( E and F ) WT and TEX264 - KO ( expressing WT or TEX264 - FLAG mutants ) HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , the cells were cultured in starvation medium lacking amino acids and serum for 9 h ( E ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of four independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( F ) .

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(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG mutants) HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).


A Transfected GFP − Atg8 homologs were immunoprecipitated with μMACS ™ from total U2OS cell extracts followed by immunoblot analysis with anti − GFP and anti − ALFY antibodies . Data are representative of two independent experiments .

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A Transfected GFP−Atg8 homologs were immunoprecipitated with μMACS™ from total U2OS cell extracts followed by immunoblot analysis with anti−GFP and anti−ALFY antibodies. Data are representative of two independent experiments.


C The indicated 35S − labelled in vitro − translated GFP − tagged constructs were incubated with GST , GST − LC3B or GST − GABARAP conjugated to glutathione Sepharose , and their binding was evaluated by autoradiography ( ARG ) . 5 % of the in vitro − translated protein ( arrow head ) used was loaded . Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue ( CBB ) staining . Data are representative of three independent experiments .

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C The indicated 35S−labelled in vitro−translated GFP−tagged constructs were incubated with GST, GST−LC3B or GST−GABARAP conjugated to glutathione Sepharose, and their binding was evaluated by autoradiography (ARG). 5% of the in vitro−translated protein (arrow head) used was loaded. Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments.


D Recombinant ALFY ( aa 2981 - 3526 ) was incubated with GST ‐ Atg8 proteins conjugated to glutathione Sepharose . The pulled ‐ down complexes were subjected to SDS - PAGE and anti ‐ ALFY and anti ‐ GST immunoblotting . 5 % of the recombinant ALFY protein used was loaded as input . Data are representative of three independent experiments .

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D Recombinant ALFY (aa 2981-3526) was incubated with GST‐Atg8 proteins conjugated to glutathione Sepharose. The pulled‐down complexes were subjected to SDS-PAGE and anti‐ALFY and anti‐GST immunoblotting. 5% of the recombinant ALFY protein used was loaded as input. Data are representative of three independent experiments.


B 35S − labelled in vitro − translated GFP − ALFY ( aa 2981 - 3526 ) wild − type and different LIR mutants were incubated with GST − GABARAP or − LC3C and binding evaluated by ARG . 10 and 2 % of the in vitro − translated proteins used were loaded to illustrate binding affinity . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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B 35S−labelled in vitro−translated GFP−ALFY (aa 2981-3526) wild−type and different LIR mutants were incubated with GST−GABARAP or −LC3C and binding evaluated by ARG. 10 and 2% of the in vitro−translated proteins used were loaded to illustrate binding affinity. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


C MBP , MBP − ALFY ( aa 3255 - 3526 ) or the ALFY − LIR mutant ( F3346A ) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP . The pulled − down complexes were subjected to SDS - PAGE and visualized by CBB staining . Data are representative of three independent experiments .

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C MBP, MBP−ALFY (aa 3255-3526) or the ALFY−LIR mutant (F3346A) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP. The pulled−down complexes were subjected to SDS-PAGE and visualized by CBB staining. Data are representative of three independent experiments.


D Thermodynamic parameters of ALFY − LIR peptide ( aa 3341 - 3354 ) to Atg8 homologs . All titrations were performed at 25 ° C as described in Supplementary Methods . ITC data were fitted to a one − site binding model . ND , not detected .

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D Thermodynamic parameters of ALFY−LIR peptide (aa 3341-3354) to Atg8 homologs. All titrations were performed at 25°C as described in Supplementary Methods. ITC data were fitted to a one−site binding model. ND, not detected.


E 35S − labelled in vitro − translated GFP or GFP − Bchs ( aa 3326 - 3341 ) were incubated with GST or GST − dAtg8a and binding evaluated by ARG . 5 % of the in vitro − translated protein used was loaded . Ponceau staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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E 35S−labelled in vitro−translated GFP or GFP−Bchs (aa 3326-3341) were incubated with GST or GST−dAtg8a and binding evaluated by ARG. 5% of the in vitro−translated protein used was loaded. Ponceau staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


D GFP − LC3B and GFP − LC3B ( Q26K / H27Y / H57D ) were immunoprecipitated with GFP − TRAP ® from total cell lysate of stably transfected HeLa FlpIn cells , followed by SDS - PAGE and immunoblotting with the indicated antibodies . Data are representative of three independent experiments .

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D GFP−LC3B and GFP−LC3B (Q26K/H27Y/H57D) were immunoprecipitated with GFP−TRAP® from total cell lysate of stably transfected HeLa FlpIn cells, followed by SDS-PAGE and immunoblotting with the indicated antibodies. Data are representative of three independent experiments.


E MBP ‐ ALFY3255 - 3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants . The pulled ‐ down complexes were subjected to SDS - PAGE and visualized by immunoblotting with anti ‐ MBP and anti ‐ GABARAP antibodies . Data are representative of three independent experiments .

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E MBP‐ALFY3255-3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants. The pulled‐down complexes were subjected to SDS-PAGE and visualized by immunoblotting with anti‐MBP and anti‐GABARAP antibodies. Data are representative of three independent experiments.


F MBP − tagged ALFY3255 - 3526 and p62168 - 391 or their corresponding LIR mutants ( F3346A and W340A , respectively ) were conjugated to amylose resin and incubated with purified GST , GABARAP , GABARAP mutant ( K24Q / Y25H / D54H ) , LC3B or LC3B mutant ( Q26K / H27Y / H57D ) . The bound proteins were visualized by immunoblotting with anti − GABARAP , anti − LC3 and anti − MBP antibodies . Data are representative of three independent experiments .

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F MBP−tagged ALFY3255-3526 and p62168-391 or their corresponding LIR mutants (F3346A and W340A, respectively) were conjugated to amylose resin and incubated with purified GST, GABARAP, GABARAP mutant (K24Q/Y25H/D54H), LC3B or LC3B mutant (Q26K/H27Y/H57D). The bound proteins were visualized by immunoblotting with anti−GABARAP, anti−LC3 and anti−MBP antibodies. Data are representative of three independent experiments.


G 35S ‐ labelled in vitro ‐ translated GFP ‐ ALFY ( aa 2981 - 3526 ) wild ‐ type and K3343A / D3344A / Y3351A mutant were incubated with GST ‐ LC3B and GABARAP and binding evaluated by ARG . 10 % of the in vitro ‐ translated proteins used was loaded . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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G 35S‐labelled in vitro‐translated GFP‐ALFY (aa 2981-3526) wild‐type and K3343A/D3344A/Y3351A mutant were incubated with GST‐LC3B and GABARAP and binding evaluated by ARG. 10% of the in vitro‐translated proteins used was loaded. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


A HeLa FlpIn GFP − GABARAP cells were treated with proteasomal inhibitor ( MG132 , 2 h ) or subjected to amino acid starvation ( EBSS , 2 h ) , before staining with anti − ALFY antibodies . Scale bar , 10 μm .

