Elastin showing more pronounced ECM accumulation in eWAT of CD169 - DTR ( HFD 16 ) when compared with ECM accumulation in WT obese ( HFD 16 ) control ( DAPI , blue ; elastin , red ; CD31 , green ) . fourth image in each row shows three channels merged . Scale bar : 50 μm . Picrosirius red - depicts excessive collagen deposits in obese ( HFD 16 ) eWAT of CD169 - DTR mice when compared with collagen deposits in WT controls . Scale bar : 250 μm . Upper panels : brightfield microscopy ; lower panels : microscopy .

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

Elastin staining showing more pronounced ECM accumulation in eWAT of CD169-DTR (HFD 16 weeks) when compared with ECM accumulation in the WT obese (HFD 16 weeks) control (DAPI, blue; elastin, red; CD31, green). The fourth image in each row shows the three channels merged. Scale bar: 50 μm. Picrosirius red-staining depicts excessive collagen deposits in obese (HFD 16 weeks) eWAT of CD169-DTR mice when compared with collagen deposits in WT controls. Scale bar: 250 μm. Upper panels: brightfield microscopy; lower panels: fluorescence microscopy.


Violin plots showing pro - inflammatory cytokine profile ( Il - 6 , Tnf - α , Il - 1β ) of 23 clusters obtained from obese ( HFD 16 ) WT CD169 - DTR eWAT . Cluster 0 : ASC1 ; Cluster 1 : Stromal ; Cluster 2 : VEC1 ; Cluster 3 : VEC2 ; Cluster 4 : NK1 ; Cluster 5 : MAC / Mono1 ; Cluster 6 : ASC2 ; Cluster 7 : B ; Cluster 8 : cDC1 ; Cluster 9 : mono - derived cDC ; Cluster 10 : Th2 / ILC2 / Treg ; Cluster 11 : MAC / Mono3 ; Cluster 12 : Th17 ; Cluster 13 : VEC2 ; Cluster 14 : CD8 ; Cluster 15 : MLCs ; Cluster 16 : CD11b + DC ; Cluster 17 : Neutrophils ; Cluster 18 : cDC2 ; Cluster 19 : cDC ; Cluster 20 : NKT ; Cluster 21 : NK2 ; Cluster 22 : Mast / Basophils . ASC : adipocyte ; VEC : vascular endothelial ; MLC : mesothelial - like ; NK : natural killer ; MAC : macrophage ; Mono : monocyte ; DC : dendritic ; NKT : natural killer T .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O

Violin plots showing the pro-inflammatory cytokine profile (Il-6, Tnf-α, and Il-1β) of all 23 cell clusters obtained from obese (HFD 16 weeks) WT and CD169-DTR eWAT. Cluster 0: ASC1; Cluster 1: Stromal cells; Cluster 2: VEC1; Cluster 3: VEC2; Cluster 4: NK1; Cluster 5: MAC/Mono1; Cluster 6: ASC2; Cluster 7: B; Cluster 8: cDC1; Cluster 9: mono-derived cDC; Cluster 10: Th2/ILC2/Treg; Cluster 11: MAC/Mono3; Cluster 12: Th17; Cluster 13: VEC2; Cluster 14: CD8; Cluster 15: MLCs; Cluster 16: CD11b+ DC; Cluster 17: Neutrophils; Cluster 18: cDC2; Cluster 19: cDC; Cluster 20: NKT; Cluster 21: NK2; Cluster 22: Mast cells/Basophils. ASC: adipocyte stem cell; VEC: vascular endothelial cell; MLC: mesothelial-like cell; NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T cells.


( A ) Confocal scanning showing localization of a functional CDKA ; 1 : mVenus fusion in wildtype ( WT ) cartoons on top highlighting changes in abundance of CDKA ; 1 : mVenus in nucleus cytoplasm during course of meiosis . region colored in beige represents cytoplasm , in green nucleoplasm , in nucleolus . Scale bar : 10 μm .

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

(A) Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wildtype (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm.


( B ) Quantitative of signal distribution of versus fraction of CDKA ; 1 : mVenus during prophase I of meiosis as revealed by live imaging Twenty at each time point were used for . Error bars represent mean ± SD two biological replicates were performed .

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(B) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD and two biological replicates were performed.


( C ) Immunolocalization of CDKA ; 1 ( green ) ASY1 ( red ) on spread chromsomes in leptotene zygotene of wild - type expressing a functional PROCDKA ; 1 : CDKA ; 1 : Strep construct . last lane shows a magnification of region marked by red rectangle . Arrowheads indicate synapsed regions of homologous chromosomes where CDKA ; 1 is no longer present . Scale bar : 5 μm .

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

(C) Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromsomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm.


( D ) Chromosome spread of wildtype hypomorphic cdka ; 1 mutant CDKA ; 1T161D . ( a , h ) zygotene or zygotene - like stages ; ( b , i ) pachytene or pachytene - like stages ; ( c , j , k ) diakinesis or dikinesis - like stages ; ( d ) metaphase I ; ( e , i , m , n ) end of meiosis I with two ( e , m ) or three ( i ) pools of chromosomes ; ( f ) metaphase II ; ( g ) tetrad . Red arrowheads indicate initiated formation of a phragmoplast . arrowheads depict mitochondria . Scale bars : 10 μm .

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

(D) Chromosome spread analysis of the wildtype and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or dikinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm.


( A ) ASY1 : GFP ASY1T142D : GFP localization in late G2 leptotene of meiocytes of wildtype CDKA ; 1T161D mutants . ASY3 : RFP , highlighting chromsomes , was used as a marker for staging of meiosis . Scale bar : 5 μm .

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

(A) ASY1:GFP and ASY1T142D:GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA;1T161D mutants. ASY3:RFP, highlighting chromsomes, was used as a marker for the staging of meiosis. Scale bar: 5 μm.


( B ) Kinase assays of CDKA ; 1 - SDS , - TAM , - CYCA3 ; 1 complexes using ASY1 puried from baculovirus - infected insect as a substrate . upper panel shows autoradiograph . control reaction without CDKA ; 1 - cyclin indicates a background activity co - purified from insect . lower panel indicates loading by coomassie briliant blue ( CBB ) . Arrowheads indicate ASY1 asterisks depict relevant cyclin used which also gets phosphorylated in . Numbers indicate relative intensities of ASY1 bands .

