( B - K ' ) 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 ' ) DSS for 36 h ( J - K ' ) were immunostained for GFP ( green ) , PH3 ( red ) DAPI ( blue ) . ( C - C ' , E - E ' , H - H ' K - K ' ) Magnification of selected areas containing PH3 + GFP ─ from B , D , G J . EB close to PH3 + ( arrow ) in C - C ' is out of focus . ( F - F ' I - I ' ) Magnification of selected areas containing PH3 + EBs ( GFP + ) from D G . arrowheads red arrows indicate PH3 in GFP ─ GFP + , respectively . ( L M ) of PH3 GFP ─ ISCs ( L ) PH3 + GFP + EBs ( M ) with given treatments . n = 21 guts for each treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 ' ' ) Dss ( F - F ' ' ) . However , PH3 expression is present in EBs with GFP ─ Pros ─ small nuclei upon P . e infection ( E - E ' ' , red arrows ) . arrows indicate GFP + with PH3 expression . ( G ) of PH3 + GFP ─ Pros ─ EBs with given treatments . n = 11 guts for each treatment . Data information : Three independent experiments were performed , error bars are ± . * * * , P < 0 . 001 ( Student ' s t - ) ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

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

O O O 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 their midguts were dissected immunostained with antibodies against GFP , β - gal Dl . Blue arrowheads indicate Dl + ISCs ; arrows indicate GFP + EBs ; red arrowheads indicate EB progeny ( LacZ + GFP ─ ) . ( D ) of Dl + ISC - like from EBs ( GFP ─ LacZ + Dl + ) in control , P . e infected midguts , n = 50 . ( E ) of Dl + ISC - like from EBs ( GFP ─ LacZ + Dl + ) in 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 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 B-MEASURED_VAR 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-MEASURED_VAR 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 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 ' ' ' ) midguts from adult 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 UAS - stg - RNAi ( C - C ' ' ' ) were dissected immunostained with antibodies against GFP , β - gal Dl . bigger GFP ─ nucleus with LacZ expression ( only red arrowheads ) indicate differentiating or mature ECs . Blue arrowheads indicate Dl + ISCs ; arrows indicate GFP + EBs ; red arrowheads indicate EB progeny ( LacZ + GFP ─ ) . ( D ) of Dl + ISC - like from EBs ( GFP ─ LacZ + Dl + ) in indicated genotypes , 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 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 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 B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

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

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

(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 ( B - C ' ' , LacZ + Pros + GFP ─ , blue arrowheads ) , but no EE or pre - EE from EBs are found in control ( A - A ' ' , arrowheads ) . ( D ) of EE from progenies of EBs with indicated genotypes or treatments . n = 9 guts for each genotype or treatment .

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 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

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

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

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

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

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

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

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

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

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 + IL - 17F + γδ T or IL - 17A ( G ) 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 of lymph node γδ T in RORγtCRE - STAT3F / F ( Cre + ) littermate control mice ( Cre − ) . In graphs , each symbol represents a mouse or experiment median . Representative dot plots showing IL - 22 IL - 17A production in γδ T ( A ) frequency of IL - 22 + γδ T ( B ) before ( steady - state ) 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 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 B-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 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

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

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 B-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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 (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 following culture with IL - 1β , IL - 23 or nothing . Each symbol represents one experiment . Open circles = Cre + ( each color represents a different culture condition ) .

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

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 of lymph node γδ T in STAT4 − / − ( − / − ) littermate control mice ( + / − ) . ( A , ) Frequency of IL - 17A + ( A ) γδ T before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . ( B ) Frequency of IL - 17F + γδ T before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis . ( C ) of mean ( MFI ) of IL - 17A in γδ T after IMQ - induced psoriasis ( D ) of mean ( MFI ) of IL - 17F in γδ T after IMQ - induced psoriasis .

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 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 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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 of lymph node γδ T in STAT4 − / − ( − / − ) littermate control mice ( + / − ) . Frequency of IL - 22 + γδ T before ( steady - state ) after ( IMQ ) IMQ - induced psoriasis .

