Figure 2A . Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low ( 0 . 2 µg ) and high ( 3 µg ) dose polySia avDP20 or PBS - vehicle . Late stage ( 10 - 11 minutes after fluorescein injection ) fundus fluorescein angiography revealed that PBS - treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20 - treated SIGLEC11 transgenic ( tg ) mice had lower levels of vessel leakage compared to PBS - injected wild type or SIGLEC11 tg mice . High polySia avDP20 dose reduced vascular leakage also in wild type animals . Representative images out of at least eight independent experiments are shown . B . Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software ( NIH ) . Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted . PolySia avDP20 - treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS - injected wild type control and SIGLEC11 tg mice . A reduction in vascular leakage was also observed in high dose polySia avDP20 - injected wild type control mice . Data show mean + / - SD . * p < 0 . 05 , * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 20 eyes ) , WT 0 . 2 µg Sia ( n = 17 eyes ) , WT 3 µg Sia ( n = 9 eyes ) , SIGLEC11 tg PBS ( n = 16 eyes ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 20 eyes ) , SIGLEC11 tg 3 µg Sia ( n = 15 eyes ) , WT PBS vs WT 3 µg Sia p = 0 . 0193 , WT 0 . 2 µg Sia vs SIGLEC11 tg 0 . 2 µg Sia p < 0 . 0001 , SIGLEC11 tg PBS vs SIGLEC11 tg 0 . 2 µg Sia p < 0 . 0001 , SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0 . 0002 . C . Anti - C5b - 9 immunostaining of RPE / choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle - injected controls . PolySia avDP20 treatment reduced MAC formation in a dose - dependent fashion independent of SIGLEC11 presence . Scale bar : 100 µm . D . Quantification of C5b - 9 fluorescence signal intensity in the laser lesions . In comparison to the high amount of MAC deposition in vehicle - injected controls , the pixel intensity is reduced in polySia avDP20 - treated eyes in a dose - dependent fashion and regardless of SIGLEC11 presence . Data show mean + / - SD . * * * p < 0 . 001 , One - way ANOVA followed by Fisher ' s LSD . WT PBS ( n = 12 laser spots ) , WT 0 . 2 µg Sia ( n = 12 laser spots ) , WT 3 µg Sia ( n = 7 laser spots ) , SIGLEC11 tg PBS ( n = 6 laser spots ) , SIGLEC11 tg 0 . 2 µg Sia ( n = 10 laser spots ) , SIGLEC11 tg 3 µg Sia ( n = 12 laser spots ) ; all statistical comparisons p < 0 . 0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0 . 2 µg Sia p = 0 . 0004 .

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

Figure 2A. Fundus fluorescein angiography was performed 48 hours after laser coagulation and intravitreal application of low (0.2 µg) and high (3 µg) dose polySia avDP20 or PBS-vehicle. Late stage (10-11 minutes after fluorescein injection) fundus fluorescein angiography revealed that PBS-treated wild type controls and humanized SIGLEC11 mice showed normal levels of vessel leakage whereas polySia avDP20-treated SIGLEC11 transgenic (tg) mice had lower levels of vessel leakage compared to PBS-injected wild type or SIGLEC11 tg mice. High polySia avDP20 dose reduced vascular leakage also in wild type animals. Representative images out of at least eight independent experiments are shown.B. Fundus fluorescein angiography pictures were exported from Heidelberg Eye Explorer Software and fluorescein leakage was quantified with ImageJ software (NIH). Pixel intensities of 6 regions of interest per picture were quantified and background fluorescence was subtracted. PolySia avDP20-treated SIGLEC11 tg animals showed reduced vascular leakage compared to PBS-injected wild type control and SIGLEC11 tg mice. A reduction in vascular leakage was also observed in high dose polySia avDP20-injected wild type control mice. Data show mean +/- SD. * p< 0.05, *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 20 eyes), WT 0.2 µg Sia (n = 17 eyes), WT 3 µg Sia (n = 9 eyes), SIGLEC11 tg PBS (n = 16 eyes), SIGLEC11 tg 0.2 µg Sia (n = 20 eyes), SIGLEC11 tg 3 µg Sia (n = 15 eyes), WT PBS vs WT 3 µg Sia p = 0.0193, WT 0.2 µg Sia vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 0.2 µg Sia p < 0.0001, SIGLEC11 tg PBS vs SIGLEC11 tg 3 µg Sia p = 0.0002.C. Anti-C5b-9 immunostaining of RPE/choroid whole mount preparations 48 hours after laser damage showed strong MAC deposition in the laser lesions of vehicle-injected controls. PolySia avDP20 treatment reduced MAC formation in a dose-dependent fashion independent of SIGLEC11 presence. Scale bar: 100 µm.D. Quantification of C5b-9 fluorescence signal intensity in the laser lesions. In comparison to the high amount of MAC deposition in vehicle-injected controls, the pixel intensity is reduced in polySia avDP20-treated eyes in a dose-dependent fashion and regardless of SIGLEC11 presence. Data show mean +/- SD. *** p< 0.001, One-way ANOVA followed by Fisher's LSD. WT PBS (n = 12 laser spots), WT 0.2 µg Sia (n = 12 laser spots), WT 3 µg Sia (n = 7 laser spots), SIGLEC11 tg PBS (n = 6 laser spots), SIGLEC11 tg 0.2 µg Sia (n = 10 laser spots), SIGLEC11 tg 3 µg Sia (n = 12 laser spots); all statistical comparisons p < 0.0001 except SIGLEC11 tg PBS versus SIGLEC11 tg 0.2 µg Sia p = 0.0004.


Figure 3A . Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11 / 16 knockout THP1 - macrophages . The levels of gene transcripts were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and concentrations of 0 . 15 µM and 1 . 5 µM of polySia avDP20 in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout line . Data show mean + / - SEM . * * p < 0 . 01 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 7 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 4 and p = 0 . 0002 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 0 . 009 , LPS / PolySia avDP20 1 . 5µM n = 5 and p = 0 . 002 . Siglec11 / 16 KO : no treatment n = 5 and p = 0 . 01 , PolySia avDP20 n = 4 and p = 0 . 122 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 1 . 0 . B . Analysis of relative TNFSF2 protein release in human control and SIGLEC11 / 16 knockout THP1 - macrophages . The released protein levels were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and concentrations of 0 . 15 µM and 1 . 5 µM of polySia avDP20 in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout line . Data show mean + / - SEM . * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 8 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 001 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 0 . 002 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 0 . 0003 . Siglec11 / 16 KO : no treatment n = 6 and p < 0 . 001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 001 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 5 and p = 1 . 0 . C . Analysis of relative VEGFA gene transcription in human control and SIGLEC11 / 16 knockout THP1 - macrophages . Gene transcripts were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and polySia avDP20 ( 0 . 15 µM and 1 . 5 µM ) in the human wild type macrophages . No response to polySia avDP20 was detectable in the knockout macrophages . Data show mean + / - SEM . * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 6 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 5 and p < 0 . 0001 , LPS n = 5 , LPS / PolySia avDP20 0 . 15µM n = 5 and p = 0 . 0002 , LPS / PolySia avDP20 1 . 5µM n = 5 and p < 0 . 0001 . Siglec11 / 16 KO : no treatment n = 5 and p = 0 . 022 , PolySia avDP20 n = 3 and p = 0 . 063 , LPS n = 4 , LPS / PolySia avDP20 0 . 15µM n = 3 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 4 and p = 1 . 0 . D . Analysis of relative VEGFA protein release in human control and SIGLEC11 / 16 knockout THP1 - macrophages . Released protein levels were reduced after 24 hours of co - treatment with LPS ( 1 µg / ml ) and polySia avDP20 ( 0 . 15 µM and 1 . 5 µM ) in the human macrophages . Data show mean + / - SEM . * p < 0 . 05 , * * * p < 0 . 001 , ANOVA followed by Bonferroni . Statistical analysis was done in relation to the LPS control . WT : no treatment n = 9 and p < 0 . 0001 , PolySia avDP20 1 . 5 µM n = 6 and p = 0 . 009 , LPS n = 9 , LPS / PolySia avDP20 0 . 15µM n = 6 and p = 0 . 043 , LPS / PolySia avDP20 1 . 5µM n = 7 and p = 0 . 0001 . Siglec11 / 16 KO : no treatment n = 9 and p = 0 . 349 , PolySia avDP20 1 . 5 µM n = 6 and p = 0 . 249 , LPS n = 7 , LPS / PolySia avDP20 0 . 15µM n = 6 and p = 1 . 0 , LPS / PolySia avDP20 1 . 5µM n = 7 and p = 1 . 0 . E . Quantification of human control and SIGLEC11 / 16 knockout THP1 - macrophages having ingested cellular debris . PolySia avDP20 ( 1 . 5 µM ) reduced the percentage of phagocytic cells having ingested drusen - like debris . No response to polySia avDP20 was detectable in the knockout macrophages . Data are presented as mean + / - SEM . n = 6 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , ANOVA followed by Bonferroni . Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0 . 028 , debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0 . 00012 . F . Prevention of superoxide release in activated human control and SIGLEC11 / 16 knockout THP1 - macrophages by polySia avDP20 . Cultured human THP1 - macrophages were stimulated with RPE cell debris or co - stimulated with debris and polySia avDP20 . Addition of debris stimulated the production of superoxide . 1 . 5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge . No response to polySia avDP20 was detectable in the knockout macrophages . Data are presented as mean + / - SEM . n = 6 . p * ≤ 0 . 05 , * * * p ≤ 0 . 001 , ANOVA followed by Bonferroni . Untreated WT macrophages vs debris treated WT macrophages p = 0 . 037 , debris treated WT macrophage vs debris plus polySia avDP20 1 . 5 µM treated WT macrophages p < 0 . 001 , debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0 . 001 , G . PolySia avDP20 ( 0 . 15 to 50 µM ) was added to normal human serum ( NHS ) to evaluate any interference with activation of the classical complement pathway by IgM . Complement activation was determined by C3b deposition . PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway . Data show mean + / - SEM . n = 3 , n . s . = not significant . H . PolySia avDP20 inhibits the alternative complement pathway . PolySia avDP20 ( 0 . 15 to 50 µM ) was added to NHS and activation of the alternative pathway was induced by LPS . C3b deposition was monitored by ELISA . Data show mean + / - SEM ( n = 4 . * p < 0 . 05 , * * p < 0 . 01 , p * * * < 0 . 001 , ANOVA followed by Bonferroni ) .

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

Figure 3A. Analysis of relative TNFSF2 gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. The levels of gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=7 and p< 0.0001, PolySia avDP20 1.5 µM n=4 and p=0.0002, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=0.009, LPS/PolySia avDP20 1.5µM n=5 and p=0.002. Siglec11/16 KO: no treatment n=5 and p=0.01, PolySia avDP20 n=4 and p=0.122, LPS n=7, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.B. Analysis of relative TNFSF2 protein release in human control and SIGLEC11/16 knockout THP1-macrophages. The released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and concentrations of 0.15 µM and 1.5 µM of polySia avDP20 in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout line. Data show mean +/- SEM. ** p < 0.01, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=8 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=0.002, LPS/PolySia avDP20 1.5µM n=4 and p=0.0003. Siglec11/16 KO: no treatment n=6 and p<0.001, PolySia avDP20 1.5 µM n=5 and p<0.001, LPS n=7, LPS/PolySia avDP20 0.15µM n=5 and p=1.0, LPS/PolySia avDP20 1.5µM n=5 and p=1.0.C. Analysis of relative VEGFA gene transcription in human control and SIGLEC11/16 knockout THP1-macrophages. Gene transcripts were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human wild type macrophages. No response to polySia avDP20 was detectable in the knockout macrophages. Data show mean +/- SEM. *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=6 and p<0.0001, PolySia avDP20 1.5 µM n=5 and p<0.0001, LPS n=5, LPS/PolySia avDP20 0.15µM n=5 and p=0.0002, LPS/PolySia avDP20 1.5µM n=5 and p<0.0001. Siglec11/16 KO: no treatment n=5 and p=0.022, PolySia avDP20 n=3 and p=0.063, LPS n=4, LPS/PolySia avDP20 0.15µM n=3 and p=1.0, LPS/PolySia avDP20 1.5µM n=4 and p=1.0.D. Analysis of relative VEGFA protein release in human control and SIGLEC11/16 knockout THP1-macrophages. Released protein levels were reduced after 24 hours of co-treatment with LPS (1 µg/ml) and polySia avDP20 (0.15 µM and 1.5 µM) in the human macrophages. Data show mean +/- SEM. * p < 0.05, *** p < 0.001, ANOVA followed by Bonferroni. Statistical analysis was done in relation to the LPS control. WT: no treatment n=9 and p<0.0001, PolySia avDP20 1.5 µM n=6 and p=0.009, LPS n=9, LPS/PolySia avDP20 0.15µM n=6 and p=0.043, LPS/PolySia avDP20 1.5µM n=7 and p=0.0001. Siglec11/16 KO: no treatment n=9 and p=0.349, PolySia avDP20 1.5 µM n= 6 and p=0.249, LPS n=7, LPS/PolySia avDP20 0.15µM n=6 and p=1.0, LPS/PolySia avDP20 1.5µM n=7 and p=1.0.E. Quantification of human control and SIGLEC11/16 knockout THP1-macrophages having ingested cellular debris. PolySia avDP20 (1.5 µM) reduced the percentage of phagocytic cells having ingested drusen-like debris. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. *p ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Debris treated WT macrophages vs debris plus polySia avDP20 treated WT macrophages p = 0.028, debris plus polySia avDP20 treated WT macrophages vs KO macrophages p = 0.00012.F. Prevention of superoxide release in activated human control and SIGLEC11/16 knockout THP1-macrophages by polySia avDP20. Cultured human THP1-macrophages were stimulated with RPE cell debris or co-stimulated with debris and polySia avDP20. Addition of debris stimulated the production of superoxide. 1.5 µM polySia avDP20 completely prevented the release of superoxide induced by debris challenge. No response to polySia avDP20 was detectable in the knockout macrophages. Data are presented as mean +/- SEM. n=6. p* ≤ 0.05, *** p ≤ 0.001, ANOVA followed by Bonferroni. Untreated WT macrophages vs debris treated WT macrophages p = 0.037, debris treated WT macrophage vs debris plus polySia avDP20 1.5 µM treated WT macrophages p < 0.001, debris plus polySia avDP20 WT macrophages vs KO macrophages p = 0.001,G. PolySia avDP20 (0.15 to 50 µM) was added to normal human serum (NHS) to evaluate any interference with activation of the classical complement pathway by IgM. Complement activation was determined by C3b deposition. PolySia avDP20 had no effect on C3b deposition induced by activation of the classical complement pathway. Data show mean +/- SEM. n = 3, n.s.= not significant.H. PolySia avDP20 inhibits the alternative complement pathway. PolySia avDP20 (0.15 to 50 µM) was added to NHS and activation of the alternative pathway was induced by LPS. C3b deposition was monitored by ELISA. Data show mean +/- SEM (n = 4. * p < 0.05, ** p < 0.01, p***<0.001, ANOVA followed by Bonferroni).


Figure 1Expression of Kit ( magenta ) largely overlaps with the expression of CD41 : YFP ( yellow ) in IAHC . Multistack reconstruction of confocal images . Scale bars : 30μm overview , 10μm magnification . ( C ) Snapshots of Movie EV1 . Time - lapse of embryonic organotypic slice culture from CD41 : YFP ( yellow ) : H2B - GFP ( blue ) reporter mouse shows the emergence of a single cell protruding from the endothelial layer into the aortic lumen . Scale bars : 50μm ( overview ) , 10μm ( magnification ) . Time expressed in hh : mm : ss . ( D ) Snapshots of Movie EV2 . Time - lapse of embryonic organotypic slice culture from CD41 : YFP ( yellow ) : H2B - GFP ( blue ) reporter mouse shows a single CD41 : YFP expressing cell that divides once on the aortic endothelium . Scale bars : 100μm ( overview ) , 20μm ( magnification ) . Time expressed in hh : mm : ss . BrdU incorporation in the Kit + IAHC upon cumulative administration . Multistack reconstruction of confocal images . Representative picture of a short BrdU pulse ( 1h ) . Scale bars : 50μm ( overview ) , 10μm ( magnification ) . ( F ) Quantification of the percentage of Kit + cells within IAHCs incorporating BrdU using confocal images . All cells ( Labelling Index = 100 % ) can be labelled after 8h , indicating the lack of quiescent cells inside the IAHCs . Graph represents mean ± SE .

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

Figure 1Expression of Kit (magenta) largely overlaps with the expression of CD41:YFP (yellow) in IAHC. Multistack reconstruction of confocal images. Scale bars: 30μm overview, 10μm magnification. (C) Snapshots of Movie EV1. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows the emergence of a single cell protruding from the endothelial layer into the aortic lumen. Scale bars: 50μm (overview), 10μm (magnification). Time expressed in hh:mm:ss. (D) Snapshots of Movie EV2. Time-lapse of embryonic organotypic slice culture from CD41:YFP (yellow): H2B-GFP (blue) reporter mouse shows a single CD41:YFP expressing cell that divides once on the aortic endothelium. Scale bars: 100μm (overview), 20μm (magnification). Time expressed in hh:mm:ss.BrdU incorporation in the Kit+ IAHC upon cumulative administration. Multistack reconstruction of confocal images. Representative picture of a short BrdU pulse (1h). Scale bars: 50μm (overview), 10μm (magnification). (F) Quantification of the percentage of Kit+ cells within IAHCs incorporating BrdU using confocal images. All cells (Labelling Index=100%) can be labelled after 8h, indicating the lack of quiescent cells inside the IAHCs. Graph represents mean±SE.


Figure 2Quantification of recombination efficiency at E10 . 5 and E11 . 5 using confocal images . Bars represents the mean ± SD of the percentage of cells expressing single colored cassettes in CD31 + endothelial cells ( n = 2 ) . Transversal section of a recombined VeCadCreER : Confetti AGM from an E10 . 5 embryo and immunostained with CD31 ( C ) ( white in image , black in label ) Multistack reconstruction of confocal images . Scale bars 30μmTransversal section of a recombined VeCadCreER : Confetti AGM from an E10 . 5 embryo and immunostained with Kit ( D ) ( white in image , black in label ) . Multistack reconstruction of confocal images . Scale bars 20 μmAnalyses of recombination in 40 clusters . Bars representing single clusters are grouped based on the number of Kit + cells . Recombination induction follows the schema in the black square . ( n = 12 embryo ) . Representative confetti recombination on clusters of different sizes . Small cluster are monoclonal ( left panel ) , while larger clusters are polyclonal ( right panel ) . Inlet shows relevant confetti channel . Kit is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bars 5 μm .

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Figure 2Quantification of recombination efficiency at E10.5 and E11.5 using confocal images. Bars represents the mean±SD of the percentage of cells expressing single colored cassettes in CD31+ endothelial cells (n=2).Transversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with CD31 (C) (white in image, black in label) Multistack reconstruction of confocal images. Scale bars 30μmTransversal section of a recombined VeCadCreER:Confetti AGM from an E10.5 embryo and immunostained with Kit (D) (white in image, black in label). Multistack reconstruction of confocal images. Scale bars 20 μmAnalyses of recombination in 40 clusters. Bars representing single clusters are grouped based on the number of Kit+ cells. Recombination induction follows the schema in the black square. (n=12 embryo).Representative confetti recombination on clusters of different sizes. Small cluster are monoclonal (left panel), while larger clusters are polyclonal (right panel). Inlet shows relevant confetti channel. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bars 5 μm.


Figure 3Detail images of Dll4 staining in Kit + cells of IAHC of different sizes . Dll4 expression inversely correlates with the size of the cluster : 2 - 5 cell IAHCs are uniformly Dll4 - positive ( upper and middle panel ) , > 5 cell IAHCs are partially Dll4 positive ( lower panel ) . Multistack reconstruction of confocal images . Scale bars : 10 μm . Quantification of Kit + Dll4 + cells using confocal images . Bars represent the percentage of Dll4 expressing cells within a cluster in each category of cluster size . Mean ± SD ( total 17 clusters analysed , n = 2 ) . Overview of Dll4 - ligand expression in the embryonic aorta at E10 . 5 . Dll4 is expressed in the majority of the endothelial layer . Scale bar :

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Figure 3Detail images of Dll4 staining in Kit+ cells of IAHC of different sizes. Dll4 expression inversely correlates with the size of the cluster: 2-5 cell IAHCs are uniformly Dll4-positive (upper and middle panel), >5 cell IAHCs are partially Dll4 positive (lower panel). Multistack reconstruction of confocal images. Scale bars: 10 μm.Quantification of Kit+ Dll4+ cells using confocal images. Bars represent the percentage of Dll4 expressing cells within a cluster in each category of cluster size. Mean±SD (total 17 clusters analysed, n=2).Overview of Dll4-ligand expression in the embryonic aorta at E10.5. Dll4 is expressed in the majority of the endothelial layer. Scale bar: 30μm


Figure 4 ( A ) Whole mount images of the aorta endothelium stained for CD31 ( white in image , black in label ) and Kit ( IAHC , cyan ) upon IgG ( left ) or αDll4 antibody treatment ( right ) . Multistack reconstruction of confocal images . Red arrows indicate IAHCs on the ventral part of the aortic wall . Asterisks indicate aspecific autofluorescence . Scale bar : 50μm . Representative IAHCs of IgG and αDll4 treated embryos . Kit is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Note the increased IAHC size in the right panel . Scale bars : 50μm ( overview ) , and 10μm ( magnification ) . Frequency of IAHC categories based on the number of Kit + cells forming a cluster . Quantification of confocal images . Bars represent the mean ± SE ( n = 6 ) Number of Kit + cells per IAHC increases following αDll4 antibody treatment . Each dot represents one cluster . Quantification on confocal images . Statistical analysis : Mann - Whitney U test . * * * * p < 0 . 0001 . Total number of IAHCs per 100μm . Quantification of confocal images . Mean ± SE . Statistical analysis : t - test . ns p > 0 . 05 ; ( n = 6 ) . ( F ) Quantification of the number of Kit + cells per 100 μm using confocal images . Bars represent the mean ± SE . Statistical analysis : t - test . ns p > 0 . 05 ; * * * p < 0 . 001 ( n = 6 ) .

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Figure 4(A)Whole mount images of the aorta endothelium stained for CD31 (white in image, black in label) and Kit (IAHC, cyan) upon IgG (left) or αDll4 antibody treatment (right). Multistack reconstruction of confocal images. Red arrows indicate IAHCs on the ventral part of the aortic wall. Asterisks indicate aspecific autofluorescence. Scale bar: 50μm.Representative IAHCs of IgG and αDll4 treated embryos. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Note the increased IAHC size in the right panel. Scale bars: 50μm (overview), and 10μm (magnification).Frequency of IAHC categories based on the number of Kit+ cells forming a cluster. Quantification of confocal images. Bars represent the mean±SE (n=6)Number of Kit+ cells per IAHC increases following αDll4 antibody treatment. Each dot represents one cluster. Quantification on confocal images. Statistical analysis: Mann-Whitney U test. ****p< 0.0001.Total number of IAHCs per 100μm. Quantification of confocal images. Mean±SE. Statistical analysis: t-test. ns p> 0.05; (n=6). (F) Quantification of the number of Kit+ cells per 100 μm using confocal images. Bars represent the mean±SE. Statistical analysis: t-test. ns p> 0.05; ***p< 0.001 (n=6).


Figure 5Experimental design : E10 . 5 embryos were injected with IgG or αDll4 , incubated for 5h and then dissected . The indicated IAHC subpopulations ( kit + CD45 + ; kit + CD45 - ; kit - CD45 - CD41 + were purified and RNA was sequenced . Experimental design and sorting gate for Kit + CD45 - cells for single cell RNAseq ( n = 483 cells ) . Violin plots of Notch family elements expression in IgG and αDll4 treated cells ( adj p val : * < 0 . 1 and * * * p < 0 . 00001 ) . Percentage of cells in each cell cycle phase extracted from single cell RNA - seq analysis . Bars represent the average ± SE ( n = 483 ) .

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Figure 5Experimental design: E10.5 embryos were injected with IgG or αDll4, incubated for 5h and then dissected. The indicated IAHC subpopulations (kit+CD45+; kit+CD45-;kit-CD45-CD41+ were purified and RNA was sequenced.Experimental design and sorting gate for Kit+CD45- cells for single cell RNAseq (n=483 cells).Violin plots of Notch family elements expression in IgG and αDll4 treated cells (adj p val: *<0.1 and *** p<0.00001).Percentage of cells in each cell cycle phase extracted from single cell RNA-seq analysis. Bars represent the average±SE (n=483).


Figure 6Bar graph represents the frequency of IAHCs containing recombined cells per embryo under the indicated conditions . Quantification of confocal images . Mean ± SE ( n = 15 for untreated , n = 7 for IgG and αDll4 treated ) . Representative images of recombined IAHCs following IgG or αDll4 conditions . Kit is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bar : 5μm . Quantification and classification of IAHC containing recombined cells in TAM - induced VeCadCreER : Confetti embryos after IgG or αDll4 treatment . Embryos treated with αDll4 contain more IAHC with colored cells . Quantification of confocal images ( n = 7 embryos ) . Representative images of recombined endothelial cells in transversal sections of the aorta . The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions . CD31 is shown in white in the image , in black in the label . Multistack reconstruction of confocal images . Scale bar : 50μm . Quantification of recombined cells in the aortic endothelium using confocal images . Mean ± SE ( n = 3 ) .

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Figure 6Bar graph represents the frequency of IAHCs containing recombined cells per embryo under the indicated conditions. Quantification of confocal images. Mean±SE (n=15 for untreated, n=7 for IgG and αDll4 treated).Representative images of recombined IAHCs following IgG or αDll4 conditions. Kit is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 5μm.Quantification and classification of IAHC containing recombined cells in TAM-induced VeCadCreER:Confetti embryos after IgG or αDll4 treatment. Embryos treated with αDll4 contain more IAHC with colored cells. Quantification of confocal images (n=7 embryos).Representative images of recombined endothelial cells in transversal sections of the aorta. The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions. CD31 is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 50μm. Quantification of recombined cells in the aortic endothelium using confocal images. Mean±SE (n=3).


Figure 7Snapshots of Movie EV5 . Time Lapse of embryonic organotypic slice from a CD41 : YFP : H2B - GFP reporter mouse showing the recruitment of CD41 : YFP negative cells ( magenta central point - tracking analyses ) migrating towards the IAHC ( CD41 : YFP + cells ) . Green central point and relative tracking identify cells with non - directional movement . Scale bar : 5μm . Time expressed in hh : mm : ss .

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Figure 7Snapshots of Movie EV5. Time Lapse of embryonic organotypic slice from a CD41:YFP: H2B-GFP reporter mouse showing the recruitment of CD41:YFP negative cells (magenta central point-tracking analyses) migrating towards the IAHC (CD41:YFP+ cells). Green central point and relative tracking identify cells with non-directional movement. Scale bar: 5μm. Time expressed in hh:mm:ss.


Figure 8Colony - forming assays using E10 . 5 AGMs derived from embryos treated with IgG or αDll4 . FACS analyses of Gfi1 + cells derived from explants treated with IgG or αDll4 for 5 hours ( C ) . Analyses of the hemogenic Gfi1 + Kit - ( D ) and the Gfi1 + Kit + ( E ) within the CD31 + CD45 - population . Percentage of reconstitution ( chimerism determined by CD45 . 1 and CD45 . 2 expression ) in peripheral blood ( PB ) . Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients . Peripheral blood was analyzed at different time points . Each dot / square represents a single transplanted mouse . Analysis of progenitors ( lin - sca1 + kit + ) and HSCs ( Lin - sca1 + kit + CD48 - CD150 + ) in the donor population in the bone marrow after 4 months of transplantation .

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Figure 8Colony-forming assays using E10.5 AGMs derived from embryos treated with IgG or αDll4.FACS analyses of Gfi1+ cells derived from explants treated with IgG or αDll4 for 5 hours (C). Analyses of the hemogenic Gfi1+Kit-(D) and the Gfi1+Kit+ (E) within the CD31+CD45- population.Percentage of reconstitution (chimerism determined by CD45.1 and CD45.2 expression) in peripheral blood (PB). Embryos treated with IgG or αDll4 were kept in explant culture for 48hours and then transplanted into irradiated recipients. Peripheral blood was analyzed at different time points. Each dot/square represents a single transplanted mouse.Analysis of progenitors (lin-sca1+kit+) and HSCs (Lin-sca1+kit+CD48-CD150+) in the donor population in the bone marrow after 4 months of transplantation.


Figure 3K . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media ( PM ) after isolation . L . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media ( DM ) . hiPSC donor 1 . M - O . Quantification of the number of Pax7 ( M ) , MyoD ( N ) and Pax7 / MyoD double - positive ( O ) cells in hskMPs and eMPs in PM and after switching to DM . hiPSC donor 1 . P . Representative immunostaining for the muscle fiber marker myosin heavy chain ( MyHC ) of eMPs maintained in DM . Q . Quantification of the fusion index of hskMPs and eMPs in PM and DM . hiPSC donor 1 . U - Z , Representative immunostainings and quantification of eMPs in DM expressing Pax7 ( V ) , MyoD ( X ) , and MyoG ( Z ) , as well as the proliferation marker Ki67 . hiPSC donor 1 .

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Figure 3K. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media (PM) after isolation. L. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media (DM). hiPSC donor 1.M-O. Quantification of the number of Pax7 (M), MyoD (N) and Pax7/MyoD double-positive (O) cells in hskMPs and eMPs in PM and after switching to DM. hiPSC donor 1.P. Representative immunostaining for the muscle fiber marker myosin heavy chain (MyHC) of eMPs maintained in DM. Q. Quantification of the fusion index of hskMPs and eMPs in PM and DM. hiPSC donor 1.U-Z, Representative immunostainings and quantification of eMPs in DM expressing Pax7 (V), MyoD (X), and MyoG (Z), as well as the proliferation marker Ki67. hiPSC donor 1.


Figure 4A . Representative immunostainings for dystrophin in cross - sections of muscles of mdx mice after transplantation of hskMPs or eMPs . Scale bars in the two left - hand side images = 300 μm . Scale bars in inserts 1 and 2 = 50 μm . B . Number of dystrophin positive fibers in mdx muscles injected with vehicle ( Ctrl ) , eMPs and hskMPs . C . Maximal distance of clusters of dystrophin positive fibers in cross - sections of mdx muscles transplanted with eMPs and hskMPs . A - C . 10 days post - transplantation . hiPSC donor 1 .

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Figure 4A. Representative immunostainings for dystrophin in cross-sections of muscles of mdx mice after transplantation of hskMPs or eMPs. Scale bars in the two left-hand side images = 300 μm. Scale bars in inserts 1 and 2 = 50 μm. B. Number of dystrophin positive fibers in mdx muscles injected with vehicle (Ctrl), eMPs and hskMPs. C. Maximal distance of clusters of dystrophin positive fibers in cross-sections of mdx muscles transplanted with eMPs and hskMPs. A-C. 10 days post-transplantation. hiPSC donor 1.


Figure 1 B - C Competitor donor derived Gr , B , CD4 T , and CD8 T cells in the peripheral blood . In the control group , competitor donor cells are WT HSCs . In the deficient group , competitor donor cells are uMT - / - or NSG HSCs Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test ) D - E WT donor derived Gr , B , CD4 T , and CD8 T cells in the peripheral blood Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test ) F - G Total production of Gr , B , CD4 T , and CD8 T cells in the peripheral blood ( derived from WT HSCs , competitor HSCs , helper whole bone marrow cells , and residual host cells ) shown as percentages of the total number of white blood cells ( WBCs ) . Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test )

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Figure 1 B-C Competitor donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood. In the control group, competitor donor cells are WT HSCs. In the deficient group, competitor donor cells are uMT-/- or NSG HSCs Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)  D-E WT donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)  F-G Total production of Gr, B, CD4 T, and CD8 T cells in the peripheral blood (derived from WT HSCs, competitor HSCs, helper whole bone marrow cells, and residual host cells) shown as percentages of the total number of white blood cells (WBCs). Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)


Figure 2 A - B Total numbers of barcoded WT clones that give rise to granulocytes ( Gr ) , B cells , CD4 T cells , and CD8 T cells Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test ) C - J Number of HSC clones that produce different amounts of Gr , B , CD4 T , and CD8 T cells . We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows : Clonal abundance = 100 % * ( Each cell population ( Gr , B , CD4T or CD8T cells ) % WBCs ) * ( Donor % Each cell population ) * ( GFP % Donor cells ) * ( number of reads for each barcode ) / ( total reads of all barcodes Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test ) K - L Production of Gr , B , CD4 T , and CD8 T cells by expanding WT clones that produce more than 0 . 1 % of WBCs in each lineage . Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 A-B Total numbers of barcoded WT clones that give rise to granulocytes (Gr), B cells, CD4 T cells, and CD8 T cells Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)  C-J Number of HSC clones that produce different amounts of Gr, B, CD4 T, and CD8 T cells. We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows: Clonal abundance =100%*(Each cell population (Gr, B, CD4T or CD8T cells) % WBCs) * (Donor % Each cell population) * (GFP % Donor cells) * (number of reads for each barcode) / (total reads of all barcodes Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)  K-L Production of Gr, B, CD4 T, and CD8 T cells by expanding WT clones that produce more than 0.1% of WBCs in each lineage. Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)


Figure 4 A - B Total amount of progenitors as a percentage of ckit and Il7rα enriched bone marrow ( BM ) cells . Shown are hematopoietic stem cells ( HSCs ) , Flk2 - and Flk2 + multipotent progenitors ( MPP ) , common lymphoid progenitors ( CLP ) , common myeloid progenitors ( CMP ) , megakaryocyte - erythroid progenitors ( MEP ) , and granulocyte - monocyte progenitors ( GMP ) Data information : Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 and * * p < 0 . 01 ( two - tailed and two - sample equal variance Student " s t - test ) C - D WT donor derived progenitors as a percentage of ckit and Il7rα enriched BM cells . Data information : Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 and * * p < 0 . 01 ( two - tailed and two - sample equal variance Student " s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A-B Total amount of progenitors as a percentage of ckit and Il7rα enriched bone marrow (BM) cells. Shown are hematopoietic stem cells (HSCs), Flk2- and Flk2+ multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), megakaryocyte-erythroid progenitors (MEP), and granulocyte-monocyte progenitors (GMP) Data information: Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)  C-D WT donor derived progenitors as a percentage of ckit and Il7rα enriched BM cells. Data information: Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)


Figure 1 ( A ) survival curves of MyD88 flies injected with different doses of A . fumigatus conidia ( c / f : conidia injected per fly ; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) . ( B ) fungal loads of single MyD88 mutant and wild - type flies ( 50 conidia injected per fly ) . ( C - D ) GFP - labeled A . fumigatus ( 50 conidia per fly ) injected in wild - type ( C ) or MyD88 mutant ( D ) flies form hyphae in the thorax of the flies ( arrows ) . Scale bars 50 μm . ( E ) expression level of Drosomycin induced by different doses of injected A . fumigatus conidia measured by RT - qPCR ; M . luteus ( OD = 10 ) represents the positive control ( pooled data of n = 3 experiments , biological replicates ) . Hayan flies are susceptible to A . fumigatus . Survival ( F ) , of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) . time course of fungal loads of single Hayan mutant flies ( 500 conidia per fly ) ( G ) , and fungal load upon death ( 500 conidia per fly ) ( H ) of Hayan mutant flies ; Hayan 0h vs . 120h , ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) .

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

Figure 1(A) survival curves of MyD88 flies injected with different doses of A. fumigatus conidia (c/f: conidia injected per fly; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).(B) fungal loads of single MyD88 mutant and wild-type flies (50 conidia injected per fly).(C-D) GFP-labeled A. fumigatus (50 conidia per fly) injected in wild-type (C) or MyD88 mutant (D) flies form hyphae in the thorax of the flies (arrows). Scale bars 50 μm.(E) expression level of Drosomycin induced by different doses of injected A. fumigatus conidia measured by RT-qPCR; M. luteus (OD=10) represents the positive control (pooled data of n=3 experiments, biological replicates).Hayan flies are susceptible to A. fumigatus. Survival (F), of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).time course of fungal loads of single Hayan mutant flies (500 conidia per fly) (G), and fungal load upon death (500 conidia per fly) (H) of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).


Figure 2 ( A ) survival of MyD88 flies injected with ∆ pptA and wild - type ( Af ) A . fumigatus conidia ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) . ( B ) fungal loads of single flies after the injection of 500 ∆ pptA conidia . ( E ) dose response of MyD88 flies after ∆ gliP ( gliotoxin ) mutant or wild - type [ ∆ akuB ] A . fumigatus infection ; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ; wild - type flies are used as a control for the dose of 250 conidia . ( F - H ) dose response of MyD88 and wild - type flies after gliotoxin ( F ) , fumagillin ( G ) , and helvolic acid ( H ) injection at the indicated concentrations ( 20 flies per condition ) .

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

Figure 2(A) survival of MyD88 flies injected with ∆pptA and wild-type (Af) A. fumigatus conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).(B) fungal loads of single flies after the injection of 500 ∆pptA conidia.(E) dose response of MyD88 flies after ∆gliP (gliotoxin) mutant or wild-type [∆akuB] A. fumigatus infection; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each; wild-type flies are used as a control for the dose of 250 conidia. (F-H) dose response of MyD88 and wild-type flies after gliotoxin (F), fumagillin (G), and helvolic acid (H) injection at the indicated concentrations (20 flies per condition).


Figure 4 ( A ) survival of MyD88 or wild - type flies to 250 injected ∆ aspf1 ( restrictocin mutant ) or wild - type [ ∆ akuB ] A . fumigatus conidia ( 20 flies per condition ) ; MyD88 : ∆ aspf1 vs . ∆ akuB ( * * * p = 0 . 0007 ) . ( B ) survival of MyD88 flies after the injection of different concentrations of restrictocin ( R ) ( 20 flies per condition ) . ( C - D ) survival of antibiotics - treated ( C ) and axenic ( D ) MyD88 mutant flies after restrictocin injection ( 20 flies per condition ) . ( E - F ) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild - type ( E ) and MyD88 ( F ) flies ; the arrowheads show the position of the 28S RNA - derived α - sarcin fragments whereas arrows on the right show its electrophoretic band position . ( H ) SDS - PAGE analysis of 35S - labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G - capped reporter RNA containing the 5 ' UTR of β - globin followed by the Renilla luciferase coding sequence ( arrow ) , in the presence of increasing concentrations ( 0 . 125 to 25 nM ) of restrictocin .

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

Figure 4(A) survival of MyD88 or wild-type flies to 250 injected ∆aspf1 (restrictocin mutant) or wild-type [∆akuB] A. fumigatus conidia (20 flies per condition); MyD88: ∆aspf1 vs. ∆akuB (***p=0.0007). (B) survival of MyD88 flies after the injection of different concentrations of restrictocin (R) (20 flies per condition ). (C-D) survival of antibiotics-treated (C) and axenic (D) MyD88 mutant flies after restrictocin injection (20 flies per condition).(E-F) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild-type (E) and MyD88 (F) flies; the arrowheads show the position of the 28S RNA-derived α-sarcin fragments whereas arrows on the right show its electrophoretic band position.(H) SDS-PAGE analysis of 35S-labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G-capped reporter RNA containing the 5'UTR of β-globin followed by the Renilla luciferase coding sequence (arrow), in the presence of increasing concentrations (0.125 to 25 nM) of restrictocin.


