( G ) Confocal microscopy of paired normal carcinoma tissues from two colon cancer . frozen sections were incubated with rabbit anti - AnnexinA1 polyclonal antibodies 5G11 mouse anti - FadA monoclonal antibodies . slides were then stained with Alexa Fluor ® 680 - conjugated donkey anti - rabbit Alexa Fluor ® 555 - conjugated goat anti - mouse , washed , covered in mounting medium containing DAPI . scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for normal tissues 400x for carcinomas . Scale bar , 50 μm . Co - localization of FadA ( red ) Annexin A1 ( green ) was observed in carcinomas but not in 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 of β - catenin expression in 10C , HCT116 DLD1 following transfection with control siRNA ( clear bars ) or ANXA1 - specific siRNA ( solid bars ) incubation with F . nucleatum 12230 at MOI of ~ 20 : 1 for indicated time periods . geometric means of treated with control siRNA at time 0 was designated as 1 . Data are mean values ± SD . experiment was performed in duplicates or triplicates 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 - of β - catenin in 10C following transfection with control siRNA or ANXA1 - specific siRNA incubation with F . nucleatum 12230 ( Fn ) at MOI of ~ 100 : 1 for 2 hrs . β - catenin was stained with Alexa Fluor ® 680 ( red ) nuclei with DAPI ( blue ) . were captured with confocal microscope at 800x magnification . - , no added . Note increased expression of β - catenin its nucleus translocation in response to F . nucleatum in control siRNA - treated , but not in ANXA1 siRNA - treated . 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 of CCND1 expression using mRNA extracted from cancerous 10C , human CRC HCT116 RKO , each grown to 50 % or 100 % confluency . Data are mean values ± . experiment was performed in triplicates repeated twice . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - ) .

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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 of Cyclin D1 , Annexin A1 , β - actin in RKO transfected with control vector or ANXA1 . Induction of Cyclin D1 was observed in response to increased Annexin A1 . 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 of E12 . 5 midbrain floor - plate ( mFP ) , midbrain roof - plate ( mRP ) , anterior ( A , adjacent anterior FP ) posterior ( P , adjacent posterior FP ) . Pbx1 is enriched in midbrain FP , together with Th Pitx3 . Lower of Pbx3 are also expressed in mFP . Gdf7 Wnt1 are restricted to 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 intermediate zone ( IZ ) marginal zones ( MZ ) , but not ventricular zone ( VZ ) , of 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 ventro - lateral part of LMX1a + domain at E10 , preceding birth of first ( TH + ) mDA neurons at E10 . 5 . At E12 . 5 , PBX1 is present in mDA neurons , but not PBX1 + are TH + . boxes indicate 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 defines a subpopulation of NURR1 + neuroblasts labels 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 is also detected in a subpopulation of NURR1 + PITX3 - postmitotic neuroblasts at E12 . 5 . Higher magnification revealed three different populations of postmitotic : neuroblasts ( NURR1 + PBX1A - PITX3 - , green ) , secondary neuroblasts ( NURR1 + PBX1A + PITX3 - , yellow / orange ) tertiary neuroblasts / mDA neurons ( NURR1 + PBX1A + PITX3 + , ) . , Dapi ( 4 ′ , 6 - diamidino - 2 - phenylindole , blue ) . 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 isoform detected in TH + NURR1 + mDA neurons at E12 . 5 .

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


( C ) Immunofluorescence microscopy of 7 - week old human VM tissue showing that mDAn are positive for PBX1 , suggesting a conserved role for this factor in development of these neurons in mice 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 substantia nigra ( SN ) ventral tegmental ( 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 left , representative immunofluorescence microscopy / confocal showing that number of TH + is reduced in Pbx1 - / - . Deletion of Pbx1 decreases proportion of NURR1 + neuroblasts that become TH + neurons at E12 . 5 , indicating that Pbx1 is required for proper maturation of NURR1 + neuroblasts . To right , of these experiments .