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 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

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

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

O O O B-MEASURED_VAR 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 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 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

(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 for SP + versus SP - synapses in UT TTX - treated neurons . AMPAR synaptic 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 number of synapses , from 2 cultures ) . * * P < 0 . 01 , * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data are represented as mean ± .

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

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 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 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

(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 of SP in lysates from COS - 7 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 GFP signals illustrate equal loading SP - shRNA expression level , respectively . ( B ) of SP expression from 6 different experiments , normalized to β - actin signal . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data represent mean ± .

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

(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 ) 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 for same conditions ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates number of , from 3 cultures ; normalized to empty vector condition ) . * * P < 0 . 01 , * * * * P < 0 . 0001 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . ( E ) Fraction of SP + synapses for same conditions . ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates number of from 3 cultures ) . * P < 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data represent mean ± .

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 B-CONTROLLED_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 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 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-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

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

O O O B-MEASURED_VAR 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 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

(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 ) showing Homer1c - GFP ( green ) immunostaining for surface AMPARs ( blue ) endogenous SP ( red ) in neurons transfected with miR - 124 or control miR - 67 ( miR - Ctrl ) , treated with TTX , or left untreated ( UT ) . Scale bar : 5 µ ( B ) 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 number of , from 3 cultures ) . * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . ( C ) Homer1c - GFP 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 number of , from 3 cultures ) . ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . ( D ) AMPAR synaptic 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 number of , from 3 cultures ) . * * * P < 0 . 001 , * P < 0 . 01 , ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . Data information : Data are represented as mean ± .

O O O O B-MEASURED_VAR 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 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 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 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 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

(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 ) showing dendrites from neurons transfected with Homer1c - DsRed ( magenta ) SEP - GluA2 constructs containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( green ) treated with TTX for 48 h , or left untreated ( UT ) . Scale bar : 10 µm . ( B ) SEP - GluA2 synaptic 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 number of , from 3 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis followed by Dunn ' s multiple comparison ) . Data information : Data are represented as mean ± .

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 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 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

(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 ) showing dendrites from neurons transfected with Homer1c - BFP ( magenta ) , GFP - SP - shRNA ( gray ) a rescue RFP - SP construct containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( geen ) treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( D ) 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 number of , from 3 cultures ) . * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant , P > 0 . 05 ( two - way ANOVA followed by Tukey ' s multiple comparison ) . Data information : Data are represented as mean ± .

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 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 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

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

O O O O B-MEASURED_VAR 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 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

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

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

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 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

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

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 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 B-MEASURED_VAR 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

(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 showing dendrites from CA3 pyramidal 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 ( n = 5 ) . Each plot represents a single spine for which SP integrated has been normalized to average SP of SP + spines in same neuron . Plots for SP - SP + spines appear in grey magenta , respectively . black dotted 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 - Student t ) . Data information : Data are represented as mean ± .

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(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 BFP - shRNA - SP ( grey ) contacted by GFP - Syp + terminals ( green ) . Arrowheads indicate putative synaptic contacts . Scale bar : 5 μm . ( G ) of SP + spines in neurons expressing RFP - SP - 3 ' UTR - WT vs - MUT depending on 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 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 number of spines from 7 pairs , 7 slices ) . * * * P < 0 . 001 , * P < 0 . 05 , ns , not significant ( Kruskall - Wallis followed by Dunn ' s multiple ) . ( I ) Cumulative probability distributions of spine head volumes for TetTx - TetTx + spines from neurons expressing RFP - SP - 3 ' UTR - WT ( SP - WT , gray green ) or RFP - SP - 3 ' UTR - MUT ( SP - MUT , gray green ) . Data information : Data are represented as mean ± .

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(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 interactions transmigration of neutrophils ( Ly - 6G + CX3CR - 1 - ; red ) cMOs ( Ly - 6G - CX3CR - 1low ; green ) to perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA , PAI - 1 , or uPA - PAI - 1 in postcapillary venules of cremaster of CX3CR - 1GFP / + mice by multi - channel in vivo microscopy . Representative still ( scale bar : 50 : µm ) quantitative data are shown ( mean ± 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 ) .

