( B ) Identification of wild - type ( WT ) , heterozygous ( HZ ) , and knockout ( KO ) mice by PCR and agarose gel electrophoresis .

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(B) Identification of wild-type (WT), heterozygous (HZ), and knockout (KO) mice by PCR and agarose gel electrophoresis.


( C ) Immunoblotting ( IB ) of ENKD1 and α - tubulin in wild - type and Enkd1 knockout mice .

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

(C) Immunoblotting (IB) of ENKD1 and α-tubulin in wild-type and Enkd1 knockout mice.


( D - F ) Immunofluorescence images ( D ) and quantification of the density of cilia ( E , n = 20 fields from three mice ) and ciliary length ( F , n = 100 cilia from three mice ) in mouse retinas stained with antibodies against acetylated α - tubulin ( Ace - α - tubulin ) and γ - tubulin and DAPI . Scale bar , 2 µm .

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

(D-F) Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α-tubulin (Ace-α-tubulin) and γ-tubulin and DAPI. Scale bar, 2 µm.


( G - H ) ERG recording images ( G ) and a - b wave amplitudes ( H , n = 5 mice ) show the difference in the ERG a - b wave amplitude between wild - type and Enkd1 knockout mice .

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

(G-H) ERG recording images (G) and a-b wave amplitudes (H, n = 5 mice) show the difference in the ERG a-b wave amplitude between wild-type and Enkd1 knockout mice.


( I - J ) VEP recording images ( I ) and N2 - P2 wave amplitudes ( J , n = 5 mice ) show the difference in the VEP N2 - P2 wave amplitude between wild - type and Enkd1 knockout mice .

(I-J) VEP recording images (I) and N2-P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2-P2 wave amplitude between wild-type and Enkd1 knockout mice.


( K - M ) Immunofluorescence images ( K ) and quantification of the percentage of ciliated cells ( L , n = 3 mice ) and ciliary length ( M , n = 100 cilia from three mice ) in mouse kidneys stained with the antibody against acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( L ) , > 200 cells from 12 images were analyzed for each mouse . Scale bar , 3 µm .

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

(K-M) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (L), >200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm.


( N - Q ) Images ( N ) and quantification of the percentage of flagellated sperm ( O , n = 9 mice ) , flagellar length ( P , n = 100 sperm from nine mice ) , and the percentage of sperm with normal motility among flagellated sperm ( Q , n = 9 mice ) for wild - type and Enkd1 knockout mice . To quantify the percentage of flagellated sperm ( O ) , > 200 sperm were analyzed for each mouse . To quantify the percentage of sperm with normal motility among flagellated sperm ( Q ) , > 200 flagellated sperm were analyzed for each mouse . In panel N , the arrows indicate two spermatozoa without tails . Scale bar , 50 µm .

O O O O O O O O O O O O O O O B-SUBCELLULAR B-CELL_TYPE O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O B-SUBCELLULAR B-CELL_TYPE O O O O O O B-ORGANISM O O O O O O B-GENEPROD O B-ORGANISM O O O O O O B-SUBCELLULAR B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O B-SUBCELLULAR B-CELL_TYPE O O O O O O B-SUBCELLULAR B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O O O O O O

(N-Q) Images (N) and quantification of the percentage of flagellated sperm (O, n = 9 mice), flagellar length (P, n = 100 sperm from nine mice), and the percentage of sperm with normal motility among flagellated sperm (Q, n = 9 mice) for wild-type and Enkd1 knockout mice. To quantify the percentage of flagellated sperm (O), >200 sperm were analyzed for each mouse. To quantify the percentage of sperm with normal motility among flagellated sperm (Q), >200 flagellated sperm were analyzed for each mouse. In panel N, the arrows indicate two spermatozoa without tails. Scale bar, 50 µm.


( R , S ) Scanning electron microscopy images of cilia ( R ) and quantification of the percentage of ciliated cells ( S , n = 9 mice ) in the mouse tracheal epithelium . To quantify the percentage of ciliated cells ( S ) , > 200 cells from six fields were analyzed for each mouse . Scale bar , 5 µm .

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

(R, S) Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), >200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm.


( A ) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum - free medium for 48 h .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O O O O O

(A) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum-free medium for 48 h.


( B - D ) Immunofluorescence images ( B ) and quantification of the percentage of ciliated cells ( C , n = 3 independent experiments ) and ciliary length ( D , n = 70 cilia from three independent experiments ) for MEFs cultured in serum - free medium and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( C ) , > 100 cells were analyzed for each experiment . Scale bar , 5 µm .

O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O B-TISSUE O O O O B-EXP_ASSAY O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(B-D) Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), >100 cells were analyzed for each experiment. Scale bar, 5 µm.


( E ) Immunoblot analysis of ENKD1 and β - actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum - free medium for 48 h .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O

(E) Immunoblot analysis of ENKD1 and β-actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum-free medium for 48 h.


Immunofluorescence images ( F ) and quantification of the percentage of ciliated cells ( G , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( G ) , > 150 cells were analyzed for each experiment . Scale bar , 5 µm .