(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 rostro - caudal of 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 showing that conditional deletion of Pbx1 Pbx3 ( cKO , Pbx1flox / flox ; Pbx3 - / - ; Th - IRES - Cre - ERT ) reduces number of PITX3 + in rostral VM , but not of NURR1 + , when compared to double heterozygous at E14 . 5 ( cHET , Pbx1flox / + ; Pbx3 - / + ; Th - IRES - Cre - ERT ) . boxes are magnified to 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 + NURR1 + is detected from anterior to posterior in cKO , but not in cHET . Pyknotic NURR1 + nuclei are observed in cKO ( arrowheads ) . boxes are magnified to 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 ) total number of TH + mDA neurons is dramatically reduced there is an increase in aCASP3 + in cKO 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 number of TH + neurons after 8 days of differentiation of hNES ( AF22 derived from induced pluripotent ) . ( C ) While number of ( Dapi + ) in both conditions did not change , number of TH + increased 2 . 3 fold in PBX1 - overexpressing .

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

(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 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 29 most regulated genes ( t - q - value < 0 . 05 ) identified by Gene Set Enrichment ( GSEA ) ranked by p - value . RPKM stands for Reads Per Kilobase of transcript per Million mapped reads . GSEA was performed on genes identified by - Seq as differentially expressed in VM at E12 . 5 compared to adjacent brain regions 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 GSEA curve showing a high correlation between cistrome 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 TSS of Pitx3 ( Pitx3 - Gbf1 ) , Onecut2and Nfe2l1 genes . No co - immunoprecipitation was detected for unrelated gene , Lrp1b ( Low Density Lipoprotein Receptor - Related 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 ) regulation of Pitx3 Onecut2 by PBX1 was confirmed in Pbx1 - / - ; Pbx3 + / - mutant at E12 . 5 , compared to wild - type ( Pbx1 + / + ) . ( F ) Representative immunofluorescence microscopy / confocal showing that decreased of PITX3 number of PITX3 + in E12 . 5 Pbx1 - / - ; Pbx3 + / - . This reduction is more evident in basal plate ( BP ) in midline ( M ) , as shown at higher magnification ( right ) . ( G ) Double ONECUT2 + TH + were detected only in lateral part of mFP in E12 . 5 Pbx1 - / - ; Pbx3 + / - . Selected areas are magnified to right . A scheme ( right ) shows distribution of in Pbx1 + / + Pbx1 - / - ; Pbx3 + / - . scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates ( dots ) per condition p - value ( T - ) 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 showing that NFE2L1 is detected in TH + mDAn 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 ) expression of NFE2L1 in TH + neurons is dramatically reduced in Pbx1 - / - ; Pbx3 + / - compared to Pbx1 + / + 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 of aCASP3 in H2O2 - treated ( 100µm for 12 hours ) TH + neurons derived from hNES differentiated for 8 days ( ) . 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 from oxidative stress induced by H2O2 ( 100µm for 12 hours ) , resulting in reduced of aCASP3 in TH + neurons ( ) .

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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 ( - blue / black , indicated by arrowheads ) in nuclei of neuromelanin + ( NM + , brown ) of SN . In contrast , PBX1 was severely reduced in PD . ( F ) Graphical representation of PBX1 in SN of NM + neurons in controls PD analyzed . Each dot in graph represents level of PBX1 in 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 PBX1 direct target , NFE2L1 , was also detected as very well defined punctuate structures in NM + of SN in control samples ( arrowheads ) , but were dramatically reduced in PD . Yellow asterisks indicate nucleoli . ( H ) of NM + neurons showing NFE2L1 in SN of control donors PD . ( I ) Graphical representation of controls PD analyzed , showing that NM + neurons with NFE2L1 + nuclei in SN of PD 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 compared with skin from healthy donors on microarray data ( No . GSE14905 ) . B Expression of ptpn11 in human PBMCs of psoriatic ( n = 14 ) normal controls ( n = 16 ) . Data information : Data are represented mean ± . P values are determined by two - Mann - Whitney U * P < 0 . 05 , * * P < 0 . 01 .

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 in skin sections of psoriatic ( n = 13 ) 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 of PBMCs lysates derived from psoriatic normal controls .