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

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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 of LFA - 1 / CD11a clustering on surface of mouse neutrophils under increasing flow conditions in vitro in 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 quantitative data are shown ( scale bar : 10 μm ; mean ± SD ; n = 3 mice ; n = 10 - 13 flow chambers , n = 122 - 222 ) ; * 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 expression of uPA ( PLAU gene ) , PAI - 1 ( SERPINE1 gene ) , neutrophil marker gene FPR1 , overall survival of breast cancer ( stages 0 1 ) with high low PLAU or SERPINE1 gene expression ( cut - off z ≥ 2 . 0 ) in 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 , NE as assessed on circulating neutrophils isolated from peripheral blood of WT mice ( saline ) or from 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 ± for n = 4 - 6 mice per group ; # p < 0 . 05 vs . saline ; t ) .

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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 cremaster muscle of WT mice 6 h after intra - scrotal injection of uPA - PAI - 1 , TNF , or saline , PECAM - 1 / CD31 + postcapillary venules ( blue ) Ly - 6G + neutrophils ( red ) are depicted . Representative 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 as assessed by ELISA , quantitative data are shown ( mean ± 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 in treated with WX - 340 a priori or therapeutically after 1 week after injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice ( mean ± 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 4T1 in lungs brain of WT mice receiving WX - 340 or vehicle therapeutically after 1 week after injection on a daily basis as assessed 14 days after intravenous injection of 4T1 by multi - channel flow cytometry , quantitative data are shown ( mean ± for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; t ) .

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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 ) , hypoxia marker CA9 ( green ) , marker SOX2 ( gray ) in human GBMs . Arrows indicate USP33 + / CA9 + / SOX2 + . 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 ) hypoxic 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 were sacrificed 2 hours post injection . Hypoxic were labeled with 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 of USP33 , HIF2α HIF1α in GSCs , NSTCs NPCs in normoxic hypoxic conditions . Hypoxia ( 5 % O2 ) relative to normoxia ( 20 % O2 ) clearly induced USP33 HIF2α expression in GSCs but not NSTCs or NPCs . mild hypoxia of 5 % O2 showed negligible induction of HIF1α in .

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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 of immunofluorescent analyses of USP33 ( red ) HIF2α ( green ) in human GBMs . Frozen sections of human GBMs were immunostained with antibodies against USP33 HIF2α , 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 of ectopic HIF2α in 293T with gradually increased amount of ectopic USP33 . 0 . 5 µg of HA - tagged HIF2α plasmids together with increased amounts of GFP - tagged USP33 plasmids ( 0 , 0 . 1 , 0 . 2 or 0 . 3 μg ) were introduced into 293T . Forty - eight hours post transfection , of ectopic HIF2α - HA GFP - USP33 were analyzed by immunoblot . Concomitantly increased of ectopic HIF2α USP33 expression indicated positive regulation of HIF2α by USP33 . G Immunoblot of ectopic HIF2α in 293T expressing ectopic wild type ( WT ) USP33 or catalytically dead ( CD ) USP33 . HA - tagged HIF2α plasmids ( 0 . 5 µg ) were introduced into 293T alone or together with GFP - tagged WT or CD USP33 plasmids ( 0 . 2 µg ) . of ectopic HIF2α - HA GFP - USP33 were analyzed 48 hours post transfection . Expression of wild type USP33 but not catalytically dead USP33 increased ectopic HIF2α .

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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 interaction between endogenous HIF2α USP33 in GSCs in response to low oxygen . GSCs were cultured under 20 % or 5 % oxygen for 24 hours before harvest for Co - IP . lysate was subjected to immunoprecipitation with anti - HIF2α antibodies . Co - immunoprecipitated products were immunoblotted with indicated antibodies . An increased interaction between HIF2α 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 interaction between endogenous HIF2α USP33 in GSCs in response to CoCl2 treatment . GSCs were treated with CoCl2 ( 300 µM ) for 12 hours before harvest for Co - IP . lysate was subjected to immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with indicated antibodies . An increased interaction between HIF2α 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 interaction between HIF2α wild - type ( WT ) or catalytically dead ( CD ) USP33 . Ectopic HA - tagged HIF2α were expressed in 293T with GFP - tagged wild - type or catalytically dead USP33 . lysates were subjected to immunoprecipitation with anti - HA antibodies . Co - immunoprecipitated products immunoblotted with indicated antibodies . catalytically dead USP33 relative to 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 of 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 , 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 interaction between endogenous HIF2α USP33 in GSCs after inhibition of ERK1 / 2 activity . GSCs were cultured under 20 % or 5 % oxygen for 24 hours , then treated with MEK inhibitor U0126 ( 10µM ) for 30 minutes to inhibit ERK1 / 2 activity , harvested for immunoprecipitation with anti - USP33 antibodies . Co - immunoprecipitated products were immunoblotted with indicated antibodies . Relative to 20 % oxygen treatment , 5 % oxygen treatment promoted interaction between HIF2α USP33 in GSCs . However , inhibition of ERK1 / 2 activity by U0126 treatment suppressed interaction between HIF2α 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 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 . lysates were subjected to immunoprecipitation with anti - HA agaroses Co - immunoprecipitated products were immunoblotted with indicated antibodies . S484A mutation largely attenuated interaction of HIF2α with USP33 , indicating that serine 484 site in HIF2α was most important phosphorylation site for interaction between HIF2α 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 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 density of 2 , 000 per well cultured for 48 hours under normoxia ( 20 % O2 ) or hypoxia ( 5 % O2 ) . Disruption of USP33 inhibited GSC tumorsphere formation in normoxia 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 titer assays showing 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 , then split into 96 well plates at of 2 , 000 per well . titer was determined by Glo luminescent viability kit ( Promega ) at indicated time points . Disruption of USP33 inhibited GSC growth in normoxia hypoxia . experiment has been repeated three times . presented curves were on three technical replicates . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ( mean ± s . e . m . ; two unpaired t - ) .

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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 of apoptotic marker 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 of 50 , 000 per well . Forty - eight hours post infection with shUSP33 or shNT lentiviruses , were stained for caspase 3 ( green ) 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 of autonomous 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 of autonomous signals of 4HRE - d2GFP ( green ) immunofluorescent of marker SOX2 ( red ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . More GFP signals were detected in SOX2 + compared with SOX2 - , 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 of immunofluorescent of apoptotic marker caspase 3 ( red ) autonomous signals of 4HRE - d2GFP ( green ) in xenografts derived from T387 GSCs transduced with 4HRE - d2GFP . GFP + population had much less apoptosis relative to GFP - population , suggesting that hypoxia response inhibited 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 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 - log - rank ) .

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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).


E , F Immunofluorescent ( E ) statistical quantifications ( F ) of proliferation marker Ki67 in xenografts derived from T387 GSCs expressing shUSP33 or shNT . Disruption of USP33 in GSCs reduced proliferation in GBM xenografts . Scale bar , 80 μm . ( n = 5 tumors for each group ; * * * , p < 0 . 001 ; mean ± s . e . m . ; two unpaired t - ) .

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E, F Immunofluorescent analysis (E) and statistical quantifications (F) of the cell proliferation marker Ki67 in xenografts derived from T387 GSCs expressing shUSP33 or shNT. Disruption of USP33 in GSCs reduced cell proliferation in GBM xenografts. Scale bar, 80 μm. (n = 5 tumors for each group; ***, p < 0.001; mean ± s.e.m.; two tailed unpaired t-test).


A . HeLa released from double thymidine synchronization in early S phase were treated with 5 - azadC for 30 min , washed collected at indicated times . Soluble - enriched fractions were immunoblotted with indicated antibodies .

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A. HeLa cells released from double thymidine synchronization in early S phase were treated with 5-azadC for 30 min, washed and collected at the indicated times. Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies.


E . HeLa / GFP - DNMT1 transfected with indicated siRNAs were processed as in ( D ) .

E. HeLa/GFP-DNMT1 cells transfected with indicated siRNAs were processed as in (D).


H . Representative of U2OS transfected with indicated siRNAs followed by mCherry - RNF4 expression plasmid . were treated with 5 - azadC in presence or absence of SUMOi , fixed 2 h later , pre - extracted co - immunostained with DNMT1 SUMO2 / 3 antibodies . Scale bar , 5 μm .

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H. Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry-RNF4 expression plasmid. Cells were treated with 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and SUMO2/3 antibodies. Scale bar, 5 μm.


J . GFP - tagged DNMT1 from extracts of HeLa / GFP - DNMT1 treated or not with 5 - azadC was immobilized on GFP - Trap agarose , subjected to stringent washing to remove non - covalently bound to GFP - DNMT1 incubated with recombinant HA - ubiquitin , E1 E2 ( UbcH5a ) enzymes RNF4 ( STUbL reaction ) at 37 ° C for 1 h . Samples were then subjected to immunoblotting to for RNF4 - dependent STUbL activity towards GFP - DNMT1 .

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J. GFP-tagged DNMT1 from extracts of HeLa/GFP-DNMT1 cells treated or not with 5-azadC was immobilized on GFP-Trap agarose, subjected to stringent washing to remove proteins non-covalently bound to GFP-DNMT1 and incubated with recombinant HA-ubiquitin, E1 and E2 (UbcH5a) enzymes and RNF4 proteins (STUbL reaction) at 37 °C for 1 h. Samples were then subjected to immunoblotting to assay for RNF4-dependent STUbL activity towards GFP-DNMT1.


A . M . HpaII was crosslinked into a plasmid to generate p4xDPC then incubated in nucleoplasmic egg extracts ( NPE ) in presence or absence of 50 μM of SUMOi . DPC pull - down under stringent conditions was performed at indicated time points recovered samples were immunoblotted for crosslinked M . HpaII ( Larsen et al , 2019 ) . Note that p4xDPC , which contains four M . HpaII DPCs , is sometimes used to increase sensitivity towards modified M . HpaII species . Identical results were obtained with plasmids containing one or four DPCs .

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A. M.HpaII was crosslinked into a plasmid to generate p4xDPC and then incubated in nucleoplasmic egg extracts (NPE) in the presence or absence of 50 μM of SUMOi. DPC pull-down under stringent conditions was performed at the indicated time points and the recovered samples were immunoblotted for crosslinked M.HpaII (Larsen et al, 2019). Note that p4xDPC, which contains four M.HpaII DPCs, is sometimes used to increase sensitivity towards modified M.HpaII species. Identical results were obtained with plasmids containing one or four DPCs.


B . p4xDPC or p4xDPCSUMO ( generated by crosslinking SUMO ∆ GG - M . HpaII ) were incubated in NPE DPC pull - down performed as in ( A ) . Note that priming M . HpaII with SUMO stimulates rapid poly - SUMOylation of DPC in NPE . Thus , this substrate was frequently used in subsequent experiments to stimulate DPC poly - SUMOylation .

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B. p4xDPC or p4xDPCSUMO (generated by crosslinking SUMO∆GG-M.HpaII) were incubated in NPE and DPC pull-down performed as in (A). Note that priming M.HpaII with SUMO stimulates rapid poly-SUMOylation of the DPC in NPE. Thus, this substrate was frequently used in subsequent experiments to stimulate DPC poly-SUMOylation.


C . p4xDPCSUMO was incubated in NPE plasmids analyzed as in ( A ) . Following plasmid pull - down , samples were split either left untreated or treated with SUMO protease Ulp1 .

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C. p4xDPCSUMO was incubated in NPE and plasmids analyzed as in (A). Following plasmid pull-down, the samples were split and either left untreated or treated with the SUMO protease Ulp1.