B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O

Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), >150 cells were analyzed for each experiment. Scale bar, 5 µm.


quantification of ciliary length ( H , n = 100 cilia from three independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free

O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O

quantification of ciliary length (H, n = 100 cilia from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium


( I , J ) Immunofluorescence images ( I ) and quantification of the percentage of ciliated cells ( J , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against Arl13b and Centrin and DAPI . To quantify the percentage of ciliated cells ( J ) , > 80 cells were analyzed for each experiment . Scale bar , 2 µm .

O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(I, J) Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), >80 cells were analyzed for each experiment. Scale bar, 2 µm.


( K , L ) Immunofluorescence images ( K ) and quantification of the percentage of ciliated cells ( L , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP , GFP - ENKD1 , GFP - ENKD1 - N , GFP - ENKD1 - M , or GFP - ENKD1 - C , cultured in serum - free medium , and stained with the antibody against acetylated α - tubulin and DAPI . The siRNA - resistant forms of ENKD1 were used for these rescue experiments . To quantify the percentage of ciliated cells ( L ) , > 120 cells were analyzed for each experiment . Scale bar , 3 µm .

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

(K, L) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP-ENKD1, GFP-ENKD1-N, GFP-ENKD1-M, or GFP-ENKD1-C, cultured in serum-free medium, and stained with the antibody against acetylated α-tubulin and DAPI. The siRNA-resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), >120 cells were analyzed for each experiment. Scale bar, 3 µm.


( A ) Immunofluorescence images of RPE1 cells cultured in normal serum medium , and stained with antibodies against ENKD1 and Centrin and DAPI . Scale bar , 1 µm .

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

(A) Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.


( B ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 1 µm .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O B-CELL_LINE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

(B) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 1 µm.


( C ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 2 µm .

(C) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 2 µm.


( D - F ) 3D - SIM super - resolution images ( D ) , quantification of the outer toroid diameters ( E , n = 20 cells from three independent experiments ) , and a model illustrating the relative positions of γ - tubulin , Ninein , ENKD1 , CEP164 , CEP912 , and Centrin ( F ) within a centriole . RPE1 cells were serum - starved and stained with antibodies against the indicated proteins . Scale bar , 1 µm .

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 O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-CELL_LINE O O B-TISSUE O O O B-EXP_ASSAY O O O O O O O O O O O O O

(D-F) 3D-SIM super-resolution images (D), quantification of the outer toroid diameters (E, n = 20 cells from three independent experiments), and a model illustrating the relative positions of γ-tubulin, Ninein, ENKD1, CEP164, CEP912, and Centrin (F) within a centriole. RPE1 cells were serum-starved and stained with antibodies against the indicated proteins. Scale bar, 1 µm.


( G ) Immunofluorescence images of RPE1 cells cultured in normal serum or serum - free medium , and stained with antibodies against ENKD1 and γ - tubulin and DAPI . Scale bar , 1 µm .

O O O B-EXP_ASSAY O O B-CELL_LINE O O O O B-TISSUE O B-TISSUE O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O

(G) Immunofluorescence images of RPE1 cells cultured in normal serum or serum-free medium, and stained with antibodies against ENKD1 and γ-tubulin and DAPI. Scale bar, 1 µm.


( H ) Immunofluorescence images of RPE1 cells cultured in serum - free medium , and stained with antibodies against ENKD1 and acetylated α - tubulin and DAPI . Scale bar , 1 µm .

O O O B-EXP_ASSAY O O B-CELL_LINE O O O B-TISSUE O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O

(H) Immunofluorescence images of RPE1 cells cultured in serum-free medium, and stained with antibodies against ENKD1 and acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( I ) Immunofluorescence images of RPE1 cells transfected with GFP - ENKD1 or GFP vector , cultured in a serum - starved condition , and stained with the antibody against acetylated α - tubulin and DAPI . Scale bar , 1 µm .

O O O B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O

(I) Immunofluorescence images of RPE1 cells transfected with GFP-ENKD1 or GFP vector, cultured in a serum-starved condition, and stained with the antibody against acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( K ) 3D - SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum - free medium ( 0 , 12 , 24 , or 48 h of serum starvation ) , and stained with antibodies against ENKD1 and acetylated α - tubulin . Scale bar , 1 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-CELL_LINE O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(K) 3D-SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum-free medium (0, 12, 24, or 48 h of serum starvation), and stained with antibodies against ENKD1 and acetylated α-tubulin. Scale bar, 1 µm.


Immunofluorescence images ( B ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI .

B-EXP_ASSAY O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O

Immunofluorescence images (B) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI.


quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot ( C ) , > 120 cells were analyzed for each experiment .

O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE 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 O O

quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot (C), >120 cells were analyzed for each experiment.


( D , E ) Immunofluorescence images ( D ) and quantification of the percentage of cells with one CEP97 dot ( E , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CEP97 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CEP97 dot ( E ) , > 150 cells were analyzed for each experiment . Scale bar , 1 µm .

O O O O O B-EXP_ASSAY 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 B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE 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

(D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm.


( F - H ) Immunoblot analysis ( F ) , immunofluorescence images ( G ) , and quantification of the percentage of cells with one CP110 dot ( H , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium for 120 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 80 cells were analyzed for each experiment . Scale bar , 1 µm .

O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY 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 B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE 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

(F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.


( A - C ) Immunoblot analysis ( A ) , immunofluorescence images ( B ) and quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in serum - free medium for 48 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 180 cells were analyzed for each experiment . Scale bar , 1 µm .

O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY 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 B-CELL_LINE O O O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE 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

(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm.


( D ) Immunoblot analysis of CP110 and β - actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum - free medium for 48 h .

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O

(D) Immunoblot analysis of CP110 and β-actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum-free medium for 48 h.


( E , F ) Immunofluorescence images ( E ) and quantification of the percentage of ciliated cells ( F , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in a serum - starved condition , and stained with antibodies against γ - tubulin and acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( F ) , > 120 cells were analyzed for each experiment . Scale bar , 10 µm .

O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.


( G , H ) Immunoprecipitation and immunoblotting ( G ) and quantification ( H , n = 3 mice ) showing the CP110 - CEP97 interaction in the retinal tissues of wild - type and Enkd1 knockout mice . The intensity of each CEP97 band was normalized to that of the β - actin band .

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

(G, H) Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110-CEP97 interaction in the retinal tissues of wild-type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β-actin band.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the CP110 - CEP97 interaction in serum - starved RPE1 cells transfected with control or ENKD1 siRNAs . The intensity of each CEP97 band was normalized to that of the β - actin band .

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(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110-CEP97 interaction in serum-starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β-actin band.


( A ) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues . Immunoprecipitation was performed with the anti - ENKD1 ( upper panel ) or anti - CP110 ( lower panel ) antibody or the corresponding control IgG .

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(A) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti-ENKD1 (upper panel) or anti-CP110 (lower panel) antibody or the corresponding control IgG.


( B - D ) Immunoprecipitation and immunoblotting showing the interaction of GFP - ENKD1 with Flag - CP110 ( B and upper panel of D ) or HA - CP110 ( C and bottom panel of D ) in HEK293T cells . Immunoprecipitation was performed with antibodies against GFP ( B and C ) , Flag ( upper panel of D ) , or HA ( bottom panel of D ) .

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(B-D) Immunoprecipitation and immunoblotting showing the interaction of GFP-ENKD1 with Flag-CP110 (B and upper panel of D) or HA-CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D).


( E ) Identification of the domains of ENKD1 mediating its interaction with CP110 .

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(E) Identification of the domains of ENKD1 mediating its interaction with CP110.


( F ) Identification of the domains of CP110 mediating its interaction with ENKD1 .

(F) Identification of the domains of CP110 mediating its interaction with ENKD1.


( H ) GST pull - down showing the interaction of purified GST - CP110 - N with purified His - ENKD1 - M .

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(H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the interaction of HA - CP110 with Flag - CEP97 in HEK293T cells transfected with the indicated plasmids . The intensity of each Flag - CEP97 band was normalized to that of the β - actin band .

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(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA-CP110 with Flag-CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag-CEP97 band was normalized to that of the β-actin band.


A - D Mesenteric blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ; PROX1 , gray ) . Asterisks indicate lymphatic valves , arrow indicates a lymphatic vessel stub , and arrowhead indicates an isolated lymphatic vessel fragmentE - H Blood vessels ( PECAM1 , green ) and lymphatic vessels ( VEGFR - 3 , red ) in the small intestinal wall .

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A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.


I , J Detection of chylous ascites ( arrows ) in the VCiΔR26 mice at P6 .

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I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.


K , L LYVE1 staining of intestinal lymphatic vessels at P6 .

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K, L LYVE1 staining of intestinal lymphatic vessels at P6.


M - T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice . Genotypes and deletion lengths are indicated in ( U ) . U Quantification of LYVE1 areas in ( M - T ) . Length of the i ∆ Vegfc gene deletion is indicated in months ( mo ) , and Vegfd indicates the VEGF - D genotype . Data are represented as mean ± SEM . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in ( M ) . * P = 0 . 003 , * * P = 0 . 001 , * * * P = 0 . 0008 , ♯ P = 0 . 0002 , § P = 0 . 0001 .

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M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.


Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in ( B - F ) ; ( I ) for the entire villus and ( II ) for the villus base . β - Gal staining pattern of the villus in wild - type ( Ctrl ) , Vegfc / LacZ ( VC ) , Vegfr3 / LacZ ( VR - 3 ) , and Vegfr2 / LacZ ( VR - 2 ) mice . Higher magnification images representing β - Gal staining of the villus base in Vegfc / LacZmice . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining reaction with smooth muscle actin ( SMA ) peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining and LYVE1 peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Surface image of Vegfc / LacZβ - Gal - stained intestine ( left ) and cross section counterstaining with PECAM1 ( right ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Immunofluorescence staining of lacteal lymphatic vessel ( LYVE1 ) , blood capillaries ( PECAM1 ) , and smooth muscle cells ( SMA ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Body weight change during seven weeks of HFD , expressed as average fold change in comparison with the starting weight . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 . Body weight comparisons at seven weeks of HFD . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT . * P = 0 . 004 ; * * P = 0 . 003 . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 .

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

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Glucose tolerance test ( GTT ) after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 014 ; * * P = 0 . 041 . n = 5 , WT ; n = 6 , VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Total fatweight , fat percentage from body composition measurements after six weeks of HFD , and weights of visceral fat ( VF ) and subcutaneous fat ( SF ) at the time of necropsy . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 006 ; * * P = 0 . 001 ; # P = 0 . 008 ; § P = 0 . 006 . n = 4 in each group .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Food consumption during the fifth week of HFD . n = 9 , WT ; n = 10 , VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Whole - mount immunofluorescence staining of blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ) in intestinal villi and intestinal wall . Quantification of the lacteal and villus length ( solid and striped color bars , respectively ) and the intestinal wallLYVE1 + area percentage from images represented in ( F ) . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 0002 ; * * P = 0 . 00007 . n = 5 , WT ; n = 6 , VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Free fatty acid ( FFA ) and cholesterol measurements from the feces after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 001 ; * * P = 0 . 007 . n = 5 , WT ; n = 6 , VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.


A . Relative antiviral activity of IFNα ( circles ) or IFNλ ( triangles ) . AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations ( ng / ml ) and induction of indicated ISGs was assessed by qPCR ( data shown is representative of four independent experiments , n = 3 - 4 ) .

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A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4).


B , C . Mice were pretreated with equivalent doses of IFNα ( 1 . 45μg / 50μl ) or IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( squares , 50μl PBS ) 24hrs prior to infection with PR8 ; weight loss and survival was assessed throughout infection ( B ) , and viral load ( C ) assessed at 4dpi ( data shown is representative of two independent experiments , n = 8 - 10 ( B ) , n = 3 ( C ) ) .

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B, C. Mice were pretreated with equivalent doses of IFNα (1.45μg/50μl) or IFNλ (2.6μg/50μl) or Veh Ctrl (squares, 50μl PBS) 24hrs prior to infection with PR8; weight loss and survival was assessed throughout infection (B), and viral load (C) assessed at 4dpi (data shown is representative of two independent experiments, n=8-10 (B), n=3 (C)).


D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; survival and weight loss was monitored ( D , data pooled from 4 independent experiments , n = 12 - 29 ) ( data representative of two independent experiments , n = 3 - 5 ) .

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D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; survival and weight loss was monitored (D, data pooled from 4 independent experiments, n=12-29) (data representative of two independent experiments, n=3-5).


D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; viral load assessed at 4dpi ( E ) ( data representative of two independent experiments , n = 3 - 5 ) .

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D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; viral load assessed at 4dpi (E) (data representative of two independent experiments, n=3-5).


A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay ( A ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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

A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay (A) (data shown is representative of two independent experiments, n=2-6).


A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed ( B ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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

A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed (B) (data shown is representative of two independent experiments, n=2-6).


C , D . Lung sections from control and infected mice treated as indicated , were stained by TUNEL for apoptotic cells at 6dpi . Quantification of TUNEL + cells in whole lung slides by Icy Spot Detector ( ICY - R3M2Y2 ) ( C ) ( data shown is pooled from three independent experiments , n = 3 - 8 ) . Red arrowheads indicate TUNEL signal ( D ) . Scale bar , 200 μM ( data shown is representative of two independent experiments , n = 3 - 4 ) .

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

C, D. Lung sections from control and infected mice treated as indicated, were stained by TUNEL for apoptotic cells at 6dpi. Quantification of TUNEL+ cells in whole lung slides by Icy Spot Detector (ICY-R3M2Y2) (C) (data shown is pooled from three independent experiments, n=3-8). Red arrowheads indicate TUNEL signal (D). Scale bar, 200 μM (data shown is representative of two independent experiments, n=3-4).


A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants ( A ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) or LPS ( AEC only ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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

A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6).


A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in Macrophage , pDC and cDC culture supernatants ( B ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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

A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in Macrophage, pDC and cDC culture supernatants (B) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) (data shown is representative of two independent experiments, n=3-6).


C . BAL samples taken from mice treated with IFNα , IFNλ or Veh Ctrl at specified time points ( data shown is representative of two independent experiments , n = 5 - 6 ) .

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C. BAL samples taken from mice treated with IFNα, IFNλ or Veh Ctrl at specified time points (data shown is representative of two independent experiments, n=5-6).


Mice were treated with IFNα ( 1 . 45μg / 50μl ) , IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( 50μl PBS ) , and whole lungs were taken at 18hrs post treatment for global analysis by Illumina . SingleColor . Mouse WG - 6 _ V2 _ 0 _ R0 _ 1127 microarrays . Samples ( n = 5 ) were normalized to the median of the vehicle control group and filtered for a fold change of 1 . 5 , yielding 553 genes differently regulated between treatments ( One way ANOVA , P < 0 . 01 , Benjamini - Hochberg multiple test correction ) , of which 429 genes are upregulated . K means clustering revealed six gene clusters , one of which encompassed genes primarily induced by IFNα4 ( A ) , while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 ( B ) . The two clusters of genes were analysed by Ingenuity Pathway Analysis ( IPA ) ( C , D ) .

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

Mice were treated with IFNα (1.45μg/50μl), IFNλ (2.6μg/50μl) or Veh Ctrl (50μl PBS), and whole lungs were taken at 18hrs post treatment for global analysis by Illumina.SingleColor.Mouse WG-6_V2_0_R0_1127 microarrays. Samples (n=5) were normalized to the median of the vehicle control group and filtered for a fold change of 1.5, yielding 553 genes differently regulated between treatments (One way ANOVA, P<0.01, Benjamini-Hochberg multiple test correction), of which 429 genes are upregulated. K means clustering revealed six gene clusters, one of which encompassed genes primarily induced by IFNα4 (A), while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 (B). The two clusters of genes were analysed by Ingenuity Pathway Analysis (IPA) (C,D).


A . HumanAEC cultures were stimulated for 4hrs with IFNα ( circles ) or IFNλ ( triangles ) at specified concentrations then assessed for stated ISG induction by qPCR ( data is representative of 2 independent experiments , n = 3 ) .

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

A. HumanAEC cultures were stimulated for 4hrs with IFNα (circles) or IFNλ (triangles) at specified concentrations then assessed for stated ISG induction by qPCR (data is representative of 2 independent experiments, n=3).


B , C . ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα ( 21 U / ml ) or IFNλ ( 1 . 2 ng / ml ) stimulation ( B ) ( data shown is pooled from 6 independent donors ) .

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

B, C. ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα (21 U/ml) or IFNλ (1.2 ng/ml) stimulation (B) (data shown is pooled from 6 independent donors).


B , C . PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ ( C ) ( data shown is pooled from 6 independent donors ) .

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

B, C. PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ (C) (data shown is pooled from 6 independent donors).


B Box plots of the Em / Ad expression ratios of GATA6 ( left ) and NKX2 - 1 ( right ) in FFPE lung tissue sections from controls ( Ctrl , n = 61 ) or lung cancer ( LC , n = 51 ) patients . Isoform - specific expression of the indicated genes was analyzed by qRT - PCR after total RNA isolation from tissue samples . Each point represents one sample .

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

B Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in FFPE lung tissue sections from controls (Ctrl, n=61) or lung cancer (LC, n=51) patients. Isoform-specific expression of the indicated genes was analyzed by qRT-PCR after total RNA isolation from tissue samples. Each point represents one sample.


C Box plots of Em / Ad of GATA6 ( top ) or NKX2 - 1 ( bottom ) show that high Em / Ad ratios in LC samples are maintained among ethnic groups ( left ) and gender ( right ) . GER , samples collected in Germany ; MEX , samples collected in Mexico .

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

C Box plots of Em/Ad of GATA6 (top) or NKX2-1 (bottom) show that high Em/Ad ratios in LC samples are maintained among ethnic groups (left) and gender (right). GER, samples collected in Germany; MEX, samples collected in Mexico.


D Box plots of Em / Ad of GATA6 ( left ) or NKX2 - 1 ( right ) in FFPE lung tissue sections from controls or LC patients . Samples were staged according to the TNM Classification ( UICC , 7th edition ) .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

D Box plots of Em/Ad of GATA6 (left) or NKX2-1 (right) in FFPE lung tissue sections from controls or LC patients. Samples were staged according to the TNM Classification (UICC, 7th edition).


A Box plots of the Em / Ad expression ratios of GATA6 ( left ) and NKX2 - 1 ( right ) in EBCs from controls ( Ctrl , n = 65 ) or lung cancer ( LC , n = 48 ) patients ( training set ) . Each point represents one sample .

O O O O O O O O O O O B-GENEPROD 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 B-TISSUE O O O O O O O O O O O O O O O O O O O O

A Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in EBCs from controls (Ctrl, n=65) or lung cancer (LC, n=48) patients (training set). Each point represents one sample.


( B ) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB - treated mMuSCs , human myoblasts and hiMPs . Red dots represent genes which display a positive fold - change in expression upon treatment with DLL4 & PDGF - BB whilst violet dots represent genes which are significantly downregulated . Differentially expressed genes required a P value of ≤ 0 . 05 to be considered significant .

O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD 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 O

(B) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant.


( C ) Heatmaps showing changes in expression of key myogenic ( MYOD , MYOGENIN ) , perivascular ( PDGFRB , NG2 , CD146 , ALPL ) and NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 & PDGF - BB in mMuSC - derived myoblasts ( left ) , human myoblasts ( middle ) and hiMPs ( right ) . Clustering was performed by genes / probes with Pearson correlation . Colour scale based on z - scores : red regions indicate high expression whilst blue regions indicate low expression . Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled .

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

(C) Heatmaps showing changes in expression of key myogenic (MYOD, MYOGENIN), perivascular (PDGFRB, NG2, CD146, ALPL) and NOTCH target (HEY1, HES1) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled.


( B ) Phase contrast images displaying morphology of untreated and treated hiMPs . Scale bar : 25 μm . ( C ) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs . Morphology was quantified as circularity ratio , where 1 = perfect circle and 0 = line ( experimental replicates = 3 ) . Statistical analysis ( paired t test ) was performed on the first quartile to enhance detection of morphological changes ( error bars : SD ) .

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

(B) Phase contrast images displaying morphology of untreated and treated hiMPs. Scale bar: 25 μm. (C) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs. Morphology was quantified as circularity ratio, where 1 = perfect circle and 0 = line (experimental replicates = 3). Statistical analysis (paired t test) was performed on the first quartile to enhance detection of morphological changes (error bars: SD).


( E ) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours . Scale bar : 75 μm . ( F ) Bar graphs quantifying EdU experiment shown in ( E ) ( experimental replicates = 3 ; error bars : SEM ) . Statistical analysis based on an unpaired t - test .

O O O B-EXP_ASSAY 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 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

(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm. (F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.


( H ) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week , induced to differentiate for 4 days in the presence or absence of γ - secretase inhibitor L685458 and immunostained for myosin heavy chain ( MyHC ) . ( I ) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes ( experimental replicates = 3 ; error bars : SEM ) . Statistical significance based on one - way ANOVA with Tukey ' s multiple comparison .

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 B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). (I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison.


( J ) Immunofluorescence panels showing human specific LAMIN A / C + ( nuclei ) and SPECTRIN ( sarcolemma ) staining in tibialis anterior muscles of immunodeficient mice ( N = 3 ) transplanted with treated ( n = 3 muscles ) and untreated ( n = 3 muscles ) N5 hiMPs . ( K ) Quantification of LAMIN A / C + grafted human cells across each muscle . Data information : full gene list for heatmaps in ( A ) and ( D ) available in Appendix Table S4 .

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

(J) Immunofluorescence panels showing human specific LAMIN A/C+ (nuclei) and SPECTRIN (sarcolemma) staining in tibialis anterior muscles of immunodeficient mice (N = 3) transplanted with treated (n = 3 muscles) and untreated (n = 3 muscles) N5 hiMPs. (K) Quantification of LAMIN A/C+ grafted human cells across each muscle. Data information: full gene list for heatmaps in (A) and (D) available in Appendix Table S4.


( F ) Representative fluorescence images of CMFDA - stained untreated and DLL4 & PDGF - BB - treated hiMPs within the top perfusion channel , 15 minutes after delivery and kept on the OrganoFlow ® . ( G ) Bar graph quantifying adhesion images in ( E ) . Statistical significance was calculated based on a paired t - test ( experimental replicates = 3 ) . Each point on the plot represents the number of adhered cells after 15 minutes within a single chip .

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

(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. (G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.


( H ) Assessment of DLL4 & PDGF - BB - treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells . Representative images showing the lower side of the trans - well membrane on which treated and untreated hiMPs ( stained with the transient dye CFDA , in green ) are simultaneously seeded on HUVECs for 8 hours . Bar graphs quantifying the average number of CFDA - positive cells / mm2 , that have migrated through the endothelial layer in each considered condition . ( experimental replicates = 3 ) . A minimum of 10 ( 1 . 5 mm2 ) fields per condition was quantified ( error bars : SEM ) . ( I ) Bar graph showing fold - change in trans - endothelial migration ( error bars : SEM ) . Statistical significance based on one - way ANOVA with Bonferroni ' s multiple comparison .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-DISEASE O B-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE 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 B-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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

(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). (I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.


( C ) HeLa cells were harvested at indicated time points and processed for western blotting probed for GBP1 and 5 protein levels , α - Tubulin ( Tubulin ) as loading control .

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

(C) HeLa cells were harvested at indicated time points and processed for western blotting probed for GBP1 and 5 protein levels, α-Tubulin (Tubulin) as loading control.


( B ) Top : Experimental outline of CRISPRa targeting of GBP1 and ASCL1 ( control ) by transient transfection with plasmids containing dCAS9 - SAM . Bottom : HeLa cells were transfected with dCas9 - SAM technology with gRNA for indicated genes , either alone or in combination with a 24h exposure to IFNγ . RNA was isolated and GBP1 mRNA expression was measured by RT - qPCR after 48h of transfection and normalized to ACTB expression . Statistical significance was determined using two - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates .

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

(B) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM. Bottom: HeLa cells were transfected with dCas9-SAM technology with gRNA for indicated genes, either alone or in combination with a 24h exposure to IFNγ. RNA was isolated and GBP1 mRNA expression was measured by RT-qPCR after 48h of transfection and normalized to ACTB expression. Statistical significance was determined using two-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.


( C ) Top : Experimental outline of CRISPRa targeting of GBP1 and ASCL1 ( control ) by transient transfection with plasmids containing dCAS9 - SAM , followed by induction with IFNγ . Bottom : RNA was isolated , GBP1 mRNA level was determined as indicted in experimental outline in top , by RT - qPCR and normalized to ACTB mRNA level . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates .

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

(C) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM, followed by induction with IFNγ. Bottom: RNA was isolated, GBP1 mRNA level was determined as indicted in experimental outline in top, by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.


( D ) Experiment as in C but with addition of IFNγ during priming . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates .

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

(D) Experiment as in C but with addition of IFNγ during priming. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.


( B ) Stable CRISPR knockouts were generated for indicated genes in HeLa cells . Knockout ( KO ) cells or their parental controls ( WT ) were induced with IFNγ for 24 hours or left untreated and prepared for SDS - PAGE and immunoblotting . Blots incubated with GBP5 antibody assess gene expression . α - Tubulin ( Tubulin ) was used as a loading control . Note that GBP5 and Tubulin blot of parental cells is as in 1C .

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

(B) Stable CRISPR knockouts were generated for indicated genes in HeLa cells. Knockout (KO) cells or their parental controls (WT) were induced with IFNγ for 24 hours or left untreated and prepared for SDS-PAGE and immunoblotting. Blots incubated with GBP5 antibody assess gene expression. α-Tubulin (Tubulin) was used as a loading control. Note that GBP5 and Tubulin blot of parental cells is as in 1C.


HeLa cells were primed with IFNγ for 24 hours , followed with IFNγ washout . After 48h , naïve and primed cells were induced by IFNγ for 1h and 3h . Cells were harvested at indicated time points and processed for CUT & RUN . Representation of processed data of CUT & RUN for STAT1 ( D ) and IRF1 ( E ) occupancy at GBP5 and GBP4 genes . Sequenced reads were mapped to the human genome ( hg38 ) , coverage data are displayed as reads per million ( RPM ) at equal scaling .

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HeLa cells were primed with IFNγ for 24 hours, followed with IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. Cells were harvested at indicated time points and processed for CUT&RUN. Representation of processed data of CUT&RUN for STAT1 (D) and IRF1 (E) occupancy at GBP5 and GBP4 genes. Sequenced reads were mapped to the human genome (hg38), coverage data are displayed as reads per million (RPM) at equal scaling.


( A ) Single - cell RNA - seq from HeLa cells for STAT1 Each dot represents STAT1 expression in one cell in naïve ( n = 91 ) , induction ( n = 90 ) and reinduction ( n = 92 ) . Error bars , SEM .

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(A) Single-cell RNA-seq from HeLa cells for STAT1 Each dot represents STAT1 expression in one cell in naïve (n=91), induction (n=90) and reinduction (n=92). Error bars, SEM.


( C ) Blot probing for STAT1 before and after IFNγ induction in STAT1 knockout ( STAT1KO ) , STAT1 rescued ( STAT1 , constitutive ) and parental control ( WT ) , to confirm knockout and rescue status . α - Tubulin ( Tubulin ) was used as a loading control .

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(C) Blot probing for STAT1 before and after IFNγ induction in STAT1 knockout (STAT1KO), STAT1 rescued (STAT1, constitutive) and parental control (WT), to confirm knockout and rescue status. α-Tubulin (Tubulin) was used as a loading control.


( D ) STAT1 rescue cells and their parental control were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A . Cell extracts were processed for western blotting and probed for GBP5 expression before and after induction and reinduction as indicated in Figure 1A . α - Tubulin ( Tubulin ) was used as a loading control .

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(D) STAT1 rescue cells and their parental control were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A. Cell extracts were processed for western blotting and probed for GBP5 expression before and after induction and reinduction as indicated in Figure 1A. α-Tubulin (Tubulin) was used as a loading control.


( G ) Cells constitutively expressing STAT1 ( as in B ) were primed with IFNγ , followed by IFNγ washout . After 48h , naïve and primed cells were induced by IFNγ for different time points ( 5 , 15 , 30 , 60 , 180 min ) . Cell extracts were prepared at indicated timepoints and processed for western blotting . Immunoblot of pSTAT1 - Y701 , and α - Tubulin ( Tubulin ) as a loading control .

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(G) Cells constitutively expressing STAT1 (as in B) were primed with IFNγ, followed by IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for different time points (5, 15, 30, 60, 180 min). Cell extracts were prepared at indicated timepoints and processed for western blotting. Immunoblot of pSTAT1-Y701, and α-Tubulin (Tubulin) as a loading control.


( A ) HeLa cells were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A with 2 days and 7 days recovery time ( primed state ) after IFNγ washout . Cell extracts were prepared at indicated timepoints , processed for western blotting and probed for STAT1 , pSTAT1 - Y701 and pSTAT1 - S727 . α - Tubulin ( Tubulin ) as a loading control .

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

(A) HeLa cells were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A with 2 days and 7 days recovery time (primed state) after IFNγ washout. Cell extracts were prepared at indicated timepoints, processed for western blotting and probed for STAT1, pSTAT1-Y701 and pSTAT1-S727. α-Tubulin (Tubulin) as a loading control.


( C ) ( H ) STAT1KO : : STAT1 - S727E and STAT1KO : : STAT1 - S727D expressing cells or their parental controls ( WT ) were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A , RNA was isolated and GBP5 mRNA level was determined by RT - qPCR and normalized to ACTB mRNA level . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean ( error bars , SD ; n = 3 biological replicates ) .

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(C) (H) STAT1KO::STAT1-S727E and STAT1KO::STAT1-S727D expressing cells or their parental controls (WT) were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A, RNA was isolated and GBP5 mRNA level was determined by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates).


( E ) Characterization of dTAG - induced destruction and recovery . STAT1 - EGFP - dTAG expressing cells were induced with IFNγ for 24h , then treated with dTAG13 ( 100 nM ) for DMSO vehicle control ( I ) or STAT1 depletion ( II ) for 24h . To determine recovery of STAT1 expression , cells were washed three times with medium and harvested 48h after IFNγ washout ( 120 hour timepoint , lane 4 ) . In Parallel , ( III ) cells were treated with dTAG13 ( 100 nM ) for 48h during 24h induction with IFNγ . Immunoblot of STAT1 - GFP - dTAG at indicated treatments confirm STAT1 depletion and recovery , respectively . α - Tubulin ( Tubulin ) was used as a loading control .

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(E) Characterization of dTAG-induced destruction and recovery. STAT1-EGFP-dTAG expressing cells were induced with IFNγ for 24h, then treated with dTAG13 (100 nM) for DMSO vehicle control (I) or STAT1 depletion (II) for 24h. To determine recovery of STAT1 expression, cells were washed three times with medium and harvested 48h after IFNγ washout (120 hour timepoint, lane 4). In Parallel, (III) cells were treated with dTAG13 (100 nM) for 48h during 24h induction with IFNγ. Immunoblot of STAT1-GFP-dTAG at indicated treatments confirm STAT1 depletion and recovery, respectively. α-Tubulin (Tubulin) was used as a loading control.


( G ) RNA was isolated and GBP5 mRNA level as indicated in ( F ) was determined by RT - qPCR , normalized to ACTB mRNA level . Statistical significance was determined using two - way ANOVA . Data are shown as mean ( error bars , SD ; n = 3 biological replicates )

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(G) RNA was isolated and GBP5 mRNA level as indicated in (F) was determined by RT-qPCR, normalized to ACTB mRNA level. Statistical significance was determined using two-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates)


A Western blot analysis STAT expression and phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ for 0 . 5 , 6 , 12 and 24 h . Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 ( Y701 ) and STAT2 ( Y689 ) and total level of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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A Western blot analysis STAT expression and phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ for 0.5, 6, 12 and 24 h. Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments . C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments .

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

B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN-induced genes (ISG). BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.


A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs . Spleens , livers or bone marrow derived macrophages ( BMDMs ) were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice . Whole cell extracts were collected and tested for levels of total Stat1 in western blot . The blots are representative of more than 3 independent experiments .

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A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs. Spleens, livers or bone marrow derived macrophages (BMDMs) were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice. Whole cell extracts were collected and tested for levels of total Stat1 in western blot. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701 . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice and stimulated for 30 min with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ . Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot . The blots are representative of more than 3 independent experiments .

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B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701. BMDMs were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice and stimulated for 30 min with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ. Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot. The blots are representative of more than 3 independent experiments.


C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

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C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ-induced genes. BMDMs of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05); the p-values were calculated using paired ratio t-test.


D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs were isolated from wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice treated with 250 IU / mL of IFNβ for 4 , 8 , 12 , 24 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test .

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D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN-induced genes (ISG). BMDMs were isolated from wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice treated with 250 IU/mL of IFNβ for 4, 8, 12, 24 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01); the p-values were calculated using paired ratio t-test.


E STAT1 and STAT2 phosphorylation in Stat1 - / - , Stat1Y701F , Stat2 - / - and Irf9 - / - macrophages . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice and treated with 250 IU / mL of IFNβ for 30 min or 6 , 12 or 24 h . The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689 . The same cell extracts were tested for total levels of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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E STAT1 and STAT2 phosphorylation in Stat1-/-, Stat1Y701F, Stat2-/- and Irf9-/- macrophages. BMDMs were isolated from wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice and treated with 250 IU/mL of IFNβ for 30 min or 6, 12 or 24 h. The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689. The same cell extracts were tested for total levels of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


A IFNβ - stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT2 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test .

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A IFNβ-stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT2-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**, p≤ 0.01); the p-values were calculated using paired ratio t-test.


B Impact of STAT2 deficiency on IFNβ - stimulated STAT1 association with the Mx2 ISRE . BMDMs of wild type ( WT ) , Stat1 - / - ( S1 ) and Stat2 - / - ( S2 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT1 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard error of mean ( SEM ) .

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B Impact of STAT2 deficiency on IFNβ -stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild type (WT), Stat1-/- (S1) and Stat2-/- (S2) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT1-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard error of mean (SEM).


C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP - reChIP . BMDMs of wild type ( WT ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with either STAT1 - specific antibody and re - immunoprecipitated with STAT2 - specific antibody or vice versa . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard deviation ( SD ) .

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C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP-reChIP. BMDMs of wild type (WT) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with either STAT1-specific antibody and re-immunoprecipitated with STAT2-specific antibody or vice versa. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD).