WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin - expressing STm pellets collected at indicated time points after STm infection were lysed , fractionated by SDS - PAGE immunoblotted for GSDMD or LC - 3 G ) . Representative immunoblots showing GSDMD full ( GSDMD FL ) , GSDMD N - terminal fragment ( GSDMD N - ter ) , LC3 - II LC3 - I bands in lysates ( G ) . H , of band intensities for GSDMD N - ter normalized to actin ( G ) LC3 - II normalized to LC3 - I actin ( H ) from three independent experiments are shown as fold change , H ) Data information : Data represent mean ± SD . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Two - Student ' s t - .

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

WT or SLC15A4feeble BMDCs unstimulated or infected with flagellin-expressing STm Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for GSDMD or LC-3 G). Representative immunoblots showing GSDMD full length (GSDMD FL), cleaved GSDMD N-terminal fragment (GSDMD N-ter), LC3-II and LC3-I bands in cell lysates (G). H, Quantification of band intensities for GSDMD N-ter normalized to actin (G) and LC3-II normalized to LC3-I and actin (H) from three independent experiments are shown as fold change , H) Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test.


Intestinal CD11c + DCs were isolated from colons of WT SLC15A4feeble mice on day 16 cultured overnight . B . pellets from three independent experiments were immunoblotted for LC3 actin . Upper panel . Representative immunoblot . Lower panel . of band intensities for LC3 - II normalized to LC3 - I actin are shown as fold induction relative to samples from untreated mice . Note that LC3 - I signal from ex vivo samples is almost undetectable . Data information : Mean is indicated . * * p < 0 . 01 . Mann - Whitney non - parametric statistical .

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

Intestinal CD11c+ DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight. B. Cell pellets from three independent experiments were immunoblotted for LC3 and actin. Upper panel. Representative immunoblot. Lower panel. Quantification of band intensities for LC3-II normalized to LC3-I and actin are shown as fold induction relative to samples from untreated mice. Note that LC3-I signal from ex vivo samples is almost undetectable. Data information: Mean is indicated. **p<0.01. Mann-Whitney non-parametric statistical test.


WT or SLC15A4feeble BMDCs expressing ASC - GFP mCherry - LC3 ( A , B were infected with flagellin - expressing STm fixed A . were analyzed by microscopy . Representative showing ASC speck ( green ) relative to mCherry - LC3 ( red ) in two infected WT SLC15A4feeble DCs each . B . of LC3 per unit in a radius of 1 μm surrounding ASC speck in 20 per type in each of 3 independent experiments . N , nucleus . Corresponding DIC show position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - Student ' s t - .

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

WT or SLC15A4feeble BMDCs expressing ASC-GFP and mCherry-LC3 (A, B were infected with flagellin-expressing STm and fixed A. Cells were analyzed by fluorescence microscopy. Representative images showing ASC speck (green) relative to mCherry-LC3 (red) in two infected WT and SLC15A4feeble DCs each. B. Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs non - transduced ( C ) were infected with flagellin - expressing STm fixed C . were stained for endogenous ASC p62 , labeled with DAPI analyzed by microscopy . Representative showing ASC speck ( red ) relative to p62 ( green ) in two infected WT SLC15A4feeble DCs each . Note p62 surrounding ASC specks in SLC15A4feeble DCs . D . of perinuclear ( within a radius of 3 μm from nucleus ) ASC specks in at least 30 per type in each of 3 independent experiments . N , nucleus . Corresponding DIC show position . Scale bar , 8 μm . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - Student ' s t - .

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

WT or SLC15A4feeble BMDCs non-transduced (C) were infected with flagellin-expressing STm and fixed C. Cells were stained for endogenous ASC and p62, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) relative to p62 (green) in two infected WT and SLC15A4feeble DCs each. Note p62 staining surrounding ASC specks in SLC15A4feeble DCs. D. Quantification of perinuclear (within a radius of 3 μm from the nucleus) ASC specks in at least 30 cells per cell type in each of 3 independent experiments. N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm. Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP ( E were infected with flagellin - expressing STm fixed ( E , were stained for endogenous ASC , labeled with DAPI analyzed by microscopy . Representative showing ASC speck ( red ) SLC15A4 - GFP ( green ) in SLC15A4feeble DCs together with non - transduced SLC15A4feeble DCs . Note perinuclear positioning of ASC specks in transduced ( E ) . N , nucleus . Dotted lines , outlines . Corresponding DIC show position . Scale bar , 8 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 B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

SLC15A4feeble BMDCs transduced with SLC15A4-GFP (E were infected with flagellin-expressing STm and fixed (E, Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) and SLC15A4-GFP (green) in SLC15A4feeble DCs together with non-transduced SLC15A4feeble DCs. Note the perinuclear positioning of ASC specks in the transduced cells (E). N, nucleus. Dotted white lines, cell outlines. Corresponding DIC images show nuclear position. Scale bar, 8 μm.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP F , were infected with flagellin - expressing STm fixed of perinuclear ( within a radius of 3 μm from nucleus ) non - perinuclear ASC specks in at least 30 per type in each of three independent experiments ( F ) . Data information : Data represent mean ± SD . * * p < 0 . 01 . Two - Student ' s t - .

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

SLC15A4feeble BMDCs transduced with SLC15A4-GFP F, were infected with flagellin-expressing STm and fixed Quantification of perinuclear (within a radius of 3 μm from the nucleus) and non-perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments (F). Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test.


SLC15A4feeble BMDCs transduced with SLC15A4 - GFP G ) were infected with flagellin - expressing STm lysed ( G ) 1 h after infection . G . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) p20 ( casp . - 1 p20 ) , LC3 - II LC3 - I , actin bands .

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

SLC15A4feeble BMDCs transduced with SLC15A4-GFP G) were infected with flagellin-expressing STm and lysed (G) 1 h after infection. G. Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), LC3-II and LC3-I, and actin bands.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) were infected with flagellin - expressing STm . ( A , B ) . pellets collected 1 h after STm infection were lysed , fractionated by SDS - PAGE immunoblotted for phospho ( P ) total mTOR , ULK1 kinase , p70 kinase S6 , FLAG actin . Representative immunoblots . Non - specific band right above FLAG - RagBQ99L is indicated with an asterisk ( A ) . of band intensities for P - mTOR normalized to total mTOR , P - ULK1 normalized to total ULK1 , P - p70 normalized to total p70 P - S6 normalized to S6 , in transduced from 3 independent experiments are shown as fold change relative to WT control ( B ) . Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - Student ' s t - .

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

WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) were infected with flagellin-expressing STm. (A, B). Cell pellets collected 1 h after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for phospho (P) and total mTOR, ULK1 kinase, p70 kinase and S6, FLAG and actin. Representative immunoblots. Non-specific band right above FLAG-RagBQ99L is indicated with an asterisk (A). Quantification of band intensities for P-mTOR normalized to total mTOR, P-ULK1 normalized to total ULK1, P-p70 normalized to total p70 and P-S6 normalized to S6, in transduced cells from 3 independent experiments are shown as fold change relative to WT control (B). Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) ( E , pellets were probed for caspase - 1 , GSDMD , actin ( E , Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) p20 ( casp . - 1 p20 ) , GSDMD full ( GSDMD FL ) GSDMD N - terminal fragment ( GSDMD N - ter ) bands .

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

WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) (E, Cell pellets were probed for caspase-1, GSDMD, actin (E, Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.


WT or SLC15A4feeble BMDCs transduced with non - target control , ATG5 or ATG7 shRNAs G ) G ) . pellets were probed for caspase - 1 , GSDMD , actin , ATG5 ATG 7 ( G ) . Representative immunoblots showing pro - caspase - 1 ( pro - casp . - 1 ) p20 ( casp . - 1 p20 ) , GSDMD full ( GSDMD FL ) GSDMD N - terminal fragment ( GSDMD N - ter ) bands .

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

WT or SLC15A4feeble BMDCs transduced with non-target control, ATG5 or ATG7 shRNAs G) G). Cell pellets were probed for caspase-1, GSDMD, actin , ATG5 and ATG 7 (G). Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands.


WT or SLC15A4feeble BMDCs transduced with constitutively active RagB ( RagBQ99L ) or metap2 ( control ) H , I ) mcherry - LC3 ( H , I ) , ( H , I ) . were stained for endogenous ASC , labeled with DAPI analyzed by microscopy . Representative showing ASC speck ( green ) LC3 ( red ) in SLC15A4feeble DCs ( H ) . of perinuclear ( within a radius of 3 μm from nucleus ) in at least 30 per type in each of three independent experiments ( I ) . N , nucleus . Corresponding DIC show position . Scale bar , 8 μ Data information : Data represent mean ± SD . * * p < 0 . 01 ; * * * p < 0 . 001 . Two - Student ' s t - .