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A HeLa FlpIn GFP−GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti−ALFY antibodies. Scale bar, 10 μm.


B GFP − tagged full − length ALFY wild − type or LC3 − interacting region ( LIR ) − mutant was expressed into Alfy − deficient MEFs using an adenovirus system . At 48 h after infection , the MEFs were cultured in normal media or EBSS for 1 . 5 h before staining with anti − LC3B or anti − GABARAP antibodies . Scale bars 10 μm .

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B GFP−tagged full−length ALFY wild−type or LC3−interacting region (LIR)−mutant was expressed into Alfy−deficient MEFs using an adenovirus system. At 48 h after infection, the MEFs were cultured in normal media or EBSS for 1.5 h before staining with anti−LC3B or anti−GABARAP antibodies. Scale bars 10 μm.


C HeLa cells were treated with control or siRNA targeting GABARAP , GABARAPL1 and GABARAPL2 . 72 h after transfection cells were immunostained with anti − LC3B , anti − ALFY and anti − p62 antibodies . Scale bars , 10 μm .

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C HeLa cells were treated with control or siRNA targeting GABARAP, GABARAPL1 and GABARAPL2. 72 h after transfection cells were immunostained with anti−LC3B, anti−ALFY and anti−p62 antibodies. Scale bars, 10 μm.


D HeLa cells were treated with control or siRNA targeting ALFY . 72 h after transfection , cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX − 100 − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments .

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D HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX−100−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.


E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X − 100 ( TX − 100 ) − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments .

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E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X−100 (TX−100)−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.


B . ERK71 mutants have elevated triacylglycerol ( TAG ) levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . C . Fat body specific depletion of ERK7 by RNAi ( BDSC 56939 ) leads to increased TAG levels ( N = 4 replicates of 10 larvae / replicate for each genotype ) .

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B. ERK71 mutants have elevated triacylglycerol (TAG) levels (N=4 replicates of ≥10 larvae/ replicate for each genotype). C. Fat body specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels (N=4 replicates of 10 larvae/replicate for each genotype).


D . Representative immunofluorescent images of lipid droplet ( LipidTOX ) and nuclear ( DAPI ) staining in control and ERK71 mutant fat bodies of third instar larvae . Scale bar : 50 µm . E . ERK71 mutant fat body cells contain more lipid droplets than control cells ( N = 30 cells for each genotype ) .

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D. Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK71 mutant fat bodies of third instar larvae. Scale bar: 50 µm. E. ERK71 mutant fat body cells contain more lipid droplets than control cells (N=30 cells for each genotype).


F . After fed [ 13C ] glucose , the ERK71 mutant fat bodies display elevated [ 13C ] TAG / PE and unlabeled TAG / PE molar ratios ( measured by mass spectrometry - based lipidomics , N = 3 replicates of 15 fat bodies / replicate , TAG = triacylglycerol , PE = phosphatidylethanolamine )

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F. After fed [13C]glucose, the ERK71 mutant fat bodies display elevated [13C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry-based lipidomics, N=3 replicates of 15 fat bodies/replicate, TAG=triacylglycerol, PE=phosphatidylethanolamine)


G . ERK7 overexpression in the fat body results in reduced TAG levels , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the TAG storage ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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G. ERK7 overexpression in the fat body results in reduced TAG levels, while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage (N=4 replicates of ≥10 larvae/replicate for each genotype).


H - ERK7 expressing , GFP - marked , fat body clone ( H , marked by yellow dotted line ; scale bar : 50 µm ) visualized by LipidTOX staining ( N = 11 for control and 4 for ERK7 overexpression ) .

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H- ERK7 expressing, GFP-marked, fat body clone (H, marked by yellow dotted line; scale bar: 50 µm) visualized by LipidTOX staining (N=11 for control and 4 for ERK7 overexpression).


ERK7 expressing , fat body clone contains less ( I ) and smaller ( J ) lipid droplets than control cells ( N = 11 for control and 4 for ERK7 overexpression ) .

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ERK7 expressing, fat body clone contains less (I) and smaller (J) lipid droplets than control cells (N=11 for control and 4 for ERK7 overexpression).


K . qRT - PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation . Expression of RP49 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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K. qRT-PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation. Expression of RP49 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


A . Fatty acid composition of total lipids in CG > control and CG > ERK7 larvae , measured by gas chromatography ( N = 3 replicates of 15 larvae / replicate for each genotype ) .

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A. Fatty acid composition of total lipids in CG>control and CG>ERK7 larvae, measured by gas chromatography (N=3 replicates of 15 larvae/replicate for each genotype).


C . Amount of glucose - derived [ 13C ] - labeled carbons incorporated into the TAG pool of control and CG > ERK7 fat bodies ( N = 3 replicates of 15 fat bodies / replicate for each genotype and diet ) .

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C. Amount of glucose-derived [13C]-labeled carbons incorporated into the TAG pool of control and CG>ERK7 fat bodies (N=3 replicates of 15 fat bodies/replicate for each genotype and diet).


D . Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels ( N = 3 replicates of 15 larvae / replicate for each genotype ) . E . Circulating glucose levels are decreased in ERK71 mutants ( N = 4 replicates of 15 larvae / replicate for each genotype ) .

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D. Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels (N=3 replicates of 15 larvae/replicate for each genotype). E. Circulating glucose levels are decreased in ERK71 mutants (N=4 replicates of 15 larvae/replicate for each genotype).


A . Four - way plot ( t - statistics ) presenting the ERK7 - dependent fasting - responsive genes ( adj . p . val < 0 . 05 ) . Dark blue indicates significant in both comparisons ; dark turquoise , significant in one comparison ; light turquoise , not significant .

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A. Four-way plot (t-statistics) presenting the ERK7-dependent fasting-responsive genes (adj.p.val < 0.05). Dark blue indicates significant in both comparisons; dark turquoise, significant in one comparison; light turquoise, not significant.


D . Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism . Color key displays scaled log2 gene expression .

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D. Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism. Color key displays scaled log2 gene expression.


E , F . Expression analysis of Seipin , Agpat3 , ATPCL , fabp , Lip4 and CG34448 by qRT - PCR . RP49 was used for normalization ( N = 3 replicates of ≥ 10 larvae / replicate for each genotype ) .

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E, F. Expression analysis of Seipin, Agpat3, ATPCL, fabp, Lip4 and CG34448 by qRT-PCR. RP49 was used for normalization (N=3 replicates of ≥10 larvae/replicate for each genotype).


A . Weight of control and ERK71 mutant larvae at 72h after egg deposition ( AED ) ( N = 4 replicates of 12 larvae / replicate for each genotype ) .