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


E catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic ( n = 25 ) normal controls ( n = 25 ) . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * 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 of skin sections of psoriatic normal controls . Scale bars : 200 μm .

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


G Quantitative PCR of Ptpn11 mRNA in 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 ± . P values are determined by two - unpaired Student ' s t - * 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 of mRNA in human PBMCs derived from psoriatic ( n = 20 ) normal healthy controls ( n = 20 ) . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * 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 ( middle ) statistical data ( bottom ) ( mean ± ) of dorsal skin . Scale bar : 100 μm . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * 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 of mRNA encoding IL - 23 / IL - 17A axis cytokines in 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 ± . P values are determined by two - unpaired Student ' s t - * 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 of of cytokines in mouse serum . Quantitative PCR of mRNA encoding psoriasis - related cytokines in 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 ± . P values are determined by two - unpaired Student ' s t - * P < 0 . 05 , * * P < 0 . 01 .

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

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 relative expression of 47 inflammation - associated genes on sample groups clusters .

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


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

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 . 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 top ten hub genes in ( F ) . ( top ) displays average expression in three groups in different myeloid subsets . Boxplots ( bottom ) show corresponding expression distribution .

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

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

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 M - Shp2 - / - mice after IMQ treatment on sequencing ( n = 3 / group ) . Colors represent high ( red ) low ( blue ) . Genes labeled in green indicate they are in 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 PMs derived from wild - type M - Shp2 - / - mice were stimulated by IMQ ( 10 µg / ml ) for indicated times . Whole 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 of skin sections from mouse . Scale bar : 100 μm .

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


E Immunoblotting of HEK293T 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 were infected with vector or SHP2 lentivirus then treated with or without R848 ( 10 µg / ml ) . Subcellular fractionation was performed , 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 of TLR7 SHP2 in isolated ER , Golgi endosome from PMA - differentiated THP - 1 with either SHP2 overexpressed ( G ) 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 healthy controls were immune - stained for TLR7 together with a marker of early endosome ( EEA1 ) or late endosome / lysosome ( LAMP1 ) prior to 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 endosome isolated from HEK293T 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 transfected with NF - κB - luciferase reporter indicated TLR7 mutant vectors then treated with IMQ ( 10 µg / ml ) for 24 h were harvested for luciferase . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * * 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 of skin sections from human . Scale bar : 100 μm .

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


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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mice ( n = 6 ) Tlr7 - Y1025D mutant Tlr7ki / wt mice ( n = 6 ) Tlr7ki / ki mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . E Representative p - p65 of 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 of microRNA expression changes in newborn mouse lungs exposed to 21 % O2 vs . 85 % O2 , at post - natal day ( P ) 5 P14 .

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

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

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


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

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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 in MLg after treatment with scrambled microRNA ( SCR ) or a miR - 34a ( MIM34a ) mimic ( n = 3 separate cultures for each group ) .

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 in MLg in vitro , maintained under 21 % O2 or 85 % O2 ( n = 3 separate cultures for each group ) .

O O O O O O O O O O O O O O O O O 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 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 in lungs of mouse pups ( n = 6 for each group ) at post - natal day ( P ) 5 , during normal ( 21 % O2 ) 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 in MLg in vitro , maintained under 21 % O2 or 85 % O2 , where had been transfected wither with a scrambled ( SCR ) antimiR , or an antimiR directed against miR - 34a ( A34a ) ( n = 3 separate cultures for each group ) .

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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 in PDGFRα + , sorted by FACS from lungs of mouse pups ( n = 4 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 .

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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 in PDGFRα + , sorted by FACS from lungs of either wild - type ( 34awt / wt ) mouse pups , or mouse pups in which miR - 34a expression in Pdgfra ‑ expressing ( 34ai ∆ PC / i ∆ PC ) at P5 ( n = 4 for each group ) .

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 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 of total number of alveoli by design - stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal aberrant alveolarization ( n = 5 for each group ) . K of mean septal thickness by design - stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal aberrant alveolarization ( n = 5 for each group ) .

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

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

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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 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 TSB2 ) during normal ( 21 % O2 ) aberrant ( 85 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments . E of total number of alveoli by design - stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 for each group ) . F of mean septal thickness by design - stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 for each group ) .

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

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

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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 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 ) aberrant ( 85 % O2 ) alveolarization ( n = 4 for each group ) .

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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 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 aberrant alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments .

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

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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 of mean septal thickness by design - 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 aberrant alveolarization ( n = 5 for each group ) .

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

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

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

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

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

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.


impact of administration of scrambled antimiR ( S ) or an antimiR directed against miR - 34a ( A34a ) on abundance of type I alveolar epithelial ( marked by aquaporin 5 , Aqp5 ) type II alveolar epithelial ( marked by pro - surfactant 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 DAPI , lines delineate tissue from airspaces , in 85 % O2 groups demarcate septa . Antibody specificity was validated by rabbit IgG isotype control antibodies . control experiments for Aqp5 Sftpc runs are illustrated here . Scale bars , 50 µm .

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

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

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

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

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

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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 ( 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 . were harvested 72h post - transfection type I IFN response was monitored by RT - qPCR of IFN - β IFIT1 expression , normalized to ACTB . Data are means ± s . d . from a representative of four biological replicate experiments .

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

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

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

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

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

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

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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 ADARhigh stratified by DHX58 in bladder cancer ( BLCA ; n = 407 ) , breast cancer ( BRCA ; n = 1099 ) sarcoma ( SARC ; n = 263 ) TCGA datasets . Median cut - offs were used for stratification logrank P values are shown .

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

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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.


A ) HT29 were treated with or without 300 nM 5 - AZA - CdR for 2 days subsequently washed transfected with indicated siRNAs . were harvested 72h post - transfection type I IFN response was analyzed by RT - qPCR of IFN - β , IFIT1 , ISG15 transcript expression , normalized to ACTB . B ) LIM1215 were treated with or without 300 nM 5 - AZA - CdR for 2 days subsequently washed transfected with indicated siRNAs . were harvested 72h post - transfection type I IFN response was analyzed as in ( A ) . C ) LIM1215 were treated with 250 nM palbociclib or a DMSO control for 7 days . Three days after treatment initiation , were transfected with indicated siRNAs cultured for an additional 72h . type I IFN response was monitored as in ( A ) . Data information : Data from three biological independent experiments are shown with mean ± s . d . . Statistical was performed using ordinary two - way ANOVA with Tukey ' s post hoc . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

A) HT29 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed by RT-qPCR analysis of IFN-β, IFIT1, and ISG15 transcript expression, normalized to ACTB. B) LIM1215 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed as in (A). C) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days. Three days after treatment initiation, cells were transfected with the indicated siRNAs and cultured for an additional 72h. The type I IFN response was monitored as in (A). Data information: Data from three biological independent experiments are shown with 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<0.001; ****, p<0.0001.


ALP . expressing Pho8Δ60p were cultured in SD + CA medium ( open bars ) shifted to SD ( − N ) medium for three hours ( filled bars ) . ALP activity of lysates was measured to estimate autophagic activity . Wild - type , TN124 ; Δapg8 mutant harboring vector , TK303 ; Δapg8 mutant harboring APG8 on a centromeric vector , TK301 ; Δapg8 mutant harboring 3 × HA - tagged APG8 plasmid , TK307 .

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ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δapg8 mutant harboring vector, TK303; Δapg8 mutant harboring APG8 on a centromeric vector, TK301; Δapg8 mutant harboring 3 × HA-tagged APG8 plasmid, TK307.


Expression of APG8 . A , lysates prepared from growing were subjected to immunoblotting with anti - Apg8p antibody . Lane 1 , wild - type ( YW5 - 1B ) ; lane 2 , Δapg8 harboring APG8 on a multicopy vector ( TK201 ) ; lane 3 , Δapg8 ( TK404 ) .

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Expression of APG8. A, The lysates prepared from the growing cells were subjected to immunoblotting with the anti-Apg8p antibody. Lane 1, wild-type cells (YW5-1B); lane 2, Δapg8 cells harboring APG8 on a multicopy vector (TK201); lane 3, Δapg8 cells (TK404).


B , wild - type ( YW5 - 1B ) were cultured in YEPD medium until 1 - 2 × 107 / ml ( 0 h ) shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , 3 , 4 . 5 , 6 h at 30 ° C . amount of Apg8p was estimated by immunoblotting with anti - Apg8p antibody . Each lane has 10 μg of total .

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B, The wild-type (YW5-1B) cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, 3, 4.5, and 6 h at 30°C. The amount of Apg8p was estimated by immunoblotting with anti-Apg8p antibody. Each lane has 10 μg of total protein.


C , YW5 - 1B were cultured in YEPD medium until 1 - 2 × 107 / ml ( 0 h ) shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , 6 h at 30 ° C , mRNA was prepared from each culture as described in Materials Methods . APG8 mRNA ACT1 mRNA were detected by Northern blotting with each specific probe . Each lane has 4 μg of total mRNA .

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C, YW5-1B cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, and 6 h at 30°C, and mRNA was prepared from each culture as described in Materials and Methods. APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe. Each lane has 4 μg of total mRNA.


D , YW5 - 1B were cultured in YEPD medium until 1 × 107 / ml at 30 ° C . Rapamycin was added to culture at a final of 0 . 2 μg / ml were further incubated in YEPD medium for two hours at 30 ° C . Western blotting was performed with anti - Apg8p antibody . Each lane has 20 μg of total .

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D, YW5-1B cells were cultured in YEPD medium until 1 × 107 cells/ml at 30°C. Rapamycin was added to the culture at a final concentration of 0.2 μg/ml and the cells were further incubated in YEPD medium for two hours at 30°C. Western blotting was performed with the anti-Apg8p antibody. Each lane has 20 μg of total protein.


Subcellular fractionation solubilization of Apg8p . wild - type ( YW5 - 1B ) growing logarithmically in YEPD medium or starved in SD ( − N ) medium for three hours were lysed . A , lysates ( T ) were centrifuged at 13 , 000 g for 15 min to generate pellet ( LSP ) supernatant ( LSS ) . LSS fraction was further centrifuged at 100 , 000 g for one hour separated to pellet ( HSP ) supernatant ( HSS ) . LSP HSP were resuspended in an equal volume of lysis buffer to original lysates . Equal volume of each sample was applied to each well , immunoblotting was performed with anti - Apg8p antibody .

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Subcellular fractionation and solubilization of Apg8p. The wild-type cells (YW5-1B) growing logarithmically in YEPD medium or starved in SD(−N) medium for three hours were lysed. A, The lysates (T) were centrifuged at 13,000 g for 15 min to generate pellet (LSP) and supernatant (LSS). LSS fraction was further centrifuged at 100,000 g for one hour and separated to pellet (HSP) and supernatant (HSS). LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates. Equal volume of each sample was applied to each well, and immunoblotting was performed with anti-Apg8p antibody.


B , lysates ( T ) were sonicated centrifuged at 100 , 000 g for one hour to separate pellet ( P ) supernatant ( S ) . distribution of CPY Apg8p was examined by immunoblotting with each specific antibody , respectively .

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B, The lysates (T) were sonicated and centrifuged at 100,000 g for one hour to separate pellet (P) and supernatant (S). The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody, respectively.


C , sonicated lysates were centrifuged at 100 , 000 g for one hour to generate pellet fraction . pellet fraction was treated with 2 % Triton X - 100 ( TX - 100 ) for 30 min on ice . sample was centrifuged at 100 , 000 g for one hour separated to supernatant ( S ) pellet ( P ) fractions . distribution of Apg8p was examined by immunoblotting with anti - Apg8p antibody .

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C, The sonicated lysates were centrifuged at 100,000 g for one hour to generate pellet fraction. The pellet fraction was treated with 2% Triton X-100 (TX-100) for 30 min on ice. The sample was centrifuged at 100,000 g for one hour and separated to supernatant (S) and pellet (P) fractions. The distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody.


Immunofluorescent of Δapg8 expressing 3 × HA - Apg8p . A , Δapg8 harboring 3 × HA - tagged APG8 plasmid , TK114 , were grown until logarithmic phase . were fixed by formaldehyde were treated with Zymolyase 100T to generate spheroplasts . spheroplasts were permeabilized by 0 . 5 % Triton X - 100 , incubated with anti - HA antibody , 16B12 , followed with FITC - conjugated anti - mouse IgG . Left , image of 3 × HA - Apg8p ; right , Nomarski image of . Arrows show punctate signals that are a little larger than tiny dot signals . Bars , 10 μm . B , Immunofluorescent of YW5 - 1B before ( 0 h ) after ( 3 h ) shift to starvation ( negative control ) . Left , image ; right , Nomarski image . Bars , 10 μm . C , were shifted to starvation for 0 , 0 . 5 , 3 h . Upper panels , of 3 × HA - Apg8p ; lower panels , Nomarski of . Bars , 5 μm .

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Immunofluorescent staining of Δapg8 cells expressing 3 × HA-Apg8p. A, The Δapg8 cells harboring 3 × HA-tagged APG8 plasmid, TK114 cells, were grown until logarithmic phase. The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts. The spheroplasts were permeabilized by 0.5% Triton X-100, and incubated with the anti-HA antibody, 16B12, followed with the FITC-conjugated anti-mouse IgG. Left, Fluorescence image of 3 × HA-Apg8p; right, Nomarski image of the cells. Arrows show punctate signals that are a little larger than tiny dot signals. Bars, 10 μm. B, Immunofluorescent staining of YW5-1B cells before (0 h) and after (3 h) shift to starvation (negative control). Left, Fluorescence image; right, Nomarski image. Bars, 10 μm. C, The cells were shifted to starvation for 0, 0.5, and 3 h. Upper panels, Fluorescence images of 3 × HA-Apg8p; lower panels, Nomarski images of the cell. Bars, 5 μm.


Change of Apg8p localization in Δpep4 after shift to starvation . Δapg8Δpep4 harboring 3 × HA - tagged APG8 plasmid , TK116 , were shifted to starvation for 0 , 0 . 5 , 1 , 3 , 6 h . Immunofluorescence microscopy was performed as described in Fig . 5 . A - E , FITC of 3 × HA - Apg8p . F - J , Nomarski of . Arrows show an autophagic . Bars , 5 μm .

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Change of Apg8p localization in Δpep4 cells after shift to starvation. The Δapg8Δpep4 cells harboring 3 × HA-tagged APG8 plasmid, TK116 cells, were shifted to starvation for 0, 0.5, 1, 3, and 6 h. Immunofluorescence microscopy was performed as described in Fig. 5. A-E, FITC staining of 3 × HA-Apg8p. F-J, Nomarski images of the cells. Arrows show an autophagic body. Bars, 5 μm.


Immunostaining image of a containing autophagic bodies in vacuole . Δapg8Δpep4 expressing 3 × HA - Apg8p , TK116 , were shifted to SD ( − N ) medium for two hours . were immunolabeled with anti - N mAb , 16B12 , followed by 10 - nm gold - conjugated goat anti - mouse IgG . AB , vacuole ; N , nucleus ; V , vacuole .

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Immunostaining image of a cell containing autophagic bodies in the vacuole. The Δapg8Δpep4 cells expressing 3 × HA-Apg8p, TK116 cells, were shifted to SD(−N) medium for two hours. Cells were immunolabeled with anti-N mAb, 16B12, followed by 10-nm gold-conjugated goat anti-mouse IgG. AB, vacuole; N, nucleus; V, vacuole.


Immuno - EM of autophagosomes . A B , Mature autophagosome . C D , Premature autophagosome . Arrows show expanded regions of intramembrane space of premature autophagosomes . TK116 were incubated in SD ( − N ) medium for one hour ( D ) , two hours ( B C ) , or three hours ( A ) . localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by incubation with 5 - nm gold - ( A D ) or 10 - nm gold - ( B C ) conjugated goat anti - mouse IgG . AP , autophagosome ; N , nucleus ; V , vacuole .

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Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti-mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.


Possible intermediate structures of autophagosome . A , A sac under construction . Arrows show gold particles . B C , Isolation membranes . B , Isolation is detected along arrows . C , small arrow shows a semicircular isolation a large arrow marks its open . D , Apg8p - residing structures gathered in close to vacuole . TK116 were incubated in SD ( − N ) medium for one hour ( A C ) , or two hours ( B D ) . localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by incubation with 5 - nm gold - ( A ) or 10 - nm gold - ( B - D ) conjugated goat anti - mouse IgG . V , vacuole .

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Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B-D) conjugated goat anti-mouse IgG. V, vacuole.


Fine of Δypt7 Δapg8 proteinase - K accession to proAPI in these mutants . A , EM image of Δypt7 starved in SD ( − N ) medium for four hours . Autophagosomes ( AP , arrows ) were accumulated . B , EM image of starved Δapg8 . C - E , representatives of structures detected in starved Δapg8 . C , Autophagosome - like structure indistinguishable from autophagosome . D E , Aberrant multivesicular structures . N , nucleus ; V , vacuole . F , Proteinase K - sensitivity of proAPI . lysates were prepared from Δypt7 Δapg8 starved in SD ( − N ) medium for 4 . 5 h . lysates were treated with 100 μg / ml proteinase K in presence or absence of Triton X - 100 . Asterisk shows mature API digestion product of proAPI by proteinase K .

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Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δypt7 cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δapg8 cell. C-E, The representatives of the membrane structures detected in the starved Δapg8 cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.


A Mpc1 Mpc2 are present under fermentative conditions , while Mpc1 Mpc3 are present under respiratory conditions . MPC subunits were detected by Western blot in mitochondria isolated from yeast grown in glucose ( YPD ) or glycerol ( YPG ) . Mitochondrial matrix aconitase ( Aco1 ) , 23 ‐ kDa translocase of inner subunit ( Tim23 ) , 70 ‐ kDa translocase of outer subunit ( Tom70 ) were used as loading controls .

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A Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls.


B Kinetics of switch from Mpc2 to Mpc3 . Yeast were grown in YPD until mid ‐ log phase then shifted to YPG medium . Whole extracts were taken at indicated time points after shift , HA ‐ tagged Mpc2 Mpc3 detected by Western blotting . Mpc3 is induced very rapidly by switch from YPD to YPG , while Mpc2 decrease only gradually .

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B Kinetics of the switch from Mpc2 to Mpc3. Yeast cells were grown in YPD until mid‐log phase and then shifted to YPG medium. Whole cell extracts were taken at the indicated time points after the shift, and HA‐tagged Mpc2 and Mpc3 detected by Western blotting. Mpc3 is induced very rapidly by switch from YPD to YPG, while Mpc2 levels decrease only gradually.


C Alternative pyruvate carrier complexes MPCFERM MPCOX are detected by BN ‐ PAGE . Isolated mitochondria expressing HA ‐ tagged MPC were run on blue native gels , MPC complexes detected with an HA ‐ specific antibody . MPCFERM consisting of Mpc1 Mpc2 is present under fermentative conditions , MPCOX consisting of Mpc1 Mpc3 is present under respiratory conditions . In addition , a of ˜ 300 kDa is observed under respiratory conditions that contains Mpc1 Mpc3 . Non ‐ specific reactivity of anti ‐ HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk ( * ) .

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C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN‐PAGE. Isolated mitochondria expressing HA‐tagged MPC proteins were run on blue native gels, and MPC complexes detected with an HA‐specific antibody. MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions, and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions. In addition, a complex of ˜300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3. Non‐specific reactivity of the anti‐HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk (*).


A Constitutive expression of either MPCFERM or MPCOX can rescue slow growth phenotype of mpc1Δmpc2Δmpc3Δ triple deletion mutant . Growth were performed with transformed with MPC1 , MPC2 , or MPC3 expression plasmids or with empty vectors in indicated combinations . A serial dilution of yeast was spotted on agar plates with glucose ‐ containing synthetic minimal medium with ( SD + AA ) or without ( SD − AA ) .

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A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids.