Figure 5A . 293T cells were transfected with poly ( I : C ) and cell lysates were prepared at the indicated times for co - immunoprecipitation analysis of endogenous HDAC6 and RIG - I . Actin was used as the loading control . B . BMDMs were isolated from HDAC6 + / + and HDAC6 - / - mice and were transfected with 5ˊppp - dsRNA . Whole cell lysates were prepared after 8 h of transfection and co - immunoprecipitation analysis of RIG - I and HDAC6 was performed . C . HeLa cells were transfected with 5ˊppp - dsRNA . Cells were stained with anti - RIG - I ( red ) and anti - HDAC6 H300 ( green ) antibodies after 8 h of transfection . Nuclei were stained with DAPI ( blue ) ( bar , 20µm ) . D . Fluorescence microscopy , green fluorescence absorbance and virus replication at 24hpi in RIG - I + / + and RIG - I - / - MEFs . Cells were transfected with si - HDAC6 and si - control for 36 h , followed infection with VSV - GFP ( MOI = 1 ) . bar , 100µm . E . HDAC6 + / + , HDAC6 - / - MEFs , and HDAC6 - / - MEFs reconstituted with HDAC6 wild - type were subjected to immunoprecipitation using an anti - RIG - I antibody and immunoblotted with anti - acetyl - lysine and anti - RIG - I antibodies . Acetyl - tubulin was measured in total lysates to determine HDAC6 deacetylase activity . F . Whole cell lysates from HDAC6 + / + and HDAC6 - / - MEFs were prepared and used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I , anti - HDAC6 , and anti - β - actin antibodies . Intensity of pull - downed RIG - I was quantified . G . Whole cell lysates from HDAC6 - / - MEFs and HDAC6 - / - MEFs reconstituted with HDAC6 wild - type and HDAC6 - CDM were prepared and used in dsRNA pull - down assays . Samples were analyzed by western blotting with anti - RIG - I and anti - HDAC6 antibodies . Intensity of pull - downed RIG - I was quantified .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control.B. BMDMs were isolated from HDAC6+/+ and HDAC6-/- mice and were transfected with 5ˊppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed.C. HeLa cells were transfected with 5ˊppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (bar, 20µm).D. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24hpi in RIG-I+/+ and RIG-I-/- MEFs. Cells were transfected with si-HDAC6 and si-control for 36 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm.E. HDAC6+/+, HDAC6-/- MEFs, and HDAC6-/- MEFs reconstituted with HDAC6 wild-type were subjected to immunoprecipitation using an anti-RIG-I antibody and immunoblotted with anti-acetyl-lysine and anti-RIG-I antibodies. Acetyl-tubulin was measured in total lysates to determine HDAC6 deacetylase activity.F. Whole cell lysates from HDAC6+/+ and HDAC6-/- MEFs were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I, anti-HDAC6, and anti-β-actin antibodies. Intensity of pull-downed RIG-I was quantified.G. Whole cell lysates from HDAC6-/- MEFs and HDAC6-/- MEFs reconstituted with HDAC6 wild-type and HDAC6-CDM were prepared and used in dsRNA pull-down assays. Samples were analyzed by western blotting with anti-RIG-I and anti-HDAC6 antibodies. Intensity of pull-downed RIG-I was quantified.


Figure 6A , B . 293T cells were transfected with the indicated expression plasmids . At 1 day post - transfection , whole cell lysates were subjected to dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with an anti - FLAG antibody . C . A luciferase assay was performed using 293T cells transfected with the IFN - βluciferase promoter and TK - Renilla together with 2 ng of RIG - I wild - type , RIG - I 858Q , RIG - I 858R , RIG - I K909Q , or RIG - I K909R mutant , followed by transfection with poly ( I : C ) ( 1 μg / ml ) for another 12 h . D . Fluorescence microscopy , green fluorescence absorbance , and virus replication at 24 hpi in RIG - I - / - MEFs . Cells were transiently transfected with empty vector , RIG - I wild - type , RIG - I K909Q , or RIG - I K909R mutant for 24 h , followed infection with VSV - GFP ( MOI = 1 ) . bar , 100µmE . HDAC6 - / - MEFs were transfected with the indicated expression plasmids . At 1 day post - transfection , whole cell lysates were prepared and used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I and anti - β - actin antibodies . F . 293T cells were transfected with 2 μg of FLAG - tagged RIG - I and 1 , 5 , or 10 μg of V5 - tagged HDAC6 . At 36 h post - transfection , whole cell lysates were prepared for co - immunoprecipitation analysis of V5 - tagged HDAC6 and acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . G . 293T cells were transfected with 2 μg of FLAG - tagged RIG - I . At 24 h post - transfection , cells were infected with VSV - GFP ( MOI = 0 . 1 ) . At 2 and 4 h post - infection , whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . Intensity of ac - 909K normalized to Flag - RIG - I was quantified . H . whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . Intensity of ac - 909K normalized to RIG - I was quantified .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B. 293T cells were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were subjected to dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with an anti-FLAG antibody.C. A luciferase assay was performed using 293T cells transfected with the IFN-βluciferase promoter and TK-Renilla together with 2 ng of RIG-I wild-type, RIG-I 858Q, RIG-I 858R, RIG-I K909Q, or RIG-I K909R mutant, followed by transfection with poly(I:C) (1 μg/ml) for another 12 h.D. Fluorescence microscopy, green fluorescence absorbance, and virus replication at 24 hpi in RIG-I-/- MEFs. Cells were transiently transfected with empty vector, RIG-I wild-type, RIG-I K909Q, or RIG-I K909R mutant for 24 h, followed infection with VSV-GFP (MOI = 1). bar, 100µmE. HDAC6-/- MEFs were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I and anti-β-actin antibodies.F. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I and 1, 5, or 10 μg of V5-tagged HDAC6. At 36 h post-transfection, whole cell lysates were prepared for co-immunoprecipitation analysis of V5-tagged HDAC6 and acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody.G. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I. At 24 h post-transfection, cells were infected with VSV-GFP (MOI = 0.1). At 2 and 4 h post-infection, whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to Flag-RIG-I was quantified.H. whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to RIG-I was quantified.


Figure 7A . The structural modeling result of RIG - I / dsRNA complex and positions of the lysine residue were presented . RIG - I helicase A and B are shown as green and pale blue ribbons . The dsRNA strands were presented as purple and yellow rods , respectively . Two lysine residues , K907 and K909 , on RIG - I that interact with dsRNA are indicated as violet sticks . K907 forms hydrogen bonding with neither 5 ´ - ppp dsRNA nor K907 . B , C . Acetylated K909 and K907 on RIG - I are indicated as green sticks . There were hydrogen bonding between NH2 of K907 and oxygen of acetyl - K909 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. The structural modeling result of RIG-I/dsRNA complex and positions of the lysine residue were presented. RIG-I helicase A and B are shown as green and pale blue ribbons. The dsRNA strands were presented as purple and yellow rods, respectively. Two lysine residues, K907 and K909, on RIG-I that interact with dsRNA are indicated as violet sticks. K907 forms hydrogen bonding with neither 5´-ppp dsRNA nor K907.B, C. Acetylated K909 and K907 on RIG-I are indicated as green sticks. There were hydrogen bonding between NH2 of K907 and oxygen of acetyl-K909.


Figure 1B - J : Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs . Antibodies against Tuj1 , Gfap and Olig2 were used to recognize neurons , astrocytes and oligodendrocytes . In D , G and J the percentage of cells positive ( mean ± SEM ) for each marker was counted over the total of DAPI positive cells ( 100 % ) . No difference between genotypes was highlighted ( Two - way ANOVA ) ; the variation through time resulted significant for the levels of Tuj1 and Gfap ( Two - way ANOVA : F ( 1 , 16 ) = 9 . 542 , p - value < 0 . 05 and F ( 1 , 10 ) = 68 . 95 , p - value < 0 . 0001 respectively ) . Sample size : n ≥ 3 wells obtained from two independent preparations ; for each well 10 random fields were acquired . Scale bars : 30 µm . P : Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14 . For each gene Two - way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) . Values are represented as mean ± SEM . Sample size : ≥ 5 samples obtained from two independent preparations .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B-J: Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs. Antibodies against Tuj1, Gfap and Olig2 were used to recognize neurons, astrocytes and oligodendrocytes. In D, G and J the percentage of cells positive (mean ± SEM) for each marker was counted over the total of DAPI positive cells (100%). No difference between genotypes was highlighted (Two-way ANOVA); the variation through time resulted significant for the levels of Tuj1 and Gfap (Two-way ANOVA: F(1,16)=9.542, p-value<0.05 and F(1,10)=68.95, p-value<0.0001 respectively). Sample size: n ≥3 wells obtained from two independent preparations; for each well 10 random fields were acquired. Scale bars: 30 µm.P: Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14. For each gene Two-way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Values are represented as mean ± SEM. Sample size: ≥5 samples obtained from two independent preparations.


Figure 2H - K : Raster plots ( H and I ) and their quantitation under basal conditions and upon 100µM 4 - AP exposure . The magnitude of the response after stimulation is expressed either as fold change ( J ) or as the averaged maximal values reached after 4 - AP exposure ( K ) . Dotted line in J represents untreated controls ( set to 1 ) . Values are represented as mean ± SEM . Student ' s t - test , * : p - value < 0 . 05 . Sample size n = 8 wells deriving from three independent preparations . L : Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points . The binary mask is representative of a neuron used to study morphology . Scale bars : 30 µm . M - Q : Each graph represents a different morphological parameter analysed at selected time point . Two - way ANOVA indicated both a significant time effect ( ( F 4 , 20 ) = 282 , 5 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 104 , 5 and p < 0 , 0001 for the total dendritic length ; ( F 4 , 20 ) = 58 , 51 and p < 0 , 0001 for the maximal dendritic length ; ( F 3 , 22 ) = 38 , 32 and p < 0 , 0001 for polarization ) and genotype effect ( ( F 1 , 20 ) = 25 , 1 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 16 , 0 and p = 0 . 005 for the total dendritic length ; ( F 1 , 20 ) = 17 , 24 and p = 0 , 005 for the maximal dendritic length ; ( F 1 , 22 ) = 18 , 76 and p < 0 . 003 for polarization ) . Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point . The dotted line in panel P represents the cut - off above which a culture is defined polarized ( Horton et al , 2006 ) . Values are represented as mean ± SEM ; * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * * : p - value < 0 . 0001 . Sample size : n ≥ 3 wells deriving from three independent preparations ; for each well we measured at least 15 cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2H-K: Raster plots (H and I) and their quantitation under basal conditions and upon 100µM 4-AP exposure. The magnitude of the response after stimulation is expressed either as fold change (J) or as the averaged maximal values reached after 4-AP exposure (K). Dotted line in J represents untreated controls (set to 1). Values are represented as mean ± SEM. Student's t-test, *: p-value<0.05. Sample size n=8 wells deriving from three independent preparations.L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.


Figure 3B , C : Five morphological parameters were measured in untreated and treated null neurons at DIV14 . One - way ANOVA indicated a significant treatment effect for the maximal dendritic length ( F = 6 , 306 ; p = 0 , 0039 ) , total dendritic length ( F = 4 , 880 ; p = 0 , 0126 ) , number of intersections ( F = 10 , 70 ; p = 0 , 0003 ) and soma area ( F = 7 , 721 ; p = 0 , 0015 ) . Tukey ' s multiple comparison test was used to assess differences between null neurons and early and late treated neurons . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 . The variation of each of the selected parameters is summarized in the radar plot ( C ) . Sample size ( panels B , C ) : n ≥ 18 wells for ctrl group and n ≥ 10 wells for the treatment groups deriving from three independent preparations ; for each well we measured at least 15 cells . Each point is representative of a single well . Values are represented as mean ± SEM . Bar graphs represent the expression values ( mean ± SEM ) of 27 genes , selected for their downregulation in null samples at DIV 14 ( p - value < 0 , 07 ; see Appendix Figure S2 ) , after CX546 administration in the two different time windows ( early in G and late in H ) . One - way ANOVA followed by Dunnett ' s multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls ( set to 1 , red dotted line ) . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 , * * * : p - value < 0 . 001 , * * * * : p - value < 0 . 0001 . Sample size : n ≥ 8 wells deriving from two independent preparations . Each point is representative of a single well .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B,C: Five morphological parameters were measured in untreated and treated null neurons at DIV14. One-way ANOVA indicated a significant treatment effect for the maximal dendritic length (F=6,306;p=0,0039), total dendritic length (F=4,880;p=0,0126), number of intersections (F=10,70; p=0,0003) and soma area (F=7,721;p=0,0015). Tukey's multiple comparison test was used to assess differences between null neurons and early and late treated neurons. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001. The variation of each of the selected parameters is summarized in the radar plot (C). Sample size (panels B,C): n≥18 wells for ctrl group and n≥10 wells for the treatment groups deriving from three independent preparations; for each well we measured at least 15 cells. Each point is representative of a single well. Values are represented as mean ± SEM.Bar graphs represent the expression values (mean ± SEM) of 27 genes, selected for their downregulation in null samples at DIV 14 (p-value <0,07; see Appendix Figure S2), after CX546 administration in the two different time windows (early in G and late in H). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls (set to 1, red dotted line). *: p-value<0.05; **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001. Sample size: n≥8 wells deriving from two independent preparations. Each point is representative of a single well.


Figure 4F , G : Violin plots represent the average values of MQAE intensity for early ( F ) or late ( G ) treated null neurons and their relative controls . One - way ANOVA followed by Tukey ' s multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons . * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 . The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles . Sample size : n ≥ 300 cells from five different embryos per genotype . I , J : In I One - way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons ( set to 100 ) , null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4 . * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 ( * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 ) ; # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine ( # : p - value < 0 . 05 ; # # : p - value < 0 . 01 ) . Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 ( expressed as 2 - ∆ ct ) . Box plot shows the median ( central band ) , the lower and upper quartiles ( boxes ) and the max and min values ( whiskers ) from at least 3 wells obtained from four different embryos . One - way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones . * * : p - value < 0 . 01 . Sample size : n ≥ 3 wells from four different embryos .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4F,G: Violin plots represent the average values of MQAE intensity for early (F) or late (G) treated null neurons and their relative controls. One-way ANOVA followed by Tukey's multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons. ***: p<0.001; ****: p <0.0001. The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles. Sample size: n≥300 cells from five different embryos per genotype.I,J: In I One-way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons (set to 100), null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4. * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001); # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine (#: p-value<0.05; ##: p-value<0.01). Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 (expressed as 2-∆ct). Box plot shows the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 3 wells obtained from four different embryos. One-way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones. **: p-value<0.01. Sample size: n≥3 wells from four different embryos.


Figure 5B : Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals . Sample size : wt = 11 , ko = 16 , ko treated with CX546 = 11 . Gehan - Breslow - Wilcoxon test was used to compare the two groups . * * : p - value < 0 . 01 . F , G : Line graph ( F ) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials . Two - way ANOVA indicated a significant effect of time ( F ( 2 , 21 ) = 5 , 490 and p = 0 , 0121 ) and genotype ( F ( 2 , 33 ) = 16 , 33 and p < 0 . 0001 ) . Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546 . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 . The scatter plot ( G ) shows latency to fall ( in seconds ) on the accelerating rotarod in the last trial . One - way ANOVA was used to compare ko mice and ko treated mice to the wt group . The Tukey ' s multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine ; * * : p - value < 0 . 01 . Values are represented as average ± SEM . Sample size : n = 6 wt treated with vehicle and n = 14 ko ( 6 treated with vehicle and 8 treated with CX546 ) . Each dot represents a single animal .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01.F,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal.


Figure 1 ( A ) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice . ( B ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 0 - 5 hours before immunoblotting with the indicated antibodies . ( C , E and G ) WT PEMs expressing GFP - FAK were incubated with either ΔinvG Salmonella ( C ) , LPS - coated beads ( E ) or E . coli ( G ) for a total of 5 hours before analysis by immunofluorescence . Cells were co - stained with antibodies recognizing LAMP1 ( red ) . Dapi was used to visualize nuclei and bacteria ( blue ) . Bars represent 10 µm . Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm . ( D , F and H ) The percent of LAMP1 - positive ( gray bars ) and LAMP1 + GFP + ( black bars ) ΔinvG Salmonella ( D ) , LPS - coated beads ( F ) and E . coli ( H ) was quantified . At least 100 bacteria were counted per condition ; N = 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice.(B) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-5 hours before immunoblotting with the indicated antibodies.(C, E and G) WT PEMs expressing GFP-FAK were incubated with either ΔinvG Salmonella (C), LPS-coated beads (E) or E. coli (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) ΔinvG Salmonella (D), LPS-coated beads (F) and E. coli (H) was quantified. At least 100 bacteria were counted per condition; N = 3.


Figure 2A ) TLR4 − / − PEMs were incubated with ΔinvG Salmonella for 0 - 5 hours before immunoblotting with the indicated antibodies . ( B ) WT PEMs were incubated with LPS - coated beads for 0 - 5 hours before immunoblotting for the indicated antibodies . ( C ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation . ( D ) WT PEMs were incubated with the orgA : : tet ; spiA : : kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs ( ΔSPI1ΔSPI2 ) for 0 - 5 hours before immunoblotting with the indicated antibodies . ( E ) WT PEMs expressing GFP - FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence . Cells were co - stained with antibodies recognizing LAMP1 ( red ) . DAPI was used to visualize nuclei and bacteria ( blue ) . Bars represent 10 µm or 2 µm in enlarged inset .

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Figure 2A) TLR4−/− PEMs were incubated with ΔinvG Salmonella for 0-5 hours before immunoblotting with the indicated antibodies.(B) WT PEMs were incubated with LPS-coated beads for 0-5 hours before immunoblotting for the indicated antibodies.(C) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.(D) WT PEMs were incubated with the orgA::tet; spiA::kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs (ΔSPI1ΔSPI2) for 0-5 hours before immunoblotting with the indicated antibodies.(E) WT PEMs expressing GFP-FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). DAPI was used to visualize nuclei and bacteria (blue). Bars represent 10 µm or 2 µm in enlarged inset.


Figure 3 ( A ) WT and FAK − / − PEMs were incubated for 0 - 5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies . ( B ) WT PEMs were pretreated for 1 hour with the FAK - kinase inhibitor PF - 228 ( 0 . 5 µm ) before incubation with ΔinvG Salmonella for 5 hours . Lysates were immunoblotted with the indicated antibodies . ( C - D ) WT or FAK − / − macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain ( C ) or LPS - coated beads ( D ) for 0 - 5 hours before immunoblotting with the indicated antibodies . In panels C and D , WT PEMs were also incubated with ΔinvG ( 5 h ) for comparison of Akt activation . Vertical white lines in panel D indicate non - contiguous lanes generated from a single exposure . E ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation . ( F ) WT PEMs expressing HA - Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours . Lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies before immunoblotting for FAK and HA . In parallel , lysates were immunoblotted for FAK , HA and ERK1 / 2 to control for the level of expression . G ) WT and FAK − / − macrophages expressing HA - Akt were stained with anti - HA and anti - LAMP1 before analysis by confocal microscopy . White boxes show enlarged regions in lower panel . Arrows indicate HA + LAMP + Salmonella evident in WT PEMs . ( H ) The percentage of ΔΣΠΙ1ΔΣΠΙ2 ( first grouping ) or ΔinvG ( second grouping ) Salmonella co - localizing with HA and LAMP - 1 5 h post - infection . At least 100 bacteria were counted per condition . Values are means ± SEM , N = 3 , * p < 0 . 05 .

O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) WT and FAK−/− PEMs were incubated for 0-5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies.(B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies.(C-D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0-5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure.E) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.(F) WT PEMs expressing HA-Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies before immunoblotting for FAK and HA. In parallel, lysates were immunoblotted for FAK, HA and ERK1/2 to control for the level of expression.G) WT and FAK−/− macrophages expressing HA-Akt were stained with anti-HA and anti-LAMP1 before analysis by confocal microscopy. White boxes show enlarged regions in lower panel. Arrows indicate HA+LAMP+ Salmonella evident in WT PEMs. (H) The percentage of ΔΣΠΙ1ΔΣΠΙ2 (first grouping) or ΔinvG (second grouping) Salmonella co-localizing with HA and LAMP-1 5 h post-infection. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05.


Figure 4 ( A ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 0 - 24 hours before immunoblotting with the indicated antibodies . ( B ) WT PEMs were incubated with Akt inhibitor AKTV / triciribine ( 10 µm ) for 30 minutes before infection with S . typhimurium strain ΔinvG for 5 hours . Lysates were immunoblotted with the indicated antibodies . ( . ( C ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3 . DAPI was used to visualize nuclei and bacteria . Bars represent 10 µm . White boxes show enlarged regions in inset panels , bars represent 2 µm . White arrowheads indicate LC3 - negative bacteria , yellow arrow heads indicate LC3 - positive bacteria ( panels c , f ) . Yellow arrowhead in panel f denotes an LC3 + cluster of bacteria . ( D ) The percentage of ΔinvG ( first grouping ) or ΔΣΠΙ1ΔΣΠΙ2 ( second grouping ) Salmonella co - localizing with LC3 at the indicated time points is displayed . At least 100 bacteria were counted per condition per time point . Values are means ± SEM , N = 3 , * p < 0 . 05 . ( E ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 ( 300 ng ) before immunoblotting with the indicated antibodies . Vertical white line indicate non - contiguous lanes generated from a single exposure . ( F ) FAK − / − PEMs were incubated with DQ - BSA - red for 1 hour before infection with S . typhimurium strain ΔinvG for 5 h . Cells were co - stained for LC3 and DAPI was used to visualize nuclei and bacteria . White boxes show enlarged regions in inset panel . G ) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin ( 4 µm ) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours . Cells were lysed before immunoblotting with the indicated antibodies . ( 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 B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-24 hours before immunoblotting with the indicated antibodies.(B) WT PEMs were incubated with Akt inhibitor AKTV/triciribine (10 µm) for 30 minutes before infection with S. typhimurium strain ΔinvG for 5 hours. Lysates were immunoblotted with the indicated antibodies. (. (C) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei and bacteria. Bars represent 10 µm. White boxes show enlarged regions in inset panels, bars represent 2 µm. White arrowheads indicate LC3-negative bacteria, yellow arrow heads indicate LC3-positive bacteria (panels c, f). Yellow arrowhead in panel f denotes an LC3+ cluster of bacteria. (D) The percentage of ΔinvG (first grouping) or ΔΣΠΙ1ΔΣΠΙ2 (second grouping) Salmonella co-localizing with LC3 at the indicated time points is displayed. At least 100 bacteria were counted per condition per time point. Values are means ± SEM, N = 3, *p<0.05.(E) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 (300 ng) before immunoblotting with the indicated antibodies. Vertical white line indicate non-contiguous lanes generated from a single exposure.(F) FAK−/− PEMs were incubated with DQ-BSA-red for 1 hour before infection with S. typhimurium strain ΔinvG for 5 h. Cells were co-stained for LC3 and DAPI was used to visualize nuclei and bacteria. White boxes show enlarged regions in inset panel.G) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin (4 µm) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours. Cells were lysed before immunoblotting with the indicated antibodies.(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.


Figure 5A ) 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 . . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) 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.. (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.


Figure 6 ( 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 . ( 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 . ( 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 . . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.(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.(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.(G) Quantitation of flow cytometric data collected from the spleens of infected mice. *p<0.05.


Figure 1A 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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.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.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.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.


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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Figure 3A , 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, 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.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.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.


Figure 4B 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 . 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 ) . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B 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.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).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.


Figure 5A , 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, 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.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.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).


Figure 1 ( 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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Figure 1(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.


Figure 2 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.


Figure 3Immunoblot 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 ) . ( 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 ) .

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Figure 3Immunoblot 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).(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).


Figure 4 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 5Immunofluorescence 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 . 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunofluorescence 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.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.(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).