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A. Weight of control and ERK71 mutant larvae at 72h after egg deposition (AED) (N=4 replicates of 12 larvae/replicate for each genotype).


B . Pupariation kinetics of control and ERK71 mutants ( N = 4 replicates of 30 larvae / replicate for each genotype ) . p < 0 . 0001 , analyzed by Log - rank test .

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B. Pupariation kinetics of control and ERK71 mutants (N=4 replicates of 30 larvae/replicate for each genotype). p<0.0001, analyzed by Log-rank test.


C . ERK71 mutants fail to suppress growth upon 24 h of starvation ( N ≥ 4 replicates of 15 larvae / replicate for each genotype ) .

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C. ERK71 mutants fail to suppress growth upon 24 h of starvation (N≥4 replicates of 15 larvae/replicate for each genotype).


D . ERK71 mutant larvae ( N = 4 replicates of 24 larvae / replicate for each genotype , p < 0 . 01 analyzed by Log - rank test ) are sensitive to nutrient deprivation .

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D. ERK71 mutant larvae (N=4 replicates of 24 larvae/replicate for each genotype, p<0.01 analyzed by Log-rank test) are sensitive to nutrient deprivation.


Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to a moderately accelerated pupariation rate ( E , p < 0 . 001 analyzed by Log - rank test , N = 8 replicates of 30 larvae / replicate for each genotype )

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Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to a moderately accelerated pupariation rate (E, p<0.001 analyzed by Log-rank test, N=8 replicates of 30 larvae/replicate for each genotype)


F . Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to an increase in larval weight at 72h AED ( F , N = 5 replicates of 15 larvae / replicate for each genotype ) .

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F. Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to an increase in larval weight at 72h AED (F, N=5 replicates of 15 larvae/replicate for each genotype).


G . A representative image showing that fat body - specific overexpression of ERK7 by the CG - GAL4 driver ( CG > ERK7 ) impairs larval growth . Scale bar : 0 . 5 mm .

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G. A representative image showing that fat body-specific overexpression of ERK7 by the CG-GAL4 driver (CG>ERK7) impairs larval growth. Scale bar: 0.5 mm.


H . Ectopic expression of ERK7 leads to delayed pupariation ( p < 0 . 0001 , analyzed by Log - rank test ) , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence pupariation rate ( N = 4 replicates of 30 larvae / replicate for each genotype ) .

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H. Ectopic expression of ERK7 leads to delayed pupariation (p<0.0001, analyzed by Log-rank test), while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence pupariation rate (N=4 replicates of 30 larvae/replicate for each genotype).


I . In contrast to ERK7 wt , kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the pupal volume ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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I. In contrast to ERK7 wt, kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the pupal volume (N=4 replicates of 10 pupae/replicate for each genotype).


A . Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus . Scale bar : 30 µm . B , C . ERK71 mutant fat bodies display increased nuclear area ( B , N > 160 cells , obtained from 5 independent fat bodies ) and increased nucleolar / nuclear area ratio ( C , N > 80 cells for nucleolus / nucleus ratio , obtained from 5 independent fat bodies ) , when compared to control fat bodies .

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A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies.


D - F . ERK7 expressing fat body clones ( D , marked by GFP , scale bar : 20 µm ) display reduced nuclear area ( E ) and reduced nucleolar / nuclear area ratio ( F ) , when compared to control cells . Nuclei were visualized by DAPI staining , nucleoli were visualized by anti - Fibrillarin antibodies ( N = 10 ) .

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D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10).


G , H . Expression analysis of Pol I ( G ) and Pol III ( H ) target RNAs by qRT - PCR . Expression of CDK7 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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G, H. Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT-PCR. Expression of CDK7 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


A . ERK7 overexpression leads to phosphorylation of PWP1 , as shown by appearance of an extra band on Phos - tag Western blot ( marked with red arrow ) . Insulin - inducible bands ( dark blue ) remain unresponsive to ERK7 .

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A. ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos-tag Western blot (marked with red arrow). Insulin-inducible bands (dark blue) remain unresponsive to ERK7.


B . Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae . Scale bar : 20 µm . C . PWP1 localization displays increased nucleolar / nuclear ratio in ERK71 mutant fat bodies compared to control ( N = 20 cells from 5 independent fat bodies per genotype ) .

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B. Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae. Scale bar: 20 µm. C. PWP1 localization displays increased nucleolar/nuclear ratio in ERK71 mutant fat bodies compared to control (N=20 cells from 5 independent fat bodies per genotype).


D . Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae . ERK7 overexpression ( GFP marked clones ) alters the subcellular localization of PWP1 . Scale bar : 30 µm . E . PWP1 localization displays reduced nuclear / cytoplasmic ratio in ERK7 overexpressing clones , compared to control cells ( N = 10 cells from at least 8 different fat bodies per genotype ) .

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D. Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae. ERK7 overexpression (GFP marked clones) alters the subcellular localization of PWP1. Scale bar: 30 µm. E. PWP1 localization displays reduced nuclear/cytoplasmic ratio in ERK7 overexpressing clones, compared to control cells (N=10 cells from at least 8 different fat bodies per genotype).


F . Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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F. Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels (N=4 replicates of ≥10 larvae/replicate for each genotype).


G . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body ( CG - GAL4 ) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . H , I . Fat body specific knockdown of PWP1 suppresses increased lipid droplet number ( H ; N > 30 cells per genotype ) and increased nuclear area ( I ; N > 45 cells per genotype ) caused by ERK7 knockdown ( BDSC 56939 ) .

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G. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG-GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. H, I. Fat body specific knockdown of PWP1 suppresses increased lipid droplet number (H; N>30 cells per genotype) and increased nuclear area (I; N>45 cells per genotype) caused by ERK7 knockdown (BDSC 56939).


A , B . Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 ( A ) and ectopic expression of ERK7 ( B ) ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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A, B. Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 (A) and ectopic expression of ERK7 (B) (N=3 replicates of 10 fat bodies/replicate for each genotype).


C . ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting ( N = 4 replicates of 10 larvae / replicate for each genotype ) .

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C. ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting (N=4 replicates of 10 larvae/replicate for each genotype).


D . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants ( sug17Δ / Df ( 2R ) Exel7123 ) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . E . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased lipid droplet number observed in ERK71 mutants ( N > 20 ) .

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D. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants (sug17Δ/Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. E. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK71 mutants (N>20).


F . Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG > ERK7 larvae ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) .

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F. Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae (N=4 replicates of ≥10 larvae/replicate for each genotype).


G . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells ( N ≥ 25 cells per genotype ) .

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G. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells (N ≥25 cells per genotype).


Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate ( H ) ( p < 0 . 001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype ) of CG > ERK7

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Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate (H) (p<0.001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype) of CG>ERK7 larvae


I . Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG > ERK7 larvae ( I ) ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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I. Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG>ERK7 larvae (I) (N=4 replicates of 10 pupae/replicate for each genotype).