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

WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) H, I) and mcherry-LC3 (H, I), (H, I). Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (green) and LC3 (red) in SLC15A4feeble DCs (H). Quantification of perinuclear (within a radius of 3 μm from the nucleus) in at least 30 cells per cell type in each of three independent experiments (I). N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μ Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test.


A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , SYCP3 ( blue ) , stained with DAPI ( upper right inset ) . Zoomed of centrosome are shown in bottom left inset . Scale bars = 5 μm .

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

A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.


B . of number of CETN3 foci per spermatocyte ( n = 30 ) at each meiotic prophase substage .

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

B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.


C . Representative of unseparated ( < 1 μm apart ) , initial separation ( 1 - 5 μm apart ) , intermediate separation ( 5 - 11 μm apart ) , pole separation ( > 11 μm apart ) of centrosomes . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , SYCP3 ( blue ) , stained with DAPI ( upper right inset ) . Zoomed of centrosome are shown in bottom top left insets . Scale bars = 5 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative images of unseparated (< 1 μm apart), initial separation (1-5 μm apart), intermediate separation (5-11 μm apart), and pole separation (>11 μm apart) of centrosomes. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.


. Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using four centrosome separation classifications P value obtained from a Kruskal - Wallis one - way ANOVA . * * P < 0 . 01 .

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

. Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications P value obtained from a Kruskal-Wallis one-way ANOVA. **P<0.01.


E . Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT ( purple ) , CETN3 ( green ) , SYCP3 ( red ) , stained with DAPI ( blue ) . Zoomed of centrosomes below each panel . Scale bars = 5 μm .

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

E. Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT (purple), CETN3 (green), and SYCP3 (red), and stained with DAPI (blue). Zoomed images of centrosomes below each panel. Scale bars = 5 μm.


A . Hematoxylin eosin of 5 - micron thick testis sections of adult control , Plk1 cHet , Plk1 cKO mice . Stars indicate examples of spermatocytes with condensed chromosomes . B . testis to ratios from control ( n = 8 ) , Plk1 cHet ( n = 4 ) , Plk1 cKO ( n = 7 ) adult mice .

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

A. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. B. Percent testis to body weight ratios from control (n = 8), Plk1 cHet (n = 4), and Plk1 cKO (n = 7) adult mice.


C . TUNEL of paraffin embedded testis sections of adult control , Plk1 cHet , Plk1 cKO mice . D . number of TUNEL positive per seminiferous tubule cross - section was quantified in control Plk1 cHet , Plk1 cKO mice . Two technical replicates were performed per genotype , 100 seminiferous tubule cross sections were analyzed per replicate .

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

C. TUNEL staining of paraffin embedded testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. D. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Plk1 cHet, and Plk1 cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.


E . Hematoxylin eosin of 5 - micron thick testis sections of adult control , Aurka cHet , Aurka cKO mice . Stars indicate examples of spermatocytes with condensed chromosomes . F . testis to ratios from control ( n = 5 ) , Aurka cHet ( n = 3 ) , Aurka cKO ( n = 5 ) adult mice .

E. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Aurka cHet, and Aurka cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. F. Percent testis to body weight ratios from control (n = 5), Aurka cHet (n = 3), and Aurka cKO (n = 5) adult mice.


G . TUNEL of paraffin embedded testis sections of adult control , Aurka cHet , Aurka cKO mice . H . number of TUNEL positive per seminiferous tubule cross - section was quantified in control Aurka cHet , Aurka cKO mice . Two technical replicates were performed per genotype , 100 seminiferous tubule cross sections were analyzed per replicate .

G. TUNEL staining of paraffin embedded testis sections of adult control, Aurka cHet, and Aurka cKO mice. H. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Aurka cHet, and Aurka cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.


I . Representative of spermatocytes containing monopolar , bipolar , multipolar spindles immunolabeled against alpha - tubulin ( red ) , centromere ( green ) , stained with DAPI . J . Spindle pole number per spermatocyte was quantified for control , Aurka cKO , Plk1 cHet , Plk1 cKO . n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment .

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

I. Representative images of spermatocytes containing monopolar, bipolar, and multipolar spindles immunolabeled against alpha-tubulin (red), centromere (green), and stained with DAPI. J. Spindle pole number per primary spermatocyte was quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO. n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment.


B . of number of CETN3 foci per spermatocyte . n = 30 for each meiotic prophase substage .

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

B. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.


C . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , SYCP3 ( blue ) , stained with DAPI ( upper right inset ) . Zoomed of centrosome are shown in bottom top left insets . Scale bars = 5 μm .

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

C. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.


D . of number of CETN3 foci per spermatocyte . n = 30 for each meiotic prophase substage .

D. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage.


E . Centrosome ( PCNT ) separation was scored per spermatocyte . Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using four centrosome separation classifications P values obtained from two - Student ' s t - comparing of unseparated centrosomes in each mouse mutant to control . n . s . ( not significant ) , * * * P < 0 . 001 .

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

E. Centrosome (PCNT) separation was scored per spermatocyte. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications P values obtained from two-tailed Student's t-test comparing the percent of unseparated centrosomes in each mouse mutant to control. n.s. (not significant), ***P<0.001 .


A . Spermatocytes were immunolabeled against CETN3 ( green ) , CEP164 ( red ) , SYCP3 ( blue ) , stained with DAPI ( upper right inset ) . Zoomed of centrosome are shown in bottom top left insets . Scale bars = 5 μm .

A. Spermatocytes were immunolabeled against CETN3 (green), CEP164 (red), SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.


B . of centrioles containing CEP164 distal appendages was quantified from three biological replicates ( n = 30 total ) in control , Aurka cKO , Plk1 cHet , Plk1 cKO spermatocytes at pachytene - stage of meiotic prophase metaphase I .

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B. The percent of centrioles containing CEP164 distal appendages was quantified from three biological replicates (n = 30 total cells) in control, Aurka cKO, Plk1 cHet, and Plk1 cKO spermatocytes at the pachytene-stage of meiotic prophase and metaphase I.


C . of gamma - tubulin positive centrosomes per metaphase I spermatocyte . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . total number of quantified for control , Aurka cKO , Plk1 cHet , Plk1 cKO mice were 137 , 169 , 198 , 198 , respectively .

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C. Quantification of gamma-tubulin positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 137, 169, 198, and 198, respectively.


D . Spermatocytes were immunolabeled against gamma - tubulin ( green ) , alpha - tubulin ( red ) , stained with DAPI ( blue ) . Plk1 Aurka cKO spermatocytes arrest at a prometaphase stage . Scale bars = 5 μm .

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D. Spermatocytes were immunolabeled against gamma-tubulin (green), alpha-tubulin (red), and stained with DAPI (blue). Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage. Scale bars = 5 μm.


Western blots of gamma - tubulin from extracts obtained from 19dpp 22dpp control , Aurka cKO , Plk1 cKO spermatocytes ( E ) . Total stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display loading .

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Western blots of gamma-tubulin from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (E). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


Western blots of gamma - tubulin from extracts from control Plk1 cHet spermatocytes at 16 , 19 22 dpp ( F ) . Total stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display loading .

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Western blots of gamma-tubulin from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (F). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


A . Spermatocytes were immunolabeled against TACC3 ( green ) , SYCP3 ( red ) , CETN3 ( magenta ) , stained with DAPI ( blue ) . Zoom of centrosomes are presented to right . Scale bars = 5 μm .

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A. Spermatocytes were immunolabeled against TACC3 (green), SYCP3 (red), CETN3 (magenta), and stained with DAPI (blue). Zoom images of the centrosomes are presented to the right. Scale bars = 5 μm.


B . of TACC3 positive centrosomes per metaphase I spermatocyte . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . total number of quantified for control , Aurka cKO , Plk1 cHet , Plk1 cKO mice were 97 , 103 , 96 , 105 , respectively .

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B. Quantification of TACC3 positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 97, 103, 96, and 105, respectively.


Western blots of TACC3 from extracts obtained from 19dpp 22dpp control , Aurka cKO , Plk1 cKO spermatocytes ( D ) . Total stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display loading .

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Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (D). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


Western blots of TACC3 from extracts from control Plk1 cHet spermatocytes at 16 , 19 22 dpp ( C ) . Total stained with 2 , 2 , 2 - Trichloroethanol ( TCE ) display loading .

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Western blots of TACC3 from Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (C). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.


E , F . Spermatocytes were immunolabeled against beta - catenin ( green ) , SYCP3 ( red ) , PCNT ( magenta ) , stained with DAPI ( blue ) . Middle columns emphasize beta - catenin signal in diplonema diakinesis spermatocytes . Scale bars = 5 μm . G . of beta - catenin positive centrosomes per spermatocyte at diakinesis . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . total number of quantified for control , Aurka cKO , Plk1 cHet , Plk1 cKO mice were 89 , 106 , 122 , 93 , respectively .

