Figure 3Human neutrophils ( PMNs , 4 × 105 / mL ) were incubated with SARS - CoV - 2 ( MOI = 0 . 1 and 1 ) with or without autologous platelets ( PLTs , 4 × 106 / mL ) for 5 h and 20 h at 37 ° C . ( a ) The image of NETs ( arrow heads : aggregated NETs ) . Human neutrophils ( PMNs , 4 × 105 / mL ) were incubated with SARS - CoV - 2 ( MOI = 0 . 1 and 1 ) with or without autologous platelets ( PLTs , 4 × 106 / mL ) for 5 h and 20 h at 37 ° C . ( b ) quantitation of NET area by citrullinated Histone H3 ( Cit - H3 ) area . Human neutrophils ( PMNs , 4 × 105 / mL ) were incubated with SARS - CoV - 2 ( MOI = 0 . 1 and 1 ) with autologous platelets ( PLTs , 4 × 106 / mL ) for 5 h and 20 h at 37 ° C . ( c ) Human platelets were pretreated with anti - CLEC2 mAb ( AYP1 , 2 . 5 μg / mL ) or isotype control for 15 min at room temperature before incubation with neutrophils and SARS - CoV - 2 stimulation . ( d ) Neutrophils ( 4 × 105 / mL ) from wild type ( WT ) mice were incubated with SARS - CoV - 2 ( MOI = 1 ) in the presence of WT platelets ( 4 × 106 / mL ) or clec2 - / - platelets ( 4 × 106 / mL ) for 5 h at 37 ° C . Human neutrophils ( PMNs , 4 × 105 / mL ) were incubated with SARS - CoV - 2 with or without autologous platelets ( PLTs , 4 × 106 / mL ) ( e & f ) Human neutrophils were stimulated with SARS - CoV - 2 spike RBD pseudotyped virus ( pseudotyped virus ) ( MOI = 0 . 1 ) in presence of autologous platelets at 37 ° C for 3 h ( e ) . Platelets were incubated with anti - CLEC2 mAb ( 2 . 5 μg / mL ) at room temperature for 15 min before incubation with neutrophils and pseudotyped virus stimulation at 37 ° C for 3 h ( f ) . The level of NET formation was determined using Cit - H3 area ( μm2 ) .

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 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 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

Figure 3Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 (MOI = 0.1 and 1) with or without autologous platelets (PLTs, 4 × 106/mL) for 5 h and 20 h at 37 °C. (a) The image of NETs (arrow heads: aggregated NETs).Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 (MOI = 0.1 and 1) with or without autologous platelets (PLTs, 4 × 106/mL) for 5 h and 20 h at 37 °C. (b) quantitation of NET area by citrullinated Histone H3 (Cit-H3) area.Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 (MOI = 0.1 and 1) with autologous platelets (PLTs, 4 × 106/mL) for 5 h and 20 h at 37 °C. (c) Human platelets were pretreated with anti-CLEC2 mAb (AYP1, 2.5 μg/mL) or isotype control for 15 min at room temperature before incubation with neutrophils and SARS-CoV-2 stimulation.(d) Neutrophils (4 × 105/mL) from wild type (WT) mice were incubated with SARS-CoV-2 (MOI = 1) in the presence of WT platelets (4 × 106/mL) or clec2-/- platelets (4 × 106/mL) for 5 h at 37 °C.Human neutrophils (PMNs, 4 × 105/mL) were incubated with SARS-CoV-2 with or without autologous platelets (PLTs, 4 × 106/mL) (e & f) Human neutrophils were stimulated with SARS-CoV-2 spike RBD pseudotyped virus (pseudotyped virus) (MOI = 0.1) in presence of autologous platelets at 37°C for 3 h (e). Platelets were incubated with anti-CLEC2 mAb (2.5 μg/mL) at room temperature for 15 min before incubation with neutrophils and pseudotyped virus stimulation at 37°C for 3 h (f). The level of NET formation was determined using Cit-H3 area (μm2).


Figure 4 ( a & b ) Human neutrophils were stimulated with SARS - CoV - 2 ( MOI = 0 . 1 and 1 ) at 37 ° C in the presence of isotype control ( hIgG1 , 3 μg / mL ) or human CLEC2 . Fc ( 3 μg / mL ) and with or without autologous platelets for 5 h ( a ) or with or without anti - SARS - CoV - 2 spike RBD antibody ( 3 μg / mL ) for 20 h ( b ) . Data are means ± sd from at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) . The expression levels of viral copy number ( d ) Data are mean ± SEM from two independent experiments ( saline : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; isotype : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; prophylactic treatment of CLEC2 . Fc treatment : n = 5 for 3 d . p . i . and 5 d . p . i . ; therapeutic treatment of CLEC2 . Fc : n = 3 for 3 d . p . i . and 5 d . p . i . ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Two - way ANOVA ) . ' d . p . i . ' stands for ' day post - infection ' . cytokines and chemokines in lung were measured by qPCR ( e ) . Data are mean ± SEM from two independent experiments ( saline : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; isotype : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; prophylactic treatment of CLEC2 . Fc treatment : n = 5 for 3 d . p . i . and 5 d . p . i . ; therapeutic treatment of CLEC2 . Fc : n = 3 for 3 d . p . i . and 5 d . p . i . ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Two - way ANOVA ) . ' d . p . i . ' stands for ' day post - infection ' .

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 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 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

Figure 4(a & b) Human neutrophils were stimulated with SARS-CoV-2 (MOI = 0.1 and 1) at 37 °C in the presence of isotype control (hIgG1, 3 μg/mL) or human CLEC2.Fc (3 μg/mL) and with or without autologous platelets for 5 h (a) or with or without anti-SARS-CoV-2 spike RBD antibody (3 μg/mL) for 20 h (b). Data are means ± sd from at least three independent experiments. *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test).The expression levels of viral copy number (d) Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n= 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). 'd.p.i.' stands for 'day post-infection'.cytokines and chemokines in lung were measured by qPCR (e). Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n= 5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Two-way ANOVA). 'd.p.i.' stands for 'day post-infection'.


Figure 5 ( a ) AAV - hACE2 mice were treated with saline and challenged with SARS - CoV - 2 ( 8 × 104 PFU ) for 5 days . Lung tissue sections were stained with DAPI ( blue ) , anti - MPO antibody ( green ) , anti - citrullinated histone H3 ( Cit - H3 ) antibody ( red ) , anti - CD42b antibody ( yellow ) and anti - CD31 antibody ( gray ) . yellow arrow : NETs ( DNA + Gr - 1 + MPO + Cit - H3 + ) ; red arrow : immunothrombosis ( NETs + thrombus ( CD42b + ) ) , Scale bar is 100 μm . b & c ) For prophylactic treatment , CLEC2 . Fc ( or vehicle ) was given at 1 h before virus challenge ; for therapeutic treatment , CLEC2 . Fc was given at 8 hours post - infection . The area of NET ( colocalization area of MPO and Cit - H3 ) ( b ) and immunothrombosis ( colocalization area of Cit - H3 and CD42b ) ( c ) were measured using MetaMorph software . Data are mean ± SEM from two independent experiments ( saline : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; isotype : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; prophylactic treatment of CLEC2 . Fc treatment : n = 5 for 3 d . p . i . and 5 d . p . i . ; therapeutic treatment of CLEC2 . Fc : n = 3 for 3 d . p . i . and 5 d . p . i . ) . * p < 0 . 05 , * * p < 0 . 01 ( Two - way ANOVA ) .