J . Addition of 20 % sucrose improves the growth rate and survival of CG > ERK7 larvae . Pupariation kinetics of CG > ERK7 larvae on 20 % yeast or 20 % yeast + 20 % sucrose diet ( p < 0 . 0001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype and diet ) .

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J. Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet (p<0.0001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype and diet).


( F ) Representative images of GFP - positive HEK293 cells at day 7 post - transduction using 1x106 TU / cell of RGD4C . TPA . GFP or RGD4C . AAVP . GFP particles . Non - targeted ( NT ) particles were included as control . Data shown are representative of five independent experiments , performed each in triplicates . Scale bar , 100 μm .

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(F) Representative images of GFP-positive HEK293 cells at day 7 post-transduction using 1x106 TU/cell of RGD4C.TPA.GFP or RGD4C.AAVP.GFP particles. Non-targeted (NT) particles were included as control. Data shown are representative of five independent experiments, performed each in triplicates. Scale bar, 100 μm.


( A ) Quantification of gene expression in HEK293 cells after transduction with 0 . 5x106 , 1x106 , and 2x106 TU / cell of RGD4C . TPA , RGD4C . AAVP or non - targeted ( NT ) particles carrying Lucia . Luciferase activity was measured at days 1 to 6 post - transduction . Data shown are representative of three independent experiments ( n = 3 ) , and are expressed as mean + SEM . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(A) Quantification of gene expression in HEK293 cells after transduction with 0.5x106, 1x106, and 2x106 TU/cell of RGD4C.TPA, RGD4C.AAVP or non-targeted (NT) particles carrying Lucia. Luciferase activity was measured at days 1 to 6 post-transduction. Data shown are representative of three independent experiments (n = 3), and are expressed as mean + SEM. One-way ANOVA with Tukey's HSD test was used for data analysis.


( B ) Representative images of FITC - labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post - inoculation ( n = 3 ) . Scale bar , 100 μm . ( C ) Particle diffusion using a vertical trans - well system partitioned by Matrigel . Samples were collected from the wells below the Matrigel at days 1 to 7 , n = 3 , technical replicates . Data are expressed as mean + SEM , independent t - test was used for data analysis .

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(B) Representative images of FITC-labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post-inoculation (n = 3). Scale bar, 100 μm. (C) Particle diffusion using a vertical trans-well system partitioned by Matrigel. Samples were collected from the wells below the Matrigel at days 1 to 7, n = 3, technical replicates. Data are expressed as mean + SEM, independent t-test was used for data analysis.


( D ) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins , then quantified by FACS at 2 and 4 hours post - transduction , 0 . 1X106TU / cell , and normalised to particle length . Data are expressed as mean + SEM of n = 3 independent experiments , independent t - test was used for data analysis .

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(D) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins, then quantified by FACS at 2 and 4 hours post-transduction, 0.1X106TU/cell, and normalised to particle length. Data are expressed as mean + SEM of n = 3 independent experiments, independent t-test was used for data analysis.


( E ) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post - transduction with 0 . 5x106 TU / cell of RGD4C . TPA and RGD4C . AAVP , using qPCR with primers specific to AAV - 2 ITRs . Data are representative of n = 3 independent experiments and expressed as mean + SEM , independent t - test was used for data analysis .

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(E) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post-transduction with 0.5x106 TU/cell of RGD4C.TPA and RGD4C.AAVP, using qPCR with primers specific to AAV-2 ITRs. Data are representative of n = 3 independent experiments and expressed as mean + SEM, independent t-test was used for data analysis.


( F ) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma - derived neural stem cells with 1x106 TU / cell . Data are expressed as mean + SEM of n = 3 independent experiments . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(F) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma-derived neural stem cells with 1x106 TU/cell. Data are expressed as mean + SEM of n = 3 independent experiments. One-way ANOVA with Tukey's HSD test was used for data analysis.


( G ) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia . Experiments were repeated three times and performed in triplicates . Data are shown as mean + SEM and are representative of one experiment . Independent t - test was used for data analysis .

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(G) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia. Experiments were repeated three times and performed in triplicates. Data are shown as mean + SEM and are representative of one experiment. Independent t-test was used for data analysis.


( B ) ELISA quantification of TNFα production in media collected on days 3 to 5 post - transduction of human primary GBM001 tumor spheres at 1x106 TU / cell of targeted ( RGD4C ) or non - targeted ( NT ) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(B) ELISA quantification of TNFα production in media collected on days 3 to 5 post-transduction of human primary GBM001 tumor spheres at 1x106 TU/cell of targeted (RGD4C) or non-targeted (NT) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis.


( C ) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C . TPA . TNFαIL2 to NOD / SCID mice bearing subcutaneous human GBM001 . Tumors and healthy organs were harvested at day 18 post - transduction and analysed using RT - qPCR to quantify the TNFαIL2 transcripts . Data shown are expressed as mean + SEM , from n = 3 mice . Two - way ANOVA was used for data analysis .

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(C) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C.TPA.TNFαIL2 to NOD/SCID mice bearing subcutaneous human GBM001. Tumors and healthy organs were harvested at day 18 post-transduction and analysed using RT-qPCR to quantify the TNFαIL2 transcripts. Data shown are expressed as mean + SEM, from n = 3 mice. Two-way ANOVA was used for data analysis.


( D ) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene , at day 18 post targeted TNFαIL2 cytokine treatment . ( E ) Analysis of tumor growth in representative mice , from D , using the tumor bioluminescence data ( total flux ) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0 .

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(D) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene, at day 18 post targeted TNFαIL2 cytokine treatment. (E) Analysis of tumor growth in representative mice, from D, using the tumor bioluminescence data (total flux) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0.


( B ) Investigation of the TPA . IL15IgK in comparison to the native TPA . IL15 in B16 . F1 and CT26 . CL25 tumor cells . The TPA DNA constructs were transfected into tumor cells , then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL - 2 . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice and performed in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data

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(B) Investigation of the TPA.IL15IgK in comparison to the native TPA.IL15 in B16.F1 and CT26.CL25 tumor cells. The TPA DNA constructs were transfected into tumor cells, then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL-2. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice and performed in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis


( C ) Targeted ( RGD4C ) and non - targeted ( NT ) TPA particles carrying IL15IgK were produced and used to transduce B16 . F1 or CT26 . CL25 cells at 0 . 5x106 or 1x106 TU / cell . Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post - transduction . RGD4C . TPA without IL15IgK ( mock ) was also included as control . Experiments were repeated twice and shown are the results from a representative experiment . Data are expressed as mean + SEM , one - way ANOVA with Tukey ' s HSD test was used for data analysis .