Figure 5 ( A - B ) survival ( A ) and fungal load ( B ) of Bom ∆ 55C ( 55C ) deficient flies compared to wild - type and MyD88 after injection of 250 conidia ( error bars represent mean ± SD of the survival of biological triplicates of 20 flies each ) ; * * * * p < 0 . 0001 . ( B ) : the fungal burden does not increase in Bom ∆ 55C - deficient flies ; wt 0h vs . 48h , * * p = 0 . 001 ; 55C 0h vs . 48h , * * p = 0 . 007 ; pooled data of n = 3 experiments , biological replicates . ( C - D ) rescue of the sensitivity of Bom ∆ 55C flies to verruculogen ( C ) or to restrictocin ( D ) by the transgenic expression of individual 55C locus genes ( caption in D also applies to ( C ) ) . 55C flies vs . BomS1 , * p = 0 . 0495 , vs . BomS6 * p = 0 . 011 for verruculogen assay ( C ) ; 55C flies vs . BomBc1 or BomS3 , * * * * p < 0 . 0001 , 55C flies vs . BomS6 , * * p = 0 . 0028 for restrictocin assay ( D ) ( 20 flies per condition ) . ( E - F ) expression levels of BomBc1 , and BomS3 measured by RT - digital PCR 48 hours after challenge ; BomBc1 PBST vs . Af , * p = 0 . 015 , PBST vs . restrictocin ( R ) , * p = 0 . 015 ; BomS3 : PBST vs . Af , * p = 0 . 02 , PBST vs . R , * p = 0 . 03 ( pooled data of n = 3 experiments , biological replicates ) .

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

Figure 5(A-B) survival (A) and fungal load (B) of Bom∆55C (55C) deficient flies compared to wild-type and MyD88 after injection of 250 conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each); ****p < 0.0001. (B): the fungal burden does not increase in Bom∆55C-deficient flies; wt 0h vs. 48h, **p=0.001; 55C 0h vs. 48h, **p=0.007; pooled data of n=3 experiments, biological replicates.(C-D) rescue of the sensitivity of Bom∆55C flies to verruculogen (C) or to restrictocin (D) by the transgenic expression of individual 55C locus genes (caption in D also applies to (C)). 55C flies vs. BomS1, *p=0.0495, vs. BomS6 *p=0.011 for verruculogen assay (C); 55C flies vs. BomBc1 or BomS3, ****p<0.0001, 55C flies vs. BomS6, **p=0.0028 for restrictocin assay (D) (20 flies per condition).(E-F) expression levels of BomBc1, and BomS3 measured by RT-digital PCR 48 hours after challenge; BomBc1 PBST vs. Af, *p=0.015, PBST vs. restrictocin (R), *p=0.015; BomS3: PBST vs. Af, *p=0.02, PBST vs. R, *p=0.03 (pooled data of n=3 experiments, biological replicates).


Figure 6 ( A - B ) tremor rate ( A ) and survival ( B ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in neurons compared to wild - type after injection of verruculogen . ( A ) Each dot corresponds to the tremor rate measured in a batch of 20 flies ; tremor rate wt vs . elav > UAS - Toll10B , * * p = 0 . 002 . ( C - D ) tremor rate ( C ) and survival ( D ) of flies ( 20 flies per condition ) overexpressing Tl [ 10B ] in glia compared to wild - type after injection of verruculogen . ( C ) Each dot corresponds to the tremor rate measured in a batch of 20 flies ; tremor rate wt vs . repo > UAS - Toll10B , * * p = 0 . 002 . ( D ) Survival wt V vs . repo > UAS - Toll10B V , * * p = 0 . 005 . ( E - G ) recovery time from tremor ( E , F ) and survival ( G ) of single flies overexpressing BomS6 ubiquitously ( E , G ) or in neurons ( F - G ) ( biological replicates ) compared to wild - type after injection of verruculogen ; in ( G ) the inset represents the survival of vehicle control groups . ( H - I ) recovery time from tremor ( H ) and survival ( I ) of single flies overexpressing BomS6 in glia ( pooled data from n = 3 experiments , biological replicates ) compared to wild - type after injection of verruculogen ; in ( I ) the inset represents the survival of vehicle control groups . Recovery time ( H ) repo > mCherry vs . repo > BomS6 , p = 0 . 058 , survival ( I ) repo > mCherry V vs . repo > BomS6 V , * p = 0 . 016 . ( J - M ) expression of BomS4 ( J ) and BomS6 ( K ) in head after verruculogen powder challenge , and BomS4 ( L ) and BomS6 ( M ) in head after A . fumigatus ( Af ) , restrictocin or M . luteus injection ( pooled data from n = 3 experiments , biological replicates ) .

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

Figure 6(A-B) tremor rate (A) and survival (B) of flies (20 flies per condition) overexpressing Tl[10B] in neurons compared to wild-type after injection of verruculogen. (A) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. elav> UAS-Toll10B, **p=0.002. (C-D) tremor rate (C) and survival (D) of flies (20 flies per condition) overexpressing Tl[10B] in glia compared to wild-type after injection of verruculogen. (C) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. repo> UAS-Toll10B, **p=0.002. (D) Survival wt V vs. repo> UAS-Toll10B V, **p=0.005.(E-G) recovery time from tremor (E,F) and survival (G) of single flies overexpressing BomS6 ubiquitously (E,G) or in neurons (F-G) (biological replicates) compared to wild-type after injection of verruculogen; in (G) the inset represents the survival of vehicle control groups.(H-I) recovery time from tremor (H) and survival (I) of single flies overexpressing BomS6 in glia (pooled data from n=3 experiments, biological replicates) compared to wild-type after injection of verruculogen; in (I) the inset represents the survival of vehicle control groups. Recovery time (H) repo>mCherry vs. repo>BomS6, p=0.058, survival (I) repo>mCherry V vs. repo>BomS6 V, *p=0.016.(J-M) expression of BomS4 (J) and BomS6 (K) in head after verruculogen powder challenge, and BomS4 (L) and BomS6 (M) in head after A. fumigatus (Af), restrictocin or M. luteus injection (pooled data from n=3 experiments, biological replicates).


Figure 1 ( A ) SPT activity and Western blot ( WB ) analysis of S1PR1 in S1pr1f / f and S1pr1ECKO endothelial cells after 4 - OHT ( 1μM , 72h ) treatment . ( n = 8 / group from 3 independent EC isolations / group ; 4 mice / EC isolation ) .

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

Figure 1(A) SPT activity and Western blot (WB) analysis of S1PR1 in S1pr1f/f and S1pr1ECKO endothelial cells after 4-OHT (1μM, 72h) treatment. (n=8/group from 3 independent EC isolations/group; 4 mice/EC isolation).


Figure 2 ( A , B ) ( A ) WB analysis of ORMDLs , NOGO - B , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) and ( B ) relative ORMDLs quantification ( n = 7 biological replicates ) . ( C ) WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of C16 : 0 - ceramide ( 300nM , 30 ' ) , with or without SKI II 1µM , 1h pre - treatment ) . ( D ) SPT activity in HUVEC in absence or presence of SEW2871 ( 300nM , 15 ' ) ( n = 7 biological replicates ) . ( E , F ) ( E ) WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of SEW2871 ( 300nM , 15 ' and 30 ' ) and ( F ) relative ORMDLs quantification ( n = 6 biological replicates ) . ( G , H ) SPT activity and WB analysis of HUVEC treated with ( G ) siCTRL and siNOGO ( 40nM , 72h ) ( n ≥ 4 / group ) , or with ( H ) siCTRL and siORMDL1 / 2 / 3 ( 40nM , 72h ) ( n = 4 biological replicates ) . ( I ) WB analysis of ORMDLs , P - p38 , p38 , P - JNK , JNK , P - ERK , ERK , P - AKT , and AKT in HUVEC lysates in absence or presence of the indicated inhibitors , with or without S1P ( 300nM , 30 ' ) .

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

Figure 2(A,B) (A) WB analysis of ORMDLs, NOGO-B, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and (B) relative ORMDLs quantification (n=7 biological replicates).(C) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of C16:0-ceramide (300nM, 30'), with or without SKI II 1µM, 1h pre-treatment). (D) SPT activity in HUVEC in absence or presence of SEW2871 (300nM, 15') (n=7 biological replicates).(E,F) (E) WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of SEW2871 (300nM, 15' and 30') and (F) relative ORMDLs quantification (n=6 biological replicates).(G,H) SPT activity and WB analysis of HUVEC treated with (G) siCTRL and siNOGO (40nM, 72h) (n≥4/group), or with (H) siCTRL and siORMDL1/2/3 (40nM, 72h) (n=4 biological replicates).(I) WB analysis of ORMDLs, P-p38, p38, P-JNK, JNK, P-ERK, ERK, P-AKT, and AKT in HUVEC lysates in absence or presence of the indicated inhibitors, with or without S1P (300nM, 30').


Figure 3 ( A ) WB analysis of ORMDL3 ( HA ) , SPTLC1 and SPTLC2 in HUVEC lysates expressing HA - ORMDL3 and treated with cycloheximide ( CHX , 10µM ) for the indicated period of time . WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) and with or without ( C ) DMOG ( 1mM , 1h pre - treatment ) , ( D ) Eeyarestatin 1 ( EER1 , 10µM , 1h pre - treatment ) , WB analysis of ORMDLs , SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) ( E ) MG132 ( 10µM , 1h pre - treatment ) . ( F ) WB analysis of HA - ORMDL3 and HA - ORMDL3 - P137A in HUVEC lysates in absence or presence of S1P ( 300nM , 30 ' ) , DMOG ( 1mM , 1h ) , EER1 ( 10µM , 1h ) , and MG132 ( 10µM , 1h ) . ( G , H ) ( G ) WB analysis of HA - ORMDL3 and HA - ORMDL3 - P137A , SPTLC1 and SPTLC2 in HUVEC lysates treated with CHX ( 10µM ) for the indicated period of time , and ( H ) relative quantification ( I ) WB analysis for Ubiquitin , HA and GFP of HEK293T transfected with GFP - ubiquitin and with the indicated HA - ORMDL3 plasmid , and immunoprecipitated with HA antibody .

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

Figure 3(A) WB analysis of ORMDL3 (HA), SPTLC1 and SPTLC2 in HUVEC lysates expressing HA-ORMDL3 and treated with cycloheximide (CHX, 10µM) for the indicated period of time.WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') and with or without (C) DMOG (1mM, 1h pre-treatment), (D) Eeyarestatin 1 (EER1, 10µM, 1h pre-treatment),WB analysis of ORMDLs, SPTLC1 and SPTLC2 in HUVEC lysates in absence or presence of S1P (300nM, 30') (E) MG132 (10µM, 1h pre-treatment).(F) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A in HUVEC lysates in absence or presence of S1P (300nM, 30'), DMOG (1mM, 1h), EER1 (10µM, 1h), and MG132 (10µM, 1h).(G,H) (G) WB analysis of HA-ORMDL3 and HA-ORMDL3-P137A, SPTLC1 and SPTLC2 in HUVEC lysates treated with CHX (10µM) for the indicated period of time, and (H) relative quantification(I) WB analysis for Ubiquitin, HA and GFP of HEK293T transfected with GFP-ubiquitin and with the indicated HA-ORMDL3 plasmid, and immunoprecipitated with HA antibody.


Figure 4SPT activity in S1pr1 , 3f / f and S1pr1 , 2ECKO mEC , in absence or presence of S1P ( 300nM , 30 ' ) ( B ) relative WB analysis for S1PR1 , ORMDLs , P - AKT and AKT . ( G ) WB analysis of ORMDLs and S1PR1 in HUVEC lysates in absence or presence of P - FTY720 ( 100nM , 30 ' )

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Figure 4SPT activity in S1pr1,3f/f and S1pr1,2ECKO mEC , in absence or presence of S1P (300nM, 30') (B) relative WB analysis for S1PR1, ORMDLs, P-AKT and AKT.(G) WB analysis of ORMDLs and S1PR1 in HUVEC lysates in absence or presence of P-FTY720 (100nM, 30') and


Figure 6 ( A - C ) ( A ) WB analysis of P - VEGFR2 ( Y1175 ) , VEGFR2 , P - IR ( Y1150 / 1151 ) , IR , P - AKT ( S473 ) , AKT , P - eNOS ( S1176 ) , and eNOS in Spns2f / f and Spns2ECKO mEC lysates in absence or presence of VEGF ( 100ng / mL , 2 ' ) or Insulin ( 1U / mL , 2 ' ) and ( B - C ) relative quantification of the indicated phospho / total protein ratios ( n ≥ 3 biological replicates ) . ( D , E ) ( D ) WB analysis of P - VEGFR2 ( Y1175 ) , VEGFR2 , P - eNOS ( S1176 ) , and eNOS in Spns2f / f and Spns2ECKO mEC lysates in absence or presence of VEGF ( 100ng / mL , 2 ' ) , and in absence or presence of Myriocin ( 300nM ) for the indicated time and ( E ) relative quantification of the indicated phospho / total protein ratios ( n ≥ 3 biological replicates ) .

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

Figure 6(A-C) (A) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-IR (Y1150/1151), IR, P-AKT (S473), AKT, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2') or Insulin (1U/mL, 2') and (B-C) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).(D,E) (D) WB analysis of P-VEGFR2 (Y1175), VEGFR2, P-eNOS (S1176), and eNOS in Spns2f/f and Spns2ECKO mEC lysates in absence or presence of VEGF (100ng/mL, 2'), and in absence or presence of Myriocin (300nM) for the indicated time and (E) relative quantification of the indicated phospho/total protein ratios (n≥3 biological replicates).


Figure 1 ( A ) Phenotype appearance of the Gmlmm1 - 1 mutant . Typical leaves ( left panel ; scale bar , 1 cm ) and whole plants ( right panel ; scale bar , 10 cm . ) were photographed 60 and 118 days after seeding , respectively . Data information : The experiments were performed three times as biological replicates , with similar results . ( B ) Enhanced resistance of Gmlmm1 - 1 and Gmlmm1 - 2 to Psg infection . Leaves from 2 - week - old soybean plants were infected with Psg and the bacteria number was determined at 0 and 4 dpi ( Mean ± SD , n = 8 , n represents sample number , * * * , p < 0 . 001 , Student ' s t - test ) . Data information : The experiments were performed three times , as biological replicates , with similar results . ( E ) Subcellular localization of GmLMM1 - GFP and GmLMM1L407H - GFP in N . benthamiana . PI4P indicates the membrane localization . Scale bar , 10 μm . GFP , green fluorescent protein . Data information : The experiments were performed three times as biological replicates , with similar results . ( F ) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro . The proteins purified from E . coli were subjected to pMIAGO phosphorylation detection assay ( upper panel ) and were detected using western blot ( lower panel ) with BIK1 - HIS as a control . GmLMM1K564E contains a mutation at the predicted ATP binding site . Data information : The experiments were performed two times ( F ) , as biological replicates , with similar results .

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

Figure 1(A) Phenotype appearance of the Gmlmm1-1 mutant. Typical leaves (left panel; scale bar, 1 cm) and whole plants (right panel; scale bar, 10 cm.) were photographed 60 and 118 days after seeding, respectively. Data information: The experiments were performed three times as biological replicates, with similar results.(B) Enhanced resistance of Gmlmm1-1 and Gmlmm1-2 to Psg infection. Leaves from 2-week-old soybean plants were infected with Psg and the bacteria number was determined at 0 and 4 dpi (Mean ± SD, n = 8, n represents sample number, ***, p < 0.001, Student's t-test). Data information: The experiments were performed three times , as biological replicates, with similar results.(E) Subcellular localization of GmLMM1-GFP and GmLMM1L407H-GFP in N. benthamiana. PI4P indicates the membrane localization. Scale bar, 10 μm. GFP, green fluorescent protein. Data information: The experiments were performed three times as biological replicates, with similar results.(F) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro. The proteins purified from E. coli were subjected to pMIAGO phosphorylation detection assay (upper panel) and were detected using western blot (lower panel) with BIK1-HIS as a control. GmLMM1K564E contains a mutation at the predicted ATP binding site. Data information: The experiments were performed two times (F), as biological replicates, with similar results.


Figure 3 ( A ) ROS accumulation in Gmlmm1 - 1 and Gmlmm1 - 2 mutant plants . Soybean leaves ( aged 3 weeks ) were subjected to ROS accumulation examination by DAB staining . Scale bar , 1 cm . Data information : The experiments were performed three times as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data . ( B ) ROS accumulation upon P . sojae treatment . The leaves ( aged 3 weeks ) were inoculated with P . sojae isolate P7076 , and DAB staining was performed at 16 hpi . The left panel was photographed under bright field ( scale bar , 1 cm ) . The middle and right panels were photographed under a microscope ( scale bar , 50 μm ) . Data information : The experiments were performed three times as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data . ( D The increased resistance of the mutants to Psp ( D ) infection . The indicated plants ( aged 2 weeks ) were infected with the indicated pathogens ( Mean ± SD , n ≥ 8 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The lesions are shown by trypan blue staining . Scale bar , 1 cm . Data information : The experiments were performed three times as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data . E ) The increased resistance of the mutants P . sojae ( E ) infection . The indicated plants ( aged 2 weeks ) were infected with the indicated pathogens ( Mean ± SD , n ≥ 8 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The lesions are shown by trypan blue staining . Scale bar , 1 cm . Data information : The experiments were performed three times , as biological replicates , with similar results . The exact number ( n ) and p values are shown in the source data .

