Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with Flag - STING ( C ) The data shown are representative of n = 3 biological replicates .

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Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with Flag-STING (C) The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) mouse peritoneal macrophages ( D ) The data shown are representative of n = 3 biological replicates .

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

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) mouse peritoneal macrophages (D) The data shown are representative of n=3 biological replicates.


Immunoblotting of the streptavidin precipitates of the mixture of biotin ( 100 μM ) or biotin - Palbociclib ( 100 μM ) THP1 cells ( E ) . The data shown are representative of n = 3 biological replicates .

Immunoblotting of the streptavidin precipitates of the mixture of biotin (100 μM) or biotin-Palbociclib (100 μM) THP1 cells (E). The data shown are representative of n=3 biological replicates.


( F ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - c - diAMP ( 2 μM ) and recombinant SUNO - tagged STING protein ( 10 μg / mL ) in the presence of DMSO and increasing concentrations of Palbociclib ( 0 , 1 , 10 μM ) . The data shown are representative of n = 3 biological replicates .

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(F) Immunoblotting of the streptavidin precipitates of the mixture of biotin-c-diAMP (2 μM) and recombinant SUNO-tagged STING protein (10 μg/mL) in the presence of DMSO and increasing concentrations of Palbociclib (0, 1, 10 μM). The data shown are representative of n=3 biological replicates.


( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - STING in the presence of DMSO and indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates .

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 B-CONTROLLED_VAR 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

(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-STING in the presence of DMSO and indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.


Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( H ) . Scale bar , 25 μm .

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

Representative immunofluorescent assay of STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (H). Scale bar, 25 μm.


STING and ERGIC in mouse peritoneal macrophages stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( I ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * * , P < 0 . 0001 .

B-MEASURED_VAR 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 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

STING and ERGIC in mouse peritoneal macrophages stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (I). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ****, P<0.0001.


( J ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STEEP and HA - STING in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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(J) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STEEP and HA-STING in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( K ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with Flag - STING and HA - TBK1 in the presence of DMSO or indicated concentrations of Palbociclib . The data shown are representative of n = 3 biological replicates .

(K) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with Flag-STING and HA-TBK1 in the presence of DMSO or indicated concentrations of Palbociclib. The data shown are representative of n=3 biological replicates.


( C ) Immunoblotting of the streptavidin precipitates of the mixture of biotin - Palbociclib ( 100 μM ) and the lysates of HEK293T cells transfected with corresponding Flag - STING mutants . The data shown are representative of n = 3 biological replicates .

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

(C) Immunoblotting of the streptavidin precipitates of the mixture of biotin-Palbociclib (100 μM) and the lysates of HEK293T cells transfected with corresponding Flag-STING mutants. The data shown are representative of n=3 biological replicates.


( D ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING or Flag - STING Y167A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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

(D) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING or Flag-STING Y167A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) ( E ) . Scale bars , 25 μm .

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

Representative immunofluorescent assay of STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) (E). Scale bars, 25 μm.


STING and ERGIC in HeLa cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 2 h in the presence of DMSO or Palbociclib ( 10 μM ) The quantification data is shown in ( F ) . The data shown are mean ± SD from indicated number of cells from 1 representative of n = 3 biological replicates . Statistical significance was determined by two - tailed Student ' s t - test . * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

B-MEASURED_VAR 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 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

STING and ERGIC in HeLa cells stimulated with 2'3' cGAMP (0.1μg/mL) for 2 h in the presence of DMSO or Palbociclib (10 μM) The quantification data is shown in (F). The data shown are mean ± SD from indicated number of cells from 1 representative of n=3 biological replicates. Statistical significance was determined by two-tailed Student's t-test. ***, P<0.001; ****, P<0.0001; ns, not significant.


( G ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - TBK1 and Flag - STING or Flag - STING Y167A . The data shown are representative of n = 3 biological replicates .

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

(G) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-TBK1 and Flag-STING or Flag-STING Y167A. The data shown are representative of n=3 biological replicates.


( H ) Immunoblotting of the lysates and anti - Flag immunoprecipitates of HEK293T cells transfected with HA - STING and Flag - STING C91A in the presence of DMSO and Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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(H) Immunoblotting of the lysates and anti-Flag immunoprecipitates of HEK293T cells transfected with HA-STING and Flag-STING C91A in the presence of DMSO and Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( B ) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; ns , not significant .

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(B) The weight of mice left untreated or treated with DSS in the absence or presence of H151 or Palbociclib. Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; ns, not significant.


The representative image of colon tissue harvested from mice of indicated groups is shown in ( C ) .

The representative image of colon tissue harvested from mice of indicated groups is shown in (C).


The length of colon is analyzed in ( D ) . Statistical significance between data sets was assessed by one - way ANOVA followed by Tukey ' s multiple comparisons post - hoc test between all groups . Values are means ± SEM , n = 5 mice per group with differences denoted by * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 length of colon is analyzed in (D). Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n = 5 mice per group with differences denoted by *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( E - F ) Representative H & E staining of the histological changes in colon tissues harvested from mice of indicated groups ( E ) . Scale bar , 200 μm . The quantification of histological score was shown in ( F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative H&E staining of the histological changes in colon tissues harvested from mice of indicated groups (E). Scale bar, 200 μm. The quantification of histological score was shown in (F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Il6 ( H ) and Il1b ( I ) in mouse colon tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

(G-I) qRT-PCR measurement of transcripts of Ifnb1 (G), Il6 (H) and Il1b (I) in mouse colon tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ****, P<0.0001; ns, not significant.