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(C) Targeted (RGD4C) and non-targeted (NT) TPA particles carrying IL15IgK were produced and used to transduce B16.F1 or CT26.CL25 cells at 0.5x106 or 1x106 TU/cell. Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post-transduction. RGD4C.TPA without IL15IgK (mock) was also included as control. Experiments were repeated twice and shown are the results from a representative experiment. Data are expressed as mean + SEM, one-way ANOVA with Tukey's HSD test was used for data analysis.


( D ) Evaluation of CTLL - 2 proliferation in the presence of media collected from B16 . F1 or CT26 . CL25 tumor cells after transduction with TPA particles carrying IL15IgK . Cells treated with a recombinant IL2 cytokine were included as a positive control . Data are representative of one experiment , expressed as mean + SEM and shown as change in CTLL - 2 number relative to treatment initiation day . Experiments were repeated twice and in triplicates , one - way ANOVA with Tukey ' s HSD test was used to analyse the data .

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(D) Evaluation of CTLL-2 proliferation in the presence of media collected from B16.F1 or CT26.CL25 tumor cells after transduction with TPA particles carrying IL15IgK. Cells treated with a recombinant IL2 cytokine were included as a positive control. Data are representative of one experiment, expressed as mean + SEM and shown as change in CTLL-2 number relative to treatment initiation day. Experiments were repeated twice and in triplicates, one-way ANOVA with Tukey's HSD test was used to analyse the data.


( A ) Tumor - bearing BALB / c mice with established subcutaneous CT26 . CL25 tumors were intravenously injected with a single dose , 5x1010 TU / mouse , of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration . Data are representative of one experiment , n = 3 , and expressed as mean + SEM . Two - way ANOVA test was used for data analysis .

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(A) Tumor-bearing BALB/c mice with established subcutaneous CT26.CL25 tumors were intravenously injected with a single dose, 5x1010 TU/mouse, of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration. Data are representative of one experiment, n = 3, and expressed as mean + SEM. Two-way ANOVA test was used for data analysis.


( B ) Safety of dose escalation regimens assessed at day 5 following TPA delivery , by evaluating the serum levels of LDH from n = 2 mice / TPA dose of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Serums from untreated mice were also analysed .

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(B) Safety of dose escalation regimens assessed at day 5 following TPA delivery, by evaluating the serum levels of LDH from n = 2 mice/TPA dose of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Serums from untreated mice were also analysed.


( C ) Representative tumor - bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C . TPA . IL15IgK ( RGD4C ) or non - targeted ( NT ) particles . The CT26 . CL25 tumor cells stably express a luciferase Luc reporter gene . Untreated mice were also included as controls ( CTRL ) .

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(C) Representative tumor-bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C.TPA.IL15IgK (RGD4C) or non-targeted (NT) particles. The CT26.CL25 tumor cells stably express a luciferase Luc reporter gene. Untreated mice were also included as controls (CTRL).


( D ) Changes in tumor viability in mice ( n = 4 ) from day 0 to day 5 post TPA administration . Tumor viability was measured by bioluminescence activity . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Kruskal - Wallis test was used for data analysis .

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(D) Changes in tumor viability in mice (n = 4) from day 0 to day 5 post TPA administration. Tumor viability was measured by bioluminescence activity. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Kruskal-Wallis test was used for data analysis.


( E ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from all experimental groups .

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(E) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from all experimental groups.


( F ) Animal weights ( n = 4 ) were monitored during therapy experiments ad expressed as mean + SEM .

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(F) Animal weights (n = 4) were monitored during therapy experiments ad expressed as mean + SEM.


( A ) The immune profile of CT26 . CL25 tumors was investigated using RT - qPCR at day 5 after TPA . IL15IgK administration . A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8 + T cell population ; NKp46 for NK cell population . Markers for cytotoxic cell - killing by CD8 + T cells ( GZMa , GZMb ) and by NK cells ( Prf1 ) were also analysed . Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma ( IFN - γ ) , and FoxP3 for Treg cells . Data shown are representative of n = 5 mice and expressed as mean + SEM . Non - parametric Mann - Whitney test was used for GZMb and TBX2 data analysis , and independent t - test to analyse data of the other markers .

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

(A) The immune profile of CT26.CL25 tumors was investigated using RT-qPCR at day 5 after TPA.IL15IgK administration. A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8+ T cell population; NKp46 for NK cell population. Markers for cytotoxic cell-killing by CD8+ T cells (GZMa, GZMb) and by NK cells (Prf1) were also analysed. Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma (IFN-γ), and FoxP3 for Treg cells. Data shown are representative of n = 5 mice and expressed as mean + SEM. Non-parametric Mann-Whitney test was used for GZMb and TBX2 data analysis, and independent t-test to analyse data of the other markers.


( B ) TPA particles encoding IL12 were used to treat immunocompetent BALB / c mice bearing subcutaneous CT26 . CL25 tumors labelled with a Luc reporter gene . Representative tumor - bearing mice at day 5 post - treatment with targeted RGD4C . TPA . IL12 ( RGD4C ) or non - targeted TPA . IL12 ( NT ) .

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(B) TPA particles encoding IL12 were used to treat immunocompetent BALB/c mice bearing subcutaneous CT26.CL25 tumors labelled with a Luc reporter gene. Representative tumor-bearing mice at day 5 post-treatment with targeted RGD4C.TPA.IL12 (RGD4C) or non-targeted TPA.IL12 (NT).


( C ) Analysis of total flux change in tumors , n = 4 mice , after IL12 immunotherapy . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Mann - Whitney test was used for data analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of total flux change in tumors, n = 4 mice, after IL12 immunotherapy. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Mann-Whitney test was used for data analysis.


( D ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from two experimental groups .

(D) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from two experimental groups.


( E ) Serial quantification of IL12 in media of B16 . F10 cells treated with RGD4C . TPA . IL12 or controls . Data are representative of one experiment , n = 3 biological repeats , and shown as mean + SD Experiments were repeated three times . One - way ANOVA with Tukey ' s HSD test was used for data analysis .

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

(E) Serial quantification of IL12 in media of B16.F10 cells treated with RGD4C.TPA.IL12 or controls. Data are representative of one experiment, n = 3 biological repeats, and shown as mean + SD Experiments were repeated three times. One-way ANOVA with Tukey's HSD test was used for data analysis.


( F ) RGD4C . TPA . IL12 immunotherapy of subcutaneous B16 . F10 tumors in C57BL / 6 mice ( n = 4 ) . Data are shown as mean + SEM independent t - test was used for data analysis . Tumor volume changes are shown over time .

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

(F) RGD4C.TPA.IL12 immunotherapy of subcutaneous B16.F10 tumors in C57BL/6 mice (n = 4). Data are shown as mean + SEM independent t-test was used for data analysis. Tumor volume changes are shown over time.