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E, F. Spermatocytes were immunolabeled against beta-catenin (green), SYCP3 (red), PCNT (magenta), and stained with DAPI (blue). Middle columns emphasize beta-catenin signal in diplonema and diakinesis spermatocytes. Scale bars = 5 μm. G. Quantification of beta-catenin positive centrosomes per spermatocyte at diakinesis. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 89, 106, 122, and 93, respectively.


A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , SYCP3 ( blue ) , stained with DAPI ( upper right inset ) . Zoomed of centrosome are shown in bottom left inset . Scale bars = 5 μm .

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A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.


B . of number of CETN3 foci per spermatocyte ( n = 30 ) at each meiotic prophase substage .

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B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.


C . Centrosome ( PCNT ) separation was assessed in Aurka ; Plk1 dKO spermatocytes . Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using four centrosome separation

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C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications


( A , B ) B - progression measured as ( A ) absolute number or ( B ) of OFP + in peripheral blood of transplanted mice ( mean ± SD , each dot represents an mouse , CTRL = 6 mice , IFNγ = 10 mice ; * * * * p ≤ 0 . 001 , Two - Way ANOVA ; mice in blue yellow ( B ) were also analyzed by single -

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(A, B) B-ALL progression measured as (A) absolute number or (B) percentage of OFP+ cells in peripheral blood of transplanted mice (mean ± SD, each dot represents an individual mouse, CTRL = 6 mice, IFNγ = 10 mice; **** p ≤ 0.001, Two-Way ANOVA; mice in blue and yellow (B) were also analyzed by single-cell RNA sequencing


( F , G ) of CD4 + CD8 + within OFP - BM ( F ) maturation state ( G ) of CD8 + lymphocytes ( CD62L - CD44 - double negative , CD62L - CD44 + effector memory , CD62L + CD44 - naive CD62L + CD44 + central memory ) in BM immune infiltrate at 12 - 17 - days after B - ( mean ± SD , each dot represents an mouse ; * * p = 0 . 005 , * * * * p ≤ 0 . 001 , Two - Way ANOVA ) .

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(F, G) Percentage of CD4+ and CD8+ cells within OFP- BM cells (F) and maturation state (G) of CD8+ lymphocytes (CD62L-CD44- double negative, CD62L-CD44+ effector memory, CD62L+CD44- naive and CD62L+CD44+ central memory) in the BM immune infiltrate at 12- and 17-days after B-ALL (mean ± SD, each dot represents an individual mouse; ** p = 0.005, **** p ≤ 0.001, Two-Way ANOVA).


( A ) Engraftment of CD45 . 2 donor lineage composition following transplantation of wild - type ( WT ) or IFNγ receptor 1 knock out ( KO ) HSPCs transduced with either control Tie2 . NGFR ( CTRL ) or Tie2 . IFNγ ( IFNγ ) LV ( mean ± SD , each dot represents an mouse , WT CTRL = 7 mice , WT IFNγ = 7 mice , KO CTRL = 7 mice , KO IFNγ = 7 mice ) .

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(A) Engraftment of CD45.2 donor cells and lineage composition following transplantation of wild-type (WT) or IFNγ receptor 1 knock out (KO) HSPCs transduced with either the control Tie2.NGFR (CTRL) or Tie2.IFNγ (IFNγ) LV (mean ± SD, each dot represents an individual mouse, WT CTRL = 7 mice, WT IFNγ = 7 mice, KO CTRL = 7 mice, KO IFNγ = 7 mice).


( C ) B - progression measured as absolute number of OFP + in peripheral blood ( mean ± SD , each dot represents an mouse ; * p = 0 . 0286 at day 10 ; p = 0 . 0174 at day 12 ; * * * p = 0 . 0002 ; * * * * p ≤ 0 . 0001 ; Ordinary Two - Way ANOVA ) .

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(C) B-ALL progression measured as absolute number of OFP+ cells in the peripheral blood (mean ± SD, each dot represents an individual mouse; * p = 0.0286 at day 10; p = 0.0174 at day 12; *** p = 0.0002; **** p ≤ 0.0001; Ordinary Two-Way ANOVA).


( E ) of CD8 + T lymphocytes within OFP - CD45 + BM ( mean ± SD , each dot represents an mouse ) .

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(E) Percentage of CD8+ T lymphocytes within OFP- CD45+ BM cells (mean ± SD, each dot represents an individual mouse).


( B , C ) Violin plots show distribution of IFNγ ( B ) MHC - II gene ( C ) signatures for each experimental condition , within each custom - type . Wilcox Rank Sum for IFNγ signature , condition IFNγ d12 vs . CTR d12 : B - p = 2 . 68x10 - 19 , B p = 0 . 5 , plasma p = 0 . 78 ; Wilcox Rank Sum for MHC - II signature , condition IFNγ d12 vs . CTRL d12 : B - p = 4 . 26x10 - 46 , B p = 1x10 - 9 , plasma p = 0 . 32 .

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(B, C) Violin plots show the distribution of IFNγ (B) and MHC-II gene (C) signatures for each experimental condition, within each custom cell-type. Wilcox Rank Sum Test for IFNγ signature, condition IFNγ d12 vs. CTR d12: B-ALL p= 2.68x10-19, B cells p=0.5, plasma cells p= 0.78; Wilcox Rank Sum Test for MHC-II signature, condition IFNγ d12 vs. CTRL d12: B-ALL p= 4.26x10-46, B cells p=1x10-9, plasma cells p= 0.32.


( F ) Expression of Mki67 transcripts in B - from CTRL d12 IFNγ d12 conditions , extracted from scRNAseq dataset ( * * * * p ≤ 0 . 0001 , Welch ' s ) .

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(F) Expression of Mki67 transcripts in B-ALL cells from CTRL d12 and IFNγ d12 conditions, extracted from the scRNAseq dataset (**** p ≤ 0.0001, Welch's test).


( I ) Loss of IFNγ response over time in B - from IFNγ group , potentially facilitated by reduced expression of Ifngr1 , Ifngr2 , Jak1 , Stat1 Irf1 ( data extracted from scRNAseq dataset statistical by Two - Way ANOVA ) .

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(I) Loss of IFNγ response over time in B-ALL cells from the IFNγ group, potentially facilitated by reduced expression of Ifngr1, Ifngr2, Jak1, Stat1 and Irf1 (data extracted from the scRNAseq dataset statistical analysis by Two-Way ANOVA).


Combination gene therapy with IFNα , TNFα IFNγ , expressed from Tie2 vector platform . CTRL ( Tie2 . NGFR ) , 2 independent experiments , n = 9 mice ; IFNα , 1 experiment , n = 2 mice ; IFNα / IFNγ , 1 experiment , n = 4 mice ; IFNα / TNFα , 1 experiment , n = 3 mice ; IFNγ / TNFα , 2 independent experiments , n = 19 mice . ( C ) of MHC II + macrophages identified by F4 / 80 expression in spleen ( mean ± SD , each dot represents an mouse ; * p = 0 . 0484 , * * p = 0 . 0049 , ordinary One - Way ANOVA ) . ( D ) of CD8 + T lymphocytes within CD45 positive OFP negative splenic ( mean ± SD , each dot represents an mouse ; * p = 0 . 0356 , ordinary One - Way ANOVA ) . (

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Combination gene therapy with IFNα, TNFα and IFNγ, expressed from the Tie2 vector platform. CTRL (Tie2.NGFR), 2 independent experiments, n = 9 mice; IFNα, 1 experiment, n = 2 mice; IFNα/IFNγ, 1 experiment, n= 4 mice; IFNα/TNFα, 1 experiment, n = 3 mice; IFNγ/TNFα, 2 independent experiments, n = 19 mice. (C) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; *p = 0.0484, **p = 0.0049, ordinary One-Way ANOVA). (D) Percentage of CD8+ T lymphocytes within CD45 positive and OFP negative splenic cells (mean ± SD, each dot represents an individual mouse; *p = 0.0356, ordinary One-Way ANOVA). (


Combination of IFNγ gene therapy with immuno - oncology drugs . CTRL ( Tie2 . NGFR ) + antibody isotype , n = 6 mice ; CTRL + αCTLA4 antibody , n = 5 mice ; CTRL + αLAG3 antibody , n = 5 mice ; CTRL + 1 - Methyltryptophan ( 1 - MT ) , n = 5 mice ; IFNγ ( Tie2 . IFNγ ) + antibody isotype , n = 7 mice ; IFNγ + αCTLA4 antibody , n = 7 mice ; IFNγ + αLAG3 antibody , n = 7 mice ; IFNγ + 1 - MT , n = 5 mice . ( F ) of MHC II + macrophages identified by F4 / 80 expression in spleen ( mean ± SD , each dot represents an mouse ; * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 as compared to CTRL isotype , ordinary One - Way ANOVA ) . ( G ) of CD8 + T lymphocytes within OFP negative BM ( mean ± SD , each dot represents an mouse ) . S

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

Combination of IFNγ gene therapy with immuno-oncology drugs. CTRL (Tie2.NGFR) + antibody isotype, n = 6 mice; CTRL + αCTLA4 antibody, n= 5 mice; CTRL + αLAG3 antibody, n = 5 mice; CTRL + 1-Methyltryptophan (1-MT), n = 5 mice; IFNγ (Tie2.IFNγ) + antibody isotype, n = 7 mice; IFNγ + αCTLA4 antibody, n = 7 mice; IFNγ + αLAG3 antibody, n = 7 mice; IFNγ +1-MT, n = 5 mice. (F) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001 as compared to CTRL isotype, ordinary One-Way ANOVA). (G) Percentage of CD8+ T lymphocytes within OFP negative BM cells (mean ± SD, each dot represents an individual mouse). S


( B ) microscopy of typical mouse small intestinal organoids under different culture conditions . Lgr5 + intestinal are labeled with GFP , which are ubiquitously present , restricted to bottom of crypts absent in CV , ENR EN , respectively . * is auto -

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(B) Fluorescence microscopy images of typical mouse small intestinal organoids under different culture conditions. Lgr5+ intestinal stem cells are labeled with GFP, which are ubiquitously present, restricted to the bottom of the crypts and absent in CV, ENR and EN, respectively. * is auto-fluorescence


( A ) Two row - matched heatmaps showing relative change in mRNA expression ( left ) expression ( right ) of significantly changing . Known markers of intestinal homeostasis are highlighted . Relative expression is shown as log2 fold change over row mean

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(A) Two row-matched heatmaps showing the relative change in mRNA expression (left) and protein expression (right) of significantly changing proteins. Known markers of intestinal homeostasis are highlighted. Relative expression is shown as log2 fold change over the row mean


( B ) Hematoxylin ( Periodic acid - Shiff ) PAS of WT ( left ) Hnf4g KO ( right ) intestine . Nuclei are visualized using Hematoxylin goblet stain positive for PAS

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(B) Hematoxylin and (Periodic acid-Shiff) PAS staining of WT (left) and Hnf4g KO (right) intestine. Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS


( C ) Alcian Blue Fast Red of WT ( left ) Hnf4g KO ( right ) small intestinal organoids . are visualized using Fast Red . Intra - extracellular mucus that is produced in goblet stain positive for Alcian Blue

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(C) Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right) small intestinal organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue


( D ) Strand specific - seq data , Hnf4g ChIP - seq in EN , promoter specific histone modification H3K4me3 over Hnf4a locus is shown . Data from different organoid cultures are color coded . - seq reads mapping to positive strand ( sense strand ) are plotted in top window of every sample , while - seq reads that map to negative strand are mirrored shown in bottom window of every sample . two isoforms of Hnf4a that are expressed are shown in bottom , together with antisense transcript Hnf4aos . " phyloP30way " track from UCSC genome browser is plotted at bottom to show conservation

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(D) Strand specific RNA-seq data, Hnf4g ChIP-seq in EN, and promoter specific histone modification H3K4me3 over the Hnf4a locus is shown. Data from different organoid cultures are color coded. RNA-seq reads mapping to the positive strand (sense strand) are plotted in the top window of every sample, while RNA-seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample. The two isoforms of Hnf4a that are expressed are shown in the bottom, together with the antisense transcript Hnf4aos. The "phyloP30way" track from the UCSC genome browser is plotted at the bottom to show sequence conservation


( E ) of amount of goblet per villus for WT Hnf4g KO intestine . Dots represent amount of goblet counted per villus . ( n = 7 to 25 villi from 11 WT mice 3 to 17 villi from 14 Hnf4g KO mice ) . P value is from two - Mann - Whitney U

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(E) Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine. Dots represent the amount of goblet cells counted per villus. (n = 7 to 25 villi from 11 WT mice and 3 to 17 villi from 14 Hnf4g KO mice). P value is from two-tailed Mann-Whitney U test


( C ) Adult of ash1 NSD mutants . In ash122 / ash121 mutants ( designated as ash1 - ) loss of Abd - B expression results in partial transformation of abdominal segments 6 5 toward segments 5 4 , which is visible from partial loss of pigmentation on tergites 5 6 ( t5 t6 ) appearance of bristles on sternite 6 ( s6 , marked with black arrows ) . loss of Ubx expression causes transformation of third thoracic to second thoracic segment visible as partial haltere ( H ) to wing third leg ( 3L ) to second leg ( 2L ) transformations . former is evident from change in haltere shape appearance of multiple bristles ( black arrowheads ) . latter is indicated by apical pre - apical bristles ( red arrows ) on tibia of third leg of ash1 mutants . These are normally present on 2L but absent on 3L ( compare to wild - type ) . Also note appearance of additional hypopleural bristles on third thoracic segment of ash1 - flies ( red arrowheads ) , which indicate its transformation towards second thoracic segment . Phenotype of NSDds46 / NSDds46 ( NSD - ) flies is indistinguishable from wild - type phenotype of double ash122 , NSDds46 / ash122 , NSDds46 ( ash1 - , NSD - ) flies is no more severe than that of single ash122 / ash121 ( ash1 - ) mutants .

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

(C) Adult phenotypes of the ash1 and NSD mutants. In ash122/ash121 mutants (designated as ash1-) the loss of Abd-B expression results in partial transformation of abdominal segments 6 and 5 toward segments 5 and 4, which is visible from the partial loss of pigmentation on tergites 5 and 6 (t5 and t6) and appearance of bristles on sternite 6 (s6, marked with black arrows). The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere (H) to wing and third leg (3L) to second leg (2L) transformations. The former is evident from the change in the haltere shape and the appearance of multiple bristles (black arrowheads). The latter is indicated by the apical and pre-apical bristles (red arrows) on the tibia of the third leg of ash1 mutants. These are normally present on 2L but absent on 3L (compare to wild-type). Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1- flies (red arrowheads), which indicate its transformation towards the second thoracic segment. Phenotype of the NSDds46/NSDds46 (NSD-) flies is indistinguishable from wild-type and the phenotype of the double ash122,NSDds46/ ash122,NSDds46 (ash1-,NSD-) flies is no more severe than that of the single ash122/ash121 (ash1-) mutants.


( D ) Ubx expression in haltere imaginal discs . expression was assayed by immunostaining with antibodies against Ubx ( red ) acetylated H3K18 ( green , positive control ) . While ash122 / ash121 ( ash1 - ) larvae show stochastic clonal loss of Ubx immunostaining in haltere discs ( yellow dashed lines ) , Set2 - larvae have uniform expression of Ubx throughout haltere disc , resembling that in wild - type larvae . Scale bars indicate 100μm .

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

(D) Ubx expression in the haltere imaginal discs. The expression was assayed by immunostaining with antibodies against Ubx (red) and acetylated H3K18 (green, positive control). While ash122/ash121 (ash1-) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs (yellow dashed lines), Set2- larvae have uniform expression of Ubx throughout the haltere disc, resembling that in the wild-type larvae. Scale bars indicate 100μm.


( A ) Adult of ash122 / ash19011 flies supplemented with transgenic constructs expressing either full - Ash1 ( Ash1FL ) or truncated variants lacking PHD ( Ash1 ∆ PHD ) or SET ( Ash1 ∆ SET ) domains . Note third leg ( L3 ) to second leg ( L2 ) , haltere ( H ) to wing ( black arrows ) , t5 to t4 t6 to t5 transformations in Ash1 ∆ PHD Ash1 ∆ SET but not in Ash1FL flies . latter are evident from partial loss of pigmentation in t6 t5 , or appearance of small bristles ( trichomas ) on t6 of Ash1 ∆ PHD Ash1 ∆ SET flies in that is normally naked ( Ash1FL , yellow dashed ) .

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

(A) Adult phenotypes of the ash122/ash19011 flies supplemented with transgenic constructs expressing either the full-length Ash1 (Ash1FL) or the truncated variants lacking the PHD (Ash1∆PHD) or the SET (Ash1∆SET) domains. Note the third leg (L3) to second leg (L2), haltere (H) to wing (black arrows), t5 to t4 and t6 to t5 transformations in the Ash1∆PHD and the Ash1∆SET but not in the Ash1FL flies. The latter are evident from the partial loss of pigmentation in t6 and t5, or the appearance of small bristles (trichomas) on t6 of the Ash1∆PHD and the Ash1∆SET flies in the area that is normally naked (Ash1FL, yellow dashed line).


( B ) Two - fold dilutions of total extracts from third instar larvae of ash122 / ash19011 mutants supplemented with various transgenic constructs wild type flies were analyzed by western blot with anti - Ash1 antibodies . Arrowhead indicates position of Ash1 . Note that transgenic are expressed at comparable level .

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

(B) Two-fold dilutions of total nuclear protein extracts from the third instar larvae of the ash122/ash19011 mutants supplemented with various transgenic constructs and wild type flies were analyzed by western blot with anti-Ash1 antibodies. Arrowhead indicates the position of Ash1 protein. Note that transgenic proteins are expressed at comparable level.


( C ) Coomassie of SDS - PAGE separated extracts from ( B ) was used to control loading .

(C) Coomassie staining of SDS-PAGE separated protein extracts from (B) was used to control the loading.


Two - fold serial dilutions of total extracts from wild - type , ash122 / ash19011 ( ash1 - ) , ash122 , NSDds46 / ash19011 , NSDds46 ( ash1 - , NSD - ) Set21 ( Set2 - ) larval brains , imaginal discs salivary were analyzed by western blot with antibodies against H3K36me1 ( A ) , H3K36me2 ( B ) H3K36me3 ( C ) . Note strong ( > 10 - fold ) reduction of H3K36me3 signal in Set2 - extract slight ( ~ 2 - fold ) reduction of H3K36me1 signal in ash1 - ash1 - , NSD - extracts . extracts from wild - type , double ash1 - , NSD - single NSD - Set2 - mutants ( right panels ) were analyzed together on same , however of H3K36me1 H3K36me3 western blots were modified to splice out marker lane between ash1 - , NSD - Set2 - extracts . Western blots with constitutively expressed BEAF - 32 were used as loading controls .

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

Two-fold serial dilutions of the total protein extracts from the wild-type, ash122/ash19011 (ash1-), ash122,NSDds46/ash19011,NSDds46 (ash1-, NSD-) and Set21 (Set2-) larval brains, imaginal discs and salivary glands were analyzed by western blot with antibodies against H3K36me1 (A), H3K36me2 (B) and H3K36me3 (C). Note the strong (>10-fold) reduction of H3K36me3 signal in the Set2- extract and the slight (~2-fold) reduction of H3K36me1 signal in the ash1- and ash1-, NSD- extracts. The protein extracts from the wild-type, double ash1-, NSD- and single NSD- and Set2- mutants (right panels) were analyzed together on the same membrane, however the images of the H3K36me1 and H3K36me3 western blots were modified to splice out the marker lane between the ash1-,NSD- and the Set2- extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls.


from wild - type ( blue bars ) , ash122 / ash19011 ( ash1 - , red bars ) transgenic ash122 / ash19011 ( Ash1FL , blue bars ; Ash1 ∆ PHD , green bars ; Ash1 ∆ SET , orange bars ) larvae was subjected to immunoprecipitation with antibodies against H3K36me1 , H3K36me2 Histograms show mean of two independent experiments ( n = 2 ) with dots indicating experimental results . An intergenic region on chromosome 3R ( intergenic ) constitutively active TBP - associated factor 4 ( Taf4 ) gene serve as controls . loss of Ash1 ChIP signal in ash1 - larvae indicates that selected genes are genuine Ash1 binding sites .

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

Chromatin from the wild-type (dark blue bars), ash122/ash19011 (ash1-, red bars) and transgenic ash122/ash19011 (Ash1FL, light blue bars; Ash1∆PHD, green bars; Ash1∆SET, orange bars) larvae was subjected to immunoprecipitation with the antibodies against H3K36me1 , H3K36me2 Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results. An intergenic region on chromosome 3R (intergenic) and the constitutively active TBP-associated factor 4 (Taf4) gene serve as controls. The loss of Ash1 ChIP signal in the ash1- larvae indicates that the selected genes are the genuine Ash1 binding sites.


from wild - type ( blue bars ) , ash122 / ash19011 ( ash1 - , red bars ) transgenic ash122 / ash19011 ( Ash1FL , blue bars ; Ash1 ∆ PHD , green bars ; Ash1 ∆ SET , orange bars ) larvae was subjected to immunoprecipitation with antibodies against Ash1 ( C ) . Histograms show mean of two independent experiments ( n = 2 ) with dots indicating experimental results . An intergenic region on chromosome 3R ( intergenic ) constitutively active TBP - associated factor 4 ( Taf4 ) gene serve as controls . loss of Ash1 ChIP signal in ash1 - larvae indicates that selected genes are genuine Ash1 binding sites .

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

Chromatin from the wild-type (dark blue bars), ash122/ash19011 (ash1-, red bars) and transgenic ash122/ash19011 (Ash1FL, light blue bars; Ash1∆PHD, green bars; Ash1∆SET, orange bars) larvae was subjected to immunoprecipitation with the antibodies against Ash1 (C). Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results. An intergenic region on chromosome 3R (intergenic) and the constitutively active TBP-associated factor 4 (Taf4) gene serve as controls. The loss of Ash1 ChIP signal in the ash1- larvae indicates that the selected genes are the genuine Ash1 binding sites.


( B ) ∆ HisC ; 12xH3K36R control ∆ HisC ; 12xWT flies show no homeotic transformations are indistinguishable from wild - type .

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) The ∆HisC; 12xH3K36R and control ∆HisC; 12xWT flies show no homeotic transformations and are indistinguishable from the wild-type.


( A ) Abd - B expression in Central Nervous System ( CNS ) of stage 16 was assayed by immunostaining with antibodies against Abd - B ( red ) . In ash122 / TM3 , Sb , e Kr : : GFP or ash19011 / TM3 , Sb , e Kr : : GFP ( heterozygous control ) , Abd - B is expressed in parasegments 14 to 10 in a gradient that recedes from posterior to anterior parasegment . In ash122 / ash19011 mutants Abd - B gradient is much steeper , with reduced of parasegments 13 12 at edge of detection in parasegments 11 - 10 . Heterozygous control ash122 / ash19011 mutant were stained together separated by strong GFP expression ( green ) in Bolwig ' s organs ( arrows ) . Here in B are oriented with anterior pole facing top scaling bars correspond to 50μm .

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

(A) The Abd-B expression in the Central Nervous System (CNS) of stage 16 embryos was assayed by immunostaining with antibodies against Abd-B (red). In the ash122/TM3, Sb, e Kr::GFP or ash19011/TM3, Sb, e Kr::GFP embryos (heterozygous control), Abd-B is expressed in parasegments 14 to 10 in a gradient that recedes from the posterior to anterior parasegment. In the ash122/ash19011 mutants the Abd-B gradient is much steeper, with reduced staining of parasegments 13 and 12 and at the edge of the detection in parasegments 11-10. Heterozygous control and ash122/ash19011 mutant embryos were stained together and separated by strong GFP expression (green) in the Bolwig's organs (white arrows). Here and in B the embryos are oriented with anterior pole facing the top and scaling bars correspond to 50μm.


( B ) Unlike ash1 mutants , homozygous for His3 . 3A , His3 . 3B , ∆ HisC deletions supplemented with 12xH3K36R transgene ( His3 . 2 - , His3 . 3 - , H3K36R ) display wild - type Abd - B immunostaining pattern ( red ) , same as control heterozygous for His3 . 3A , ∆ HisC deletions . In this experiment , immunostaining with antibodies against acetylated H3K18 ( green ) served as positive control .

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

(B) Unlike ash1 mutants, embryos homozygous for His3.3A, His3.3B, ∆HisC deletions and supplemented with 12xH3K36R transgene (His3.2-,His3.3-,H3K36R) display the wild-type Abd-B immunostaining pattern (red), the same as the control embryos heterozygous for His3.3A, and ∆HisC deletions. In this experiment, immunostaining with antibodies against acetylated H3K18 (green) served as positive control.


( D ) Reverse transcription quantitative PCR ( RT - qPCR ) measurement of Abd - B expression in His3 . 2 - , His3 . 3 - , H3K36R . Expression of Abd - B in stage 16 , which are homozygous for His3 . 3A , His3 . 3B , ∆ HisC deletions carry 12xH3K36R transgene ( mutant ) , is not reduced compared to their wild - type counterparts ( control 1 ) or heterozygous for His3 . 3A , ∆ HisC deletions ( control 2 ) . Histograms show mean of two independent experiments ( n = 2 ) with dots indicating experimental results .

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

(D) Reverse transcription and quantitative PCR (RT-qPCR) measurement of Abd-B expression in His3.2-,His3.3-,H3K36R embryos. Expression of Abd-B in stage 16 embryos, which are homozygous for His3.3A, His3.3B, and ∆HisC deletions and carry 12xH3K36R transgene (mutant), is not reduced compared to their wild-type counterparts (control 1) or embryos heterozygous for His3.3A, and ∆HisC deletions (control 2). Histograms show the mean of the two independent experiments (n=2) with dots indicating individual experimental results.


A , B . Comparisons of RNAseq ChIPseq data . Wt ( A ) ∆ rnjA ( B ) . x - axis shows relative RNAP occupancy at a given gene ( normalized ChIPseq coverage ) . y - axis shows relative abundance of transcript ( normalized RNAseq coverage ; each dot is one gene ) . genes are color - coded , ranging from yellow ( low abundance ) to red ( high abundance ) in wt . ∆ rnjA has higher RNAP occupancy mainly among genes with less abundant transcripts . color coding in ( B ) reveals no dramatic overall changes in vertical direction ( abundance ) . If anything , some of low abundance transcripts decreased further in level in mutant . main difference ( mostly among low abundance transcripts ) is their shift in horizontal direction to right ( towards higher occupancy with RNAP ) . Data represent mean values of three independent experiments .

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

A, B. Comparisons of RNAseq and ChIPseq data. Wt strain (A) and ∆rnjA (B). The x-axis shows the relative RNAP occupancy at a given gene (normalized ChIPseq coverage). The y-axis shows the relative abundance of the transcript (normalized RNAseq coverage; each dot is one gene). The genes are color-coded, ranging from yellow (low RNA abundance) to red (high RNA abundance) in wt. The ∆rnjA strain has higher RNAP occupancy mainly among the genes with less abundant transcripts. The color coding in (B) reveals no dramatic overall changes in the vertical direction (RNA abundance). If anything, some of the low abundance transcripts decreased further in level in the mutant strain. The main difference (mostly among the low abundance transcripts) is their shift in the horizontal direction to the right (towards higher occupancy with RNAP). Data represent mean values of three independent experiments.


C . Relative expression of sigma factors in wt ( LK1371 ) versus ΔrnjA ( LK1381 ) . Wt of each sigma factor were set as 1 ( indicated with horizontal ) .

O O O O O O O O O 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. Relative expression of all sigma factors in wt (LK1371) versus ΔrnjA (LK1381). Wt levels of each sigma factor were set as 1 (indicated with the horizontal line).


D . Sigma - dependent genes correlation with expression of sigma factors ( significantly changed ) in ΔrnjA ( LK1381 ) . most down - regulated sigma factor was sigD almost sigD - dependent genes were downregulated . A similar trend is visible for of sigma factors their respective dependent genes . x - axis shows expression of each sigma factor , y - axis shows expression of genes for each sigma regulon . violet ( blue ) : regression .

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

D. Sigma-dependent genes and correlation with expression of sigma factors (significantly changed) in ΔrnjA (LK1381). The most down-regulated sigma factor was sigD and almost all sigD-dependent genes were downregulated. A similar trend is visible for the rest of sigma factors and their respective dependent genes. The x-axis shows expression of each sigma factor, the y-axis shows expression of genes for each sigma regulon. The violet (dark blue) line: regression line.


Average gene profiles of normalized - seq RNAP ChIPseq coverages from wt ( solid lines ) ∆ rnjA ( dashed lines ) strains were plotted for gene classes I - IV ( n = 762 , 315 , 553 1654 genes , respectively ) . Open reading frames were rescaled to 1 kb , upstream downstream regions of 0 . 2 kb were also included in plots . Data represent mean values of 3 independent experiments . pie - chart shows overall distribution of class I - IV other genes .

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

Average gene profiles of normalized RNA-seq and RNAP ChIPseq coverages from wt (solid lines) and ∆rnjA (dashed lines) strains were plotted for gene classes I-IV (n = 762, 315, 553 and 1654 genes, respectively). Open reading frames were rescaled to 1 kb, upstream and downstream regions of 0.2 kb were also included in the plots. Data represent mean values of 3 independent experiments. The pie-chart shows the overall distribution of class I-IV and other genes.


A . SIM of exponential B . subtilis . RNase J1 was fused to GFP ( green ) , RNAP to mCherry ( red ) . graph shows relative intensities at midsection ( along long axis ) ; SIM of is below . Yellow indicates colocalization of two .

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

A. SIM of exponential B. subtilis cells. RNase J1 was fused to GFP (green), RNAP to mCherry (red). The graph shows relative fluorescence intensities at the cell midsection (along the long axis); SIM of the cell is below. Yellow indicates colocalization of the two proteins.


B . Pull - down with RNase J1 - 8xHis tag RNAP - 10xHis tag detection of with antibodies . Lanes 1 , 2 - RNase J1 - 8xHis was used to pull down RNAP ; lanes 3 , 4 - RNAP - 10xHis was used to pull down RNase J1 ; lanes 5 , 6 - purified were used as markers ; lanes 7 , 8 - strains without His - tagged were used as negative controls to demonstrate specificity of interaction . M , molecular marker ; EX , exponential phase ; STA , stationary phase .

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

B. Pull-down with RNase J1-8xHis tag and RNAP-10xHis tag and detection of the proteins with antibodies. Lanes 1, 2 - RNase J1-8xHis was used to pull down RNAP; lanes 3, 4 - RNAP-10xHis was used to pull down RNase J1; lanes 5, 6 - purified proteins were used as markers; lanes 7, 8 - strains without His-tagged proteins were used as negative controls to demonstrate the specificity of the interaction. M, molecular size marker; EX, exponential phase; STA, stationary phase.


C . Pull - down with RNase J1 - 8xHis tag from stationary phase samples then either were ( lane 9 ) or were not ( lane 10 ) treated with RNase A to detect whether interaction was via . Lanes 11 , 12 - purified were used as markers . experiment was performed three times ( biological replicates ) with same result .

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

C. Pull-down with RNase J1-8xHis tag from stationary phase cells The samples then either were (lane 9) or were not (lane 10) treated with RNase A to detect whether the interaction was via RNA. Lanes 11, 12 - purified proteins were used as markers. The experiment was performed three times (biological replicates) with the same result.


B . Representative data - polyacrylamide gel ( experiment was performed 3x with same results ) . Lane 1 , full ( 33 nt ) labeled ; lane 2 , same as lane 1 but it included also incubation with RNase J1 ( J1 ) , 17 - 18 nt long fragments ( RNase J1 stopped by RNAP ; indicated with asterisks ) < 5 nt fragments ( released from TC into buffer - indicative of TC disassembly ) are shown ; lane 3 , same as lane 1 but included also incubation with RNase R ( R ) ; lanes 4 5 , TCs were denatured by heat prior to RNase addition to demonstrate activity cleavage patterns of both enzymes ; lanes 6 7 , same as lanes 2 3 but buffer was washed off ( TCs were retained by streptavidin beads ) to demonstrate which fragments were associated with TC ; lanes 8 , 9 ( M1 , M2 ) Mw marker generated by treating 30 nt with alkali formamide ( M1 - 4 min treatment , M2 - 7 min treatment ) . As reported in ( Costanzo et al . , 2016 ) [ references therein ] , cleavage by alkali or formamide leaves phosphate group of attacked phosphodiester bond bound at 3 ' , initially in 2 ' , 3 ' cyclic form ( upper band in band couples ) . This successively opens ( lower band in band couples ) yielding a double - banded pattern for short oligoribonucleotides .

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

B. Representative primary data - polyacrylamide gel (the experiment was performed 3x with the same results). Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), 17-18 nt long fragments (RNase J1 stopped by RNAP; indicated with asterisks) and <5 nt fragments (RNA released from TC into buffer - indicative of TC disassembly) are shown; lane 3, the same as lane 1 but included also incubation with RNase R (R); lanes 4 and 5, TCs were denatured by heat prior to RNase addition to demonstrate the activity and cleavage patterns of both enzymes; lanes 6 and 7, the same as lanes 2 and 3 but the buffer was washed off (TCs were retained by streptavidin beads) to demonstrate which RNA fragments were associated with TC; lanes 8, 9 (M1, M2) Mw marker generated by treating the 30 nt RNA with alkali and formamide (M1-4 min treatment, M2-7 min treatment). As reported in (Costanzo et al., 2016) [and references therein], the cleavage by alkali or formamide leaves the phosphate group of the attacked phosphodiester bond bound at 3', initially in the 2',3' cyclic form (upper band in the band couples). This successively opens (lower band in the band couples) yielding a double-banded pattern for short oligoribonucleotides.


A . data - polyacrylamide gel - a representative result . For description of bands / fragments see next panel legend . Asterisks indicate STUBS ( 16 - 19 nt ) . B . A schematic representation of quantitation of fragments . OUTSIDE ( 16 - 30 nt ) are fragments that originated by digestion of full by RNases that were stopped either before reaching or at point of reaching RNAP ; STUBS ( 16 - 19 nt ) are fragments that originated by digestion of full that were stopped at point of reaching RNAP ; INSIDE ( < 16 nt ) are fragments ( oligonucleotides ) that could be only generated after disassembly of by torpedo mechanism . red color indicates parts of that were digested . C . Quantitation of three independent experiments . " Full " indicates remaining undigested . " OUTSIDE " , " INSIDE " are fragments as explained in ( B ) indicated in ( A ) . bars represent 100 % ( fragments ) . black - grey - boxes indicate of each fragment group ( in % ) . error bars indicate ± for each group of fragments calculated from three biological replicates .

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A. Primary data - polyacrylamide gel - a representative result. For description of bands/fragments see next panel legend. Asterisks indicate STUBS (16-19 nt). B. A schematic representation of quantitation of fragments. OUTSIDE (16-30 nt) are RNA fragments that originated by digestion of the full length RNA by RNases that were stopped either before reaching or at the point of reaching RNAP; STUBS (16-19 nt) are RNA fragments that originated by digestion of the full length RNA that were stopped at the point of reaching RNAP; INSIDE (< 16 nt) are RNA fragments (oligonucleotides) that could be only generated after the disassembly of the complex by the torpedo mechanism. The red color indicates parts of RNA that were digested. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments as explained in (B) and indicated in (A). The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments calculated from three biological replicates.


A . TCs were assembled on - scaffolds ( was biotinylated attached to streptavidin coated magnetic beads ) ; TCs were then divided into three tubes challenged with buffer ( " - " , mock treatment ) or RNase J1 of Xrn1 . Dissociation of RNAP from was monitored by detecting RNAP with anti - β antibody in two fractions : B - beads ( RNAP still bound to ) , R - released ( free in buffer ) . gel strip shows representative data ( Western blot ) . graph shows averages ( bars ) of two experiments ( biological replicates ) error bars show range . combined signal for B + R for each treatment was set as 100 % .

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A. TCs were assembled on DNA-RNA scaffolds (DNA was biotinylated and attached to streptavidin coated magnetic beads); TCs were then divided into three tubes and challenged with buffer ("-", mock treatment) or RNase J1 of Xrn1. Dissociation of RNAP from DNA was monitored by detecting RNAP with anti-β antibody in two fractions: B - beads (RNAP still bound to DNA), R - released (free in buffer). The gel strip shows representative primary data (Western blot). The graph shows averages (the bars) of two experiments (biological replicates) and the error bars show the range. The combined signal for B+R for each treatment was set as 100 %.


B . data - polyacrylamide gel . RNAP from E . coli . Lane 1 , full ( 33 nt ) labeled ; lane 2 , same as lane 1 but it included also incubation with RNase J1 ( J1 ) , lane 3 , same as lane 1 but included also incubation with Xrn1 . C . Quantitation of three independent experiments . " Full " indicates remaining undigested . " OUTSIDE " , " INSIDE " are fragments bars represent 100 % ( fragments ) . black - grey - boxes indicate of each fragment group ( in % ) . error bars indicate ± for each group of

O O O O O O O O O O O O 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 O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Primary data - polyacrylamide gel. RNAP from E. coli. Lane 1, the full length (33 nt) labeled RNA; lane 2, the same as lane 1 but it included also incubation with RNase J1 (J1), lane 3, the same as lane 1 but included also incubation with Xrn1. C. Quantitation of three independent experiments. "Full length" indicates the remaining undigested RNA. "OUTSIDE", "INSIDE" are fragments The bars represent 100 % (all fragments). The black-grey-white boxes indicate the percentage of each fragment group (in %). The error bars indicate ± SEM for each group of fragments


A . Exponential of indicated strains ( below bars ) were plated onto LB agar either were , or were not UV - irradiated . After overnight incubation , CFU were counted . UV sensitivity of mutant strains ( KO ) was then calculated as between irradiated vs non - irradiated normalized to this from wt As a consequence , wt is 1 . experiment was conducted 4x ( biological replicates ) bars show geometric mean . p - values , shown above bars , were computed using two - unpaired t - for logarithms of ratios . error bars show ± ( computed on log scale ) .

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A. Exponential cells of indicated strains (below the bars) were plated onto LB agar and either were, or were not UV-irradiated. After overnight incubation, CFU were counted. UV sensitivity of the mutant strains (KO) was then calculated as the ratio between irradiated vs non-irradiated cells and normalized to this ratio from the wt strain As a consequence, the wt ratio is 1. The experiment was conducted 4x (biological replicates) and the bars show the geometric mean. The p-values, shown above the bars, were computed using two-tailed unpaired t-test for logarithms of the ratios. The error bars show ±SEM (computed on the log scale).


B . Relative expression ( mRNA ) of helD , mfd , rho , greA , nusA , nusB , nusG in ∆ rnjA ( normalized to wt [ set as 1 ] ) .

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B. Relative expression (mRNA) of helD, mfd, rho, greA, nusA, nusB, and nusG in the ∆rnjA strain (normalized to wt [set as 1]).


( A ) HeLa were infected with Listeria for 0 . 5 - 4 h , lysates were analysed by blotting using indicated antibodies .

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(A) HeLa cells were infected with Listeria for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies.


( B ) HeLa left unstimulated ( CTR ) or infected with Listeria for 1 or 3 h , analysed by IF using antibodies against mTOR LAMP2 .

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(B) HeLa cells left unstimulated (CTR) or infected with Listeria for 1 or 3 h, analysed by IF using antibodies against mTOR and LAMP2.


( C ) of Listeria ‐ infected displaying mTOR localization to LAMP2 + vesicles . Values are means ± s . e . m . n = 3 . * * * P0 . 001 * * P0 . 01 over uninfected .

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(C) Percentage of Listeria‐infected cells displaying mTOR localization to LAMP2+ vesicles. Values are means±s.e.m. n=3. ***P0.001 and **P0.01 over uninfected.


( D , E ) HeLa were infected with Listeria for 0 . 5 - 4 h , lysates were analysed by blotting using indicated antibodies .

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(D, E) HeLa cells were infected with Listeria for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies.


( F ) qPCR of ATF3 induction following infection of HeLa with Listeria for 1 - 4 h . Values are means ± s . e . m . n = 3 . Scale bars : 10 μm . Source data for this figure is available on online supplementary information page .

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(F) qPCR analysis of ATF3 induction following infection of HeLa cells with Listeria for 1-4 h. Values are means±s.e.m. n=3. Scale bars: 10 μm.Source data for this figure is available on the online supplementary information page.


( A ) HeLa were infected with Listeria wild ‐ type ( WT ) or LLO / PlcA / PlcB triple knockout ( TKO ) mutant for 0 . 5 - 4 h , lysates were analysed by blotting using indicated antibodies .

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(A) HeLa cells were infected with Listeria wild‐type (WT) or the LLO/PlcA/PlcB triple knockout (TKO) mutant strain for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies.


( B ) qPCR of ATF3 IL ‐ 8 induction following infection of HeLa with Listeria WT or TKO for 1 - 4 h . Values are means ± s . e . m . n = 3 .

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(B) qPCR analysis of ATF3 and IL‐8 induction following infection of HeLa cells with Listeria WT or TKO for 1-4 h. Values are means±s.e.m. n=3.


( C ) HeLa left unstimulated ( CTR ) or infected with Listeria TKO for 1 h , analysed by IF using antibodies against mTOR LAMP2 .

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(C) HeLa cells left unstimulated (CTR) or infected with Listeria TKO for 1 h, analysed by IF using antibodies against mTOR and LAMP2.


( D , E ) of infected with either WT or TKO Listeria strains displaying one or several LAMP2 + ( D ) or NDP52 + ( E ) Listeria vesicles . Values are means ± s . e . m . n = 3 . * P0 . 05 ; * * P0 . 01 . Scale bars : 10 μm . Source data for this figure is available on online supplementary information page .

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(D, E) Percentage of cells infected with either WT or TKO Listeria strains displaying one or several LAMP2+ (D) or NDP52+ (E) Listeria vesicles. Values are means±s.e.m. n=3. *P0.05; **P0.01. Scale bars: 10 μm.Source data for this figure is available on the online supplementary information page.


( A ) qPCR of ATF3 induction following infection of HeLa with WT , TKO , PlcA / B − or LLO − Listeria strains for 1 - 4 h . Values are means ± s . e . m . n = 3 .

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(A) qPCR analysis of ATF3 induction following infection of HeLa cells with WT, TKO, PlcA/B− or LLO− Listeria strains for 1-4 h. Values are means±s.e.m. n=3.


( B ) HeLa were infected with WT , PlcA / B − or LLO − Listeria strains for 0 . 5 - 4 h , lysates were analysed by blotting using indicated antibodies .

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(B) HeLa cells were infected with WT, PlcA/B− or LLO− Listeria strains for 0.5-4 h, and lysates were analysed by blotting using indicated antibodies.


( C ) HeLa left unstimulated ( CTR ) or infected with WT , PlcA / B − or LLO − Listeria strains for 1 h , analysed by IF using antibodies against mTOR LAMP2 . arrow shows a Listeria vacuole positive for mTOR LAMP2 , arrowheads indicate mTOR ‐ LAMP2 ‐ positive late endosomes or lysosomes .

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(C) HeLa cells left unstimulated (CTR) or infected with WT, PlcA/B− or LLO− Listeria strains for 1 h, analysed by IF using antibodies against mTOR and LAMP2. The arrow shows a Listeria vacuole positive for mTOR and LAMP2, and arrowheads indicate mTOR‐ and LAMP2‐positive late endosomes or lysosomes.


( D , E ) of infected with WT , PlcA / B − or LLO − Listeria strains displaying one or several LAMP2 + ( D ) or NDP52 + ( E ) Listeria vesicles . Values are means ± s . e . m . n = 3 . * * P0 . 01 .

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(D, E) Percentage of cells infected with WT, PlcA/B− or LLO− Listeria strains displaying one or several LAMP2+ (D) or NDP52+ (E) Listeria vesicles. Values are means±s.e.m. n=3. **P0.01.


( F ) of infected with WT Listeria , in presence ( black bars ) or absence ( bars ) of rapamycin , displaying one or several GFP ‐ LC3 + Listeria autophagosomes . Values are means ± s . e . m . n = 2 . Scale bars : 5 μm . Source data for this figure is available on online supplementary information page .

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(F) Percentage of cells infected with WT Listeria, in the presence (black bars) or absence (white bars) of rapamycin, displaying one or several GFP‐LC3+ Listeria autophagosomes. Values are means±s.e.m. n=2. Scale bars: 5 μm.Source data for this figure is available on the online supplementary information page.


( A ) HeLa left unstimulated ( CTR ) or infected with Listeria WT for 1 - 4 h , or with Listeria TKO for 4 h with analysed by IF using an antibody against NDP52 .

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(A) HeLa cells left unstimulated (CTR) or infected with Listeria WT for 1-4 h, or with Listeria TKO for 4 h and with analysed by IF using an antibody against NDP52.


( B ) Higher magnification view of selected from ( A ) showing that NDP52 + granules generally form at vicinity of Listeria .

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(B) Higher magnification view of the selected area from (A) showing that NDP52+ granules generally form at the vicinity of cytosolic Listeria.


( C ) of infected with Listeria WT for 1 - 4 h , or with Listeria TKO for 4 h , displaying one or several NDP52 + granules . Values are means ± s . e . m . n = 3 .

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(C) Percentage of cells infected with Listeria WT for 1-4 h, or with Listeria TKO for 4 h, displaying one or several NDP52+ granules. Values are means±s.e.m. n=3.


( D ) of infected for 4 h with different Listeria mutants as indicated displaying one or several NDP52 + granules . Values are means ± s . e . m . n = 3 .

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(D) Percentage of cells infected for 4 h with different Listeria mutants as indicated displaying one or several NDP52+ granules. Values are means±s.e.m. n=3.


( E ) HeLa infected with Listeria WT , PlcA / B − or PlcA / B − ectopically expressing PlcA for 3 h , analysed by IF using an antibody against NDP52 .

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(E) HeLa cells infected with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA for 3 h, analysed by IF using an antibody against NDP52.


( F , G ) of infected for 3 h with Listeria WT , PlcA / B − or PlcA / B − ectopically expressing PlcA displaying one or several NDP52 + granules ( F ) or entrapped in NDP52 + autophagosomes ( G ) . Values are means ± s . e . m . n = 2 . Scale bars : 5 μm .

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(F, G) Percentage of cells infected for 3 h with Listeria WT, PlcA/B− or PlcA/B− ectopically expressing PlcA displaying one or several NDP52+ granules (F) or bacteria entrapped in NDP52+ autophagosomes (G). Values are means±s.e.m. n=2. Scale bars: 5 μm.


( A ) HeLa transfected with GFP ‐ LC3 infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52 .

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(A) HeLa cells transfected with GFP‐LC3 and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52.


( B ) Murine embryonic fibroblasts ( MEFs ) from WT or ATG16L1 ‐ deficient ( ATG16L1 KO ) mice infected with Listeria WT for 4 h were analysed by IF using an antibody against ubiquitinated ( Ubi ) .

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(B) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected with Listeria WT for 4 h were analysed by IF using an antibody against ubiquitinated proteins (Ubi).


( C , D ) HeLa infected with Listeria WT for 4 h analysed by IF using antibodies against NDP52 WIPI ‐ 2 ( C ) or Galectin ‐ 8 ( Gal ‐ 8 ) calreticulin ( D ) .

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(C, D) HeLa cells infected with Listeria WT for 4 h analysed by IF using antibodies against NDP52 and WIPI‐2 (C) or Galectin‐8 (Gal‐8) and calreticulin (D).


( E ) HeLa infected with Listeria WT for 4 h , in absence or presence of Wortmannin added during last hour of infection , analysed by IF using antibodies against NDP52 WIPI ‐ 2 . Scale bars : 5 μm for panels except ( D ) ( 1 . 5 μm ) .

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(E) HeLa cells infected with Listeria WT for 4 h, in the absence or presence of Wortmannin added during the last hour of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. Scale bars: 5 μm for all panels except (D) (1.5 μm).


( A ) HeLa were transfected with GFP ‐ RFP ‐ LC3 either were left uninfected ( t = 0 ) or were infected with Listeria WT or PlcA / B − for 1 - 4 h . GFP + RFP + GFP − RFP + appear yellow red , respectively . of infected with Listeria WT or PlcA / B − for 1 - 4 h displaying GFP − RFP + red were quantified . Values are means ± s . e . m . n = 3 . * * P0 . 01 ; * * * P0 . 001 .

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(A) HeLa cells were transfected with GFP‐RFP‐LC3 and either were left uninfected (t=0) or were infected with Listeria WT or PlcA/B− for 1-4 h. GFP+ RFP+ and GFP− RFP+ puncta appear yellow and red, respectively. Percentage of cells infected with Listeria WT or PlcA/B− for 1-4 h displaying GFP− RFP+ red puncta were quantified. Values are means±s.e.m. n=3. **P0.01; ***P0.001.


( B ) HeLa left unstimulated ( CTR ) or infected with Listeria WT or PlcA / B − for 4 h were lysed PI3P measured by competitive ELISA .

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(B) HeLa cells left unstimulated (CTR) or infected with Listeria WT or PlcA/B− for 4 h were lysed and PI3P levels measured by competitive ELISA.


( C ) HeLa transfected with GFP ‐ PKCδ infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52 . arrow indicates an NDP52 + granule .

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(C) HeLa cells transfected with GFP‐PKCδ and infected with Listeria WT for 4 h were analysed by IF using an antibody against NDP52. The arrow indicates an NDP52+ granule.


( D ) HeLa infected with Listeria PlcA / B − for 3 h , in absence or presence of Wortmannin added during last 10 min of infection , analysed by IF using antibodies against NDP52 WIPI ‐ 2 . arrow indicates an NDP52 + granule . Scale bars : 5 μm .

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

(D) HeLa cells infected with Listeria PlcA/B− for 3 h, in the absence or presence of Wortmannin added during the last 10 min of infection, analysed by IF using antibodies against NDP52 and WIPI‐2. The arrow indicates an NDP52+ granule. Scale bars: 5 μm.


( A , B ) HeLa infected for 4 h with Listeria WT expressing GFP ( WT GFP ) , Listeria PlcA / B − expressing mCherry ( PlcA / B − mCherry ) , or both Listeria strains at a 1 : 1 , were analysed by IF using an antibody against NDP52 ( A ) . ( B ) of of displaying NDP52 + PlcA / B − mCherry from experiments described in ( A ) , in infected with either PlcA / B − mCherry alone or WT GFP PlcA / B − mCherry ( co ‐ infection ) . Values are means ± s . e . m . n = 3 . * * P0 . 01 .

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

(A, B) HeLa cells infected for 4 h with Listeria WT expressing GFP (WT GFP), Listeria PlcA/B− expressing mCherry (PlcA/B− mCherry), or both Listeria strains at a 1:1 ratio, were analysed by IF using an antibody against NDP52 (A). (B) Quantification of the percentage of cells displaying NDP52+ PlcA/B− mCherry from the experiments described in (A), in cells infected with either PlcA/B− mCherry alone or WT GFP and PlcA/B−mCherry (co‐infection). Values are means±s.e.m. n=3. **P0.01.