I . Representative of U2OS transfected with Myc - PIAS4 expression plasmid that were left untreated or exposed to 5 - azadC in presence or absence of SUMOi , fixed 2 h later , pre - extracted co - immunostained with DNMT1 Myc antibodies . Scale bar , 5 μm .

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I. Representative images of U2OS cells transfected with Myc-PIAS4 expression plasmid that were left untreated or exposed to 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and Myc antibodies. Scale bar, 5 μm.


J . HeLa or HeLa / GFP - DNMT1 left untreated or exposed to 5 - azadC for 30 min were lysed subjected to GFP IP under stringent conditions . After extensive washing , IPs were incubated with an equal amount ( 800 μg ) of whole lysate of HeLa transfected with Myc - PIAS4 expression construct ( Fig . EV2J ) , washed immunoblotted with antibodies to Myc , SUMO2 / 3 GFP . * , cross - reactive band .

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J. HeLa or HeLa/GFP-DNMT1 cells left untreated or exposed to 5-azadC for 30 min were lysed and subjected to GFP IP under stringent conditions. After extensive washing, individual IPs were incubated with an equal amount (800 μg) of whole cell lysate of HeLa cells transfected with Myc-PIAS4 expression construct (Fig. EV2J), washed and immunoblotted with antibodies to Myc, SUMO2/3 and GFP. *, cross-reactive band.


K . HeLa / GFP - DNMT1 transfected with previously validated siRNAs targeting established SUMO E3 ligases ( Fig . EV2L Methods section ) were treated with 5 - azadC for 30 min , collected subjected to GFP immunoprecipitation under denaturing conditions , immunoblotted with antibodies to SUMO1 , SUMO2 / 3 , ubiquitin GFP .

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K. HeLa/GFP-DNMT1 cells transfected with previously validated siRNAs targeting established SUMO E3 ligases (Fig. EV2L and Methods section) were treated with 5-azadC for 30 min, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with antibodies to SUMO1, SUMO2/3, ubiquitin and GFP.


B . p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in presence [ α - 32P ] dATP . Where indicated , 50 μM of SUMOi was added to extracts . Reaction samples were analyzed by native agarose gel electrophoresis . RI , replication intermediates ; OC , open circular ; SC , supercoiled . Red arrowheads indicate OC molecules that have not yet undergone repair . C . Samples in ( B ) were recovered by stringent DPC pull - down immunoblotted for crosslinked M . HpaII .

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B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair. C. Samples in (B) were recovered by stringent DPC pull-down and immunoblotted for crosslinked M.HpaII.


B . p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in presence [ α - 32P ] dATP . Where indicated , 50 μM of SUMOi was added to extracts . Reaction samples were analyzed by native agarose gel electrophoresis . RI , replication intermediates ; OC , open circular ; SC , supercoiled . Red arrowheads indicate OC molecules that have not yet undergone repair .

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B. p2xDPCLeads or p2xDPCSUMOLeads were replicated in egg extracts in the presence [α-32P]dATP. Where indicated, 50 μM of SUMOi was added to the extracts. Reaction samples were analyzed by native agarose gel electrophoresis. RI, replication intermediates; OC, open circular; SC, supercoiled. Red arrowheads indicate OC molecules that have not yet undergone repair.


J . As in ( I ) , but using RNF4 - depleted CSF - arrested extracts reconstituted with recombinant RNF4 WT or C130A from ( H ) .

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J. As in (I), but using RNF4-depleted CSF-arrested extracts reconstituted with recombinant RNF4 WT or C130A from (H).


L . p4xDPCSUMO was subjected to sequential addition of NPE CSF - arrested extract as in ( I ) . CSF - arrested extracts from ( K ) were supplemented with recombinant RNF4 , DPC plasmid was recovered by DPC pull - down at indicated time points immunoblotted against M . HpaII .

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L. p4xDPCSUMO was subjected to sequential addition of NPE and CSF-arrested extract as in (I). CSF-arrested extracts from (K) were supplemented with recombinant RNF4, and the DPC plasmid was recovered by DPC pull-down at the indicated time points and immunoblotted against M.HpaII.


D . HeLa transfected with indicated siRNAs synchronized in early S phase by double thymidine block were - labeled with 5 - azadC in late S phase as outlined in ( A ) . were then collected at indicated times after 5 - azadC withdrawal analyzed by flow cytometry . Data are representative of three independent experiments . Proportion of with G2 / M content is indicated .

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D. HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse-labeled with 5-azadC in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.


E . HeLa transfected with indicated siRNAs were treated with 5 - azadC / or SUMOi in late S phase as outlined in ( A ) . were then collected at indicated times after 5 - azadC withdrawal analyzed by flow cytometry . Data are representative of three independent experiments . Proportion of with G2 / M content is indicated .

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E. HeLa cells transfected with indicated siRNAs were treated with 5-azadC and/or SUMOi in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.


F . HeLa transfected with indicated siRNAs were - labeled or not with 5 - azadC for 30 min in late S phase according to experimental setup in ( A ) . Following 5 - azadC withdrawal were incubated with nocodazole , collected at indicated times immunoblotted with indicated antibodies .

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F. HeLa cells transfected with indicated siRNAs were pulse-labeled or not with 5-azadC for 30 min in late S phase according to the experimental setup in (A). Following 5-azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies.


H . Immunoblot of siRNA - treated HeLa that were exposed to indicated genotoxic agents collected 1 h later .

H. Immunoblot analysis of siRNA-treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.


B . Representative time - lapse sequences of mitotic progression in in ( A ) . NEBD corresponds to t = 0 . arrowheads indicate undergoing division . Scale bar , 10 μm .

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B. Representative time-lapse sequences of mitotic progression in cells in (A). NEBD corresponds to t=0. White arrowheads indicate cells undergoing division. Scale bar, 10 μm.


E . HeLa transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block . Six h after release from block , were - labeled with 5 - azadC for 30 min . Mitotic were isolated by shake - off 8 h later , washed incubated or not with MPS1 inhibitor ( MPS1i ) collected at indicated times . were then processed for immunoblotting with indicated antibodies .

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E. HeLa cells transfected with RNF4 siRNA were synchronized in early S phase by double thymidine block. Six h after release from the block, cells were pulse-labeled with 5-azadC for 30 min. Mitotic cells were isolated by shake-off 8 h later, washed and incubated or not with MPS1 inhibitor (MPS1i) and collected at the indicated times. Cells were then processed for immunoblotting with indicated antibodies.


( A - B ) Spinal cord sections from 5 - month - old WT Tmem106b - / - mice were stained with anti - neurofascin , anti - LAMP1 anti - Cath D antibodies . Accumulation of LAMP1 CathD positive vacuoles at distal end of AIS was observed in Tmem106b - / - motor neurons . Representative from three independent mice were shown . Scale bar = 10 µm . n = 3 . Data presented as mean ± . Unpaired Student ' s t . * * * * , p < 0 . 0001 .

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

(A-B) Spinal cord sections from 5-month-old WT and Tmem106b-/- mice were stained with anti- neurofascin, anti- LAMP1 and anti-Cath D antibodies. Accumulation of LAMP1 and CathD positive vacuoles at the distal end of AIS was observed in Tmem106b-/- motor neurons. Representative images from three independent mice were shown. Scale bar = 10 µm. n=3. Data presented as mean ± SEM. Unpaired Student's t test. ****, p<0.0001.


( C - F ) Western blot of p62 , ubiquitin ( Ub ) , TDP - 43 p - TDP - 43 in RIPA - urea - soluble fractions from brain ( C E ) spinal cord ( D F ) of 16 - month - old WT Tmem106b - / - mice . n = 5 . Data presented as mean ± . Unpaired Student ' s t . * , p < 0 . 05 , * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

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

(C-F) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from brain (C and E) and spinal cord (D and F) of 16-month-old WT and Tmem106b-/- mice. n=5. Data presented as mean ± SEM. Unpaired Student's t test. *, p<0.05, ***, p<0.001, ****, p<0.0001.