( G ) Wild - type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity . Kinase assays of either wild - type and mutant PI4KB ( 10 nM ) were carried out with 100 % PI lipid vesicles ( 0 . 5 mg / L ) , 100 μM ATP , and PI4KB ( 300 nM ) on Golgi - mimic vesicles ( 0 . 5 mg / mL ) with 10 μM ATP . The data was normalized to the kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(G) Wild-type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity. Kinase assays of either wild-type and mutant PI4KB (10 nM) were carried out with 100% PI lipid vesicles (0.5 mg/L), 100 μM ATP, and PI4KB (300 nM) on Golgi-mimic vesicles (0.5 mg/mL) with 10 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( H ) FLH409AAA - c10orf76 mutant shows decreased affinity for PI4KB . His - pulldown assays of 6xHis - c10orf76 ( 1 μM ) with wild - type PI4KB ( 1 μM ) . Samples washed a total of 4 times .

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(H) FLH409AAA-c10orf76 mutant shows decreased affinity for PI4KB. His-pulldown assays of 6xHis-c10orf76 (1 μM) with wild-type PI4KB (1 μM). Samples washed a total of 4 times.


( I ) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced . Kinase assay of PI4KB ( 40 nM ) and a concentration curve of c10orf76 ( 3 . 9 nM - 2μM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(I) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced. Kinase assay of PI4KB (40 nM) and a concentration curve of c10orf76 (3.9 nM-2μM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( A ) Transfections of HEK293 cells revealed that both wild - type GFP - PI4KB and RL494EA GFP - PI4KB localize to the Golgi .

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(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi.


( B ) WT c10orf76 also localized to the Golgi , however , the PI4KB binding deficient mutant of c10orf76 ( FLH409AAA ) predominantly localized to the cytosol .

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(B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol.


( D ) Mitochondria recruitment experiment with wild - type PI4KB and c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after rapamycin ( 100 nM ) treatment ( bottom ) . Middle : mRFP - FKBP12 - PI4KB at high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 at a high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) .

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(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).


( F ) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after ( bottom ) rapamycin treatment . Middle : mRFP - FKBP12 - PI4KB ( RL494EA ) is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 is located in the cytosol before ( top ) and after ( bottom ) rapamycin induction .

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(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.


( A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


B ) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers ( B ) . ( C ) Western blot of GBF1 , PI4KB and B - Actin levels in both wild - type and c10orf76 KO HAP1 cells . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( D ) Markers of Arf1 activation - the coatomer proteins COPIα / γ and βCOP act as a readout for GTP - bound Arf1 , while the native coatomer was detected with the CM1 antibody . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( A ) Viral infection assays determining viral titers by end - point titration at 0 hours and 8 hours in HAP1 wild - type or c10orf76 knockout cells . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild - type titres at 8 hours using a one - way ANOVA . * * , P < 0 . 01 ; N . S . , P > 0 . 05 .

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(A) Viral infection assays determining viral titers by end-point titration at 0 hours and 8 hours in HAP1 wild-type or c10orf76 knockout cells. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild-type titres at 8 hours using a one-way ANOVA. **, P<0.01; N.S., P>0.05.


( B ) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild - type and PI4KB knockout cells upon transfection of wild - type PI4KB , the complex - disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Values were statistically evaluated compared to the GalT control using a one - way ANOVA . * * , P < 0 . 01 ; * , P < 0 . 05 ; N . S . , P > 0 . 05 .

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(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.


Overall structure of 2019 - nCoV RBD bound with ACE2 . ACE2 is colored green . 2019 - nCoV RBD core is colored cyan and RBM is colored red . Disulfide bonds in the 2019 - nCoV RBD are shown as stick and indicated by yellow arrows . The N - terminal helix of ACE2 responsible for binding is labeled .

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Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.


Alignment of the 2019 - nCoV RBD ( core in cyan and RBM in red ) and SARS - CoV RBD ( core in orange and RBM in blue ) structures .

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Alignment of the 2019-nCoV RBD (core in cyan and RBM in red) and SARS-CoV RBD (core in orange and RBM in blue) structures.


Structural alignment of 2019 - nCoV RBD / ACE2 and SARS - CoV RBD / ACE2 complexes . 2019 - nCoV RBD is colored cyan and red , its interacting ACE2 is colored green . SARS - CoV RBD is colored orange and blue , its interacting ACE2 is colored salmon . The PDB code for SARS - CoV RBD / ACE2 complex : 2AJF .

Structural alignment of 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 complexes. 2019-nCoV RBD is colored cyan and red, its interacting ACE2 is colored green. SARS-CoV RBD is colored orange and blue, its interacting ACE2 is colored salmon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.


Contacting residues shown as stick at the 2019 - nCoV RBD / ACE2 and SARS - CoV RBD / ACE2 interfaces . Positions in both RBDs involved in ACE2 binding are indicated by red labels .

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Contacting residues shown as stick at the 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 interfaces. Positions in both RBDs involved in ACE2 binding are indicated by red labels.


( B - K ' ) The Drosophila posterior midguts containing Su ( H ) - Gal4 > UAS - CD8 : GFP ( Su ( H ) > GFP ) with sucrose ( Suc , control ) ( B - C ' ) , P . e for 36 h ( D - F ' ) , Ecc15 for 36 h ( G - I ' ) and DSS for 36 h ( J - K ' ) were immunostained for GFP ( green ) , PH3 ( red ) and DAPI ( blue ) . ( C - C ' , E - E ' , H - H ' and K - K ' ) Magnification of selected areas containing PH3 + GFP ─ cells from B , D , G and J . The EB cell close to the PH3 + cell ( white arrow ) in C - C ' is out of focus . ( F - F ' and I - I ' ) Magnification of selected areas containing PH3 + EBs ( GFP + ) from D and G . White arrowheads and red arrows indicate PH3 in GFP ─ and GFP + cells , respectively . ( L and M ) Quantification of PH3 GFP ─ ISCs ( L ) and PH3 + GFP + EBs ( M ) with given treatments . n = 21 guts for each treatment .

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(B-K') The Drosophila posterior midguts containing Su(H)-Gal4>UAS-CD8:GFP (Su(H)>GFP) with sucrose (Suc, control) (B-C'), P.e for 36 h (D-F'), Ecc15 for 36 h (G-I') and DSS for 36 h (J-K') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (C-C', E-E', H-H' and K-K') Magnification of selected areas containing PH3+GFP─ cells from B, D, G and J. The EB cell close to the PH3+ cell (white arrow) in C-C' is out of focus. (F-F' and I-I') Magnification of selected areas containing PH3+ EBs (GFP+) from D and G. White arrowheads and red arrows indicate PH3 in GFP─ and GFP+ cells, respectively. (L and M) Quantification of PH3 GFP─ ISCs (L) and PH3+GFP+ EBs (M) with given treatments. n = 21 guts for each treatment.


( D - F ' ' ) When the ISCs were marked by esgGal4 , su ( H ) - Gal80 > UAS - GFP , PH3 expression was found in GFP + ISCs with feeding of Suc ( control ) ( D - D ' ' ) , P . e ( E - E ' ' ) and Dss ( F - F ' ' ) . However , PH3 expression is present in EBs with GFP ─ Pros ─ and small nuclei upon P . e infection ( E - E ' ' , red arrows ) . The white arrows indicate GFP + cells with PH3 expression . ( G ) Quantification of PH3 + GFP ─ Pros ─ EBs with given treatments . n = 11 guts for each treatment . Data information : Three independent experiments were performed , and error bars are ± SEM . * * * , P < 0 . 001 ( Student ' s t - test ) ( G ) .

