( C ) S1P2 expression in S1P2 knockout HBMEC using CRISPR / Cas9 ( left panel ) . Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC . From left to right , p value of K79 is 0 . 0018 , p value of K160 is 0 . 0091 , p value of K161 is 0 . 0054 , p value of K181 is 0 . 00040 , p value of K226 is 0 . 00032 , p value of K237 is 0 . 040 , p value of K238 is 0 . 041 p value of P539 is 0 . 0025 .

O O O B-MEASURED_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-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

(C) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR/Cas9 (left panel). Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC. From left to right, p value of K79 is 0.0018, p value of K160 is 0.0091, p value of K161 is 0.0054, p value of K181 is 0.00040, p value of K226 is 0.00032, p value of K237 is 0.040, p value of K238 is 0.041 and p value of P539 is 0.0025.


( D ) GBS K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control .

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

(D) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control.


( E ) recovered from blood brain of wild type ( n = 5 ) S1P2 - / - mice ( n = 5 ) infected with K79 for 1h .

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

(E) Bacterial counts recovered from the blood and brain of wild type (n = 5) and S1P2-/- mice (n = 5) infected with strain K79 for 1h.


( A ) Serine phosphorylation of cPLA2α in response to GBS K79 in HBMEC pretreated with gefitinib or vehicle control in EGFR knockout HBMEC .

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

(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC.


( B ) Serine phosphorylation of cPLA2α in response to GBS K79 in control , EGFR knockout S1P2 knockout HBMEC .

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

(B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC.


( C ) recovered from blood brain in wild type mice ( n = 5 ) CysLT2 - / - mice ( n = 5 ) infected with K79 for 1h .

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) Bacterial counts recovered from the blood and brain in wild type mice (n = 5) and CysLT2-/- mice (n = 5) infected with strain K79 for 1h.


( D ) CysLT1 expression in CysLT1 knockdown HBMEC using shRNA ( left panel ) relative invasion frequency of 8 GBS isolates in CysLT1 knockdown control HBMEC . t - . From left to right , p value of K79 is 0 . 0039 , p value of K160 is 0 . 0082 , p value of K161 is 0 . 00091 , p value of K181 is 0 . 0087 , p value of K226 is 0 . 00020 , p value of K237 is 0 . 0039 , p value of K238 is 0 . 00066 , p value of P539 is 1 . 76E - 05 ( right panel ) .

O O O B-MEASURED_VAR O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA (left panel) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC. t-test. From left to right, p value of K79 is 0.0039, p value of K160 is 0.0082, p value of K161 is 0.00091, p value of K181 is 0.0087, p value of K226 is 0.00020, p value of K237 is 0.0039, p value of K238 is 0.00066, p value of P539 is 1.76E-05 (right panel).


( E ) GBS K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control .

(E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control.


( F ) recovered from blood brain in wild type mice ( n = 7 ) CysLT1 - / - mice ( n = 7 ) infected with K79 for 1h .

(F) Bacterial counts recovered from the blood and brain in wild type mice (n = 7) and CysLT1-/- mice (n = 7) infected with strain K79 for 1h.


( G ) Ezrin expression in ezrin knockdown HBMEC using shRNA ( left panel ) relative invasion frequency of GBS K79 in ezrin knockdown control HBMEC ( right panel ) .

O O O B-MEASURED_VAR O O B-CONTROLLED_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

(G) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel).


( H Ezrin phosphorylation in HBMEC pre - treated with CysLT1 antagonist ( montelukast ) infected with GBS K79 for 60 min ( left panel )

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

(H Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel)


I ) ezrin phosphorylation in homogenates of brain capillaries of CysLT1 - / - control mice infected with GBS K79 for 1h ( right panel ) .

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

I) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1-/- and control mice infected with GBS strain K79 for 1h (right panel).


( J ) Intracellular K79 co - localization with EGFR ezrin in control S1P2 knockout HBMEC ( as shown by arrows , cyan ) . of co - localization was calculated by counting numbers of GBS co - localized intracellular GBS from at least three representative fields , expressing as numbers of co - localized GBS / GBS x 100 .

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

(J) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100.


( A ) Survival of wild type mice receiving drug administration for 7 days after infection with GBS K79 . Data are presented as a Kaplan - Maier plot with a log rank used to compare of survival between groups , CFX group n = 18 , CFX + MONT group n = 10 * p = 0 . 015 . ( CFX : ceftriaxone ; MONT : montelukast ) .

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

(A) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79. Data are presented as a Kaplan-Maier plot with a log rank test used to compare percentage of survival between the groups, CFX group n = 18, and CFX + MONT group n = 10*p = 0.015. (CFX: ceftriaxone; MONT: montelukast).


( B ) Nissl of hippocampus CA1 , CA2 & CA3 regions ( low magnification ) cortex region ( higher magnification ) ( left ) histograms showing number of Nissl - stained bodies ( right ) Scale bar , 50 μm . Data represent means ± from three independent experiments , with statistical by One - way ANOVA , p value between control group Ceftriaxone group is 0 . 00021 , p value between Ceftriaxone group Ceftriaxone + Montelukast group is 0 . 013 .

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

(B) Nissl staining of the hippocampus CA1, CA2 & CA3 regions (low magnification) and cortex region (higher magnification) (left) and histograms showing the number of Nissl-stained bodies (right) Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value between control group and Ceftriaxone group is 0.00021, p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0.013.


( C - D ) Representative immunofluorescence ( left ) ( right ) of TUNEL bodies , astrocyte ( GFAP ) , microglia ( Iba1 ) in cortex of wild type mice after 7 days of therapy . Scale bar , 50 μm . Data represent means ± from three independent experiments , with statistical by One - way ANOVA , p value of TUNEL bodies between CFX group MONT + CFX group is 0 . 0012 , p value of astrocyte between CFX group MONT + CFX group is 0 . 0068 , p value of microglia between CFX group MONT + CFX group is 0 . 0029 . ( E ) Y - maze for spatial learning memory of different group of wild type mice . total duration of time spent at novel arm ( left ) of entries to novel arm of Y - maze ( right ) . Data represent means ± was statistically analyzed by One - way ANOVA , p value of total duration of time spent at novel arm between control group ( n = 6 ) CFX group ( n = 5 ) is 0 . 00012 , p value between CFX group ( n = 5 ) MONT + CFX group ( n = 6 ) is 0 . 00017 ; p value of total duration of % of entry to novel arm between control group ( n = 6 ) CFX group ( n = 5 ) is 0 . 0102 , p value between CFX group ( n = 5 ) MONT + CFX group ( n = 6 ) is 0 . 0216 .

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

(C-D) Representative immunofluorescence images (left) and quantification (right) of TUNEL bodies, astrocyte (GFAP), microglia (Iba1) in cortex of wild type mice after 7 days of therapy. Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value of TUNEL bodies between CFX group and MONT + CFX group is 0.0012, p value of astrocyte between CFX group and MONT + CFX group is 0.0068, and p value of microglia cells between CFX group and MONT + CFX group is 0.0029. (E) Y-maze test for spatial learning and memory of different group of wild type mice. The total duration of time spent at novel arm (left) and the percentage of entries to novel arm of Y-maze (right). Data represent the means ± SEM was statistically analyzed by One-way ANOVA, p value of total duration of time spent at novel arm between control group (n = 6) and CFX group (n = 5) is 0.00012, p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.00017; p value of total duration of % of entry to novel arm between control group (n = 6) and CFX group (n = 5) is 0.0102, and p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.0216.


Wip1 represses gene responses induced by Nodal or BMP signaling in caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in absence of reverse transcriptase . WE , whole . Co AC , uninjected control caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , Xnr1 ( 50 pg )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), Xnr1 (50 pg)


Wip1 represses gene responses induced by Nodal or BMP signaling in caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in absence of reverse transcriptase . WE , whole . Co AC , uninjected control caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , BMP4 ( 100 pg ) .

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), BMP4 (100 pg).


Wip1 represses gene responses induced by Nodal or BMP signaling in caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in absence of reverse transcriptase . WE , whole . Co AC , uninjected control caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng )

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

Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng)


( D wt Wip1 , but not Wip1 ( D277A ) , down - regulates activity of Activin - or BMP - responsive promoters in HEK293T . FAST1 , were co - transfected as indicated for efficient activation of promoters . Data are expressed as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

O O O B-CONTROLLED_VAR 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 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

(D wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. FAST1, were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


E ) wt Wip1 , but not Wip1 ( D277A ) , down - regulates activity of Activin - or BMP - responsive promoters in HEK293T . Smad1 Smad4 were co - transfected as indicated for efficient activation of promoters . Data are expressed as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

O O O B-CONTROLLED_VAR 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-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

E) wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. Smad1 and Smad4 were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( F - Q ) Dorsal ( F - N ) or ventral ( O - Q ) injection of wt Wip1 abrogates in vivo expression of mesodermal markers at stage 10 . 25 as analyzed by in situ hybridization . are shown in vegetal views with dorsal to top . Arrowheads denote absence of expression of mesodermal markers in injected region of . Control , an injected with LacZ only . amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) LacZ ( 100 pg ) . Scale bar , 150 μm .

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 B-CONTROLLED_VAR 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

(F-Q) Dorsal (F-N) or ventral (O-Q) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10.25 as analyzed by in situ hybridization. Embryos are shown in vegetal views with dorsal to the top. Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos. Control, an embryo injected with LacZ only. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng) and LacZ (100 pg). Scale bar, 150 μm.


( A , Nodal - induced gene responses are enhanced further in absence of Wip1 as analyzed by RT - PCR . caps ere treated with - ) , no injection of Co MO , Wip1 MO / or Wip1 mRNA . dose of reagent injected : Xnr1 ( 5 pg ) , Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) Wip1 ( 1 ng ) .

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O

(A, Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps ere treated with -), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Xnr1 (5 pg), Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


B ) Activin / Nodal - induced gene responses are enhanced further in absence of Wip1 as analyzed by RT - PCR . caps in ( B ) were treated with Activin ( 5 ng / ml ) . ( - ) , no injection of Co MO , Wip1 MO / or Wip1 mRNA . dose of reagent injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) Wip1 ( 1 ng ) .

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

B) Activin/Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps in (B) were treated with Activin protein (5 ng/ml). (-), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).


( C ) Knockdown of Wip1 up - regulates activity of Activin / Nodal - responsive promoter . HEK293T transfected with Co siRNA ( 50 nM ) or Wip1 siRNA ( 50 nM ) were stimulated with Activin ( 10 ng / ml ) for 10 h then subjected to luciferase assays . Data are expressed as mean ± ( n = 3 biological replicates ) . * * * P < 0 . 001 by unpaired Student ' s t - .

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

(C) Knockdown of Wip1 up-regulates the activity of Activin/Nodal-responsive promoter. HEK293T cells transfected with Co siRNA (50 nM) or Wip1 siRNA (50 nM) were stimulated with Activin protein (10 ng/ml) for 10 h and then subjected to luciferase assays. Data are expressed as the mean ± SEM (n=3 biological replicates). ***P<0.001 by unpaired Student's t-test.


Depletion of Wip1 promotes epidermal differentiation at expense of neural fate . ( - ) , no injection of Noggin mRNA . amount of MO mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G J ; 30 ng for F I ) Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

O O B-CONTROLLED_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 B-CONTROLLED_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

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. (-), no injection of Noggin mRNA. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


Depletion of Wip1 promotes epidermal differentiation at expense of neural fate . in ( E - J ) are shown in dorsal views with anterior to top . amount of MO mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G J ; 30 ng for F I ) Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm .

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 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

Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. Embryos in (E-J) are shown in dorsal views with anterior to the top. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.


( K , Activin induction of p21 is up - regulated upon Wip1 knockdown . HEK293T were transfected with Co siRNA or Wip1 siRNA 48 h after siRNA transfection , were stimulated with Activin ( 10 ng / ml ) for 3 to 12 h as indicated t = 0 in ( K ) is 48 h after siRNA transfection . P - Smad2 serves as an indicator of active Activin / Nodal signaling . Arrows ndicate non - specific bands .

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

(K, Activin induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with Activin (10 ng/ml) for 3 to 12 h as indicated t=0 in (K) is 48 h after siRNA transfection. P-Smad2 serves as an indicator of active Activin/Nodal signaling. Arrows ndicate non-specific bands.


L ) TGF - β induction of p21 is up - regulated upon Wip1 knockdown . HEK293T were transfected with Co siRNA or Wip1 siRNA 48 h after siRNA transfection , were stimulated with increasing of TGF - β1 ( 10 , 20 ng / ml ) for 3 h .

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

L) TGF-β induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with increasing concentration of TGF-β1 (10, 20 ng/ml) for 3 h.


( A , Effects of gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T were transfected or not with wt Wip1 , Wip1 ( D277A ) , as indicated subjected to CCK - 8 assays before ( 0 h ) after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - . n . s . , not significant .

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(A, Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with wt Wip1, Wip1(D277A), as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


B ) Effects of gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T were transfected or not with Co siRNA or Wip1 siRNA as indicated subjected to CCK - 8 assays before ( 0 h ) after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - . n . s . , not significant .

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B) Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.


Depletion of Wip1 augments TGF - β - induced migration invasion of MDA - MB231 breast cancer . MDA - MB231 transfected with Co siRNA or Wip1 siRNA were scratched with a 20 - μL pipette tip then incubated in absence or presence of TGF - β1 for indicated times ( C , D )

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Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20-μL pipette tip and then incubated in the absence or presence of TGF-β1 for the indicated times (C, D)


Depletion of Wip1 augments TGF - β - induced migration invasion of MDA - MB231 breast cancer . MDA - MB231 transfected with Co siRNA or Wip1 siRNA were subjected to transwell with or without TGF - β1 for 20 h ( E , F ) .

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Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF-β1 for 20 h (E, F).


( A - D ) Knockdown of Wip1 expands expression of dorsal mesodermal markers . injected dorsally with Co MO ( 40 ng ) or Wip1 MO ( 40 ng ) were subjected to in situ hybridization at stage 10 . 25 . are shown in dorso - vegetal views with dorsal to top . Scale bar , 150 μm .

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(A-D) Knockdown of Wip1 expands the expression of dorsal mesodermal markers. Embryos injected dorsally with Co MO (40 ng) or Wip1 MO (40 ng) were subjected to in situ hybridization at stage 10.25. Embryos are shown in dorso-vegetal views with dorsal to the top. Scale bar, 150 μm.


( E - I ) Morphological of Wip1 morphants . were injected radially in marginal zone with Co MO ( 80 ng ) , Wip1 MO ( 80 ng ) , wt Wip1 ( 1 ng ) Wip1 ( D277A ) mRNA as indicated cultured to tadpole stages . are shown in lateral views with anterior to left . Un Co , uninjected control . Scale bar , 1 mm . ( J ) of shown in ( E - I ) . Severe defects include microcephaly , shortened kinked axis , no eye . Mild defects indicate normal axis with malformed eyes .

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(E-I) Morphological phenotypes of Wip1 morphants. Embryos were injected radially in the marginal zone with Co MO (80 ng), Wip1 MO (80 ng), wt Wip1 (1 ng) and Wip1(D277A) mRNA as indicated and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Un Co, uninjected control. Scale bar, 1 mm. (J) Quantification of the phenotypes shown in (E-I). Severe defects include microcephaly, shortened and kinked body axis, and no eye. Mild defects indicate normal body axis with malformed eyes.


( A Wip1 Smad4 form an endogenous . lysates of HEK293T , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) ( - ) , no treatment with ligand . IgG , control IgG . An arrow in ( A ) indicates non - specific bands .

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(A Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) (-), no treatment with protein ligand. IgG, control IgG. An arrow in (A) indicates non-specific bands.


B ) Wip1 Smad4 form an endogenous . lysates of HEK293T , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) vice versa ( B ) . ( - ) , no treatment with ligand . IgG , control IgG .

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B) Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) and vice versa (B). (-), no treatment with protein ligand. IgG, control IgG.


( C ) Upon ligand stimulation , Wip1 forms a with Smad4 in nucleus . HeLa were treated or not with TGF - β1 ( 40 ng / ml ) for 2 h , fixed then subjected to proximity ligation assays with antibodies against Wip1 Smad4 . Blue : DAPI ; Red : PLA signal . Scale bar , 20 μm . ( D ) Quantitative of Wip1 / Smad4 interactions shown in ( C ) . Data are represented as mean ± ( n = 3 biological replicates ) . Twenty were analyzed per sample . * * * P < 0 . 001 by Student ' s t - .