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 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

Figure 5(a) AAV-hACE2 mice were treated with saline and challenged with SARS-CoV-2 (8 × 104 PFU) for 5 days. Lung tissue sections were stained with DAPI (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (Cit-H3) antibody (red), anti-CD42b antibody (yellow) and anti-CD31 antibody (gray). yellow arrow: NETs (DNA+Gr-1+MPO+Cit-H3+); red arrow: immunothrombosis (NETs + thrombus (CD42b+)), Scale bar is 100 μm.b & c) For prophylactic treatment, CLEC2.Fc (or vehicle) was given at 1 h before virus challenge; for therapeutic treatment, CLEC2.Fc was given at 8 hours post-infection. The area of NET (colocalization area of MPO and Cit-H3) (b) and immunothrombosis (colocalization area of Cit-H3 and CD42b) (c) were measured using MetaMorph software. Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n=5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). *p <0.05, **p < 0.01 (Two-way ANOVA).


Figure 6a , b ) AAV - hACE2 mice were treated with saline and challenged with SARS - CoV - 2 ( 8 × 104 PFU ) for 5 days . Samples were collected at day 5 post - infection , and the collagen were stained with Picro Sirius red ( a ) , while collagen deposition was quantified using MetaMorph software and presented as area ( μm2 ) ( b ) . The images were captured by light microscopy ( upper panel ) and polarized light microscopy ( lower panel ) . Data are mean ± SEM from two independent experiments ( saline : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; isotype : n = 3 for 3 d . p . i . , n = 4 for 5 d . p . i . ; prophylactic treatment of CLEC2 . Fc treatment : n = 5 for 3 d . p . i . and 5 d . p . i . ; therapeutic treatment of CLEC2 . Fc : n = 3 for 3 d . p . i . and 5 d . p . i . ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Two - way ANOVA ) .

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

Figure 6a, b) AAV-hACE2 mice were treated with saline and challenged with SARS-CoV-2 (8 × 104 PFU) for 5 days. Samples were collected at day 5 post-infection, and the collagen were stained with Picro Sirius red (a), while collagen deposition was quantified using MetaMorph software and presented as area (μm2) (b). The images were captured by light microscopy (upper panel) and polarized light microscopy (lower panel). Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n=5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). ***p < 0.001, ****p < 0.0001 (Two-way ANOVA).


Figure 1B . Scatter plot illustrates individual mouse TSPO - PET values derived from a whole brain volume of interest . 8 - 15 female mice per group at an average age of 11 . 1 ± 1 . 6 months ( Grn - / - ( n = 8 ) , Trem2 - / - ( n = 9 ) , Double - / - ( n = 10 ) , WT ( n = 15 ) ) . Data represent mean ± SD . For statistical analysis one - way ANOVA with Tukey post hoc test was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant .

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

Figure 1B. Scatter plot illustrates individual mouse TSPO-PET values derived from a whole brain volume of interest. 8-15 female mice per group at an average age of 11.1 ± 1.6 months (Grn-/- (n = 8), Trem2-/- (n = 9), Double-/- (n = 10), WT (n= 15)). Data represent mean ± SD. For statistical analysis one-way ANOVA with Tukey post hoc test was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ns, not significant.


Figure 2B . Volcano plot presentation of the differently expressed transcripts in FCRLS - and CD11b - positive Grn - / - ( n = 6 ) in comparison to WT ( n = 6 ) microglia isolated from 6 - month - old male mice . 35 out of 65 analyzed genes are significantly changed more than 20 % , with 22 genes upregulated ( purple ) and 13 genes downregulated ( blue ) . Data information : For statistical analysis the unpaired , two - tailed student ` s t - test was performed Statistical significance was set at * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 000 ; ns , not significant .

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

Figure 2B. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Grn-/- (n=6) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 35 out of 65 analyzed genes are significantly changed more than 20%, with 22 genes upregulated (purple) and 13 genes downregulated (blue). Data information: For statistical analysis the unpaired, two-tailed student`s t-test was performed Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.000; ns, not significant.


Figure 3B . AlphaLISA - mediated quantification of p - Syk in human macrophages with a dose titration treatment of Ab1 and Ab2 with or without liposomes for 5 min . IC50 and maximal inhibition ( max ) are indicated by a dotted line . Data represent the mean ± SEM ( n = 3 independent experiments ) . Data information : Data represent mean ± SEM . For statistical analysis of patient samples in comparison to HC , the unpaired , two - tailed student ` s t - test was performed . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; ns , not significantG . Western blot of TREM2 in lysates and media of cultured human macrophages isolated from GRN - FTLD # 1 and HC # 1 upon treatment with Ab1 and Ab2 . An isotype antibody was used as a negative control . ADAM protease inhibition ( GM ) does not further increase mTREM2 levels in GRN - FTLD patients . Equal amounts of protein were loaded . GAPDH was used as loading control .

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 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

Figure 3B. AlphaLISA-mediated quantification of p-Syk in human macrophages with a dose titration treatment of Ab1 and Ab2 with or without liposomes for 5 min. IC50 and maximal inhibition (max) are indicated by a dotted line. Data represent the mean ± SEM (n=3 independent experiments). Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significantG. Western blot of TREM2 in lysates and media of cultured human macrophages isolated from GRN-FTLD #1 and HC #1 upon treatment with Ab1 and Ab2. An isotype antibody was used as a negative control. ADAM protease inhibition (GM) does not further increase mTREM2 levels in GRN-FTLD patients. Equal amounts of protein were loaded. GAPDH was used as loading control.


Figure 4A . Western blot of PGRN in whole - cell lysates of WT and GRN - / - hiMGL . Actin was used as loading control . B . Western blot of TREM2 in whole - cell lysates of WT and GRN - / - hiMGL . Mature ( mTREM ) and immature TREM2 ( imTREM2 ) are indicated . Actin was used as loading control . E . ELISA - mediated quantification of sTREM2 in conditioned media of GRN - / - hiMGL upon treatment with Ab1 and Ab2 ( 20 μg / ml , 40 μg / ml ) ( n = 3 , biological replicates ) . An isotype antibody ( 10 μg / ml ) was used as a negative control . F . AlphaLISA - mediated quantification of p - Syk levels in GRN - / - hiMGL upon treatment with Ab1 and Ab2 ( 5 μg / ml , 10 μg / ml ) with liposomes ( 1 mg / ml ) for 5 min . ( n = 8 , biological replicates ) . Data represent mean ± SEM . For statistical analysis in E and F one - way ANOVA with Dunnett ` s post hoc test , was used to compare untreated , Ab1 , and Ab2 ( 20 µg / ml and 40µg / ml ) conditions to the isotype treated condition . H . Uptake assay for fluorescent myelin . GRN - / - hiMGL phagocytose significantly more myelin as compare to WT hiMGL . This is reversed upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 ( n = 4 , biological replicates ) .

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

Figure 4A. Western blot of PGRN in whole-cell lysates of WT and GRN-/- hiMGL. Actin was used as loading control. B. Western blot of TREM2 in whole-cell lysates of WT and GRN-/- hiMGL. Mature (mTREM) and immature TREM2 (imTREM2) are indicated. Actin was used as loading control.E. ELISA-mediated quantification of sTREM2 in conditioned media of GRN-/- hiMGL upon treatment with Ab1 and Ab2 (20 μg/ml, 40 μg/ml) (n = 3, biological replicates). An isotype antibody (10 μg/ml) was used as a negative control. F. AlphaLISA-mediated quantification of p-Syk levels in GRN-/- hiMGL upon treatment with Ab1 and Ab2 (5 μg/ml, 10 μg/ml) with liposomes (1 mg/ml) for 5 min. (n = 8, biological replicates). Data represent mean ± SEM. For statistical analysis in E and F one-way ANOVA with Dunnett`s post hoc test, was used to compare untreated, Ab1, and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition.H. Uptake assay for fluorescent myelin. GRN-/- hiMGL phagocytose significantly more myelin as compare to WT hiMGL. This is reversed upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 (n=4, biological replicates).


Figure 5E . Transcript levels of DAM gene transcripts significantly altered in GRN - / - hiMGL , treated with Ab1 or Ab2 in comparison to isotype treatment from the data set in A , normalized to the mean of the WT hiMGL samples ( n = 4 , biological replicates ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Dunnett ` s post hoc test was used to compare Ab1 and Ab2 ( 20 µg / ml and 40µg / ml ) conditions to the isotype treated condition , Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , * * * * * , p < 0 . 00001 , and ns , not significant .

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

Figure 5E. Transcript levels of DAM gene transcripts significantly altered in GRN-/- hiMGL, treated with Ab1 or Ab2 in comparison to isotype treatment from the data set in A, normalized to the mean of the WT hiMGL samples (n=4, biological replicates). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Dunnett`s post hoc test was used to compare Ab1 and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition, Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, *****, p < 0.00001, and ns, not significant.


Figure 6A . Western blot of CatD in total brain lysates from 14 - month - old female WT , Grn - / - , Trem2 - / - , and Double - / - mice . CatD maturation variants are indicated ( sc : single chain ; hc : heavy chain ; n = 3 ) . B . , C . Quantification of CatD variants in A normalized to WT ( n = 3 per genotype ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ' s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant . D . , E . Catalytic activity of CatD in brain lysates from female 6 - month - old ( n = 4 per genotype ) ( D ) or 14 - month - old ( n = 3 per genotype ) ( E ) Grn - / - , Trem2 - / - , Double - / - mice normalized to WT . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ' s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant . F . Immunohistochemical analysis of lipofuscin ( green ) in coronal brain sections . Representative images of thalamus are shown . Scalebars = 50 μm . G . Quantification of lipofuscin autofluorescence . Five images per mouse were taken , means were normalized to WT samples ( n = 3 per genotype , female ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ' s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant .

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 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 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

Figure 6A. Western blot of CatD in total brain lysates from 14-month-old female WT, Grn-/-, Trem2-/-, and Double-/- mice. CatD maturation variants are indicated (sc: single chain; hc: heavy chain; n=3). B., C. Quantification of CatD variants in A normalized to WT (n=3 per genotype). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.D., E. Catalytic activity of CatD in brain lysates from female 6-month-old (n=4 per genotype) (D) or 14-month-old (n=3 per genotype) (E) Grn-/-, Trem2-/-, Double-/- mice normalized to WT. Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.F. Immunohistochemical analysis of lipofuscin (green) in coronal brain sections. Representative images of thalamus are shown. Scalebars = 50 μm. G. Quantification of lipofuscin autofluorescence. Five images per mouse were taken, means were normalized to WT samples (n=3 per genotype, female). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.


Figure 7A - D . Volcano plot presentation of lipids and metabolites upregulated ( purple ) or downregulated ( blue ) in total brain homogenates from 6 - month - old male Grn - / - ( A , n = 5 ) , Trem2 - / - ( B , n = 5 ) , Double - / - ( C , n = 5 ) mice in comparison to WT ( n = 4 ) , and Double - / - in comparison to Grn - / - mice ( D ) . Counts for each sample were normalized to the mean value of WT followed by a log2 transformation ( n = 4 - 5 per genotype ) . Analyte values were adjusted with an FDR < 10 % to exclude type I errors in null hypothesis testing . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ` s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant . E - G . Abundance of BMP species and glucosylsphingosine ( GlcSph ) in total brain of 6 - month - old Grn - / - , Trem2 - / - , Double - / - and WT mice ( n = 4 - 5 per genotype ) . H . Glucocerebrosidase ( GCase ) activity in whole brain lysates from 6 - month - old male Grn - / - , Trem2 - / - , Double - / - and WT mice . The linear increase of fluorescence signal was measured and then normalized to WT mice ( n = 3 - 6 per genotype ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ` s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant .

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 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

Figure 7A-D. Volcano plot presentation of lipids and metabolites upregulated (purple) or downregulated (blue) in total brain homogenates from 6-month-old male Grn-/- (A, n=5), Trem2-/- (B, n=5), Double-/- (C, n=5) mice in comparison to WT (n=4), and Double-/- in comparison to Grn-/- mice (D). Counts for each sample were normalized to the mean value of WT followed by a log2 transformation (n=4-5 per genotype). Analyte values were adjusted with an FDR <10% to exclude type I errors in null hypothesis testing. Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey`s post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at ***, p < 0.001; ****, p < 0.0001, and ns, not significant.E-G. Abundance of BMP species and glucosylsphingosine (GlcSph) in total brain of 6-month-old Grn-/-, Trem2-/-, Double-/- and WT mice (n=4-5 per genotype). H. Glucocerebrosidase (GCase) activity in whole brain lysates from 6-month-old male Grn-/-, Trem2-/-, Double-/- and WT mice. The linear increase of fluorescence signal was measured and then normalized to WT mice (n=3-6 per genotype). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey`s post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at ***, p < 0.001; ****, p < 0.0001, and ns, not significant.


Figure 8A . Immunohistochemical analysis of synaptophysin ( SPH , pink ) and VGAT ( yellow ) in coronal brain sections . Representative images of thalamus are shown . Scalebars = 50 μm . F . Morphological analysis of cortical microglia . Representative maximum intensity projections of confocal z - stack images showing IBA1 + microglial cells of female WT , Grn - / - , Trem2 - / - and Double - / - mice ( scalebar = 50 µm ) . Arrows point to individual microglia , which are shown as three - dimensional reconstruction , scalebar = 10 µm .

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 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

Figure 8A. Immunohistochemical analysis of synaptophysin (SPH, pink) and VGAT (yellow) in coronal brain sections. Representative images of thalamus are shown. Scalebars = 50 μm.F. Morphological analysis of cortical microglia. Representative maximum intensity projections of confocal z-stack images showing IBA1+ microglial cells of female WT, Grn-/-, Trem2-/- and Double-/- mice (scalebar = 50 µm). Arrows point to individual microglia, which are shown as three-dimensional reconstruction, scalebar = 10 µm.


Figure 9A . Immunoassay - based quantification of neurofilament - light chain ( NfL ) protein levels in CSF of 6 - month - old Grn - / - ( n = 4 ) , Trem2 - / - ( n = 4 ) , Double - / - ( n = 4 ) and WT ( n = 5 ) male mice . B . Immunoassay - based quantification of NfL levels in CSF of 14 - month - old Grn - / - , Trem2 - / - , Double - / - and WT female mice ( n = 3 per genotype ) . Data information : Data in A , B represent mean ± SEM . For statistical analysis in A - B two - way ANOVA with Dunnett ` s post hoc test was used the unpairedD . Transcript levels of all significantly changed genes in Double - / - vs . Grn - / - brain mRNA of 6 - month - old and 14 - month - old mice analyzed in C . Grin3b was under detection limit in the 14 - month - old cohort . Transcript expression is normalized to the mean of the WT cohort . Data in D represent mean ± SEM . in D two - tailed student ` s t - test was performed . F . Bar graph illustrates individual FDG - PET values derived from a whole brain volume of interest . Data represent mean ± SD . 8 - 15 female mice per group at an average age of 10 . 7 ± 1 . 5 months ( Grn - / - n = 8 , Trem2 - / - n = 10 , Double - / - n = 10 , WT n = 15 ) were used . Data in F two - tailed student ` s t - test was 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 9A. Immunoassay-based quantification of neurofilament-light chain (NfL) protein levels in CSF of 6-month-old Grn-/- (n=4), Trem2-/-(n=4), Double-/- (n=4) and WT (n=5) male mice. B. Immunoassay-based quantification of NfL levels in CSF of 14-month-old Grn-/-, Trem2-/-, Double-/- and WT female mice (n=3 per genotype). Data information: Data in A, B represent mean ± SEM. For statistical analysis in A-B two-way ANOVA with Dunnett`s post hoc test was used the unpairedD. Transcript levels of all significantly changed genes in Double-/- vs. Grn-/- brain mRNA of 6-month-old and 14-month-old mice analyzed in C. Grin3b was under detection limit in the 14-month-old cohort. Transcript expression is normalized to the mean of the WT cohort. Data in D represent mean ± SEM. in D two-tailed student`s t-test was performed.F. Bar graph illustrates individual FDG-PET values derived from a whole brain volume of interest. Data represent mean ± SD. 8-15 female mice per group at an average age of 10.7 ± 1.5 months (Grn-/- n = 8, Trem2-/- n = 10, Double-/- n = 10, WT n = 15) were used. Data in F two-tailed student`s t-test was performed.


Figure 1A HEK293T were transfected with NF - kBluciferase reporter plasmid and FLAG - tagged full - length CARD14 ( FL ) or CARD14sh ( short ) at the indicated concentrations and analyzed 24 h later . ( A ) NF - kB reporter gene expression . HEK293T were transfected with NF - kB luciferase reporter plasmid and FLAG - tagged full - length CARD14 ( FL ) or CARD14sh ( short ) at the indicated concentrations and analyzed 24 h later . ( B ) IL - 8 secretion . Values are the mean of triplicates ± s . e . P - values are compared against empty vector treatment . Significance levels , ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 by student t test . Unless otherwise stated , p - values are p < 0 . 001 . HEK293T were transfected with NF - kB luciferase reporter plasmid and FLAG - tagged full - length CARD14 ( FL ) or CARD14sh ( short ) at the indicated concentrations and analyzed 24 h later . ( C ) Immunoblotting for c - Jun , phospho - c - Jun , JNK , phospho - JNK , p38 and phospho - p38 as indicated . Actin was used as a loading control . Data are representative of two independent experiments .

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 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 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

Figure 1A HEK293T were transfected with NF-kBluciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (A) NF-kB reporter gene expression.HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (B) IL-8 secretion. Values are the mean of triplicates ± s.e. P-values are compared against empty vector treatment. Significance levels, ns P>0.05, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Unless otherwise stated, p-values are p<0.001.HEK293T were transfected with NF-kB luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (C) Immunoblotting for c-Jun, phospho-c-Jun, JNK, phospho-JNK, p38 and phospho-p38 as indicated. Actin was used as a loading control. Data are representative of two independent experiments.


Figure 2 ( A ) MALT1 interacts with CARD14sh in a BCL10 - dependent manner . HEK293T cells were transfected with FLAG - CARD14sh , Myc - MALT1 and FLAG - BCL10 as indicated . 24 h later , MALT1 was immunoprecipitated ( IP ) from cell lysates with anti - MALT1 . CARD14sh and BCL10 co - immunoprecipitation with MALT1 was detected by immunoblotting with anti - FLAG . Immunoprecipitated MALT1 was detected by immunoblotting with anti - myc . The closed arrowhead shows the position of CARD14 , the double arrowhead shows the position of BCL10 . The asterisk indicates a non - specific band . Total expression levels of transfected proteins are shown by immunoblotting of a fraction of the cell lysates with the indicated antibodies ( bottom panel ) . ( B ) MALT1 co - expression potentiates CARD14sh - induced JNK and p38 MAP kinase activation . HEK293T cells were transfected with the indicated concentrations of FLAG - tagged CARD14sh , with or without MALT1 ( 30ng ) . Transfection with FLAG - TRAF6 and FLAG - CARD11 ( L232LI ) were used as a positive control . c - Jun accumulation , JNK and p38 phosphorylation were analyzed by immunoblotting with the antibodies indicated . Actin was used as a loading control . ( C ) MALT1 co - expression potentiates NF - kB activation by pathogenic CARD14 mutants . HEK293T were transfected with NF - kB reporter plasmid and wild - type ( WT ) FLAG - CARD14 or the indicated psoriasis - associated CARD14 mutants ( 15ng ) , with or without MALT1 ( 15ng ) . Luciferase activity in cell lysates was measured after 24 h . Values are the mean of triplicates ± s . e . Significance levels , * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 by student t test . Expression of transfected proteins was verified by western blotting ( bottom panel ) . ( D ) CARD14 ( E138A ) mutation enhances MALT1 binding . HEK293T cells were transfected with wild - type ( WT ) FLAG - CARD14sh or the indicated CARD14sh mutants and Myc - MALT1 . 24 h later , MALT1 was immunoprecipitated ( IP ) from cell lysates with anti - MALT1 and CARD14 co - immunoprecipitation was detected by immunoblotting with anti - FLAG . Immunoprecipitated MALT1 was detected by immunoblotting with anti - MALT1 . The asterisk indicates a non - specific band . Immunoprecipitation with a non - relevant antibody ( rIgG ) was used as a negative control ( first lane ) . Total expression levels of transfected proteins are shown by immunoblotting of total cell lysates ( bottom panel ) . Data are representative of two independent experiments .

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 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 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

Figure 2(A) MALT1 interacts with CARD14sh in a BCL10-dependent manner. HEK293T cells were transfected with FLAG-CARD14sh, Myc-MALT1 and FLAG-BCL10 as indicated. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1. CARD14sh and BCL10 co-immunoprecipitation with MALT1 was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-myc. The closed arrowhead shows the position of CARD14, the double arrowhead shows the position of BCL10. The asterisk indicates a non-specific band. Total expression levels of transfected proteins are shown by immunoblotting of a fraction of the cell lysates with the indicated antibodies (bottom panel).(B) MALT1 co-expression potentiates CARD14sh-induced JNK and p38 MAP kinase activation. HEK293T cells were transfected with the indicated concentrations of FLAG-tagged CARD14sh, with or without MALT1 (30ng). Transfection with FLAG-TRAF6 and FLAG-CARD11(L232LI) were used as a positive control. c-Jun accumulation, JNK and p38 phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control.(C) MALT1 co-expression potentiates NF-kB activation by pathogenic CARD14 mutants. HEK293T were transfected with NF-kB reporter plasmid and wild-type (WT) FLAG-CARD14 or the indicated psoriasis-associated CARD14 mutants (15ng), with or without MALT1 (15ng). Luciferase activity in cell lysates was measured after 24 h. Values are the mean of triplicates ± s.e. Significance levels, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Expression of transfected proteins was verified by western blotting (bottom panel).(D) CARD14(E138A) mutation enhances MALT1 binding. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14sh or the indicated CARD14sh mutants and Myc-MALT1. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1 and CARD14 co-immunoprecipitation was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-MALT1. The asterisk indicates a non-specific band. Immunoprecipitation with a non-relevant antibody (rIgG) was used as a negative control (first lane). Total expression levels of transfected proteins are shown by immunoblotting of total cell lysates (bottom panel). Data are representative of two independent experiments.


Figure 3 ( A ) MALT1 expression is essential for CARD14sh - induced NF - kB activation . MALT1 - deficient HEK293T cells were transfected with NF - kB reporter plasmid and the indicated expression plasmids . Luciferase activity in cell lysates was measured 24 h later . CARD11 ( L232LI ) and TRAF6 transfection were used as MALT1 - dependent and MALT1 - independent controls , respectively . Values are the mean of triplicates ± s . e . Expression of the transfected plasmids was verified by immunoblotting ( lower panel ) . ( B ) MALT1 expression is essential for CARD14sh - induced JNK activation . MALT1 - deficient HEK293T cells were transfected with FLAG - CARD14sh ( 30ng ) either alone or together with either wild - type ( MALT1WT ) or catalytically inactive MALT1 ( MALT1C / A ) as indicated . c - Jun accumulation and JNK phosphorylation were analyzed by immunoblotting with the antibodies indicated . Actin was used as a loading control . ( C ) MALT1 deficiency inhibits CARD14 - induced gene expression in HaCaT keratinocytes . HaCaT cells were transfected with scrambled ( scr ) or MALT1 - targeting siRNA prior to doxycycline - induced CARD14 expression as described in materials and methods . IL - 8 and MCP - 1 concentration in the cell supernatants was measured 8 h ( IL - 8 ) or 24 h ( MCP - 1 ) later by ELISA . Values are the mean of triplicates ± s . e . Doxycycline - induced CARD14 expression and MALT1 knock - down were verified by immunoblotting ( right panel ) . Data are representative of two independent experiments .

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 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 O O O O O O O O O O O O O O O O O O O O O O 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) MALT1 expression is essential for CARD14sh-induced NF-kB activation. MALT1-deficient HEK293T cells were transfected with NF-kB reporter plasmid and the indicated expression plasmids. Luciferase activity in cell lysates was measured 24 h later. CARD11(L232LI) and TRAF6 transfection were used as MALT1-dependent and MALT1-independent controls, respectively. Values are the mean of triplicates ± s.e. Expression of the transfected plasmids was verified by immunoblotting (lower panel).(B) MALT1 expression is essential for CARD14sh-induced JNK activation. MALT1-deficient HEK293T cells were transfected with FLAG-CARD14sh (30ng) either alone or together with either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A) as indicated. c-Jun accumulation and JNK phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control.(C) MALT1 deficiency inhibits CARD14-induced gene expression in HaCaT keratinocytes. HaCaT cells were transfected with scrambled (scr) or MALT1-targeting siRNA prior to doxycycline-induced CARD14 expression as described in materials and methods. IL-8 and MCP-1 concentration in the cell supernatants was measured 8 h (IL-8) or 24 h (MCP-1) later by ELISA. Values are the mean of triplicates ± s.e. Doxycycline-induced CARD14 expression and MALT1 knock-down were verified by immunoblotting (right panel). Data are representative of two independent experiments.


Figure 4 ( A ) CARD14 signaling induces CYLD and A20 cleavage . HEK293T cells were transfected with different concentrations of FLAG - CARD14 , with or without MALT1 as indicated . CARD11 ( L232LI ) transfection was used as a positive control . Endogenous CYLD and A20 processing was analyzed by immunoblotting . CYLD and A20 cleavage fragments are indicated by an arrow . ( B ) CARD14 signaling induces CYLD cleavage after R324 . HEK293T cells were transfected with different concentrations of FLAG - CARD14sh and either wild - type ( CYLDWT ) or non - cleavable CYLD ( CYLDR324A ) . CYLD processing was analyzed by immunoblotting . The CYLD cleavage fragment is indicated by an arrow . ( C ) Expression of catalytically active MALT1 is necessary for CARD14 - induced CYLD cleavage . MALT1 - deficient HEK293T cells in which MALT1 expression was restored by transfection with different concentrations of either wild - type ( MALT1WT ) or catalytically inactive MALT1 ( MALT1C / A ) , were co - transfected with FLAG - CARD14sh and CYLD as indicated . CYLD processing was analyzed by immunoblotting . The CYLD cleavage fragment is indicated by an arrow . ( D ) Psoriasis - associated CARD14 mutation enhances activation of MALT1 proteolytic activity . HEK293T cells were transfected with wild - type ( WT ) FLAG - CARD14 or different psoriasis - associated CARD14 mutants ( 20ng ) , with or without MALT1 ( 20 ng ) as indicated . Endogenous CYLD and A20 processing , as well as IκBα phosphorylation , were analyzed by immunoblotting . The CYLD and A20 cleavage fragments are indicated by an arrow . ( E ) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14 - induced gene expression in HaCaT keratinocytes . CARD14 expression was induced by doxycycline ( dox ) treatment in the absence or presence of different concentrations of mepazine as indicated . The next morning , cell culture medium was refreshed and IL - 8 and MCP - 1 concentration in the cell supernatant was measured 8 h later by ELISA . TNF stimulation ( MALT1 - independent ) was used as a negative control . Values are the mean of triplicates ± s . e . Data are representative of two independent experiments . Significance levels , * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 by student t test .

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 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 O O O O O O 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 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

Figure 4(A) CARD14 signaling induces CYLD and A20 cleavage. HEK293T cells were transfected with different concentrations of FLAG-CARD14, with or without MALT1 as indicated. CARD11(L232LI) transfection was used as a positive control. Endogenous CYLD and A20 processing was analyzed by immunoblotting. CYLD and A20 cleavage fragments are indicated by an arrow.(B) CARD14 signaling induces CYLD cleavage after R324. HEK293T cells were transfected with different concentrations of FLAG-CARD14sh and either wild-type (CYLDWT) or non-cleavable CYLD (CYLDR324A). CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow.(C) Expression of catalytically active MALT1 is necessary for CARD14-induced CYLD cleavage. MALT1-deficient HEK293T cells in which MALT1 expression was restored by transfection with different concentrations of either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A), were co-transfected with FLAG-CARD14sh and CYLD as indicated. CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow.(D) Psoriasis-associated CARD14 mutation enhances activation of MALT1 proteolytic activity. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14 or different psoriasis-associated CARD14 mutants (20ng), with or without MALT1 (20 ng) as indicated. Endogenous CYLD and A20 processing, as well as IκBα phosphorylation, were analyzed by immunoblotting. The CYLD and A20 cleavage fragments are indicated by an arrow.(E) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14-induced gene expression in HaCaT keratinocytes. CARD14 expression was induced by doxycycline (dox) treatment in the absence or presence of different concentrations of mepazine as indicated. The next morning, cell culture medium was refreshed and IL-8 and MCP-1 concentration in the cell supernatant was measured 8 h later by ELISA. TNF stimulation (MALT1-independent) was used as a negative control. Values are the mean of triplicates ± s.e. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.


Figure 5 ( A - B ) CYLD and A20 inhibit CARD14sh - induced NF - kB activation . HEK293T cells were transfected with NF - kB reporter plasmid and FLAG - tagged CARD14sh ( 30ng ) , with or without the indicated concentrations of CYLD ( A ) or A20 ( B ) . Luciferase activity in cell lysates was measured 24 h later . Closed arrowheads indicate full length A20 and CYLD ; arrows indicate cleaved A20 and CYLD . Data are representative of three independent experiments . ( C ) A20 and ABIN - 1 inhibit CARD14 - induced NF - kB activation . HEK293T cells were transfected with NF - kB reporter plasmid , A20 ( 3 ng ) or ABIN1 ( 15ng ) and the indicated concentrations of FLAG - tagged CARD14sh . Luciferase activity in cell lysates was measured 24 h later . Values are the mean of triplicates ± s . e . Protein expression of transfected plasmids was verified by western blot and is shown in the accompanying panels . ( D ) A20 inhibits CARD14 - induced NF - kB activation through its ZnF domains . HEK293T cells were transfected with NF - kB reporter plasmid , FLAG - CARD14sh ( 20ng ) and wild - type ( WT ) or mutant A20 expression plasmids ( 3ng ) as indicated . Luciferase activity in cell lysates was measured after 24 h . CARD14 and A20 expression was measured by immunoblotting ( bottom panel ) . Data are representative of two independent experiments .

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 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 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

Figure 5(A-B) CYLD and A20 inhibit CARD14sh-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid and FLAG-tagged CARD14sh (30ng), with or without the indicated concentrations of CYLD (A) or A20 (B). Luciferase activity in cell lysates was measured 24 h later. Closed arrowheads indicate full length A20 and CYLD; arrows indicate cleaved A20 and CYLD. Data are representative of three independent experiments.(C) A20 and ABIN-1 inhibit CARD14-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid, A20 (3 ng) or ABIN1 (15ng) and the indicated concentrations of FLAG-tagged CARD14sh. Luciferase activity in cell lysates was measured 24 h later. Values are the mean of triplicates ± s.e. Protein expression of transfected plasmids was verified by western blot and is shown in the accompanying panels.(D) A20 inhibits CARD14-induced NF-kB activation through its ZnF domains. HEK293T cells were transfected with NF-kB reporter plasmid, FLAG-CARD14sh (20ng) and wild-type (WT) or mutant A20 expression plasmids (3ng) as indicated. Luciferase activity in cell lysates was measured after 24 h. CARD14 and A20 expression was measured by immunoblotting (bottom panel). Data are representative of two independent experiments.


Figure 6 ( A ) CARD14 ( E138A ) induces cytokine and chemokine expression in human primary keratinocytes . Cells were transfected with empty vector ( EV ) or CARD14 ( E138A ) as indicated . 24 h later , the secretion of 36 different human cytokines , chemokines , and acute phase proteins was analyzed via a multiplex antibody array . Quantification was done using ImageJ software and is expressed as relative CARD14 / EV values for a selected number of proteins ( bottom panel ) . ( B ) MALT1 deficiency inhibits CARD14 ( E138A ) - induced cytokine and chemokine expression in human primary keratinocytes . Cells were transfected with scrambled ( scr ) or MALT1 - targeting siRNA as indicated , 48 h later followed by transfection with CARD14 ( E138A ) . After 24 h , cytokine and chemokine expression was analyzed and is expressed as described in ( A ) . ( C ) Pathogenic CARD14 mutation enhances MALT1 - dependent CARD14 - induced IL - 8 expression in human primary keratinocytes . Cells were transfected with scrambled ( scr ) or MALT1 - targeting siRNA as indicated , 48 h later followed by transfection with CARD14 wild - type ( WT ) , CARD14 ( G117S ) or CARD14 ( E138A ) as indicated . TNF treatment was used as a control . After 24 h , IL - 8 concentration in the cell supernatant was analyzed via ELISA . Values are the mean of triplicates ± s . e . Expression levels of MALT1 and CARD14 variants is verified by immunoblotting ( bottom panel ) . ( D ) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14 - induced IL - 8 production and A20 processing in human primary keratinocytes . Cells were transfected with different CARD14 variants in the presence or absence of mepazine as indicated . TNF treatment was used as a control . 24 h later , IL - 8 levels in the cell supernatant were measured by ELISA . Values are the mean of triplicates ± s . e . Expression of different CARD14 variants and CARD14 - induced processing of endogenous A20 were analyzed by immunoblotting . Data are representative of two independent experiments . Significance levels , * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 by student t test .

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

Figure 6(A) CARD14(E138A) induces cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with empty vector (EV) or CARD14(E138A) as indicated. 24 h later, the secretion of 36 different human cytokines, chemokines, and acute phase proteins was analyzed via a multiplex antibody array. Quantification was done using ImageJ software and is expressed as relative CARD14/EV values for a selected number of proteins (bottom panel).(B) MALT1 deficiency inhibits CARD14(E138A)-induced cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14(E138A). After 24 h, cytokine and chemokine expression was analyzed and is expressed as described in (A).(C) Pathogenic CARD14 mutation enhances MALT1-dependent CARD14-induced IL-8 expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14 wild-type (WT), CARD14(G117S) or CARD14(E138A) as indicated. TNF treatment was used as a control. After 24 h, IL-8 concentration in the cell supernatant was analyzed via ELISA. Values are the mean of triplicates ± s.e. Expression levels of MALT1 and CARD14 variants is verified by immunoblotting (bottom panel).(D) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14-induced IL-8 production and A20 processing in human primary keratinocytes. Cells were transfected with different CARD14 variants in the presence or absence of mepazine as indicated. TNF treatment was used as a control. 24 h later, IL-8 levels in the cell supernatant were measured by ELISA. Values are the mean of triplicates ± s.e. Expression of different CARD14 variants and CARD14-induced processing of endogenous A20 were analyzed by immunoblotting. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.


Figure 1 ( A ) Substrate - dependent nitrogenase activity , measured through reduction of acetylene into ethylene , in isolated B . diazoefficiens bacteroids upon supplementation of malate , succinate , arginine , and nodule extract . Data represents the mean and standard error of the mean of at least eight independent replciates . ( B ) Fold change in intracellular ATP level in isolated B . diazoefficiens bacteroids upon supplementation of succinate , arginine , and nodule extract . The values obtained for each of the 4 individual measurements ( points ) together with the regression trajectory of each experiment is shown .

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

Figure 1(A) Substrate-dependent nitrogenase activity, measured through reduction of acetylene into ethylene, in isolated B. diazoefficiens bacteroids upon supplementation of malate, succinate, arginine, and nodule extract. Data represents the mean and standard error of the mean of at least eight independent replciates.(B) Fold change in intracellular ATP level in isolated B. diazoefficiens bacteroids upon supplementation of succinate, arginine, and nodule extract. The values obtained for each of the 4 individual measurements (points) together with the regression trajectory of each experiment is shown.


Figure 3 ( A ) The aerial part of M . truncatula upon infection with S . meliloti ∆ argI1 , ∆ argI2 , ∆ argI1 , 2 and the dicarboxylate transport mutant ∆ dctAB were smaller than those inoculated with the wild type strain , highlighting the importance of arginine catabolism for nitrogen fixation ( B ) Cross sections of nodules bearing S . meliloti WT or arginine catabolism mutant ∆ argI1 , ∆ argI2 reveals the yellowish color of non - functional nodules induced by the ∆ argI1 ∆ argI2 double deletion mutant . ( C ) Bacteroid ultrastructure across nodule sections determined by scanning electron microscopy indicates the presence of hollow nodules with aberrant cell morphology in the ∆ argI1 ∆ argI2 double deletion mutant defective in arginine catabolism . ( D ) Nitrogenase activity in M . truncatula nodules inoculated with S . meliloti strains defective in arginine catabolism . Data points are the mean of at least 30 plants measured after 8 weeks post inoculation ; error bars indicate standard error of the mean .

