( h ) Quantification of pre - mRNA co - precipitated with Prp5 and U2 . For each lane in ( g ) , pre - mRNA co - precipitated with Prp5 ( Wild - type or GAR ) was normalized against the Prp5 ( WT / GAR ) band in the same lane ( detected by Anti - FLAG ) [ ( intensity of pre - mRNA ) / ( intensity of WT / GAR Prp5 ) ] . Lanes 2 , 3 , and 4 ( see ( g ) ) were then further normalized against lane 1 ( see ( g ) ) [ ( the value of normalized pre - mRNA from lane 2 , 3 or 4 in ( g ) ) / ( the value of normalized pre - mRNA from lane 1 in ( g ) ) ] . The numbers at the bottom of ( h ) correspond to the lane numbers of ( g ) . Quantification was based on three independent experiments .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Quantification of pre-mRNA co-precipitated with Prp5 and U2. For each lane in (g), pre-mRNA co-precipitated with Prp5 (Wild-type or GAR) was normalized against the Prp5 (WT/GAR) band in the same lane (detected by Anti-FLAG) [(intensity of pre-mRNA) / (intensity of WT/GAR Prp5)]. Lanes 2,3,and 4 (see (g)) were then further normalized against lane 1 (see (g)) [(the value of normalized pre-mRNA from lane 2, 3 or 4 in (g)) / (the value of normalized pre-mRNA from lane 1 in (g))]. The numbers at the bottom of (h) correspond to the lane numbers of (g). Quantification was based on three independent experiments.


( a ) Prp5 ' s ATPase activity assay using synthetic U2 . The 5 ' sequence of S . cerevisiae U2 is shown ( top ) . The three ψs ( 35 , 42 and 44 ) as well as the branch site recognition sequence ( underlined ) are indicated . In the ATPase assay ( bottom ) , no RNA ( lane 3 ) , or an equal amount of LiCl - precipitated RNA ( lane 1 ) or tRNA ( lane 2 ) , a short U2 fragment ( nts 1 - 76 ) ( lane 4 ) , a fully pseudouridylated short U2 fragment ( nts 1 - 76 ) ( lane 5 ) , a long U2 fragment ( nts 1 - 120 ) ( lane 6 ) , or a fully pseudouridylated long U2 fragment ( nts 1 - 120 ) ( lane 7 ) , was used . The un - hydrolyzed ATP and hydrolyzed product ADP are indicated . Lanes 1 and 2 , lanes 3 - 5 , and lanes 6 and 7 are from separate gels .

O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(a) Prp5's ATPase activity assay using synthetic U2. The 5' sequence of S. cerevisiae U2 is shown (top). The three ψs (35, 42 and 44) as well as the branch site recognition sequence (underlined) are indicated. In the ATPase assay (bottom), no RNA (lane 3), or an equal amount of LiCl-precipitated RNA (lane 1) or tRNA (lane 2), a short U2 fragment (nts 1-76) (lane 4), a fully pseudouridylated short U2 fragment (nts 1-76) (lane 5), a long U2 fragment (nts 1-120) (lane 6), or a fully pseudouridylated long U2 fragment (nts 1-120) (lane 7), was used. The un-hydrolyzed ATP and hydrolyzed product ADP are indicated. Lanes 1 and 2, lanes 3-5, and lanes 6 and 7 are from separate gels.


( b ) Quantification of Prp5 ' s ATPase activity activated by in vitro transcribed U2 . Prp5 ' s ATPase activity shown in ( a ) was calculated using the formula ( ADP ) / ( ADT + ATP ) . The numbers at the bottom of ( b ) correspond to the lane numbers of ( a ) . Quantification was based on three independent experiments .

O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O 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 Prp5's ATPase activity activated by in vitro transcribed U2. Prp5's ATPase activity shown in (a) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (b) correspond to the lane numbers of (a). Quantification was based on three independent experiments.


( c ) U2 - Prp5 binding assay . FLAG - Prp5 and a radiolabeled short ( nts 1 - 76 ) ( lanes 1 and 2 ) or long ( nts 1 - 120 ) ( lanes 5 and 6 ) U2 fragment , or FLAG - Prp5 and a radiolabeled fully pseudouridylated short ( nts 1 - 76 ) ( lanes 3 and 4 ) or long ( nts 1 - 120 ) ( lanes 7 and 8 ) U2 fragment , were incubated under the conditions used for ATPase activity assay . Anti - FLAG IP was then carried out . Co - precipitated ( lanes 2 , 4 , 6 and 8 ) and un - precipitated ( lanes 1 , 3 , 5 and 7 ) U2 RNAs were analyzed by electrophoresis . The U2 band is indicated .

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O 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-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O O O O

(c) U2-Prp5 binding assay. FLAG-Prp5 and a radiolabeled short (nts 1-76) (lanes 1 and 2) or long (nts 1-120) (lanes 5 and 6) U2 fragment, or FLAG-Prp5 and a radiolabeled fully pseudouridylated short (nts 1-76) (lanes 3 and 4) or long (nts 1-120) (lanes 7 and 8) U2 fragment, were incubated under the conditions used for ATPase activity assay. Anti-FLAG IP was then carried out. Co-precipitated (lanes 2, 4, 6 and 8) and un-precipitated (lanes 1, 3, 5 and 7) U2 RNAs were analyzed by electrophoresis. The U2 band is indicated.


( d ) Quantification of Prp5 - U2 binding . Relative intensities of U2 bands shown in ( c ) were calculated [ setting the un - precipitated uridine - containing U2 ( lane 1 and 5 ) to 1 ] . The numbers at the bottom of ( d ) correspond to the lane numbers of ( c ) . Quantification was based on three independent experiments .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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) Quantification of Prp5-U2 binding. Relative intensities of U2 bands shown in (c) were calculated [setting the un-precipitated uridine-containing U2 (lane 1 and 5) to 1]. The numbers at the bottom of (d) correspond to the lane numbers of (c). Quantification was based on three independent experiments.


( e ) Prp5 ' s ATPase activity assay using cellular U2 . U2 RNA isolated from the wild - type strain ( lanes 2 and 4 ) or from the snR81Δ pus1Δ strain ( lanes 6 and 8 ) was used for the ATPase activity assay . In odd - numbered lanes , no RNA was added . Both the wild - type Prp5 ( lanes 1 - 4 ) and the mutant Prp5 ( GAR ) ( lanes 5 - 8 ) were tested .

O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

(e) Prp5's ATPase activity assay using cellular U2. U2 RNA isolated from the wild-type strain (lanes 2 and 4) or from the snR81Δ pus1Δ strain (lanes 6 and 8) was used for the ATPase activity assay. In odd-numbered lanes, no RNA was added. Both the wild-type Prp5 (lanes 1-4) and the mutant Prp5 (GAR) (lanes 5-8) were tested.


( f ) Quantification of Prp5 ' s ATPase activity activated by U2 from cells . Prp5 ' s ATPase activity shown in ( e ) was calculated using the formula ( ADP ) / ( ADT + ATP ) . The numbers at the bottom of ( f ) correspond to the lane numbers of ( e ) . Quantification was based on four independent experiments .

O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O 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) Quantification of Prp5's ATPase activity activated by U2 from cells. Prp5's ATPase activity shown in (e) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (f) correspond to the lane numbers of (e). Quantification was based on four independent experiments.


( g ) U2 level in the ATPase activity reactions . RNAs from lanes 2 and 4 in ( c ) were recovered , and U2 level was measured using primer - extension . Lanes 1 - 5 , a titration of U2 snRNA . Lane 6 , U2 from the wild - type strain [ lane 2 of ( c ) ] . Lane 7 , U2 from the snR81Δ pus1Δ strain [ lane 4 of ( c ) ] .

O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O

(g) U2 level in the ATPase activity reactions. RNAs from lanes 2 and 4 in (c) were recovered, and U2 level was measured using primer-extension. Lanes 1-5, a titration of U2 snRNA. Lane 6, U2 from the wild-type strain [lane 2 of (c)]. Lane 7, U2 from the snR81Δ pus1Δ strain [lane 4 of (c)].


( a ) Native gel analysis of pre - splicing complexes . Pre - splicing complex assembly was carried out in the test tube with Prp5 - reconstituted cell extracts and labeled pre - mRNA . Lane 1 , Prp5 - depleted wild - type cell extract . Lane 2 , Prp5 - depleted wild - type cell extract , plus wild - type Prp5 . Lane 3 , Prp5 - depleted snR81Δ pus1Δ cell extract , plus wild - type Prp5 . Lane 4 , Prp5 - depleted wild - type cell extract , plus mutant Prp5 ( GAR ) . Lane 5 , Prp5 - depleted snR81Δ pus1Δ cell extract , plus mutant Prp5 ( GAR ) . The pre - splicing complexes A and B are indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O

(a) Native gel analysis of pre-splicing complexes. Pre-splicing complex assembly was carried out in the test tube with Prp5-reconstituted cell extracts and labeled pre-mRNA. Lane 1, Prp5-depleted wild-type cell extract. Lane 2, Prp5-depleted wild-type cell extract, plus wild-type Prp5. Lane 3, Prp5-depleted snR81Δ pus1Δ cell extract, plus wild-type Prp5. Lane 4, Prp5-depleted wild-type cell extract, plus mutant Prp5 (GAR). Lane 5, Prp5-depleted snR81Δ pus1Δ cell extract, plus mutant Prp5 (GAR). The pre-splicing complexes A and B are indicated.


( b ) DMS in vivo probing . After being exposed to DMS , yeast cells were lysed , total RNA collected , and primer - extension analysis carried out ( left panel ) . Lane 1 , cells that were not exposed to DMS . Lane 2 , wild - type cells exposed to DMS . Lane 3 , snR81Δ pus1Δ cells exposed to DMS . Lane 4 , snR81Δ pus1Δ cells , transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44 , and exposed to DMS . Lane 5 , snR81Δ pus1Δ cells , transformed with a plasmid carrying a control guide RNA gene with random guide sequences , and exposed to DMS . The nucleotides in parentheses are DMS - modified C and A residues that are one nucleotide after the actual primer - extension stops ( indicated by the arrows ) . A partial U2 sequence ( along with the BSL structure ) is shown ( right panel ) . DMS - modified adenosine and cytosine in the BSL are also indicated .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

(b) DMS in vivo probing. After being exposed to DMS, yeast cells were lysed, total RNA collected, and primer-extension analysis carried out (left panel). Lane 1, cells that were not exposed to DMS. Lane 2, wild-type cells exposed to DMS. Lane 3, snR81Δ pus1Δ cells exposed to DMS. Lane 4, snR81Δ pus1Δ cells, transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44, and exposed to DMS. Lane 5, snR81Δ pus1Δ cells, transformed with a plasmid carrying a control guide RNA gene with random guide sequences, and exposed to DMS. The nucleotides in parentheses are DMS-modified C and A residues that are one nucleotide after the actual primer-extension stops (indicated by the arrows). A partial U2 sequence (along with the BSL structure) is shown (right panel). DMS-modified adenosine and cytosine in the BSL are also indicated.


( c ) Residue accessibility quantification . Relative accessibility ( relative band intensity ) was quantified based on three independent experiments . The intensity of every band in each lane was normalized against the C46 band ( the least changed band ; compare lanes 2 - 5 in ( b ) ) in its respective lane [ ( intensity of a band ) / ( intensity of C46 in the same lane ) ] . Each C46 - normailzed band in lanes 3 - 5 ( refer to ( b ) ) was then further normalized against its respective counterpart in lane 2 [ ( the value of a C46 - normalized band in lanes 3 - 5 ) / ( the value of its counterpart in lane 2 ) ] . The error bars represent the standard deviations of the measurements .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Residue accessibility quantification. Relative accessibility (relative band intensity) was quantified based on three independent experiments. The intensity of every band in each lane was normalized against the C46 band (the least changed band; compare lanes 2-5 in (b)) in its respective lane [(intensity of a band) / (intensity of C46 in the same lane)]. Each C46-normailzed band in lanes 3-5 (refer to (b)) was then further normalized against its respective counterpart in lane 2 [(the value of a C46-normalized band in lanes 3-5) / (the value of its counterpart in lane 2)]. The error bars represent the standard deviations of the measurements.


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( A ) Percentage body weight loss Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

B-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (A) Percentage body weight loss Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( B ) Relative amount of PR8 viral NP mRNA , by qPCR Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

B-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (B) Relative amount of PR8 viral NP mRNA, by qPCR Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( C ) Histology of lungs by H & E staining with pathology scores . Scale bars = 200 μm Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

B-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (C) Histology of lungs by H&E staining with pathology scores. Scale bars = 200 μm Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( D ) Absolute number of live CD45 + cells in whole lungs and BAL , by flow cytometry Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Statistical significanc was determined with unpaired student ' s T -

B-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (D) Absolute number of live CD45+ cells in whole lungs and BAL, by flow cytometry Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M Statistical significanc was determined with unpaired student's T-test


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( E ) Level of LDH in BAL , by LDH cytotoxicity assay Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

B-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (E) Level of LDH in BAL, by LDH cytotoxicity assay Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( F ) Expression of PDPN in lungs , by western blot Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M

B-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (F) Expression of PDPN in lungs, by western blot Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( G ) Total protein in BAL , by Bradford assay . ( H ) Concentration of albumin in BAL , by ELISA Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M . Statistical significanc was determined with unpaired student ' s T -

B-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (G) Total protein in BAL, by Bradford assay. (H) Concentration of albumin in BAL, by ELISA Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M. Statistical significanc was determined with unpaired student's T-test


( A ) Absolute cell counts from lungs of sub - lethally influenza virus infected mice following analysis by flow cytometry . After gating out alveolar macrophages by forward and side scatter , immune cells are defined as follows : Neutrophil ( Ly6G + CD11b + SiglecF - Ly6Cint ) , Eosinophil ( SiglecF + CD11c - / + F4 / 80 - CD64 - ) , Monocyte ( Ly6G - CD11b + MHCII - SiglecF - ) , Natural killer cell ( NK , B220 - / intNK1 . 1 + ) , B cell ( B220hiNK1 . 1 - ) , CD8 + T cell ( TCRβ + CD8 + ) , CD4 + T cell ( TCRβ + CD4 + ) Data information : Data are representative of three independent experiments with 3 - 4 mice per group and represented as mean ± S . E . M . Statistical significance in ( A ) was determined with unpaired student ' s T - test . n . s . = not significant

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-ORGANISM I-ORGANISM O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O 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) Absolute cell counts from lungs of sub-lethally influenza virus infected mice following analysis by flow cytometry. After gating out alveolar macrophages by forward and side scatter, immune cells are defined as follows: Neutrophil (Ly6G+CD11b+SiglecF-Ly6Cint), Eosinophil (SiglecF+CD11c-/+F4/80-CD64-), Monocyte (Ly6G-CD11b+MHCII-SiglecF-), Natural killer cell (NK, B220-/intNK1.1+), B cell (B220hiNK1.1-), CD8+ T cell (TCRβ+CD8+), CD4+ T cell (TCRβ+CD4+) Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M. Statistical significance in (A) was determined with unpaired student's T-test. n.s.= not significant


( B ) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub - lethal influenza virus infection . Numbers in plots indicate frequency of gated immune cells in each throughput Data information : Data are representative of three independent experiments with 3 - 4 mice per group and represented as mean ± S . E . M

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE O B-TISSUE O O O O O O O O O B-ORGANISM I-ORGANISM O O B-EXP_ASSAY O 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-ORGANISM O O O O O O O O O O O O

(B) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub-lethal influenza virus infection. Numbers in plots indicate frequency of gated immune cells in each throughput Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M


Mice were superinfected as described in ( A ) ( B ) Survival of mice following bacterial superinfection Data information : Data from ( B ) are combined from two independent experiments , n = 13 mice per experimental group All data are represented as mean ± S . E . M . Statistical significance was determined by logrank test in (

B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (B) Survival of mice following bacterial superinfection Data information: Data from (B) are combined from two independent experiments, n=13 mice per experimental group All data are represented as mean ± S.E.M. Statistical significance was determined by logrank test in (B


Mice were superinfected as described in ( A ) ( C ) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group All data are represented as mean ± S . E . M Statistical significance wa determined b Mann - Whitney U test in (

B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (C) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M Statistical significance wa determined b Mann-Whitney U test in (C


Mice were superinfected as described in ( A ) ( D ) Absolute immune cell counts in lungs , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group . All data are represented as mean ± S . E . M . Statistical significanc determined b unpaired student ' s T - test in (

B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (D) Absolute immune cell counts in lungs, assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significanc determined b unpaired student's T-test in (D


Mice were superinfected as described in ( A ) ( E ) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) or following influenza virus infection ( + Flu ) Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . All data are represented as mean ± S . E . M . Statistical significance was determined b Mann - Whitney U tes n . s . = not

B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (E) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu) or following influenza virus infection (+Flu) Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significance was determined b Mann-Whitney U tes n.s. = not significant


Mice were superinfected as described in ( A ) ( F ) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S . pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . All data are represented as mean ± S . E .

B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (F) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S. pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M


Mice were superinfected as described in ( A ) ( G ) Absolute neutrophil and eosinophil cell count in lungs infected as described in ( E ) , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group All data are represented as mean ± S . E . M . Statistical significance wa determined b ) , unpaired student ' s T - test i ( G ( E ) . n . s . = not

B-ORGANISM O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (G) Absolute neutrophil and eosinophil cell count in lungs infected as described in (E), assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M. Statistical significance wa determined b ), unpaired student's T-test i (G (E). n.s. = not significant


( A ) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub - lethal influenza virus infection as determined by protein bead array Data information : Data in ( A ) are obtained from one independent experiment with 4 mice per group , and results of selected analytes IFNγ , CXCL10 , IL - 6 , IL - 13 and IL - 17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student ' s T - test . n . s . = not

O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(A) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub-lethal influenza virus infection as determined by protein bead array Data information: Data in (A) are obtained from one independent experiment with 4 mice per group, and results of selected analytes IFNγ, CXCL10, IL-6, IL-13 and IL-17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A . ( B ) Concentrations of IL - 17 and IL - 22 in BAL of mice , as determined by ELISA Data informatio Data in ( B are representative of two independent experiments with 3 - 4 mice per group . All data are represented as mean ± S . E . M . Significance i was determined by unpaired student ' s T - test . n . s . = not

B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A. (B) Concentrations of IL-17 and IL-22 in BAL of mice, as determined by ELISA Data informatio Data in (B are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significance i was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A ( C ) Concentrations of IL - 17 and IL - 22 in BAL of mice 12 h after with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) , or following superinfection ( + Flu ) , assessed by ELISA Data information Dat i ( C ) are representative of two independent experiments with 3 - 4 mice per group . All data are represented as mean ± S . E . M . Significanc was determined by unpaired student ' s T - test . n . s . = not

B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (C) Concentrations of IL-17 and IL-22 in BAL of mice 12 h after with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu), or following superinfection (+Flu), assessed by ELISA Data information Dat i (C) are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significanc was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A . ( A ) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL - 17 or αIL - 22 blocking antibody . Control wild - type and T - bet - / - groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined with Kruskal - Wallis test followed by Dunn " s multiple comparisons test n . s . = not

B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A. (A) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL-17 or αIL-22 blocking antibody. Control wild-type and T-bet-/- groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined with Kruskal-Wallis test followed by Dunn"s multiple comparisons test n.s. = not significant


Mice were superinfected as described in Fig 3A ( B ) T - bet - / - mice were administered αIL - 17 or IgG isotype control antibody every other day from day - 2 post - secondary bacterial infection and monitored for survival Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined wit logrank test in (

B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (B) T-bet-/- mice were administered αIL-17 or IgG isotype control antibody every other day from day -2 post-secondary bacterial infection and monitored for survival Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit logrank test in (B


Mice were superinfected as described in Fig 3A ( C ) Absolute neutrophil counts in the lungs following administration of αIL - 17 or IgG isotype control antibody , as determined by flow cytometry Data information Dat are representative of two independent experiments with 3 - 4 in ( C mice per group All data are represented as mean ± S . E . M . Statistical significance was determined wit unpaired - students t - test in (

B-ORGANISM O O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (C) Absolute neutrophil counts in the lungs following administration of αIL-17 or IgG isotype control antibody, as determined by flow cytometry Data information Dat are representative of two independent experiments with 3-4 in (C mice per group All data are represented as mean ± S.E.M. Statistical significance was determined wit unpaired-students t-test in (C


Mice were superinfected as described in Fig 3A ( D ) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion . Control T - bet - / - mice were administered with IgG isotype control antibody Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined wit Mann - Whitney U test in (

B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (D) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion. Control T-bet-/- mice were administered with IgG isotype control antibody Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit Mann-Whitney U test in (D


Mice were superinfected as described in Fig 3A ( E ) mRNA expression of antimicrobial peptides in lungs following administration of αIL - 17 , IL - 22 or IgG control antibody , as determined by qPCR Data information Dat are representative of two independent experiments wit 4 - 6 in ( E ) mice per group . All data are represented as mean ± S . E . M . Statistical significance was determined wit one - way ANOVA followed by Tukey " s multiple comparisons test in ( E ) . n . s . = not

B-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (E) mRNA expression of antimicrobial peptides in lungs following administration of αIL-17, IL-22 or IgG control antibody, as determined by qPCR Data information Dat are representative of two independent experiments wit 4-6 in (E) mice per group. All data are represented as mean ± S.E.M. Statistical significance was determined wit one-way ANOVA followed by Tukey"s multiple comparisons test in (E). n.s. = not signif


Mice were superinfected as described in Fig 3A ( A ) Frequency of IFNγ + and IL - 17 + immune cells following intracellular cytokine staining and flow cytometry Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M . Statistical significanc was determined by unpaired student ' s T -

B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (A) Frequency of IFNγ+ and IL-17+ immune cells following intracellular cytokine staining and flow cytometry Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc was determined by unpaired student's T-test


Mice were superinfected as described in Fig 3A ( B ) Representative flow cytometry plots of total and IL - 17 + lymphocyte subsets on day 3 post - secondary bacterial infected lungs following intracellular cytokine staining . Numbers in plots indicate the frequency of each IL - 17 + cell subset over total numbers of respective cell - types Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M

B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (B) Representative flow cytometry plots of total and IL-17+ lymphocyte subsets on day 3 post-secondary bacterial infected lungs following intracellular cytokine staining. Numbers in plots indicate the frequency of each IL-17+ cell subset over total numbers of respective cell-types Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M


Mice were superinfected as described in Fig 3A ( C ) Frequency over total IL - 17 + cells and absolute numbers of each IL - 17 producing lymphocyte subset on days 0 , 1 and 3 post - secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M . Statistical significanc i ( C ) was determined by unpaired student ' s T -

B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (C) Frequency over total IL-17+ cells and absolute numbers of each IL-17 producing lymphocyte subset on days 0, 1 and 3 post-secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc i (C) was determined by unpaired student's T-test


Clustered heatmap of the whole chemical genomics screen ; each subsection belongs to an individual strain ' s KO library . Grey cells indicate missing values .

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

Clustered heatmap of the whole chemical genomics screen; each subsection belongs to an individual strain's KO library. Grey cells indicate missing values.


S - score scatterplots for each pairwise strain comparison , highlighting conserved phenotypes ( black points ) , significant changes ( red points ) and gene - condition relationships for which no call can reliably be made ( grey points ) . " r " , pearson ' s r value .

O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

S-score scatterplots for each pairwise strain comparison, highlighting conserved phenotypes (black points), significant changes (red points) and gene-condition relationships for which no call can reliably be made (grey points). "r", pearson's r value.


Fraction of deletion phenotypes in each strain conserved with other stains in pairwise , three - way and four - way comparisons . Error bars represent standard deviation for all pairwise and three - way comparisons . Only a four - way comparison is possible for each strain so no error bars are represented in these cases .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fraction of deletion phenotypes in each strain conserved with other stains in pairwise, three-way and four-way comparisons. Error bars represent standard deviation for all pairwise and three-way comparisons. Only a four-way comparison is possible for each strain so no error bars are represented in these cases.


Gene exclusiveness : a measure of each gene ' s propensity to change its chemical genomics profile across strains . The top 10 genes ' names are reported .

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

Gene exclusiveness: a measure of each gene's propensity to change its chemical genomics profile across strains. The top 10 genes' names are reported.


Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons.


Wild type fitness of each strain relative to S288C across the same conditions as in panel A ; each dot represents a specific replicate where colony sizes were measured .

O 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-EXP_ASSAY I-EXP_ASSAY O O O

Wild type fitness of each strain relative to S288C across the same conditions as in panel A; each dot represents a specific replicate where colony sizes were measured.


Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C ; each dot represents a strain - condition replicate as in panel B . The conditions maltose , glycerol , and NaCl are highlighted .

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Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C; each dot represents a strain-condition replicate as in panel B. The conditions maltose, glycerol, and NaCl are highlighted.


Changes in gene deletion phenotypes for growth on osmotic stress conditions . The top heatmap contains genes belonging to the HOG pathway , while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS . Significant growth phenotypes are marked with " - " .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

Changes in gene deletion phenotypes for growth on osmotic stress conditions. The top heatmap contains genes belonging to the HOG pathway, while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS. Significant growth phenotypes are marked with "-".


Changes in deletion phenotypes for growth on glycerol and maltose . The top heatmap contains the MAL genes , while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains . Significant growth phenotypes are marked with " - " .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

Changes in deletion phenotypes for growth on glycerol and maltose. The top heatmap contains the MAL genes, while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains. Significant growth phenotypes are marked with "-".


S - score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening . B ) Number of variants significantly associated ( p - value < 1E - 6 ) with phenotypic variation in each growth condition . C ) Number of associated variants that overlap ( i . e . are in a 3kbp window ) with a conditionally essential gene in the same condition , in any of the four KO libraries .

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S-score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening. B) Number of variants significantly associated (p-value < 1E-6) with phenotypic variation in each growth condition. C) Number of associated variants that overlap (i.e. are in a 3kbp window) with a conditionally essential gene in the same condition, in any of the four KO libraries.


Manhattan plots showing examples of overlaps between associated variants and the KO screening . The top plot shows the associations between variants and growth in the natural isolates as a function of the - log10 of the association p - value , while the four bottom plots show the strength of the KO phenotypes across the four yeast strains , as a function of the - log10 of the corrected S - score p - value . Sections shaded in grey indicate the overlap between associations and KO data . Position in the yeast chromosome is reported in kilobase units .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O 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-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O 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-ORGANISM B-SUBCELLULAR O O O O O O

Manhattan plots showing examples of overlaps between associated variants and the KO screening. The top plot shows the associations between variants and growth in the natural isolates as a function of the -log10 of the association p-value, while the four bottom plots show the strength of the KO phenotypes across the four yeast strains, as a function of the -log10 of the corrected S-score p-value. Sections shaded in grey indicate the overlap between associations and KO data. Position in the yeast chromosome is reported in kilobase units.


C . RADX interacts with ssDNA . Extracts of U2OS cells stably expressing GFP - RADX WT or mutants were incubated with biotin - coupled ssDNA oligo immobilized on Streptavidin beads , washed extensively and immunoblotted with GFP and RPA1 antibodies .

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C. RADX interacts with ssDNA. Extracts of U2OS cells stably expressing GFP-RADX WT or mutants were incubated with biotin-coupled ssDNA oligo immobilized on Streptavidin beads, washed extensively and immunoblotted with GFP and RPA1 antibodies.


D . ssDNA - bound Streptavidin beads incubated with cell extracts as in ( C ) were washed with buffer containing indicated salt concentrations prior to immunoblotting .

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D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting.


E . Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in ( C ) and immunoblotted with RADX and RPA1 antibodies .

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E. Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in (C) and immunoblotted with RADX and RPA1 antibodies.


F . Immunoblot analysis of RADX expression in human cell lines . See also Fig EV1B .

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F. Immunoblot analysis of RADX expression in human cell lines. See also Fig EV1B.


G . Representative images from in situ proximity ligation assays ( PLAs ) in BrdU - labeled U2OS and GFP / GFP - RADX cell lines ( Fig EV1E , F ) using GFP and BrdU antibodies under native conditions . Scale bar , 10 μm .

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G. Representative images from in situ proximity ligation assays (PLAs) in BrdU-labeled U2OS and GFP/GFP-RADX cell lines (Fig EV1E,F) using GFP and BrdU antibodies under native conditions. Scale bar, 10 μm.


I . Soluble and chromatin - enriched fractions of U2OS cell lines stably expressing GFP - RADX alleles were immunoblotted with indicated antibodies .

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I. Soluble and chromatin-enriched fractions of U2OS cell lines stably expressing GFP-RADX alleles were immunoblotted with indicated antibodies.


A . Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU .

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A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU.


B . U2OS cells treated with HU in the presence or absence of ATR inhibitor ( ATRi ) were fractionated and immunoblotted with indicated antibodies . Relative RADX levels on chromatin were quantified and normalized to histone H3 . See also Fig EV2A .

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B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A.


C . Representative images of U2OS cells stably expressing indicated GFP - RADX alleles that were subjected to laser microirradiation , fixed with methanol / acetone immediately afterwards and immunostained with γH2AX antibody .

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C. Representative images of U2OS cells stably expressing indicated GFP-RADX alleles that were subjected to laser microirradiation, fixed with methanol/acetone immediately afterwards and immunostained with γH2AX antibody.


D . As in ( C ) , except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody .

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D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody.


E . Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS / GFP - RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation .

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E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation.


A . Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines ( RADXΔ ) .

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A. Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines (RADXΔ).


C . Cells in ( A ) were labeled with consecutive pulses of CldU and IdU as shown in ( B ) . Replication fork speeds were calculated as length of labeled track divided by pulse time ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


D . Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from ( C ) . Concordance is 100 % , representing equal rates of bidirectional elongation for both daughter forks ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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D. Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from (C). Concordance is 100%, representing equal rates of bidirectional elongation for both daughter forks (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


E . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers from ( C ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


F . Proportion of new origins ( IdU - only tracks ) among DNA fibers in ( C ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

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F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


H . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers from cells labeled as in ( G ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


I . Proportion of restarted forks ( CldU - and IdU - positive tracks ) among DNA fibers in ( G ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


A . Replication fork speeds in U2OS or U2OS / GFP - RADX cells transfected with control ( − ) or RADX ( # 6 ) siRNA targeting the 3 ' UTR and labeled with CldU and IdU as in Fig 3B ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


B . Fork symmetry in cells treated as in ( A ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


C . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers in ( A ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


D . Proportion of new origins ( IdU - only tracks ) among DNA fibers in ( A ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


E . Parental U2OS and derivative lines stably expressing GFP - RADX alleles or all three RPA isoforms at near - endogenous levels ( U2OS / Super - RPA ) ( Fig EV4A ) were transfected with control ( − ) or RADX siRNA . Replication fork speeds were determined as in ( A ) ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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E. Parental U2OS and derivative lines stably expressing GFP-RADX alleles or all three RPA isoforms at near-endogenous levels (U2OS/Super-RPA) (Fig EV4A) were transfected with control (−) or RADX siRNA. Replication fork speeds were determined as in (A) (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


F . Replication fork speeds in HCT116 cells with indicated genotypes transfected with control ( − ) , RPA1 ( 0 . 2 nM concentration [ 17 ] ) or RAD51 siRNAs ( Fig EV4B ) and labeled with CldU and IdU as in Fig 3B ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


G . Fork symmetry among DNA fibers in ( F ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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G. Fork symmetry among DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


H . New origin firing among DNA fibers in ( F ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


B . Replication fork degradation ( IdU / CldU ratio ) in U2OS cells transfected with indicated siRNAs and processed as in ( A ) ( bars , mean ; n = 200 fibers , pooled from two independent experiments , per condition ) .

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B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition).


C . As in ( B ) , except fork degradation was analyzed in U2OS / GFP - RADX cell lines .

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C. As in (B), except fork degradation was analyzed in U2OS/GFP-RADX cell lines.


D . U2OS or U2OS / GFP - RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co - immunostained with RPA1 and γ - H2AX antibodies and analyzed by QIBC . Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation ( Fig EV5A ) , reflecting replication fork catastrophe [ 17 ] , is indicated ( bars , mean ; n = 5 independent experiments ( ≥ 3000 cells analyzed per condition ) ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired t - test ) .

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D. U2OS or U2OS/GFP-RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co-immunostained with RPA1 and γ-H2AX antibodies and analyzed by QIBC. Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation (Fig EV5A), reflecting replication fork catastrophe [17], is indicated (bars, mean; n=5 independent experiments (≥3000 cells analyzed per condition); *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, unpaired t-test).


E . As in ( D ) , but using U2OS or U2OS / Super - RPA cells exposed or not to HU ( n = 6 independent experiments ) . See also Fig EV5B .

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E. As in (D), but using U2OS or U2OS/Super-RPA cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B.


F . Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h ( mean ± SEM ; n = 3 independent experiments ) . LQ ( Linear Quadratic ) model was fitted to the fractional survival data , using non - linear least square method . Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment ( * No overlap of 95 % confidence interval with siCTRL ) .

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F. Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h (mean±SEM;n=3 independent experiments). LQ (Linear Quadratic) model was fitted to the fractional survival data, using non-linear least square method. Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment (*No overlap of 95% confidence interval with siCTRL).


G . As in ( F ) , except cells were treated with indicated doses of HU ( mean ± SEM ; n = 3 independent experiments ) .

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G. As in (F), except cells were treated with indicated doses of HU (mean±SEM;n=3 independent experiments).


H . As in ( F ) , using U2OS and U2OS / Super - RPA cells ( mean ± SEM ; n = 3 independent experiments ) .

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H. As in (F), using U2OS and U2OS/Super-RPA cells (mean±SEM;n=3 independent experiments).


A Turbidity and bright - field microscope images of indicated groups ( left ) and the quantitative analysis of turbidity of samples were measured by absorbance at 600 nm ( right ) . 10 μM cGAS , 5 μM G3BP1 , 5 μM BSA and 100 nM dsDNA ( 60 bp ) were used in these assays . n = 3 biological replicates . Data information : Representative images are shown Error bars , mean with s . d . , * P < 0 . 05 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . Scale bars , 10

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A Turbidity and bright-field microscope images of indicated groups (left) and the quantitative analysis of turbidity of samples were measured by absorbance at 600 nm (right). 10 μM cGAS, 5 μM G3BP1, 5 μM BSA and 100 nM dsDNA (60 bp) were used in these assays. n = 3 biological replicates. Data information: Representative images are shown Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. Scale bars, 10 μm


F FRAP analysis of DNA - induced droplets of cGAS ( 1 μM dsDNA , 10 μM cGAS - mCherry ) , the yellow dotted circle indicated the region of photobleaching . G FRAP curve of ( F ) . n = 3 biological replicates . H FRAP analysis of cGAS - G3BP1 droplets . 10 μM cGAS - mCherry and 5 μM G3BP1 were used . The yellow dotted circle indicated the region of photobleaching . I FRAP curve of ( H ) . n = 3 biological replicates . Data information : Representative images are shown ( A , Error bars , mean with s . d . , * P < 0 . 05 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . FRAP , Fluorescence recovery after photobleaching . Scale bars , 2 μm ( F and H ) .

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F FRAP analysis of DNA-induced droplets of cGAS (1 μM dsDNA, 10 μM cGAS-mCherry), the yellow dotted circle indicated the region of photobleaching. G FRAP curve of (F). n = 3 biological replicates. H FRAP analysis of cGAS-G3BP1 droplets. 10 μM cGAS-mCherry and 5 μM G3BP1 were used. The yellow dotted circle indicated the region of photobleaching. I FRAP curve of (H). n = 3 biological replicates. Data information: Representative images are shown (A, Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. FRAP, Fluorescence recovery after photobleaching. Scale bars, 2 μm (F and H).


B Immunofluorescent staining of cGAS in both WT and G3BP1 - / - U937 cells . 3D images were reconstituted with Leica LAS X software . C Quantitative analysis of total cGAS puncta number ( left ) and volume ( right ) per cell of ( B ) . n = 51 cells . D Data information : Representative images are shown Scale bars , 5 μm . Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . WT , wild - type .

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B Immunofluorescent staining of cGAS in both WT and G3BP1-/- U937 cells. 3D images were reconstituted with Leica LAS X software. C Quantitative analysis of total cGAS puncta number (left) and volume (right) per cell of (B). n = 51 cells. D Data information: Representative images are shown Scale bars, 5 μm. Hoechst (blue), nuclear staining. Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type.


F ELISA analysis of the secreted IFN - β in HT - DNA - treated WT or G3BP1 - / - U937 cells . n = 3 biological replicates . Data information : Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . WT , wild - type . HT - DNA , Herring testes DNA .

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F ELISA analysis of the secreted IFN-β in HT-DNA-treated WT or G3BP1-/- U937 cells. n = 3 biological replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. HT-DNA, Herring testes DNA.


G cGAMP production in ISD - treated WT or G3BP1 - / - U937 cells were analyzed by LC - MS / MRM . ND , not detected . n = 3 technical replicates . Data information : Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . WT , wild - type . ISD , interferon stimulatory DNA .

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G cGAMP production in ISD-treated WT or G3BP1-/- U937 cells were analyzed by LC-MS/MRM. ND, not detected. n = 3 technical replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. ISD, interferon stimulatory DNA.


B Fluorescence intensity distribution on cGAS - G3BP1 droplets . A quantitative analysis of a representative cGAS - G3BP1 droplet is shown . Along the white line on the merged image , the fluorescence intensity of both G3BP1 and cGAS channels were recorded . Data information : Representative images are shown Scale bars , 10 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP were used in the assays .

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B Fluorescence intensity distribution on cGAS-G3BP1 droplets. A quantitative analysis of a representative cGAS-G3BP1 droplet is shown. Along the white line on the merged image, the fluorescence intensity of both G3BP1 and cGAS channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP were used in the assays.


D cGAS , G3BP1 and Cy5 - dsDNA were incubated . A quantitative analysis of a representative cGAS - DNA droplet is shown . Along the white line on the merged image , the fluorescence intensity of G3BP1 , cGAS and dsDNA channels were recorded . Data information : Representative images are shown Scale bars , 10 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP , 2 μM dsDNA were used in the assays .

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D cGAS, G3BP1 and Cy5-dsDNA were incubated. A quantitative analysis of a representative cGAS-DNA droplet is shown. Along the white line on the merged image, the fluorescence intensity of G3BP1, cGAS and dsDNA channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsDNA were used in the assays.


F cGAS , G3BP1 and Cy5 - dsRNA were incubated . Similar quantification analysis as in D was performed . Data information : Representative images are shown Scale bars , 5 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP , 2 μM dsRNA were used in the assays .

O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O

F cGAS, G3BP1 and Cy5-dsRNA were incubated. Similar quantification analysis as in D was performed. Data information: Representative images are shown Scale bars, 5 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsRNA were used in the assays.


A , B Recombinant cGAS ( 10 μM ) and the indicated amounts of Cy5 - labeled dsDNA were incubated , with or without G3BP1 ( 2 μM ) . The DNA - induced cGAS - DNA liquid droplets were analyzed with time - lapse imaging . Data information : Representative images are shown Scale bars , 10

O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

A, B Recombinant cGAS (10 μM) and the indicated amounts of Cy5-labeled dsDNA were incubated, with or without G3BP1 (2 μM). The DNA-induced cGAS-DNA liquid droplets were analyzed with time-lapse imaging. Data information: Representative images are shown Scale bars, 10 μm


D cGAMP production assays . Recombinant cGAS ( 10 μM ) was incubated with the indicated amounts of dsDNA , with or without G3BP1 ( 10 μM ) . The produced cGAMP was quantitatively analyzed by LC - MS / MRM . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * * P < 0 . 01 , two - tailed t - test .

O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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 cGAMP production assays. Recombinant cGAS (10 μM) was incubated with the indicated amounts of dsDNA, with or without G3BP1 (10 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.


G qPCR analysis of IFNB mRNA levels in WT and G3BP1 - / - U937 cells transfected with dsDNA as indicated . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * * P < 0 . 01 , two - tailed t - test .

O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G qPCR analysis of IFNB mRNA levels in WT and G3BP1-/- U937 cells transfected with dsDNA as indicated. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.


C cGAS - G3BP1 interaction analysis . Full - length and truncated G3BP1 was pulled - down using anti - Flag M2 beads . The cGAS - G3BP1 interaction was further analyzed with immunoblotting ( IB ) . 2 μg recombinant full - length cGAS and 2 μg each of truncated G3BP1 were used in the assay .

O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O

C cGAS-G3BP1 interaction analysis. Full-length and truncated G3BP1 was pulled-down using anti-Flag M2 beads. The cGAS-G3BP1 interaction was further analyzed with immunoblotting (IB). 2 μg recombinant full-length cGAS and 2 μg each of truncated G3BP1 were used in the assay.


D , Time - lapse imaging of DNA - induced LLPS of cGAS , in the presence of full length or truncated G3BP1 ( 2 μM ) n = 3 biological replicates . 10 μM cGAS , 100 nM dsDNA were used in the assay Data information : Representative images are shown Scale bars , 10

O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

D, Time-lapse imaging of DNA-induced LLPS of cGAS, in the presence of full length or truncated G3BP1 (2 μM) n = 3 biological replicates.10 μM cGAS, 100 nM dsDNA were used in the assay Data information: Representative images are shown Scale bars, 10 μm


A Bright - field microscope images of indicated groups ( left ) and a quantitative analysis of total area of droplets ( right ) . n = 3 biological replicates . 10 μM cGAS , 5 μM G3BP1 and 50 μM KCl , 50 μM MgCl2 , 50 μM ZnCl2 were used . Data information : Representative images are shown Scale bars , 10 μm . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O 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-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Bright-field microscope images of indicated groups (left) and a quantitative analysis of total area of droplets (right). n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 50 μM KCl, 50 μM MgCl2, 50 μM ZnCl2 were used. Data information: Representative images are shown Scale bars, 10 μm. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


C Fluorescent images and analysis of DNA - induced cGAS LLPS in the presence of the indicated reagent . n = 3 biological replicates . 10 μM cGAS , 50 μM of each ion and 100 nM dsDNA were used in these assays . Data information : Representative images are shown Scale bars , 10 μm . The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

O O O O O O B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescent images and analysis of DNA-induced cGAS LLPS in the presence of the indicated reagent. n = 3 biological replicates. 10 μM cGAS, 50 μM of each ion and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


D Fluorescent images and analysis of DNA - induced LLPS of cGAS in the presence of different amounts of ZnCl2 . n = 3 biological replicates . 10 μM cGAS and 100 nM dsDNA were used in the assay . Data information : Representative images are shown Scale bars , 10 μm . The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescent images and analysis of DNA-induced LLPS of cGAS in the presence of different amounts of ZnCl2. n = 3 biological replicates. 10 μM cGAS and 100 nM dsDNA were used in the assay. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


E Fluorescent images and analysis of DNA - induced LLPS of cGAS in the presence of G3BP1 ( 2 μM ) and / or ZnCl2 ( 20 μM ) as indicated . n = 3 biological replicates . Data information : Representative images are shown Scale bars , 10 μm . The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescent images and analysis of DNA-induced LLPS of cGAS in the presence of G3BP1 (2 μM) and/or ZnCl2 (20 μM) as indicated. n = 3 biological replicates. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


A Fluorescent images of cGAS - G3BP1 droplets in the presence or absence of 10 μM EGCG ( left ) . The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background ( right ) , n = 3 biological replicates . 45 μM cGAS - mCherry and 10 μM G3BP1 - mEGFP were used in these assays . Data information : Representative images are shown Scale bars , 10 μm Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescent images of cGAS-G3BP1 droplets in the presence or absence of 10 μM EGCG (left). The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background (right), n = 3 biological replicates. 45 μM cGAS-mCherry and 10 μM G3BP1-mEGFP were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


D Fluorescent images of DNA - induced LLPS of cGAS , with or without G3BP1 , in the absence or presence of 10 μM EGCG ( left ) . The partition coefficient was calculated as in ( A ) , n = 3 biological replicates . 10 μM cGAS , 5 μM G3BP1 and 100 nM dsDNA were used in these assays . Data information : Representative images are shown Scale bars , 10 μm Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant .

O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescent images of DNA-induced LLPS of cGAS, with or without G3BP1, in the absence or presence of 10 μM EGCG (left). The partition coefficient was calculated as in (A), n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


E cGAMP production assays . Recombinant cGAS ( 10 μM ) and G3BP1 ( 10 μM ) were incubated , in the presence or absence of dsDNA ( 200 nM ) , with or without EGCG ( 20 μM ) . The produced cGAMP was quantitatively analyzed by LC - MS / MRM . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant .

O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cGAMP production assays. Recombinant cGAS (10 μM) and G3BP1 (10 μM) were incubated, in the presence or absence of dsDNA (200 nM), with or without EGCG (20 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


F cGAS - G3BP1 interaction analysis . cGAS was precipitated using anti - cGAS antibodies in U937 cells treated with EGCG as indicated . IgG served as control . The cGAS - G3BP1 interaction was further analyzed by immunoblotting . Data information : Representative images are shown WCL , whole cell lysate . IP , immunoprecipitation .

O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O

F cGAS-G3BP1 interaction analysis. cGAS was precipitated using anti-cGAS antibodies in U937 cells treated with EGCG as indicated. IgG served as control. The cGAS-G3BP1 interaction was further analyzed by immunoblotting. Data information: Representative images are shown WCL, whole cell lysate. IP, immunoprecipitation.


A . Quantitative real - time PCR analysis of PKCδ mRNA levels in FACS sorted Lin - , LT - HSC , ST - HSC , MPP , L - S - K + , GMP , CMP , MEP and CLP subsets from C56BL / 6 wild - type ( 6 - to 9 - week old ) mice bone marrow . Levels of PKCδ expression were normalized to an internal control gene ( β - actin ) . Expression of PKCδ is shown relative to Lineage negative ( Lin - ) cells whose expression was arbitrarily set to 1 ( n = 4 mice analyzed for each population ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with Sidak ' s multiple comparisons test ( A )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantitative real-time PCR analysis of PKCδ mRNA levels in FACS sorted Lin-, LT-HSC, ST-HSC, MPP, L-S-K+, GMP, CMP, MEP and CLP subsets from C56BL/6 wild-type (6-to 9-week old) mice bone marrow. Levels of PKCδ expression were normalized to an internal control gene (β-actin). Expression of PKCδ is shown relative to Lineage negative (Lin-) cells whose expression was arbitrarily set to 1 (n=4 mice analyzed for each population). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with Sidak's multiple comparisons test (A)


( B ) FACS plots depict the percentage of live , Lin - Sca1 + c - Kit + ( LSK ) ( left panel ) , and primitive CD34 - Flt3 - , CD34 + Flt3 - and CD34 + Flt3 + HSPCs among LSK cells ( right panel ) .

O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O O

(B) FACS plots depict the percentage of live, Lin-Sca1+c-Kit+ (LSK) (left panel), and primitive CD34-Flt3-, CD34+Flt3- and CD34+Flt3+ HSPCs among LSK cells (right panel).


( C ) Bar charts show the average frequency ( left ) and absolute number ( right ) ± SEM of the indicated populations , analyzed from 2 femurs and 2 tibias per mouse ( n = 10 mice analyzed for each genotype ) . All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice .

O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

(C) Bar charts show the average frequency (left) and absolute number (right) ± SEM of the indicated populations, analyzed from 2 femurs and 2 tibias per mouse (n=10 mice analyzed for each genotype). All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.


D . FACS plots show percentages of ' SLAM - code ' based stem and progenitor cells in the LSK population . Bar graphs show the average frequency ( left ) and absolute numbers ( right ) ± SEM of LT - HSC per 2 femurs and 2 tibias . ( n = 10 mice analyzed for each genotype ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice .

O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

D. FACS plots show percentages of 'SLAM-code' based stem and progenitor cells in the LSK population. Bar graphs show the average frequency (left) and absolute numbers (right) ± SEM of LT-HSC per 2 femurs and 2 tibias. (n=10 mice analyzed for each genotype). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.


E . FACS plots illustrate gating and frequencies of myeloid progenitors ( MP ) , granulocyte - monocyte progenitor ( GMP ) , common myeloid progenitor ( CMP ) , and megakaryocyte - erythroid progenitor ( MEP ) population . Bar graphs show the frequencies ( left ) and absolute numbers ( right ) of L - S - K + MP and GMP , CMP and MEP populations in the BM of WT ( n = 12 ) and PKCδ KO ( n = 12 ) . Data information : All data are presented as mean ± SEM . * p < 0 . 05 ; * * p < 0 . 01 , and * * * p < 0 . 001 by with one - way ANOVAs with two - tailed Student ' s unpaired t - test analysis for comparison of WT to PKCδ KO mice .

O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-ORGANISM O

E. FACS plots illustrate gating and frequencies of myeloid progenitors (MP), granulocyte-monocyte progenitor (GMP), common myeloid progenitor (CMP), and megakaryocyte-erythroid progenitor (MEP) population. Bar graphs show the frequencies (left) and absolute numbers (right) of L-S-K+ MP and GMP, CMP and MEP populations in the BM of WT (n=12) and PKCδ KO (n=12). Data information: All data are presented as mean±SEM. *p<0.05; **p<0.01, and ***p<0.001 by with one-way ANOVAs with two-tailed Student's unpaired t-test analysis for comparison of WT to PKCδ KO mice.