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(C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm. (D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test.


( E ) Wip1 interferes with ability of Smad4 to bind to Smad2 . HEK293T co - transfected with indicated constructs were treated or not with TGF - β1 for 1 h then harvested . An arrow denotes Myc - tagged Wip1 .

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(E) Wip1 interferes with the ability of Smad4 to bind to Smad2. HEK293T cells co-transfected with the indicated constructs were treated or not with TGF-β1 for 1 h and then harvested. An arrow denotes Myc-tagged Wip1 protein.


( F ) Wip1 has a negative effect on accumulation of Smad4 . HEK293T were transfected with Co siRNA or Wip1 siRNA , treated with TGF - β1 ( 40 ng / ml , 1 h ) 48 h after siRNA transfection subjected to / fractionation western blotting . β - tubulin lamin A / C serve as markers , respectively . C , cytoplasm ; N , nucleus . experiment was repeated two times with similar results . ( G ) of of Smad4 normalized to those of Smad4 shown in ( F ) .

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(F) Wip1 has a negative effect on nuclear accumulation of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA, treated with TGF-β1 (40 ng/ml, 1 h) 48 h after siRNA transfection and subjected to nuclear/cytoplasmic fractionation and western blotting analysis. β-tubulin and lamin A/C serve as cytoplasmic and nuclear markers, respectively. C, cytoplasm; N, nucleus. The experiment was repeated two times with similar results. (G) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in (F).


( A ) Overexpression of Wip1 down - regulates Smad4 phosphorylation at Thr277 . MOCK - or Flag - Wip1 - transfected HEK293T were treated with 1 h of FGF2 ( 10 ng / ml ) , followed by addition of U0126 ( 40 μM ) harvesting for western blotting at indicated times . - FGF2 , no treatment with FGF2 .

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(A) Overexpression of Wip1 down-regulates Smad4 phosphorylation at Thr277. MOCK- or Flag-Wip1-transfected HEK293T cells were treated with 1 h pulse of FGF2 (10 ng/ml), followed by addition of U0126 (40 μM) and harvesting for western blotting at the indicated times. - FGF2, no treatment with FGF2.


( B ) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control Wip1 - depleted HEK293T , showing that knockdown of Wip1 up - regulates Smad4 phosphorylation at MAPK site . A bracket denotes phospho - Smad4 .

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(B) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1-depleted HEK293T cells, showing that knockdown of Wip1 up-regulates Smad4 phosphorylation at the MAPK site. A bracket denotes phospho-Smad4.


( C ) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase . Myc - tagged Smad4 was expressed in HEK293T , which were then treated or not with FGF2 ( 40 ng / ml , 2 h ) , Myc - Smad4 was immunoprecipitated using anti - Myc antibody subsequently incubated with or without recombinant human Wip1 ( 0 . 5 μg ) in absence or presence of Mg2 + as indicated .

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(C) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay. Myc-tagged Smad4 was expressed in HEK293T cells, which were then treated or not with FGF2 (40 ng/ml, 2 h), and the Myc-Smad4 was immunoprecipitated using anti-Myc antibody and subsequently incubated with or without recombinant human Wip1 protein (0.5 μg) in the absence or presence of Mg2+ as indicated.


( D ) FGF - induced destabilization of Smad4 is enhanced further in Wip1 - depleted cap as analyzed by western blotting . amount of reagent injected : Myc - Smad4 ( 400 pg ) , FGF8 ( 100 pg ) , Wnt8 ( 400 pg ) , Co MO ( 60 ng ) Wip1 MO ( 60 ng ) .

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(D) FGF-induced destabilization of Smad4 is enhanced further in Wip1-depleted animal cap cells as analyzed by western blotting. The amount of reagent injected: Myc-Smad4 (400 pg), FGF8 (100 pg), Wnt8 (400 pg), Co MO (60 ng) and Wip1 MO (60 ng).


( E ) Polyubiquitination of wt Smad4 is promoted in Wip1 - silenced . HEK293T were transfected with indicated combinations of Flag - ubiquitin , Co siRNA , Wip1 siRNA , Myc - Smad4 Myc - Smad4 ( T277A ) then treated with FGF2 ( 10 ng / ml , 4 h ) in presence of proteasome inhibitor MG132 , Myc - Smad4 its phosphorylation - resistant mutant were immunoprecipitated with anti - Myc antibody . Ubiquitin - conjugated Smad4 ( Smad4 - Ubn ) was detected by western blotting with anti - Flag antibody .

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(E) Polyubiquitination of wt Smad4 is promoted in Wip1-silenced cells. HEK293T cells were transfected with the indicated combinations of Flag-ubiquitin, Co siRNA, Wip1 siRNA, Myc-Smad4 and Myc-Smad4(T277A) and then treated with FGF2 (10 ng/ml, 4 h) in the presence of the proteasome inhibitor MG132, and Myc-Smad4 and its phosphorylation-resistant mutant were immunoprecipitated with anti-Myc antibody. Ubiquitin-conjugated Smad4 (Smad4-Ubn) was detected by western blotting with anti-Flag antibody.


( F ) Wip1 prolongs half - life of Smad4 . HEK293T were transfected with Co siRNA or Wip1 siRNA then treated with FGF2 along with cycloheximide ( CHX , 100 ng / ml ) for indicated times harvested for western blotting . An arrow indicates non - specific bands .

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(F) Wip1 prolongs the half-life of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide (CHX, 100 ng/ml) for the indicated times and harvested for western blotting. An arrow indicates non-specific bands.


( A - J ) injected dorsally with wt Wip1 ( 1 ng ) , wt Smad4 ( 1 ng ) , Smad4 ( T277A ) ( 1 ng ) Smad4 ( T277E ) ( 1 ng ) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10 . 25 . Control , control injected with LacZ mRNA only . Scale bar , 150 μm . ( K ) of results from in situ hybridization in ( A - J ) .

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(A-J) Embryos injected dorsally with wt Wip1 (1 ng), wt Smad4 (1 ng), Smad4(T277A) (1 ng) and Smad4 (T277E) (1 ng) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10.25. Control, control embryo injected with LacZ mRNA only. Scale bar, 150 μm. (K) Quantification of the results from in situ hybridization analysis in (A-J).


( L ) Wip1 - mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho - mimetic acid at MAPK site . ( - ) , no co - injection of wt or mutated Smad4 .

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(L) Wip1-mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho-mimetic amino acid at the MAPK site. (-), no co-injection of wt or mutated Smad4.


( M ) MDA - MB231 MDA - MB468 were transfected with wt Wip1 alone or with wt Smad4 or Smad4 ( T277A ) then subjected to CCK - 8 assays to measure growth . Data are presented as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

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(M) MDA-MB231 and MDA-MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4(T277A) and then subjected to CCK-8 assays to measure cell growth. Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


( N ) Anchorage - independent colony - formation assays were performed in MDA - MB231 MDA - MB468 transfected with Wip1 or negative control constructs . Scale bar , 100 μm . ( O ) of assays shown in ( N ) . number of colonies larger than 50 μm in diameter was scored . Data are expressed as mean ± ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - .

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(N) Anchorage-independent colony-formation assays were performed in MDA-MB231 and MDA-MB468 cells transfected with Wip1 or negative control constructs. Scale bar, 100 μm. (O) Quantification of the assays shown in (N). The number of colonies larger than 50 μm in diameter was scored. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


a - e , Scanning electron microscopy ( ) of fly eyes expressing DTS7 with or without indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . a , Normal eyes in DTS7 flies reared at 22 ° C . b , Rough eyes in DTS7 flies reared at 28 ° C . c , d , Degeneration was suppressed by expression of Drosophila HDAC6 ( dHDAC6 ; c ) or human HDAC6 ( hHDAC6 ; d ) , but not a catalytically dead mutant of hHDAC6 ( cat . mut . ; e ) .

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a-e, Scanning electron microscopy (SEM) images of fly eyes expressing DTS7 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. a, Normal eyes in DTS7 flies reared at 22 °C. b, Rough eyes in DTS7 flies reared at 28 °C. c, d, Degeneration was suppressed by expression of Drosophila HDAC6 (dHDAC6; c) or human HDAC6 (hHDAC6; d), but not a catalytically dead mutant of hHDAC6 (cat. mut.; e).


f - j , of fly eyes expressing AR52 with or without indicated HDAC6 transgenes . Lower panels , magnification of ommatidia . f , Normal eyes in AR52 flies reared without ( - ) DHT . g , Rough eyes in AR52 flies reared with ( + ) DHT . Degeneration was suppressed by expression of Drosophila HDAC6 ( h ) or human HDAC6 ( i ) , but not a catalytically dead mutant of human HDAC6 ( j ) .

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f-j, SEM images of fly eyes expressing AR52 with or without the indicated HDAC6 transgenes. Lower panels, magnification of ommatidia. f, Normal eyes in AR52 flies reared without (-) DHT. g, Rough eyes in AR52 flies reared with (+) DHT. Degeneration was suppressed by expression of Drosophila HDAC6 (h) or human HDAC6 (i), but not a catalytically dead mutant of human HDAC6 (j).


k - p , Detection of UPS reporter in imaginal eye discs from third - instar larvae by confocal microscopy . High level was found in flies expressing GFP ( k , positive control ) , but was barely detectable in control flies expressing CL1 - GFP ( l , negative control ) . CL1 - GFP accumulates in DTS7flies with - dependent proteasome impairment ( compare m to n ) in AR52flies with ligand - dependent degeneration ( compare o to p ) . retinal of 200 to > 1 , 000 flies of each genotype were examined . Quantitative analyses of eye proteasome impairment are presented in Supplementary Figs S2 S3 , respectively . ( DHT , dihydrotestosterone . )

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k-p, Detection of UPS reporter in imaginal eye discs from third-instar larvae by confocal microscopy. High level fluorescence was found in flies expressing GFP (k, positive control), but fluorescence was barely detectable in control flies expressing CL1-GFP (l, negative control). CL1-GFP accumulates in DTS7flies with temperature-dependent proteasome impairment (compare m to n) and in AR52flies with ligand-dependent degeneration (compare o to p). The retinal phenotypes of 200 to >1,000 flies of each genotype were examined. Quantitative analyses of eye phenotypes and proteasome impairment are presented in Supplementary Figs S2 and S3, respectively. (DHT, dihydrotestosterone.)


a - e , Representative examples of autophagic vacuoles detected by in retinal sections used to generate quantitative data shown in ( f ) . a , An autophagosome ( red arrow ) containing contents in a photoreceptor neuron from an AR52fly reared on DHT . b , Higher magnification of autophagosome in a . c , Multiple autophagolysosomes ( red arrows ) containing dense , amorphous material from an AR52fly reared on DHT . d , A juxtanuclear multilamellar ( red arrow ) from a DTS7fly reared at 28 ° C . e , A multivesicular ( red arrow ) from a DTS7fly reared at 28 ° C . f , A significant increase in frequency of neurons with autophagic figures in DTS7 flies reared at 28 ° C compared to those reared at 22 ° C , in AR52 flies reared on DHT compared to those reared off DHT . Data show mean ± s . d . , n = 59 - 82 neurons in 5 sections per condition . No accumulation of autophagic figures was found in AR12 flies .

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

a-e, Representative examples of autophagic vacuoles detected by TEM in retinal sections used to generate the quantitative data shown in (f). a, An autophagosome (red arrow) containing cytoplasmic contents in a photoreceptor neuron from an AR52fly reared on DHT. b, Higher magnification of the autophagosome in a. c, Multiple autophagolysosomes (red arrows) containing dense, amorphous material from an AR52fly reared on DHT. d, A juxtanuclear multilamellar body (red arrow) from a DTS7fly reared at 28 °C. e, A multivesicular body (red arrow) from a DTS7fly reared at 28 °C. f, A significant increase in the frequency of neurons with autophagic figures in DTS7 flies reared at 28 °C compared to those reared at 22 °C, and in AR52 flies reared on DHT compared to those reared off DHT. Data show mean ± s.d., n = 59-82 neurons in 5 sections per condition. No accumulation of autophagic figures was found in AR12 flies.


g - l , of fly eyes expressing indicated transgenes . RNAi knockdown ( KD ) of atg6 atg12 enhances degeneration in DTS7 flies reared at 28 ° C ( compare h , i to g ) AR52 flies reared on DHT ( compare k , l to j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye are presented in Supplementary Fig . S2 . ( N , nucleus ; Rh , rhabdomere . )

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

g-l, SEM images of fly eyes expressing the indicated transgenes. RNAi knockdown (KD) of atg6 and atg12 enhances degeneration in DTS7 flies reared at 28 °C (compare h, i to g) and AR52 flies reared on DHT (compare k, l to j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2. (N, nucleus; Rh, rhabdomere.)


a , b , d , Western blots from flies expressing indicated transgenes . a , Steady state of AR52 are reduced in flies overexpressing Drosophila HDAC6 , but are elevated in flies in which atg6 or atg12 has been knocked down . b , Western blots showing temporal profile of AR52 monomer high molecular aggregate after a brief of expression . AR52 became detectable by 2 . 5 h after treatment with RU486 , reached a peak at 10 h , then slowly decayed . c , A logarithmic plot of AR52 / actin ratios was used to determine of best fit by regression ( y = Ae - Kx ) . R2 = 0 . 9117 ( AR52 - DHT ) , R2 = 0 . 7808 ( AR52 + DHT ) , R2 = 0 . 9719 ( HDAC6 + ( AR52 - DHT ) ) , R2 = 0 . 9644 ( HDAC6 + ( AR52 + DHT ) ) . Half - life was determined by slope of best fit with equation t1 / 2 = 0 . 693 / K . Half - life of AR52 in vivo was reduced ∼ 2 - fold in flies co - expressing Drosophila HDAC6 did not differ significantly in presence ( broken lines ) or absence ( solid lines ) of DHT . Plots of mean AR52 / actin ratios are shown in Supplementary Fig . S12 . d , Flies co - expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b , but AR decayed at an accelerated rate . Exogenous Drosophila HDAC6 was detected by immunoblot against V5 epitope .

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

a, b, d, Western blots from flies expressing the indicated transgenes. a, Steady state levels of AR52 protein are reduced in flies overexpressing Drosophila HDAC6, but are elevated in flies in which atg6 or atg12 has been knocked down. b, Western blots showing the temporal profile of AR52 protein monomer and high molecular weight aggregate levels after a brief pulse of expression. AR52 protein became detectable by 2.5 h after treatment with RU486, reached a peak at 10 h, and then slowly decayed. c, A logarithmic plot of AR52/actin ratios was used to determine the line of best fit by regression analysis (y = Ae-Kx). R2 = 0.9117 (AR52 - DHT), R2 = 0.7808 (AR52 + DHT), R2 = 0.9719 (HDAC6 + (AR52 - DHT)), R2 = 0.9644 (HDAC6 + (AR52 + DHT)). Half-life was determined by the slope of the best fit line with the equation t1/2 = 0.693/K. Half-life of AR52 in vivo was reduced ∼2-fold in flies co-expressing Drosophila HDAC6 and did not differ significantly in the presence (broken lines) or absence (solid lines) of DHT. Plots of the mean AR52/actin ratios are shown in Supplementary Fig. S12. d, Flies co-expressing Drosophila HDAC6 showed a nearly identical profile of induced expression as in b, but AR protein decayed at an accelerated rate. Exogenous Drosophila HDAC6 was detected by immunoblot against the V5 epitope.


a - l , of fly eyes expressing indicated transgenes . rough eye caused by proteasome mutation ( a ) or by expression of polyQ - expanded AR ( b ) were both suppressed by rearing flies on TOR inhibitor rapamycin ( c , d ) . e , f , Rapamycin failed to suppress degeneration in an autophagy - deficient background created by knockdown of atg12 , confirming that rescue by rapamycin is autophagy - dependent . g , h , Rapamycin also failed to suppress degeneration when HDAC6 were knocked down , demonstrating that suppression via TOR pathway is HDAC6 - dependent . k , j , HDAC6 failed to suppress degeneration in an autophagy - deficient background , confirming that rescue by HDAC6 is dependent on autophagy ( compare k , l to i , j ) . 200 to > 1 , 000 fly eyes of each genotype were examined . Quantitative analyses of eye are presented in Supplementary Fig . S2 .

O O O 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 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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

a-l, SEM images of fly eyes expressing the indicated transgenes. The rough eye phenotypes caused by proteasome mutation (a) or by expression of polyQ-expanded AR (b) were both suppressed by rearing flies on the TOR inhibitor rapamycin (c, d). e, f, Rapamycin failed to suppress degeneration in an autophagy-deficient background created by knockdown of atg12, confirming that rescue by rapamycin is autophagy-dependent. g, h, Rapamycin also failed to suppress degeneration when HDAC6 levels were knocked down, demonstrating that suppression via the TOR pathway is HDAC6-dependent. k, j, HDAC6 failed to suppress degeneration in an autophagy-deficient background, confirming that rescue by HDAC6 is dependent on autophagy (compare k, l to i, j). 200 to >1,000 fly eyes of each genotype were examined. Quantitative analyses of eye phenotypes are presented in Supplementary Fig. S2.


A - L HeLa stably expressing FLAG - tagged UNC13D ( F - UNC13D ) ( A B ) , UNC13D stable knock - down ( KD ) ( C , E , G , I K ) or knock out ( KO ) ( D , F , H , J L ) HeLa , their control were infected with VACV ( A , C D ) , HSV ( E F ) , or SeV ( K L ) , or they were transfected with plasmid ( pDNA ) ( 1 μg / mL ) ( B , G - J ) for indicated time , before immunoblotting with anti - Flag , - UNC13D , - p - TBK1 , - p - IRF3 , - STING , - p - P65 , - p - IκB - α , - actin or - tubulin antibody .

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

A - L HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (A and B), UNC13D stable knock-down (KD) (C, E, G, I and K) or knock out (KO) ( D, F, H, J and L) HeLa cells, and their control cells were infected with VACV (A, C and D), HSV (E and F), or SeV (K and L), or they were transfected with plasmid DNA (pDNA) (1 μg/mL) (B, G-J) for the indicated time, before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -p-P65, -p-IκB-α, -actin or -tubulin antibody.


UNC13D stable knock - down ( KD ) HeLa their control were transfected with plasmid ( 1 μg / mL ) for indicated time . UNC13D , IFN - β IL6 mRNA were measured by qPCR ( M - 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 O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

UNC13D stable knock-down (KD) HeLa cells and their control cells were transfected with plasmid DNA (1 μg/mL) for the indicated time. UNC13D, IFN-β and IL6 mRNA levels were measured by qPCR (M-O).


UNC13D stable knock - down ( KD ) HeLa their control were transfected with plasmid ( 1 μg / mL ) infected with SeV ( Q ) for indicated time . secretion of IFN - β was measured by bioassay ( P Q ) .

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 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

UNC13D stable knock-down (KD) HeLa cells and their control cells were transfected with plasmid DNA (1 μg/mL) infected with SeV (Q) for the indicated time. The secretion of IFN-β was measured by bioassay (P and Q).


R , S UNC13D knock - out ( KO ) HeLa their control were infected with GFP - HSV for 18 h or 24 h . GFP expression was visualized ( left ) green was determined by ImageJ .

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

R, S UNC13D knock-out (KO) HeLa cells and their control cells were infected with GFP-HSV for 18 h or 24 h. GFP expression was visualized (left) and the green fluorescence intensity was determined by ImageJ.


A HEK293T were transfected with indicated plasmids ( 5 μg each ) for 24 h . Whole lysates ( WCL ) were examined , lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies , followed by immunoblotting ( IB ) with anti - HA - Flag antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (IB) with anti-HA and -Flag antibody.


B - E HeLa stably expressing FLAG - tagged UNC13D ( F - UNC13D ) ( B ) , UNC13D stable knock - down ( KD ) ( D ) or knock - out ( KO ) ( C E ) HeLa , their control were stimulated with cGAMP ( 100 nM ) for 30 min ( B C ) or for indicated time ( D E ) before immunoblotting with anti - Flag , - UNC13D , - p - TBK1 , - p - IRF3 , - STING , - GAPDH , - actin or - tubulin antibody .

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

B - E HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) (B), UNC13D stable knock-down (KD) (D) or knock-out (KO) (C and E) HeLa cells, and their control cells were stimulated with cGAMP (100 nM) for 30 min (B and C) or for the indicated time (D and E) before immunoblotting with anti-Flag, -UNC13D, -p-TBK1, -p-IRF3, -STING, -GAPDH, -actin or -tubulin antibody.


F - H HEK293T were transfected with indicated plasmids ( 5 μg each ) for 24 h ( F G ) . UNC13D knock - out ( KO ) control HeLa were stimulated with cGAMP ( 100 nM ) for 30 min ( H ) . Whole lysates ( WCL ) were examined , lysates were immunoprecipitated ( IP ) with anti - HA antibodies ( F G ) , IgG anti - STING antibodies ( H ) , followed by immunoblotting ( IB ) with anti - HA , - Flag antibody or indicated antibodies .

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 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 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

F- H HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h (F and G). UNC13D knock-out (KO) and control HeLa cells were stimulated with cGAMP (100 nM) for 30 min (H). Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies (F and G), IgG and anti-STING antibodies (H), followed by immunoblotting (IB) with anti-HA, -Flag antibody or the indicated antibodies.


UNC13D knock - out ( KO ) HeLa HeLa stably expressing FLAG - tagged UNC13D ( F - UNC13D ) control HeLa were treated with cGAMP ( 100 nM ) for 30 min or SeV for 12 h . Confocal microscopy was used to determine localization of endogenous STING ( red ) , ERGIC marker ERGIC - 53 ( green ) nuclei ( blue ) .

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

UNC13D knock-out (KO) HeLa cells and HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. Confocal microscopy was used to determine the localization of endogenous STING (red), ERGIC marker ERGIC-53 (green) and nuclei (blue).


UNC13D knock - out ( KO ) HeLa HeLa stably expressing FLAG - tagged UNC13D ( F - UNC13D ) control HeLa were treated with cGAMP ( 100 nM ) for 30 min or SeV for 12 h . co - localization of STING ERGIC was analyzed using ImageJ ( J ) .

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

UNC13D knock-out (KO) HeLa cells and HeLa cells stably expressing FLAG-tagged UNC13D (F-UNC13D) and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. The co-localization of STING and ERGIC was analyzed using ImageJ (J).


K , L UNC13D stable knock - down ( KD ) HeLa control HeLa were treated with cGAMP ( 100 nM ) for 30 min or SeV for 12 h . aggregation of endogenous STING ( red ) was examined via confocal microscopy ( K ) . Scale bar : 25 μm . red was determined by ImageJ ( L ) .

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 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

K, L UNC13D stable knock-down (KD) HeLa cells and control HeLa cells were treated with cGAMP (100 nM) for 30 min or SeV for 12 h. The aggregation of endogenous STING (red) was examined via confocal microscopy (K). Scale bar: 25 μm. The red fluorescence intensity was determined by ImageJ (L).


A UNC13D knock - out HeLa stably expressing FLAG - tagged UNC13D were stimulated with cGAMP ( 100 nM ) or left unstimulated for indicated time . Whole lysates ( WCL ) were examined , lysates were immunoprecipitated ( IP ) with IgG anti - Flag antibodies , followed by immunoblotting ( IB ) with indicated antibodies .

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

A UNC13D knock-out HeLa cells stably expressing FLAG-tagged UNC13D were stimulated with cGAMP (100 nM) or left unstimulated for the indicated time. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with IgG and anti-Flag antibodies, followed by immunoblotting (IB) with the indicated antibodies.


UNC13D knock - out HeLa stably expressing FLAG - tagged UNC13D were pretreated with BFA ( 20 μM ) or left untreated for 2 h , followed by stimulation with cGAMP ( 100 nM ) ( or left unstimulated ) for 30 min . Whole lysates ( WCL ) were examined , lysates were immunoprecipitated ( IP ) with IgG anti - Flag antibodies , followed by immunoblotting ( IB ) with indicated antibodies ( E ) .

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

UNC13D knock-out HeLa cells stably expressing FLAG-tagged UNC13D were pretreated with BFA (20 μM) or left untreated for 2 h, followed by stimulation with cGAMP (100 nM) (or left unstimulated) for 30 min. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with IgG and anti-Flag antibodies, followed by immunoblotting (IB) with the indicated antibodies (E).


F HEK293T were transfected for 24h with IFN - β - luciferase reporter plasmid , pRL - TK indicated expression plasmids , followed by dual - luciferase reporter assays . expression of plasmids was examined by western blotting with anti - Flag , - HA or - tubulin antibody .

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

F HEK293T cells were transfected for 24h with IFN-β-luciferase reporter plasmid, pRL-TK and the indicated expression plasmids, followed by dual-luciferase reporter assays. The expression of plasmids was examined by western blotting with anti-Flag, -HA or -tubulin antibody.


H Wild - type HeLa were pretreated with BFA ( 20 μM ) or left untreated for 2 h before stimulation with plasmid ( 1 μg / ml ) for 18h or cGAMP ( 100 nM ) for 30 min ( or left unstimulated ) . lysates were separated by native ( top ) or SDS ( bottom ) PAGE analyzed by immunoblotting ( IB ) with anti - p - TBK1 , - p - IRF3 , - STING or - tubulin antibody .

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

H Wild-type HeLa cells were pretreated with BFA (20 μM) or left untreated for 2 h before stimulation with plasmid DNA (1 μg/ml) for 18h or cGAMP (100 nM) for 30 min (or left unstimulated). Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-p-TBK1, -p-IRF3, -STING or -tubulin antibody.


UNC13D stable knock - down ( KD ) HeLa control were transfected with plasmid ( pDNA ) ( 1 μg / mL ) for indicated time . lysates were separated by native ( top ) or SDS ( bottom ) PAGE analyzed by immunoblotting ( IB ) with anti - UNC13D , - p - TBK1 , - p - IRF3 , - STING or - tubulin antibody .

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

UNC13D stable knock-down (KD) HeLa cells and control cells were transfected with plasmid DNA (pDNA) (1 μg/mL) for the indicated time. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -p-TBK1, -p-IRF3, -STING or -tubulin antibody.


UNC13D knock - out ( KO ) ( J ) HeLa control were transfected with plasmid ( pDNA ) ( 1 μg / mL ) for indicated time . lysates were separated by native ( top ) or SDS ( bottom ) PAGE analyzed by immunoblotting ( IB ) with anti - UNC13D , - p - TBK1 , - p - IRF3 , - STING or - tubulin antibody .

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

UNC13D knock-out (KO) (J) HeLa cells and control cells were transfected with plasmid DNA (pDNA) (1 μg/mL) for the indicated time. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -p-TBK1, -p-IRF3, -STING or -tubulin antibody.


K , L HEK293T were transfected with HA - tagged UNC13D , VN / VC - STING or vector for 24 h , followed by stimulation with cGAMP ( 100 nM ) ( or left unstimulated ) for 30 min . Nucleus marker DAPI ( blue ) , VN / VC - STING ( green ) HA - UNC13D ( red ) were examined via confocal microscopy ( K ) . green was determined by ImageJ ( L ) . n = 5 biological replicates .

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

K, L HEK293T cells were transfected with HA-tagged UNC13D, VN/VC-STING or vector for 24 h, followed by stimulation with cGAMP (100 nM) (or left unstimulated) for 30 min. Nucleus marker DAPI (blue), VN/VC-STING (green) and HA-UNC13D (red) were examined via confocal microscopy (K). The green fluorescence intensity was determined by ImageJ (L). n = 5 biological replicates.


HEK293T were transfected with indicated plasmids ( 5 μg each ) for 24 h . Whole lysates ( WCL ) were examined , lysates were immunoprecipitated ( IP ) with indicated antibodies , followed by immunoblotting ( IB ) with anti - HA or Flag antibody .

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

HEK293T cells were transfected with the indicated plasmids (5 μg each) for 24 h. Whole cell lysates (WCL) were examined, and cell lysates were immunoprecipitated (IP) with the indicated antibodies, followed by immunoblotting (IB) with anti-HA or Flag antibody.


D UNC13D knock - out ( KO ) HeLa , UNC13D knock - out HeLa stably expressing FLAG - tagged wild - type UNC13D ( UNC13D RE ) FHL3 mutation ( FHL3 mut ) , control were stimulated with cGAMP ( 100 nM ) ( or left unstimulated ) for 30 min . lysates were separated by native ( top ) or SDS ( bottom ) PAGE analyzed by immunoblotting ( IB ) with anti - UNC13D , - Flag , - p - IRF3 , - STING or - tubulin antibody .

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D UNC13D knock-out (KO) HeLa cells, UNC13D knock-out HeLa cells stably expressing FLAG-tagged wild-type UNC13D (UNC13D RE) and FHL3 mutation (FHL3 mut), and control cells were stimulated with cGAMP (100 nM) (or left unstimulated) for 30 min. Cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed by immunoblotting (IB) with anti-UNC13D, -Flag, -p-IRF3, -STING or -tubulin antibody.


Top : Schematic of primer pairs designed to discriminate between wild - type knock - in alleles . Bottom : Representative genotyping of Ccnd1 + / + , Ccnd1 + / KI , Ccnd1KI / KI mice validating specificity of primer pairs somatic insertion of targeting construct .

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Top: Schematic of primer pairs designed to discriminate between wild-type and knock-in alleles. Bottom: Representative genotyping of Ccnd1+/+, Ccnd1+/KI, and Ccnd1KI/KI mice validating specificity of the primer pairs and somatic insertion of the targeting construct.


PCR of transcript b expression in organs harvested from Ccnd1 + / + Ccnd1KI / KI mice , demonstrating production of transcript b specifically in KI mice . Gapdh serves as a control ( NTC , Non - template control ) .

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PCR analysis of transcript b expression in organs harvested from Ccnd1+/+ and Ccnd1KI/KI mice, demonstrating production of transcript b specifically in KI mice. Gapdh serves as a control (NTC, Non-template control).


Immunoblot from parallel samples in ( C ) utilizing antisera specific to 33 generated by human CCND1 intron 4 . Lamin B serves as a control . Arrow indicates cyclin D1b band .

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Immunoblot from parallel samples in (C) utilizing antisera specific to the 33 amino acids generated by human CCND1 intron 4. Lamin B serves as a control. Arrow indicates the cyclin D1b band.


Neonates from 2 independent litters of Ccnd1 + / KI × Ccnd1 + / KI crosses were sacrificed at birth genotyped . Mice were organized by genotype total measured . Mass of neonates was calculated at birth average mass quantified ( n = at least 4 mice per group ) . Mice were weighed weekly , growth is plotted as average mass of each genotype / week ( left : , right : , n > 5 per gender genotype ) .

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Neonates from 2 independent litters of Ccnd1+/KI × Ccnd1+/KI crosses were sacrificed at birth and genotyped. Mice were organized by genotype and total size measured.Mass of neonates was calculated at birth and average mass quantified (n = at least 4 mice per group).Mice were weighed weekly, and growth is plotted as the average mass of each genotype/week (left: male, right: female, n > 5 per gender and genotype).


Mice were held by tails analyzed for leg clasping phenotype . Representative of Ccnd1 + / + Ccnd1KI / KI - matched mice are shown ( n > 10 for each group ) .

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Mice were held by the tails and analyzed for the leg clasping phenotype. Representative images of Ccnd1+/+ and Ccnd1KI/KI age-matched mice are shown (n > 10 for each group).


Top : whole - lysates were generated from eyes of mice of each genotype analyzed for expression of + / + cyclin D1 ( cyclin D1a ) KI / KI cyclin D1b via immunoblot using antisera specific to each isoform . Bottom : Representative H & amp ; amp ; E of retinal tissue from Ccnd1 + / + Ccnd1KI / KI mice showing layers of retina . ganglion layer inner plexiform layer ( G + IPL ) , inner layer ( INL ) , outer layer ( ONL ) were quantified ( right ) . were taken at 400 × magnification .

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Top: Individual whole-cell lysates were generated from the eyes of mice of each genotype and analyzed for expression of +/+ cyclin D1 (cyclin D1a) and KI/KI cyclin D1b via immunoblot using antisera specific to each isoform. Bottom: Representative H&amp;E staining of retinal tissue from Ccnd1+/+ and Ccnd1KI/KI mice showing individual layers of the retina. The ganglion cell layer and inner plexiform layer (G + IPL), inner nuclear layer (INL), and outer nuclear layer (ONL) were quantified (right). Images were taken at 400× magnification.


Indicated passage - matched MAF lines were grown to 70 % confluency then harvested for extraction . Top : qPCR of cyclin D1 transcript using primer pairs common to both transcript a transcript b . Bottom : Immunoblot of cyclin D1 in Ccnd1 + / + Ccnd1KI / KMAF lines using an antibody common to both isoforms .

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Indicated passage-matched MAF lines were grown to 70% confluency and then harvested for RNA and protein extraction. Top: qPCR analysis of cyclin D1 transcript using primer pairs common to both transcript a and transcript b. Bottom: Immunoblot of cyclin D1 levels in Ccnd1+/+ and Ccnd1KI/KMAF lines using an antibody common to both isoforms.


of - free mice post - injection of 1 million mixed with Matrigel ( 1 : 1 ) into flanks of nude mice over a period of 10 months ( n = 10 per genotype ) .

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Percentage of tumor-free mice post-injection of 1 million cells mixed with Matrigel (1:1 ratio) into the flanks of nude mice over a period of 10 months (n = 10 per genotype).


Indicated tumors were harvested 3 after detectable formation , fixed in formalin , stained with H & amp ; amp ; E . Tumors were analyzed for features of malignancy by a board - certified veterinary pathologist . Arrows indicate specific - associated features noted . Top panels were taken at 40 × magnification , lower panels were taken at 400 × magnification .

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Indicated tumors were harvested 3 weeks after detectable tumor formation, fixed in formalin, and stained with H&amp;E. Tumors were analyzed for features of malignancy by a board-certified veterinary pathologist. Arrows indicate the specific tumor-associated features noted. Top panels were taken at 40× magnification, and lower panels were taken at 400× magnification.


Ccnd1 + / + or Ccnd1KI / KI MAF lines were stably transfected with either cyclin D1a ( + / + ) , cyclin D1b ( KI / KI ) or vector control constructs . After selection with puromycin , lines were assessed for induction of cyclin D1a in + / + cyclin D1b in KI / KI lines via immunoblot .

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Ccnd1+/+ or Ccnd1KI/KI MAF lines were stably transfected with either cyclin D1a (+/+), cyclin D1b (KI/KI) or vector control constructs. After selection with puromycin, individual lines were assessed for the induction of cyclin D1a in the +/+ line and cyclin D1b in KI/KI lines via immunoblot.


One million from indicated stable lines were injected subcutaneously into flanks of nude mice monitored for formation over a period of 365 days . Indicated time points represent time of palpable detection are plotted as % of - free mice over time .

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One million cells from the indicated stable cell lines were injected subcutaneously into the flanks of nude mice and monitored for tumor formation over a period of 365 days. Indicated time points represent time of palpable tumor detection and are plotted as % of tumor-free mice over time.


Sections from KI / KI vec KI / KI - D1b tumors were stained for proliferative marker Ki67 . Three random fields from each were quantified for Ki67 positivity , are plotted as mean of Ki67 - positive / total number for each .

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Sections from KI/KI vec and KI/KI-D1b tumors were stained for the proliferative marker Ki67. Three random fields from each tumor were quantified for Ki67 positivity, and are plotted as the mean ratio of Ki67-positive cells/total cell number for each individual tumor.


Passage - matched Ccnd1 + / + or Ccnd1KI / KI MAF lines were infected with lentivirus containing vector control or h - Ras constructs . were selected with puromycin for 14 days assayed for β - galactosidase activity . DAPI serves as a control for number . were taken at 200 × magnification with insets taken at 400 × magnification .

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Passage-matched Ccnd1+/+ or Ccnd1KI/KI MAF lines were infected with lentivirus containing vector control or h-Ras constructs. Cells were selected with puromycin for 14 days and assayed for β-galactosidase activity. DAPI serves as a control for cell number. Images were taken at 200× magnification with insets taken at 400× magnification.


indicated lines were grown in complete media assayed for h - Ras expression via immunoblot . Lamin B serves as a loading control .

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The indicated cell lines were grown in complete media and assayed for h-Ras expression via immunoblot. Lamin B serves as a loading control.


were plated in soft agar allowed to grow for a period of 3 . Plates were then washed , fixed , stained with 0 . 01 % crystal violet . Colonies greater than 50 μm were counted are plotted ( top ) . Representative of colony growth are shown for each . were taken at 40 × with insets taken at 200 × magnification .

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Cells were plated in soft agar and allowed to grow for a period of 3 weeks. Plates were then washed, fixed, and stained with 0.01% crystal violet. Colonies greater than 50 μm were counted and are plotted (top). Representative images of colony growth are shown for each cell line. Images were taken at 40× with insets taken at 200× magnification.


Indicated MAF lines were injected subcutaneously into flanks of nude mice as in ( A ) , incidence is plotted as % of - free mice over time ( n = 10 ) .

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Indicated MAF lines were injected subcutaneously into the flanks of nude mice as in (A), and tumor incidence is plotted as % of tumor-free mice over time (n = 10).


Indicated tumors were harvested , fixed , stained for proliferative marker mKi67 . Three random fields from each were counted for mKi67 positivity , are plotted as a % of total number , n = 5 ( right : representative ) . were taken at 400 × magnification .

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Indicated tumors were harvested, fixed, and stained for the proliferative marker mKi67. Three random fields from each tumor were counted for mKi67 positivity, and are plotted as a % of total cell number, n = 5 (right: representative images). Images were taken at 400× magnification.


Ccnd1 + / + Ccnd1KI / KIMAF lines were arrested in G1 , S , G2 / M phases of cycle expression of cyclin D1 isoforms determined by immunoblot . cyclin A2 cyclin B1 serve as phase - specific cycle controls .

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Ccnd1+/+ and Ccnd1KI/KIMAF lines were arrested in G1, S, and G2/M phases of the cell cycle and expression of cyclin D1 isoforms determined by immunoblot. cyclin A2 and cyclin B1 serve as phase-specific cell cycle controls.


were plated in serum - proficient media lysates generated from each genetic . D1 cyclins were immunoblotted to verify genetic identity along with other essential cycle components . Lamin B serves as a control .

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Cells were plated in serum-proficient media and protein lysates generated from each genetic line. Individual D1 cyclins were immunoblotted to verify genetic identity along with other essential cell cycle components. Lamin B serves as a control.


Indicated lines were immunoprecipitated for CDK4 analyzed for their ability to incorporate 32P - ATP into full - RB substrate . Radioactive were normalized to CDK4 activity of + / + lines adjusted for efficiency of CDK4 pull down as determined by densitometry . Data is representative of three independent biological replicates .

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Indicated cells lines were immunoprecipitated for CDK4 and analyzed for their ability to incorporate 32P-ATP into full-length RB substrate. Radioactive counts were normalized to CDK4 activity of +/+ cell lines and adjusted for efficiency of CDK4 pull down as determined by densitometry. Data is representative of three independent biological replicates.


Indicated lines were plated in serum - proficient ( 10 % ) or serum - deficient ( 1 % ) media total number counted at 24 , 48 , 72 h in biological triplicate .

Indicated cell lines were plated in serum-proficient (10%) or serum-deficient (1%) media and total cell number counted at 24, 48, and 72 h in biological triplicate.


Ccnd1 + / + Ccnd1KI / KI MAF lines were transfected with a validated pool of siRNA directed against N - terminus of murine cyclin D1 transcript or control siRNA in full serum . 48 h post - transfection were harvested analyzed for biochemical markers of cycle kinetics via immunoblot . GAPDH Lamin B serve as controls .

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Ccnd1+/+ and Ccnd1KI/KI MAF lines were transfected with a validated pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or control siRNA in full serum. 48 h post-transfection cells were harvested and analyzed for biochemical markers of cell cycle kinetics via immunoblot. GAPDH and Lamin B serve as controls.


were treated as ( E ) , incubated in indicated serum for 48 h . were treated with BrdU for 1 h prior to harvesting then stained for BrdU incorporation . Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence data is represented as positive / total number .

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Cells were treated as (E), and incubated in the indicated serum concentration for 48 h. Cells were treated with BrdU for 1 h prior to harvesting and then stained for BrdU incorporation. Three random fields from each of three biological triplicates were counted for BrdU incorporation via immunofluorescence and data is represented as percent positive/total cell number.


Ccnd1 + / + Ccnd1KI / KI were transfected with a pool of siRNA directed against N - terminus of murine cyclin D1 transcript or scramble control for 48 h ( as in Figure5 ) . were then fixed stained for markers of double - strand breaks ( p - H2AX 53BP1 , 400 × objective ) . Total number of foci for each was quantified represented as % of with > 10 foci / ( p - H2AX ) or % of with > 2 foci / ( 53BP1 ) . Error bars represent ± .

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Ccnd1+/+ and Ccnd1KI/KI cells were transfected with a pool of siRNA directed against the N-terminus of the murine cyclin D1 transcript or scramble control for 48 h (as in Figure5). Cells were then fixed and stained for markers of double-strand breaks (p-H2AX and 53BP1, 400× objective). Total number of foci for each cell was quantified and represented as the % of cells with > 10 foci/cell (p-H2AX) or % of cells with > 2 foci/cell (53BP1). Error bars represent ± SEM.


Ccnd1 + / + Ccnd1KI / KIMAF lines were grown in serum - proficient media for 24 h probed for expression of auto - modified PARP1 via immunoblot . Gapdh cyclin D1b serve as controls .

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Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h and probed for expression of auto-modified PARP1 via immunoblot. Gapdh and cyclin D1b serve as controls.


were plated as in ( B ) treated with 0 . 5 μM etoposide for 3 h . were harvested then probed for markers of damage via immunoblot . Lamin B serves as a control .

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Cells were plated as in (B) and treated with 0.5 μM etoposide for 3 h. Cells were harvested and then probed for markers of DNA damage via immunoblot. Lamin B serves as a control.


Indicated MAF lines were treated as in ( A ) harvested 48 h post - transfection . were analyzed for total PAR via immunoblot . cyclin D1 serve as siRNA validation controls .

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Indicated MAF lines were treated as in (A) and harvested 48 h post-transfection. Cells were analyzed for total PAR levels via immunoblot. cyclin D1 levels serve as siRNA validation controls.


cyclin D1b expression was induced in prostate cancer LNCaP ( previously described ( Augello et al , 2014 ) ) stained for p - H2AX 53BP1 foci as in ( A ) via immunofluorescence ( 400 × objective ) . Total number of foci / is reported for LNCaP vector control cyclin D1b - expressing isogenic pairs in biological triplicate .

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cyclin D1b expression was induced in the prostate cancer cell line LNCaP (previously described (Augello et al, 2014)) and stained for p-H2AX and 53BP1 foci as in (A) via immunofluorescence (400× objective). Total number of foci/cell is reported for LNCaP vector control and cyclin D1b-expressing isogenic pairs in biological triplicate.


Isogenic pairs from ( E ) were grown in serum - proficient media for 24 h , harvested , probed for total PAR via immunoblot .

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Isogenic pairs from (E) were grown in serum-proficient media for 24 h, harvested, and probed for total PAR levels via immunoblot.


Ccnd1 + / + Ccnd1KI / KIMAF lines were grown in serum - proficient media for 24 h . were then treated with control DMSO or 2 . 5 μM of PARP inhibitor ABT - 888 . One hour post - treatment , indicated lines were treated with 5 Gy of radiation . were then allowed to recover for 48 h , after which BrdU was added for 1 h , then harvested for bivariate flow cytometry . Representative traces for each condition are shown ( left ) BrdU incorporation of biological triplicates was quantified ( right ) .

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Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right).


were plated treated as in ( A ) . Forty - eight hours post - treatment , were fixed stained for markers of senescence ( β - galactosidase activity ) . positive for ( blue ) were quantified for each condition ( 400 × magnification ) reported as a of total population ( right ) . Plates treated in parallel with both IR ABT - 888 were harvested at 48 h via 1 × trypsin re - plated in serum - proficient media . were allowed to grow for 96 h were then stained with crystal violet ( 4 × objective , with inset at 200 × magnification , boxes highlight of magnified ) .

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Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).


B - B2 Largely non ‐ overlapping distribution of AVP + , oxytocin + secretagogin + ( sgcn + ) neurons in magnocellular PVN . mousesupraoptic nucleus ( SON ) harbored vasopressin + oxytocin + but not secretagogin + neurons . Open arrowheads pinpoint single ‐ labeled neurons . Solid arrowhead denotes AVP / secretagogin dual ‐ labeling . lv , lateral ventricle ; PVNm , magnocellular part of paraventricular nucleus ; scgn , secretagogin . B3 , B4 Secretagogin + neurons had smaller somatic diameters than AVP + or oxytocin + neurons yet without a difference in their diameter quotient , a measure of ovoid profiles ( * P 0 . 05 , Student ' s t ‐ ) .

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B-B2 Largely non‐overlapping distribution of AVP+, oxytocin+ and secretagogin+ (sgcn+) neurons in the magnocellular PVN. The mousesupraoptic nucleus (SON) harbored vasopressin+ and oxytocin+ but not secretagogin+neurons. Open arrowheads pinpoint single‐labeled neurons. Solid arrowhead denotes AVP/secretagogin dual‐labeling. lv, lateral ventricle; PVNm, magnocellular part of the paraventricular nucleus; scgn, secretagogin.B3, B4 Secretagogin+neurons had smaller somatic diameters than AVP+ or oxytocin+neurons yet without a difference in their diameter quotient, a measure of ovoid profiles (*P 0.05, Student's t‐test).


C - D3 Terminal ‐ like profiles in posterior pituitary ( arrowheads in C ) suggesting that secretagogin can co ‐ exist , even if infrequently with oxytocin . Solanum tuberosum lectin ( Sol . tub ) was used to identify blood vessels .

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C-D3 Terminal‐like profiles in the posterior pituitary (arrowheads in C) suggesting that secretagogin can co‐exist, even if infrequently with oxytocin. Solanum tuberosum lectin (Sol. tub) was used to identify blood vessels.


A Secretagogin ( scgn ) distribution at ultrastructural level as revealed by pre ‐ embedding silver ‐ enhanced immunogold labeling . Secretagogin ( arrowheads ) was localized to membranous organelles in perikarya ( A ) , particularly plasmalemma ( A1 ) endoplasmic reticulum ( ER ) in neuronal soma ( s ; A2 ) . Open rectangles in ( A ) denote location of insets . Semi ‐ transparent shading is used to visually dissociate subcellular compartments in ( A - C ) . B Pre ‐ embedding secretagogin labeling ( arrowheads ) was also seen in dendrite ( d ) segments . C In axo ‐ dendritic terminals ( ax ) , secretagogin was closely associated with synaptic vesicles along plasmalemma ( arrowheads ) . D Quantitative of subcellular secretagogin distribution upon electron microscopy detection of silver ‐ enhanced gold particles . Particles were considered as bound when they were at 50 nm of a ( plasma or endomembrane ; i . e . , secretory vesicle , Golgi or endoplasmic reticulum ) . In soma of PVNneurons , significantly more particles were found in cytosol as along plasma ( * * P 0 . 01 ) . In contrast , association predominated in axonal nerve endings in median eminence ( * P 0 . 05 ) . Note that a significant proportion of particles was found adjacent to endomembranes in subcellular compartments studied .

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A Secretagogin (scgn) distribution at the ultrastructural level as revealed by pre‐embedding silver‐enhanced immunogold labeling. Secretagogin (arrowheads) was localized to membranous organelles in the perikarya (A), particularly the plasmalemma (A1) and endoplasmic reticulum (ER) in neuronal soma (s; A2). Open rectangles in (A) denote the location of insets. Semi‐transparent shading is used to visually dissociate subcellular compartments in (A-C).B Pre‐embedding secretagogin labeling (arrowheads) was also seen in dendrite (d) segments.C In axo‐dendritic terminals (ax), secretagogin was closely associated with synaptic vesicles along the plasmalemma (arrowheads).D Quantitative analysis of subcellular secretagogin distribution upon electron microscopy detection of silver‐enhanced gold particles. Particles were considered as membrane bound when they were at 50 nm of a membrane (plasma membrane or endomembrane; i.e., secretory vesicle, Golgi or endoplasmic reticulum). In the soma of PVNneurons, significantly more particles were found in the cytosol as along the plasma membrane (**P 0.01). In contrast, membrane association predominated in axonal nerve endings in the median eminence (*P 0.05). Note that a significant proportion of particles was found adjacent to endomembranes in all subcellular compartments studied.