The percentage of B220low CD5 + leukocytes in the liver at different time points is illustrated . Besides representative dot plots show the selection of B220low CD5 + cells in the liver . Cells were pre - gated on single cells , living cells and CD45 + leukocytes . Shown are mean values with ± SD . Significant differences between groups were tested by ordinary one - way ANOVA with Šídák ' s post - hoc test . Data are from at least 10 independent experiments for individual time points , comprising n = 5 - 12 animals per time point and genotype . X = no surviving TCL1 x Siglecg - / - animals at the age of 60 weeks . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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

The percentage of B220low CD5+ leukocytes in the liver at different time points is illustrated. Besides representative dot plots show the selection of B220low CD5+ cells in the liver. Cells were pre-gated on single cells, living cells and CD45+ leukocytes. Shown are mean values with ±SD. Significant differences between groups were tested by ordinary one-way ANOVA with Šídák's post-hoc test. Data are from at least 10 independent experiments for individual time points, comprising n=5-12 animals per time point and genotype. X = no surviving TCL1 x Siglecg-/- animals at the age of 60 weeks. *p<0.05, **p<0.01, ****p<0.0001.


The survival of TCL1 x Siglecg - / - mice compared to TCL1 mice is illustrated with the Kaplan - Meier survival curve . ( n = 81 for TCL1 , n = 101 for TCL1 x Siglecg - / - ) . The groups were compared for significant differences via log - rank 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

The survival of TCL1 x Siglecg-/- mice compared to TCL1 mice is illustrated with the Kaplan-Meier survival curve. (n=81 for TCL1, n=101 for TCL1 x Siglecg-/-). The groups were compared for significant differences via log-rank test.


To monitor the progression of the CLL - like population , blood was collected from the animals every four weeks up to an age of 48 weeks and analysed by flow cytometry . Genotypes of the mice are : controls ( Siglecg - R26ki / ki ) , Siglec - G overexpressing mice ( Siglecg - R26ki / ki mb1cre ) , transgenic TCL1 controls ( TCL1 x Siglecg - R26ki / ki ) and transgenic TCL1 Siglec - G overexpressing mice ( TCL1 x Siglecg - R26ki / ki mb1cre ) . Representative FACSplots show examples of stainingsof B220lowCD5 + B cells ( CLL - like cells ) and the selected gate for B )

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

To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 48 weeks and analysed by flow cytometry. Genotypes of the mice are: controls (Siglecg-R26ki/ki), Siglec-G overexpressing mice (Siglecg-R26ki/ki mb1cre), transgenic TCL1 controls (TCL1 x Siglecg-R26ki/ki) and transgenic TCL1 Siglec-G overexpressing mice (TCL1 x Siglecg-R26ki/ki mb1cre). Representative FACSplots show examples of stainingsof B220lowCD5+B cells (CLL-like cells) and the selected gate for B)


To monitor the progression of the CLL - like population , blood was collected from the animals every four weeks up to an age of 48 weeks and analysed by flow cytometry . Genotypes of the mice are : controls ( Siglecg - R26ki / ki ) , Siglec - G overexpressing mice ( Siglecg - R26ki / ki mb1cre ) , transgenic TCL1 controls ( TCL1 x Siglecg - R26ki / ki ) and transgenic TCL1 Siglec - G overexpressing mice ( TCL1 x Siglecg - R26ki / ki mb1cre ) . The diagram displays the percentage of B220lowCD5 + cells in respect to lymphocytesover a time periodof 48 weeks . Shown are mean values with ± SD . Significant differences between groups were determined by one - way ANOVA with Kruskal - Wallis test ( no normal distribution ) and corrected for multiple comparison with Dunn ' s test . All time pointswere tested separately . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Summarized data from more than 20 independent experiments with n = 5 - 20 animals per time pointand genotype are shown .

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

To monitor the progression of the CLL-like population, blood was collected from the animals every four weeks up to an age of 48 weeks and analysed by flow cytometry. Genotypes of the mice are: controls (Siglecg-R26ki/ki), Siglec-G overexpressing mice (Siglecg-R26ki/ki mb1cre), transgenic TCL1 controls (TCL1 x Siglecg-R26ki/ki) and transgenic TCL1 Siglec-G overexpressing mice (TCL1 x Siglecg-R26ki/ki mb1cre). The diagram displays the percentage of B220lowCD5+cells in respect to lymphocytesover a time periodof 48 weeks. Shown are mean values with ±SD. Significant differences between groups were determined by one-way ANOVA with Kruskal-Wallis test (no normal distribution) and corrected for multiple comparison with Dunn's test. All time pointswere tested separately. *p<0.05, **p<0.01, ***p<0.001. Summarized data from more than 20 independent experiments with n=5-20 animals per time pointand genotype are shown.


( C ) Absolute cell numbers of B220low CD5 + lymphocytes at 36 and 48 weeks of age in the spleen are shown as mean values with ± SD . Significant differences between groups were tested by one - way ANOVA with Kruskal - Wallis test and corrected for multiple comparison with Dunn ' s test . The different time points were tested separately . n = 8 - 13 animals per time point and genotype . ( D ) Representative dot plots show selection and percentages of B220low CD5 + B cells in the spleen pre - gated on single cells , living cells and lymphocytes . Data are from at least 10 independent experiments for individual time points . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . n = 48 for TCL1 x Siglecg - R26ki / ki , n = 52 for TCL1 x Siglecg - R26ki / ki

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

(C) Absolute cell numbers of B220low CD5+ lymphocytes at 36 and 48 weeks of age in the spleen are shown as mean values with ±SD. Significant differences between groups were tested by one-way ANOVA with Kruskal-Wallis test and corrected for multiple comparison with Dunn's test. The different time points were tested separately. n=8-13 animals per time point and genotype. (D) Representative dot plots show selection and percentages of B220low CD5+ B cells in the spleen pre-gated on single cells, living cells and lymphocytes. Data are from at least 10 independent experiments for individual time points. *p<0.05, **p<0.01, ***p<0.001. n=48 for TCL1 x Siglecg-R26ki/ki, n= 52 for TCL1 x Siglecg-R26ki/ki mb1cre


( E ) The percentage of B220low CD5 + lymphocytes in the liver at 36 and 48 weeks of age is illustrated . Shown are mean values with ± SD . Significant differences between groups were tested by ordinary one - way ANOVA with Šídák ' s post - hoc test . n = 7 - 14 animals per time point and genotype . ( F ) Representative dot plots for the selection of B220low CD5 + cells in the liver . Cells were pre - gated on single cells , living cells and CD45 + leukocytes . Data are from at least 10 independent experiments for individual time points . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . n = 48 for TCL1 x Siglecg - R26ki / ki , n = 52 for TCL1 x Siglecg - R26ki / ki

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

(E)The percentage of B220low CD5+ lymphocytes in the liver at 36 and 48 weeks of age is illustrated. Shown are mean values with ±SD. Significant differences between groups were tested by ordinary one-way ANOVA with Šídák's post-hoc test. n=7-14 animals per time point and genotype. (F) Representative dot plots for the selection of B220low CD5+ cells in the liver. Cells were pre-gated on single cells, living cells and CD45+ leukocytes. Data are from at least 10 independent experiments for individual time points. *p<0.05, **p<0.01, ***p<0.001. n=48 for TCL1 x Siglecg-R26ki/ki, n= 52 for TCL1 x Siglecg-R26ki/ki mb1cre


( G ) The differences in survival are shown as Kaplan - Meier plots and log - rank test n = 59 for TCL1 x Siglecg - R26ki / ki , n = 57 for TCL1 x Siglecg - R26ki / ki mb1cre ) .

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

(G) The differences in survival are shown as Kaplan-Meier plots and log-rank test n=59 for TCL1 x Siglecg-R26ki/ki , n=57 for TCL1 x Siglecg-R26ki/ki mb1cre).


To measure calcium influx cells were loaded with Indo - 1 . Shown is the mean calcium concentration as ratio of bound to unbound Indo - 1 versus time in s . First the basal level was monitored for 50 s in Krebs - Ringer solution . Subsequently , the BCR was stimulated with 13 µg / ml anti - IgM F ( ab ) 2 ( shown by the black arrow ) . Shown is one representative result of four independent experiments . Mice were between 36 and 48 weeks of age . To measure intracellular signaling splenic cells were pre - gated on single , living cells and lymphocytes , then B220 + cells , including B220low CD5 + lymphocytes were selected .

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

To measure calcium influx cells were loaded with Indo-1. Shown is the mean calcium concentration as ratio of bound to unbound Indo-1 versus time in s. First the basal level was monitored for 50 s in Krebs-Ringer solution. Subsequently, the BCR was stimulated with 13 µg/ml anti-IgM F(ab)2 (shown by the black arrow). Shown is one representative result of four independent experiments. Mice were between 36 and 48 weeks of age. To measure intracellular signaling splenic cells were pre-gated on single, living cells and lymphocytes, then B220+ cells, including B220low CD5+ lymphocytes were selected.


TCL1 and TCL1 x Siglecg - / - B cells were analyzed by intracellular staining with phopho - specific antibodies ( or total protein detecting antibody Bcl2 ) .

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TCL1 and TCL1 x Siglecg-/- B cells were analyzed by intracellular staining with phopho-specific antibodies (or total protein detecting antibody Bcl2).


TCL1 x Siglecg - R26ki / ki or TCL1 x Siglecg - R26ki / ki mb1 - cre B cells were analyzed by intracellular staining with phopho - specific antibodies ( or total protein detecting antibody Bcl2 ) .

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

TCL1 x Siglecg-R26ki/ki or TCL1 x Siglecg-R26ki/ki mb1-cre B cells were analyzed by intracellular staining with phopho-specific antibodies (or total protein detecting antibody Bcl2).


Peripheral blood B cells of CLL patients are pre - gated on single , living B lymphocytes ( CD19 + ) . The mean fluorescence intensity ( MFI ) of surface Siglec - 10 is given from 35 IgV - mutated ( left ) and 18 IgV - unmutated CLL cases ( right ) , always including tumor cells ( CD20lowCD5high ) and normal residual B cells ( CD20highCD5 - ) as paired samples .

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

Peripheral blood B cells of CLL patients are pre-gated on single, living B lymphocytes (CD19+). The mean fluorescence intensity (MFI) of surface Siglec-10 is given from 35 IgV-mutated (left) and 18 IgV-unmutated CLL cases (right), always including tumor cells (CD20lowCD5high) and normal residual B cells (CD20highCD5-) as paired samples.


The transcript expression ( normalized counts from bulk mRNA sequencing ) of SIGLEC10 is given from five healthy donors , discriminating naïve CD5 - ( five donors ) , mature CD5 + 86 donors ) and CD27 + memory B cell ( eight donors ) subsets , from CLL tumor cells ( n = 9 ) and from five paired normal residual B ( NRB ) cells ( n = 5 ) .

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

The transcript expression (normalized counts from bulk mRNA sequencing) of SIGLEC10 is given from five healthy donors, discriminating naïve CD5- (five donors), mature CD5+ 86 donors) and CD27+ memory B cell (eight donors) subsets, from CLL tumor cells (n=9) and from five paired normal residual B (NRB) cells (n=5).


A Frequency of MN leukemic cells expressing the immature marker cKit and mature myeloid marker Ly6G in the peripheral blood following 5 days of treatment ( n = 9 - 10 mice / group ; mice with < 2 % tumor burden in any condition were censored from the analysis ) . Data information : data in A are presented as mean ± s . d . ; P values were calculated using a one - tailed Student ' s unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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A Frequency of MN leukemic cells expressing the immature marker cKit and mature myeloid marker Ly6G in the peripheral blood following 5 days of treatment (n = 9-10 mice/group; mice with < 2% tumor burden in any condition were censored from the analysis). Data information: data in A are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


B Absolute number of MN cells in the peripheral blood quantified by flow cytometry . Grey bars denote treatment ( n = 11 - 12 mice / group ) . Data information : Dox - doxycycline .

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B Absolute number of MN cells in the peripheral blood quantified by flow cytometry. Grey bars denote treatment (n = 11-12 mice/group). Data information: Dox - doxycycline.


C Kaplan - Meier survival curve of leukemic mice ( n = 11 - 12 mice / group , median survival is 16 . 5 for vehicle and not reached for AG636 and doxycycline , P < 0 . 0001 by Log - rank test ) . Data information : Dox - doxycycline .

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C Kaplan-Meier survival curve of leukemic mice (n = 11-12 mice/group, median survival is 16.5 for vehicle and not reached for AG636 and doxycycline, P < 0.0001 by Log-rank test). Data information: Dox - doxycycline.


D Representative FACS plots showing differentiation induced by AG636 and doxycycline . Data information : Dox - doxycycline .

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D Representative FACS plots showing differentiation induced by AG636 and doxycycline. Data information: Dox - doxycycline.


E Spleen weights of MN tumor - bearing mice ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - tailed Student ' s unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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E Spleen weights of MN tumor-bearing mice (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


F Absolute number of MN cells in the bone marrow ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - tailed Student ' s unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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

F Absolute number of MN cells in the bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


G Frequency ( as percentage of all MN cells ) of LSCs ( CD11blowcKithighFcgR + ) and differentiated cells ( CD182 + Ly6G + ) in the bone marrow ( n = 3 - 6 mice / group ) . Data information : data are presented as mean ± s . d . ; P values were calculated using a one - tailed Student ' s unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Dox - doxycycline .

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

G Frequency (as percentage of all MN cells) of LSCs (CD11blowcKithighFcgR+) and differentiated cells (CD182+Ly6G+) in the bone marrow (n = 3-6 mice/group). Data information: data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.


H May - Grunwald - Giemsa - stained cytospins of sorted MN cells showing myeloid differentiation . Data information : Dox - doxycycline .

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H May-Grunwald-Giemsa-stained cytospins of sorted MN cells showing myeloid differentiation. Data information: Dox - doxycycline.


I Quantification of functionally - define LSCs using a limiting dilution assay calculated by ELDA ( n = 4 - 8 mice / group ) .

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I Quantification of functionally-define LSCs using a limiting dilution assay calculated by ELDA (n = 4-8 mice/group).


A Spleen weights of RUNX1 - RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - tailed Student ' s unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 .

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

A Spleen weights of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


B - C Absolute number of leukemic cells in the bone marrow ( B ) and spleens ( C ) of RUNX1 - RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - tailed Student ' s unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 .

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

B-C Absolute number of leukemic cells in the bone marrow (B) and spleens (C) of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days. Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


D - E Frequency of leukemic cells expressing the immature marker cKit and mature myeloid marker CD11b in the bone marrow and spleens of RUNX1 - RUNX1T1 ( D ) or I1DN ( E ) tumor bearing mice treated with AG636 or vehicle for 4 days ( n = 6 mice / group ) . Data information : n = 5 - 6 mice / group for RUNX1 - RUNX1T1 model , n = 4 mice / group for I1DN model ; data are presented as mean ± s . d . ; P values were calculated using a one - tailed Student ' s unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 .

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

D-E Frequency of leukemic cells expressing the immature marker cKit and mature myeloid marker CD11b in the bone marrow and spleens of RUNX1-RUNX1T1 (D) or I1DN (E) tumor bearing mice treated with AG636 or vehicle for 4 days (n = 6 mice/group). Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.


Various bone marrow populations were quantified by flow cytometry in mice treated with AG636 or vehicle for 1 or 4 days ( acute treatment ) or for 4 cycles followed by 4 weeks off treatment ( post - recovery ) . Data information : n = 4 mice / group for acute treatment , n = 5 - 6 mice for prolonged treatment and recovery ; data are presented as mean ± s . d . ; P values were calculated using a two - tailed Student ' s unpaired t - test ; only comparisons that meet the threshold of P < 0 . 05 are shown . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 .

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

Various bone marrow populations were quantified by flow cytometry in mice treated with AG636 or vehicle for 1 or 4 days (acute treatment) or for 4 cycles followed by 4 weeks off treatment (post-recovery). Data information: n = 4 mice/group for acute treatment, n = 5-6 mice for prolonged treatment and recovery; data are presented as mean ± s.d.; P values were calculated using a two-tailed Student's unpaired t-test; only comparisons that meet the threshold of P < 0.05 are shown. *P < 0.05, **P < 0.01, ***P < 0.001


E - H Peripheral blood red blood cells ( E ) , platelets ( F ) , myeloid cells ( G ) and lymphoid cells ( H ) quantified in mice treated with AG636 or vehicle . Grey bars denote treatment .

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

E-H Peripheral blood red blood cells (E), platelets (F), myeloid cells (G) and lymphoid cells (H) quantified in mice treated with AG636 or vehicle. Grey bars denote treatment.


RNA sequencing performed on cKithighCD11blow MN cells sorted from AG636 , doxycycline or vehicle treated mice ( n = 3 mice / group ) . Gene set enrichment analysis showing common and differential enrichment of biological pathways in gene expression data from AG636 or doxycycline treated animals . Gene sets are from C2 : CGP and Reactome sub - collections in MSigDB database ( B ) .

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RNA sequencing performed on cKithighCD11blow MN cells sorted from AG636, doxycycline or vehicle treated mice (n = 3 mice/group). Gene set enrichment analysis showing common and differential enrichment of biological pathways in gene expression data from AG636 or doxycycline treated animals. Gene sets are from C2:CGP and Reactome sub-collections in MSigDB database (B).


C RNA sequencing performed on cKit + CD11b - RUNX1 - RUNX1T1 or I1DN cells sorted from mice treated with AG636 or vehicle for 1 day ( n = 3 mice / group ) . Bar code plots showing enrichment of selected pathways .

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C RNA sequencing performed on cKit+CD11b- RUNX1-RUNX1T1 or I1DN cells sorted from mice treated with AG636 or vehicle for 1 day (n = 3 mice/group). Bar code plots showing enrichment of selected pathways.


E - F qPCR showing down - regulation of genes encoding ribosomal proteins in MN cells ( E ) and human AML cell lines ( F ) treated for 24 hours with AG636 ( n = 3 biological replicates for each cell line ) . Data information : data in E , F are presented as mean ± s . e . m . ; P values were calculated using a one - tailed Student ' s unpaired t - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ;

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E-F qPCR showing down-regulation of genes encoding ribosomal proteins in MN cells (E) and human AML cell lines (F) treated for 24 hours with AG636 (n = 3 biological replicates for each cell line). Data information: data in E, F are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;


G - H Polysome profiling of MN cells treated in vitro with AG636 or vehicle for 24h . Representative trace ( G ) and quantification of sub - polysome and heavy polysome fractions ( H ) determined by measuring area under the curve ( AUC ) ( n = 4 biological replicates ) . Data information : data in are presented as mean ± s . e . m . ; P values were calculated using a one - tailed Student ' s unpaired t - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ;

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

G-H Polysome profiling of MN cells treated in vitro with AG636 or vehicle for 24h. Representative trace (G) and quantification of sub-polysome and heavy polysome fractions (H) determined by measuring area under the curve (AUC) (n = 4 biological replicates). Data information: data in are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;


I Nascent protein synthesis quantified using the AHA incorporation assay in MN or MOLM13 cells treated with AG636 for 24 hours or cycloheximide for 1 hour . Cells that were cultured in the absence of AHA served as a negative control . ( n = 3 biological replicates ) . Data information : data in I are presented as mean ± s . e . m . ; P values were calculated using a one - tailed Student ' s unpaired t - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; CHX - cycloheximide .

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

I Nascent protein synthesis quantified using the AHA incorporation assay in MN or MOLM13 cells treated with AG636 for 24 hours or cycloheximide for 1 hour. Cells that were cultured in the absence of AHA served as a negative control. (n = 3 biological replicates). Data information: data in I are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; CHX - cycloheximide.


C Rank plots of ChIPseq enrichment scores of transcription factors at the promoter regions ( + 1000 bp to - 50 bp from TSS ) of genes within the indicated gene sets extracted from ENCODE 3 ( n = 24 genes for translation genes , n = 707 genes for AG636 down - regulated genes , n = 4237 for the other genes ) .

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C Rank plots of ChIPseq enrichment scores of transcription factors at the promoter regions (+1000 bp to -50 bp from TSS) of genes within the indicated gene sets extracted from ENCODE 3 (n = 24 genes for translation genes, n = 707 genes for AG636 down-regulated genes, n = 4237 for the other genes).


D ChIPqPCR showing the binding of YY1 at the promoter regions of selected translation genes in MN cells treated with AG636 or DMSO . For each replicate enrichment was normalized to YY1 pull - down in DMSO treated cells ( n = 2 biological replicates ) . Data information : data in D are presented as mean ± s . e . m . ; P values were calculated using a two - tailed Student ' s unpaired t - test in D ; * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D ChIPqPCR showing the binding of YY1 at the promoter regions of selected translation genes in MN cells treated with AG636 or DMSO. For each replicate enrichment was normalized to YY1 pull-down in DMSO treated cells (n = 2 biological replicates). Data information: data in D are presented as mean ± s.e.m.; P values were calculated using a two-tailed Student's unpaired t-test in D ; *P < 0.05, ***P < 0.001, ****P < 0.0001.


E Time - course showing global downregulation of O - GlcNAcylation and YY1 expression in MN cells treated with AG636 . The experiment was repeated twice with similar results .

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

E Time-course showing global downregulation of O-GlcNAcylation and YY1 expression in MN cells treated with AG636. The experiment was repeated twice with similar results.


F Western blot of YY1 expression in human AML cell lines treated with AG636 for 24 hours .

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F Western blot of YY1 expression in human AML cell lines treated with AG636 for 24 hours.


G qPCR showing down - regulation of genes encoding ribosomal proteins that are putative YY1 targets in Cas9 expressing MN cells treated with AG636 or DMSO ( left ) ; or transduced with sgRNAs targeting YY1 or control sgRNAs ( right ) ( n = 2 biological replicates ) . Data information : data in G are presented as mean ± s . e . m . ; P values were calculated using a one - tailed Student ' s unpaired t - test in G ; * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

G qPCR showing down-regulation of genes encoding ribosomal proteins that are putative YY1 targets in Cas9 expressing MN cells treated with AG636 or DMSO (left); or transduced with sgRNAs targeting YY1 or control sgRNAs (right) (n = 2 biological replicates). Data information: data in G are presented as mean ± s.e.m.; P values were calculated using a one-tailed Student's unpaired t-test in G; *P < 0.05, ***P < 0.001, ****P < 0.0001.


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H Barcode plots showing downregulation of the Reactome Translation gene set in YY1 knockout pro B cells and human melanoma cells upon YY1 knockdown


G Proliferative competition assays in MN cells transduced with individual sgRNAs and cultured in AG636 or DMSO ( n = 2 biological replicates ) . Data information : data in G represented as mean ± s . e . m . ; P values were calculated using one - tailed Student ' s unpaired t - test ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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G Proliferative competition assays in MN cells transduced with individual sgRNAs and cultured in AG636 or DMSO (n = 2 biological replicates). Data information: data in G represented as mean ± s.e.m.; P values were calculated using one-tailed Student's unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001.


A - B Proliferative competition assays in AML cells transduced with indicated sgRNAs and cultured in AG636 or DMSO ( n = 2 biological replicates ) . Data information : data represented as mean ± s . e . m ; P values were calculated using one - tailed Student ' s unpaired t - test in A and B ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * P < 0 . 0001 .

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

A-B Proliferative competition assays in AML cells transduced with indicated sgRNAs and cultured in AG636 or DMSO (n = 2 biological replicates). Data information: data represented as mean ± s.e.m; P values were calculated using one-tailed Student's unpaired t-test in A and B ; *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.


F AML cells were treated with AG636 , Roscovitine or the combination for 17 days . Cumulative live cell counts are shown ( n = 2 biological replicates ) . Data information : data represented as mean ± s . e . m ; P values were calculated using Brown - Forsythe and Welch ANOVA test in F ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * P < 0 . 0001 .

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F AML cells were treated with AG636, Roscovitine or the combination for 17 days. Cumulative live cell counts are shown (n = 2 biological replicates). Data information: data represented as mean ± s.e.m; P values were calculated using Brown-Forsythe and Welch ANOVA test in F; *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.


A . Coomassie Blue stained SDS - PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C - terminally FLAG - tagged Elp1 .

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A. Coomassie Blue stained SDS-PAGE gel of yeast Elongator isolated by FLAG affinity purification and glycerol gradient ultracentrifugation from a strain encoding C-terminally FLAG-tagged Elp1.


B . Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation ( Lane 6 of Fig 1A ) . Circled particles correspond to intact Elongator . White scale bar corresponds to 50 nm .

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B. Representative segment of an electron micrograph of negatively stained Elongator after stabilization by gradient fixation (Lane 6 of Fig 1A). Circled particles correspond to intact Elongator. White scale bar corresponds to 50 nm.


C . Representative 2D class averages of the full Elongator complex from a total of 17 , 074 particles . All class averages shown in this figure have side width of 500 Å .

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C. Representative 2D class averages of the full Elongator complex from a total of 17,074 particles. All class averages shown in this figure have side width of 500 Å.


D . Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure .

D. Representative 2D class averages of the Elp123 subcomplex prepared through a high salt dissociation procedure.


E . Six evenly - sized densities are arranged in one Elongator lobe . A projection of the Elp456 hexamer X - ray structure ( PDB code : 4A8J ) is shown for comparison .

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E. Six evenly-sized densities are arranged in one Elongator lobe. A projection of the Elp456 hexamer X-ray structure (PDB code: 4A8J) is shown for comparison.


F . Analysis of Elp2 localization using a C - terminal MBP tag . Class averages of untagged Elongator is shown for comparison . White arrowheads point towards additional densities in the tagged complex .

F. Analysis of Elp2 localization using a C-terminal MBP tag. Class averages of untagged Elongator is shown for comparison. White arrowheads point towards additional densities in the tagged complex.


G . Elp3 localization analysis using a C - terminal MBP tag , denoted by the white arrowhead .

G. Elp3 localization analysis using a C-terminal MBP tag, denoted by the white arrowhead.


H . Mapping of Elp1 by incubating purified Elongator containing an Elp1 C - terminal FLAG tag with α - FLAG antibody . The additional antibody density is denoted by the white arrowhead .

H. Mapping of Elp1 by incubating purified Elongator containing an Elp1 C-terminal FLAG tag with α-FLAG antibody. The additional antibody density is denoted by the white arrowhead.


I . Three dimensional reconstruction of the intact Elongator complex at 25Å .

I. Three dimensional reconstruction of the intact Elongator complex at 25Å.


J . Schematic of the approximate subunit locations based on 2D EM analysis .

J. Schematic of the approximate subunit locations based on 2D EM analysis.


K . Inset showing the cavity formed through the centre of the putative Elp456 density . Black scale bars in this figure correspond to 50 Å .

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K. Inset showing the cavity formed through the centre of the putative Elp456 density. Black scale bars in this figure correspond to 50 Å.


A . Number of peptides detected of each Elongator subunit purified through Elp4 - FLAG pulldown from yeast lacking ELP1 , ELP2 , or ELP3 as a fraction of the number of Elp4 peptides . Individual data points are represented as circles , while the averages are represented as bars .

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A. Number of peptides detected of each Elongator subunit purified through Elp4-FLAG pulldown from yeast lacking ELP1, ELP2, or ELP3 as a fraction of the number of Elp4 peptides. Individual data points are represented as circles, while the averages are represented as bars.


B . Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate ( DSS ) and analyzed by mass spectrometry . Schematic representation of the major crosslinking connectivity of Elp123 is shown .

B. Purified Elongator was incubated with the primary amine crosslinker disuccinimidyl suberate (DSS) and analyzed by mass spectrometry. Schematic representation of the major crosslinking connectivity of Elp123 is shown.


C . Major crosslinking connectivity of the Elp456 complex .

C. Major crosslinking connectivity of the Elp456 complex.


B . Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast . The strains were then plated onto - Ura media containing caffeine . The pRS416 vector is used for these experiments .

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B. Plasmids containing ELP2 and the four loop mutants were transformed into elp2Δ yeast. The strains were then plated onto -Ura media containing caffeine. The pRS416 vector is used for these experiments.


C . Growth curve analysis of loop mutants in - Ura media with and without K . lactis toxin zymocin . Shaded regions correspond to the SEM at each time point . N = 3 .

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C. Growth curve analysis of loop mutants in -Ura media with and without K. lactis toxin zymocin. Shaded regions correspond to the SEM at each time point. N=3.


D . Lysates of Elp4 - FLAG - containing yeast expressing the Elp2 loop mutants were incubated with α - FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α - HA and α - FLAG

D. Lysates of Elp4-FLAG-containing yeast expressing the Elp2 loop mutants were incubated with α-FLAG resin and eluted with 3xFLAG peptides then analyzed by Western Blot using α-HA and α-FLAG antibody


A . Fitting of Elp123 structures into the 3D reconstruction of Elongator . The homology structure of the Elp1 N - terminal double WD40 - domains , the homology structure of Elp3 based on the X - ray structure of D . mccartyi , the X - ray structure of Elp1 C - terminal dimer ( 5CQS ) , and the X - ray structure of Elp2 ( 4XFV ) . Scale bar corresponds to 50 Å , and applies to panels A - C .

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A. Fitting of Elp123 structures into the 3D reconstruction of Elongator. The homology structure of the Elp1 N-terminal double WD40-domains, the homology structure of Elp3 based on the X-ray structure of D. mccartyi, the X-ray structure of Elp1 C-terminal dimer (5CQS), and the X-ray structure of Elp2 (4XFV). Scale bar corresponds to 50 Å, and applies to panels A-C.


B . Fitting of the Elp456 heterohexameric ATPase ring ( 4A8J ) into the six - membered ring from the larger Elongator lobe .

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B. Fitting of the Elp456 heterohexameric ATPase ring (4A8J) into the six-membered ring from the larger Elongator lobe.


C . Size comparison of a representative tRNA molecule ( 1VTQ ) and the cavity observed through the middle of the Elp456 hexamer .

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C. Size comparison of a representative tRNA molecule (1VTQ) and the cavity observed through the middle of the Elp456 hexamer.


D . Alternate views of the Elongator model showing the subunit crosslinking patterns .

D. Alternate views of the Elongator model showing the subunit crosslinking patterns.


A . CXMS and EM data , combined with the X - ray structures of Elp1 C - terminus ( 5CQS ) , Elp2 ( 4XFV ) , Elp456 ( 4A8J ) , and homology models of the Elp1 tandem WD - 40 domains and Elp3 , were used to estimate the relative positions of each subunit to generate a multi - scale model of Elongator subunit organization .

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A. CXMS and EM data, combined with the X-ray structures of Elp1 C-terminus (5CQS), Elp2 (4XFV), Elp456 (4A8J), and homology models of the Elp1 tandem WD-40 domains and Elp3, were used to estimate the relative positions of each subunit to generate a multi-scale model of Elongator subunit organization.


B . Side view of the Elongator architectural model highlighting the cavity ( red outline ) spanning through the centre of the Elp456 hexamer .

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B. Side view of the Elongator architectural model highlighting the cavity (red outline) spanning through the centre of the Elp456 hexamer.


C . Comparison of the Elp123 conformations of the intact Elongator complex ( gold ) and the Elp123 subcomplex ( cyan ) . The Elp456 - unloaded lobe of Elongator and one lobe of Elp123 were aligned .

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C. Comparison of the Elp123 conformations of the intact Elongator complex (gold) and the Elp123 subcomplex (cyan). The Elp456-unloaded lobe of Elongator and one lobe of Elp123 were aligned.


A . Silver - stained SDS - PAGE of Elongator glycerol gradient fractions purified using low salt ( 75 mM NaCl ) buffer . The fraction containing the most Elp4 , Elp5 , and Elp6 is designated by an asterisk ( * ) and the corresponding fraction from a glutaraldehyde - containing glycerol gradient is used for EM analysis .

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A. Silver-stained SDS-PAGE of Elongator glycerol gradient fractions purified using low salt (75 mM NaCl) buffer. The fraction containing the most Elp4, Elp5, and Elp6 is designated by an asterisk (*) and the corresponding fraction from a glutaraldehyde-containing glycerol gradient is used for EM analysis.


B . Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations ( 150 mM and 75 mM NaCl , respectively ) . Densities resembling a second copy of Elp456 is indicated by white triangles .

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B. Comparison of class averages of Elongator purified in buffer containing regular or low salt concentrations (150 mM and 75 mM NaCl, respectively). Densities resembling a second copy of Elp456 is indicated by white triangles.


D . Coomassie - stained SDS - PAGE of rElp456 glycerol gradient fractions .

D. Coomassie-stained SDS-PAGE of rElp456 glycerol gradient fractions.


E . Coomassie - stained SDS - PAGE of the Elongator mixed with rElp456 glycerol gradient fractions . The fraction containing the most intact complexes is indicated with an asterisk ( * ) , and the corresponding fraction from a glutaraldehyde - containing glycerol is used for EM analysis .

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E. Coomassie-stained SDS-PAGE of the Elongator mixed with rElp456 glycerol gradient fractions. The fraction containing the most intact complexes is indicated with an asterisk (*), and the corresponding fraction from a glutaraldehyde-containing glycerol is used for EM analysis.


F . Comparison of class averages of natively purified Elongator with and without rElp456 incubation . Densities resembling a second copy of Elp456 are indicated by white triangles .

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F. Comparison of class averages of natively purified Elongator with and without rElp456 incubation. Densities resembling a second copy of Elp456 are indicated by white triangles.


ivMCs induce differentiation of co - cultured intestinal organoids as determined by qRT - PCR for stem cell ( Lgr5 , Olfm4 ) and proliferation ( Ki67 , Pcna ) and differentiation markers ( Muc2 - goblet cells , Krt20 - differentiated epithelial cells , Sim and Alpi - enterocytes ) . Enhanced terminal enterocytic differentiation was checked by villus center markers ( Fabp1 , Fabp2 , G6pc , Leap2 ) and villus tip markers ( Ada , Lama3 , Lgals3 , Cdh1 ) . ( Expression levels normalized to Ubiquitin B ( Ubb ) , ENR parallel set as 1 , error bars show sd , each dot represents one technical replicate of one representative experiment ) . If n = 2 technical replicates , error bars , and mean are not shown .

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ivMCs induce differentiation of co-cultured intestinal organoids as determined by qRT-PCR for stem cell (Lgr5, Olfm4) and proliferation (Ki67, Pcna) and differentiation markers (Muc2-goblet cells, Krt20-differentiated epithelial cells, Sim and Alpi-enterocytes). Enhanced terminal enterocytic differentiation was checked by villus center markers (Fabp1, Fabp2, G6pc, Leap2) and villus tip markers (Ada, Lama3, Lgals3, Cdh1). (Expression levels normalized to Ubiquitin B (Ubb), ENR parallel set as 1, error bars show sd, each dot represents one technical replicate of one representative experiment). If n = 2 technical replicates, error bars, and mean are not shown.


The impact of individual Bmp ligands on the growth of freshly established intestinal organoids . ( After 96 hours in 500 ng / ml of indicated Bmp . Brightfield images . Scale bar , 200 μm ) Proliferation of freshly established intestinal organoids cultivated with indicated Bmp for 96 hours determined by quantification of EdU incorporation ( % of EdU positive cells ) . Each dot represents one cultured organoid . Average value for each treatment indicated by line , error bars show sd .

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

The impact of individual Bmp ligands on the growth of freshly established intestinal organoids. (After 96 hours in 500 ng/ml of indicated Bmp. Brightfield images. Scale bar, 200 μm) Proliferation of freshly established intestinal organoids cultivated with indicated Bmp for 96 hours determined by quantification of EdU incorporation (% of EdU positive cells). Each dot represents one cultured organoid. Average value for each treatment indicated by line, error bars show sd.


Relative expression of differentiation ( Muc2 - goblet cells , Krt20 - differentiated epithelial cells , Sim and Alpi - enterocytes ) and stem ( Lgr5 , Olfm4 ) markers in intestinal crypts upon 96 hours cultivation with indicated Bmp ( 500 ng / ml ) . ( qRT - PCR , expression levels normalized to Ubiquitin B ( Ubb ) , error bars show sd , Untreated parallel set as 1 , each dot represents one technical replicate of one representative experiment . ) .

Relative expression of differentiation (Muc2-goblet cells, Krt20-differentiated epithelial cells, Sim and Alpi-enterocytes) and stem (Lgr5, Olfm4) markers in intestinal crypts upon 96 hours cultivation with indicated Bmp (500 ng/ml). (qRT-PCR, expression levels normalized to Ubiquitin B (Ubb), error bars show sd, Untreated parallel set as 1, each dot represents one technical replicate of one representative experiment.).


Heatmap showing changes in expression of indicated genes after 24 hours of indicated Bmp ( 500 ng / ml ) treatment in the intestinal organoids . Hierarchical clustering of Bmp treatments shows differences in gene regulation by Bmp2 vs . Bmp4 . ( n = at least 3 per each treatment and / or control , top 10 differentially expressed genes per each treatment were selected and are shown . Z - score was calculated using standard formula z = ( x - μ ) / σ ) .

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Heatmap showing changes in expression of indicated genes after 24 hours of indicated Bmp (500 ng/ml) treatment in the intestinal organoids. Hierarchical clustering of Bmp treatments shows differences in gene regulation by Bmp2 vs. Bmp4. (n= at least 3 per each treatment and/or control, top 10 differentially expressed genes per each treatment were selected and are shown. Z-score was calculated using standard formula z = (x - μ) / σ).


Whereas Bmp4 triggers expression of villus center genes connected to lipid metabolism , Bmp2 preferentially activates the expression of villus tip genes . ( Graphs show normalized counts determined by RNAseq , n = at least 3 for each treatment , timepoint : 24 hours , error bars denote sd ) .

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Whereas Bmp4 triggers expression of villus center genes connected to lipid metabolism, Bmp2 preferentially activates the expression of villus tip genes. (Graphs show normalized counts determined by RNAseq, n= at least 3 for each treatment, timepoint: 24 hours, error bars denote sd).


Bmp2 treatment induces the expression of the villus tip markers Nt5e ( Cd73 protein ) . ( Graph shows normalized counts determined by RNAseq , n = at least 3 for each treatment , timepoint : 24 hours , error bars denote sd ) .

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Bmp2 treatment induces the expression of the villus tip markers Nt5e (Cd73 protein). (Graph shows normalized counts determined by RNAseq, n= at least 3 for each treatment, timepoint: 24 hours, error bars denote sd).


Expression of Cd73 ( Nt5e gene ) ( green ) and Fabp2 ( red ) upon the Bmp2 and Bmp4 treatment in the intestinal organoids . Immunocytochemistry , DAPI ( blue ) counterstains nuclei , timepoint : 48 hours . ( Scale bar , 100 μm ) .

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Expression of Cd73 (Nt5e gene) (green) and Fabp2 (red) upon the Bmp2 and Bmp4 treatment in the intestinal organoids. Immunocytochemistry, DAPI (blue) counterstains nuclei, timepoint: 48 hours. (Scale bar,100 μm).


Quantification of Cd73 ( Nt5e gene ) and Fabp2 immunostainings determined as the ratio of specific signal to DAPI ratio . ( Each dot represents one organoid , timepoint : 48 hours , n = at least 20 organoids , graphs show : min . , 1st quartile , median , 3th quartile , max . , p values counted using t - test ) .

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Quantification of Cd73 (Nt5e gene) and Fabp2 immunostainings determined as the ratio of specific signal to DAPI ratio. (Each dot represents one organoid, timepoint: 48 hours, n= at least 20 organoids, graphs show: min.,1st quartile, median, 3th quartile, max., p values counted using t-test).


Relative Ada enzymatic activity in intestinal organoids after Bmp2 and Bmp4 treatment ( Each dot represents individual measurement , timepoint : 48 hours , sd . ) .

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Relative Ada enzymatic activity in intestinal organoids after Bmp2 and Bmp4 treatment (Each dot represents individual measurement, timepoint: 48 hours, sd.).


Tgfβ induces the expression of villus tip genes and represses villus center genes . ( Graphs show normalized counts determined by RNAseq , n = 3 for each treatment , timepoint : 24 hours , error bars denote sd . ) .

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Tgfβ induces the expression of villus tip genes and represses villus center genes. (Graphs show normalized counts determined by RNAseq, n=3 for each treatment, timepoint: 24 hours, error bars denote sd.).


Four days treatment with LDN - 193189 - 4HCl ( 50 mg / kg ) reduces the expression of Ada ( green ) protein at the villus tips . ( Immunohistochemistry , DAPI ( blue ) counterstains nuclei . E - cadherin ( white ) marks epithelial cells , scale bar , 100 μm ) .

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Four days treatment with LDN-193189-4HCl (50 mg/kg) reduces the expression of Ada (green) protein at the villus tips. (Immunohistochemistry, DAPI (blue) counterstains nuclei. E-cadherin (white) marks epithelial cells, scale bar, 100 μm).


Quantification of Ada signal range at the villus tip as visualised in B . ( Each dot represents one villus , n = at least 20 villi , graphs show : min . , 1st quartile , median , 3th quartile , max . , p values counted using t - test ) .

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Quantification of Ada signal range at the villus tip as visualised in B. (Each dot represents one villus, n= at least 20 villi, graphs show: min.,1st quartile, median, 3th quartile, max., p values counted using t-test).


Changes in the expression of villus tip and villus center genes after LDN - 193189 - 4HCl ( 150 mg / kg ) treatment determined by qRT - PCR normalized to β - actin ( β - act ) . ( Each dot represents one technical replicate of one representative experiment , error bars show sd . ) .

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Changes in the expression of villus tip and villus center genes after LDN-193189-4HCl (150 mg/kg) treatment determined by qRT-PCR normalized to β-actin (β-act). (Each dot represents one technical replicate of one representative experiment, error bars show sd.).


Induction of shRNA against Smad4 upon the doxycycline ( + Dox ) treatment ( 3 days ) followed by Bmp2 treatment for additional 24 hours . shRNA activation is visualised by the expression of coupled TdTomato ( red ) , scale bar , 200 μm .

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Induction of shRNA against Smad4 upon the doxycycline (+Dox) treatment (3 days) followed by Bmp2 treatment for additional 24 hours. shRNA activation is visualised by the expression of coupled TdTomato (red), scale bar, 200 μm.


Smad4 knock - down ( shRNA - ON ) alleviates Bmp2 - induced differentiation marked by relative expression of villus tip genes . ( qRT - PCR , expression levels normalized to Ubiquitin B ( Ubb ) , each dot represents one technical replicate of one representative experiment , error bars show sd , untreated parallel set as 1 , treatment as in 6A ) .

Smad4 knock-down (shRNA-ON) alleviates Bmp2-induced differentiation marked by relative expression of villus tip genes. (qRT-PCR, expression levels normalized to Ubiquitin B (Ubb), each dot represents one technical replicate of one representative experiment, error bars show sd, untreated parallel set as 1, treatment as in 6A).


Long term ( 4 weeks ) organoid cultivation in the presence of Bmp2 ( 10 ng / ml ) enriches the expression of villus tip genes . ( qRT - PCR , expression levels normalized to Ubiquitin B ( Ubb ) , each dot represents one technical replicate of one representative experiment , error bars show sd , untreated parallel set as 1 , P2 - collected 4 days after 2nd passage , P4 - collected 4 days after 4th passage ) .

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Long term (4 weeks) organoid cultivation in the presence of Bmp2 (10 ng/ml) enriches the expression of villus tip genes. (qRT-PCR, expression levels normalized to Ubiquitin B (Ubb), each dot represents one technical replicate of one representative experiment, error bars show sd, untreated parallel set as 1, P2 - collected 4 days after 2nd passage, P4 -collected 4 days after 4th passage).


qRT - PCR for villus tip genes and villus center genes in freshly established organoids cultivated 24 or 96 hours with Bmp2 ( 500 ng / ml ) , Wnt5a ( 500 ng / ml ) or both Bmp2 + Wnt5a ( each 500 ng / ml ) . Whereas Bmp2 induces villus tip genes by its own , it can act synergistically with Wnt5a . For the short - term cultivation , the effect is specific for villus tip genes ( notice the different scale in left graphs ) . ( Expression levels normalized to Hmbs , untreated parallel set as 1 , each dot represents one technical replicate of one representative experiment , error bars show sd ) . If n = 2 technical replicates , error bars and mean are not shown .

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qRT-PCR for villus tip genes and villus center genes in freshly established organoids cultivated 24 or 96 hours with Bmp2 (500 ng/ml), Wnt5a (500 ng/ml) or both Bmp2 + Wnt5a (each 500 ng/ml). Whereas Bmp2 induces villus tip genes by its own, it can act synergistically with Wnt5a. For the short-term cultivation, the effect is specific for villus tip genes (notice the different scale in left graphs). (Expression levels normalized to Hmbs, untreated parallel set as 1, each dot represents one technical replicate of one representative experiment, error bars show sd). If n = 2 technical replicates, error bars and mean are not shown.


Expression of E - cadherin ( red ) and Bmp2 , Bmp4 , Wnt5a , Bmp2 + Wnt5a treatment in the intestinal organoids . Immunocytochemistry , DAPI ( blue ) counterstains nuclei . ( Immunocytochemistry , timepoint : 24 hours , 500 ng / ml each protein , scale bar , 200 μm ) .

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Expression of E-cadherin (red) and Bmp2, Bmp4, Wnt5a, Bmp2 + Wnt5a treatment in the intestinal organoids. Immunocytochemistry, DAPI (blue) counterstains nuclei. (Immunocytochemistry, timepoint: 24 hours, 500 ng/ml each protein, scale bar, 200 μm).


( a ) Instant Blue stained SDS - polyacrylamide gel of Scc1wt cohesin tetramers used in ( b ) .

(a) Instant Blue stained SDS-polyacrylamide gel of Scc1wt cohesin tetramers used in (b).


( b ) Cohesin loading assay . Scc1wt cohesin tetramer was incubated with nicked circular ( CN ) or 1x , 2x or 4x concentration of linearised ( L ) plasmid DNA , immunoprecipitated with anti - Scc1 antibodies , washed with high salt buffer and then eluted with Scc1 peptide . Recovered DNA was separated by agarose gel electrophoresis and stained with GelRedDNA stain . Input DNA = 7 % .

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(b) Cohesin loading assay. Scc1wt cohesin tetramer was incubated with nicked circular (CN) or 1x, 2x or 4x concentration of linearised (L) plasmid DNA, immunoprecipitated with anti-Scc1 antibodies, washed with high salt buffer and then eluted with Scc1 peptide. Recovered DNA was separated by agarose gel electrophoresis and stained with GelRedDNA stain. Input DNA = 7 %.


( c ) Silver stainedSDS - polyacrylamide gel of non - cleaved and cleaved Scc1Halo - TEVcohesin tetramers used in ( d ) .

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(c) Silver stainedSDS-polyacrylamide gel of non-cleaved and cleaved Scc1Halo-TEVcohesin tetramers used in (d).


( d ) Non - cleaved and cleaved Scc1Halo - TEVcohesin tetramer was incubated with nicked circular plasmid DNA and processed as in ( b ) . Input DNA = 4 % .

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(d) Non-cleaved and cleaved Scc1Halo-TEVcohesin tetramer was incubated with nicked circular plasmid DNA and processed as in (b). Input DNA = 4 %.


( e ) Kymograph of Scc1GFP - TEVcohesin bound to doubly tethered λ - DNA in cohesin binding buffer and washed with 750 mM NaCl buffer + Sytox Orange . XhoI flow in induced DNA cleavage and cohesin release . Flow in from top and scale bar = 5 µm in this and all subsequent kymographs .

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(e) Kymograph of Scc1GFP-TEVcohesin bound to doubly tethered λ-DNA in cohesin binding buffer and washed with 750 mM NaCl buffer + Sytox Orange. XhoI flow in induced DNA cleavage and cohesin release. Flow in from top and scale bar = 5 µm in this and all subsequent kymographs.


( f ) Kymographs of Scc1GFP - TEVcohesin and Scc1GFPcohesin bound to doubly tethered λ - DNA and washed with 750 mM NaCl buffer . TEV protease flow in released Scc1GFP - TEVbut not Scc1GFP from DNA .

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(f) Kymographs of Scc1GFP-TEVcohesin and Scc1GFPcohesin bound to doubly tethered λ-DNA and washed with 750 mM NaCl buffer. TEV protease flow in released Scc1GFP-TEVbut not Scc1GFP from DNA.


( a ) Kymograph of a single DNA - bound Scc1Halo - TMR - TEVcohesin complex after 750 mM NaCl buffer wash . ( b ) Distribution of background subtracted EMCCD counts of Scc1Halo - TMR - TEVcohesin complexes immobilized on the coverslip . Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively . n = 290 regions with fluorescent molecules , 228 regions with background . ( c ) Photobleaching kinetics of a single DNA - bound Scc1Halo - TMR - TEVcohesin complex . ( d ) Diffusion coefficients of Smc1 / 3 wild type or K38A ATP binding deficient ' KA ' forms of Scc1Halo - TMR - TEV - cohesin in the presence or absence of ATP . Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period .

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(a) Kymograph of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex after 750 mM NaCl buffer wash. (b) Distribution of background subtracted EMCCD counts of Scc1Halo-TMR-TEVcohesin complexes immobilized on the coverslip. Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively. n = 290 regions with fluorescent molecules, 228 regions with background. (c) Photobleaching kinetics of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex. (d) Diffusion coefficients of Smc1/3 wild type or K38A ATP binding deficient 'KA' forms of Scc1Halo-TMR-TEV-cohesin in the presence or absence of ATP. Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period.


( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( a ) TetO - DNA - bound TetRHalo - TMR .

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(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (a) TetO-DNA-bound TetRHalo-TMR.


( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( b ) DNA - bound Halo - TMREcoRIE111Q .

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(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (b) DNA-bound Halo-TMREcoRIE111Q.


( a - c ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin diffusing past ( c ) a DNA - bound TMR - labelled nucleosome .

(a - c) Kymograph of salt-resistant Scc1GFP-TEV-cohesin diffusing past (c) a DNA-bound TMR-labelled nucleosome.


( d ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin failing to bypass DNA - bound QDotEcoRIE111Q . DNA was stained with Sytox Orange .

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(d) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound QDotEcoRIE111Q. DNA was stained with Sytox Orange.


( a ) Kymograph of two Halo - TMRT7 RNAP transcription elongation events . ( b , c ) Kymographs showing ( b ) Halo - TMRT7 RNAP transcription stalling following removal of NTPs and ( c ) subsequent resumption after NTP flow in .

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(a) Kymograph of two Halo-TMRT7 RNAP transcription elongation events. (b, c) Kymographs showing (b) Halo-TMRT7 RNAP transcription stalling following removal of NTPs and (c) subsequent resumption after NTP flow in.


( d ) Kymograph of Halo - TMRT7 RNAP ( diameter ~ 8 nm excluding HaloTag ) displacing Scc1GFP - TEV - cohesin following resumption of transcription in T7 reaction buffer + NTPs .

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(d) Kymograph of Halo-TMRT7 RNAP (diameter ~ 8 nm excluding HaloTag) displacing Scc1GFP-TEV-cohesin following resumption of transcription in T7 reaction buffer + NTPs.


( e ) Kymograph of Halo - TMRT7 RNAP constraining translocation of salt - resistant Scc1GFP - TEV - cohesin .

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(e) Kymograph of Halo-TMRT7 RNAP constraining translocation of salt-resistant Scc1GFP-TEV-cohesin.


( a ) Representative field of view showing Halo - TMRCTCF bound to pPlat - 4xCTCF following 75 mM NaCl , 75 mM KCl buffer wash . Arrows denote CTCF bound at predicted site of 4xCTCF array .

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(a) Representative field of view showing Halo-TMRCTCF bound to pPlat-4xCTCF following 75 mM NaCl, 75 mM KCl buffer wash. Arrows denote CTCF bound at predicted site of 4xCTCF array.


( b ) Kymograph of salt - resistant Scc1GFP - TEV - cohesin failing to bypass DNA - bound Halo - TMRCTCF . DNA was post - stained with Sytox Green .

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(b) Kymograph of salt-resistant Scc1GFP-TEV-cohesin failing to bypass DNA-bound Halo-TMRCTCF. DNA was post-stained with Sytox Green.


Western blot analysis of lysates from cerebellum of control and FXS patients ' ' FXS ' ' ( n = 4 ) . Extract of pCI - hDGKk - transfected Hela cells ( hDGKk ) was used as antibody specificity control and size marker . Representative images of immunoblots probed with antibodies against the indicated proteins are shown . GAPDH was the loading control . Quantification of Western blots is shown on right . Protein amounts of DGKk are normalized to GAPDH and presented as fold change relative to control .

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Western blot analysis of lysates from cerebellum of control and FXS patients ''FXS'' (n = 4). Extract of pCI-hDGKk-transfected Hela cells (hDGKk) was used as antibody specificity control and size marker. Representative images of immunoblots probed with antibodies against the indicated proteins are shown. GAPDH was the loading control. Quantification of Western blots is shown on right. Protein amounts of DGKk are normalized to GAPDH and presented as fold change relative to control.


C - D ) Immunoblots and quantification of lysates from Cos - 1 cells transfected with plasmid pCI - mDGKk - HA or non - transfected ( NT ) and pre - transfected 24h before with siRNA control ( siC ) or against FMRP ( siFMR1 ) . GAPDH was used as a loading control . For quantification , the DGKk and FMRP signals were normalized against GAPDH signal and presented relative to the signal for siC treated cells ( C ) . Immunoblots and quantification of lysates from Cos - 1 cells transfected with pCI ( empty plasmid ) , pCI - HA - ∆ N - DGKk , or pCI - mDGKk - HA , and pre - treated with siRNA control ( siC ) or against FMRP ( siFMR1 ) ( D ) . Quantifications as in C .

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C-D) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-mDGKk-HA or non-transfected (NT) and pre-transfected 24h before with siRNA control (siC) or against FMRP (siFMR1). GAPDH was used as a loading control. For quantification, the DGKk and FMRP signals were normalized against GAPDH signal and presented relative to the signal for siC treated cells (C). Immunoblots and quantification of lysates from Cos-1 cells transfected with pCI (empty plasmid), pCI-HA-∆N-DGKk, or pCI-mDGKk-HA, and pre-treated with siRNA control (siC) or against FMRP (siFMR1) (D). Quantifications as in C.


A ) HeLa cells transfected with indicated plasmids expressing mouse ( m ) or human ( h ) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag ( red ) and Dapi staining ( blue ) . ∆ C1 - hDGKk construct bears deletion of phorbol ester C1 domain ( 328 - 449 ) .

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A) HeLa cells transfected with indicated plasmids expressing mouse (m) or human (h) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag (red) and Dapi staining (blue). ∆C1-hDGKk construct bears deletion of phorbol ester C1 domain (328-449).


B ) Immunoblots and quantification of lysates from Cos - 1 cells transfected with plasmid pCI - HA - ∆ N - DGKk , untransfected ( NT ) , or plasmid pCI control , with the indicated amount of plasmid ( µg ) and after 24h , incubated with puromycin ( 20µg / ml , 30 min ) to measure rates of protein synthesis . GAPDH was used as a loading control . 0 indicates no puromycin treatment , - / + indicates treatment without or with DGK inhibitor ( DGKi ) 3 µM R59022 and 0 . 2 µM R59949 at 6 µM , 15 min . Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions .

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B) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-HA-∆N-DGKk, untransfected (NT), or plasmid pCI control, with the indicated amount of plasmid (µg) and after 24h, incubated with puromycin (20µg/ml, 30 min) to measure rates of protein synthesis. GAPDH was used as a loading control. 0 indicates no puromycin treatment, -/+ indicates treatment without or with DGK inhibitor (DGKi) 3 µM R59022 and 0.2 µM R59949 at 6 µM, 15 min. Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions.


C ) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV ( days in vitro ) with 10e9 VG / ml of AAVRh10 - GFP or AAVRh10 - ∆ N - DGKk and assessed after 5 days using anti - MAP2 and anti - HA for ∆ N - DGKk or direct 488 nm excitation for GFP . DAPI was used to visualize nuclei on merged images .

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C) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV (days in vitro) with 10e9 VG/ml of AAVRh10-GFP or AAVRh10-∆N-DGKk and assessed after 5 days using anti-MAP2 and anti-HA for ∆N-DGKk or direct 488 nm excitation for GFP. DAPI was used to visualize nuclei on merged images.


D ) Representative immunoblots of lysates from WT and Fmr1 - KO cortical neurons transduced with AAVRh10 - ∆ N - DGKk ( AAV ∆ N - DGKk ) , at the indicated titers ( VG / ml culture volume ) and quantification of phosphorylation and total levels of eIF4E . GAPDH was used as a loading control . For quantification , the phospho - protein signal was normalized first against total protein signal and is presented relative to the signal for WT culture .

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D) Representative immunoblots of lysates from WT and Fmr1-KO cortical neurons transduced with AAVRh10-∆N-DGKk (AAV ∆N-DGKk), at the indicated titers (VG/ml culture volume) and quantification of phosphorylation and total levels of eIF4E. GAPDH was used as a loading control. For quantification, the phospho-protein signal was normalized first against total protein signal and is presented relative to the signal for WT culture.


B ) Viral titers ( viral genome copy VG per cell ) determined by qPCR in cortical , hippocampal and rest of brain areas of Fmr1 - KO mice treated with saline solution ( Vehicle ) , AAVPHP . eB - ∆ N - DGKk ( PHP . eB ) , AAVRh10 - ∆ N - DGKk ( Rh10 ) 8 weeks after injections . Data are mean ± SEM . Each dot represents an individual mouse .

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B) Viral titers (viral genome copy VG per cell) determined by qPCR in cortical, hippocampal and rest of brain areas of Fmr1-KO mice treated with saline solution (Vehicle), AAVPHP.eB-∆N-DGKk (PHP.eB), AAVRh10-∆N-DGKk (Rh10) 8 weeks after injections. Data are mean ± SEM. Each dot represents an individual mouse.


C ) Immunoblots and quantification of ∆ N - DGKk protein in lysates from brain areas of mice treated as in B . GAPDH was used as a loading control . Densitogram of ∆ N - DGKk expression is presented as change relative to vehicle conditions .

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C) Immunoblots and quantification of ∆N-DGKk protein in lysates from brain areas of mice treated as in B. GAPDH was used as a loading control. Densitogram of ∆N-DGKk expression is presented as change relative to vehicle conditions.


D ) Representative coronal brain sections processed for detection of ∆ N - DGKk using immunohistochemistry on Fmr1 - KO mice treated with indicated treatment , 8 weeks post - injections , counter stained with eosin hematoxylin . Adjacent sections were immunolabelled with NeuN . 3 mice per genotype were processed . The sections shown are between Bregma levels - 1 . 50 mm and - 1 . 80 mm . Scale bar is 2 mm . Magnifications of regions of cortex ( c ) , hippocampus ( h ) , and striatum ( s ) are shown in side panels , scale bar 200 µm .

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D) Representative coronal brain sections processed for detection of ∆N-DGKk using immunohistochemistry on Fmr1-KO mice treated with indicated treatment, 8 weeks post-injections, counter stained with eosin hematoxylin. Adjacent sections were immunolabelled with NeuN. 3 mice per genotype were processed. The sections shown are between Bregma levels -1.50 mm and -1.80 mm. Scale bar is 2 mm. Magnifications of regions of cortex (c), hippocampus (h), and striatum (s) are shown in side panels, scale bar 200 µm.


E ) Total phosphatidic acid ( PA ) level measure by mass spectrometry in cortex of WT mice treated with saline solution ( WT - S ) and Fmr1 - KO mice treated with saline ( Fmr1 - S ) , AAVPHP . eB - ∆ N - DGKk ( Fmr1 - PHP . eB ) , AAVRh10 - ∆ N - DGKk ( Fmr1 - Rh10 ) 8 weeks after injections . Data are expressed as mol % of total lipids and represented as median with interquartile range with minimum and maximum values . Statistics : one - way ANOVA and Tukey ' s multiple comparisons test , n = 8 individual animals , except for WT - S and Fmr1 - S n = 11 . * * P < 0 . 01 , * * * P < 0 . 001 .

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E) Total phosphatidic acid (PA) level measure by mass spectrometry in cortex of WT mice treated with saline solution (WT-S) and Fmr1-KO mice treated with saline (Fmr1-S), AAVPHP.eB-∆N-DGKk (Fmr1-PHP.eB), AAVRh10-∆N-DGKk (Fmr1-Rh10) 8 weeks after injections. Data are expressed as mol % of total lipids and represented as median with interquartile range with minimum and maximum values. Statistics: one-way ANOVA and Tukey's multiple comparisons test, n=8 individual animals, except for WT-S and Fmr1-S n=11. **P < 0.01, ***P < 0.001.


F ) Total diacylglycerol ( DAG ) level in cortex measured as in E ) .

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F) Total diacylglycerol (DAG) level in cortex measured as in E).


A ) Elevated Plus Maze . Percentage of time spent in open arms and number of entries in open , closed and total ( open + closed ) arms . ( n = 12 for time in open arm , n = 24 for arm entries ) .

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A) Elevated Plus Maze. Percentage of time spent in open arms and number of entries in open, closed and total (open+closed) arms. (n = 12 for time in open arm, n=24 for arm entries).


B ) Novel object recognition . Locomotor activity ( distance in m ) in the whole arena and percentage of time spent in the center during 15 min habituation . Locomotor activity during the acquisition and retention trials . Recognition index . ( n = 24 ) .

B) Novel object recognition. Locomotor activity (distance in m) in the whole arena and percentage of time spent in the center during 15 min habituation. Locomotor activity during the acquisition and retention trials. Recognition index. (n = 24).


C ) Social recognition . Number of entries in the two side compartments during preference session ( left ) , social preference ( percentage of exploration of a congener vs an object ) ( center ) and social memory ( percentage of exploration of a novel vs familiar congener ) ( right ) . ( n = 24 ) .

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C) Social recognition. Number of entries in the two side compartments during preference session (left), social preference (percentage of exploration of a congener vs an object) (center) and social memory (percentage of exploration of a novel vs familiar congener) (right). (n=24).


A , B . MDCK - SIAT1 cells co - incubated with Luc - WSN / 33 and MH - S CM fractions . Replication was quantified by luminescence . ( A ) Replication curves display average luminescence reads from 5 wells per condition ( measured at 7 . 5 min intervals - displayed here at 30 min intervals ) from 1 experiment representative of 7 independent experiments . ( B ) Mean calculated area under the luminescence curves ( AUC ) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments .

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A, B. MDCK-SIAT1 cells co-incubated with Luc-WSN/33 and MH-S CM fractions. Replication was quantified by luminescence. (A) Replication curves display average luminescence reads from 5 wells per condition (measured at 7.5 min intervals - displayed here at 30 min intervals) from 1 experiment representative of 7 independent experiments. (B) Mean calculated area under the luminescence curves (AUC) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments.


C . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with flow cytometry - quantified MH - S AM - EVs at specified EV : cell ratios .

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C. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with flow cytometry-quantified MH-S AM-EVs at specified EV:cell ratios.


D . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with CM fractions of J2 retrovirus - immortalized primary mouse AMs .

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D. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with CM fractions of J2 retrovirus-immortalized primary mouse AMs.


E . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with primary rat AM - derived EVs .

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E. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with primary rat AM-derived EVs.


F . Luc - WSN / 33 replication in MDCK - SIAT1 cells incubated with human THP - 1 macrophage - derived EVs .

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F. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with human THP-1 macrophage-derived EVs.


A , B . Luc - WSN / 33 replication co - incubated with MH - S EVs was assessed in ( A ) human A549 and ( B ) mouse MLE - 12 AECs . Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments .

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A, B. Luc-WSN/33 replication co-incubated with MH-S EVs was assessed in (A) human A549 and (B) mouse MLE-12 AECs. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments.


C . MLE - 12 AECs incubated with MH - S EVs infected with PR / 8 influenza . Replication was quantified by RT - qPCR at 12 h post infection . Data represent mean M gene transcripts normalized to β actin from 3 independent experiments .

C. MLE-12 AECs incubated with MH-S EVs infected with PR/8 influenza. Replication was quantified by RT-qPCR at 12 h post infection. Data represent mean M gene transcripts normalized to β actin from 3 independent experiments.


E Total lung RNA was isolated from mice on day 1 post infection and virus was quantified by RT - qPCR . Data represent mean M gene transcripts from individual mice normalized to β actin from 3 independent experiments ( 4 - 5 mice per group ) .

E Total lung RNA was isolated from mice on day 1 post infection and virus was quantified by RT-qPCR. Data represent mean M gene transcripts from individual mice normalized to β actin from 3 independent experiments (4-5 mice per group).


A . EVs isolated from MH - S AMs and A549 AECs were co - incubated in parallel with Luc - WSN / 33 ( MOI = 0 . 5 ) in MDCK - SIAT1 cells . Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments .

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A. EVs isolated from MH-S AMs and A549 AECs were co-incubated in parallel with Luc-WSN/33 (MOI = 0.5) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments.


B . EVs isolated from MH - S cells treated with increasing concentrations of CSE were co - incubated with Luc - WSN / 33 in MLE - 12 cells . Data represent mean luminescence at 12 h post infection from individual wells normalized to the mean of untreated AM - EVs from 3 independent experiments per condition .

B. EVs isolated from MH-S cells treated with increasing concentrations of CSE were co-incubated with Luc-WSN/33 in MLE-12 cells. Data represent mean luminescence at 12 h post infection from individual wells normalized to the mean of untreated AM-EVs from 3 independent experiments per condition.


A . MLE - 12 AECs co - incubated with the listed WT strains ( MOI = 0 . 4 ) plus AM - EVs ( grey bars , EV : cell = 1 ) . Data represent mean M gene transcripts at 12 h post infection relative to the mean of the corresponding untreated strain ( white bars ) , each normalized to β actin from 2 - 4 independent experiments per strain .

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A. MLE-12 AECs co-incubated with the listed WT strains (MOI = 0.4) plus AM-EVs (grey bars, EV:cell = 1). Data represent mean M gene transcripts at 12 h post infection relative to the mean of the corresponding untreated strain (white bars), each normalized to β actin from 2-4 independent experiments per strain.


C . MHS EVs were co - incubated with the listed Luc - expressing strains plus AM - EVs ( grey bars , EV : cell = 2 ) in MDCK - SIAT1 cells . Data represent mean luminescence AUC from individual wells normalized to the means of the corresponding untreated strain ( white bars ) from 3 independent experiments per strain .

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C. MHS EVs were co-incubated with the listed Luc-expressing strains plus AM-EVs (grey bars, EV:cell = 2) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the means of the corresponding untreated strain (white bars) from 3 independent experiments per strain.


A . MDCK - SIAT1 cells were pretreated with MH - S EVs ( EV : cell = 2 , ' EV pretreat ' ) or control for 8 h , washed , and subsequently incubated with Luc - WSN / 33 ( MOI = 1 ) . Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments .

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A. MDCK-SIAT1 cells were pretreated with MH-S EVs (EV:cell = 2, 'EV pretreat') or control for 8 h, washed, and subsequently incubated with Luc-WSN/33 (MOI = 1). Data represent mean luminescence AUC from individual wells normalized to the means of the control condition from 3 independent experiments.


MLE - 12 cells were incubated with WT PR / 8 + AM - EVs at 4 ° C ( to permit surface binding but not entry ) for 1 h , washed , and processed for ( B ) RNA isolation ( B ) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition , each normalized to β actin from 3 independent experiments .

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MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (B) RNA isolation (B) Data represent mean M gene transcripts relative to the means of the corresponding untreated condition, each normalized to β actin from 3 independent experiments.


C . MLE - 12 cells were incubated with WT PR / 8 + AM - EVs at 4 ° C ( to permit surface binding but not entry ) for 1 h , washed , and processed for ( C ) immunofluorescence microscopy . ( C ) Cells in both conditions were fixed and stained with anti - NP or isotype control Ab . Images are representative of two independent experiments . Scale bars = 50

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C. MLE-12 cells were incubated with WT PR/8 + AM-EVs at 4 °C (to permit surface binding but not entry) for 1 h, washed, and processed for (C) immunofluorescence microscopy. (C) Cells in both conditions were fixed and stained with anti-NP or isotype control Ab. Images are representative of two independent experiments. Scale bars = 50 μm


D . A549 cells were incubated with WSN / 33 or HK / 14 + AM - EVs for 40 min , washed , fixed , and permeabilized . Cells were subsequently stained with Anti - NP ( green ) , Anti - Rab - 7 ( purple ) Abs , and DAPI ( blue ) . Images shown are representative of 8 independent experiments . Scale bars = 5

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D. A549 cells were incubated with WSN/33 or HK/14 + AM-EVs for 40 min, washed, fixed, and permeabilized. Cells were subsequently stained with Anti-NP (green), Anti-Rab-7 (purple) Abs, and DAPI (blue). Images shown are representative of 8 independent experiments. Scale bars = 5 μm


E , F . Co - localization analysis of ( E ) NP : DAPI and ( F ) NP : Rab7 for each condition was performed on Z - stack confocal images images of WSN / 33 ( black ) or HK / 14 ( red ) infected A549s . Data represent the mean NP : DAPI co - localization ratios and the mean NP : Rab7 Mander ' s co - localization coefficient for individual cells from these 8 experiments .

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E, F. Co-localization analysis of (E) NP:DAPI and (F) NP:Rab7 for each condition was performed on Z-stack confocal images images of WSN/33 (black) or HK/14 (red) infected A549s. Data represent the mean NP:DAPI co-localization ratios and the mean NP:Rab7 Mander's co-localization coefficient for individual cells from these 8 experiments.


A - D . Replication assessed by luminescence following 10 nM Baf - A1 addition at the indicated times following synchronized uptake of bound Luc - expressing virions . ( A , B ) Shown are examples from 1 experiment representative of a total of 3 - 11 independent experiments per strain . Data represent mean luminescence AUC normalized to the mean of the corresponding strain incubated in 0 . 1 % DMSO ( dashed line ) . ( C , D ) The kinetics of endosomal escape by Luc - WSN / 33 and HK / 14 strains are depicted by the loss of inhibition by high dose Baf - A1 between ( C ) 0 - 20 min and ( D ) 60 - 80 min . Data represent mean slope ( change in % inhibition / time interval ) from 5 wells per plate from 3 - 11 independent experiments .

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A-D. Replication assessed by luminescence following 10 nM Baf-A1 addition at the indicated times following synchronized uptake of bound Luc-expressing virions. (A, B) Shown are examples from 1 experiment representative of a total of 3-11 independent experiments per strain. Data represent mean luminescence AUC normalized to the mean of the corresponding strain incubated in 0.1% DMSO (dashed line). (C, D) The kinetics of endosomal escape by Luc-WSN/33 and HK/14 strains are depicted by the loss of inhibition by high dose Baf-A1 between (C) 0-20 min and (D) 60-80 min. Data represent mean slope (change in % inhibition/time interval) from 5 wells per plate from 3-11 independent experiments.


Shown are dose response curves ( left ) and corresponding IC50 values ( right ) for effects of the endosomal acidification inhibitors Baf - A1 on replication of Luc - expressing strains . Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3 - 5 independent experiments ( mean of 3 wells per condition ) for each strain and inhibitor concentration . Bar graphs depict mean IC50 values from these experiments .

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Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Baf-A1 on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.


F . Shown are dose response curves ( left ) and corresponding IC50 values ( right ) for effects of the endosomal acidification inhibitors Chl on replication of Luc - expressing strains . Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3 - 5 independent experiments ( mean of 3 wells per condition ) for each strain and inhibitor concentration . Bar graphs depict mean IC50 values from these experiments .

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F. Shown are dose response curves (left) and corresponding IC50 values (right) for effects of the endosomal acidification inhibitors Chl on replication of Luc-expressing strains. Curves display mean luminescence AUC expressed as a percentage of replication of corresponding strains in control conditions from 3-5 independent experiments (mean of 3 wells per condition) for each strain and inhibitor concentration. Bar graphs depict mean IC50 values from these experiments.


A . Data are mean 525em ratio from 488 / 445 nm excitation intensities of internalized FDx particles vs . pH of clamping buffer fit to a standard curve from two independent experiments .

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A. Data are mean 525em ratio from 488/445 nm excitation intensities of internalized FDx particles vs. pH of clamping buffer fit to a standard curve from two independent experiments.


B . Representative images of PBS - and DR - EV - treated MDCK - SIAT1 cells . Scale bars for full size images = 50 μm . Scale bars for cropped images = 10 μm . FDx particles are shown green and DR - EV particles are shown purple . White arrows are FDx endosomes containing DR - EVs .

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B. Representative images of PBS- and DR-EV-treated MDCK-SIAT1 cells. Scale bars for full size images = 50 μm. Scale bars for cropped images = 10 μm. FDx particles are shown green and DR-EV particles are shown purple. White arrows are FDx endosomes containing DR-EVs.