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(D-F'') When the ISCs were marked by esgGal4, su(H)-Gal80>UAS-GFP, PH3 expression was found in GFP+ ISCs with feeding of Suc (control) (D-D''), P.e (E-E'') and Dss (F-F''). However, PH3 expression is present in EBs with GFP─Pros─ and small nuclei upon P.e infection (E-E'', red arrows). The white arrows indicate GFP+ cells with PH3 expression. (G) Quantification of PH3+GFP─Pros─ EBs with given treatments. n = 11 guts for each treatment. Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (G).


( D - N ' ) The Drosophila midguts expressing Su ( H ) ts > GFP with control ( D - E ' ) , UAS - RasV12 for 6 days ( F - H ' ) , EGFRA887T for 4 days ( I - K ' ) and UAS − λTop for 5 days ( L - N ' ) were immunostained for GFP ( green ) , PH3 ( red ) and DAPI ( blue ) . ( E - E ' , G - G ' , J - J ' and M - M ' ) Magnification of selected areas containing PH3 + GFP ─ cells from D , F , I and L . ( H - H ' , K - K ' and N - N ' ) Magnification of selected areas containing PH3 + GFP + cells from F , I and L . White arrowheads and red arrows indicate PH3 in GFP ─ ISCs and GFP + EBs , respectively . ( O , P ) Quantification of PH3 + GFP ─ ( P ) and PH3 + GFP + ( O ) in midguts with indicated genotypes . n = 13 guts for each genotype . ( Q ) Quantification of PH3 + EBs ( GFP + ) with the given genotypes in response to P . e infection . n = 11 guts for each genotype .

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(D-N') The Drosophila midguts expressing Su(H)ts>GFP with control (D-E'), UAS-RasV12 for 6 days (F-H'), EGFRA887T for 4 days (I-K') and UAS−λTop for 5 days (L-N') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (E-E', G-G', J-J' and M-M') Magnification of selected areas containing PH3+GFP─ cells from D, F, I and L. (H-H', K-K' and N-N') Magnification of selected areas containing PH3+GFP+ cells from F, I and L. White arrowheads and red arrows indicate PH3 in GFP─ ISCs and GFP+ EBs, respectively. (O, P) Quantification of PH3+GFP─ (P) and PH3+GFP+ (O) in midguts with indicated genotypes. n = 13 guts for each genotype. (Q) Quantification of PH3+ EBs (GFP+) with the given genotypes in response to P. e infection. n = 11 guts for each genotype.


( B - C ' ' ' ) Flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ were fed with Suc ( B - B ' ' ' ) or P . e ( C - C ' ' ' ) for 36h and their midguts were dissected and immunostained with antibodies against GFP , β - gal and Dl . Blue arrowheads indicate Dl + ISCs ; white arrows indicate GFP + EBs ; and red arrowheads indicate EB progeny cells ( LacZ + GFP ─ ) . ( D ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the control , P . e infected midguts , n = 50 . ( E ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the control with Suc or P . e , or knockdown of EGFR with Suc or P . e infection , n = 50 .

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

(B-C''') Flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ were fed with Suc (B-B''') or P.e (C-C''') for 36h and their midguts were dissected and immunostained with antibodies against GFP, β-gal and Dl. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control, P. e infected midguts, n=50. (E) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control with Suc or P. e, or knockdown of EGFR with Suc or P. e infection, n=50.


( A - C ' ' ' ) The midguts from adult female flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ with ( B - B ' ' ' ) or without ( A - A ' ' ' ) UAS - RasV12 or with UAS - RasV12 and UAS - stg - RNAi ( C - C ' ' ' ) were dissected and immunostained with antibodies against GFP , β - gal and Dl . The bigger GFP ─ nucleus with LacZ expression ( only red arrowheads ) indicate differentiating or mature ECs . Blue arrowheads indicate Dl + ISCs ; white arrows indicate GFP + EBs ; and red arrowheads indicate EB progeny cells ( LacZ + GFP ─ ) . ( D ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the indicated genotypes , n = 50 .

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(A-C''') The midguts from adult female flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ with (B-B''') or without (A-A''') UAS-RasV12 or with UAS-RasV12 and UAS-stg-RNAi (C-C''') were dissected and immunostained with antibodies against GFP, β-gal and Dl. The bigger GFP─ nucleus with LacZ expression (only red arrowheads) indicate differentiating or mature ECs. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the indicated genotypes, n=50.


( C - E ' ' ) The representative twin clones from midguts when EGFRA887T is overexpressed in EBs ( D - D ' ' , red and green arrows ) or adult flies were fed with P . e for 36h ( E - E ' ' , red and green arrows ) , but no twin clone is found in the control ( C - C ' ' ) . ( F ) Quantification of different types of twin clones in the control midguts , or in the midguts with EGFRA887T overexpression or P . e infection . n = 10 guts for each genotype .

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(C-E'') The representative twin clones from midguts when EGFRA887T is overexpressed in EBs (D-D'', red and green arrows) or adult flies were fed with P.e for 36h (E-E'', red and green arrows), but no twin clone is found in the control (C-C''). (F) Quantification of different types of twin clones in the control midguts, or in the midguts with EGFRA887T overexpression or P.e infection. n = 10 guts for each genotype.


( A - C ' ' ) ISCs from EBs upon RasV12 expression or P . e infection could differentiate to generate EE or pre - EE cells ( B - C ' ' , LacZ + Pros + GFP ─ , blue arrowheads ) , but no EE or pre - EE cells from EBs are found in the control ( A - A ' ' , white arrowheads ) . ( D ) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments . n = 9 guts for each genotype or treatment .

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(A-C'') ISCs from EBs upon RasV12 expression or P. e infection could differentiate to generate EE or pre-EE cells (B-C'', LacZ+Pros+GFP─, blue arrowheads), but no EE or pre-EE cells from EBs are found in the control (A-A'', white arrowheads). (D) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments. n = 9 guts for each genotype or treatment.


Flow cytometric analysis of γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse and line the median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 using Mann - Whitney test . Numbers of γδT17 cells in the LN ( A ) and skin ( B ) before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments .

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Flow cytometric analysis of γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse and line the median. *p < 0.05, **p < 0.01, ***p < 0.001 using Mann-Whitney test. Numbers of γδT17 cells in the LN (A) and skin (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments.


Representative micrographs indicating ear skin sections stained for H & E before and after IMQ - induced psoriasis ( scale bar = 100μm ) . Quantification of epidermal thickening from H & E stained sections . Steady - state : n = 8 ; 4 experiments , IMQ : n = 11 - 12 ; 4 experiments . Each symbol is the average thickening from 5 micrographs measured in μm .

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Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.


Flow cytometric analysis of γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . In graphs , each symbol represents a mouse and line the median . Numbers of γδT17 cells in the LN and skin ( staining as in Fig EV1 ) before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 6 ; 3 experiments , IMQ : n = 11 ; 4 experiments .

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Flow cytometric analysis of γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). In graphs, each symbol represents a mouse and line the median. Numbers of γδT17 cells in the LN and skin (staining as in Fig EV1) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 11; 4 experiments.


