( E ) Expression of Fam210a mRNA in the heart of miR - 574 - / - and WT mice from 3 biological replicates .

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(E) Expression of Fam210a mRNA in the heart of miR-574-/- and WT mice from 3 biological replicates.


( F ) Expression of FAM210A protein in the heart of miR - 574 - / - and WT mice . Protein intensity was quantified in the right panel ( n = 6 per group ) .

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(F) Expression of FAM210A protein in the heart of miR-574-/- and WT mice. Protein intensity was quantified in the right panel (n=6 per group).


( H ) Dual luciferase reporter assays with co - transfection of miR - 574 - 5p mimics and FLuc - Fam210a 3 ' UTR bearing wild type and mutant seed sequences . The assays were performed in three biological replicates .

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(H) Dual luciferase reporter assays with co-transfection of miR-574-5p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.


( I ) Dual luciferase reporter assays with co - transfection of miR - 574 - 3p mimics and FLuc - Fam210a 3 ' UTR bearing wild type and mutant seed sequences . The assays were performed in three biological replicates .

(I) Dual luciferase reporter assays with co-transfection of miR-574-3p mimics and FLuc-Fam210a 3'UTR bearing wild type and mutant seed sequences. The assays were performed in three biological replicates.


( B ) The expression level of FAM210A mRNA in 15 human organs from the GTEx Portal database . TPM : transcripts per million of reads . The central band shows the median value and the box indicates the first and third quartiles . The sample size of human samples used for quantification in GTEx Portal database ( from testis to pancreas ) : n = 361 / 429 / 432 / 555 / 803 / 240 / 432 / 226 / 406 / 578 / 241 / 202 / 85 / 328 / 755 .

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(B) The expression level of FAM210A mRNA in 15 human organs from the GTEx Portal database. TPM: transcripts per million of reads. The central band shows the median value and the box indicates the first and third quartiles. The sample size of human samples used for quantification in GTEx Portal database (from testis to pancreas): n=361/429/432/555/803/240/432/226/406/578/241/202/85/328/755.


( C ) Cellular localization of FAM210A in primary mouse ACMs detected by IF . Scale bar : 30 μm .

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(C) Cellular localization of FAM210A in primary mouse ACMs detected by IF. Scale bar: 30 μm.


( D ) Cellular localization of recombinant FAM210A in human AC16 CM cells . FAM210A - 3xFlag expression plasmid was transfected into AC16 cells for 48 hrs . Scale bar : 40 μm .

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(D) Cellular localization of recombinant FAM210A in human AC16 CM cells. FAM210A-3xFlag expression plasmid was transfected into AC16 cells for 48 hrs. Scale bar: 40 μm.


( E ) Immunoblot detection of FAM210A in isolated cytoplasmic and mitochondrial fractions from mouse hearts . VDAC1 and tubulin are used as fraction controls . Cyto : cytoplasm ; Mito : mitochondria . A representative image is shown from three biological replicates .

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(E) Immunoblot detection of FAM210A in isolated cytoplasmic and mitochondrial fractions from mouse hearts. VDAC1 and tubulin are used as fraction controls. Cyto: cytoplasm; Mito: mitochondria. A representative image is shown from three biological replicates.


( F ) Sub - organelle localization of FAM210A in isolated mouse cardiac mitochondria . TOM20 and ATP5B are used as the outer membrane ( OM ) and mitoplast ( MP ) markers , respectively .

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(F) Sub-organelle localization of FAM210A in isolated mouse cardiac mitochondria. TOM20 and ATP5B are used as the outer membrane (OM) and mitoplast (MP) markers, respectively.


( B ) Top list of FAM210A - interacting proteins in IP - MS ( immunoprecipitation - mass spectrometry ) analysis . PSM : peptide spectrum match .

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(B) Top list of FAM210A-interacting proteins in IP-MS (immunoprecipitation-mass spectrometry) analysis. PSM: peptide spectrum match.


C ) Confocal imaging of co - localization of FAM210A - 3xFLAG and endogenous EF - Tu by immunostaining . Scale bar : 40 μm .

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C) Confocal imaging of co-localization of FAM210A-3xFLAG and endogenous EF-Tu by immunostaining. Scale bar: 40 μm.


( D ) Left panel : IP - IB validates FAM210A - interacting proteins using pre - immune IgG and FAM210A antibodies . Right panel : IP - IB validates FAM210A - interacting proteins in HEK293T cell lysates subjected to IP using pre - immune IgG and anti - FLAG antibodies . A representative image is shown in three biological replicates .

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(D) Left panel: IP-IB validates FAM210A-interacting proteins using pre-immune IgG and FAM210A antibodies. Right panel: IP-IB validates FAM210A-interacting proteins in HEK293T cell lysates subjected to IP using pre-immune IgG and anti-FLAG antibodies. A representative image is shown in three biological replicates.


( E ) Mapping of interacting domains in FAM210A for binding EF - Tu .

(E) Mapping of interacting domains in FAM210A for binding EF-Tu.


( F , G ) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells : ( F ) siRNA knockdown of FAM210A . ( G ) FAM210A overexpression .

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(F,G) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (F) siRNA knockdown of FAM210A. (G) FAM210A overexpression.


( A ) miR - 574 - 5p and miR - 574 - 3p overexpression reduced FAM210A mRNA expression in AC16 cells ( n = 3 in each group ) . ( B ) miR - 574 - 5p and miR - 574 - 3p inhibition increased FAM210A mRNA expression in AC16 cells ( n = 3 in each group ) .

(A) miR-574-5p and miR-574-3p overexpression reduced FAM210A mRNA expression in AC16 cells (n=3 in each group). (B) miR-574-5p and miR-574-3p inhibition increased FAM210A mRNA expression in AC16 cells (n=3 in each group).


( C , D ) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells : ( C ) miR - 574 - 5p or miR - 574 - 3p overexpression . ( D ) Transfection of anti - miR - 574 - 5p or anti - miR - 574 - 3p inhibitor .

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(C,D) Protein expression of mitochondrial ETC component genes was measured in AC16 cells after following treatments in comparison with control cells: (C) miR-574-5p or miR-574-3p overexpression. (D) Transfection of anti-miR-574-5p or anti-miR-574-3p inhibitor.


( E , F ) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment ( 10 μM for 24 hr ) with overexpression of miR - 574 - 5p or miR - 574 - 3p . > 120 cells / group were quantified in ( E ) and n = 4 biological replicates in ( F ) . The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group .

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(E,F) Mitochondrial membrane potential and ATP production in AC16 cells under ISO treatment (10 μM for 24 hr) with overexpression of miR-574-5p or miR-574-3p. >120 cells/group were quantified in (E) and n=4 biological replicates in (F). The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.


( G , H ) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR - 574 - / - mice under ISO treatment . Scale bar : 10 μm . > 110 cells / group were quantified in bottom panel of ( G ) and n = 5 in ( H ) .

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(G,H) Mitochondrial membrane potential and ATP production in primary mouse ACMs isolated from WT and miR-574-/- mice under ISO treatment. Scale bar: 10 μm. >110 cells/group were quantified in bottom panel of (G) and n=5 in (H).


( I ) transfection of miR - 574 - 5p / 3p in the absence or presence of FAM210A overexpression . All the Western blot images were provided as representative data from 3 - 4 biologically replicated experiments .

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(I) transfection of miR-574-5p/3p in the absence or presence of FAM210A overexpression. All the Western blot images were provided as representative data from 3-4 biologically replicated experiments.


( J ) Increased protein expression of FAM210A and ND1 in miR - 574 KO hearts compared to WT hearts upon TAC surgery . N = 5 hearts ( ~ 110 - 150 CM cells ) were measured per group . The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group .

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(J) Increased protein expression of FAM210A and ND1 in miR-574 KO hearts compared to WT hearts upon TAC surgery. N=5 hearts (~110-150 CM cells) were measured per group. The dashed line in the violin plot shows medium value for the group and the dotted lines represent two quartile lines in each group.