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 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 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 3(A) The aerial part of M. truncatula upon infection with S. meliloti ∆argI1, ∆argI2, ∆argI1,2 and the dicarboxylate transport mutant ∆dctAB were smaller than those inoculated with the wild type strain, highlighting the importance of arginine catabolism for nitrogen fixation(B) Cross sections of nodules bearing S. meliloti WT or arginine catabolism mutant ∆argI1, ∆argI2 reveals the yellowish color of non-functional nodules induced by the ∆argI1 ∆argI2 double deletion mutant.(C) Bacteroid ultrastructure across nodule sections determined by scanning electron microscopy indicates the presence of hollow nodules with aberrant cell morphology in the ∆argI1 ∆argI2 double deletion mutant defective in arginine catabolism.(D) Nitrogenase activity in M. truncatula nodules inoculated with S. meliloti strains defective in arginine catabolism. Data points are the mean of at least 30 plants measured after 8 weeks post inoculation; error bars indicate standard error of the mean.


Figure 4 ( B ) Heat map representing enzymatic activities participating in the arginine transamination network with relative enzyme activities highlighted as a color map Rows are annotated on the left with the abbreviated names of the substrates and on the right with the corresponding EC numbers . Columns are annotated by corresponding enzyme names . ( C ) Recombinant enzymes involved in arginine catabolism were combined into a single reaction mixture using arginine and pyruvate as substrates . Substrate ( S ) consumption into the products ( P ) succinate and alanine , including their intermediates ( I1 , I2 ) , was determined along the time series using LC - MS / MS . ( D ) The relative metabolite abundance along the reaction time - course is highlighted as a color

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

Figure 4(B) Heat map representing enzymatic activities participating in the arginine transamination network with relative enzyme activities highlighted as a color map Rows are annotated on the left with the abbreviated names of the substrates and on the right with the corresponding EC numbers. Columns are annotated by corresponding enzyme names.(C) Recombinant enzymes involved in arginine catabolism were combined into a single reaction mixture using arginine and pyruvate as substrates. Substrate (S) consumption into the products (P) succinate and alanine, including their intermediates (I1,I2), was determined along the time series using LC-MS/MS.(D) The relative metabolite abundance along the reaction time-course is highlighted as a color map


Figure 1 ( B ) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 ( Casparian strip integrity factor 2 ) ligand treatment . Lignin and cellulosic ( unmodified ) cell walls are stained with Basic Fuchsin and Calcofluor White , shown in magenta and white respectively . Schematics are indicating the position of optical sections in a 3D illustration . For each condition , at least 10 roots were tested and showed similar results in two independent experiments . White arrows indicate sites of excess lignification on the cortex facing ( outer ) side . Scale Bar = 5 µm . Data information : " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface . ( C , D ) Localization of SGN1 - Citrine and lignin deposition patterns in pCASP1 : : SGN1 - Citrine lines in wild - type ( Col ) and different mutant backgrounds ( sgn1 , sgn3 , cif1 cif2 ) ( C ) . myrpalm - SGN1 - Citrine localization and lignin deposition patterns in pCASP1 : : myrpalm - SGN1 - Citrine lines ( D ) . Lignin ( Basic Fuchsin ) and cell walls ( Calcoflour White ) are shown in magenta and white respectively . For this experiment , two or three independent lines were tested . From each transgenic line , 2 positions from 12 roots were observed and representative pictures are shown in the figure . Schematics are indicating the position of optical sections in a 3D illustration . White arrows in ( D ) highlight excess lignification on the pericycle - facing ( inner ) side . Scale bars = 5 µm . Data information : " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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 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

Figure 1(B) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex facing (outer) side. Scale Bar = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.(C,D) Localization of SGN1-Citrine and lignin deposition patterns in pCASP1::SGN1-Citrine lines in wild-type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm-SGN1-Citrine localization and lignin deposition patterns in pCASP1::myrpalm-SGN1-Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcoflour White) are shown in magenta and white respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle-facing (inner) side. Scale bars = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


Figure 2 ( A ) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1 - GFP domain at around 10 cells after onset of CASP1 - GFP expression in 5 - day - old seedlings . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to maximum or minimum values no further than 1 . 5 times IQR ( interquartile range , the distance between the first and third quartiles ) . One - way ANOVA was performed followed by Tukey ' s test . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA and Tukey ' s test , 12 roots in total were observed for each condition in two independent assays ) . ( B ) Propidium iodide ( PI ) penetration assay in the presence or the absence of CIF2 . CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to the maximum or minimum values no further than 1 . 5 times IQR . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA and Tukey ' s test . During two independent experiments , 10 roots in total were tested for each condition ) . ( C ) [ γ - 32P ] ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1 . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence and equal loading of recombinant proteins . Note that a kinase - dead SGN1 variant was used to avoid auto - phosphorylation activity of SGN1 . Also note that trigger factor represents a very big tag protein , accounting for the high migration of TF - SGN1 . Representative result of three independent experiments is shown .

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 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 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

Figure 2(A) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1-GFP domain at around 10 cells after onset of CASP1-GFP expression in 5-day-old seedlings. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to maximum or minimum values no further than 1.5 times IQR (interquartile range, the distance between the first and third quartiles). One-way ANOVA was performed followed by Tukey's test. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test, 12 roots in total were observed for each condition in two independent assays).(B) Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition).(C) [γ-32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase-dead SGN1 variant was used to avoid auto-phosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF-SGN1. Representative result of three independent experiments is shown.


Figure 3 ( A ) Localization of Citrine - RBOHD ( left ) or Citrine - RBOHF ( right ) in endodermal ( en ) cells . Both proteins were expressed under the control of pCASP1 , an endodermis - specific promoter . Representative pictures are shown , 2 positions from 10 roots for each transgenic line were inspected . Scale bar = 5 µm . ( B ) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2 - hour 100 nM CIF2 peptide treatment . Arrowheads indicate excess lignification . Pictures are shown as overviews ( maximum projection ) or median sections . Lignin and cell walls are shown with magenta ( stained with Basic Fuchsin ) and gray ( stained with Calcofluor White ) respectively . Representative pictures are shown , 12 roots ( overview ) and 2 positions in 12 roots ( median section ) were inspected . Scale bars = 20 µm ( lignin overviews ) , 5 µm ( median sections ) . " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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

Figure 3(A) Localization of Citrine-RBOHD (left) or Citrine-RBOHF (right) in endodermal (en) cells. Both proteins were expressed under the control of pCASP1, an endodermis-specific promoter. Representative pictures are shown, 2 positions from 10 roots for each transgenic line were inspected. Scale bar = 5 µm.(B) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2-hour 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White) respectively. Representative pictures are shown, 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 µm (lignin overviews), 5 µm (median sections). "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


Figure 4 ( A ) Co - visualization of pCASP1 : : SGN1 - mCherry and pCASP1 : : SGN3 - GFP . Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain ( Arrow ) . Representative picture is shown , 3 - 6 roots in 4 different transgenic lines were inspected . Scale bar = 10 µm . " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface . ( B , C ) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours . Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in ( sky blue and creme boxes in C and black boxes in D ) . Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment . Scale bars = 500 nm . ( D ) In situ H2O2 detection at Casparian strips in WT , sgn3 , sgn1 , rbohF , rbohD and rbohDF with or without 24 - hour treatment of 1 µM CIF2 . Brackets and arrowheads indicate Casparian strips ( seen as uniformly whitish cell wall areas ) and H2O2 production sites ( black area ) respectively . Scale bar = 1000 nm . ( E ) Quantification of ROS production as a number of dark pixels area ( n = 23 - 44 sites from 5 roots of each condition in one experiment . ) . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to the maximum or minimum values no further than 1 . 5 times IQR . Different letters mean significant statistical differences . ( p < 0 . 01 , One - way ANOVA and Tukey ' s test . )

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 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 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

Figure 4(A) Co-visualization of pCASP1::SGN1-mCherry and pCASP1::SGN3-GFP. Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain (Arrow). Representative picture is shown, 3-6 roots in 4 different transgenic lines were inspected. Scale bar = 10 µm. "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.(B,C) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours. Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in (sky blue and creme boxes in C and black boxes in D). Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment. Scale bars = 500 nm.(D) In situ H2O2 detection at Casparian strips in WT, sgn3, sgn1, rbohF, rbohD and rbohDF with or without 24-hour treatment of 1 µM CIF2. Brackets and arrowheads indicate Casparian strips (seen as uniformly whitish cell wall areas) and H2O2 production sites (black area) respectively. Scale bar = 1000 nm.(E) Quantification of ROS production as a number of dark pixels area (n = 23 - 44 sites from 5 roots of each condition in one experiment.). In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters mean significant statistical differences. (p<0.01, One-way ANOVA and Tukey's test.)


Figure 5 ( A ) [ γ - 32P ] ATP radioactive in vitro kinase assay of TF - SGN1 against GST - N - terminal cytoplasmic domains of RBOHD or F . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence and equal loading of recombinant proteins . Experiments were done independently three times with similar results . ( B ) HEK293Tcell based NOX activation assay . Cells were transfected with the indicated plasmid combinations . The phosphatase inhibitor Calyculin A was added directly before the start of the measurements . Each data point represents the mean of six wells analyzed in parallel , bars indicate S . D . . Experiments were repeated three

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 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

Figure 5(A) [γ-32P]ATP radioactive in vitro kinase assay of TF-SGN1 against GST-N-terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results.(B) HEK293Tcell based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel, bars indicate S.D.. Experiments were repeated three times


Figure 6 ( A ) CASP1 - GFP and lignin deposition in WT , sgn3 , rbohD , rbohF and rbohDF . CASP1 - GFP and lignin ( fuchsin ) are presented in green and magenta respectively . Pictures were obtained from more than 10 roots from each background with similar results . Scale bar = 10 µm . ( B ) Time lapse imaging of single - or cotreatment of 10 nM CIF2 with 25 µM cycloheximide ( CHX ) on CASP1 - GFP in cif1 cif2 . Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium . Scale bar = 10 µm ( See also movie EV1 ) . ( C ) Quantification of ( B ) . Relative numbers of holes in CASP1 - GFP domain after single - or co - treatment with CIF2 or CHX from the pictures in ( B ) . Bars are S . D . * indicates statistical significance from all other conditions ( p < 0 . 01 ) after one - way ANOVA and Tukey test . 6 roots in total for each condition were observed during two independent tests . ( D ) Fold - change of 930 genes ( p < 0 . 05 and log2 ( Fold change ) ≥ 1 or ≤ - 1 at least one time point in one genotype ) after CIF2 treatment at indicated time points in WT , cif1 , 2 and sgn3 . Degree of the fold changes are shown in color code as indicated . ( E ) Relative expression levels of PER15 to CLATHRIN control in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( F ) Fold changes of PER15 in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistical significance differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( G ) Relative expression levels of PER49 to CLATHRIN control in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( H ) Fold changes of PER49 in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( I ) Relative fold changes of PER15 in pCASP1 : : SGN1 - Citrine and pCASP1 : : myrpalm - SGN1 - Citrine compared to the expression level in WT . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( J ) Relative fold changes of PER49 in pCASP1 : : SGN1 - Citrine and pCASP1 : : myrpalm - SGN1 - Citrine compared to the expression level in WT . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 6(A) CASP1-GFP and lignin deposition in WT, sgn3, rbohD, rbohF and rbohDF. CASP1-GFP and lignin (fuchsin) are presented in green and magenta respectively. Pictures were obtained from more than 10 roots from each background with similar results. Scale bar = 10 µm.(B) Time lapse imaging of single- or cotreatment of 10 nM CIF2 with 25 µM cycloheximide (CHX) on CASP1-GFP in cif1 cif2. Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium. Scale bar = 10 µm (See also movie EV1). (C) Quantification of (B). Relative numbers of holes in CASP1-GFP domain after single- or co-treatment with CIF2 or CHX from the pictures in (B). Bars are S.D. * indicates statistical significance from all other conditions (p<0.01) after one-way ANOVA and Tukey test. 6 roots in total for each condition were observed during two independent tests. (D) Fold-change of 930 genes (p<0.05 and log2(Fold change) ≥1 or ≤ -1 at least one time point in one genotype) after CIF2 treatment at indicated time points in WT, cif1,2 and sgn3. Degree of the fold changes are shown in color code as indicated.(E) Relative expression levels of PER15 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (F)Fold changes of PER15 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistical significance differences (p < 0.01, ANOVA and Tukey test). (G) Relative expression levels of PER49 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (H) Fold changes of PER49 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (I)Relative fold changes of PER15 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (J)Relative fold changes of PER49 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).


Fig 1 ( A ) Macrophages were exposed to increasing concentrations of cell - free RNase / DNase I treated iodixanol velocity gradient purified HIV for 24 h , harvested , lysed and analyzed for endogenous LC3B , SQSTM1 , and ACTB by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 3 . ( B ) Macrophages were exposed to increasing concentrations of cell - free RNase / DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A , harvested , lysed and analyzed for endogenous LC3B , SQSTM1 , and ACTB by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 3 . ( C ) Macrophages were exposed to mock , infectious , AT - 2 - inactivated , or RNase / DNase I treated AT - 2 - inactivated iodixanol velocity gradient purified HIVBa - L for 24 h . Cells were then harvested , lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O

Fig 1(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.(B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.(C) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h. Cells were then harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


Fig 2A ) Macrophages were transduced with non - specific scrambled shRNA ( shNS ) , or TLR8 shRNA ( shTLR8 ) and analyzed for TLR8 expression . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 . ( B ) Macrophages transduced with shNS or shTLR8 from ( A ) were exposed to infectious HIV ( + ) , AT - 2 - inactivated HIV ( AT ) , or RNase / DNase I treated AT - 2 - inactivated HIV ( R ) or mock infected ( - ) for 24 h , harvested , lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 . ( C ) Representative fluorescence microscopy images of HIV - infected macrophages which were fixed , permeabilized then stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) and antibody to LC3B ( green ) , SQSTM1 ( magenta ) , HIV p24 and p17 ( red ) . Co - localization of HIV and LC3B is represented by orange pixels , and co - localization of HIV , SQSTM1 , and LC3B is represented by cream pixels . Scale bars indicate 10 μm . ( D ) Macrophages were transduced with shNS or ATG13 shRNA ( shATG13 ) and analyzed for ATG13 expression . Bottom , a representative blot is shown . Top , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 . ( E ) Macrophages transduced with shNS or shATG13 from ( D ) were exposed to infectious HIV , 5 μg / mL ssRNA40 , 5 μg / mL ssRNA41 , or 100 nmol / L sirolimus for 24 h , harvested , lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 4 . ( F ) Macrophages were pretreated with 100 nmol / L bafilomycin A1 then exposed to mock , infectious , or RNase / DNase I treated AT - 2 - inactivated purified HIV , LyoVec , 5 μg / mL ssRNA41 , 5 μg / mL ssRNA40 , or 100 nmol / L sirolimus for 24 h , harvested , lysed and analyzed for endogenous LC3B , CYP27B1 and VDR by Western blotting . Left , a representative blot is shown . Right , densitometric analysis of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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 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 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 O O O 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

Fig 2A) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.(B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.(C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm.(D) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.(E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.(F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.