Representative micrographs indicating ear skin sections stained for H & E before and after IMQ - induced psoriasis ( scale bar = 100μm ) . Quantification of epidermal thickening from H & E stained sections . Steady - state : n = 6 ; 3 experiments , IMQ : n = 8 ; 3 experiments . Each symbol is the average thickening from 5 micrographs measured in μm .

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

Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm). Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 6; 3 experiments, IMQ: n = 8; 3 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.


Flow cytometric analysis of lymph node γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment and line the median . Representative dot plots showing IL - 17A ( A ) or IL - 17F ( D ) and IFNγ production in γδ T cells before ( steady - state ) and after IMQ - induced psoriasis . Numbers in gate indicate % positive cells ; numbers outside the gate indicate mean fluorescence intensity of IL - 17A or IL - 17F . ( B , E ) Frequency of IL - 17A + ( B ) and IL - 17F + ( E ) γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . ( C , F ) Quantification of mean fluorescence intensity ( MFI ) of IL - 17A ( C ) and IL - 17F ( F ) staining in γδ T cells after IMQ - induced psoriasis ( each color represents a different experiment ) .

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Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-17A (A) or IL-17F (D) and IFNγ production in γδ T cells before (steady-state) and after IMQ-induced psoriasis. Numbers in gate indicate % positive cells; numbers outside the gate indicate mean fluorescence intensity of IL-17A or IL-17F. (B, E) Frequency of IL-17A+ (B) and IL-17F+ (E) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C, F) Quantification of mean fluorescence intensity (MFI) of IL-17A (C) and IL-17F (F) staining in γδ T cells after IMQ-induced psoriasis (each color represents a different experiment).


Frequency of IL - 17A + and IL - 17F + γδ T cells or IL - 17A ( G ) and IL - 17F ( H ) MFI following culture with IL - 1β , IL - 23 or nothing . Each symbol represents one experiment .

Frequency of IL-17A+ and IL-17F+ γδ T cells or IL-17A (G) and IL-17F (H) MFI following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment.


Flow cytometric analysis of lymph node γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment and line the median . Representative dot plots showing IL - 22 and IL - 17A production in γδ T cells ( A ) and frequency of IL - 22 + γδ T cells ( B ) before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . Steady - state : n = 6 ; 3 experiments , IMQ : n = 8 - 9 ; 3 experiments .

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

Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (A) and frequency of IL-22+ γδ T cells (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 8-9; 3 experiments.


Flow cytometric analysis of lymph node γδ T cells in RORγtCRE - STAT3F / F ( Cre + ) and littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment and line the median . Representative dot plots showing IL - 22 and IL - 17A production in γδ T cells ( C ) and frequency of IL - 22 + γδ T cells ( D ) following culture without ( − ) or with 40ng / ml recombinant IL - 23 ( n = 9 - 10 , 3 experiments ) .

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Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. Representative dot plots showing IL-22 and IL-17A production in γδ T cells (C) and frequency of IL-22+ γδ T cells (D) following culture without (−) or with 40ng/ml recombinant IL-23 (n = 9-10, 3 experiments).


Frequency of IL - 22 + γδ T cells following culture with IL - 1β , IL - 23 or nothing . Each symbol represents one experiment . Open circles = Cre + ( each color represents a different culture condition ) .

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Frequency of IL-22+ γδ T cells following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment. Open circles = Cre+ (each color represents a different culture condition).


Flow cytometric analysis of lymph node γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . ( A , ) Frequency of IL - 17A + ( A ) γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . ( B ) Frequency of IL - 17F + γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis . ( C ) Quantification of mean fluorescence intensity ( MFI ) of IL - 17A staining in γδ T cells after IMQ - induced psoriasis ( D ) Quantification of mean fluorescence intensity ( MFI ) of IL - 17F staining in γδ T cells after IMQ - induced psoriasis .

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

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). (A, ) Frequency of IL-17A+ (A) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (B) Frequency of IL-17F+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C) Quantification of mean fluorescence intensity (MFI) of IL-17A staining in γδ T cells after IMQ-induced psoriasis (D) Quantification of mean fluorescence intensity (MFI) of IL-17F staining in γδ T cells after IMQ-induced psoriasis.


Flow cytometric analysis of lymph node γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . Frequency of IL - 22 + γδ T cells before ( steady - state ) and after ( IMQ ) IMQ - induced psoriasis .

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Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis.


Flow cytometric analysis of lymph node γδ T cells in STAT4 − / − ( − / − ) and littermate control mice ( + / − ) . Frequency of IL - 22 + γδ T cells following culture without ( − ) or with 40ng / ml recombinant IL -

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

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). Frequency of IL-22+ γδ T cells following culture without (−) or with 40ng/ml recombinant IL-23


( A ) Homer1c - GFP signal ( magenta ) and immunostaining for surface AMPARs ( cyan ) and SP ( green ) in neurons treated for 48h with TTX , or untreated ( UT ) . Scale bars : 10 µm ( low magnification ) , 5 µm ( insets ) . Dotted lines indicate the outline of dendrites . Arrowheads indicate SP + synapses . ( B ) Percentage of SP + synapses for untreated ( UT ) and TTX - treated neurons ( dot plots represent different cells ; UT : n = 26 ; TTX : n = 33 , n indicates the number of cells , from 2 cultures ) . % SP + spines : * * P = 0 . 019 ( Mann Whitney test ) . Data information : Data are represented as mean ± SEM .

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(A) Homer1c-GFP signal (magenta) and immunostaining for surface AMPARs (cyan) and SP (green) in neurons treated for 48h with TTX, or untreated (UT). Scale bars: 10 µm (low magnification), 5 µm (insets). Dotted lines indicate the outline of dendrites. Arrowheads indicate SP+ synapses. (B) Percentage of SP+ synapses for untreated (UT) and TTX-treated neurons (dot plots represent different cells; UT: n = 26; TTX: n = 33, n indicates the number of cells, from 2 cultures). % SP+ spines: **P = 0.019 (Mann Whitney test). Data information: Data are represented as mean ± SEM.


( C ) AMPAR synaptic fluorescence intensity for SP + versus SP - synapses in UT and TTX - treated neurons . AMPAR synaptic fluorescence intensity was normalized to SP - or small synapses , respectively , from UT condition . ( UT : SP - , n = 1455 , SP + , n = 516 ; TTX : SP - , n = 1529 , SP + , n = 724 , n indicates the number of synapses , from 2 cultures ) . * * P < 0 . 01 , * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

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(C) AMPAR synaptic fluorescence intensity for SP+ versus SP- synapses in UT and TTX-treated neurons. AMPAR synaptic fluorescence intensity was normalized to SP- or small synapses, respectively, from UT condition. (UT: SP-, n = 1455, SP +, n = 516; TTX: SP-, n = 1529, SP+, n = 724, n indicates the number of synapses, from 2 cultures). **P < 0.01, ****P < 0.0001, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Example images of a FRAP experiment : SP - and SP + spines from the same cultured hippocampal neuron transfected with SEP - GluA2 ( gray and color - coded ) + RFP - SP ( red ) 10s before , and 0 , 370 , 750 s after the photobleaching . Scale bars : 1 µm . Dotted lines represent the outline of dendrites . Arrowheads indicate dendritic spines which were targeted for photobleaching . ( B ) SEP - GluA2 fluorescence intensity at SP - vs SP + spines . Each pair of dot plots represents average fluorescence for SP - and SP + spines from a same neuron ( 4 cultures ) . * * P = 0 . 0061 ( two - tailed paired Student ' s t test ) . ( C ) Quantification of FRAP dynamics for SP - vs SP + spines . Recovery curves represent SEP - GluA2 fluorescence average per cell ( n = 14 cells , from 4 cultures ) . The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test . The F test indicates that the traces are best fitted by two divergent models ( P < 0 . 0001 ) . ( D ) Quantification of the recovery fraction 750 s after the photobleaching . Each pair of dot plots represents average recovery for SP - and SP + spines from a same neuron ( 4 cultures ) . * * P = 0 . 0067 ( Wilcoxon matched - pairs signed rank test ) . Data information : Data are represented as mean ± SEM .

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(A) Example images of a FRAP experiment: SP- and SP+ spines from the same cultured hippocampal neuron transfected with SEP-GluA2 (gray and color-coded) + RFP-SP (red) 10s before, and 0, 370, 750 s after the photobleaching. Scale bars: 1 µm. Dotted lines represent the outline of dendrites. Arrowheads indicate dendritic spines which were targeted for photobleaching. (B) SEP-GluA2 fluorescence intensity at SP- vs SP+ spines. Each pair of dot plots represents average fluorescence for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0061 (two-tailed paired Student's t test). (C) Quantification of FRAP dynamics for SP- vs SP+ spines. Recovery curves represent SEP-GluA2 fluorescence average per cell (n = 14 cells, from 4 cultures). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by two divergent models (P < 0.0001). (D) Quantification of the recovery fraction 750 s after the photobleaching. Each pair of dot plots represents average recovery for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0067 (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.


( A ) Immunoblot analysis of SP in lysates from COS - 7 cells which were untransfected , transfected with RFP - SP + empty vector or co - transfected with SP - shRNA - GFP + RFP - SP ( knock - down ) or shRNA - resistant RFP - SP ( rescue ) . β - actin and GFP signals illustrate equal protein loading and SP - shRNA expression level , respectively . ( B ) Quantification of SP expression from 6 different experiments , normalized to the β - actin signal . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data represent mean ± SEM .

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(A) Immunoblot analysis of SP in lysates from COS-7 cells which were untransfected, transfected with RFP-SP + empty vector or co-transfected with SP-shRNA-GFP + RFP-SP (knock-down) or shRNA-resistant RFP-SP (rescue). β-actin and GFP signals illustrate equal protein loading and SP-shRNA expression level, respectively. (B) Quantification of SP expression from 6 different experiments, normalized to the β-actin signal. *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( C ) Homer - DsRed ( green ) and immunostained endogenous SP ( red ) in dendrites from neurons transfected with either empty vector , scramble shRNA or SP - shRNA with GFP reporter ( blue ) . Scale bar : 10 µm . ( D ) Average SP intensity for same conditions ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates the number of cells , from 3 cultures ; normalized to empty vector condition ) . * * P < 0 . 01 , * * * * P < 0 . 0001 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . ( E ) Fraction of SP + synapses for same conditions . ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates the number of cells from 3 cultures ) . * P < 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data represent mean ± SEM .

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(C) Homer-DsRed (green) and immunostained endogenous SP (red) in dendrites from neurons transfected with either empty vector, scramble shRNA or SP-shRNA with GFP reporter (blue). Scale bar: 10 µm. (D) Average SP intensity for same conditions (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells, from 3 cultures; normalized to empty vector condition). **P < 0.01, ****P < 0.0001 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (E) Fraction of SP+ synapses for same conditions. (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells from 3 cultures). *P < 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( F ) Homer1c - DsRed ( red ) and immunostained surface AMPARs ( green ) in dendrites from neurons transfected with either SP - shRNA - GFP or empty vector ( EV ) with GFP reporter ( blue ) and treated with TTX or left untreated ( UT ) . Scale bar : 5 µm . ( G ) AMPAR synaptic fluorescence intensity normalized to untreated empty vector ( EV ) condition ( EV : UT , n = 42 , TTX , n = 33 ; SP - shRNA : UT , n = 30 , TTX , n = 31 , n indicates the number of cells , from 3 cultures ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data represent mean ± SEM .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O 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 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

(F) Homer1c-DsRed (red) and immunostained surface AMPARs (green) in dendrites from neurons transfected with either SP-shRNA-GFP or empty vector (EV) with GFP reporter (blue) and treated with TTX or left untreated (UT). Scale bar: 5 µm. (G) AMPAR synaptic fluorescence intensity normalized to untreated empty vector (EV) condition (EV: UT, n = 42, TTX, n = 33; SP-shRNA: UT, n = 30, TTX, n = 31, n indicates the number of cells, from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( A ) Micrographs showing Homer1c - GFP ( green ) and immunostaining for surface AMPARs ( blue ) and endogenous SP ( red ) in neurons transfected with miR - 124 or control miR - 67 ( miR - Ctrl ) , and treated with TTX , or left untreated ( UT ) . Scale bar : 5 µ ( B ) Percentage of SP + synapses for same conditions as in ( A ) ( miR - Ctrl : UT , n = 25 , TTX , n = 26 ; miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates the number of cells , from 3 cultures ) . * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . ( C ) Homer1c - GFP intensity for same condition as in ( A ) , normalized to untreated miR - Ctrl ( miR - Ctrl : UT , n = 25 , TTX , n = 26 ; miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates the number of cells , from 3 cultures ) . ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . ( D ) AMPAR synaptic fluorescence intensity for same condition as in ( A ) , normalized to untreated miR - Ctrl ( miR - Ctrl : UT , n = 25 , TTX , n = 26 , miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates the number of cells , from 3 cultures ) . * * * P < 0 . 001 , * P < 0 . 01 , ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

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

(A) Micrographs showing Homer1c-GFP (green) and immunostaining for surface AMPARs (blue) and endogenous SP (red) in neurons transfected with miR-124 or control miR-67 (miR-Ctrl), and treated with TTX, or left untreated (UT). Scale bar: 5 µ (B) Percentage of SP+ synapses for same conditions as in (A) (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ****P < 0.0001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (C) Homer1c-GFP intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (D) AMPAR synaptic fluorescence intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26, miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ***P < 0.001, *P < 0.01, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Micrographs showing dendrites from neurons transfected with Homer1c - DsRed ( magenta ) and SEP - GluA2 constructs containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( green ) and treated with TTX for 48 h , or left untreated ( UT ) . Scale bar : 10 µm . ( B ) SEP - GluA2 synaptic fluorescence intensity for each condition , normalized to GluA2 - 3 ' UTR - WT untreated neurons ( 3 ' UTR - WT : UT , n = 29 , TTX , n = 37 ; 3 ' UTR - MUT : UT , n = 18 , TTX , n = 14 ; n indicates the number of cells , from 3 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Micrographs showing dendrites from neurons transfected with Homer1c-DsRed (magenta) and SEP-GluA2 constructs containing wild-type (WT) or mutated (MUT) 3'UTR (green) and treated with TTX for 48 h, or left untreated (UT). Scale bar: 10 µm. (B) SEP-GluA2 synaptic fluorescence intensity for each condition, normalized to GluA2-3'UTR-WT untreated neurons (3'UTR-WT: UT, n = 29, TTX, n = 37; 3'UTR-MUT: UT, n = 18, TTX, n = 14; n indicates the number of cells, from 3 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( C ) Micrographs showing dendrites from neurons transfected with Homer1c - BFP ( magenta ) , GFP - SP - shRNA ( gray ) and a rescue RFP - SP construct containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( geen ) and treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( D ) Percentage of SP + synapses for each condition ( 3 ' UTR - WT : UT , n = 26 , TTX , n = 28 ; 3 ' UTR - MUT : UT , n = 26 , TTX , n = 24 ; n indicates the number of cells , from 3 cultures ) . * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

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

(C) Micrographs showing dendrites from neurons transfected with Homer1c-BFP (magenta), GFP-SP-shRNA (gray) and a rescue RFP-SP construct containing wild-type (WT) or mutated (MUT) 3'UTR (geen) and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (D) Percentage of SP+ synapses for each condition (3'UTR-WT: UT, n = 26, TTX, n = 28; 3'UTR-MUT: UT, n = 26, TTX, n = 24; n indicates the number of cells, from 3 cultures). **P < 0.01, ***P < 0.001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.


( E ) Micrographs showing Homer1c - GFP ( magenta ) and immunostaining for surface AMPARs ( cyan ) and endogenous SP ( green ) in neurons transfected with or without 50 nM SP TSB - LNA and treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( F ) Percentage of SP + synapses for each condition ( Control : UT , n = 36 , TTX , n = 52 ; SP TSB - LNA : UT , n = 62 , TTX , n = 29 ; n indicates the number of cells from 3 cultures ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . ( G ) AMPAR synaptic fluorescence intensity for each condition , normalized to control untreated neurons ( Control : UT , n = 30 , TTX , n = 36 ; SP TSB - LNA : UT , n = 37 , TTX , n = 20 ; n indicates the number of cells from 2 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing Homer1c-GFP (magenta) and immunostaining for surface AMPARs (cyan) and endogenous SP (green) in neurons transfected with or without 50 nM SP TSB-LNA and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (F) Percentage of SP+ synapses for each condition (Control: UT, n = 36, TTX, n = 52; SP TSB-LNA: UT, n = 62, TTX, n = 29; n indicates the number of cells from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (G) AMPAR synaptic fluorescence intensity for each condition, normalized to control untreated neurons (Control: UT, n = 30, TTX, n = 36; SP TSB-LNA: UT, n = 37, TTX, n = 20; n indicates the number of cells from 2 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Micrographs showing puro - PLA staining of newly synthesized SP ( green ) in neurons immunostained for MAP - 2 ( magenta ) and DAPI ( blue ) and treated with TTX for 24 h or left untreated . The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody . Scale bars : 30 µm ( large view ) , 5 µm ( insets ) .

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

(A) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons immunostained for MAP-2 (magenta) and DAPI (blue) and treated with TTX for 24 h or left untreated. The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody. Scale bars: 30 µm (large view), 5 µm (insets).


( C ) Density of SP puro - PLA puncta , normalized to the untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n represents the number of cells , from 3 cultures ) . * P < 0 . 05 ( Mann - Whitney test ) . ( D ) SP puro - PLA cluster fluorescence intensity normalized to the untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n indicates the number of cells , from 3 cultures ) . * * * * P < 0 . 0001 ( Mann - Whitney test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Density of SP puro-PLA puncta, normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n represents the number of cells, from 3 cultures). *P < 0.05 (Mann-Whitney test). (D) SP puro-PLA cluster fluorescence intensity normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n indicates the number of cells, from 3 cultures). ****P < 0.0001 (Mann-Whitney test). Data information: Data are represented as mean ± SEM.


( E ) Micrographs showing puro - PLA staining of newly synthesized SP ( green ) in neurons transfected with Homer1c - GFP ( magenta ) and treated with TTX for 24 h or left untreated . Scale bar : 5 µm . Arrowheads indicate puro - PLA + spines . ( F ) Percentage of SP puro - PLA + synapses ( UT , n = 26 , TTX , n = 33 ; n indicates the number of cells , from 2 cultures ) . * * P < 0 . 01 , ns , not significant ( Mann - Whitney test ) . ( G ) Homer1c - GFP integrated fluorescence intensity at SP puro - PLA - vs SP puro - PLA + synapses ( n = 32 cells , from 2 cultures ) . * * * * P < 0 . 0001 , ns , not significant ( Wilcoxon matched - pairs signed rank test ) . Data information : Data are represented as mean ± SEM .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons transfected with Homer1c-GFP (magenta) and treated with TTX for 24 h or left untreated. Scale bar: 5 µm. Arrowheads indicate puro-PLA+ spines. (F) Percentage of SP puro-PLA+ synapses (UT, n = 26, TTX, n = 33; n indicates the number of cells, from 2 cultures). **P < 0.01, ns, not significant (Mann-Whitney test). (G) Homer1c-GFP integrated fluorescence intensity at SP puro-PLA- vs SP puro-PLA+ synapses (n = 32 cells, from 2 cultures). ****P < 0.0001, ns, not significant (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.


( A ) Confocal images showing dendrites from CA3 pyramidal cells transfected with RFP - SP ( green ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR + SP - shRNA - BFP ( magenta ) . Arrowheads indicate SP + spines . Scale bar : 10 µm . ( B ) Graph plot showing spine head volume vs SP integrated intensity ( n = 5 cells ) . Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP + spines in the same neuron . Plots for SP - and SP + spines appear in grey and magenta , respectively . The black dotted line represents linear regression ( R2 = 0 . 25 , P = 0 . 0004 ) . ( C ) Paired data showing spine head volume for SP - vs SP + spines . Each pair of plot represents average values for SP - vs SP + spines for a given neuron . * P = 0 . 0031 ( two - tailed Student t test ) . Data information : Data are represented as mean ± SEM .

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

(A) Confocal images showing dendrites from CA3 pyramidal cells transfected with RFP-SP (green) containing wild-type (WT) or mutated (MUT) 3'UTR + SP-shRNA-BFP (magenta). Arrowheads indicate SP+ spines. Scale bar: 10 µm. (B) Graph plot showing spine head volume vs SP integrated intensity (n = 5 cells). Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP+ spines in the same neuron. Plots for SP- and SP+ spines appear in grey and magenta, respectively. The black dotted line represents linear regression (R2 = 0.25, P = 0.0004). (C) Paired data showing spine head volume for SP- vs SP+ spines. Each pair of plot represents average values for SP- vs SP+ spines for a given neuron. *P = 0.0031 (two-tailed Student t test). Data information: Data are represented as mean ± SEM.


( F ) Higher magnification of dendritic spines from neurons transfected with rescue RFP - SP ( magenta ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR and BFP - shRNA - SP ( grey ) and contacted by GFP - Syp + terminals ( green ) . Arrowheads indicate putative synaptic contacts . Scale bar : 5 μm . ( G ) Percentage of SP + spines in neurons expressing RFP - SP - 3 ' UTR - WT vs - MUT and depending on the apposition or not to a GFP - Syp + terminal ( SP - 3 ' UTR - WT : TetTx - , n = 220 , TetTx + , n = 26 ; SP - 3 ' UTR - MUT : TetTx - , n = 616 , TetTx + , n = 25 ; n indicates the number of spines from 5 - 7 pairs ) . ( H ) Spine head volume in same conditions as in ( G ) ( SP - 3 ' UTR - WT : TetTx - , n = 26 , TetTx + , n = 26 spines from 5 pairs , 4 slices ; SP - 3 ' UTR - MUT : TetTx - , n = 25 , TetTx + , n = 25 ; n indicates the number of spines from 7 pairs , 7 slices ) . * * * P < 0 . 001 , * P < 0 . 05 , ns , not significant ( Kruskall - Wallis test followed by Dunn ' s multiple test ) . ( I ) Cumulative probability distributions of spine head volumes for TetTx - and TetTx + spines from neurons expressing RFP - SP - 3 ' UTR - WT ( SP - WT , light gray and light green ) or RFP - SP - 3 ' UTR - MUT ( SP - MUT , dark gray and dark green ) . Data information : Data are represented as mean ± SEM .

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

(F) Higher magnification of dendritic spines from neurons transfected with rescue RFP-SP (magenta) containing wild-type (WT) or mutated (MUT) 3'UTR and BFP-shRNA-SP (grey) and contacted by GFP-Syp+ terminals (green). Arrowheads indicate putative synaptic contacts. Scale bar: 5 μm. (G) Percentage of SP+ spines in neurons expressing RFP-SP-3'UTR-WT vs -MUT and depending on the apposition or not to a GFP-Syp+ terminal (SP-3'UTR-WT: TetTx-, n = 220, TetTx+, n = 26; SP-3'UTR-MUT: TetTx-, n = 616, TetTx+, n = 25; n indicates the number of spines from 5-7 pairs). (H) Spine head volume in same conditions as in (G) (SP-3'UTR-WT: TetTx-, n = 26, TetTx+, n = 26 spines from 5 pairs, 4 slices; SP-3'UTR-MUT: TetTx-, n = 25, TetTx+, n = 25; n indicates the number of spines from 7 pairs, 7 slices). ***P < 0.001, *P < 0.05, ns, not significant (Kruskall-Wallis test followed by Dunn's multiple test). (I) Cumulative probability distributions of spine head volumes for TetTx- and TetTx+ spines from neurons expressing RFP-SP-3'UTR-WT (SP-WT, light gray and light green) or RFP-SP-3'UTR-MUT (SP-MUT, dark gray and dark green). Data information: Data are represented as mean ± SEM.


( c ) Intravascular endothelial cell interactions and transmigration of neutrophils ( Ly - 6G + CX3CR - 1 - ; red ) and cMOs ( Ly - 6G - CX3CR - 1low ; green ) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA , PAI - 1 , or uPA - PAI - 1 in postcapillary venules of the cremaster of CX3CR - 1GFP / + mice by multi - channel in vivo microscopy . Representative still images ( scale bar : 50 : µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; * p < 0 . 05 vs . uPA ; § p < 0 . 05 vs . PAI - 1 ; One - way ANOVA ) .

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

(c) Intravascular endothelial cell interactions and transmigration of neutrophils (Ly-6G+ CX3CR-1-; red) and cMOs (Ly-6G- CX3CR-1low; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI-1, or uPA-PAI-1 in postcapillary venules of the cremaster of CX3CR-1GFP/+ mice by multi-channel in vivo microscopy. Representative still images (scale bar: 50: µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. uPA; §p<0.05 vs. PAI-1; One-way ANOVA).


( b ) Expression of PECAM - 1 / CD31 ( red ) and ICAM - 1 / CD54 or VCAM - 1 / CD106 ( green ) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA - PAI - 1 or saline , representative images ( scale bar : 50 µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; t test ) .

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(b) Expression of PECAM-1/CD31 (red) and ICAM-1/CD54 or VCAM-1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA-PAI-1 or saline, representative images (scale bar: 50 µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; t test).


( g ) Quantitative analysis of LFA - 1 / CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E - selectin / CD62E + ICAM - 1 / CD54 , E - selectin / CD62E + ICAM - 1 / CD54 + KC , or E - selectin / CD62E + ICAM - 1 / CD54 + KC + uPA - PAI - 1 as assessed by spinning disk confocal microscopy , representative images and quantitative data are shown ( scale bar : 10 μm ; mean ± SD ; n = 3 mice ; n = 10 - 13 flow chambers , n = 122 - 222 cells ) ; * p < 0 . 05 vs . E - selectin / CD62E + ICAM - 1 / CD54 ; One - way ANOVA ) .

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(g) Quantitative analysis of LFA-1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E-selectin/CD62E + ICAM-1/CD54, E-selectin/CD62E + ICAM-1/CD54 + KC, or E-selectin/CD62E + ICAM-1/CD54 + KC + uPA-PAI-1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 μm; mean±SD; n=3 mice; n=10-13 flow chambers, n =122-222 cells); *p<0.05 vs. E-selectin/CD62E + ICAM-1/CD54; One-way ANOVA).


( b ) Correlation analyses of RNA expression levels of uPA ( PLAU gene ) , PAI - 1 ( SERPINE1 gene ) , and the neutrophil marker gene FPR1 , and overall survival of breast cancer patients ( stages 0 and 1 ) with high and low PLAU or SERPINE1 gene expression levels ( cut - off z ≥ 2 . 0 ) in the METABRIC breast cancer cohort .

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(b) Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI-1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cut-off z≥ 2.0) in the METABRIC breast cancer cohort.


( c ) Surface expression of MMP - 9 , VEGF , and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice ( saline ) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA - PAI - 1 ( uPA - PAI - 1 ) by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; # p < 0 . 05 vs . saline ; t test ) .

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(c) Surface expression of MMP-9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA-PAI-1 (uPA-PAI-1) by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; #p<0.05 vs. saline; t test).


( f ) Formation of NETs ( histone H3 + ; green ) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra - scrotal injection of uPA - PAI - 1 , TNF , or saline , PECAM - 1 / CD31 + postcapillary venules ( blue ) and Ly - 6G + neutrophils ( red ) are depicted . Representative images are shown ( scale bar : 50 µm ) .

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(f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).


( a ) Effect of compound WX - 340 on binding of recombinant murine uPA to PAI - 1 protein as assessed by ELISA , quantitative data are shown ( mean ± SEM for n = 3 experiments per group ; # p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) .

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(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA).


( d ) Relative development rates and tumor weight in animals treated with WX - 340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) .

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(d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA).


( f ) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX - 340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; t test ) .

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(f) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX-340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).


C Immunofluorescent analyses of USP33 ( red ) , the hypoxia marker CA9 ( green ) , and the stem cell marker SOX2 ( gray ) in human primary GBMs . Arrows indicate the USP33 + / CA9 + / SOX2 + cells . Scale bar , 80 µm .

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C Immunofluorescent analyses of USP33 (red), the hypoxia marker CA9 (green), and the stem cell marker SOX2 (gray) in human primary GBMs. Arrows indicate the USP33+/CA9+/SOX2+ cells. Scale bar, 80 µm.


E Immunofluorescent analyses of USP33 ( red ) and the hypoxic cell population labeled by pimonidazole ( green ) in intracranial xenografts derived from T3359 GSCs . Mice bearing intracranial xenografts were injected intravenously ( tail vein ) with 60 mg / kg of pimonidazole and were sacrificed 2 hours post injection . Hypoxic cells were labeled with the FITC - conjugated mouse monoclonal anti - pimonidazole antibody . Scale bar , 80 µm .

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E Immunofluorescent analyses of USP33 (red) and the hypoxic cell population labeled by pimonidazole (green) in intracranial xenografts derived from T3359 GSCs. Mice bearing intracranial xenografts were injected intravenously (tail vein) with 60 mg/kg of pimonidazole and were sacrificed 2 hours post injection. Hypoxic cells were labeled with the FITC-conjugated mouse monoclonal anti-pimonidazole antibody. Scale bar, 80 µm.


G Immunoblot analysis of USP33 , HIF2α and HIF1α protein levels in GSCs , NSTCs and NPCs in normoxic and hypoxic conditions . Hypoxia ( 5 % O2 ) relative to normoxia ( 20 % O2 ) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs . The mild hypoxia of 5 % O2 showed negligible induction of HIF1α in all cells .

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G Immunoblot analysis of USP33, HIF2α and HIF1α protein levels in GSCs, NSTCs and NPCs in normoxic and hypoxic conditions. Hypoxia (5% O2) relative to normoxia (20% O2) clearly induced USP33 and HIF2α expression in GSCs but not NSTCs or NPCs. The mild hypoxia of 5% O2 showed negligible induction of HIF1α in all cells.


A , Representative images of immunofluorescent analyses of USP33 ( red ) and HIF2α ( green ) in human primary GBMs . Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α , and counterstained with DAPI to show nuclei ( blue ) . Scale bar , 40 μm . ( n = 5 sections ; mean ± s . e . m . ) .

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A, Representative images of immunofluorescent analyses of USP33 (red) and HIF2α (green) in human primary GBMs. Frozen sections of human GBMs were immunostained with antibodies against USP33 and HIF2α, and counterstained with DAPI to show nuclei (blue). Scale bar, 40 μm. (n = 5 sections; mean ± s.e.m.).


F Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33 . 0 . 5 µg of HA - tagged HIF2α plasmids together with the increased amounts of GFP - tagged USP33 plasmids ( 0 , 0 . 1 , 0 . 2 or 0 . 3 μg ) were introduced into 293T cells . Forty - eight hours post transfection , levels of ectopic HIF2α - HA and GFP - USP33 were analyzed by immunoblot analysis . Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33 . G Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild type ( WT ) USP33 or the catalytically dead ( CD ) USP33 . HA - tagged HIF2α plasmids ( 0 . 5 µg ) were introduced into 293T cells alone or together with GFP - tagged WT or CD USP33 plasmids ( 0 . 2 µg ) . Levels of ectopic HIF2α - HA and GFP - USP33 were analyzed 48 hours post transfection . Expression of the wild type USP33 but not the catalytically dead USP33 increased the ectopic HIF2α protein levels .

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F Immunoblot analysis of ectopic HIF2α protein levels in 293T cells with gradually increased amount of ectopic USP33. 0.5 µg of HA-tagged HIF2α plasmids together with the increased amounts of GFP-tagged USP33 plasmids (0, 0.1, 0.2 or 0.3 μg) were introduced into 293T cells. Forty-eight hours post transfection, levels of ectopic HIF2α-HA and GFP-USP33 were analyzed by immunoblot analysis. Concomitantly increased levels of ectopic HIF2α and USP33 expression indicated the positive regulation of HIF2α protein by USP33. G Immunoblot analysis of ectopic HIF2α protein levels in 293T cells expressing ectopic wild type (WT) USP33 or the catalytically dead (CD) USP33. HA-tagged HIF2α plasmids (0.5 µg) were introduced into 293T cells alone or together with GFP-tagged WT or CD USP33 plasmids (0.2 µg). Levels of ectopic HIF2α-HA and GFP-USP33 were analyzed 48 hours post transfection. Expression of the wild type USP33 but not the catalytically dead USP33 increased the ectopic HIF2α protein levels.


A , Co - immunoprecipitation ( Co - IP ) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to low oxygen . GSCs were cultured under 20 % or 5 % oxygen for 24 hours before harvest for the Co - IP . Cell lysate was subjected to immunoprecipitation with anti - HIF2α antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . An increased interaction between HIF2α and USP33 was detected in GSCs cultured under 5 % oxygen relative to 20 % oxygen .

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A, Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to low oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-HIF2α antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs cultured under 5% oxygen relative to 20% oxygen.


D Co - immunoprecipitation ( Co - IP ) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to CoCl2 treatment . GSCs were treated with CoCl2 ( 300 µM ) for 12 hours before harvest for the Co - IP . Cell lysate was subjected to immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . An increased interaction between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia .

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D Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to CoCl2 treatment. GSCs were treated with CoCl2 (300 µM) for 12 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-USP33 antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia.


G Co - immunoprecipitation to detect the interaction between HIF2α and the wild - type ( WT ) or the catalytically dead ( CD ) USP33 . Ectopic HA - tagged HIF2α were expressed in 293T cells with GFP - tagged wild - type or catalytically dead USP33 . Cell lysates were subjected to immunoprecipitation with anti - HA antibodies . Co - immunoprecipitated products immunoblotted with the indicated antibodies . The catalytically dead USP33 relative to the wild type USP33 showed a stronger affinity to HIF2α .

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G Co-immunoprecipitation to detect the interaction between HIF2α and the wild-type (WT) or the catalytically dead (CD) USP33. Ectopic HA-tagged HIF2α were expressed in 293T cells with GFP-tagged wild-type or catalytically dead USP33. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies. Co-immunoprecipitated products immunoblotted with the indicated antibodies. The catalytically dead USP33 relative to the wild type USP33 showed a stronger affinity to HIF2α.


A Immunoblot analysis of the serine / threonine phosphorylation ( pS / T ) status of HIF2α in GSCs in response to 5 % oxygen . GSCs were cultured under 20 % or 5 % oxygen for 24 hours , and then harvested for immunoprecipitation with anti - HIF2α antibodies or IgG control . Immunoprecipitated endogenous HIF2α were immunoblotted with anti - pan - pS / T antibodies . An increased pS / T of HIF2α was detected in GSCs cultured under 5 % oxygen relative to 20 % oxygen .

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A Immunoblot analysis of the serine/threonine phosphorylation (pS/T) status of HIF2α in GSCs in response to 5% oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours, and then harvested for immunoprecipitation with anti-HIF2α antibodies or IgG control. Immunoprecipitated endogenous HIF2α were immunoblotted with anti-pan-pS/T antibodies. An increased pS/T of HIF2α was detected in GSCs cultured under 5% oxygen relative to 20% oxygen.


D Co - immunoprecipitation to detect the interaction between endogenous HIF2α and USP33 in GSCs after inhibition of ERK1 / 2 activity . GSCs were cultured under 20 % or 5 % oxygen for 24 hours , then treated with the MEK inhibitor U0126 ( 10µM ) for 30 minutes to inhibit ERK1 / 2 activity , and harvested for immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with the indicated antibodies . Relative to 20 % oxygen treatment , 5 % oxygen treatment promoted the interaction between HIF2α and USP33 in GSCs . However , inhibition of ERK1 / 2 activity by U0126 treatment suppressed the interaction between HIF2α and USP33 .

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D Co-immunoprecipitation to detect the interaction between endogenous HIF2α and USP33 in GSCs after inhibition of ERK1/2 activity. GSCs were cultured under 20% or 5% oxygen for 24 hours, then treated with the MEK inhibitor U0126 (10µM) for 30 minutes to inhibit ERK1/2 activity, and harvested for immunoprecipitation with anti-USP33 antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. Relative to 20% oxygen treatment, 5% oxygen treatment promoted the interaction between HIF2α and USP33 in GSCs. However, inhibition of ERK1/2 activity by U0126 treatment suppressed the interaction between HIF2α and USP33.


G Co - immunoprecipitation to determine the serine phosphorylation sites in human HIF2α responsible for its binding to USP33 . Ectopic GFP - tagged USP33 along with HA - tagged wild type HIF2α or HIF2α mutants with indicated serine to alanine point mutations were expressed in 293T cells . Cell lysates were subjected to immunoprecipitation with anti - HA agaroses Co - immunoprecipitated products were immunoblotted with the indicated antibodies . The S484A mutation largely attenuated the interaction of HIF2α with USP33 , indicating that the serine 484 site in HIF2α was the most important phosphorylation site for the interaction between HIF2α and USP33 .

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G Co-immunoprecipitation to determine the serine phosphorylation sites in human HIF2α responsible for its binding to USP33. Ectopic GFP-tagged USP33 along with HA-tagged wild type HIF2α or HIF2α mutants with indicated serine to alanine point mutations were expressed in 293T cells. Cell lysates were subjected to immunoprecipitation with anti-HA agaroses Co-immunoprecipitated products were immunoblotted with the indicated antibodies. The S484A mutation largely attenuated the interaction of HIF2α with USP33, indicating that the serine 484 site in HIF2α was the most important phosphorylation site for the interaction between HIF2α and USP33.


B Representative images of tumorsphere formation of T387 GSCs expressing USP33 - targeting shRNA ( shUSP33 ) or non - targeting shRNA ( shNT ) . Twenty - four hours after lentiviral infection , T387 GSCs expressing shUSP33 or shNT were planted in 96 well plates at the density of 2 , 000 cells per well and cultured for 48 hours under normoxia ( 20 % O2 ) or hypoxia ( 5 % O2 ) . Disruption of USP33 inhibited GSC tumorsphere formation in normoxia and dramatically suppressed GSC tumorsphere formation in hypoxia . Scale bar , 200 μm .

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B Representative images of tumorsphere formation of T387 GSCs expressing USP33-targeting shRNA (shUSP33) or non-targeting shRNA (shNT). Twenty-four hours after lentiviral infection, T387 GSCs expressing shUSP33 or shNT were planted in 96 well plates at the density of 2,000 cells per well and cultured for 48 hours under normoxia (20% O2) or hypoxia (5% O2). Disruption of USP33 inhibited GSC tumorsphere formation in normoxia and dramatically suppressed GSC tumorsphere formation in hypoxia. Scale bar, 200 μm.


E Cell titer assays showing cell growth of T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia . GSCs were transduced with shNT or shUSP33 through lentiviral infection for 24 hours , and then split into 96 well plates at the concentration of 2 , 000 cells per well . Cell titer was determined by the Glo luminescent cell viability assay kit ( Promega ) at the indicated time points . Disruption of USP33 inhibited GSC growth in normoxia and hypoxia . The experiment has been repeated three times . The presented curves were based on three technical replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( mean ± s . e . m . ; two tailed unpaired t - test ) .

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E Cell titer assays showing cell growth of T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia. GSCs were transduced with shNT or shUSP33 through lentiviral infection for 24 hours, and then split into 96 well plates at the concentration of 2,000 cells per well. Cell titer was determined by the Glo luminescent cell viability assay kit (Promega) at the indicated time points. Disruption of USP33 inhibited GSC growth in normoxia and hypoxia. The experiment has been repeated three times. The presented curves were based on three technical replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (mean ± s.e.m.; two tailed unpaired t-test).


F Representative immunofluorescent staining of the apoptotic marker cleaved caspase 3 in T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia . GSCs were attached to hESC - Qualified Matrix ( Corning ) in 6 - well plates at the concentration of 50 , 000 cells per well . Forty - eight hours post infection with shUSP33 or shNT lentiviruses , cells were stained for cleaved caspase 3 ( green ) and nuclei ( blue ) . Scale bar , 80 μm .

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F Representative immunofluorescent staining of the apoptotic marker cleaved caspase 3 in T387 GSCs expressing shUSP33 or shNT in normoxia or hypoxia. GSCs were attached to hESC-Qualified Matrix (Corning) in 6-well plates at the concentration of 50,000 cells per well. Forty-eight hours post infection with shUSP33 or shNT lentiviruses, cells were stained for cleaved caspase 3 (green) and nuclei (blue). Scale bar, 80 μm.


C , Representative images of autonomous fluorescent signals of 4HRE - d2GFP in xenografts derived from or shUSP33 - or shNT - expressing T387 GSCs transduced with 4HRE - d2GFP . Scale bar , 200 μm .

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C, Representative images of autonomous fluorescent signals of 4HRE-d2GFP in xenografts derived from or shUSP33- or shNT- expressing T387 GSCs transduced with 4HRE-d2GFP. Scale bar, 200 μm.


E , Representative images of autonomous fluorescent signals of 4HRE - d2GFP ( green ) and immunofluorescent staining of the stem cell marker SOX2 ( red ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . More GFP signals were detected in the SOX2 + cells compared with the SOX2 - cells , suggesting a stronger hypoxia response in GSCs relative to NSTCs . Scale bar , 200 μm .

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E, Representative images of autonomous fluorescent signals of 4HRE-d2GFP (green) and immunofluorescent staining of the stem cell marker SOX2 (red) in xenografts derived from T387 GSCs transduced with 4HRE-d2GFP. More GFP signals were detected in the SOX2+ cells compared with the SOX2- cells, suggesting a stronger hypoxia response in GSCs relative to NSTCs. Scale bar, 200 μm.


G , Representative images of immunofluorescent staining of the apoptotic marker cleaved caspase 3 ( red ) and autonomous fluorescent signals of 4HRE - d2GFP ( green ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . The GFP + population had much less apoptosis relative to the GFP - population , suggesting that hypoxia response inhibited cell apoptosis . Scale bar , 200 μm .

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G, Representative images of immunofluorescent staining of the apoptotic marker cleaved caspase 3 (red) and autonomous fluorescent signals of 4HRE-d2GFP (green) in xenografts derived from T387 GSCs transduced with 4HRE-d2GFP. The GFP+ population had much less apoptosis relative to the GFP- population, suggesting that hypoxia response inhibited cell apoptosis. Scale bar, 200 μm.


B Kaplan - Meier survival curves of mice bearing GBMs derived from T387 GSCs expressing shUSP33 or shNT from ( A ) . Disruption of USP33 extended the survival of mice bearing GSC - derived GBM tumors . ( n = 5 mice for each group ; shUSP33 # 42 vs shNT , p = 0 . 0185 ; shUSP33 # 45 vs shNT , p = 0 . 0027 ; two - tailed log - rank test ) .

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B Kaplan-Meier survival curves of mice bearing GBMs derived from T387 GSCs expressing shUSP33 or shNT from (A). Disruption of USP33 extended the survival of mice bearing GSC-derived GBM tumors. (n= 5 mice for each group; shUSP33#42 vs shNT, p = 0.0185; shUSP33#45 vs shNT, p = 0.0027; two-tailed log-rank test).