( A - C ) qRT - PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages ( A ) , THP1 cells ( B ) and human peripheral blood mononuclear cells ( PBMC ) ( C ) that were left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib ( 0 . 1 , 1 , 10 μM ) . The data are presented as fold induction of Ifnb1 / Gapdh transcripts . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by one - way ANOVA followed by Dunnett ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 .

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(A-C) qRT-PCR measurement of Ifnb1 transcripts in mouse peritoneal macrophages (A), THP1 cells (B) and human peripheral blood mononuclear cells (PBMC) (C) that were left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) for 4 h in the presence of DMSO or indicated concentrations of Palbociclib (0.1, 1, 10 μM). The data are presented as fold induction of Ifnb1/Gapdh transcripts. The data shown are mean±SEM of n=3 biological replicates. Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. *, P<0.05; ****, P<0.0001.


( D ) MTS detection of the viability of mouse peritoneal macrophages ( PM ) , THP1 and PBMC cells left untreated ( DMSO ) or treated with Palbociclib ( 1 μM , 10 μM ) for 4 h . The data shown are mean ± SEM from n = 3 biological replicates . Statistical significance was determined by one - way ANOVA . ns , not significant .

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(D) MTS detection of the viability of mouse peritoneal macrophages (PM), THP1 and PBMC cells left untreated (DMSO) or treated with Palbociclib (1 μM, 10 μM) for 4 h. The data shown are mean ± SEM from n=3 biological replicates. Statistical significance was determined by one-way ANOVA. ns, not significant.


( E - F ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( E ) and THP1 cells ( F ) stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates .

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(E-F) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (E) and THP1 cells (F) stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.


( G - H ) qRT - PCR measurement of Il1b ( G ) and Il6 ( H ) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant . ( I - J ) qRT - PCR measurement of Il1b ( I ) and Il6 ( J ) transcripts in THP1 cells left unstimulated or stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are mean ± SEM of n = 3 biological replicates . Statistical significance was determined by two - way ANOVA followed by Tukey ' s post hoc test . * , P < 0 . 05 ; * * * * , P < 0 . 0001 ; ns , not significant .

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(G-H) qRT-PCR measurement of Il1b (G) and Il6 (H) transcripts in mouse peritoneal macrophages left unstimulated or stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant. (I-J) qRT-PCR measurement of Il1b (I) and Il6 (J) transcripts in THP1 cells left unstimulated or stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are mean ± SEM of n=3 biological replicates. Statistical significance was determined by two-way ANOVA followed by Tukey's post hoc test. *, P<0.05; ****, P<0.0001 ; ns, not significant.


( K - L ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( K ) and THP1 ( L ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) for indicated times . The data shown are representative of n = 3 biological replicates .

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(K-L) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (K) and THP1 (L) cells stimulated with 2'3' cGAMP (0.1μg/mL) in the presence of DMSO or Palbociclib (10 μM) for indicated times. The data shown are representative of n=3 biological replicates.


( M - N ) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages ( M ) and THP1 ( N ) cells stimulated with 2 ' 3 ' cGAMP ( 0 . 1 μg / mL ) in the presence of DMSO or Palbociclib ( 10 μM ) . The data shown are representative of n = 3 biological replicates .

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(M-N) Immunoblotting of indicated proteins in the lysates of mouse peritoneal macrophages (M) and THP1 (N) cells stimulated with 2'3' cGAMP (0.1 μg/mL) in the presence of DMSO or Palbociclib (10 μM). The data shown are representative of n=3 biological replicates.


( A ) A real - time OR - ID assay showing the association - dissociation curves of surface - immobilized Palbociclib with recombinant STING protein . The data shown are representative of n = 3 biological replicates .

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(A) A real-time OR-ID assay showing the association-dissociation curves of surface-immobilized Palbociclib with recombinant STING protein. 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 ) 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 .

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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 .

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(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 .

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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 .

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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 .

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(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 .

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(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 .

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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 .

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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 .

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(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 .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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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 .

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(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 .

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(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 .

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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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

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 .

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

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 .

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

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 .

O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

(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 .

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

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 .

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 .

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

(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.