( B ) ANXA1 mRNA levels in Apcmin / + mouse colonic tumors and normal colonic tissues as measured by real - time qPCR . Each symbol represents one mouse . Horizontal lines represent mean values . * p < 0 . 05 , * * p < 0 . 01 ( two - way ANOVA ) .

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(B) ANXA1 mRNA levels in Apcmin/+ mouse colonic tumors and normal colonic tissues as measured by real-time qPCR. Each symbol represents one mouse. Horizontal lines represent mean values. *p<0.05, **p<0.01 (two-way ANOVA).


( C ) Positive correlation between fadA gene copy numbers ( x - axis ) and ANXA1 mRNA levels ( y - axis ) in F . nucleatum - induced Apcmin / + mouse colonic tumors ( n = 34 ; Pearson ' s correlation ) . Each dot represents the average of qPCR results performed in

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(C) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y- axis) in F. nucleatum-induced Apcmin/+ mouse colonic tumors (n=34; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates


( D ) Abundance of fadA in the paired normal and adenocarcinoma tissues from human CRC patients ( n = 18 ) expressed as ratio of fadA over total 16S rRNA genes determined by qPCR . Each symbol shows the average of duplicate qPCR results from one patient . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - test ) .

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(D) Abundance of fadA in the paired normal and adenocarcinoma tissues from human CRC patients (n=18) expressed as ratio of fadA over total 16S rRNA genes determined by qPCR. Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test).


( E ) ANXA1 mRNA levels in paired normal and adenocarcinoma tissues from CRC patients ( n = 18 ) . Each symbol shows the average of duplicate qPCR results from one patient . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - test ) .

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(E) ANXA1 mRNA levels in paired normal and adenocarcinoma tissues from CRC patients (n=18). Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test).


( F ) Positive correlation between fadA gene copy numbers ( x - axis ) and ANXA1 mRNA levels ( y - axis ) in human colorectal adenocarcinoma tissues ( n = 18 ; Pearson ' s correlation ) . Each dot represents the average of qPCR results performed in duplicates .

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(F) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y-axis) in human colorectal adenocarcinoma tissues (n=18; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates.


( G ) Confocal microscopy analysis of paired normal and carcinoma tissues from two colon cancer patients . The frozen sections were incubated with rabbit anti - AnnexinA1 polyclonal antibodies and 5G11 mouse anti - FadA monoclonal antibodies . The slides were then stained with Alexa Fluor ® 680 - conjugated donkey anti - rabbit and Alexa Fluor ® 555 - conjugated goat anti - mouse , washed , and covered in mounting medium containing DAPI . The scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for the normal tissues and 400x for the carcinomas . Scale bar , 50 μm . Co - localization of FadA ( red ) and Annexin A1 ( green ) was observed in carcinomas but not in the paired normal tissues .

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(G) Confocal microscopy analysis of paired normal and carcinoma tissues from two colon cancer patients. The frozen sections were incubated with rabbit anti-AnnexinA1 polyclonal antibodies and 5G11 mouse anti-FadA monoclonal antibodies. The slides were then stained with Alexa Fluor®680-conjugated donkey anti-rabbit and Alexa Fluor®555-conjugated goat anti-mouse, washed, and covered in mounting medium containing DAPI. The scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for the normal tissues and 400x for the carcinomas. Scale bar, 50 μm. Co-localization of FadA (red) and Annexin A1 (green) was observed in carcinomas but not in the paired normal tissues.


( A ) Flow cytometry analysis of β - catenin expression in 10C , HCT116 and DLD1 cells following transfection with control siRNA ( clear bars ) or ANXA1 - specific siRNA ( solid bars ) and incubation with F . nucleatum 12230 at MOI of ~ 20 : 1 for indicated time periods . The geometric means of cells treated with control siRNA at time 0 was designated as 1 . Data are mean values ± SD . The experiment was performed in duplicates or triplicates and repeated 1 - 3 times . * * p < 0 . 01 , * * * p < 0 . 001 ( two - way ANOVA ) .

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(A) Flow cytometry analysis of β-catenin expression in 10C, HCT116 and DLD1 cells following transfection with control siRNA (clear bars) or ANXA1-specific siRNA (solid bars) and incubation with F. nucleatum 12230 at MOI of ~20:1 for indicated time periods. The geometric means of cells treated with control siRNA at time 0 was designated as 1. Data are mean values ± SD. The experiment was performed in duplicates or triplicates and repeated 1-3 times. **p<0.01, *** p<0.001 (two-way ANOVA).


( B ) Immuno - staining of β - catenin in 10C cells following transfection with control siRNA or ANXA1 - specific siRNA and incubation with F . nucleatum 12230 ( Fn ) at MOI of ~ 100 : 1 for 2 hrs . β - catenin was stained with Alexa Fluor ® 680 ( red ) and the nuclei with DAPI ( blue ) . The images were captured with confocal microscope at 800x magnification . - , no bacteria added . Note the increased expression of β - catenin and its nucleus translocation in response to F . nucleatum in control siRNA - treated cells , but not in ANXA1 siRNA - treated cells . The experiment was repeated twice . Scale bar , 200 nm .

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(B) Immuno-staining of β-catenin in 10C cells following transfection with control siRNA or ANXA1-specific siRNA and incubation with F. nucleatum 12230 (Fn) at MOI of ~100:1 for 2 hrs. β-catenin was stained with Alexa Fluor®680 (red) and the nuclei with DAPI (blue). The images were captured with confocal microscope at 800x magnification. -, no bacteria added. Note the increased expression of β-catenin and its nucleus translocation in response to F. nucleatum in control siRNA-treated cells, but not in ANXA1 siRNA-treated cells. The experiment was repeated twice. Scale bar, 200 nm.


( C ) Real - time qPCR analysis of CCND1 expression using mRNA extracted from the cancerous 10C , and human CRC cells HCT116 and RKO , each grown to 50 % or 100 % confluency . Data are mean values ± SEM . The experiment was performed in triplicates and repeated twice . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - test ) .

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(C) Real-time qPCR analysis of CCND1 expression using mRNA extracted from the cancerous 10C, and human CRC cells HCT116 and RKO, each grown to 50% or 100% confluency. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. *p<0.05, ***p<0.001 (student's t-test).


( D ) Western - blot analysis of Cyclin D1 , Annexin A1 , and β - actin in RKO cells transfected with control vector or ANXA1 . Induction of Cyclin D1 was observed in response to increased Annexin A1 . The experiment was repeated twice .

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(D) Western-blot analysis of Cyclin D1, Annexin A1, and β-actin in RKO cells transfected with control vector or ANXA1. Induction of Cyclin D1 was observed in response to increased Annexin A1. The experiment was repeated twice.


( A ) Tru - Seq RNA sequence analysis of E12 . 5 midbrain floor - plate ( mFP ) , midbrain roof - plate ( mRP ) , anterior ( A , adjacent anterior FP ) and posterior ( P , adjacent posterior FP ) . Pbx1 is enriched in the midbrain FP , together with Th and Pitx3 . Lower levels of Pbx3 are also expressed in the mFP . Gdf7 and Wnt1 are restricted to the mRP at E12 . 5 .

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(A) Tru-Seq RNA sequence analysis of E12.5 midbrain floor-plate (mFP), midbrain roof-plate (mRP), anterior (A, adjacent anterior FP) and posterior (P, adjacent posterior FP). Pbx1 is enriched in the midbrain FP, together with Th and Pitx3. Lower levels of Pbx3 are also expressed in the mFP. Gdf7 and Wnt1 are restricted to the mRP at E12.5.


( B ) Pbx1 is expressed in the intermediate zone ( IZ ) and marginal zones ( MZ ) , but not the ventricular zone ( VZ ) , of the mFP at E12 . 5 , as detected by in situ hybridization .

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(B) Pbx1 is expressed in the intermediate zone (IZ) and marginal zones (MZ), but not the ventricular zone (VZ), of the mFP at E12.5, as detected by in situ hybridization.


( C ) PBX1 is first detected in the ventro - lateral part of the LMX1a + domain at E10 , preceding the birth of the first ( TH + ) mDA neurons at E10 . 5 . At E12 . 5 , PBX1 is present in all mDA neurons , but not all PBX1 + cells are TH + . White boxes indicate the area showed in higher magnification ( right ) .

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(C) PBX1 is first detected in the ventro-lateral part of the LMX1a+ domain at E10, preceding the birth of the first (TH+) mDA neurons at E10.5. At E12.5, PBX1 is present in all mDA neurons, but not all PBX1+ cells are TH+. White boxes indicate the area showed in higher magnification (right).


( D ) At E11 . 5 , PBX1 protein defines a subpopulation of NURR1 + neuroblasts and labels all NURR1 + TH + mDA neurons .

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(D) At E11.5, PBX1 protein defines a subpopulation of NURR1+ neuroblasts and labels all NURR1+TH+ mDA neurons.


( E ) PBX1 co - localizes with PITX3 and is also detected in a subpopulation of NURR1 + PITX3 - postmitotic neuroblasts at E12 . 5 . Higher magnification revealed three different populations of postmitotic cells : primary neuroblasts ( NURR1 + PBX1A - PITX3 - cells , green ) , secondary neuroblasts ( NURR1 + PBX1A + PITX3 - cells , yellow / orange ) and tertiary neuroblasts / mDA neurons ( NURR1 + PBX1A + PITX3 + cells , white ) . Nuclear staining , Dapi ( 4 ′ , 6 - diamidino - 2 - phenylindole , blue ) . All scale bars , 20µm .

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(E) PBX1 co-localizes with PITX3 and is also detected in a subpopulation of NURR1+PITX3- postmitotic neuroblasts at E12.5. Higher magnification revealed three different populations of postmitotic cells: primary neuroblasts (NURR1+PBX1A-PITX3-cells, green), secondary neuroblasts (NURR1+PBX1A+PITX3-cells, yellow/orange) and tertiary neuroblasts/mDA neurons (NURR1+PBX1A+PITX3+cells, white). Nuclear staining, Dapi (4′,6-diamidino-2-phenylindole, blue). All scale bars, 20µm.


( A ) PBX1A is the isoform detected in the TH + NURR1 + mDA neurons at E12 . 5 .

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(A) PBX1A is the isoform detected in the TH+NURR1+mDA neurons at E12.5.


( C ) Immunofluorescence microscopy analysis of 7 - week old human VM tissue showing that all mDAn are positive for PBX1 , suggesting a conserved role for this factor in the development of these neurons in mice and humans .

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(C) Immunofluorescence microscopy analysis of 7-week old human VM tissue showing that all mDAn are positive for PBX1, suggesting a conserved role for this factor in the development of these neurons in mice and humans.


( D ) PBX1 is also present in TH + adult mDA neurons of the substantia nigra ( SN ) and ventral tegmental area ( VTA ) .

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(D) PBX1 is also present in TH+ adult mDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA).


( A ) To the left , representative immunofluorescence microscopy / confocal images showing that the number of TH + cells is reduced in Pbx1 - / - embryos . Deletion of Pbx1 decreases the proportion of NURR1 + neuroblasts that become TH + neurons at E12 . 5 , indicating that Pbx1 is required for the proper maturation of NURR1 + neuroblasts . To the right , quantification of these experiments .

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(A) To the left, representative immunofluorescence microscopy/confocal images showing that the number of TH+cells is reduced in Pbx1-/- embryos. Deletion of Pbx1 decreases the proportion of NURR1+neuroblasts that become TH+neurons at E12.5, indicating that Pbx1 is required for the proper maturation of NURR1+neuroblasts. To the right, quantification of these experiments.


( B ) Ectopic PBX3 is detected in all rostro - caudal levels of the VM in Pbx1 - / - mice at E12 . 5 , indicating a repressive function of PBX1 on Pbx3 expression .

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(B) Ectopic PBX3 is detected in all rostro-caudal levels of the VM in Pbx1-/- mice at E12.5, indicating a repressive function of PBX1 on Pbx3 expression.


( C ) Representative immunofluorescence microscopy / confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 ( cKO , Pbx1flox / flox ; Pbx3 - / - ; Th - IRES - Cre - ERT ) reduces the number of PITX3 + cells in rostral VM levels , but not of NURR1 + cells , when compared to double heterozygous at E14 . 5 ( cHET , Pbx1flox / + ; Pbx3 - / + ; Th - IRES - Cre - ERT ) . White boxes are magnified to the right .

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(C) Representative immunofluorescence microscopy/confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 (cKO, Pbx1flox/flox;Pbx3-/-;Th-IRES-Cre-ERT) reduces the number of PITX3+cells in rostral VM levels, but not of NURR1+cells, when compared to double heterozygous at E14.5 (cHET, Pbx1flox/+;Pbx3-/+;Th-IRES-Cre-ERT). White boxes are magnified to the right.


( D ) At E18 . 5 , a drastic reduction of TH + , PITX3 + and NURR1 + cells is detected from anterior to posterior levels in cKO embryos , but not in cHET . Pyknotic NURR1 + nuclei are observed in cKO embryos ( arrowheads ) . White boxes are magnified to the right .

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(D) At E18.5, a drastic reduction of TH+, PITX3+ and NURR1+cells is detected from anterior to posterior levels in cKO embryos, but not in cHET. Pyknotic NURR1+nuclei are observed in cKO embryos (arrowheads). White boxes are magnified to the right.


( E ) The total number of TH + mDA neurons is dramatically reduced and there is an increase in aCASP3 + cells in cKO embryos compared to cHET mice at E18 . 5 .

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(E) The total number of TH+ mDA neurons is dramatically reduced and there is an increase in aCASP3+ cells in cKO embryos compared to cHET mice at E18.5.


( B ) Representative immunofluorescence microscopy / confocalimages showing that PBX1 - overexpression increases the number of TH + neurons after 8 days of differentiation of hNES cells ( AF22 line derived from induced pluripotent stem cells ) . ( C ) While the number of cells ( Dapi + ) in both conditions did not change , the number of TH + cells increased 2 . 3 fold in PBX1 - overexpressing cells .

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(B) Representative immunofluorescence microscopy/confocalimages showing that PBX1-overexpression increases the number of TH+neurons after 8 days of differentiation of hNES cells (AF22 line derived from induced pluripotent stem cells). (C) While the number of cells (Dapi+) in both conditions did not change, the number of TH+cells increased 2.3 fold in PBX1-overexpressing cells.


( D ) TH + cells derived from the PBX1 - overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3 . All scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates per condition and p - value ( T - test ) are indicated in the graphs .

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(D) TH+cells derived from the PBX1-overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates per condition and p-value (T-test) are indicated in the graphs.


( B ) Schematic of the track of PBX1 ChIP - seq compared to IgGChIP - seq as a control on different loci with peaks identified by HOMER . Each locus represents a 20kb chromosome region .

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(B) Schematic of the track of PBX1 ChIP-seq compared to IgGChIP-seq as a control on different loci with peaks identified by HOMER. Each locus represents a 20kb chromosome region.


( C ) List of the 29 most regulated genes ( t - test q - value < 0 . 05 ) identified by Gene Set Enrichment Analysis ( GSEA ) and ranked by p - value . RPKM stands for Reads Per Kilobase of transcript per Million mapped reads . GSEA was performed on genes identified by RNA - Seq as differentially expressed in the VM at E12 . 5 compared to adjacent brain regions and identified by PBX1Chip - Seq in proximity to transcription starting sites .

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(C) List of the 29 most regulated genes (t-test q-value <0.05) identified by Gene Set Enrichment Analysis (GSEA) and ranked by p-value. RPKM stands for Reads Per Kilobase of transcript per Million mapped reads. GSEA was performed on genes identified by RNA-Seq as differentially expressed in the VM at E12.5 compared to adjacent brain regions and identified by PBX1Chip-Seq in proximity to transcription starting sites.


( D ) Graphic representation of these top 29 genes ( red dots ) in the GSEA curve showing a high correlation between the cistrome and transcriptome ( p - value < 0 . 001 , q - value < 0 . 005 ) .

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(D) Graphic representation of these top 29 genes (red dots) in the GSEA curve showing a high correlation between the cistrome and transcriptome (p-value <0.001, q-value<0.005).


( E ) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 ( Pitx3 - Gbf1 ) , Onecut2and Nfe2l1 genes . No co - immunoprecipitation was detected for the unrelated gene , Lrp1b ( Low Density Lipoprotein Receptor - Related Protein 1B ) .

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(E) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 (Pitx3-Gbf1), Onecut2and Nfe2l1 genes. No co-immunoprecipitation was detected for the unrelated gene, Lrp1b (Low Density Lipoprotein Receptor-Related Protein 1B).


( F , G ) The regulation of Pitx3 and Onecut2 by PBX1 was confirmed in Pbx1 - / - ; Pbx3 + / - mutant embryos at E12 . 5 , compared to wild - type ( Pbx1 + / + ) littermates . ( F ) Representative immunofluorescence microscopy / confocal images showing that decreased levels of PITX3 and number of PITX3 + cells in E12 . 5 Pbx1 - / - ; Pbx3 + / - embryos . This reduction is more evident in the basal plate ( BP ) and in the midline ( M ) , as shown at higher magnification ( right ) . ( G ) Double ONECUT2 + TH + cells were detected only in the lateral part of the mFP in E12 . 5 Pbx1 - / - ; Pbx3 + / - embryos . Selected areas are magnified to the right . A scheme ( right ) shows the distribution of cells in Pbx1 + / + and Pbx1 - / - ; Pbx3 + / - embryos . All scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates ( dots ) per condition and p - value ( T - test ) are indicated in each graph .

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(F, G) The regulation of Pitx3 and Onecut2 by PBX1 was confirmed in Pbx1-/-;Pbx3+/- mutant embryos at E12.5, compared to wild-type (Pbx1+/+) littermates. (F) Representative immunofluorescence microscopy/confocal images showing that decreased levels of PITX3 and number of PITX3+cells in E12.5 Pbx1-/-;Pbx3+/- embryos. This reduction is more evident in the basal plate (BP) and in the midline (M), as shown at higher magnification (right). (G) Double ONECUT2+TH+cells were detected only in the lateral part of the mFP in E12.5 Pbx1-/-;Pbx3+/- embryos. Selected areas are magnified to the right. A scheme (right) shows the distribution of cells in Pbx1+/+ and Pbx1-/-;Pbx3+/- embryos. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates (dots) per condition and p-value (T-test) are indicated in each graph.


( A ) Representative immunofluorescence microscopy / confocal images showing that NFE2L1 is detected in TH + mDAn and neuroblasts at E12 . 5 .

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(A) Representative immunofluorescence microscopy/confocal images showing that NFE2L1 is detected in TH+mDAn and neuroblasts at E12.5.


( B ) The expression of NFE2L1 in TH + neurons is dramatically reduced in Pbx1 - / - ; Pbx3 + / - compared to Pbx1 + / + embryos at E12 .

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(B) The expression of NFE2L1 in TH+neurons is dramatically reduced in Pbx1-/-;Pbx3+/- compared to Pbx1+/+ embryos at E12.5


( C ) shRNA lentiviruses against NFE2L1 increased the levels of aCASP3 in H2O2 - treated ( 100µm for 12 hours ) TH + neurons derived from hNES cells differentiated for 8 days ( percentage ) . White arrows indicate double TH + aCASP3 + neurons .

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(C) shRNA lentiviruses against NFE2L1 increased the levels of aCASP3 in H2O2-treated (100µm for 12 hours) TH+neurons derived from hNES cells differentiated for 8 days (percentage). White arrows indicate double TH+aCASP3+neurons.


( D ) PBX1 overexpression protected TH + neurons derived from hNES cells from the oxidative stress induced by H2O2 ( 100µm for 12 hours ) , resulting in reduced levels of aCASP3 in TH + neurons ( percentage ) .

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(D) PBX1 overexpression protected TH+neurons derived from hNES cells from the oxidative stress induced by H2O2 (100µm for 12 hours), resulting in reduced levels of aCASP3 in TH+neurons (percentage).


( E ) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining ( dark - blue / black , indicated by arrowheads ) in the nuclei of neuromelanin + cells ( NM + , cytosolic brown aggregates ) of the SN . In contrast , PBX1 staining was severely reduced in PD patients . ( F ) Graphical representation of PBX1 intensity levels in the SN of NM + neurons in all individual controls and PD patients analyzed . Each dot in the graph represents the level of PBX1 in the nuclei of one single NM + neuron .

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(E) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining (dark-blue/black, indicated by arrowheads) in the nuclei of neuromelanin+cells (NM+, cytosolic brown aggregates) of the SN. In contrast, PBX1 staining was severely reduced in PD patients. (F) Graphical representation of PBX1 intensity levels in the SN of NM+ neurons in all individual controls and PD patients analyzed. Each dot in the graph represents the level of PBX1 in the nuclei of one single NM+neuron.


( G ) Strong immunoreactivity of the PBX1 direct target , NFE2L1 , was also detected as very well defined nuclear punctuate structures in NM + cells of the SN in control samples ( arrowheads ) , but were dramatically reduced in PD patients . Yellow asterisks indicate the nucleoli . ( H ) Percentage of NM + neurons showing nuclear NFE2L1 staining in the SN of control donors and PD patients . ( I ) Graphical representation of all individual controls and PD patients analyzed , showing that NM + neurons with NFE2L1 + nuclei in the SN of PD patients are less than 10 % or are totally absent .

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(G) Strong immunoreactivity of the PBX1 direct target, NFE2L1, was also detected as very well defined nuclear punctuate structures in NM+cells of the SN in control samples (arrowheads), but were dramatically reduced in PD patients. Yellow asterisks indicate the nucleoli. (H) Percentage of NM+ neurons showing nuclear NFE2L1 staining in the SN of control donors and PD patients. (I) Graphical representation of all individual controls and PD patients analyzed, showing that NM+neurons with NFE2L1+nuclei in the SN of PD patients are less than 10% or are totally absent.


A Expression of PTPN11 ( gene encoding SHP2 ) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data ( No . GSE14905 ) . B Expression of ptpn11 in human PBMCs of psoriatic patients ( n = 14 ) and normal controls ( n = 16 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 .

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A Expression of PTPN11 (gene encoding SHP2) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905). B Expression of ptpn11 in human PBMCs of psoriatic patients (n=14) and normal controls (n=16). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.


C Representative SHP2 staining in skin sections of psoriatic patients ( n = 13 ) and normal controls ( n = 5 ) . Scale bars : 200 μm .

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C Representative SHP2 staining in skin sections of psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm.


D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls .

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D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls.


E The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients ( n = 25 ) and normal controls ( n = 25 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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E The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n=25) and normal controls (n=25). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


F Representative p - ERK staining of skin sections of psoriatic patients and normal controls . Scale bars : 200 μm .

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F Representative p-ERK staining of skin sections of psoriatic patients and normal controls. Scale bars: 200 μm.


G Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ - treated or non - treated dorsal back of C57BL / 6 mice at day 5 ( n = 6 / group ) . Data were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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G Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5 (n=6/group). Data were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


H Representative histological sections of IMQ - treated or non - treated dorsal back of C57BL / 6 mice at day 5 . Scale bar : 100 μm .

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H Representative histological sections of IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5. Scale bar: 100 μm.


I Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients ( n = 20 ) and normal healthy controls ( n = 20 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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I Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients (n=20) and normal healthy controls (n=20). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


C57BL / 6 mice ( n = 6 / group ) were treated with indicated dose of SHP099 or vehicle for 4 days . A Phenotypic presentation ( top ) , H & E staining ( middle ) and statistical data ( bottom ) ( mean ± SEM ) of dorsal skin . Scale bar : 100 μm . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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C57BL/6 mice (n=6/group) were treated with indicated dose of SHP099 or vehicle for 4 days. A Phenotypic presentation (top), H&E staining (middle) and statistical data (bottom) (mean ± SEM) of dorsal skin. Scale bar: 100 μm. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


Quantitative PCR analysis of mRNA encoding IL - 23 / IL - 17A axis cytokines in the dorsal skin of C57BL / 6 mice treated with indicated dose of SHP099 or vehicle for 4 days . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099 or vehicle for 4 days. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


ELISA quantification of protein levels of cytokines in mouse serum . Quantitative PCR analysis of mRNA encoding psoriasis - related cytokines in the dorsal skin of C57BL / 6 mice treated with indicated dose of SHP099 or vehicle for 4 days . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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ELISA quantification of protein levels of cytokines in mouse serum. Quantitative PCR analysis of mRNA encoding psoriasis-related cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099 or vehicle for 4 days. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


Violin plot showed the relative expression of 47 inflammation - associated genes based on sample groups and cell clusters .

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Violin plot showed the relative expression of 47 inflammation-associated genes based on sample groups and cell clusters.


D Gene set enrichment analysis ( GSEA ) of the NF - κB signaling pathway .

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D Gene set enrichment analysis (GSEA) of the NF-κB signaling pathway.


E Volcano plots of DEGs that were upregulated ( red ) or downregulated ( blue ) in myeloid cells . Examples of DEGs were labeled .

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E Volcano plots of DEGs that were upregulated (red) or downregulated (blue) in myeloid cells. Examples of DEGs were labeled.


G Expression patterns of the top ten hub genes in ( F ) . Heatmap ( top ) displays the average expression in the three groups and in different myeloid cell subsets . Boxplots ( bottom ) show the corresponding expression distribution .

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G Expression patterns of the top ten hub genes in (F). Heatmap (top) displays the average expression in the three groups and in different myeloid cell subsets. Boxplots (bottom) show the corresponding expression distribution.


Representative images ( A and E ) , histological sections of dorsal back ( B and F ) and clinical scores ( C and G ) and quantitative PCR analysis of mRNA levels in dorsal skin ( D and H ) from wild - type ( n = 6 ) and M - Shp2 - / - mice or DC - Shp2 - / - mice ( n = 6 ) treated with IMQ for 4 days . Scale bar : 100 μm . Left : H & E ( hematoxylin and eosin ) staining data ; right : statistical data ( mean ± SEM ) . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values were calculated by Tukey multiple comparison test ( B , D , F , H ) or Bonferroni multiple comparison test ( C , G ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant .

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Representative images (A and E), histological sections of dorsal back (B and F) and clinical scores (C and G) and quantitative PCR analysis of mRNA levels in dorsal skin (D and H) from wild-type (n=6) and M-Shp2-/- mice or DC-Shp2-/- mice (n=6) treated with IMQ for 4 days. Scale bar: 100 μm. Left: H&E (hematoxylin and eosin) staining data; right: statistical data (mean ± SEM). Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values were calculated by Tukey multiple comparison test (B, D, F, H) or Bonferroni multiple comparison test (C, G). *P<0.05, **P<0.01, ns, not significant.


A Heat map showing mRNA expression in peritoneal macrophages derived from wild - type and M - Shp2 - / - mice after IMQ treatment based on RNA sequencing ( n = 3 / group ) . Colors represent high ( red ) and low ( blue ) intensity . Genes labeled in green indicate they are in the NF - κB signaling .

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A Heat map showing mRNA expression in peritoneal macrophages derived from wild-type and M-Shp2-/- mice after IMQ treatment based on RNA sequencing (n=3/group). Colors represent high (red) and low (blue) intensity. Genes labeled in green indicate they are in the NF-κB signaling.


D BMDMs and PMs derived from wild - type and M - Shp2 - / - mice were stimulated by IMQ ( 10 µg / ml ) for indicated times . Whole cell lysates were subjected to Western blotting .

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D BMDMs and PMs derived from wild-type and M-Shp2-/- mice were stimulated by IMQ (10 µg/ml) for indicated times. Whole cell lysates were subjected to Western blotting.


E Representative p - p65 staining of skin sections from mouse . Scale bar : 100 μm .

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E Representative p-p65 staining of skin sections from mouse. Scale bar: 100 μm.


E Immunoblotting of HEK293T cells co - transfected for 48 h with GFP - TLR7 , plus HA - SHP2 , HA - SHP2 mutant vectors , followed by immunoprecipitation with anti - GFP beads .

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E Immunoblotting of HEK293T cells co-transfected for 48 h with GFP-TLR7, plus HA-SHP2, HA-SHP2 mutant vectors, followed by immunoprecipitation with anti-GFP beads.


F PMA - differentiated THP - 1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 ( 10 µg / ml ) . Subcellular fractionation was performed , and cytoplasmic and cell membrane proteins were probed by respective antibodies .

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F PMA-differentiated THP-1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 (10 µg/ml). Subcellular fractionation was performed, and cytoplasmic and cell membrane proteins were probed by respective antibodies.


G , H Immunoblot analysis of TLR7 and SHP2 in the isolated ER , Golgi and endosome from PMA - differentiated THP - 1 cells with either SHP2 overexpressed ( G ) and SHP2 deficiency ( H ) .

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G, H Immunoblot analysis of TLR7 and SHP2 in the isolated ER, Golgi and endosome from PMA-differentiated THP-1 cells with either SHP2 overexpressed (G) and SHP2 deficiency(H).


I Skin sections from psoriatic patients and healthy controls were immune - stained for TLR7 together with a marker of the early endosome ( EEA1 ) or late endosome / lysosome ( LAMP1 ) prior to analysis by confocal microscopy , showing TLR7 localization in different organelles ( arrows ) . Scale bar : 50 μm .

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I Skin sections from psoriatic patients and healthy controls were immune-stained for TLR7 together with a marker of the early endosome (EEA1) or late endosome/lysosome (LAMP1) prior to analysis by confocal microscopy, showing TLR7 localization in different organelles (arrows). Scale bar: 50 μm.


B , Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP - TLR7 or GFP - TLR7 mutant vectors .

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B, Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP-TLR7 or GFP-TLR7 mutant vectors.


G HEK293T cells transfected with NF - κB - luciferase reporter and indicated TLR7 mutant vectors and then treated with IMQ ( 10 µg / ml ) for 24 h were harvested for luciferase assay . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * * P < 0 . 01 , ns , not significant .

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G HEK293T cells transfected with NF-κB-luciferase reporter and indicated TLR7 mutant vectors and then treated with IMQ (10 µg/ml) for 24 h were harvested for luciferase assay. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test **P<0.01, ns, not significant.


I Representative p - TLR7 Y1024 staining of skin sections from human . Scale bar : 100 μm .

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I Representative p-TLR7 Y1024 staining of skin sections from human. Scale bar: 100 μm.


Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . A Phenotypic presentation ( top ) and H & E staining ( bottom ) of dorsal skin . Scale bar : 100 μm . Left : H & E staining data ; right : statistical data ( mean ± SEM ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 .

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

Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. A Phenotypic presentation (top) and H&E staining (bottom) of dorsal skin. Scale bar: 100 μm. Left: H&E staining data; right: statistical data (mean ± SEM). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.


Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . D Quantitative PCR analysis of mRNA encoding IL - 23 / IL - 17A axis cytokines and other psoriasis - related cytokines in the dorsal skin . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 .

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

Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. D Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines and other psoriasis-related cytokines in the dorsal skin. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.


Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . E Representative p - p65 staining of the dorsal skin . Scale bars : 100 μm .

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Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. E Representative p-p65 staining of the dorsal skin. Scale bars: 100 μm.


A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21 % O2 vs . 85 % O2 , at post - natal day ( P ) 5 and P14 .

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

A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21% O2 vs. 85% O2, at post-natal day (P)5 and P14.


B Quantitative RT - PCR detection of microRNA - 34a / b / c - 5p family members in the lung over the course of normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization . C Quantitative RT - PCR detection of microRNA - 34a / b / c - 3p family members in the lung over the course of normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization .

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

B Quantitative RT-PCR detection of microRNA-34a/b/c-5p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization. C Quantitative RT-PCR detection of microRNA-34a/b/c-3p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization.


A Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) and miR - 34a - / - mice during normal and aberrant alveolarization ( scale bar , 50 µm ) . B Quantification of total number of alveoli by design - based stereology in wild - type ( WT ) and miR ‑ 34a - / - mice ( 34a - / - ) during normal and aberrant alveolarization . C Quantification of mean septal thickness by design - based stereology in wild - type ( WT ) and miR ‑ 34a - / - mice ( 34a - / - ) during normal and aberrant alveolarization .

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

A Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34a-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). B Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization. C Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization.


D Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) and miR - 34bc - / - mice during normal and aberrant alveolarization ( scale bar , 50 µm ) . E Quantification of total number of alveoli by design - based stereology in wild - type ( WT ) and miR ‑ 34bc - / - mice ( 34bc - / - ) during normal and aberrant alveolarization . F Quantification of mean septal thickness by design - based stereology in wild - type ( WT ) and miR ‑ 34bc - / - mice ( 34bc - / - ) during normal and aberrant alveolarization .

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

D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34bc-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). E Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization. F Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization.


G Localization of miR - 34a expression by β - galactosidase activity staining in the developing lungs of P14 miR - 34a : : lacZ + / + mice that were undergoing normal or aberrant alveolarization ( scale bar , 50 µm ; larger and some additional images are presented in Appendix Fig S2 ) .

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

G Localization of miR-34a expression by β-galactosidase activity staining in the developing lungs of P14 miR-34a::lacZ+/+ mice that were undergoing normal or aberrant alveolarization (scale bar, 50 µm; larger and some additional images are presented in Appendix Fig S2).


B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA ( SCR ) or a miR - 34a ( MIM34a ) mimic ( n = 3 separate cell cultures for each group ) .

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B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA (SCR) or a miR-34a (MIM34a) mimic (n = 3 separate cell cultures for each group).


C Quantitative RT - PCR detection of miR - 34a / b / c - 5p levels in MLg cells in vitro , maintained under 21 % O2 or 85 % O2 ( n = 3 separate cell cultures for each group ) .

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C Quantitative RT-PCR detection of miR-34a/b/c-5p levels in MLg cells in vitro, maintained under 21% O2 or 85% O2 (n = 3 separate cell cultures for each group).


D Immunoblot detection of PDGFRα levels in the lungs of mouse pups ( n = 6 animals for each group ) at post - natal day ( P ) 5 , during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization .

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D Immunoblot detection of PDGFRα levels in the lungs of mouse pups (n = 6 animals for each group) at post-natal day (P)5, during normal (21% O2) and aberrant (85% O2) alveolarization.


E Immunoblot detection of PDGFRα levels in MLg cells in vitro , maintained under 21 % O2 or 85 % O2 , where cells had been transfected wither with a scrambled ( SCR ) antimiR , or an antimiR directed against miR - 34a ( A34a ) ( n = 3 separate cell cultures for each group ) .

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

E Immunoblot detection of PDGFRα levels in MLg cells in vitro, maintained under 21% O2 or 85% O2, where cells had been transfected wither with a scrambled (SCR) antimiR, or an antimiR directed against miR-34a (A34a) (n = 3 separate cell cultures for each group).


F Quantitative RT - PCR detection of miR - 34a - 5p levels in PDGFRα + cells , sorted by FACS from the lungs of mouse pups ( n = 4 animals for each group ; data from an independent repetition are provided in Appendix Fig S5 ) at P5 , maintained under 21 % O2 or 85 % O2 from birth .

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

F Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of mouse pups (n = 4 animals for each group; data from an independent repetition are provided in Appendix Fig S5) at P5, maintained under 21% O2 or 85% O2 from birth.


H Quantitative RT - PCR detection of miR - 34a - 5p levels in PDGFRα + cells , sorted by FACS from the lungs of either wild - type ( 34awt / wt ) mouse pups , or mouse pups in which miR - 34a expression in Pdgfra ‑ expressing cells ( 34ai ∆ PC / i ∆ PC ) at P5 ( n = 4 animals for each group ) .

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

H Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of either wild-type (34awt/wt) mouse pups, or mouse pups in which miR-34a expression in Pdgfra‑expressing cells (34ai∆PC/i∆PC) at P5 (n = 4 animals for each group).


I Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 during aberrant ( 85 % O2 ) alveolarization , compared with 34ai ∆ PC / i ∆ PC during normal ( 21 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of observations made in four other experiments . J Quantification of total number of alveoli by design - based stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal and aberrant alveolarization ( n = 5 animals for each group ) . K Quantification of mean septal thickness by design - based stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

I Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14 during aberrant (85% O2) alveolarization, compared with 34ai∆PC/i∆PC during normal (21% O2) alveolarization (scale bar, 50 µm). Data are representative of observations made in four other experiments. J Quantification of total number of alveoli by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group). K Quantification of mean septal thickness by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group).


B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR ( SCR ) and miR - 34a ( MIM34a ) mimics , and probing for PDGFRα and c - Kit as TSB ‑ dependent and TSB ‑ independent target readouts , respectively . Protein loading equivalence was controlled by βactin levels . Note : PDGFRα , βactin , and c - Kit were all probed on the same membrane ; hence a single βactin immunoblot is presented . Data are representative of three experiments .

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

B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and c-Kit as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and c-Kit were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.


C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR ( SCR ) and miR - 34a ( MIM34a ) mimics , and probing for PDGFRα and SIRT1 as TSB ‑ dependent and TSB ‑ independent target readouts , respectively . Protein loading equivalence was controlled by βactin levels . Note : PDGFRα , βactin , and SIRT1 were all probed on the same membrane ; hence a single βactin immunoblot is presented . Data are representative of three experiments .

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

C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and SIRT1 as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and SIRT1 were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.


D Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled target site blocker ( SCR ) or a cocktail of both target site blockers ( TSB1 and TSB2 ) during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments . E Quantification of total number of alveoli by design - based stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) . F Quantification of mean septal thickness by design - based stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) .

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

D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled target site blocker (SCR) or a cocktail of both target site blockers (TSB1 and TSB2) during normal (21% O2) and aberrant (85% O2) alveolarization (scale bar, 50 µm). Data are representative of three or more experiments. E Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). F Quantification of mean septal thickness by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).


G Quantitative analysis of PDGFRα + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) .

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

G Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).


H Quantitative analysis of PDGFRα + / αSMA + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) .

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

H Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).


B Quantitative RT - PCR detection of miR - 34a - 5p levels in wild - type mouse pups at post - natal day ( P ) 14 that had been treated either with a scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization ( n = 4 animals for each group ) .

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

B Quantitative RT-PCR detection of miR-34a-5p levels in wild-type mouse pups at post-natal day (P)14 that had been treated either with a scrambled antimiR (S), or antimiR-34a (A34a), during normal (21% O2) and aberrant (85% O2) alveolarization (n = 4 animals for each group).


C Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments .

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

C Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (scale bar, 50 µm). Data are representative of three or more experiments.


D Quantification of total number of alveoli by design - based stereology in wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant lung alveolarization ( n = 5 animals for each group ) .

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

D Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant lung alveolarization (n = 5 animals for each group).


E Quantification of mean septal thickness by design - based stereology in in wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

E Quantification of mean septal thickness by design-based stereology in in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).


F Quantitative analysis of PDGFRα + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

F Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).


G Quantitative analysis of PDGFRα + / αSMA + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

G Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).


A Mice expressing nuclear ‑ localized GFP under the control of the Pdgfra promoter were maintained under normoxic ( 21 % O2 ) or hyperoxic ( 85 % O2 ) conditions , and lungs were harvested , processed and immunostained for Ki67 to determine proliferation status . DAPI staining revealed nuclei of all cells present in the section . Low - magnification images from individual channels are presented to the right of the merged ( large ) image first row of images . The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image , as well as visualization of a single Ki67 + , GFP + cell ( white arrowhead ) in all three channels separately .

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

A Mice expressing nuclear‑localized GFP under the control of the Pdgfra promoter were maintained under normoxic (21% O2) or hyperoxic (85% O2) conditions, and lungs were harvested, processed and immunostained for Ki67 to determine proliferation status. DAPI staining revealed nuclei of all cells present in the section. Low-magnification images from individual channels are presented to the right of the merged (large) image first row of images. The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image, as well as visualization of a single Ki67+,GFP+ cell (white arrowhead) in all three channels separately.


B The number of PDGFRα + cells in four microscopic fields was assessed for co - staining with an anti - Ki67 antibody to reveal proliferating cells . P values were calculated by unpaired Student ' s t ‑ test ( n = 4 fields for each group , trends are representative of those observed in two other experiments ) .

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

B The number of PDGFRα+ cells in four microscopic fields was assessed for co-staining with an anti-Ki67 antibody to reveal proliferating cells. P values were calculated by unpaired Student's t‑test (n=4 fields for each group, trends are representative of those observed in two other experiments).


C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild - type mice that were treated with an isotype - matched control IgG used for the Ki67 staining experiments . Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining . Scale bar : 100 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; GFP , green fluorescent protein .

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

C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild-type mice that were treated with an isotype-matched control IgG used for the Ki67 staining experiments. Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining. Scale bar: 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.


The impact of administration of scrambled antimiR ( S ) or an antimiR directed against miR - 34a ( A34a ) on the abundance of type I alveolar epithelial cells ( marked by aquaporin 5 , Aqp5 ) and type II alveolar epithelial cells ( marked by pro - surfactant protein C , Sftpc ) was assessed in 3 - µm sections of paraffin ‑ embedded lung tissue from P5 mice undergoing normal ( 21 % O2 ) or aberrant ( 85 % O2 ) lung alveolarization . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole . In the DAPI images , white lines delineate tissue from airspaces , and in the 85 % O2 groups demarcate septa . Antibody specificity was validated by rabbit IgG isotype control primary antibodies . The control experiments for the Aqp5 and Sftpc staining runs are illustrated here . Scale bars , 50 µm .

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

The impact of administration of scrambled antimiR (S) or an antimiR directed against miR-34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro-surfactant protein C, Sftpc) was assessed in 3-µm sections of paraffin‑embedded lung tissue from P5 mice undergoing normal (21% O2) or aberrant (85% O2) lung alveolarization. DAPI, 4′,6-diamidino-2-phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O2 groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 µm.


D ) HEK293 cells were genetically engineered to knockout RIG - I , MDA5 , or both , and subsequently subjected to retroviral transduction to stably express FLAG - LGP2 or an empty vector ( EV ) . Correct gene editing and intact type I IFN responsiveness was validated by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) . * , non - specific band .

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

D) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band.


E ) Cells generated in ( D ) were transfected with siCtrl or siADAR1 . The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post - transfection as in ( B ) . Data are means ± s . d . from a representative of four biological replicate experiments .

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

E) Cells generated in (D) were transfected with siCtrl or siADAR1. The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post-transfection as in (B). Data are means ± s.d. from a representative of four biological replicate experiments.


F ) Cells generated in ( D ) were transfected with siCtrl ( siC ) or siADAR1 ( siA ) . Protein lysates were prepared 78h post - transfection , followed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) .

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

F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).


G ) HEK293 WT cells and FLAG - LGP2 - expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence . Cells were fixed , permeabilized and stained 72h post - transfection with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 450 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of three biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analyses were performed using unpaired two - tailed Mann - Whitney U tests . ns , not significant ; * * * * , p < 0 . 0001 .

O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained 72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>450 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.


A ) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR / Cas9 . Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness , respectively . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) .

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

A) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR/Cas9. Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness, respectively. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3).


B ) ADAR1 - knockout HEK293 cells ( clone 1 ) were co - transfected with an empty vector ( EV ) or a FLAG - LGP2 - encoding vector ( LGP2 ) combined with a vector encoding GFP - tagged ADAR1 p110 or p150 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 expression , normalized to ACTB . Data are means ± s . d . from a representative of four biological replicate experiments .

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

B) ADAR1-knockout HEK293 cells (clone 1) were co-transfected with an empty vector (EV) or a FLAG-LGP2-encoding vector (LGP2) combined with a vector encoding GFP-tagged ADAR1 p110 or p150. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 expression, normalized to ACTB. Data are means ± s.d. from a representative of four biological replicate experiments.


C ) ADAR1 - knockout cells ( clone 1 ) were transfected as in ( B ) . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 4 ) .

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

C) ADAR1-knockout cells (clone 1) were transfected as in (B). Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=4).


A ) ADAR1 - knockout HEK293 cells ( clone 1 ) were modified with a lentiviral - based inducible system to express FLAG - LGP2 WT or a FLAG - LGP2 RNA binding mutant ( K138E / R490E / K634E , denoted as " KRK mutant " ) in a doxycycline - regulated manner . Cells were treated 72h with doxycycline ( dox ) . Protein lysates were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 3 ) . iEV = inducible empty vector ; iLGP2 = inducible LGP2 .

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

A) ADAR1-knockout HEK293 cells (clone 1) were modified with a lentiviral-based inducible system to express FLAG-LGP2 WT or a FLAG-LGP2 RNA binding mutant (K138E/R490E/K634E, denoted as "KRK mutant") in a doxycycline-regulated manner. Cells were treated 72h with doxycycline (dox). Protein lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies (n=3). iEV = inducible empty vector; iLGP2 = inducible LGP2.


C ) Cells generated in ( A ) were plated on coverslips and treated with or without doxycycline for 72h . Cells were fixed , permeabilized and stained with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 500 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of two biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analysis was performed using a Kruskal - Wallis test with a Dunn ' s post hoc test for multiple comparisons . ns , not significant ; * * * * , p < 0 . 0001 .

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 B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells generated in (A) were plated on coverslips and treated with or without doxycycline for 72h. Cells were fixed, permeabilized and stained with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>500 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of two biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analysis was performed using a Kruskal-Wallis test with a Dunn's post hoc test for multiple comparisons. ns, not significant; ****, p<0.0001.


E ) MDA5 - knockout HEK293 cells , were transfected with an ADAR1 - targeting siRNA ( siADAR1 ) or a control siRNA ( siCtrl ) and 8h later with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of MDA5 . Cells were harvested 72h post - siRNA transfection and the type I IFN response was monitored by RT - qPCR analysis of IFIT1 and ISG15 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of two biological replicate experiments .

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

E) MDA5-knockout HEK293 cells, were transfected with an ADAR1-targeting siRNA (siADAR1) or a control siRNA (siCtrl) and 8h later with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of MDA5. Cells were harvested 72h post-siRNA transfection and the type I IFN response was monitored by RT-qPCR analysis of IFIT1 and ISG15 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of two biological replicate experiments.


B ) HEK293 WT or ADAR1 - knockout cells ( clone 1 ) were transiently transfected with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of LGP2 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of three biological replicate experiments .

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

B) HEK293 WT or ADAR1-knockout cells (clone 1) were transiently transfected with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of LGP2. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of three biological replicate experiments.


C ) Cells were treated as in ( B ) . Protein lysates were prepared and analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) . The dotted line indicates the juxtaposition of two non - adjacent lanes .

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 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

C) Cells were treated as in (B). Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3). The dotted line indicates the juxtaposition of two non-adjacent lanes.


A ) Kaplan - Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer ( BLCA ; n = 407 ) , breast cancer ( BRCA ; n = 1099 ) and sarcoma ( SARC ; n = 263 ) TCGA datasets . Median cut - offs were used for patient stratification and logrank test P values are shown .

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

A) Kaplan-Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer (BLCA; n=407), breast cancer (BRCA; n=1099) and sarcoma (SARC; n=263) TCGA datasets. Median cut-offs were used for patient stratification and logrank test P values are shown.


B ) CAL27 cells transduced with doxycycline - inducible shRNAs targeting ADAR1 or GFP ( negative control ) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 ( siLGP2 # 1 or # 2 ) or a control siRNA ( siCtrl ) . To determine cell confluency at 120h post - transfection , cells were fixed , stained with Crystal Violet , and imaged . Images of a representative experiment are shown ( n = 3 ) . C ) Crystal Violet was extracted from stained cells ( B ) and the dye intensity was quantified using a colorimetric assay ( OD590 ) . OD590 values of doxycycline - treated cells were normalized to the OD 590 values of untreated cells . Quantification of data from three independent experiments is shown as mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 .

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

B) CAL27 cells transduced with doxycycline-inducible shRNAs targeting ADAR1 or GFP (negative control) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 (siLGP2 #1 or #2) or a control siRNA (siCtrl). To determine cell confluency at 120h post-transfection, cells were fixed, stained with Crystal Violet, and imaged. Images of a representative experiment are shown (n=3). C) Crystal Violet was extracted from stained cells (B) and the dye intensity was quantified using a colorimetric assay (OD590). OD590 values of doxycycline-treated cells were normalized to the OD590 values of untreated cells. Quantification of data from three independent experiments is shown as mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.