p<0.05.


( E ) Hematoxylin and eosin ( H & amp ; amp ; E ) staining of spleen isolated from WT and FAKΔmyeloid mice 5 days post - infection with S . typhimurium strain SL1344 . Arrows indicate areas of leukocyte infiltration . Lesion indicated by yellow arrow is outlined in the higher magnification panel to the right . Bars represent 200 µm and 50 µm .

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(E) Hematoxylin and eosin (H&amp;E) staining of spleen isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Arrows indicate areas of leukocyte infiltration. Lesion indicated by yellow arrow is outlined in the higher magnification panel to the right. Bars represent 200 µm and 50 µm.


. ( F ) Gr - 1 immunostaining of spleens isolated from WT and FAKΔmyeloid mice 5 days post - infection with S . typhimurium strain SL1344 . Panels b and d are enlarged sections indicated by black boxes in panels a and c . Gr - 1 - positive cells appear red - brown .

. (F) Gr-1 immunostaining of spleens isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Panels b and d are enlarged sections indicated by black boxes in panels a and c. Gr-1-positive cells appear red-brown.


( G ) Quantitation of flow cytometric data collected from the spleens of infected mice . * p < 0 . 05 .

(G) Quantitation of flow cytometric data collected from the spleens of infected mice. *p<0.05.


A RNF123 suppresses the SeV - and EMCV - induced but not the cGAS and STING - mediated activation of the IFN - βpromoter . 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 cGAS ( 50 ng ) and STING ( 50 ng ) or empty vector ( 100 ng ) , and 0 , 20 , 50 , or 100 ng ( wedges ) of RNF123 for 12 h , and then were infected with SeV or EMCV or left uninfected . Luciferase assays were performed after 18 h and results are presented relative to the luciferase activity in control cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNF123 suppresses the SeV- and EMCV-induced but not the cGAS and STING-mediated activation of the IFN-βpromoter. 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 cGAS (50 ng) and STING (50 ng) or empty vector (100 ng), and 0, 20, 50, or 100 ng (wedges) of RNF123 for 12 h, and then were infected with SeV or EMCV or left uninfected. Luciferase assays were performed after 18 h and results are presented relative to the luciferase activity in control cells.


B RNF123 inhibits the SeV - , but not the cGAS and STING - induced activation of the NF - κB promoter . HEK293T cells ( 3 × 105 ) were transfected with the NF - κB reporter plasmid ( 100 ng ) , the pRL - TK Renilla luciferase plasmid ( 100 ng ) , the expression plasmids for cGAS ( 50 ng ) and STING ( 50 ng ) , or empty vector ( 100 ng ) , and 0 , 20 , 50 or 100 ng ( wedges ) of RNF123 for 12 h , and were then infected for 24 h with SeV or left uninfected , followed by analysis as described in A .

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B RNF123 inhibits the SeV-, but not the cGAS and STING-induced activation of the NF-κB promoter. HEK293T cells (3×105) were transfected with the NF-κB reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmids for cGAS (50 ng) and STING (50 ng), or empty vector (100 ng), and 0, 20, 50 or 100 ng (wedges) of RNF123 for 12 h, and were then infected for 24 h with SeV or left uninfected, followed by analysis as described in A.


C , D RNF123 inhibits the SeV - induced , but not the cGAS and STING - mediated RANTES and IFN - β transcription . The transfection and treatment were as in A . Total RNA was isolated and RT - PCR ( C ) was performed using the indicated primers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated RANTES and IFN-β transcription. The transfection and treatment were as in A. Total RNA was isolated and RT-PCR (C) was performed using the indicated primers.


C , D RNF123 inhibits the SeV - induced , but not the cGAS and STING - mediated RANTES and IFN - β transcription . The transfection and treatment were as in A . Total RNA was isolated and Q - PCR ( D ) was performed using the indicated primers .

C, D RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated RANTES and IFN-β transcription. The transfection and treatment were as in A. Total RNA was isolated and Q-PCR (D) was performed using the indicated primers.


E RNF123 inhibits the SeV - induced , but not the cGAS and STING - mediated IFN - β secretion . The transfection and treatment were as in A . Bioassays for type I interferon in the supernatants of HEK293T cells were performed and the results are presented relative to untreated control cells .

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

E RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated IFN-β secretion. The transfection and treatment were as in A. Bioassays for type I interferon in the supernatants of HEK293T cells were performed and the results are presented relative to untreated control cells.


F Overexpression of RNF123 enhances NDV - eGFP replication . HEK293T cells ( 1 × 105 ) were transfected with the expression plasmids for Flag - RNF123 or empty vector ( 2 µg ) . At 24 h after transfection , cells 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Overexpression of RNF123 enhances NDV-eGFP replication. HEK293T cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells 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.


G RNF123 enhances EMCV replication . HeLa cells ( 1 × 105 ) were transfected with the expression plasmids for Flag - RNF123 or empty vector ( 2 µg ) . At 24 h after transfection , cells were infected with EMCV or given DMEM with no virus . At 18 h after infection , viral replication was determined by VP1 expression , which was analyzed by western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNF123 enhances EMCV replication. HeLa cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells were infected with EMCV or given DMEM with no virus. At 18 h after infection, viral replication was determined by VP1 expression, which was analyzed by western blot.


A HEK293T cells with stable knockdown of RNF123 were generated using shRNA . Western blot was performed with anti - RNF123 , anti - VISA ( as a control ) , and anti - GAPDH antibodies after infection with five lentiviruses encoding a shRNA control ( GFPsh ) and four shRNAs targeting RNF123 ( RNF123 sh # 1 , # 2 , # 3 , and # 4 ) in HEK293T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293T cells with stable knockdown of RNF123 were generated using shRNA. Western blot was performed with anti-RNF123, anti-VISA (as a control), and anti-GAPDH antibodies after infection with five lentiviruses encoding a shRNA control (GFPsh) and four shRNAs targeting RNF123 (RNF123 sh #1, #2, #3, and #4) in HEK293T cells.


B , C Knockdown of RNF123 facilitated the activation of IFN - β and NF - κB promoters induced by SeV and EMCV but not by cGAS and STING . Stable HEK293T cells with GFPsh or RNF123 shRNAs ( 2 × 105 ) were transfected with the indicated reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , cGAS ( 50 ng ) , and STING ( 50 ng ) , or empty vector ( 100 ng ) . At 12 h after transfection , they were infected for 12 h with SeV or EMCV or left uninfected , followed by analysis as described in the legend of Fig 1A .

B, C Knockdown of RNF123 facilitated the activation of IFN-β and NF-κB promoters induced by SeV and EMCV but not by cGAS and STING. Stable HEK293T cells with GFPsh or RNF123 shRNAs (2×105) were transfected with the indicated reporter plasmid (100 ng), pRL-TK Renilla luciferase plasmid (100 ng), cGAS (50 ng), and STING (50 ng), or empty vector (100 ng). At 12 h after transfection, they were infected for 12 h with SeV or EMCV or left uninfected, followed by analysis as described in the legend of Fig 1A.


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 .

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

D, E 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 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 .

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 cells 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 - 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- 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 - 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 .

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 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 .

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

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 cells ( 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 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 .

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

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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 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 .

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

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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 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 .

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

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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 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 .

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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 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 .

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 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 .

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 the indicated primers after HEK293T cells were infected with six lentiviruses encoding an shRNA control ( GFPsh ) and five shRNAs targeting kpc2 ( Kpc2 # 1 - # 5 ) .

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

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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 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 .

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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 . The methods of transfection and 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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O 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 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).


( C , D ) qRT - PCR analysis of Il2 and Ifng mRNA in CD4 + T cells from LN and OB mice ( C ) or from aged lean ( AL , 17 - month - old ) and aged obese ( AO , 17 - month - old ) mice ( D ) , then stimulated with TCR ( 1 / 1 μg / ml ) for 6 h . For AL and AO mice , mice were fed with a normal diet for 17 months , then divided into AL ( ≤ 35 g ) and AO ( ≥ 45 g ) mice based on body weight ( n = 3 or 4 , biological replicates ) . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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(C, D) qRT-PCR analysis of Il2 and Ifng mRNA in CD4+ T cells from LN and OB mice (C) or from aged lean (AL, 17-month-old) and aged obese (AO, 17-month-old) mice (D), then stimulated with TCR (1/1 μg/ml) for 6 h. For AL and AO mice, mice were fed with a normal diet for 17 months, then divided into AL (≤ 35 g) and AO (≥ 45 g) mice based on body weight (n = 3 or 4, biological replicates). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


( J - L ) BM12 / SJL mice ( recipient , R ) were immunized with CD4 + T cells from LN and OB mice ( donor , D ) , and then intraperitoneally injected once every day with 2 - DG ( 200 mg / kg ) or PBS for 3 weeks . Enzyme - linked immunosorbent assay ( ELISA ) of anti - dsDNA , anti - ssDNA , and anti - histone IgG or IgM in serum from the immunized mice at the indicated time ( J ) . Immunofluorescent analysis of anti - nuclear antibody ( ANA ) in serum ( top ) and IgG deposition in the kidney ( bottom ) ( n = 4 or 5 , biological replicates ) . Scale bar : 50 μm . Data are presented as immunofluorescent images ( K ) and quantification of bar graphs ( L ) . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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(J-L) BM12/SJL mice (recipient, R) were immunized with CD4+ T cells from LN and OB mice (donor, D), and then intraperitoneally injected once every day with 2-DG (200 mg/kg) or PBS for 3 weeks. Enzyme-linked immunosorbent assay (ELISA) of anti-dsDNA, anti-ssDNA, and anti-histone IgG or IgM in serum from the immunized mice at the indicated time (J). Immunofluorescent analysis of anti-nuclear antibody (ANA) in serum (top) and IgG deposition in the kidney (bottom) (n = 4 or 5, biological replicates). Scale bar: 50 μm. Data are presented as immunofluorescent images (K) and quantification of bar graphs (L). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


( F ) qRT - PCR analysis of the transcriptional levels of indicated genes in fatty acid metabolism in CD4 + T cells ex vivo from LN and OB mice ( n = 3 or 4 , biological replicates ) . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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(F) qRT-PCR analysis of the transcriptional levels of indicated genes in fatty acid metabolism in CD4+ T cells ex vivo from LN and OB mice (n = 3 or 4, biological replicates). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


( L , M ) ECAR measurements of glycolysis of naïve CD4 + T cell from LN and OB mice treated with vehicle or etomoxir ( Eto , 150 μM ) that stimulated with α - CD3 / CD28 ( TCR , 1 / 1 μg / ml ) for 24 h ( n = 3 , biological replicates ) . The statistical results are presented as a bar graph ( M ) . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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(L, M) ECAR measurements of glycolysis of naïve CD4+ T cell from LN and OB mice treated with vehicle or etomoxir (Eto, 150 μM) that stimulated with α-CD3/CD28 (TCR, 1/1 μg/ml) for 24 h (n = 3, biological replicates). The statistical results are presented as a bar graph (M). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


( S ) BM12 / SJL mice ( recipient , R ) were adoptively transferred with CD4 + T cells from WT and Cpt1a - KO lean or obese mice ( donors , D ) to induce inflammation . The anti - nuclear antibody ( ANA ) in serum ( top , scale bar : 50 μm ) and the kidney IgG deposition ( bottom , scale bar : 50 μm ) were visualized using immunofluorescence ( n = 4 or 5 , biological replicates ) . Data are presented as Immunofluorescent images ( left ) and quantification bar graphs ( right ) . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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(S) BM12/SJL mice (recipient, R) were adoptively transferred with CD4+ T cells from WT and Cpt1a-KO lean or obese mice (donors, D) to induce inflammation. The anti-nuclear antibody (ANA) in serum (top, scale bar: 50 μm) and the kidney IgG deposition (bottom, scale bar: 50 μm) were visualized using immunofluorescence (n = 4 or 5, biological replicates). Data are presented as Immunofluorescent images (left) and quantification bar graphs (right). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


Immunoblot analysis of cytoplasmic and nuclear NF - AT levels ( C ) , that activated by PMA / Ionomycin ( P / I , 10 / 100 ng / ml ) for the indicated time points .

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Immunoblot analysis of cytoplasmic and nuclear NF-AT levels (C), that activated by PMA/Ionomycin (P/I, 10/100 ng/ml) for the indicated time points.


( R ) Endogenous ubiquitination of Usp9x in WT and Goliath - deficient CD4 + T cells that were left unstimulated ( - ) or stimulated ( + ) with α - CD3 / 28 ( 5 / 5 μg / ml ) .

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(R) Endogenous ubiquitination of Usp9x in WT and Goliath-deficient CD4+ T cells that were left unstimulated (-) or stimulated (+) with α-CD3/28 (5/5 μg/ml).


( S ) Co - IP analysis of the interactions between CaN and Usp9x or NFATc2 and CaN in WT and Goliath - KO CD4 + T cells that were left unstimulated ( - ) or stimulated ( + ) with α - CD3 / 28 ( 5 / 5 μg / ml ) .

(S) Co-IP analysis of the interactions between CaN and Usp9x or NFATc2 and CaN in WT and Goliath-KO CD4+ T cells that were left unstimulated (-) or stimulated (+) with α-CD3/28 (5/5 μg/ml).


( A , B ) Immunoblot analysis of cytoplasmic and nuclear NF - AT levels in WT and Goliath - KO naïve CD4 + T cells from lean ( LN ) or obese ( OB ) mice that were stimulated by α - CD3 / 28 ( 5 / 5 μg / ml ) or PMA / ionomycin ( P / I , 10 / 100 ng / ml ) .

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(A, B) Immunoblot analysis of cytoplasmic and nuclear NF-AT levels in WT and Goliath-KO naïve CD4+ T cells from lean (LN) or obese (OB) mice that were stimulated by α-CD3/28 (5/5 μg/ml) or PMA/ionomycin (P/I, 10/100 ng/ml).


( C - F ) ECAR measurements of glycolysis in WT and Goliath - deficient naïve CD4 + T cells treated with DMSO or fenofibrate ( FF , 5 μM ) ( C , D ) , or isolated from LN or OB mice ( E , F ) that stimulated by α - CD3 / 28 ( 1 / 1 μg / ml ) for 24 h ( n = 3 , biological replicates ) . The statistical results are presented as bar graphs ( D , F ) . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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(C-F) ECAR measurements of glycolysis in WT and Goliath-deficient naïve CD4+ T cells treated with DMSO or fenofibrate (FF, 5 μM) (C, D), or isolated from LN or OB mice (E, F) that stimulated by α-CD3/28 (1/1 μg/ml) for 24 h (n = 3, biological replicates). The statistical results are presented as bar graphs (D, F). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


( J - L ) BM12 / SJL mice ( recipient , R ) were adoptively transferred with CD4 + T cells from WT and Goliath - KO lean or obese mice ( donors , D ) to induce inflammation . The anti - dsDNA , anti - ssDNA and anti - histone IgG or IgM in serum were examined by ELISA ( J ) . ANA in serum ( top , scale bar : 50 μm ) and the kidney IgG deposition ( bottom , scale bar : 50 μm ) were visualized using immunofluorescence ( n = 3 , biological replicates ) . Data are presented as Immunofluorescent images ( K ) and quantification bar graphs ( L ) . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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(J-L) BM12/SJL mice (recipient, R) were adoptively transferred with CD4+ T cells from WT and Goliath-KO lean or obese mice (donors, D) to induce inflammation. The anti-dsDNA, anti-ssDNA and anti-histone IgG or IgM in serum were examined by ELISA (J). ANA in serum (top, scale bar: 50 μm) and the kidney IgG deposition (bottom, scale bar: 50 μm) were visualized using immunofluorescence (n = 3, biological replicates). Data are presented as Immunofluorescent images (K) and quantification bar graphs (L). Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


Immunofluorescence analysis of Goliath protein expression in CD4 + T cells from lean and obese mice as the indicated treatments . Data are presented as immunofluorescent images ( A , and quantification bar graphs ( B , Etomoxir ( Eto ) 150 μM for 6 h ; Scale bar : 2 μM . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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Immunofluorescence analysis of Goliath protein expression in CD4+ T cells from lean and obese mice as the indicated treatments. Data are presented as immunofluorescent images (A, and quantification bar graphs (B, Etomoxir (Eto) 150 μM for 6 h; Scale bar: 2 μM. Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, **p < 0.01, ***p < 0.001. Error bars represent SD.


Immunofluorescence analysis of Goliath protein expression in CD4 + T cells from mice as the indicated treatments . Data are presented as immunofluorescent images C ) Etomoxir ( Eto ) , 150 μM for 6 h ; MG132 , 10 μM for the last 4 h . Scale bar : 2 μM .

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Immunofluorescence analysis of Goliath protein expression in CD4+ T cells from mice as the indicated treatments. Data are presented as immunofluorescent images C) Etomoxir (Eto), 150 μM for 6 h; MG132, 10 μM for the last 4 h. Scale bar: 2 μM.


Immunofluorescence analysis of Goliath protein expression in CD4 + T cells from mice as the indicated treatments . quantification bar graphs D ) . Etomoxir ( Eto ) , 150 μM for 6 h ; MG132 , 10 μM for the last 4 h . Data information : All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

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

Immunofluorescence analysis of Goliath protein expression in CD4+ T cells from mice as the indicated treatments. quantification bar graphs D). Etomoxir (Eto), 150 μM for 6 h; MG132, 10 μM for the last 4 h. Data information: All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, **p < 0.01, ***p < 0.001. Error bars represent SD.


( L ) Endogenous K48 - linked ubiquitination of HA - Goliath in GOLIATH - overexpressed Jurkat T cell with or without Cpt1a knockdown .

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

(L) Endogenous K48-linked ubiquitination of HA-Goliath in GOLIATH-overexpressed Jurkat T cell with or without Cpt1a knockdown.


( M ) Endogenous ubiquitination of HA - Goliath with indicated treatment in GOLIATH - knockdown Jurkat T cell reconstituted with HA - tagged GOLIATH ( WT ) or Ring - truncated GOLIATH ( ΔR ) .

O O O O O O O O O O O O O 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) Endogenous ubiquitination of HA-Goliath with indicated treatment in GOLIATH-knockdown Jurkat T cell reconstituted with HA-tagged GOLIATH (WT) or Ring-truncated GOLIATH (ΔR).


( E , F ) Endogenous ubiquitination of Usp9x ( E ) and CaN ( F ) in primary CD4 + T cells treated with DMSO or Gonib32 , then stimulated with ( + ) or without ( - ) PMA / ionomycin ( P / I , 10 / 100 ng / ml ) .

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

(E, F) Endogenous ubiquitination of Usp9x (E) and CaN (F) in primary CD4+ T cells treated with DMSO or Gonib32, then stimulated with (+) or without (-) PMA/ionomycin (P/I, 10/100 ng/ml).


( G , H ) Immunoblot analysis of the interaction of Usp9x and CaN , NF - AT and CaN ( G ) , and NFATc1 / 2 in nuclear extract ( NE ) and cytoplasmic extract ( CE ) ( H ) in primary CD4 + T cells treated with Gonib32 , then stimulated with ( + ) or without ( - ) P / I ( 10 / 100 ng / ml ) .

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

(G, H) Immunoblot analysis of the interaction of Usp9x and CaN, NF-AT and CaN (G), and NFATc1/2 in nuclear extract (NE) and cytoplasmic extract (CE) (H) in primary CD4+ T cells treated with Gonib32, then stimulated with (+) or without (-) P/I (10/100 ng/ml).


ECAR measurements of glycolysis and proliferation analysis in lean and obese naïve CD4 + T cells treated with DMSO or Gonib32 ( 2 μM ) upon α - CD3 / CD28 ( 1 / 1 µg / ml ) stimulation for 24 h ( ECAR ) ( I , J ) ( n = 3 , biological replicates ) . The statistical results are presented as a bar graph (

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

ECAR measurements of glycolysis and proliferation analysis in lean and obese naïve CD4+ T cells treated with DMSO or Gonib32 (2 μM) upon α-CD3/CD28 (1/1 µg/ml) stimulation for 24 h (ECAR) (I, J) (n = 3, biological replicates). The statistical results are presented as a bar graph (J


( M - O ) BM12 / SJL mice ( recipient , R ) were immunized with CD4 + T cells from lean and obese mice ( donors , D ) , then were injected with Gonib32 ( 50 mg / kg ) or vehicle control ( 5 % DMSO + 95 % saline ) once a day for 3 weeks . Anti - dsDNA , anti - ssDNA , and anti - histone IgG or IgM in serum were measured using ELISA ( M ) . Immunofluorescent analysis of anti - nuclear antibody in serum ( top ) and IgG deposition in the kidney ( bottom ) ( n = 4 or 5 , biological replicates ) . Data are presented as immunofluorescent images ( N ) and quantification bar graphs ( O ) . All data are representative of at least 3 individual experiments . Statistics , two - tailed Student ' s t test ; ns : not significant , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . Error bars represent SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-O) BM12/SJL mice (recipient, R) were immunized with CD4+ T cells from lean and obese mice (donors, D), then were injected with Gonib32 (50 mg/kg) or vehicle control (5% DMSO + 95% saline) once a day for 3 weeks. Anti-dsDNA, anti-ssDNA, and anti-histone IgG or IgM in serum were measured using ELISA (M). Immunofluorescent analysis of anti-nuclear antibody in serum (top) and IgG deposition in the kidney (bottom) (n = 4 or 5, biological replicates). Data are presented as immunofluorescent images (N) and quantification bar graphs (O). All data are representative of at least 3 individual experiments. Statistics, two-tailed Student's t test; ns: not significant, *p < 0.5, **p < 0.01, ***p < 0.001. Error bars represent SD.


A . Optical coherence tomography ( OCT ) horizontal B - scan and thickness map of neovascular age related macular degeneration ( NV AMD ) patient with active lesions ( 1 ) , cystoid macular edema ( 2 ) , subretinal fibrin deposits ( 3 ) and serous retinal detachment ( 4 ) . B . Optical coherence tomography ( OCT ) horizontal B - scan and thickness map of control patient with a medium sized , stage 3 , full thickness macular hole ( 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Optical coherence tomography (OCT) horizontal B-scan and thickness map of neovascular age related macular degeneration (NV AMD) patient with active lesions (1), cystoid macular edema (2), subretinal fibrin deposits (3) and serous retinal detachment (4). B. Optical coherence tomography (OCT) horizontal B-scan and thickness map of control patient with a medium sized, stage 3, full thickness macular hole (5).


C - G . Vitreous humor analyzed by ELISA for VEGF - A ( C ) ; n = 7 ( Ctrl ) , 9 ( NV AMD ) , SEMA3A ( D ) ; n = 10 ( Ctrl ) , 10 ( NV AMD ) , TGFβ ( E ) ; n = 8 ( Ctrl ) , 7 ( NV AMD ) , PDGF - BB ( F ) ; n = 5 ( Ctrl ) , 6 ( NV AMD ) , PGF ( G ) : n = 5 ( Ctrl ) , 5 ( NV AMD ) . Dots represent concentrations of individual patient samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. Vitreous humor analyzed by ELISA for VEGF-A (C); n = 7 (Ctrl), 9 (NV AMD), SEMA3A (D); n = 10 (Ctrl), 10 (NV AMD), TGFβ (E); n = 8 (Ctrl), 7 (NV AMD), PDGF-BB (F); n = 5 (Ctrl),6 (NV AMD), PGF (G): n = 5 (Ctrl), 5 (NV AMD). Dots represent concentrations of individual patient samples.


H . Representative confocal images of IBA - 1 - stained mononuclear phagocytes on choroidal flat mounts from LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D7 and D14 . Examples of macrophage quantification ( yellow stars ) are presented in side panels . Scale bar : 20μm . I , J . Total number of IBA - 1 - positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 ( I ) and D14 ( J ) ; n = 19 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 25 burns ( D7 LysM - Cre / Nrp1fl / fl ) n = 37 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 23 burns ( D7 LysM - Cre / Nrp1fl / fl ) , 3 - 5 mice with ~ 4 burns per eye .

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

H. Representative confocal images of IBA-1-stained mononuclear phagocytes on choroidal flat mounts from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D7 and D14. Examples of macrophage quantification (yellow stars) are presented in side panels. Scale bar: 20μm. I, J. Total number of IBA-1-positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 (I) and D14 (J); n = 19 burns (D7 LysM-Cre/Nrp1+/+), n = 25 burns (D7 LysM-Cre/Nrp1fl/fl) n = 37 burns (D7 LysM-Cre/Nrp1+/+), n = 23 burns (D7 LysM-Cre/Nrp1fl/fl), 3-5 mice with ~4 burns per eye.


K . Compilation of representative compressed Z - stack confocal images of FITC - dextran - labeled CNV and IB4 - stained laser impact area from LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D14 . Scale bar : 20μm . L - N . Quantification of area of FITC - dextran - labeled CNV ( L ) , isolectin B4 ( IB4 ) - stained laser impact area ( M ) and the ratio of FITC / IB4 per laser - burn ( N ) relative to LysM - Cre / Nrp1 + / + at D14 ; n = 23 burns ( LysM - Cre / Nrp1 + / + ) , n = 27 burns ( LysM - Cre / Nrp1fl / fl ) .

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

K. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm. L-N. Quantification of area of FITC-dextran-labeled CNV (L), isolectin B4 (IB4 )-stained laser impact area (M) and the ratio of FITC/IB4 per laser-burn (N) relative to LysM-Cre/Nrp1+/+ at D14; n = 23 burns (LysM-Cre/Nrp1+/+), n = 27 burns ( LysM-Cre/Nrp1fl/fl).


A - D . mRNA expression of inflammation markers relative to LysM - Cre / Nrp1 + / + in mouse RPE - choroid - sclera complexes at D3 for Il1b ( A ) ; n = 10 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Il6 ( B ) ; n = 7 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Vegfa ( C ) ; n = 10 ( LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl ) , Tnf ( D ) ; n = 5 ( LysM - Cre / Nrp1 + / + ) , n = 10 ( LysM - Cre / Nrp1fl / fl ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. mRNA expression of inflammation markers relative to LysM-Cre/Nrp1+/+ in mouse RPE-choroid-sclera complexes at D3 for Il1b (A); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Il6 (B); n = 7 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Vegfa (C); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Tnf (D); n = 5 (LysM-Cre/Nrp1+/+), n = 10 (LysM-Cre/Nrp1fl/fl).


K , L . mRNA expression relative to LysM - Cre / Nrp1 + / + of inflammation markers in mouse BMDMs for Il1b ( K ) ; n = 4 ( LysM - Cre / Nrp1 + / + ) , n = 5 ( LysM - Cre / Nrp1fl / fl ) and Il6 ( L ) ; n = 3 ( LysM - Cre / Nrp1 + / + ) , n = 2 ( LysM - Cre / Nrp1fl / fl )

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

K, L. mRNA expression relative to LysM-Cre/Nrp1+/+ of inflammation markers in mouse BMDMs for Il1b (K); n = 4 (LysM-Cre/Nrp1+/+), n = 5 (LysM-Cre/Nrp1fl/fl) and Il6 (L); n = 3 (LysM-Cre/Nrp1+/+), n = 2 (LysM-Cre/Nrp1fl/fl)


P . Heatmap ( left ) and enrichment plot ( right ) of GO Angiogenesis gene set enrichment analysis ( GSEA ) of wildtype and LysM - Cre / Nrp1fl / fl peritoneal macrophages ; n = 2 . NES , normalized enrichment score ; FDR , false discovery rate .

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

P. Heatmap (left) and enrichment plot (right) of GO Angiogenesis gene set enrichment analysis (GSEA) of wildtype and LysM-Cre/Nrp1fl/fl peritoneal macrophages; n = 2. NES, normalized enrichment score; FDR, false discovery rate.


E . Compilation of representative compressed Z - stack confocal images of FITC - dextran - labeled CNV and isolectin B4 ( IB4 ) - stained laser impact area from Vehicle and NRP1 - derived trap treated wildtype mice . Scale bar : 20μm . F - H . Quantification of area of FITC - dextran - labeled CNV ( F ) , IB4 - stained laser impact area ( G ) and the ratio of FITC / IB4 per laser - burn ( H ) relative to Vehicle at D14 ; n = 24 burns ( Vehicle ) , n = 26 burns ( NRP1 - derived trap ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and isolectin B4 (IB4)-stained laser impact area from Vehicle and NRP1-derived trap treated wildtype mice. Scale bar: 20μm. F-H. Quantification of area of FITC -dextran-labeled CNV (F), IB4-stained laser impact area (G) and the ratio of FITC/IB4 per laser-burn (H) relative to Vehicle at D14; n = 24 burns (Vehicle), n = 26 burns (NRP1-derived trap).


J - L . Quantification of area of FITC - dextran - labeled CNV ( J ) , isolectin B4 ( IB4 ) - stained laser impact area ( K ) and the ratio of FITC / IB4 per laser - burn ( L ) relative to LysM - Cre / Nrp1 + / + + Vehicle in Vehicle and NRP1 - derived trap treated LysM - Cre / Nrp1 + / + and LysM - Cre / Nrp1fl / fl mice at D14 ; n = 16 burns ( LysM - Cre / Nrp1 + / + + Vehicle ) , n = 16 burns ( LysM - Cre / Nrp1 + / + + Trap ) , n = 13 burns ( LysM - null / Nrp1fl / fl + Vehicle ) , n = 19 burns ( LysM - Cre / Nrp1fl / fl + Trap ) .

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

J-L. Quantification of area of FITC-dextran-labeled CNV (J), isolectin B4 (IB4)-stained laser impact area (K) and the ratio of FITC/IB4 per laser-burn (L) relative to LysM-Cre/Nrp1+/+ + Vehicle in Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14; n = 16 burns (LysM-Cre/Nrp1+/+ + Vehicle), n = 16 burns (LysM-Cre/Nrp1+/+ + Trap), n = 13 burns (LysM-null/Nrp1fl/fl + Vehicle) , n = 19 burns (LysM-Cre/Nrp1fl/fl + Trap).


A Up , Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD - / - fibroblasts ( Left ) or in wt and Nfe2l2 - / - fibroblasts ( Right ) . Two representative clones , with their corresponding controls , are shown per genotype . H2B was used as negative control . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . n ≥ 3 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 O O O O O O O

A Up, Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD-/- fibroblasts (Left) or in wt and Nfe2l2-/- fibroblasts (Right). Two representative clones, with their corresponding controls, are shown per genotype. H2B was used as negative control. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). n ≥ 3 independent experiments.


B Up , Representative western blot from kidney whole cell extracts showing H2AX protein levels in age - matched wt and junD - / - mice . Mice are 2 months old ( young ) and 18 months old ( old ) mice , respectively . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . N ≥ 5 mice per age and per genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Up, Representative western blot from kidney whole cell extracts showing H2AX protein levels in age-matched wt and junD-/- mice. Mice are 2 months old (young) and 18 months old (old) mice, respectively. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 5 mice per age and per genotype.


C Up , Representative western blot from kidney whole cell extracts , showing H2AX protein levels in 24 months old wt and junD - / - mice , either untreated ( - ) or treated with the anti - oxidant agent , N - acetyl - cysteine ( NAC ) . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) . N ≥ 4 mice per treatment and per genotype .

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

C Up, Representative western blot from kidney whole cell extracts, showing H2AX protein levels in 24 months old wt and junD-/- mice, either untreated (-) or treated with the anti-oxidant agent, N-acetyl-cysteine (NAC). Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 4 mice per treatment and per genotype.


D , E Left , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and Kap1 phosphorylation ( P - Kap1 ) in wt and junD - / - fibroblasts ( D ) or in wt and Nfe2l2 - / - fibroblasts ( E ) , after H2O2 exposure for the indicated times ( hours , h ) . Right , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown on the left ) . n = 3 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 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 O

D,E Left, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and Kap1 phosphorylation (P-Kap1) in wt and junD-/- fibroblasts (D) or in wt and Nfe2l2-/- fibroblasts (E), after H2O2 exposure for the indicated times (hours, h). Right, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown on the left). n = 3 independent experiments.


F , H Up , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and P - Kap1 in wt and junD - / - fibroblasts , following 2 Gy of γ - irradiation ( F ) for the indicated times ( hours , h ) . Down , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown upper ) . n = 3 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 O O O O O O O O O O O O O O O

F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, following 2 Gy of γ-irradiation (F) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.


G , I Up , Representative γ - H2AX immunofluorescence ( green ) in wt and junD - / - fibroblasts before ( - ) or after γ - irradiation ( 2Gy , 45 min ) ( G ) . Blue signal corresponds to DAPI staining . Down , box - plots of large γ - H2AX foci per nuclei ( diameter > 0 . 8 μm ) . At least 50 nuclei per genotype have been used for quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,I Up, Representative γ-H2AX immunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after γ-irradiation (2Gy, 45 min) (G). Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.


F , H Up , Representative western blots from whole cell extract showing γ - H2AX , H2AX protein levels and P - Kap1 in wt and junD - / - fibroblasts , after camptothecin ( CPT ) treatment ( H ) for the indicated times ( hours , h ) . Down , Bar plots showing γ - H2AX and H2AX protein levels , as well as γ - H2AX / H2AX ratio as assessed by densitometry analysis of western blots ( as shown upper ) . n = 3 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 B-CONTROLLED_VAR 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 O O O

F,H Up, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and P-Kap1 in wt and junD-/- fibroblasts, after camptothecin (CPT) treatment (H) for the indicated times (hours, h). Down, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown upper). n = 3 independent experiments.


G , I Up , Representative γ - H2AXimmunofluorescence ( green ) in wt and junD - / - fibroblasts before ( - ) or after after camptothecin ( CPT ) treatment for 1h ( I ) . . Blue signal corresponds to DAPI staining . Down , box - plots of large γ - H2AX foci per nuclei ( diameter > 0 . 8 μm ) . At least 50 nuclei per genotype have been used for quantification .

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

G,I Up, Representative γ-H2AXimmunofluorescence (green) in wt and junD-/- fibroblasts before (-) or after after camptothecin (CPT) treatment for 1h (I).. Blue signal corresponds to DAPI staining. Down, box-plots of large γ-H2AX foci per nuclei (diameter > 0.8 μm). At least 50 nuclei per genotype have been used for quantification.


A Bar plot showing the doubling time of wt and junD - / - fibroblasts .

A Bar plot showing the doubling time of wt and junD-/- fibroblasts.


B Representative cell cycle distribution of asynchronous wt and junD - / - fibroblasts .

B Representative cell cycle distribution of asynchronous wt and junD-/- fibroblasts.


C Bar plots showing the percentage of wt and junD - / - fibroblasts in G1 , S and G2 phases of the cell cycle , following serum stimulation for the indicated times ( hours , h ) , after 48h serum starvation .

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C Bar plots showing the percentage of wt and junD-/- fibroblasts in G1, S and G2 phases of the cell cycle, following serum stimulation for the indicated times (hours, h), after 48h serum starvation.


D Representative views of alkaline comet assays from wt and junD - / - fibroblasts either untreated or after 10Gy γ - irradiation for the indicated times ( minutes , min ) . E Alkaline comet assays from wt and junD - / - fibroblasts at basal state . Left , Box - plots showing tail moments at basal state in wt and junD - / - fibroblasts . Right , Distribution of wt and junD - / - fibroblasts expressed as percentage of cells ( % ) according to categorized tail moments . At basal state , 16 % of junD - / - cells exhibit tail moment higher than 5 , while it is the case for only 3 % of wt cells . F Alkaline comet assays from wt and junD - / - fibroblasts after 10Gy γ - irradiation . Box - plots showing tail moments at basal state in wt and junD - / - fibroblasts , following 10Gy γ - irradiation for the indicated times ( minutes , min ) . G Distribution of wt and junD - / - fibroblasts expressed as percentage of cells ( % ) according to categorized tail moments , following 10Gy γ - irradiation for the indicated times ( minutes , min ) .

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D Representative views of alkaline comet assays from wt and junD-/- fibroblasts either untreated or after 10Gy γ-irradiation for the indicated times (minutes, min).E Alkaline comet assays from wt and junD-/- fibroblasts at basal state. Left, Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts. Right, Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments. At basal state, 16% of junD-/- cells exhibit tail moment higher than 5, while it is the case for only 3% of wt cells.F Alkaline comet assays from wt and junD-/- fibroblasts after 10Gy γ-irradiation. Box-plots showing tail moments at basal state in wt and junD-/- fibroblasts, following 10Gy γ-irradiation for the indicated times (minutes, min).G Distribution of wt and junD-/- fibroblasts expressed as percentage of cells (%) according to categorized tail moments, following 10Gy γ-irradiation for the indicated times (minutes, min).


H Surviving fraction of wt and junD - / - fibroblasts 7 days after 2Gy or 5Gy γ - irradiation , as assessed by clonogenic assay . Surviving fraction is expressed as percentage ( % ) of number of colonies after irradiation normalized with respect to untreated conditions .

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H Surviving fraction of wt and junD-/- fibroblasts 7 days after 2Gy or 5Gy γ-irradiation, as assessed by clonogenic assay. Surviving fraction is expressed as percentage (%) of number of colonies after irradiation normalized with respect to untreated conditions.


I Surviving fraction of wt and junD - / - fibroblasts either transfected with an empty vector ( + Ctl ) or H2AX - expressing ( + H2AX ) vector . Surviving fraction is expressed as percentage ( % ) of alive cells , 72 hours post - transfection .

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I Surviving fraction of wt and junD-/- fibroblasts either transfected with an empty vector (+ Ctl) or H2AX-expressing (+ H2AX) vector. Surviving fraction is expressed as percentage (%) of alive cells, 72 hours post-transfection.


A Up , Representative western blot from chromatin - free fraction or whole cell extracts , as indicated , showing H2AX protein levels in wt and junD - / - fibroblasts . Different amounts of loaded proteins and exposures ( low - or high - exp ) are presented , H2AX being easier to detect when associated with chromatin . Down , Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown upper ) .

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A Up, Representative western blot from chromatin-free fraction or whole cell extracts, as indicated, showing H2AX protein levels in wt and junD-/- fibroblasts. Different amounts of loaded proteins and exposures (low- or high-exp) are presented, H2AX being easier to detect when associated with chromatin. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).


B Up , Representative western blot from chromatin - free fraction extracts showing H2AX protein levels in wt and junD - / - fibroblasts either untreated ( - ) or after 8h treatment ( + ) with proteasome inhibitor MG132 . Jun is used as positive control for treatment efficiency . Down , Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown upper ) . C Bar plot showing stabilization of H2AX protein in wt and junD - / - fibroblasts , as assessed by densitrometry analysis of western blots ( as shown in B ) . Data are expressed as percentage ( % ) of H2AX protein levels after treatment with MG132 with respect to untreated conditions .

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

B Up, Representative western blot from chromatin-free fraction extracts showing H2AX protein levels in wt and junD-/- fibroblasts either untreated (-) or after 8h treatment (+) with proteasome inhibitor MG132. Jun is used as positive control for treatment efficiency. Down, Bar plots showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown upper).C Bar plot showing stabilization of H2AX protein in wt and junD-/- fibroblasts, as assessed by densitrometry analysis of western blots (as shown in B). Data are expressed as percentage (%) of H2AX protein levels after treatment with MG132 with respect to untreated conditions.


D Representative western blots from whole cell extracts showing HA - tagged ubiquitin ( Left ) or Flag - tagged H2AX ( Right ) protein levels . wt and junD - / - fibroblasts were co - transfected with vectors encoding HA - tagged ubiquitin and Flag - tagged wild - type H2AX ( WT ) , or K13 - , K15 - , K119 - , or 9K - H2AX mutants . Flag - tagged H2AX proteins were then immunoprecipitated with Flag - specific antibody and incubated either with HA - specific antibody ( Left ) or Flag - specific antibody ( Right ) . Two different exposures ( low or high exp ) are presented . * stands for the immunoglobulins .

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D Representative western blots from whole cell extracts showing HA-tagged ubiquitin (Left) or Flag-tagged H2AX (Right) protein levels. wt and junD-/- fibroblasts were co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were then immunoprecipitated with Flag-specific antibody and incubated either with HA-specific antibody (Left) or Flag-specific antibody (Right). Two different exposures (low or high exp) are presented. * stands for the immunoglobulins.


E Left , Representative western blots showing H2AX - WT or H2AX - K119 protein levels ( revealed using Flag - specific antibody ) from whole cell extracts in wt and junD - / - fibroblasts . Cells were first transfected with Flag - tagged wild - type H2AX ( H2AX - WT ) or K119 H2AX mutant ( H2AX - K119 ) and next treated with cycloheximide ( CHX ) for the indicated times ( hours , h ) . Right , Bar graph showing H2AX - WT and H2AX - K119 protein half - life in wt and junD - / - fibroblasts , as indicated . Protein half - life has been calculated from the degradation curve of H2AX protein ( based on densitometry analysis of western blots , as shown on the Left ) by extrapolating its linear part .

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

E Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels (revealed using Flag-specific antibody) from whole cell extracts in wt and junD-/- fibroblasts. Cells were first transfected with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and next treated with cycloheximide (CHX) for the indicated times (hours, h). Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in wt and junD-/- fibroblasts, as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.


F Left , Representative western blots showing H2AX - WT protein levels ( revealed using Flag - specific antibody ) from whole cell extracts in junD - / - fibroblasts co - transfected with Flag - tagged H2AX - WT construct and either non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) , RNF8 ( siRNF8 ) or BMI1 ( siBMI1 ) . Efficiency of each siRNA has been verified and reached 60 % of inhibition , in average , as shown for RNF168 ( Up ) . Protein extracts were processed following cycloheximide ( CHX ) treatment for the indicated times ( hours , h ) . Right , Bar graph showing the percentage ( % ) of decrease of H2AX - WT protein levels after 4h treatment with CHX ( t = 4 ) with respect to untreated conditions ( t = 0 ) in junD - / - fibroblasts transfected with specific siRNA , as indicated .

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

F Left, Representative western blots showing H2AX-WT protein levels (revealed using Flag-specific antibody) from whole cell extracts in junD-/- fibroblasts co-transfected with Flag-tagged H2AX-WT construct and either non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), RNF8 (siRNF8) or BMI1 (siBMI1). Efficiency of each siRNA has been verified and reached 60% of inhibition, in average, as shown for RNF168 (Up). Protein extracts were processed following cycloheximide (CHX) treatment for the indicated times (hours, h). Right, Bar graph showing the percentage (%) of decrease of H2AX-WT protein levels after 4h treatment with CHX (t=4) with respect to untreated conditions (t=0) in junD-/- fibroblasts transfected with specific siRNA, as indicated.


G Left , Representative western blots showing H2AX - WT or H2AX - K119 protein levels from whole cell extracts in junD - / - fibroblasts transfected either with Flag - tagged wild - type H2AX ( H2AX - WT ) or K119 H2AX mutant ( H2AX - K119 ) and co - transfected with non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) . Cells were treated with cycloheximide ( CHX ) for the indicated times ( hours , h ) before protein extraction . Right , Bar graph showing H2AX - WT and H2AX - K119 protein half - life in junD - / - fibroblasts transfected with non - targeting siRNA ( siCtrl ) or siRNA targeting RNF168 ( siRNF168 ) , as indicated . Protein half - life has been calculated from the degradation curve of H2AX protein ( based on densitometry analysis of western blots , as shown on the Left ) by extrapolating its linear part .

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

G Left, Representative western blots showing H2AX-WT or H2AX-K119 protein levels from whole cell extracts in junD-/- fibroblasts transfected either with Flag-tagged wild-type H2AX (H2AX-WT) or K119 H2AX mutant (H2AX-K119) and co-transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168). Cells were treated with cycloheximide (CHX) for the indicated times (hours, h) before protein extraction. Right, Bar graph showing H2AX-WT and H2AX-K119 protein half-life in junD-/- fibroblasts transfected with non-targeting siRNA (siCtrl) or siRNA targeting RNF168 (siRNF168), as indicated. Protein half-life has been calculated from the degradation curve of H2AX protein (based on densitometry analysis of western blots, as shown on the Left) by extrapolating its linear part.


H Representative western blot showing RNF168 , Flag - tagged H2AX ( H2AX - FLAG ) and endogenous H2AX ( H2AX ) protein levels from whole cell extracts in wt and junD - / - fibroblasts . Cells were first co - transfected with vectors encoding HA - tagged ubiquitin and Flag - tagged wild - type H2AX ( WT ) , or K13 - , K15 - , K119 - , or 9K - H2AX mutants . Flag - tagged H2AX proteins were next immunoprecipitated with Flag - specific antibody and incubated either with RNF168 - specific antibody ( Up ) or H2AX - specific antibody ( Down ) .

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H Representative western blot showing RNF168, Flag-tagged H2AX (H2AX-FLAG) and endogenous H2AX (H2AX) protein levels from whole cell extracts in wt and junD-/- fibroblasts. Cells were first co-transfected with vectors encoding HA-tagged ubiquitin and Flag-tagged wild-type H2AX (WT), or K13-, K15-, K119-, or 9K-H2AX mutants. Flag-tagged H2AX proteins were next immunoprecipitated with Flag-specific antibody and incubated either with RNF168-specific antibody (Up) or H2AX-specific antibody (Down).


A Up , Representative views of H2AXimmunostaining from 117 BC patients of Luminal - A ( LumA , N = 33 ) , HER2 ( N = 37 ) and Triple - Negative ( TN , N = 47 ) subtypes . Scale bars = 100 μm . Down , representative views at high magnification of H2AXimmunostaining focused on the Fibroblasts ( Left ) or Epithelium ( Right ) cells . Scale bars = 25 μm . B Scatter plots of H2AX histological score ( Hscore = staining intensity ( 0 to 4 ) x % of positive cells quantified from H2AXimmunostaining ( as shown in A ) in Fibroblasts ( Left ) or Epithelial ( Right ) cells from LumA , HER2 and TNbreasttumours . C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells ( as shown in B ) , in HER2 ( left ) and TN ( right ) breasttumours .

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A Up, Representative views of H2AXimmunostaining from 117 BC patients of Luminal-A (LumA, N=33), HER2 (N=37) and Triple-Negative (TN, N=47) subtypes. Scale bars = 100 μm. Down, representative views at high magnification of H2AXimmunostaining focused on the Fibroblasts (Left) or Epithelium (Right) cells. Scale bars = 25 μm.B Scatter plots of H2AX histological score (Hscore = staining intensity (0 to 4) x % of positive cells quantified from H2AXimmunostaining (as shown in A) in Fibroblasts (Left) or Epithelial (Right) cells from LumA, HER2 and TNbreasttumours.C Correlation plots between H2AX Hscores evaluated in fibroblasts and in epithelial cells (as shown in B), in HER2 (left) and TN (right) breasttumours.


D Representative views of smooth muscle actin ( SMA ) immunostaining from BC patients ( N = 29 ) of Luminal - A ( LumA , N = 10 ) , HER2 ( N = 10 ) and TN ( N = 9 ) subtypes . Scale bars = 100 μm . Zoom views are focused on the fibroblast compartment . Scale bars = 25 μm . E Scatter plots of SMA histological score ( Hscore ) evaluated from SMA immunostaining ( as shown in D ) . F Correlation plot between SMA and H2AX Hscores in stromal compartment .

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D Representative views of smooth muscle actin (SMA) immunostaining from BC patients (N=29) of Luminal-A (LumA, N=10), HER2 (N=10) and TN (N=9) subtypes. Scale bars = 100 μm. Zoom views are focused on the fibroblast compartment. Scale bars = 25 μm.E Scatter plots of SMA histological score (Hscore) evaluated from SMA immunostaining (as shown in D).F Correlation plot between SMA and H2AX Hscores in stromal compartment.


G Scatter plot of the mitotic index , evaluated by pathologists , in LumA , HER2 , and TN BC patients , as indicated .

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G Scatter plot of the mitotic index, evaluated by pathologists, in LumA, HER2, and TN BC patients, as indicated.


H Absence of correlation between mitotic index and epithelial H2AX Hscore , in LumA HER2 and TN BC .

H Absence of correlation between mitotic index and epithelial H2AX Hscore, in LumA HER2 and TN BC.


B Left , Representative views of H2AX and γ - H2AX immunostaining in TN tumours ( N = 22 ) before and after successive cycles of chemotherapy . Scale bars = 100 μm . Zoom views are focused on the epithelial cells . Scale bars = 25 μm . Right , Scatter plots of H2AX , γ - H2AX histological scores ( Hscores ) and γ - H2AX / H2AX ratio evaluated from immunostaining in the epithelium , before and after chemotherapy .

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B Left, Representative views of H2AX and γ-H2AX immunostaining in TN tumours (N = 22) before and after successive cycles of chemotherapy. Scale bars = 100 μm. Zoom views are focused on the epithelial cells. Scale bars = 25 μm. Right, Scatter plots of H2AX, γ-H2AX histological scores (Hscores) and γ-H2AX/H2AX ratio evaluated from immunostaining in the epithelium, before and after chemotherapy.


C Correlation plot between Cisplatin IC50 ( half maximal inhibitory concentration ) and H2AX protein level defined by western blots in BC cell lines ( MCF7 , BT474 , MDA361 , SKBr3 , MDA453 , MDA231 , HCC70 ) . Correlation coefficients σ and P - value are based on Spearman ' s rank correlation test .

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C Correlation plot between Cisplatin IC50 (half maximal inhibitory concentration) and H2AX protein level defined by western blots in BC cell lines (MCF7, BT474, MDA361, SKBr3, MDA453, MDA231, HCC70). Correlation coefficients σ and P-value are based on Spearman's rank correlation test.


D Up , Representative western - blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non - targeting siRNA ( siCtrl ) or an siRNA directed against H2AX ( siH2AX ) . Actin is used as internal control for protein loading . Down , Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots ( as shown above ) .

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D Up, Representative western-blot showing H2AX protein levels from whole cell extracts in HCC70 TN BC cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX). Actin is used as internal control for protein loading. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above).


E Bar plot showing the percentage ( % ) of apoptotic cells as assessed by flow - cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non - targeting siRNA ( siCtrl ) or an siRNA directed against H2AX ( siH2AX ) , and next treated with cisplatin for 72 hrs .

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E Bar plot showing the percentage (%) of apoptotic cells as assessed by flow-cytometry analysis of annexin V and DAPI staining in HCC70 cell line transfected with non-targeting siRNA (siCtrl) or an siRNA directed against H2AX (siH2AX), and next treated with cisplatin for 72 hrs.


F Box plot showing the impact of H2AX decrease on the therapeutic response of TN BC patients ( N = 22 ) . Reduction of H2AX protein level was assessed by the difference of H2AX Hscores before and after chemotherapy ( as evaluated in B ) , for each patient ( values in Log2 ) . Only patients who have experienced an incomplete response have been taken into account . After treatment H2AX staining was evaluated only on residual tumour cells . The therapeutic response was monitored by pathologists through evaluation of tumour residual mass and detection of tumour cells inside lymph nodes .

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F Box plot showing the impact of H2AX decrease on the therapeutic response of TN BC patients (N = 22). Reduction of H2AX protein level was assessed by the difference of H2AX Hscores before and after chemotherapy (as evaluated in B), for each patient (values in Log2). Only patients who have experienced an incomplete response have been taken into account. After treatment H2AX staining was evaluated only on residual tumour cells. The therapeutic response was monitored by pathologists through evaluation of tumour residual mass and detection of tumour cells inside lymph nodes.


G Kaplan - Meier curves showing specific overall survival ( Left ) and progression - free survival ( Right ) of TN patients with respect to major ( N = 15 ) or minor ( N = 7 ) H2AX protein level decrease , observed upon chemotherapy . Iterative analyses were performed on ΔLog2 H2AX Hscore to find optimal threshold that maximally discriminates the major and minor H2AX decrease ( respectively 2 / 3 and 1 / 3 of patients ) .

G Kaplan-Meier curves showing specific overall survival (Left) and progression-free survival (Right) of TN patients with respect to major (N=15) or minor (N=7) H2AX protein level decrease, observed upon chemotherapy. Iterative analyses were performed on ΔLog2 H2AX Hscore to find optimal threshold that maximally discriminates the major and minor H2AX decrease (respectively 2/3 and 1/3 of patients).