Kaplan - Meier survival curves stratified by the patient MICA - 129 genotype for all patients ( n = 446 ) . The survival curve is displayed for the first 66 months ; 7 . 6 % of the patients were followed longer . Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2 . The HR indicates the risk per MICA - 129Met allele carried by the patients ( additive risk model ) . The numbers of patients carrying the three genotypes and the number of events ( in brackets ) are indicated .

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Kaplan-Meier survival curves stratified by the patient MICA-129 genotype for all patients (n = 446). The survival curve is displayed for the first 66 months; 7.6% of the patients were followed longer. Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2. The HR indicates the risk per MICA-129Met allele carried by the patients (additive risk model). The numbers of patients carrying the three genotypes and the number of events (in brackets) are indicated.


Kaplan - Meier survival curves for patients receiving a graft matched for the MICA - 129 genotype ( n = 404 ) .

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Kaplan-Meier survival curves for patients receiving a graft matched for the MICA-129 genotype (n = 404).


Kaplan - Meier survival curves for patients who experienced aGVDH ( any grade , n = 244 ) .

Kaplan-Meier survival curves for patients who experienced aGVDH (any grade, n = 244).


Kaplan - Meier survival curves for patients not experiencing aGVHD ( n = 189 ) . The HR indicates the risk of patients carrying two MICA - 129Met alleles ( recessive risk model ) .

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Kaplan-Meier survival curves for patients not experiencing aGVHD (n = 189). The HR indicates the risk of patients carrying two MICA-129Met alleles (recessive risk model).


Kaplan - Meier survival curves for patients who did not receive a T - cell - depleting treatment with ATG ( n = 193 ) . Kaplan - Meier survival curves for patients treated with ATG ( n = 250 ) .

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Kaplan-Meier survival curves for patients who did not receive a T-cell-depleting treatment with ATG (n = 193).Kaplan-Meier survival curves for patients treated with ATG (n = 250).


Kaplan - Meier survival curves for patients with the MICA - 129Val / Val genotype ( n = 229 ) stratified by treatment with ATG .

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Kaplan-Meier survival curves for patients with the MICA-129Val/Val genotype (n = 229) stratified by treatment with ATG.


Kaplan - Meier survival curves for patients carrying one or two MICA - 129Met alleles ( n = 214 ) stratified by treatment with ATG .

Kaplan-Meier survival curves for patients carrying one or two MICA-129Met alleles (n = 214) stratified by treatment with ATG.


The linear regression of MICA expression intensity and binding of a recombinant NKG2D - Fc fusion protein both determined as MFI by flow cytometry is displayed for L - MICA - 129Met ( n = 79 , left panel ) and L - MICA - 129Val clones ( n = 81 , right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

The linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met (n = 79, left panel) and L-MICA-129Val clones (n = 81, right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


Purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells ( 106 ) were stimulated with immobilized MICA - 129Met - mIgG2a - Fc or MICA - 129Val - mIgG2a - Fc or OVA - mIgG2a - Fc fusion proteins ( 10 μg / ml ) for 3 , 10 , or 30 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb , an anti - phospho - SRC family ( Tyr419 ) kinases Ab , and an anti - β - actin mAb as a loading control . The arrow points toward phosphorylated SRC family kinases . Blots obtained from three independent experiments were analyzed by densitometry , and the means plus SD of the ratio between phospho - SRC family kinase and β - actin signals is displayed . The difference between NK cells stimulated for 10 min by MICA - 129Met - Fc or MICA - 129Val - Fc proteins was assessed by t - test .

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

Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (106) were stimulated with immobilized MICA-129Met-mIgG2a-Fc or MICA-129Val-mIgG2a-Fc or OVA-mIgG2a-Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases.Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t-test.


Purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days , 106 ) were incubated with the SRC kinase inhibitor PP2 ( 25 μM ) , the vehicle DMSO , or medium only ( Ø ) for 30 min before being added to immobilized MICA - 129Met - Fc , MICA - 129Val - Fc , or OVA - Fc fusion proteins ( 10 μg / ml ) for 10 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb and an anti - β - actin mAb as a loading control . The blot is representative for two independent experiments .

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

Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.


In parallel , degranulation of the NK cells was measured by anti - CD107a staining in flow cytometry . The difference between DMSO - and PP2 - treated cells with respect to CD107a + cells and the MFI of CD107a is indicated in the histograms . The results are representative for two independent experiments .

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In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.


The degranulation ( CD107a expression ) of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells in response to immobilized MICA - 129Met - Fc and MICA - 129Val - Fc , and as negative control , OVA - Fc fusion proteins was determined by flow cytometry after 1 h ( n = 3 ) . Displayed are means and SD of the MFI ( left panel ) and the percentage of CD107a + cells ( right panel ) . Differences between MICA - 129Met - Fc - and MICA - 129Val - Fc - induced NK - cell degranulation were analyzed by two - way ANCOVA adjusted for MICA protein concentration , and the respective P - values are indicated .

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

The degranulation (CD107a expression) of purified IL-2-stimulated (100 U/ml for 4 days) NK cells in response to immobilized MICA-129Met-Fc and MICA-129Val-Fc, and as negative control, OVA-Fc fusion proteins was determined by flow cytometry after 1 h (n = 3). Displayed are means and SD of the MFI (left panel) and the percentage of CD107a+ cells (right panel). Differences between MICA-129Met-Fc- and MICA-129Val-Fc-induced NK-cell degranulation were analyzed by two-way ANCOVA adjusted for MICA protein concentration, and the respective P-values are indicated.


The degranulation of IL - 2 - stimulated LAK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 27 ) or L - MICA - 129Val clones ( n = 27 ) for 1 h was determined by flow cytometry . CD107a cell surface expression was analyzed after gating on CD56 + NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of CD107a on NK cells ( upper panels ) or the proportion of CD107a + NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

The degranulation of IL-2-stimulated LAK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 27) or L-MICA-129Val clones (n = 27) for 1 h was determined by flow cytometry. CD107a cell surface expression was analyzed after gating on CD56+ NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of CD107a on NK cells (upper panels) or the proportion of CD107a+ NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L - MICA - 129Met and an L - MICA - 129Val clone . L - con cells served as a negative and K562 cells as a positive control . The means of specific lysis of triplicates plus SD at different E : T ratios ( 200 : 1 to 3 : 1 ) were measured in an 51chromium - release assay . The MICA expression intensity and the binding of a recombinant NKG2D - Fc fusion protein to the target cells were determined in parallel by flow cytometry , and the MFIs are indicated .

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

A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L-MICA-129Met and an L-MICA-129Val clone. L-con cells served as a negative and K562 cells as a positive control. The means of specific lysis of triplicates plus SD at different E:T ratios (200:1 to 3:1) were measured in an 51chromium-release assay. The MICA expression intensity and the binding of a recombinant NKG2D-Fc fusion protein to the target cells were determined in parallel by flow cytometry, and the MFIs are indicated.


The IFNγ expression of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells exposed for 4 h to L - MICA - 129Met ( n = 23 ) or L - MICA - 129Val clones ( n = 23 ) was determined by flow cytometry . The IFNγ expression was analyzed after gating on CD56brightCD16 − NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16 − NK cells ( upper panels ) or the proportion of IFNγ + CD56brightCD16 − NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

The IFNγ expression of purified IL-2-stimulated (100 U/ml for 4 days) NK cells exposed for 4 h to L-MICA-129Met (n = 23) or L-MICA-129Val clones (n = 23) was determined by flow cytometry. The IFNγ expression was analyzed after gating on CD56brightCD16−NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16−NK cells (upper panels) or the proportion of IFNγ+CD56brightCD16−NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


The IFNγ release of purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days ) co - cultured with L - MICA - 129Met ( n = 34 ) or L - MICA - 129Val clones ( n = 32 ) for 24 h was measured in the supernatant by ELISA . In parallel , the MICA expression intensity on target cells was determined by flow cytometry . The linear regressions of IFNγ release ( pg / ml ) by NK cells and MICA expression on targets ( MFI ) are displayed for the L - MICA - 129Met clones ( left panel ) and the L - MICA - 129Val clones ( right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

The IFNγ release of purified IL-2-stimulated NK cells (100 U/ml for 4 days) co-cultured with L-MICA-129Met (n = 34) or L-MICA-129Val clones (n = 32) for 24 h was measured in the supernatant by ELISA. In parallel, the MICA expression intensity on target cells was determined by flow cytometry. The linear regressions of IFNγ release (pg/ml) by NK cells and MICA expression on targets (MFI) are displayed for the L-MICA-129Met clones (left panel) and the L-MICA-129Val clones (right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


NKG2D expression on purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 25 ) or L - MICA - 129Val clones ( n = 25 ) for 0 , 4 , and 24 h was analyzed by flow cytometry . NK cells ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating of CD3 − CD56 + cells . The means and SD of the percentage of NKG2D + NK cells ( left panel ) and of the MFI of NKG2D ( right panel ) are shown . Differences between the groups were analyzed by repeated measures ANOVA , and P - values for pairwise comparisons are indicated .

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

NKG2D expression on purified IL-2-stimulated NK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 25) or L-MICA-129Val clones (n = 25) for 0, 4, and 24 h was analyzed by flow cytometry. NK cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating of CD3−CD56+ cells. The means and SD of the percentage of NKG2D+ NK cells (left panel) and of the MFI of NKG2D (right panel) are shown. Differences between the groups were analyzed by repeated measures ANOVA, and P-values for pairwise comparisons are indicated.


MACS - separated CD8 + T cells were cultured in triplicate on an immobilized anti - CD3 mAb ( 0 . 005 μg / ml [ upper panel ] or 0 . 01 μg / ml [ lower panel ] ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , and OVA - Fc proteins at various concentrations ( 1 . 0 , 0 . 5 , 0 . 1 , 0 . 0 μg / ml ) . After 72 h , 25 % of the supernatant was harvested and IL - 2 concentrations were measured by ELISA . The harvested medium was replaced by the same volume containing 1 μCi 3H - labeled thymidine . After 12 h , the plates were completely harvested and the DNA - bound radioactivity was determined . The means and SD of the stimulation index ( SI ) are displayed ( n = 4 ) . Significant differences between MICA - 129Met / Val - Fc and OVA - Fc proteins were found when the antigen - specific signal ( anti - CD3 ) was limited ( * P < 0 . 05 , t - test ; upper left panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0372 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0366 ; upper right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0499 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0192 ; 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0164 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0357 ; lower left panel : 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0287 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0232 ; lower right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0171 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0484 ) .

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

MACS-separated CD8+ T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed (n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (*P < 0.05, t-test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484).


Purified CFSE - stained CD8 + T cells were stimulated by immobilized anti - CD3 ( 0 . 005 μg / ml ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , OVA - Fc proteins , or co - stimulatory mAb ( anti - CD28 , anti - NKG2D ) or an isotype control ( mIgG1 ) . The proliferation of CD3 + CD8 + T cells was assessed at 60 h by flow cytometry . Results of a representative out of 6 experiments are displayed . Untreated CFSE - stained CD8 + T cells are included for comparison . The percentage of proliferating cells and MFI for CFSE are indicated . The MFI of CFSE in unstimulated CD8 + T cells ( control ) was set to 100 % in individual experiments ( n = 6 ) , and the relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( * P = 0 . 0277 , Wilcoxon test ) between MICA - 129Met - Fc versus MICA - 129Val - Fc and OVA - Fc proteins were found at slightly higher anti - CD3 concentrations ( 0 . 1 and 0 . 05 μg / ml ) than at later time points ( see A ) . Anti - CD28 and anti - NKG2D mAb were used in parallel as a positive control , mean + SD are shown , and significant differences ( * P = 0 . 0277 , Wilcoxon test ) to the isotype control ( mIgG1 ) are indicated ( n = 6 ) .

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Purified CFSE-stained CD8+T cells were stimulated by immobilized anti-CD3 (0.005 μg/ml) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, OVA-Fc proteins, or co-stimulatory mAb (anti-CD28, anti-NKG2D) or an isotype control (mIgG1). The proliferation of CD3+CD8+T cells was assessed at 60 h by flow cytometry. Results of a representative out of 6 experiments are displayed. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (*P = 0.0277, Wilcoxon test) between MICA-129Met-Fc versus MICA-129Val-Fc and OVA-Fc proteins were found at slightly higher anti-CD3 concentrations (0.1 and 0.05 μg/ml) than at later time points (see A).Anti-CD28 and anti-NKG2D mAb were used in parallel as a positive control, mean + SD are shown, and significant differences (*P = 0.0277, Wilcoxon test) to the isotype control (mIgG1) are indicated (n = 6).


NKG2D expression on purified CD8 + T cells exposed to L - MICA - 129Met ( n = 19 ) or L - MICA - 129Val clones ( n = 19 ) for 0 , 4 , and 24 h was analyzed by flow cytometry . CD8 + T cells ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3 + CD8 + T cells . The means and SD of the MFI of NKG2D ( left panel ) and of the percentage of NKG2D + CD8 + T cells ( right panel ) are displayed . Differences between the groups were analyzed by repeated measures ANOVA , and the P - values are indicated .

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

NKG2D expression on purified CD8+T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.


Purified CD8 + T cells were cultured on plate - bound anti - NKG2D ( 1 μg / ml ) or isotype control ( mIgG1 ) for 24 h before the NKG2D expression was measured by flow cytometry . Means and SD of MFI ( upper panel ) and percentage of NKG2D + cells ( lower panel ) are shown ( n = 6 ) . Differences between the groups were analyzed by t - tests , and the P - values are indicated .

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

Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.


These CD8 + T cells were subsequently CFSE - stained and cultured on plates coated with anti - CD3 ( 0 . 005 μg / ml ) in combination with anti - CD28 ( 0 . 5 μg / ml ) as a positive control or anti - NKG2D ( 0 . 5 μg / ml ) . Proliferation was measured after 60 h by flow cytometry . Untreated CFSE - stained CD8 + T cells are included for comparison . The percentage of proliferating cells and the MFI for CFSE are indicated . The MFI of CFSE in unstimulated CD8 + T cells ( control ) was set to 100 % in individual experiments ( n = 6 ) , and the relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( Wilcoxon test ) between CD8 + T cells pre - exposed to anti - NKG2D and isotype control ( mIgG1 ) were found in these experiments at anti - CD3 concentrations of 0 . 01 and 0 . 005 μg / ml .

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

These CD8+T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.


A - D Appearance of autophagic structures ( marked with GFP - Atg8a ) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1 , Atg5 , Atg6 , Atg7 or Atg12 . All constructs are expressed in the epidermis under the control of the A58 - Gal4 driver . ( A ) Time points from movies of wounded epidermis . The wounds have closed by 2 h in all cases . ( B ) Higher magnification of the areas marked by magenta boxes at t = 120 min . ( C ) Quantification of the appearance of GFP - Atg8a puncta in the imaged area ( 10000 µm2 ) 3 control larvae , shown for each individual larva . ( D ) Quantification of number of GFP - Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure ; n = 4 - 10 larvae each genotype , for the detail see Data analysis .

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

A-D Appearance of autophagic structures (marked with GFP-Atg8a) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1, Atg5, Atg6, Atg7 or Atg12. All constructs are expressed in the epidermis under the control of the A58-Gal4 driver. (A) Time points from movies of wounded epidermis. The wounds have closed by 2 h in all cases. (B) Higher magnification of the areas marked by magenta boxes at t=120 min. (C) Quantification of the appearance of GFP-Atg8a puncta in the imaged area (10000 µm2) 3 control larvae, shown for each individual larva. (D) Quantification of number of GFP-Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure; n=4-10 larvae each genotype, for the detail see Data analysis.


Control of epidermal autophagy by TOR signalling . ( A ) Epidermis of third instar larvae expressing the autophagosome marker GFP - Atg8a together with constructs for up - or down - regulating the autophagy pathway in the epidermis . Healthy epidermis contains few autophagosomes , but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes . ( B ) Higher magnification of the areas marked by magenta boxes in ( A ) .

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Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A).


Control of epidermal autophagy by TOR signalling . ( C ) Quantification of Atg8a puncta in an area of 10000 µm2 , n = 6 - 8 larvae each genotype . We assumed unequal sample size and unequal variances and calculations were performed . Values are presented as box plots . Box plot elements are : center line , median ; box limits , upper and lower quartiles ; whiskers , 1 . 5x interquartile range ; points , outliers .

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Control of epidermal autophagy by TOR signalling. (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.


A - D Fluorescence loss in photobleaching ( FLIP ) to test free cytoplasmic GFP motility within the epidermis . A small area ( magenta circle ; 179 or 1098 µm2 ) was laser - illuminated for the indicated times ( 3 or 20 min ) in control or Atg1 - expressing epidermis also expressing free GFP . ( A ) Snapshots before bleaching and at 3 points of recovery . ( B ) Kymographs along the broken line in ( A ) during recovery . ( C , D ) Quantification of fluorescence recovery after bleaching , shown separately for 3 and 20 min bleaching protocols .

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

A-D Fluorescence loss in photobleaching (FLIP) to test free cytoplasmic GFP motility within the epidermis. A small area (magenta circle; 179 or 1098 µm2) was laser-illuminated for the indicated times (3 or 20 min) in control or Atg1-expressing epidermis also expressing free GFP. (A) Snapshots before bleaching and at 3 points of recovery. (B) Kymographs along the broken line in (A) during recovery. (C, D) Quantification of fluorescence recovery after bleaching, shown separately for 3 and 20 min bleaching protocols.


B - D Atg1 - induced syncytium formation is abolished when Atg5 function is downregulated ( compare to rows 2 and 3 in Fig . 5A , B ) ; same representation as shown in Fig . 5A - D .

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B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.


C , D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy . ( C ) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493 / 503 to mark lipid accumulations . ( D ) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions . A - D , n = 7 - 20 larvae each genotype .

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C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.


( a , b ) Bacteria in the spleens of Slamf1 − / − Rag1 − / − and Rag1 − / − BALB / c mice ( a ) or Slamf1 − / − and Slamf1 + / + BALB / c mice ( b ) 48 h after intraperitoneal injection of virulent S . typhimurium 14028s or attenuated S . typhimurium SseB − . CFU , colony - forming units ; ND , not detectable . Data are representative of four independent experiments ( mean and s . d . ) .

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(a,b) Bacteria in the spleens of Slamf1−/−Rag1−/− and Rag1−/− BALB/c mice (a) or Slamf1−/− and Slamf1+/+ BALB/c mice (b) 48 h after intraperitoneal injection of virulent S. typhimurium 14028s or attenuated S. typhimurium SseB−. CFU, colony-forming units; ND, not detectable. Data are representative of four independent experiments (mean and s.d.).


( c - e ) Killing of bacteria by peritoneal macrophages from Slamf1 + / + and Slamf1 − / − BALB / c mice exposed to S . typhimurium SseB − ( c ) , E . coli F18 ( d ) or S . aureus ( e ) , assessed by gentamycin assay . Data are representative of five independent experiments ( mean and s . d . ) .

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(c-e) Killing of bacteria by peritoneal macrophages from Slamf1+/+ and Slamf1−/− BALB/c mice exposed to S. typhimurium SseB− (c), E. coli F18 (d) or S. aureus (e), assessed by gentamycin assay. Data are representative of five independent experiments (mean and s.d.).


( f ) Uptake of bacteria by Slamf1 + / + ( red solid lines ) or Slamf1 − / − ( black solid lines ) BALB / c peritoneal macrophages incubated at 37 ° C with E . coli - eGFP or S . typhimurium - eGFP or by Slamf1 + / + macrophages incubated for 60 min at 4 ° C with the bacteria ( dotted lines ) . Right , mean fluorescence intensity ( MFI ) . Data are representative of three independent experiments ( mean and s . d . ) .

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(f) Uptake of bacteria by Slamf1+/+ (red solid lines) or Slamf1−/− (black solid lines) BALB/c peritoneal macrophages incubated at 37 °C with E. coli-eGFP or S. typhimurium-eGFP or by Slamf1+/+ macrophages incubated for 60 min at 4 °C with the bacteria (dotted lines). Right, mean fluorescence intensity (MFI). Data are representative of three independent experiments (mean and s.d.).