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

(B) Kinase assays of CDKA;1-SDS, -TAM, and -CYCA3;1 complexes using ASY1 puried from baculovirus-infected insect cells as a substrate. The upper panel shows the autoradiograph. The control reaction without CDKA;1-cyclin complex indicates a background activity co-purified from insect cells. The lower panel indicates protein loading by coomassie briliant blue (CBB) staining. Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay. Numbers indicate the relative intensities of ASY1 bands.


( C ) upper panel shows a phos - tag gel of ASY11 - 300 ASY11 - 300 / T142V ; T184V with without CDKA ; 1 - SDS kinase complexes using an anti - MBP antibody . lower panel denotes loading of CDKA ; 1 using an anti - Strep antibody . Arrowheads represent as indicated .

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

(C) The upper panel shows a phos-tag gel analysis of ASY11-300 and ASY11-300/T142V;T184V with and without CDKA;1-SDS kinase complexes using an anti-MBP antibody. The lower panel denotes loading of CDKA;1 using an anti-Strep antibody. Arrowheads represent the proteins as indicated.


( B ) Localization patterns of different ASY1 : GFP variants together with ASY3 : RFP ( for staging of zygotene pachytene ) in a asy1 mutant background during prophase I . Bar : 5

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(B) Localization patterns of different ASY1:GFP variants together with ASY3:RFP (for staging of zygotene and pachytene) in a asy1 mutant background during prophase I. Bar: 5 μ


( C ) Signal distribution profiles of ASY1 : GFP ASY1T142V : GFP at leptotene many small peaks with low amplitude in ASY1T142V : GFP indicate diffused localization as opposed to clear peaks seen in wildtype .

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(C) Signal distribution profiles of ASY1:GFP and ASY1T142V:GFP at leptotene The many small peaks with low amplitude in ASY1T142V:GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype.


( D , E ) Immunolocalization of ASY1 ( D ) ZYP1 ( E ) in ASY1 : GFP ( asy1 ) ASY1T142V : GFP ( asy1 ) using anti - GFP anti - ZYP1 antibodies , respectively . was stained with DAPI ( blue ) . Scale bars : 5 μm .

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(D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.


( A ) Yeast two - hybrid interaction assays of ASY3 with different ASY1 variants . Monomeric GFP ( mGFP ) fused with AD ( activating domain ) BD ( binding domain ) were used as controls . Yeast harboring both AD BD plasmids were grown on synthetic medium supplied with glucose in absence of Leu Trp ( - L / T , left panel ) , on Synthetic Drop - out ( SD ) medium in absence of Leu , Trp , His ( - L / T / H , middle panel ) , on SD medium in absence of Leu , Trp , His Ade ( - L / T / H / A , right panel ) . Yeast were incubated until OD600 = 1 then diluted 10 - , 100 - 1000 - fold for assays .

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

(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays.


( B ) GST pull down of ASY3 with different ASY1 variants . numbers above bands show relative of bands . input pull down fractions were analyzed by immuno - blotting with anti - GST ( ASY3 ) anti - MBP ( ASY1 ) antibodies . ( C ) of pull down fractions of ASY1 as shown in ( B ) . band in pull down of ASY11 - 300 / T142V ; T184V at a Triton X - 100 of 0 . 5 % was defined as 1 . relative amount of ASY1 in pull down fractions was normalized by band of pulled - down ASY3 fraction . average band of ASY1 at different concentrations of Triton X - 100 used was plotted . Asteriks indicate significant difference ( two - t - , p < 0 . 05 ) . Error bars represent mean ± SD two biological replicates were performed . .

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

(B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies. (C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed..


( A ) Localization patterns of ASY1T142V ; T184V : GFP together with ASY3 - RFP in wildtype pch2 mutants . Please note that of ASY1T142V ; T184V : GFP in pch2 mutants were taken with increased sensitivity for a better visibility . Scale bar : 5 μm .

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(A) Localization patterns of ASY1T142V;T184V:GFP together with ASY3-RFP in the wildtype and pch2 mutants. Please note that images of ASY1T142V;T184V:GFP in pch2 mutants were taken with increased sensitivity for a better visibility. Scale bar: 5 μm.


( B ) Seed sets ( mean ± SD , n = 5 ) of WT , asy1 , pch2 , asy1 pch2 , ASY1T142V ; T184V : GFP ( asy1 ) ASY1T142V ; T184V : GFP ( asy1 pch2 ) . Asteriks indicate significant differences ( two - t - , P < 0 . 01 ) ns depicts no significant difference . Scale bar : 2 mm .

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(B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm.


( C ) Localization patterns of ASY1 : GFP , ASY1T142V ; T184V : GFP , ASY11 - 570 : GFP in wildtype ( WT ) / or in pch2 mutant at early prophase I . Scale bars : 5 μm .

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(C) Localization patterns of ASY1:GFP, ASY1T142V;T184V:GFP, and ASY11-570:GFP in the wildtype (WT) and/or in pch2 mutant plants at early prophase I. Scale bars: 5 μm.


( D ) Localization pattern of PCH2 : GFP together with ASY1 : RFP in meiocytes of wildtype . Arrowheads indicate chromosomal regions where ASY1 removal was concomitant with localization of PCH2 . Scale bar : 10 μm .

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(D) Localization pattern of PCH2:GFP together with ASY1:RFP in the male meiocytes of wildtype. Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2. Scale bar: 10 μm.


( B ) Interaction assays of different ASY1 fragments ( with without closure motif ) .

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(B) Interaction assays of different ASY1 fragments (with and without the closure motif).


( C ) Interaction of ASY1 closure motif ( ASY1571 - 596 ) with different ASY1 HORMA domain variants .

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(C) Interaction analysis of the ASY1 closure motif (ASY1571-596) with different ASY1 HORMA domain variants.


( A ) GalNAc - T expression in human liver ( map data for human liver was kindly provided by " Gene Expression Applications research group at Illumina " , http : / / www . ebi . ac . uk / arrayexpress / experiments / E - MTAB - 513 / ) HepG2 wild type quantified by RNAseq ( n = 2 , bars representing mean with SD ) .

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(A) GalNAc-T expression levels in human liver (The body map data for human liver was kindly provided by the "Gene Expression Applications research group at Illumina", http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/) and HepG2 wild type cells quantified by RNAseq (n=2, bars representing mean with SD).


( B ) VVA lectin immunoblot of secretomes from HepG2 SC , SCΔT1 , SCΔT2 SCΔT3 .

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(B) VVA lectin immunoblot of secretomes from HepG2 SC, SCΔT1, SCΔT2 and SCΔT3.


Histograms showing distribution of M / L quantitation ratios of monoglycosylated peptides identified in ( A ) HepG2SCΔT1 , ( B ) ΔT2 ( C ) + T3 . Glycopeptides with M / L < - 1 are colored red glycopeptides with a M / L > + 1 are colored green . ( D ) Venn diagram showing distribution of candidates for isoform - specific sites among HepG2SCΔT1 , T2 + T3 applying a log10 ( + / - 1 ) cut - off ( excluding sites identified in both TCL SEC for each isoform ) , ( E ) TCL alone ( F ) SEC alone .

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

Histograms showing the distribution of M/L quantitation ratios of monoglycosylated peptides identified in (A) HepG2SCΔT1, (B) ΔT2 and (C) +T3. Glycopeptides with M/L ratio < -1 are colored red and glycopeptides with a M/L ratio > +1 are colored green. (D) Venn diagram showing the distribution of candidates for isoform-specific sites among HepG2SCΔT1, T2 and +T3 applying a log10 (+/-1) cut-off (excluding sites identified in both TCL and SEC for each isoform), and (E) TCL alone and (F) SEC alone.


human NK ( n = 19 different donors per condition ) were co - incubated with either no target ( NK ( - ) ) , autologous CD4 T ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) at an effector : target of 1 : 2 . NK degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( A ) Representative histograms ( overlay ) displaying CD107a expression on NK after culture with described culture conditions ( NK ( - ) , Mock , HIV ) . For HIV - 1 co - culture condition , subsequent analyses of antigen expression were conducted by gating non - responsive ( CD107a - ) responsive ( CD107a + ) NK subsets . ( B ) Relative frequency of CD107a + NK ( y - axis ) after culture in described conditions ( x - axis ) ( n = 19 different donors ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O O O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (A) Representative histograms (overlay) displaying CD107a expression on NK cells after culture with the described culture conditions (NK(-), Mock, HIV). For the HIV-1 co-culture condition, subsequent analyses of antigen expression were conducted by gating non-responsive (CD107a-) and responsive (CD107a+) NK cell subsets. (B) Relative frequency of CD107a+ NK cells (y-axis) after culture in the described conditions (x-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


human NK ( n = 19 different donors per condition ) were co - incubated with either no target ( NK ( - ) ) , autologous CD4 T ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) at an effector : target of 1 : 2 . NK degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( C ) HIV - specific response of bulk NK displayed as points ( p . p . ) CD107a + NK after normalization to mock CD4 T ( y - axis ) ( n = 19 different donors ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (C) HIV-specific response of bulk NK cells displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


human NK ( n = 19 different donors per condition ) were co - incubated with HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( E ) Representative histograms ( overlay ) showing TRAIL surface expression as on NK after co - incubation with HIV - 1 - infected CD4 T . Histograms show TRAIL expression for CD107a + CD107a - NK subsets ( solid lines ) as well as their corresponding FMO controls ( dotted lines ) . Gate defining TRAIL + is indicated as well . ( F ) Left panel : Relative frequency of TRAIL + ( y - axis ) of CD107a + CD107a - NK subsets ( x - axis ) after co - incubation with HIV - 1 - infected CD4 T . Right panel : Difference in relative frequency of TRAIL + ( y - axis ) between CD107a + CD107a - displayed as p . p . ( n = 19 different donors ) . Data information : Wilcoxon signed - rank . Adjustment for multiplicity : Benjamini Hochberg false discovery rate ( FDR ) F , left panel : ) ; Bonferroni ( F , right panel Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Lines connecting CD107a - CD107a + refer to measured values of same donor .

B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-CELL_TYPE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with HIV-1-infected (NL4-3) CD4 T cells (HIV) (E) Representative histograms (overlay) showing TRAIL surface expression as fluorescence intensity on NK cells after co-incubation with HIV-1-infected CD4 T cells. Histograms show TRAIL expression for CD107a+ and CD107a- NK cell subsets (solid lines) as well as their corresponding FMO controls (dotted lines). Gate defining TRAIL+ cells is indicated as well. (F) Left panel: Relative frequency of TRAIL+ cells (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells. Right panel: Difference in relative frequency of TRAIL+ cells (y-axis) between CD107a+ and CD107a- cells displayed as p.p. (n = 19 different donors). Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: Benjamini and Hochberg false discovery rate (FDR) F, left panel:); Bonferroni (F, right panel Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


human NK ( n = 19 different donors per condition ) were co - incubated with HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( G ) Left panel : TRAIL surface expression ( y - axis ) of CD107a + CD107a - NK subsets ( x - axis ) after co - incubation with HIV - 1 - infected CD4 T displayed as median . Right panel : Differences in TRAIL surface expression ( y - axis ) between CD107a + CD107a - displayed as fold - difference ( n = 19 different donors ) . Data information : Wilcoxon signed - rank . Adjustment for multiplicity : ; Bonferroni G ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Lines connecting CD107a - CD107a + refer to measured values of same donor .

B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

Primary human NK cells (n = 19 different donors per condition) were co-incubated with HIV-1-infected (NL4-3) CD4 T cells (HIV) (G) Left panel: TRAIL surface expression (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells displayed as median fluorescence intensity. Right panel: Differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells displayed as fold-difference (n = 19 different donors). Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: ; Bonferroni G). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


Comparison of CD107a expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( A ) Relative frequency of CD107a + ( y - axis ) of predefined NK subsets ( x - axis ) . Left panel displays data for bulk NK , middle panel for CD56Dim CD56Bright NK , right panel for uneducated , KIR - educated NKG2A - educated ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of CD107a expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (A) Relative frequency of CD107a+ cells (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of CD107a expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( B ) HIV - specific responses of predefined NK sub - populations ( x - axis ) displayed as points ( p . p . ) CD107a + NK after normalization to mock CD4 T ( y - axis ) ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of CD107a expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (B) HIV-specific responses of predefined NK cell sub-populations (x-axis) displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of TRAIL expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( C ) TRAIL expression ( y - axis ) of predefined NK subsets ( x - axis ) . Left panel displays data for bulk NK , middle panel for CD56Dim CD56Bright NK , right panel for uneducated , KIR - educated NKG2A - educated . Expression is displayed as median ( MdFI ) ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of TRAIL expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (C) TRAIL expression levels (y-axis) of predefined NK cell subsets (x-axis). Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as median fluorescence intensity (MdFI) (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Comparison of TRAIL expression between various NK subsets after co - incubation with either no target present ( - ) , autologous CD4 T ( Mock ) , or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T ( HIV ) ( n = 13 different donors per condition ) . Predefined NK subsets comprise CD56Dim , CD56Bright , KIR - educated , defined as expressing at least one self - inhibitory KIR ( 2DL1 / L2 / L3 , 3DL1 ) negative for NKG2A , NKG2A - educated , expressing NKG2A but lacking self - inhibitory KIR , uneducated , lacking self - inhibitory KIR NKG2A altogether . ( D ) Comparison of TRAIL expression ( y - axis ) between CD107a - CD107a + of predefined NK subsets . Left panel displays data for bulk NK , middle panel for CD56Dim CD56Bright NK , right panel for uneducated , KIR - educated NKG2A - educated . Expression is displayed as MdFI . ( n = 13 different donors ) . ( E ) Fold - differences in TRAIL surface expression ( y - axis ) between CD107a + CD107a - of pre - defined NK sub - populations ( x - axis ) . ( n = 13 different donors ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Lines connecting CD107a - CD107a + refer to measured values of same donor .

O O B-GENEPROD B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

Comparison of TRAIL expression levels between various NK cell subsets after co-incubation with either no target cells present (-), autologous CD4 T cells (Mock), or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) (n = 13 different donors per condition). Predefined NK cell subsets comprise CD56Dim, CD56Bright, KIR-educated cells, defined as expressing at least one self-inhibitory KIR (2DL1/L2/L3, 3DL1) and negative for NKG2A, NKG2A-educated cells, expressing NKG2A but lacking self-inhibitory KIR, and uneducated cells, lacking self-inhibitory KIR and NKG2A altogether. (D) Comparison of TRAIL expression levels (y-axis) between CD107a- and CD107a+ cells of predefined NK cell subsets. Left panel displays data for bulk NK cells, middle panel for CD56Dim and CD56Bright NK cells, right panel for uneducated, KIR-educated and NKG2A-educated cells. Expression is displayed as MdFI. (n = 13 different donors). (E) Fold-differences in TRAIL surface expression (y-axis) between CD107a+ and CD107a- cells of pre-defined NK cell sub-populations (x-axis). (n = 13 different donors). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.


human CD4 T from eight different donors ( n = 8 ) were separately infected with lab - adapted HIV - 1 strains . Combined expression of DR4 DR5 was assessed by flow cytometry four days after infection . Uninfected CD4 T were determined as CD4 - positive HIV - 1 p24 - negative , infected as CD4 - negative p24 - positive . ( A ) Histograms ( overlay ) of one representative donor displaying combined DR4 / 5 surface expression on CD4 T previously incubated with either NL4 - 3 , JR - CSF , WITO , CH198 , or CH236 . Expression is displayed as ( x - axis ) . ( B ) Comparison of combined surface expression of DR4 / 5 between mock ( circles ) , uninfected ( grey circles ) infected CD4 T ( orange circles ) , from left to right : Mock : n = 8 ; NL4 - 3 : n = 8 ; JR - CSF : n = 8 ; WITO : n = 7 ; CH198 : n = 7 ; CH236 : n = 8 ( 7 - 8 different donors per condition ) . Expression is displayed as relative ( RFI ) after normalization to respective secondary - AB - only control ( y - axis ) . Data information Wilcoxon signed - rank . Values for non - infected infected of same donor are connected with lines .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary human CD4 T cells from eight different donors (n = 8) were separately infected with lab-adapted and primary HIV-1 strains. Combined expression of DR4 and DR5 was assessed by flow cytometry four days after infection. Uninfected CD4 T cells were determined as CD4-positive and HIV-1 p24-negative, infected cells as CD4-negative and p24-positive. (A) Histograms (overlay) of one representative donor displaying combined DR4/5 surface expression on CD4 T cells previously incubated with either NL4-3, JR-CSF, WITO, CH198, or CH236. Expression is displayed as fluorescence intensity (x-axis). (B) Comparison of the combined surface expression of DR4/5 between mock (white circles), uninfected (grey circles) and infected CD4 T cells (orange circles), from left to right: Mock: n = 8; NL4-3: n = 8; JR-CSF: n = 8; WITO: n = 7; CH198: n = 7; CH236: n = 8 (7-8 different donors per condition). Expression is displayed as relative fluorescence intensity (RFI) after normalization to the respective secondary-AB-only control (y-axis). Data information Wilcoxon signed-rank test. Values for non-infected and infected cells of the same donor are connected with lines.


Degranulation of human NK after co - culture with various target in presence or absence of αTRAIL ( A ) Comparison of CD107a expression after co - culture with 721 . 221 target with either 10 µg / ml αTRAIL or isotype control ( Iso ) using flow cytometry ( n = 10 different donors per condition ) . Each data point represents mean of 2 technical replicates . Effector : target was 1 : 1 . Left panel : Concatenated density plot depicting CD107a expression as for one representative donor . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) . ( B ) Box plots display inhibition of degranulation ( y - axis ) after co - culture with 721 . 221 target in presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 10 different donors per condition ) . ( C ) Comparison of CD107a expression after co - culture with autologous HIV - I - infected CD4 T with either 10 µg / ml αTRAIL or isotype control ( Iso ) using flow cytometry ( n = 9 different donors per condition ) Each data point represents mean of 2 technical replicates . Left panel : Concatenated density plot depicting CD107a expression as for one representative donor . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) . ( D ) Box plots display inhibition of degranulation ( y - axis ) after co - culture with autologous HIV - I - infected CD4 T in presence of either isotype or αTRAIL as relative reduction compared to no antibody ( n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Bar graphs represent mean associated whiskers display SD .

