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 .

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

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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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (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 ) . 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) , 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 .

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

O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the 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 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 ) , and SYCP3 ( red ) , and stained with DAPI ( blue ) . Zoomed images of centrosomes below each panel . 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 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 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 .

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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 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 .

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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 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 .

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 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 .

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 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 .

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

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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 ) , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . Quantification of the 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) , 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 .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) , and stained with DAPI ( blue ) . Plk1 and 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 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 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 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 .

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

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 ) , and stained with DAPI ( blue ) . Zoom images of the centrosomes are presented to the right . 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

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 . 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 .

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

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

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 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 .

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

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 ) , 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 .

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

E, F. 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 ) , 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 .

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

O O O O O O O O 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 . 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

O O O O O O O O O O O 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. 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 - 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

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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 ) 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 ) .

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

(F, 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 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 ) .

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

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

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


( 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 .

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

(B, C) 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 - ALL cells from CTRL d12 and IFNγ d12 conditions , extracted from the scRNAseq dataset ( * * * * p ≤ 0 . 0001 , Welch ' s test ) .

(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 - 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 ) .

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

(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 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 ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) 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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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

(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 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

O O O O O O O O O O O O O O O O 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) 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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

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

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

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

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

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

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

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


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 .

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

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.


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 .

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


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 .

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

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


( 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 .

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(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 , 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 .

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(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 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 .

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(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 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 .

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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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 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 % .

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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 . 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

O 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 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

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 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 ) .

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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 , and nusG in the ∆ rnjA strain ( 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 cells were infected with Listeria for 0 . 5 - 4 h , and 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 cells left unstimulated ( CTR ) or infected with Listeria for 1 or 3 h , analysed by IF using antibodies against mTOR and 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 ) 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 .

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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 cells were infected with Listeria for 0 . 5 - 4 h , and 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 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 .

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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 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 .

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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 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 .

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

(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 ) 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 .

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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 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 .

(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 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 .

(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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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

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


( 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 .

(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 ) 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 .

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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 cells 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 ) 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 .

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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 cells transfected with GFP ‐ LC3 and 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 proteins ( 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 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 ) .

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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 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 ) .

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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 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 .

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

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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 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 .

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(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 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 .

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(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.


( C ) Murine embryonic fibroblasts ( MEFs ) from WT or ATG16L1 ‐ deficient ( ATG16L1 KO ) mice infected for 4 h with WT GFP or Listeria PlcA / B − Listeria , either separately ( Single ) or in combination ( Mix ) were lysed and colony forming units ( CFUs ) determined on agar plates containing appropriate selection markers . Each point represents the value from individual independent experiments . * * P0 . 01 ; * P0 . 05 . Scale bars : 2 . 5 μm .

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(C) Murine embryonic fibroblasts (MEFs) from WT or ATG16L1‐deficient (ATG16L1 KO) mice infected for 4 h with WT GFP or Listeria PlcA/B− Listeria, either separately (Single) or in combination (Mix) were lysed and colony forming units (CFUs) determined on agar plates containing appropriate selection markers. Each point represents the value from individual independent experiments. **P0.01; *P0.05. Scale bars: 2.5 μm.


METTL14 is arginine methylated at its C - terminal IDR in cells . HEK293 cells expressing Flag - tagged FL or C - terminal IDR - truncated ( 1 - 400 ) METTL14 were lysed and immunoprecipitated with an anti - Flag antibody . Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA . the black triangles indicate arginine methylated - METTL14 ; open triangles indicate the immunoprecipitated METTL14 proteins .

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METTL14 is arginine methylated at its C-terminal IDR in cells. HEK293 cells expressing Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14 were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA. the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins.


The C - terminal IDR of METTL14 is essential for its interaction with PRMT1 . HEK293 cells were transfected with GFP - tagged PRMT1 and Flag - tagged FL or C - terminal IDR - truncated ( 1 - 400 ) METTL14 . IP was performed using an anti - GFP antibody , and Western blot analysis was performed using anti - GFP and anti - Flag antibodies .

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The C-terminal IDR of METTL14 is essential for its interaction with PRMT1. HEK293 cells were transfected with GFP-tagged PRMT1 and Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14. IP was performed using an anti-GFP antibody, and Western blot analysis was performed using anti-GFP and anti-Flag antibodies.


Knockdown of PRMT1 expression reduces METTL14 arginine methylation . HEK293 cells were transfected with control siRNA ( siCtrl ) and the siRNA targeting PRMT1 ( siPRMT1 ) . METTL14 was immunoprecipitated from these cells , and its methylation level was detected by Western blot analysis using an anti - ASYM26 antibody . * indicates the location of the IgG heavy chain .

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Knockdown of PRMT1 expression reduces METTL14 arginine methylation. HEK293 cells were transfected with control siRNA (siCtrl) and the siRNA targeting PRMT1 (siPRMT1). METTL14 was immunoprecipitated from these cells, and its methylation level was detected by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG heavy chain.


Arginine methylation of the C - terminal IDR enhances the RNA methylation activity of the METTL14 / METTL3 complex in vitro . In vitro RNA methylation assays were performed by incubating biotin - labeled RNA substrates with METTL3 / METTL14 methyltransferase complexes containing WT , hypomethylated ( MS023 ) , and arginine methylation - deficient ( RK ) mutant METTL14 in various concentrations ( 10 - 100 nM ) . The methylation status of the METTL3 / METTL14 complex was confirmed by Western blot analysis using an anti - ADMA antibody . Coomassie staining shows the purification of the enzyme complex . Enzymatic activity was measured in counts per minute ( c . p . m . ) using a scintillation counter . Data from three replicates were analyzed by Student ' s t - test and shown as mean ± SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant .

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

Arginine methylation of the C-terminal IDR enhances the RNA methylation activity of the METTL14/METTL3 complex in vitro. In vitro RNA methylation assays were performed by incubating biotin-labeled RNA substrates with METTL3/METTL14 methyltransferase complexes containing WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 in various concentrations (10-100 nM). The methylation status of the METTL3/METTL14 complex was confirmed by Western blot analysis using an anti-ADMA antibody. Coomassie staining shows the purification of the enzyme complex. Enzymatic activity was measured in counts per minute (c.p.m.) using a scintillation counter. Data from three replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01; ns, not significant.


Co - IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023 - treated HEK293 cells . Cells were treated with either DMSO or MS023 ( 1 μM ) for 48 h before they were lysed . IP was performed using control IgG and METTL14 antibodies , respectively . Western blot analysis was performed using anti - RNAPII , anti - ADMA , anti - METTL14 , and anti - METTL3 antibodies . Co - IP assays were performed to examine the involvement of RNA in the METTL14 - RNAPII interaction . Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP . Western blot analysis was performed using anti - RNAPII , anti - METTL3 , and anti - METTL14 antibodies .

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Co-IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023-treated HEK293 cells. Cells were treated with either DMSO or MS023 (1 μM) for 48 h before they were lysed. IP was performed using control IgG and METTL14 antibodies, respectively. Western blot analysis was performed using anti-RNAPII, anti-ADMA, anti-METTL14, and anti-METTL3 antibodies.   Co-IP assays were performed to examine the involvement of RNA in the METTL14-RNAPII interaction. Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP. Western blot analysis was performed using anti-RNAPII, anti-METTL3, and anti-METTL14 antibodies.