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

Figure 3(A) ROS accumulation in Gmlmm1-1 and Gmlmm1-2 mutant plants. Soybean leaves (aged 3 weeks) were subjected to ROS accumulation examination by DAB staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.(B) ROS accumulation upon P. sojae treatment. The leaves (aged 3 weeks) were inoculated with P. sojae isolate P7076, and DAB staining was performed at 16 hpi. The left panel was photographed under bright field (scale bar, 1 cm). The middle and right panels were photographed under a microscope (scale bar, 50 μm). Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.(D The increased resistance of the mutants to Psp (D) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.E) The increased resistance of the mutants P. sojae (E) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm. Data information: The experiments were performed three times , as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.


Figure 4 ( A ) Flg22 - induced ROS burst in Gmlmm1 - 1 , Gmlmm1 - 2 and Williams 82 . The indicated leaves ( aged 2 weeks ) were subjected to flg22 - induced ROS examination , and the peak relative luminescence unit ( RLU ) value was recorded ( Mean ± SEM , n ≥ 12 , n represents sample number ) . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . ( B ) Flg22 - induced ROS burst in the CRISPR lines C9 - 24 - 13 and DN50 . The indicated leaves ( aged 2 weeks ) were subjected to flg22 - induced ROS examination , and the relative RLU value was recorded ( Mean ± SEM , n ≥ 9 , n represents sample number ) . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . ( C ) Suppression of flg22 - induced ROS production in Arabidopsis by GmLMM1 . The GmLMM1 gene was introduced into Arabidopsis WT ( Col - 0 ) plants by Agrobacterium - mediated transformation . Two independent T2 lines were subjected to flg22 - induced ROS examination ( Mean ± SD , n ≥ 8 , n represents sample number , * * * , p < 0 . 001 , Student ' s t - test ) . The protein expression is shown . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . Suppression of flg22 - induced ( D ) ROS production in N . benthamiana by GmLMM1 . The indicated constructs were transiently expressed by Agrobacterium - mediated transient expression for 2 days and subjected to flg22 - induced ROS examination . GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity . GFP was used as a control ( Mean ± SD , n = 6 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The protein expression is shown in the lower panel . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . E ) Suppression of chitin - induced ( E ) ROS production in N . benthamiana by GmLMM1 . The indicated constructs were transiently expressed by Agrobacterium - mediated transient expression for 2 days and subjected to ROS examination . GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity . GFP was used as a control ( Mean ± SD , n = 6 , n represents sample number , * * , p < 0 . 01 , * * * , p < 0 . 001 , Student ' s t - test ) . The protein expression is shown in the lower panel . Data information : All the experiments were performed three times ( biological replicates ) with similar results . The exact n , SEM values and p values are shown in the source data . ( F ) Inhibition of XEG1 - induced cell death in N . benthamiana by GmLMM1 . The indicated constructs were transiently expressed in N . benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector . The cell death phenotypes were visualized 5 days later . Protein expression of GmLMM1 - HA and GmLMM1K564E - HA is shown in the lower panel .

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

Figure 4(A) Flg22-induced ROS burst in Gmlmm1-1, Gmlmm1-2 and Williams 82. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the peak relative luminescence unit (RLU) value was recorded (Mean ± SEM, n ≥ 12, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.(B) Flg22-induced ROS burst in the CRISPR lines C9-24-13 and DN50. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the relative RLU value was recorded (Mean ± SEM, n ≥ 9, n represents sample number). Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.(C) Suppression of flg22-induced ROS production in Arabidopsis by GmLMM1. The GmLMM1 gene was introduced into Arabidopsis WT (Col-0) plants by Agrobacterium-mediated transformation. Two independent T2 lines were subjected to flg22-induced ROS examination (Mean ± SD, n ≥8, n represents sample number, ***, p < 0.001, Student's t-test). The protein expression is shown. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.Suppression of flg22-induced (D) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.E) Suppression of chitin-induced (E) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel. Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.(F) Inhibition of XEG1-induced cell death in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed in N. benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector. The cell death phenotypes were visualized 5 days later. Protein expression of GmLMM1-HA and GmLMM1K564E-HA is shown in the lower panel.


Figure 5 ( A ) Identification of GmLMM1 - interacting proteins by IP - MS analysis . GmLMM1 - FLAG was transiently expressed in Arabidopsis protoplasts , purified by anti - FLAG M2 agarose , and subjected to mass spectrometry analysis . The complete list is shown in the source data . ( B ) Interactions of GmLMM1 with PRR receptors . The indicated constructs were transiently expressed in N . benthamiana leaves and subjected to luciferase complementation assay . GmFLS2 ( Gm08G083300 ) is the closest homolog of FLS2 in soybean . GmILPA1 was used as a negative control . The protein interaction intensity is shown by the relative luminescence unit ( RLU ) ( Mean ± SD , n ≥ 6 , n represents sample number , p < 0 . 05 , Student ' s t - test , different letters indicate signiﬁcant difference ) . Immunoblots show the levels of protein expression . Data information : The experiments were performed three times as biological replicates , with similar results . ( C ) Co - IP of GmLMM1 with NbFLS2 and GmFLS2 . The indicated constructs were transiently expressed in N . benthamiana by Agrobacterium - mediated transient expression for 2 days and the protein interactions were examined by Co - IP assay . Data information : The experiments were performed three times as biological replicates , with similar results . ( D ) GmLMM1 interacts with NbRXEG1 in N . benthamiana plants . Data information : The experiments were performed three times as biological replicates , with similar results . ( E ) Interaction of GmLMM1 with NbBAK1 , GmBAK1a , and GmBAK1b . The experiment and statistical testing were performed as in ( B ) . Immunoblots show the levels of protein expression . Data information : The experiments were performed three times as biological replicates , with similar results . ( F ) GmLMM1 interacts with NbBAK1 , GmBAK1a , and GmBAK1b . The indicated constructs were transiently expressed in N . benthamiana and the interactions were examined by Co - IP assays . Data information : The experiments were performed two times ( F ) , as biological replicates , with similar results .

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Figure 5(A) Identification of GmLMM1-interacting proteins by IP-MS analysis. GmLMM1-FLAG was transiently expressed in Arabidopsis protoplasts, purified by anti-FLAG M2 agarose, and subjected to mass spectrometry analysis. The complete list is shown in the source data.(B) Interactions of GmLMM1 with PRR receptors. The indicated constructs were transiently expressed in N. benthamiana leaves and subjected to luciferase complementation assay. GmFLS2 (Gm08G083300) is the closest homolog of FLS2 in soybean. GmILPA1 was used as a negative control. The protein interaction intensity is shown by the relative luminescence unit (RLU) (Mean±SD, n ≥ 6, n represents sample number, p<0.05, Student's t-test, different letters indicate signiﬁcant difference). Immunoblots show the levels of protein expression. Data information: The experiments were performed three times as biological replicates, with similar results.(C) Co-IP of GmLMM1 with NbFLS2 and GmFLS2. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days and the protein interactions were examined by Co-IP assay. Data information: The experiments were performed three times as biological replicates, with similar results.(D) GmLMM1 interacts with NbRXEG1 in N. benthamiana plants. Data information: The experiments were performed three times as biological replicates, with similar results.(E) Interaction of GmLMM1 with NbBAK1, GmBAK1a, and GmBAK1b. The experiment and statistical testing were performed as in (B). Immunoblots show the levels of protein expression. Data information: The experiments were performed three times as biological replicates, with similar results.(F) GmLMM1 interacts with NbBAK1, GmBAK1a, and GmBAK1b. The indicated constructs were transiently expressed in N. benthamiana and the interactions were examined by Co-IP assays. Data information: The experiments were performed two times (F), as biological replicates, with similar results.