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

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αTRAIL (A) Comparison of CD107a expression after co-culture with 721.221 target cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 10 different donors per condition). Each data point represents the mean of 2 technical replicates. Effector:target ratio was 1:1. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis). (B) Box plots display inhibition of degranulation (y-axis) after co-culture with 721.221 target cells in presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 10 different donors per condition). (C) Comparison of CD107a expression after co-culture with autologous HIV-I-infected CD4 T cells with either 10 µg/ml αTRAIL or isotype control (Iso) using flow cytometry (n = 9 different donors per condition) Each data point represents the mean of 2 technical replicates. Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity for one representative donor. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis). (D) Box plots display inhibition of degranulation (y-axis) after co-culture with autologous HIV-I-infected CD4 T cells in the presence of either isotype or αTRAIL as relative reduction compared to no antibody (n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of human NK after co - culture with various target in presence or absence of αDR4 / 5 . ( E ) Representative histograms ( overlay , left panel ) bar graphs ( n = 3 independent experiments , right panel ) showing combined surface expression of DR4 DR5 on 721 . 221 . Each data point represents mean of 3 technical replicates . Data information : Wilcoxon signed - rank . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Bar graphs represent mean associated whiskers display SD .

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

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αDR4/5. (E) Representative histograms (overlay, left panel) and bar graphs (n = 3 independent experiments, right panel) showing the individual and combined surface expression of DR4 and DR5 on 721.221 cells. Each data point represents the mean of 3 technical replicates. Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of human NK after co - culture with various target in presence or absence of αDR4 / 5 . ( F ) Comparison of CD107a expression after co - culture with 721 . 221 target in presence of either αDR4 / 5 ( 10 µg / ml each ) , or 20 µg / ml isotype control ( Iso ) using flow cytometry ( n = 9 different donors per condition ) . Effector : target was 1 : 1 . Box plots showing relative frequency of CD107a + NK ( y - axis ) . ( G ) Box plots display inhibition of degranulation after co - culture with 721 . 221 target in presence of either isotype or αDR4 / 5 as relative reduction compared to no antibody ( n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank . Adjustment for multiple comparisons was performed using Bonferroni . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points . Bar graphs represent mean associated whiskers display SD .

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

Degranulation of primary human NK cells after co-culture with various target cells in the presence or absence of αDR4/5. (F) Comparison of CD107a expression after co-culture with 721.221 target cells in the presence of either αDR4/5 (10 µg/ml each), or 20 µg/ml isotype control (Iso) using flow cytometry (n = 9 different donors per condition). Effector:target ratio was 1:1. Box plots showing relative frequency of CD107a+ NK cells (y-axis). (G) Box plots display inhibition of degranulation after co-culture with 721.221 target cells in presence of either isotype or αDR4/5 as relative reduction compared to no antibody (n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. Adjustment for multiple comparisons was performed using Bonferroni. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points. Bar graphs represent the mean and the associated whiskers display the SD.


Degranulation of human NK after incubation with plate - coated antibodies ( A ) Comparison of CD107a expression after incubation in either uncoated wells ( PBS ) or wells coated with αTRAIL , αNKG2D , αNKp46 , or isotype using flow cytometry ( n = 12 different donors per condition ) . Left panel : Concatenated density plot depicting CD107a expression as ( y - axis ) for one representative donor 10 µg / ml antibody . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) after incubation with plate - coated antibodies of different concentrations ( x - axis ) . ( B ) Comparison of CD107a expression after incubation with plate - coated DR4 , DR5 or human IgG using flow cytometry ( n = 11 different donors per condition ) . Left panel : Concatenated density plot depicting CD107a expression as ( y - axis ) for one representative donor 10 µg / ml . Right panel : Box plots showing relative frequency of CD107a + NK ( y - axis ) after incubation with plate - coated of different concentrations ( x - axis ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Spearman rank . ( A , B , Each data point represents mean of 2 technical replicates . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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

Degranulation of primary human NK cells after incubation with plate-coated antibodies (A) Comparison of CD107a expression after incubation in either uncoated wells (PBS) or wells coated with αTRAIL, αNKG2D, αNKp46, or isotype using flow cytometry (n = 12 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml antibody concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated antibodies of different concentrations (x-axis). (B) Comparison of CD107a expression after incubation with plate-coated DR4 protein, DR5 protein or human IgG using flow cytometry (n = 11 different donors per condition). Left panel: Concatenated density plot depicting CD107a expression as fluorescence intensity (y-axis) for one representative donor and 10 µg/ml protein concentration. Right panel: Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. (A, B, Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Degranulation of human NK after incubation with plate - coated antibodies ( C ) Comparison of granzyme B release after incubation with various stimuli ( 10 µg / ml each ) . Box plots showing Granzyme B in supernatant as determined by ELISA ( left panel : n = 8 different donors per condition , right panel : n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Spearman rank . C , Each data point represents mean of 2 technical replicates . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (C) Comparison of granzyme B release after incubation with various stimuli (10 µg/ml each). Box plots showing Granzyme B concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. C, Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Degranulation of human NK after incubation with plate - coated antibodies ( D ) Correlation between relative frequency of CD107a + NK granzyme B ( n = 53 , data points obtained from A , B , C , 11 different donors ) .

O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated antibodies (D) Correlation analysis between relative frequency of CD107a+ NK cells and granzyme B concentration (n = 53, data points obtained from A, B, and C, 11 different donors).


Degranulation of human NK after incubation with plate - coated whole . ( E ) Comparison of CD107a expression after incubation with plate - coated DcR1 , osteoprotegerin ( OPG ) or human IgG using flow cytometry ( n = 9 different donors ) . Box plots showing relative frequency of CD107a + NK ( y - axis ) after incubation with plate - coated of different concentrations ( x - axis ) . Data information : Wilcoxon signed - rank adjusted for multiple comparisons ( Bonferroni ) . Spearman rank . E ) Each data point represents mean of 2 technical replicates . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Degranulation of primary human NK cells after incubation with plate-coated whole proteins. (E) Comparison of CD107a expression after incubation with plate-coated DcR1 protein, osteoprotegerin (OPG) or human IgG using flow cytometry (n = 9 different donors). Box plots showing relative frequency of CD107a+ NK cells (y-axis) after incubation with plate-coated proteins of different concentrations (x-axis). Data information: Wilcoxon signed-rank test adjusted for multiple comparisons (Bonferroni). Spearman rank analysis. E) Each data point represents the mean of 2 technical replicates. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( A ) Comparison of IFNγ production ( y - axis ) after incubation with plate - coated antibodies or ( 10 µg / ml each ) ( x - axis ) . Box plots showing IFNγ in supernatant as determined by ELISA ( left panel : n = 8 different donors per condition , right panel : n = 9 different donors per condition ) . Data information : Wilcoxon signed - rank . ( A , Samples were acquired in duplicate mean was calculated for each donor condition . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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

(A) Comparison of IFNγ production (y-axis) after incubation with plate-coated antibodies or proteins (10 µg/ml each) (x-axis). Box plots showing IFNγ concentration in the supernatant as determined by ELISA (left panel: n = 8 different donors per condition, right panel: n = 9 different donors per condition). Data information: Wilcoxon signed-rank test. (A, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( B ) Comparison of IFNγ in supernatant after co - culture of NK with 721 . 221 target in presence or absence of either 10 µg / ml αTRAIL or isotype control ( Iso ) using ELISA ( n = 8 different donors per condition ) . Effector : target was 1 : 1 . Left panel : Box plots showing IFNγ ( y - axis ) for described culture conditions ( x - axis ) . Right panel : Box plots display inhibition of IFNγ production by NK ( y - axis ) in presence of either isotype or αTRAIL as relative reduction compared to no antibody . Data information : Wilcoxon signed - rank . B , Samples were acquired in duplicate mean was calculated for each donor condition . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of IFNγ levels in the supernatant after co-culture of NK cells with 721.221 target cells in the presence or absence of either 10 µg/ml αTRAIL or isotype control (Iso) using ELISA (n = 8 different donors per condition). Effector:target ratio was 1:1. Left panel: Box plots showing IFNγ levels (y-axis) for the described culture conditions (x-axis). Right panel: Box plots display inhibition of IFNγ production by NK cells (y-axis) in presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. B, Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( C ) Representative contour plots showing conjugate formation between NK 721 . 221 target as determined by flow cytometry . Plots showing three distinct populations at beginning of co - culture ( 0 min ) after 50 min : single 721 . 221 ( CT Orange - positive ) , single NK ( CT Violet - positive ) conjugates ( double - positive ) . Relative frequency of NK in conjugates is calculated on total number of gated NK . ( D ) Comparison of conjugate formation after co - culture between NK 722 . 221 after 0 min after 50 min in presence or absence of 10 µg / ml αTRAIL or isotype control ( n = 8 different donors ) . Left panel : Box plots showing relative frequency of NK in conjugates ( y - axis ) for different time points conditions ( x - axis ) . Right panel : Box plots display inhibition of conjugate formation ( y - axis ) in presence of either isotype or αTRAIL as relative reduction compared to no antibody . Data information : Wilcoxon signed - rank . D ) Samples were acquired in duplicate mean was calculated for each donor condition . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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

(C) Representative contour plots showing conjugate formation between NK cells and 721.221 target cells as determined by flow cytometry. Plots showing three distinct populations at the beginning of the co-culture (0 min) and after 50 min: single 721.221 cells (CT Orange-positive), single NK cells (CT Violet-positive) and conjugates (double-positive). Relative frequency of NK cells in conjugates is calculated based on the total number of gated NK cells. (D) Comparison of conjugate formation after co-culture between NK cells and 722.221 cells after 0 min and after 50 min in the presence or absence of 10 µg/ml αTRAIL or isotype control (n = 8 different donors). Left panel: Box plots showing relative frequency of NK cells in conjugates (y-axis) for different time points and conditions (x-axis). Right panel: Box plots display inhibition of conjugate formation (y-axis) in the presence of either isotype or αTRAIL as relative reduction compared to no antibody. Data information: Wilcoxon signed-rank test. D) Samples were acquired in duplicate and the mean was calculated for each donor and condition. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


( E ) Expression of phosphorylated signaling . Upper panel : Concatenated contour plots of one donor depicting expression of phosphorylated signaling Syk , p38 MAPK , Akt PLC - γ2 for following culture conditions controls ( x - axis : left to right ) : FMO , PBS , isotype , αTRAIL , αNKp46 , αNKG2D , αCD16 . Lower panel : Bar graphs showing of p - Syk + , p - p38 MAPK + , p - Akt + p - PLC - γ2 + NK after 30 min of stimulation ( n = 3 different donors ) . Bar graphs represent median , whiskers display minimum maximum data points .

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

(E) Expression levels of phosphorylated signaling proteins. Upper panel: Concatenated contour plots of one donor depicting the expression of phosphorylated signaling proteins Syk, p38 MAPK, Akt and PLC-γ2 for the following culture conditions and controls (x-axis: left to right): FMO, PBS, isotype, αTRAIL, αNKp46, αNKG2D, αCD16. Lower panel: Bar graphs showing the percentage of p-Syk+, p-p38 MAPK+, p-Akt+ and p-PLC-γ2+ NK cells after 30 min of stimulation (n = 3 different donors). Bar graphs represent the median, whiskers display minimum and maximum data points.


Lysis of different target in co - culture with NK was quantified in various cytotoxicity assays . ( A ) Left panel : Representative contour plots showing depletion of 721 . 221 target in presence of NK . Middle panel : of target remaining ( y - axis ) after co - culture with NK in presence of either αTRAIL or isotype control , in reference to target kept alone . Right panel : Box plots displaying difference of target remaining ( y - axis ) between αTRAIL isotype conditions displayed as p . p . ( n = 12 different donors ) . Each data point represents mean of at least 2 technical replicates . Data information : Wilcoxon signed - rank . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for donors in each batch . Lines connect each data value of NK condition with their designated " No NK " control . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (A) Left panel: Representative contour plots showing depletion of 721.221 target cells in the presence of NK cells. Middle panel: Percentage of target cells remaining (y-axis) after co-culture with NK cells in the presence of either αTRAIL or isotype control, in reference to target cells kept alone. Right panel: Box plots displaying difference of target cells remaining (y-axis) between αTRAIL and isotype conditions displayed as p.p. (n = 12 different donors). Each data point represents the mean of at least 2 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Lysis of different target in co - culture with NK was quantified in various cytotoxicity assays . ( B ) Left panel : Representative contour plots showing of . 221 - DR4 / 5KO ( control ) . 221 - Cas9 ( target ) in presence or absence of NK . Middle panel : between . 221 - DR4 / 5KO . 221 - Cas9 ( y - axis ) in presence or absence of NK . Right panel : Specific lysis of . 221 - Cas9 displayed as % ( n = 12 different donors ) . Each data point represents mean of at least 3 technical replicates . Data information : Wilcoxon signed - rank . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for donors in each batch . Lines connect each data value of NK condition with their designated " No NK " control . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (B) Left panel: Representative contour plots showing the percentage of .221-DR4/5KO (control) and .221-Cas9 cells (target) in the presence or absence of NK cells. Middle panel: Ratio between .221-DR4/5KO and .221-Cas9 cells (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of .221-Cas9 cells displayed as % (n = 12 different donors). Each data point represents the mean of at least 3 technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


Lysis of different target in co - culture with NK was quantified in various cytotoxicity assays . ( C ) Left panel : Representative contour plots showing of Raji - pSIP ( control ) Raji - DR5 + + ( target ) in presence or absence of NK . Middle panel : between Raji - pSIP Raji - DR5 + + ( y - axis ) in presence or absence of NK . Right panel : Specific lysis of Raji - DR5 + + displayed as % ( n = 12 different donors ) . Each data point represents mean of at least three technical replicates . Data information : Wilcoxon signed - rank . Experiments were performed in four batches with three different donors each . " No NK " control samples served as a reference for donors in each batch . Lines connect each data value of NK condition with their designated " No NK " control . Box plots represent median 25 % / 75 % percentile . Whiskers indicate minimum maximum data points .

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

Lysis of different target cells in co-culture with NK cells was quantified in various cytotoxicity assays. (C) Left panel: Representative contour plots showing the percentage of Raji-pSIP (control) and Raji-DR5++ (target) in the presence or absence of NK cells. Middle panel: Ratio between Raji-pSIP and Raji-DR5++ (y-axis) in the presence or absence of NK cells. Right panel: Specific lysis of Raji-DR5++ cells displayed as % (n = 12 different donors). Each data point represents the mean of at least three technical replicates. Data information: Wilcoxon signed-rank test. Experiments were performed in four batches with three different donors each. "No NK" control samples served as a reference for all donors in each batch. Lines connect each data value of the NK cell condition with their designated "No NK" control. Box plots represent the median and 25%/75% percentile. Whiskers indicate minimum and maximum data points.


A . Immunoblot of of indicated in lysates from non - infected ( ni ) infected wild - type iBMDMs for 60 or 120 min .

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

A. Immunoblot analysis of the levels of the indicated proteins in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.


C . Immunoblot of Klf4 tubulin in lysates from non - infected ( ni ) infected wild - type iBMDMs for 60 or 120 min .

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C. Immunoblot analysis of Klf4 and tubulin levels in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.


H . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type stat6 - / - iBMDMs for 60 or 120 min .

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H. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type and stat6-/- iBMDMs for 60 or 120 min.


D . Immunofluorescence confocal microscopy of colocalization of Kp52145 harbouring pFPV25 . 1Cm , cresyl violet dye in wild - type ( WT ) stat6 - / - macrophages . were taken 90 min post infection . are representative of duplicate coverslips of three independent experiments . E . of Kp52145 harbouring pFPV25 . 1Cm co - localization with cresyl violet over a time course . Values are given as mean of Kp52145 co - localizing with marker ± . number of infected counted per time in three independent experiments are indicated in figure .

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

D. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and stat6-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. E. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.


A . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , tlr2 - / - , tlr4 - / - tlr2 / 4 - / - iBMDMs for 60 or 120 min . B . Immunoblot of Arg1and tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , tlr2 - / - , tlr4 - / - tlr2 / 4 - / - iBMDMs for 60 or 120 min .

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

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min. B. Immunoblot analysis of Arg1and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), tlr2-/-, tlr4-/- and tlr2/4-/- iBMDMs for 60 or 120 min.


F . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , myd88 - / - , tram / trif - / - for 60 or 120 min . G . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , myd88 - / - , tram / trif - / - for 60 or 120 min .

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

F. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min. G. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), myd88-/-, tram/trif-/- for 60 or 120 min.


A . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or ifnar1 - / - for 60 or 120 min . B . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or ifnar1 - / - for 60 or 120 min .

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

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min. B. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1-/- for 60 or 120 min.


K . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or irf3 - / - for 60 or 120 min . L Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or irf31 - / - for 60 or 120 min .

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

K. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf3-/- for 60 or 120 min. L Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf31-/- for 60 or 120 min.


A . Immunoblot of phospho - CREB ( pCREB ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or tlr4 - / - for 60 or 120 min . B . Immunoblot of phospho - CREB ( pCREB ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or myd88 - / - for 60 or 120 min .

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

A. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or tlr4-/- for 60 or 120 min. B. Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or myd88-/- for 60 or 120 min.


D . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or il10 - / - for 60 or 120 min .

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

D. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.


F . Immunoblot of Arg1 tubulin in lysates from non - infected ( ni ) infected wild - type ( WT ) , or il10 - / - for 60 or 120 min .

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

F. Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10-/- for 60 or 120 min.


L . Immunofluorescence confocal microscopy of colocalization of Kp52145 harbouring pFPV25 . 1Cm , cresyl violet dye in wild - type ( WT ) il10 - / - macrophages . were taken 90 min post infection . are representative of duplicate coverslips of three independent experiments . M . of Kp52145 harbouring pFPV25 . 1Cm co - localization with cresyl violet over a time course in wild - type ( WT ) il10 - / - macrophages . Values are given as mean of Kp52145 co - localizing with marker ± . number of infected counted per time in three independent experiments are indicated in figure .

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

L. Immunofluorescence confocal microscopy of the colocalization of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and il10-/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments. M. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course in wild-type (WT) and il10-/- macrophages. Values are given as mean percentage of Kp52145 co-localizing with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure.


A . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from wild - type macrophages non - infected ( ni ) , or infected with Kp52145 or LPS O - polysaccharide mutant , 52145 - Δglf , for 60 or 120 min .

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

A. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the LPS O-polysaccharide mutant, strain 52145-Δglf, for 60 or 120 min.


C . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from wild - type macrophages non - infected ( ni ) , or infected with Kp52145 or CPS mutant , 52145 - ΔwcaK2 , for 60 or 120 min .

O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O

C. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from wild-type macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.


A . arg1 mRNA were assessed by qPCR in hM - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h . D . ido mRNA were assessed by qPCR in hM - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h .

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A. arg1 mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. D. ido mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h.


B . il10 mRNA were assessed by qPCR in M - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h . C . chi3l mRNA were assessed by qPCR in hM - CSF - treated PBMCs from 6 donors non - infected ( ni ) or infected Kp52145 for 1 , 3 or 5 h .

O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O

B. il10 mRNA levels were assessed by qPCR in M-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. C. chi3l mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h.


E . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from PMA - treated THP - 1 macrophages non - infected ( ni ) , or infected with Kp52145 for 60 or 120

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

E. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 for 60 or 120 min


O . Immunoblot of phospho - STAT6 ( pSTAT6 ) tubulin in lysates from PMA - treated THP - 1 macrophages non - infected ( ni ) , or infected with Kp52145 or CPS mutant , 52145 - ΔwcaK2 , for 60 or 120 min .

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

O. Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-ΔwcaK2, for 60 or 120 min.


SARS - CoV - 2 . For weigh loss record , hACE2 mice ( n = 7 ) WT mice ( n = 3 ) were experimentally challenged intranasally with SARS - CoV - 2 , ACE2 - Mock mice ( n = 3 ) were used as control . then loss was recorded over 14 days ( a , ANOVA , * * * p < 0 . 001 ) .

O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SARS-CoV-2.For weigh loss record, hACE2 mice (n=7) and WT mice (n=3) were experimentally challenged intranasally with SARS-CoV-2, and the ACE2-Mock mice (n=3) were used as control. And then the weight loss was recorded over 14 days (a, ANOVA, ***p<0.001).


To screen replication , 12 mice were infected in each group , 3 mice per group were sacrificed their major organs harvested for viral load titer at 1 dpi , 3 dpi , 5 dpi 7 dpi respectively . distribution of SARS - CoV - 2 in organs of ACE2 - HB - 01 mice was detected by qRT - PCR ( b ) .

O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

To screen virus replication, 12 mice were infected in each group, and 3 mice per group were sacrificed and their major organs harvested for viral load and virus titer at 1 dpi, 3 dpi, 5 dpi and 7 dpi respectively. The distribution of SARS-CoV-2 in the primary organs of ACE2-HB-01 mice was detected by qRT-PCR (b).


titers of lungs were determined on Vero E6 ( c , unpaired t - , * * * p < 0 . 001 )

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Virus titers of lungs were determined on Vero E6 cells (c, unpaired t-test, ***p<0.001)


isolated from lungs of ACE2 - HB - 01 mice at 3 dpi was observed by electron microscope ( d ) .

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the virus isolated from lungs of ACE2-HB-01 mice at 3 dpi was observed by electron microscope (d).


specific IgG against SARS - CoV - 2 was detected at 21 dpi by ELISA ( e , unpaired t - , * * * p < 0 . 001 ) .

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The specific IgG against SARS-CoV-2 was detected at 21 dpi by ELISA (e, unpaired t-test, ***p<0.001).


mice . a . Gross pathology histopathology of lungs from WT - HB - 01 mice ( 3 dpi ) , ACE2 - Mock mice ( 3 dpi ) ACE2 - HB - 01 mice ( 3 dpi 5 dpi ) . Postmortem examinations showed focal red lesions ( red arrow ) throughout dorsal of right middle lobe of lung at 3dpi . lesions progressed into multifocally scattered - reddish purple areas palpable nodules ( red arrow ) throughout right lobe of lung at 5 dpi . Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa ( black arrows ) infiltration of lymphocytes ( red arrows ) . swollen degenerative alveolar macrophages ( green arrows ) are scattered within alveolar cavities at 3dpi 5 dpi .

O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-ORGANISM 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-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O O B-DISEASE I-DISEASE O O B-TISSUE I-TISSUE O O O O O O B-CELL_TYPE O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE O O O O O

mice.a. Gross pathology and histopathology of lungs from WT-HB-01 mice (3 dpi), ACE2-Mock mice (3 dpi) and ACE2-HB-01 mice (3 dpi and 5 dpi). Postmortem examinations showed focal dark red lesions (red arrow) throughout the dorsal of the right middle lobe of the lung at 3dpi. The lesions progressed into multifocally scattered-dark reddish purple areas and palpable nodules (red arrow) throughout the right lobe of the lung at 5 dpi. Histopathological observation indicated that moderate interstitial pneumonia with thickened alveolar septa (black arrows) and infiltration of lymphocytes (red arrows). The swollen and degenerative alveolar macrophages (green arrows) are scattered within the alveolar cavities at 3dpi and 5 dpi.


b . Immunohistochemical examination of lungs of each group . sequential sections were stained by HE , respectively . viral antigens were observed in alveolar macrophage ( green arrows ) , alveolar epithelia ( blue arrows ) , degenerative being desquamated bronchial epithelial ( black arrows ) . Black bar = 100 µm , red bar = 50 µm .

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b. Immunohistochemical examination of lungs of each group. The sequential sections were stained by HE and IHC, respectively. The viral antigens were observed in the alveolar macrophage (green arrows), the alveolar epithelia (blue arrows), and the degenerative and being desquamated bronchial epithelial cells (black arrows). Black bar = 100 µm, red bar = 50 µm.


Immunofluorescence of viral antigens in lungs of SARS - CoV - 2 - infected hACE2 mice . Co - localization of SARS - CoV - 2 S hACE2 receptor in hACE2 mouse lungs , sections were incubated with anti - SARS - CoV - 2 S antibody , anti - human ACE2 antibody , DAPI . lung sections of ACE2 - Mock mice ( a - d ) . lung sections of ACE2 - HB - 01 mice ( e - h ) . arrows showed viral S ( f ) hACE2 ( g ) , respectively , yellow arrow showed merge of viral S hACE2 ( h ) . bar = 25 µm .

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Immunofluorescence analysis of viral antigens in lungs of SARS-CoV-2-infected hACE2 mice.Co-localization of SARS-CoV-2 S protein and hACE2 receptor in hACE2 mouse lungs, the sections were incubated with anti-SARS-CoV-2 S protein antibody, anti-human ACE2 antibody, and DAPI. The lung sections of ACE2-Mock mice (a-d). The lung sections of ACE2-HB-01 mice (e-h). The white arrows showed the viral S protein (f) and hACE2 (g), respectively, the yellow arrow showed the merge of viral S protein and hACE2 (h). White bar=25 µm.


( C ) Clonal lines expressing p53 - Venus were engineered to monitor p53 expression dynamics ( " input " ) two canonically regulated p53 target promoters expressing mCherry ( " output " ) in response to Nutlin - 3 treatment .

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(C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment.


( D ) Representative phase contrast yellow ( indicating p53 - Venus ) at indicated time points for a exposed to " natural dynamics " Nutlin - 3 dosing regimen .

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(D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the "natural dynamics" Nutlin-3 dosing regimen.