( B - F ) Representative H & E staining of the histological changes in heart , stomach , tongue and thigh muscle tissues harvested from mice of indicated groups ( B ) . Scale bar , 200 μm . The quantification of histological score was shown in ( C - F ) . Mann - Whitney U - test was used for statistical analysis of two independent groups . Values represent means ± SEM , each symbol indicates the value of 1 mouse from n = 5 mice per group . * , P < 0 . 05 ; * * , P < 0 . 01 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative H&E staining of the histological changes in heart, stomach, tongue and thigh muscle tissues harvested from mice of indicated groups (B). Scale bar, 200 μm. The quantification of histological score was shown in (C-F). Mann-Whitney U-test was used for statistical analysis of two independent groups. Values represent means ± SEM, each symbol indicates the value of 1 mouse from n = 5 mice per group. *, P<0.05; **, P<0.01; ns, not significant.


( G - I ) qRT - PCR measurement of transcripts of Ifnb1 ( G ) , Cxcl10 ( H ) and Il6 ( I ) in mouse stomach tissues harvested from mice of indicated groups . The data shown are mean ± SEM of n = 5 mice from 1 of 3 biological replicates with similar results . Each symbol indicates the value of 1 mouse from n = 5 mice per group . Statistical significance was determined by one - way ANOVA test followed by Tukey ' s multiple comparisons post - hoc test between all groups . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ; ns , not significant .

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

(G-I) qRT-PCR measurement of transcripts of Ifnb1(G), Cxcl10 (H) and Il6 (I) in mouse stomach tissues harvested from mice of indicated groups. The data shown are mean ± SEM of n=5 mice from 1 of 3 biological replicates with similar results. Each symbol indicates the value of 1 mouse from n = 5 mice per group. Statistical significance was determined by one-way ANOVA test followed by Tukey's multiple comparisons post-hoc test between all groups. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.


( A ) Heatmap showing viability measurements for the 192 samples ( rows ) subject to each of 17 microenvironmental stimuli ( columns ) . The data are shown on a robust z - score scale , i . e . , the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row . Limits were applied to scaling factor for optimal visualisation . Red indicates increased viability , blue indicates decreased viability . Samples are annotated for genetic features , sex and pre - treatment status .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heatmap showing viability measurements for the 192 samples (rows) subject to each of 17 microenvironmental stimuli (columns). The data are shown on a robust z-score scale, i.e., the logarithm of the relative viability measurements were scaled by the median absolute deviation within each row. Limits were applied to scaling factor for optimal visualisation. Red indicates increased viability, blue indicates decreased viability. Samples are annotated for genetic features, sex and pre-treatment status.


Logarithm of the relative viability after treatment with Resiquimod ( A , n = 169 ) , IL4 ( B , n = 174 ) stratified by trisomy 12 , and in A , also by IGHV status . P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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

Logarithm of the relative viability after treatment with Resiquimod (A, n=169), IL4 (B, n=174) stratified by trisomy 12, and in A, also by IGHV status. P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Logarithm of the relative viability after treatment with TGFβ ( C , n = 174 ) stratified by trisomy 12 , P - values from Student ' s t - test ( two - sided , non - paired ) . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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

Logarithm of the relative viability after treatment with TGFβ (C, n=174) stratified by trisomy 12, P-values from Student's t-test (two-sided, non-paired). The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


( A ) Graphical representation of the four drug - stimulus interaction categories . Categories are defined according to the nature of the interaction ( synergistic or antagonistic ) , and whether the viability is increased or decreased by the stimulus ( positive or negative ) . x - axis shows treatment type , y - axis shows viability with each treatment . Red and blue points and lines depict a representative treatment response pattern for given interaction type . Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug - stimulus ( i . e . additive effects ) , black horizontal lines represent the measured viability after combinatorial treatment . The difference between the black and blue lines indicates the impact of the interaction on expected viability .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graphical representation of the four drug-stimulus interaction categories. Categories are defined according to the nature of the interaction (synergistic or antagonistic), and whether the viability is increased or decreased by the stimulus (positive or negative). x-axis shows treatment type, y-axis shows viability with each treatment. Red and blue points and lines depict a representative treatment response pattern for given interaction type. Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug-stimulus (i.e. additive effects), black horizontal lines represent the measured viability after combinatorial treatment. The difference between the black and blue lines indicates the impact of the interaction on expected viability.


( D - I ) Examples of drug - stimulus interactions , for each category . Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus . Each line represents one patient sample linked across treatments . Black horizontal lines in each single treatment indicate the expected viability based on linear modelling . Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus ( blue ) , and the expected viability accounting for the additive effect and the interaction ( black ) . ( D + E ) Ibrutinib , a clinically used BTK inhibitor , is blocked by IL4 and IFNγ . ( F ) The JAK inhibitor pyridone - 6 inhibits the pro - survival effect of sCD40L + IL4 stimulation . ( G ) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro - survival effect not observed in either single treatment . ( H ) TLR agonists , including CpG ODN ( shown ) increase sensitivity to BTK inhibition by ibrutinib , despite increasing viability as single treatments . ( I ) Soluble anti - IgM sensitises CLL samples to HSP90 inhibition by luminespib .

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

(D - I) Examples of drug-stimulus interactions, for each category. Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus. Each line represents one patient sample linked across treatments. Black horizontal lines in each single treatment indicate the expected viability based on linear modelling. Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus (blue), and the expected viability accounting for the additive effect and the interaction (black). (D+E) Ibrutinib, a clinically used BTK inhibitor, is blocked by IL4 and IFNγ. (F) The JAK inhibitor pyridone-6 inhibits the pro-survival effect of sCD40L + IL4 stimulation. (G) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro-survival effect not observed in either single treatment. (H) TLR agonists, including CpG ODN (shown) increase sensitivity to BTK inhibition by ibrutinib, despite increasing viability as single treatments. (I) Soluble anti-IgM sensitises CLL samples to HSP90 inhibition by luminespib.


Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( C ) fludarabine + CpG ODN and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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

Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (C) fludarabine + CpG ODN and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Beeswarm - boxplots of the natural logarithm of the relative viability of 169 CLL samples , for ( D ) ibrutinib + IL4 single and combinatorial treatments , faceted by IGHV status and trisomy 12 status . P - values from paired Student ' s t - test . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

Beeswarm-boxplots of the natural logarithm of the relative viability of 169 CLL samples, for (D) ibrutinib + IL4 single and combinatorial treatments, faceted by IGHV status and trisomy 12 status. P-values from paired Student's t-test . The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


( A Mean ( A ) pSTAT6 staining intensity in CLL - infiltrated ( n = 100 for pSTAT and tumour - free lymph node biopsies ( n = 98 for pSTAT6 after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

(A Mean (A) pSTAT6 staining intensity in CLL-infiltrated (n=100 for pSTAT and tumour-free lymph node biopsies (n=98 for pSTAT6 after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


B ) Mean ( B ) pIRAK4 staining intensity in CLL - infiltrated ( n = 100 for pIRAK4 ) and tumour - free lymph node biopsies n = 100 for pIRAK4 ) after background subtraction ( y axis ) , p - values from Student ' s t - test . Each dot represents the mean of all cells in the tissue microarray cores per patient sample . The central bar , boxes and whiskers of the plot represent the median , first and third quartiles , and 1 . 5 - times IQR , respectively .

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B) Mean (B) pIRAK4 staining intensity in CLL-infiltrated (n=100 for pIRAK4) and tumour-free lymph node biopsies n=100 for pIRAK4) after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.


Example images of immunohistochemistry sections : ( C + D ) pSTAT6 levels in ( C ) CLL - infiltrated and ( D ) tumour - free samples . Scale bar defines 20µm in all images .

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Example images of immunohistochemistry sections: (C + D) pSTAT6 levels in (C) CLL-infiltrated and (D) tumour-free samples. Scale bar defines 20µm in all images.


Example images of immunohistochemistry sections : ( E + F ) pIRAK4 levels in ( E ) CLL - infiltrated and ( F ) tumour - free samples . Scale bar defines 20µm in all images .

Example images of immunohistochemistry sections: (E + F) pIRAK4 levels in (E) CLL-infiltrated and (F) tumour-free samples. Scale bar defines 20µm in all images.


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 .

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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 ) .

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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.


C . Measured pH of FDx endosomes in PBS - ( left bar ) and DR - EV - treated ( right bar ) MDCK - SIAT1 cells . The pH of FDx endosomes containing DR - EVs in DR - EV - treated cells ( middle bar ) ( corresponding to white arrows in Fig 7B ) . Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments .

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C. Measured pH of FDx endosomes in PBS- (left bar) and DR-EV-treated (right bar) MDCK-SIAT1 cells. The pH of FDx endosomes containing DR-EVs in DR-EV-treated cells (middle bar) (corresponding to white arrows in Fig 7B). Data are the means of individual data points representing the average calculated pH of each endosome population within 1 hpf in each of 5 independent experiments.


( A ) Dually labeled D . discoideum amoebae producing MCS components fused to GFP , and either calnexin ( CnxA ) - mCherry ( pAW012 ) , P4C - mCherry ( pWS032 ) , or AmtA - mCherry were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) , fixed with 4 % PFA , and imaged by confocal fluorescence microscopy . Merged images are shown . Scale bars : 3 μm ( insets : 1 μm ) .

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(A) Dually labeled D. discoideum amoebae producing MCS components fused to GFP, and either calnexin (CnxA)-mCherry (pAW012), P4C-mCherry (pWS032), or AmtA-mCherry were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed with 4 % PFA, and imaged by confocal fluorescence microscopy. Merged images are shown. Scale bars: 3 μm (insets: 1 μm).


( B - D ) The Pearson ' s correlation coefficients were generated using Coloc 2 from Fiji ( ImageJ ) and are shown for MCS components fused to GFP with respect to ( B ) CnxA - mCherry ( n = 146 ) , ( C ) P4C - mCherry ( n = 138 ) , or ( D ) AmtA - mCherry ( n = 159 ) . Data represent mean and SEM of three independent biological replicates ( statistics refer to JR32 - infected D . discoideum producing GFP - Sac1 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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(B-D) The Pearson's correlation coefficients were generated using Coloc 2 from Fiji (ImageJ) and are shown for MCS components fused to GFP with respect to (B) CnxA-mCherry (n=146), (C) P4C-mCherry (n=138), or (D) AmtA-mCherry (n=159). Data represent mean and SEM of three independent biological replicates (statistics refer to JR32-infected D. discoideum producing GFP-Sac1; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing mCherry - Vap ( pSV048 ) and OSBP8 - GFP ( pMIB89 ) or GFP - OSBP11 ( pMIB39 ) were infected ( MOI 5 , 1 - 8 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm ( insets : 1 μm ) .

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(A) Dually labeled D. discoideum Ax3 producing mCherry-Vap (pSV048) and OSBP8-GFP (pMIB89) or GFP-OSBP11 (pMIB39) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm (insets: 1 μm).


( B ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry - Vap ( n = 100 - 150 each per time point ) . Data represent mean and SEM of three independent biological replicates ( * * P < 0 . 01 , Student ` s t - test ) .

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(B) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for OSBP8 or OSBP11 fused to GFP with respect to mCherry-Vap (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (**P<0.01, Student`s t-test).


( A ) D . discoideum Ax3 , or the ∆ vap , ∆ osbG , ∆ osbH , or ∆ osbK mutant strains producing GFP ( pDM317 ) or GFP fusions of the MCS components were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) . To improve clarity , the different mutant strains are shown in separate graphs , each depicting the same data for Ax3 / GFP .

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(A) D. discoideum Ax3, or the ∆vap, ∆osbG, ∆osbH, or ∆osbK mutant strains producing GFP (pDM317) or GFP fusions of the MCS components were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP; *P<0.05; **P<0.01; ***P<0.001, Student`s t-test). To improve clarity, the different mutant strains are shown in separate graphs, each depicting the same data for Ax3/GFP.


( B ) D . discoideum Ax3 producing CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 1 ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to JR32 infected D . discoideum Ax3 producing GFP - Sac1 ; * * * P < 0 . 001 , Student ` s t - test ) .

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(B) D. discoideum Ax3 producing CnxA-GFP (pAW016), GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 1) with mCherry-producing L. pneumophila JR32 (pNP102), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to JR32 infected D. discoideum Ax3 producing GFP-Sac1; ***P<0.001, Student`s t-test).


( C ) RAW 264 . 7 macrophages were treated with increasing concentrations of OSW - 1 ( 0 - 100 nM , 1 - 60 h ) , infected ( MOI 1 ) with GFP - producing L . pneumophila JR32 ( pNT28 ) , and intracellular replication was assessed by relative fluorescence units ( RFU ) . Mean and SEM of three independent biological replicates are shown ( statistics refer to infected cells treated with 0 nM OSW - 1 ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(C) RAW 264.7 macrophages were treated with increasing concentrations of OSW-1 (0-100 nM, 1-60 h), infected (MOI 1) with GFP-producing L. pneumophila JR32 (pNT28), and intracellular replication was assessed by relative fluorescence units (RFU). Mean and SEM of three independent biological replicates are shown (statistics refer to infected cells treated with 0 nM OSW-1; *P<0.05; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and CnxA - GFP ( pAW016 ) , or Ax3 producing P4C - mCherry and either GFP - Sac1 ( pLS037 ) , GFP - Sac1 _ C383S ( pSV015 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 2 - 16 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B ) LCV area was measured using ImageJ ( n = 100 - 200 per condition from 3 independent biological replicates ) . Means and SEM of single cells are shown ( * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and CnxA-GFP (pAW016), or Ax3 producing P4C-mCherry and either GFP-Sac1 (pLS037), GFP-Sac1_C383S (pSV015) or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B) LCV area was measured using ImageJ (n=100-200 per condition from 3 independent biological replicates). Means and SEM of single cells are shown (**P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and GFP - Sac1 ( pLS037 ) were infected ( MOI 5 , 2 - 16 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B , C ) Imaging flow cytometry ( IFC ) analysis of dually labeled D . discoideum Ax3 , ∆ vap , ∆ osbG , ∆ osbH , ∆ osbK or ∆ sey1 mutants producing P4C - mCherry ( pWS032 ) and either GFP - Sac1 ( pLS037 ) or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Data information ( B , C , : Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(A) Dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and GFP-Sac1 (pLS037) were infected (MOI 5, 2-16 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B, C) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3, ∆vap, ∆osbG, ∆osbH, ∆osbK or ∆sey1 mutants producing P4C-mCherry (pWS032) and either GFP-Sac1 (pLS037) or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Data information (B, C, : Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( D ) Dually labeled D . discoideum Ax3 producing GFP fusions of MCS components and either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) were infected ( MOI 5 , 2 h ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( E ) IFC analysis of dually labeled D . discoideum Ax3 producing GFP fusions of MCS components and either mCherry - Sac1 ( pSV044 ) or mCherry - Sac1 _ ΔTMD ( pSV045 ) , infected ( MOI 5 , 2 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Quantification of ( B ) P4C - mCherry ( C ) Sac1 - GFP or ( E ) GFP fusions of MCS components localizing to LCVs at 2 h p . i . Due to the lower resolution of IFC , " LCVs " designates the LCV limiting membrane and tightly attached ER . Number of events per sample , n = 5000 . Data information E ) : Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(D) Dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045) were infected (MOI 5, 2 h) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) IFC analysis of dually labeled D. discoideum Ax3 producing GFP fusions of MCS components and either mCherry-Sac1 (pSV044) or mCherry-Sac1_ΔTMD (pSV045), infected (MOI 5, 2 h) with mPlum-producing L. pneumophila JR32 (pAW014). Quantification of (B) P4C-mCherry (C) Sac1-GFP or (E) GFP fusions of MCS components localizing to LCVs at 2 h p.i. Due to the lower resolution of IFC, "LCVs" designates the LCV limiting membrane and tightly attached ER. Number of events per sample, n = 5000. Data information E): Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing GFP - D4H * ( pSV046 ) or P4C - GFP ( pWS034 ) and P4C - mCherry ( pWS032 ) , CnxA - mCherry ( pAW012 ) , or AmtA - mCherry were infected ( MOI 5 , 15 - 120 min ) with mCerulean - producing L . pneumophila JR32 ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( B - C ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for ( B ) AmtA - mCherry with respect to P4C - GFP or GFP - D4H * ( n = 100 - 150 per time point ) , and ( C ) GFP - D4H * with respect to P4C - mCherry or CnxA - mCherry ( n = 100 - 150 each per time point ) . Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 , Student ` s t - test ) .

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

(A) Dually labeled D. discoideum Ax3 producing GFP-D4H* (pSV046) or P4C-GFP (pWS034) and P4C-mCherry (pWS032), CnxA-mCherry (pAW012), or AmtA-mCherry were infected (MOI 5, 15 - 120 min) with mCerulean-producing L. pneumophila JR32 (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (B-C) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for (B) AmtA-mCherry with respect to P4C-GFP or GFP-D4H* (n=100-150 per time point), and (C) GFP-D4H* with respect to P4C-mCherry or CnxA-mCherry (n=100-150 each per time point). Data represent mean and SEM of three independent biological replicates (*P<0.05, Student`s t-test).


( D ) Single labeled D . discoideum Ax3 producing either CnxA - GFP ( pAW016 ) or P4C - GFP ( pWS034 ) were infected ( MOI 5 , 15 - 120 min ) with mCherry - producing L . pneumophila JR32 ( pNP102 ) , fixed with 4 % PFA and stained with filipin . Merged images for the analyzed time points are shown . Scale bars : 3 μm . ( E ) The Pearson ' s correlation coefficient was generated using Coloc 2 from Fiji ( ImageJ ) and is shown for filipin with respect to CnxA - GFP or P4C - GFP ( n = 80 - 90 each per time point ) . Data represent mean and SEM of the means of three independent biological replicates ( * * * P < 0 . 001 , Student ` s t - test ) .

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

(D) Single labeled D. discoideum Ax3 producing either CnxA-GFP (pAW016) or P4C-GFP (pWS034) were infected (MOI 5, 15-120 min) with mCherry-producing L. pneumophila JR32 (pNP102), fixed with 4 % PFA and stained with filipin. Merged images for the analyzed time points are shown. Scale bars: 3 μm. (E) The Pearson's correlation coefficient was generated using Coloc 2 from Fiji (ImageJ) and is shown for filipin with respect to CnxA-GFP or P4C-GFP (n=80-90 each per time point). Data represent mean and SEM of the means of three independent biological replicates (***P<0.001, Student`s t-test).


( A ) Dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) and either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) were infected ( MOI 5 , 1 - 8 h ) with mCerulean - producing L . pneumophila JR32 , ΔlepB or ΔsidC ( pNP99 ) and fixed with 4 % PFA . Merged images for the analyzed time points are shown . Scale bars : 3 μm .

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

(A) Dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034) were infected (MOI 5, 1-8 h) with mCerulean-producing L. pneumophila JR32, ΔlepB or ΔsidC (pNP99) and fixed with 4 % PFA. Merged images for the analyzed time points are shown. Scale bars: 3 μm.


( C ) Imaging flow cytometry ( IFC ) analysis of dually labeled D . discoideum Ax3 producing P4C - mCherry ( pWS032 ) and either CnxA - GFP ( pAW016 ) , GFP - Sac1 ( pLS037 ) , or GFP - Sac1 _ ΔTMD ( pSV034 ) , infected ( MOI 5 , 1 - 8 h ) with mPlum - producing L . pneumophila JR32 ( pAW014 ) . Number of events per sample , n = 5000 . Data represent mean and SEM of three independent biological replicates ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ` s t - test ) .

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

(C) Imaging flow cytometry (IFC) analysis of dually labeled D. discoideum Ax3 producing P4C-mCherry (pWS032) and either CnxA-GFP (pAW016), GFP-Sac1 (pLS037), or GFP-Sac1_ΔTMD (pSV034), infected (MOI 5, 1-8 h) with mPlum-producing L. pneumophila JR32 (pAW014). Number of events per sample, n = 5000. Data represent mean and SEM of three independent biological replicates (*P<0.05; **P<0.01; ***P<0.001, Student`s t-test).


( E ) Representative western blot of scr or miR - 30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control . Densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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

(E) Representative western blot of scr or miR-30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( F ) Representative western blot of scr or miR - 29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control . Densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

(F) Representative western blot of scr or miR-29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control . Densitometry analysis of n = 3 western blots . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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

(C) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control. Densitometry analysis of n=3 western blots. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction . GAPDH was used a loading control ( n = 3 ) . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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

(B) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction. GAPDH was used a loading control (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( D ) Representative western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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

(D) Representative western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( F ) Representative western blot of DLL4 levels in scr or miR - 30c transfected TREx cells at 0 , 24 and 48 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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

(F) Representative western blot of DLL4 levels in scr or miR-30c transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3 Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( J ) Representative western blot of DLL4 levels in scr or miR - 30c antagomiR transfected TREx cells at 0 , 24 and 48 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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

(J) Representative western blot of DLL4 levels in scr or miR-30c antagomiR transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control , densitometry analysis is n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

O O O O O O O B-MEASURED_VAR 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

(B) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control, densitometry analysis is n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 levels at 0 , 24 and 48 hours post induction in scr and DLL4 KD TREx cells , GAPDH was used as a loading control and densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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

(C) Representative western blot of ORF65 levels at 0, 24 and 48 hours post induction in scr and DLL4 KD TREx cells, GAPDH was used as a loading control and densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


B ChIP gel images showing the relative enrichment by H3K4me3 in controls ( n = 9 ) and PD patients ( n = 18 ) . PCR amplified a 188 - bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum . Mouse IgG ( mIgG ) was used as control and the bands were normalized by unbiased amplification from respective inputs . Data information : Data represent mean ± standard error of the mean .

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 B-MEASURED_VAR 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

B ChIP gel images showing the relative enrichment by H3K4me3 in controls (n=9) and PD patients (n= 18). PCR amplified a 188-bp region of SNCA from intron 1 where H3K4me3 peak was at its optimum. Mouse IgG (mIgG) was used as control and the bands were normalized by unbiased amplification from respective inputs. Data information: Data represent mean ± standard error of the mean.


C The normalized levels of α - synuclein in the entire cohort were divided into high ( n = 12 ) and low levels . The cut off value for determining the threshold was set at the median from all the subjects . The high levels of α - synuclein were plotted with corresponding H3K4me3 values of those subjects . The corresponding ID of each sample is shown next to each point in the graph . There was a significant correlation between high levels of α - synuclein and H3K4me3 . Data information : Data represent mean ± standard error of the mean . Spearman rank correlation test was performed in ( C ) . Two tailed p value was calculated with Fisher ' s exact p .

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

C The normalized levels of α-synuclein in the entire cohort were divided into high (n = 12) and low levels. The cut off value for determining the threshold was set at the median from all the subjects. The high levels of α-synuclein were plotted with corresponding H3K4me3 values of those subjects. The corresponding ID of each sample is shown next to each point in the graph. There was a significant correlation between high levels of α-synuclein and H3K4me3. Data information: Data represent mean ± standard error of the mean. Spearman rank correlation test was performed in (C). Two tailed p value was calculated with Fisher's exact p.


D The graph represents the statistically significant difference of relative H3K4me3 enrichment between PD ( n = 7 ) and controls ( n = 6 ) . Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 ( p = 0 . 01 ) compared to controls . * P < 0 . 05 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . Two - tailed p - values were calculated for all . Data information : Data represent mean ± standard error of the mean .

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 B-MEASURED_VAR 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

D The graph represents the statistically significant difference of relative H3K4me3 enrichment between PD (n=7) and controls (n=6). Neuronal nuclei from PD brain samples show significantly higher enrichment of H3K4me3 at SNCA intron 1 (p=0.01) compared to controls. *P < 0.05. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated for all. Data information: Data represent mean ± standard error of the mean.


The corresponding α - synuclein levels C ) were evaluated in SH - SY5Y cells expressing dCas9 - 5xGCN4 with ( sgA - dCas9 ) or without scFV - sfGFP - JARID1A ( sgA - dCas9 - JA ) . C α - SYN levels in SH - SY5Y cells were also evaluated using western blot . The levels of α - SYN was normalized to β - actin . Significant reduction of α - SYN was observed in cells expressing JARID1A . Three independent repeats were performed . Percentage of reduction is shown in the graph . * * P < 0 . 01 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . Two - tailed p - values were calculated . Data information : All data are presented as mean ± SEM .

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

The corresponding α-synuclein levels C) were evaluated in SH-SY5Y cells expressing dCas9-5xGCN4 with (sgA-dCas9) or without scFV-sfGFP-JARID1A (sgA-dCas9-JA). C α-SYN levels in SH-SY5Y cells were also evaluated using western blot. The levels of α-SYN was normalized to β-actin. Significant reduction of α-SYN was observed in cells expressing JARID1A. Three independent repeats were performed. Percentage of reduction is shown in the graph. **P <0.01. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. Two-tailed p-values were calculated. Data information: All data are presented as mean ± SEM.


E ChIP on H3K4me3 at the SNCA promoter between sPD1 - 1 lines after locus - specific epigenomic modulation . Differentiated cells were transiently transfected with either sgA - dCas9 5xGCN4 - scFV - JARID1A or sg A - dCas9 5xGCN4 - scFV - empty backbone vectors . A significant reduction in H3K4me3 at the SNCA promoter was observed in cells transfected with JARID1A . Data information : Three independent repeats were performed for both ChIP experiments . * P < 0 . 05 ; * * * * P < 0 . 0001 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . One - tailed p - values were calculated for ChIP

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

E ChIP on H3K4me3 at the SNCA promoter between sPD1-1 lines after locus-specific epigenomic modulation. Differentiated cells were transiently transfected with either sgA-dCas9 5xGCN4-scFV-JARID1A or sg A-dCas9 5xGCN4-scFV-empty backbone vectors. A significant reduction in H3K4me3 at the SNCA promoter was observed in cells transfected with JARID1A. Data information: Three independent repeats were performed for both ChIP experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. One-tailed p-values were calculated for ChIP analysis


F Western blot analysis of α - synuclein ( α - SYN ) levels in the cells under the same conditions as analyzed in ( E ) . A significant decrease ( 56 - 66 % ) in α - SYN levels was observed in cells transfected with JARID1A . The normalized and relative expression of α - SYN in JARID1A transfected cells are shown as a percentage of control ( transfected by empty backbone vector ) . Data information : Three independent repeats were performed for western blot experiments . * P < 0 . 05 ; * * * * P < 0 . 0001 . Data were analyzed using non - parametric t - test followed by Mann - Whitney post - hoc corrections . two - tailed p - value was calculated for western blot experiments . All data are presented as mean ± SEM .

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

F Western blot analysis of α-synuclein (α-SYN) levels in the cells under the same conditions as analyzed in (E). A significant decrease (56-66%) in α-SYN levels was observed in cells transfected with JARID1A. The normalized and relative expression of α-SYN in JARID1A transfected cells are shown as a percentage of control (transfected by empty backbone vector). Data information: Three independent repeats were performed for western blot experiments. *P < 0.05; ****P<0.0001. Data were analyzed using non-parametric t-test followed by Mann-Whitney post-hoc corrections. two-tailed p-value was calculated for western blot experiments. All data are presented as mean ± SEM.


( L - N ) B16N parental , O1C44 , and O1DN cells were subcutaneously injected in the right flank of syngeneic C57Bl6 mice revealing the presence of O1C44 ( but not B16N - WT ) cells in abdominal organs and within the peritoneal wall ( L ) . ( M ) H & E stains of the peritoneal wall revealing the presence of melanoma cells in the peritoneal wall . ( N ) Quantitative measurement of degree of invasion within and through the peritoneal wall . Data was analyzed using the Kruskal - Wallis Rank Sum test ( P < 0 . 001 ) . Within all panels , differences established through multiple comparisons are labeled as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(L-N) B16N parental, O1C44, and O1DN cells were subcutaneously injected in the right flank of syngeneic C57Bl6 mice revealing the presence of O1C44 (but not B16N-WT) cells in abdominal organs and within the peritoneal wall (L). (M) H&E stains of the peritoneal wall revealing the presence of melanoma cells in the peritoneal wall. (N) Quantitative measurement of degree of invasion within and through the peritoneal wall. Data was analyzed using the Kruskal-Wallis Rank Sum test (P < 0.001). Within all panels, differences established through multiple comparisons are labeled as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( D - F ) Confluent layers of WM983 cells treated as indicated were ' scratched ' followed by a 20 hour incubation at 37 ° C with 5 % CO2 . ( D ) Representative images of cells after being scratched after being incubated for 0 , 10 or 20 hours in the absence ( - ) or presence ( + ) of 2uM IA65 . ( E - F ) The cell - free area was quantified by ImageJ ( N ≥ 8 biological replicates ) ; data displayed are mean ± SEM . Data was analyzed by 2 - way ANOVA . In the absence of IA65 , SOCE suppression effect was p < 0 . 05 ; effect of time was p < 0 . 0001 ; interaction was p < 0 . 05 . In the presence of IA65 , SOCE suppression effect was p > 0 . 05 ; effect of time was p < 0 . 0001 ; interaction was p > 0 . 05 . Within all panels , differences established through multiple comparisons are labeled as * 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

(D-F) Confluent layers of WM983 cells treated as indicated were 'scratched' followed by a 20 hour incubation at 37°C with 5% CO2. (D) Representative images of cells after being scratched after being incubated for 0, 10 or 20 hours in the absence (-) or presence (+) of 2uM IA65. (E-F) The cell-free area was quantified by ImageJ (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed by 2-way ANOVA. In the absence of IA65, SOCE suppression effect was p<0.05; effect of time was p < 0.0001; interaction was p<0.05. In the presence of IA65, SOCE suppression effect was p>0.05; effect of time was p < 0.0001; interaction was p>0.05. Within all panels, differences established through multiple comparisons are labeled as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


WM983 and UACC257 human melanoma cells were transfected with Perfringolysin O ( PFO ) - mCherry D4H cholesterol sensor and plated on glass coverslips . Cells were exposed to UV ( 0 or 175 J / m2 ) and incubated for 0 , 2 or 5 weeks as indicated . ( H ) Representative cell images . ( I ) Fluorescence intensity from cells analyzed in ( H ) was measured and quantified using LASX analysis software ( N ≥ 8 biological replicates ) ; data displayed are mean ± SEM . Data was analyzed using 1 - way ANOVA with multiple comparisons ( WM983 , p < 0 . 001 ; UACC257 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 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 O O O O O O O O O O O O O O O O O O O O O

WM983 and UACC257 human melanoma cells were transfected with Perfringolysin O (PFO)-mCherry D4H cholesterol sensor and plated on glass coverslips. Cells were exposed to UV (0 or 175 J/m2) and incubated for 0, 2 or 5 weeks as indicated. (H) Representative cell images. (I) Fluorescence intensity from cells analyzed in (H) was measured and quantified using LASX analysis software (N≥8 biological replicates); data displayed are mean±SEM. Data was analyzed using 1-way ANOVA with multiple comparisons (WM983, p < 0.001; UACC257 p > 0.05). **p<0.01, ***p<0.001, ****p<0.0001.


O1C78 WM983 cells expressing the PFO - mCherry D4H cholesterol sensor were examined as in panels H and I ( N ≥ 8 biological replicates ) ; data displayed are mean ± SEM .

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O1C78 WM983 cells expressing the PFO-mCherry D4H cholesterol sensor were examined as in panels H and I (N≥8 biological replicates); data displayed are mean±SEM.


Patient surgical explants were received from the biosample repository of FCCC . Cells were isolated from tumors using 50 μM Medicons and the Medimachine system and grown for 3 days before experimentation to eliminate non - melanoma cells . ( A - B ) Plated control or UV irradiated ( 175J / m2 ) cells were then incubated overnight on glass coverslips prior to Fura2 loading and administration of Tg in Ca2 + - free media for 15 minutes . ( A ) Representative examples of SOCE responses in patient samples following the addition of Ca2 + ( 1 mM ) . Each trace represents 5 to 10 individual cells ; solid line represents the mean and the dotted line represents SEM . ( B ) Each dot represents SOCE in cells obtained from a different patient sample ( n = 20 ; data displayed as mean ± SEM ) . The effect of UV on SOCE was determined by one way ANOVA ( p > 0 . 05 ) . ( C ) Comparison of SOCE in cells collected from both primary tumors and metastatic lymph nodes from the same patient ( from patients analyzed in panels A , 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Patient surgical explants were received from the biosample repository of FCCC. Cells were isolated from tumors using 50 μM Medicons and the Medimachine system and grown for 3 days before experimentation to eliminate non-melanoma cells. (A-B) Plated control or UV irradiated (175J/m2) cells were then incubated overnight on glass coverslips prior to Fura2 loading and administration of Tg in Ca2+-free media for 15 minutes. (A) Representative examples of SOCE responses in patient samples following the addition of Ca2+ (1 mM). Each trace represents 5 to 10 individual cells; solid line represents the mean and the dotted line represents SEM. (B) Each dot represents SOCE in cells obtained from a different patient sample (n=20; data displayed as mean±SEM). The effect of UV on SOCE was determined by one way ANOVA (p > 0.05). (C) Comparison of SOCE in cells collected from both primary tumors and metastatic lymph nodes from the same patient (from patients analyzed in panels A, B).


( D ) SOCE in primary patient samples exhibiting NRAS ( 8 of 27 samples ) , BRAF ( 6 of 27 samples ) , TERT promoter ( 6 of 27 samples ) , NF1 ( 5 of 27 samples ) , and CDKN2A ( 5 of 27 samples ) alterations were compared to SOCE in samples that expressed WT or the unaltered gene . Data displayed as mean ± SEM .

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(D) SOCE in primary patient samples exhibiting NRAS (8 of 27 samples), BRAF (6 of 27 samples), TERT promoter (6 of 27 samples), NF1 (5 of 27 samples), and CDKN2A (5 of 27 samples) alterations were compared to SOCE in samples that expressed WT or the unaltered gene. Data displayed as mean±SEM.


( E ) Single cells from patient samples were treated with or without the indicated drug in the presence of Tg in nominally Ca2 + - free media for 15 minutes prior to the addition of Ca2 + ( 1 mM ) . The number on each bar represents the number of patient specimens tested . Data were compared against their matched control analyzed by the unpaired T test ( Tak475 , p = 0 . 015 ; compactin , p = 0 . 0049 ; IA65 , p = 0 . 0390 ) .

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(E) Single cells from patient samples were treated with or without the indicated drug in the presence of Tg in nominally Ca2+-free media for 15 minutes prior to the addition of Ca2+ (1 mM). The number on each bar represents the number of patient specimens tested. Data were compared against their matched control analyzed by the unpaired T test (Tak475, p = 0.015; compactin, p = 0.0049; IA65, p = 0.0390).


( F ) Patient cells were placed in Matrigel transwell inserts in OptiMEM reduced serum media with or without TAK475 ( 10uM ) , compactin ( 2μM ) , IA65 ( 2μM ) or OSMI1 ( 40μM ) ; DMEM with 10 % FBS was used as a chemoattractant . Crystal violet - stained invaded cells were measured 20 hrs after initiation of assay . Each dot represents 1 patient sample . Data analyzed by 2 - way ANOVA ( cholesterol inhibition was p < 0 . 0001 ; patient samples were p < 0 . 001 ; interaction was p < 0 . 01 ) . For IA65 , ( inhibition was p < 0 . 0001 ; patient samples were p < 0 . 01 ; interaction was p < 0 . 0001 ) . For OSMI1 , ( inhibition was p < 0 . 05 ; patient samples were p > 0 . 05 ; interaction was p < 0 . 05 ) . * 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

(F) Patient cells were placed in Matrigel transwell inserts in OptiMEM reduced serum media with or without TAK475 (10uM), compactin (2μM), IA65 (2μM) or OSMI1 (40μM); DMEM with 10% FBS was used as a chemoattractant. Crystal violet-stained invaded cells were measured 20 hrs after initiation of assay. Each dot represents 1 patient sample. Data analyzed by 2-way ANOVA (cholesterol inhibition was p < 0.0001; patient samples were p < 0.001; interaction was p < 0.01). For IA65, (inhibition was p < 0.0001; patient samples were p < 0.01; interaction was p < 0.0001). For OSMI1, (inhibition was p < 0.05; patient samples were p > 0.05; interaction was p < 0.05). *p<0.05, **p<0.01, ****p<0.0001.


ZO - 1 and GAPDH protein levels assessed in Epi cells by Western blot of whole protein extracts .

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ZO-1 and GAPDH protein levels assessed in Epi cells by Western blot of whole protein extracts.


ZO - 1 quantification of IF images performed using the Fiji software and bar - plotted as normalized integrated densities per cell ( grey dots ) with bars corresponding to Mean ± Standard Error of the Mean ( SEM ) for at least 11 high - field units of at least 100 cells ; * P - value < 0 . 05 ( Student ' s t - test , N = 11 ) .

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ZO-1 quantification of IF images performed using the Fiji software and bar-plotted as normalized integrated densities per cell (grey dots) with bars corresponding to Mean ± Standard Error of the Mean (SEM) for at least 11 high-field units of at least 100 cells; * P-value < 0.05 (Student's t-test, N=11).


LMS ( red ) and HMS ( black ) gene expression quantification in Epi cells by random - primed RT - qPCR relative to RPL11 and represented as log2 fold change to CTR . Each dot corresponds to an independent replicate , bars represent Mean ± Standard Deviation values .

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LMS (red) and HMS (black) gene expression quantification in Epi cells by random-primed RT-qPCR relative to RPL11 and represented as log2 fold change to CTR. Each dot corresponds to an independent replicate, bars represent Mean ± Standard Deviation values.


Metagene profiling within the 10 kb window around TSS of H3K27ac , LSD1 and H3K4me2 over IgG in Epi cells expressing none ( CTR ) or different HOTAIR variants ( HOT , HOTΔP and HOTΔL ) . bp stands for base pairs .

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

Metagene profiling within the 10 kb window around TSS of H3K27ac, LSD1 and H3K4me2 over IgG in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs.


Heatmap of unsupervised hierarchical clustering based on H3K27ac mean values at gene promoters showing separation of two phenotypic groups .

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Heatmap of unsupervised hierarchical clustering based on H3K27ac mean values at gene promoters showing separation of two phenotypic groups.


Metagene profiling of H3K27ac , LSD1 and H3K4me2 over IgG within the 5 kb window of enhancers in Epi cells expressing none ( CTR ) or different HOTAIR variants ( HOT , HOTΔP and HOTΔL ) . bp stands for base pairs .

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

Metagene profiling of H3K27ac, LSD1 and H3K4me2 over IgG within the 5 kb window of enhancers in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs.