( 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 .

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

(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 .

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

(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 .

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

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 - .

O O O O O O O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 ) .

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

(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 .

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

(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 ) .

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

(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 .

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

(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 .

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

(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 ) .

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

(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 - .

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

(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 ) .

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

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 . )

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

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 B-SUBCELLULAR I-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 B-ORGANISM B-TISSUE O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O B-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-ORGANISM B-GENEPROD 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 B-ORGANISM B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O B-TISSUE 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 .

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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 ) .

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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 ) .

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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 .

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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 .

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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 .

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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 B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD 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 ) .

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

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

B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD 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 .

O B-GENEPROD O O O O O O B-CELL_LINE O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-DISEASE O O B-DISEASE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O

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 .

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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.


A - A3 Biocytin ‐ filled ( arrow ) neuron immunonegative for secretagogin yet containing AVP / oxytocin ( a mixture of magnocellular markers was used in triple ‐ labeling experiments ) . Scale bars : 50 μm ( A ) , 8 μm ( A3 ) . lv , lateral ventricle ; PVN , paraventricular nucleus .

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A-A3 Biocytin‐filled (arrow) neuron immunonegative for secretagogin yet containing AVP/oxytocin (a mixture of magnocellular markers was used in triple‐labeling experiments). Scale bars: 50 μm (A), 8 μm (A3). lv, lateral ventricle; PVN, paraventricular nucleus.


B - B2 Secretagogin + biocytin ‐ filled neuron ( arrow ) lacking AVP / oxytocin immunosignal . Scale bars : 8 μm .

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B-B2 Secretagogin+ biocytin‐filled neuron (arrow) lacking AVP/oxytocin immunosignal. Scale bars: 8 μm.


D Electrophysiology characteristics of magnocellular parvocellular neurons . waveform of repetitive action potential firing is shown on left , while differential channel characteristics are depicted on right . Examples of secretagogin + neurons are shown in red .

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D Electrophysiology characteristics of magnocellular and parvocellular neurons. The waveform of repetitive action potential firing is shown on the left, while differential channel characteristics are depicted on the right. Examples of secretagogin+neurons are shown in red.


A Differential clustering on secretagogin ( Scgn ) , neuropeptide , hormone hormone receptor mRNA expression . Secretagogin ‐ expressing ( + ) neurons typically contained corticotropin ‐ releasing hormone ( Crh ) Nr3c1 mRNA transcripts . A1 Clusters of gene transcripts from 130 reveal phenotypic segregation of PVN neurons . Increasing mRNA copy numbers were depicted by a color gradient from deep blue ( not detected ) to red ( high numbers ) . Secretagogin + neurons are indicated by red arrows .

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A Differential clustering based on secretagogin (Scgn), neuropeptide, hormone and hormone receptor mRNA expression. Secretagogin‐expressing(+) neurons typically contained corticotropin‐releasing hormone (Crh) and Nr3c1 mRNA transcripts.A1 Clusters of gene transcripts from 130 cells reveal the phenotypic segregation of PVN neurons. Increasing mRNA copy numbers were depicted by a color gradient from deep blue (not detected) to red (high numbers). Secretagogin+ neurons are indicated by red arrows.


C , C1 Secretagogin co ‐ localization with GFP in PVN ( solid arrowheads ) of adult CRH ‐ GFP ( BAC ) reporter mice ( Alon et al , ) . Open arrowhead pinpoints a single ‐ labeled neuron .

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C, C1 Secretagogin co‐localization with GFP in the PVN (solid arrowheads) of adult CRH‐GFP (BAC) reporter mice (Alon et al, ). Open arrowhead pinpoints a single‐labeled neuron.


D - I3 We found secretagogin ( scgn ) co ‐ expressed with CRH in both neuronal soma ( solid arrowheads ; D1 - D3 ) axon terminal ‐ like specializations ( arrowheads ; E - E2 ) in PVN . Likewise , secretagogin + neurons harbored , yet infrequently , tyrosine hydroxylase ( TH ; solid arrowheads ; F - F3 ) , GABA ( G - G2 ) , somatostatin ( Sst ; H - H3 ) galanin ( I - I2 ) . Open rectangles denote general location of insets . Open arrowheads indicate lack of co ‐ localization .

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D-I3 We found secretagogin (scgn) co‐expressed with CRH in both neuronal soma (solid arrowheads; D1-D3) and axon terminal‐like specializations (arrowheads; E-E2) in the PVN. Likewise, secretagogin+ neurons harbored, yet infrequently, tyrosine hydroxylase (TH; solid arrowheads; F-F3), GABA (G-G2), somatostatin (Sst; H-H3) and galanin (I-I2). Open rectangles denote the general location of insets. Open arrowheads indicate the lack of co‐localization.


A - A2 Secretagogin co ‐ existed in majority of CRH + nerve endings in median eminence ( ME ) . Open rectangle denotes general location of ( B , B1 ) .

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A-A2 Secretagogin co‐existed in the majority of CRH+ nerve endings in the median eminence (ME). Open rectangle denotes the general location of (B, B1).


B , B1 Large axon terminals in median eminence were immunopositive for secretagogin with silver ‐ intensified immunogold particles ( open arrowheads ) associated with axonal dense ‐ vesicles ( see Fig D for quantitative data ) . Solid arrowheads denote silver deposit particles proximal to plasmalemma .

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B, B1 Large axon terminals in the median eminence were immunopositive for secretagogin with silver‐intensified immunogold particles (open arrowheads) associated with axonal membrane and dense‐core vesicles (see Fig D for quantitative data). Solid arrowheads denote silver deposit particles proximal to the plasmalemma.


C Immunoprecipitation using an anti ‐ secretagogin antibody in Ca2 + ‐ free Ca2 + ‐ loaded conditions was subtractively used to decipher Ca2 + ‐ dependent interactions . Silver ‐ gel is shown . C1 Ontology classification of 99 hits on function assignments . Unbiased MALDI ‐ TOF proteomics was used to identify interacting . most abundant hits ( Supplementary Table S3 is referred to for details on ) are implicated in vesicle fusion , trafficking , transport formation regulation of vesicle exocytosis . " Other " refers to a group of without known function .

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C Immunoprecipitation using an anti‐secretagogin antibody in Ca2+‐free and Ca2+‐loaded conditions was subtractively used to decipher the Ca2+‐dependent protein interactions. Silver‐staining gel is shown.C1 Ontology classification of the 99 protein hits based on primary function assignments. Unbiased MALDI‐TOF proteomics was used to identify interacting proteins. The most abundant hits (Supplementary Table S3 is referred to for details on individual proteins) are proteins implicated in vesicle fusion, trafficking, transport and formation and the regulation of vesicle exocytosis. "Other" refers to a group of proteins without known function.


E - E3 Rab3 , a family of vesicular fusion transport ( Schluter et al , ) , was found co ‐ localized with secretagogin ( arrowheads ) in median eminence . Neuronal soma in PVN lacked appreciable co ‐ localization . Note that neither our MALDI ‐ TOF nor our histochemical probing allowed precise identification of Rab3C ‐ E family members .

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E-E3 Rab3, a family of vesicular fusion and transport proteins (Schluter et al, ), was found co‐localized with secretagogin (arrowheads) in the median eminence. Neuronal soma in the PVN lacked appreciable co‐localization. Note that neither our MALDI‐TOF analysis nor our histochemical probing allowed the precise identification of individual Rab3C‐E family members.


A , A1 siRNA ‐ mediated secretagogin ( scgn ) knockdown in cultured hypothalamic neurons , as indicated by reduced secretagogin immunoreactivity ( A ) decreased CRH content in culture medium ( A1 ) .

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A, A1 siRNA‐mediated secretagogin (scgn) knockdown in cultured hypothalamic neurons, as indicated by reduced secretagogin immunoreactivity (A) and decreased CRH content in the culture medium (A1).


B - B2 Transient overexpression of secretagogin in immortalized CRH ‐ expressing mHypoE ‐ N44 hypothalamic significantly reduced CRHimmunofluorescence microscopy in secretagogin + bodies , indirectly supporting enhanced CRH release . experiments were performed in triplicate . Scale bar : 50 nm .

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B-B2 Transient overexpression of secretagogin in immortalized CRH‐expressing mHypoE‐N44 hypothalamic cells significantly reduced CRHimmunofluorescence microscopy intensity in secretagogin+cell bodies, indirectly supporting enhanced CRH release. All experiments were performed in triplicate. Scale bar: 50 nm.


C , C1 siRNA ‐ mediated in vivo silencing of secretagogin mRNA expression in PVN provoked somatic CRH accumulation ( arrowheads ) . Scale bar : 150 μm . C2 Quantitative demonstrating significantly increased somatic CRH contents . Note that somatic secretagogin remained unchanged , which we interpret as data on a neuronal contingent not affected by siRNA silencing . lack of secretagogin / CRH co ‐ localization suggests that secretagogin expression fell below detection threshold in many CRH + neurons .

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C, C1 siRNA‐mediated in vivo silencing of secretagogin mRNA expression in the PVN provoked somatic CRH accumulation (arrowheads). Scale bar: 150 μm.C2 Quantitative analysis demonstrating significantly increased somatic CRH contents. Note that somatic secretagogin levels remained unchanged, which we interpret as data on a neuronal contingent not affected by siRNA silencing. The lack of secretagogin/CRH co‐localization suggests that secretagogin expression fell below detection threshold in many CRH+ neurons.


D , D1 data points show maximal CRH ( gray scale arbitrary unit ( a . u . ) expression ) in PVN neurons that have low or no secretagogin expression after siRNA infusion .

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D, D1 Individual data points show maximal CRH fluorescence intensity (gray scale arbitrary unit (a.u.) expression) in PVN neurons that have low or no secretagogin expression after siRNA infusion.


A , A1 Paraventricular secretagogin ‐ immunoreactive ( scgn + ) neurons weakly expressed CRH ( open arrowheads ) but not c ‐ fos under control conditions . Scale bars : 300 μm ( A ) , 25 μm ( A1 ) .

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A, A1 Paraventricular secretagogin‐immunoreactive (scgn+) neurons weakly expressed CRH (open arrowheads) but not c‐fos under control conditions. Scale bars: 300 μm (A), 25 μm (A1).


B , B1 Stress induced by subcutaneous injection of formalin ‐ triggered co ‐ expression of c ‐ fos CRH in secretagogin + paraventricular neurons ( arrowheads ) . Scale bars : 300 μm ( B ) , 25 μm ( B1 ) .

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B, B1 Stress induced by subcutaneous injection of formalin‐triggered co‐expression of c‐fos and CRH in secretagogin+ paraventricular neurons (arrowheads). Scale bars: 300 μm (B), 25 μm (B1).


C , D In vivo siRNA ‐ mediated silencing of secretagogin expression in PVN occluded stress ‐ induced surge of serum ACTH ( C ) significantly reduced increase in plasma corticosterone ( D ) . * P 0 . 05 versus control .

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C, D In vivo siRNA‐mediated silencing of secretagogin expression in the PVN occluded the stress‐induced surge of serum ACTH levels (C) and significantly reduced the increase in plasma corticosterone (D). *P 0.05 versus control.


Differential expression for ACE2 expression changes in treated vs control samples was performed with level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . Volcano plots showing log fold - change ( X - axis ) uncorrected negative log10P value ( Y - axis ) of ACE2 expression changes are given for ( A ) 48 antihypertensive drugs enrichment of positive / negative ACE2 expression regulators in different drug classes on their mechanism of action ( MOA ) was tested with GSEA method as implemented in R package fgsea ( Segushichev , 2016 ; Methods ) , enrichment significance ( negative log10 P values ) is shown in bar plots in ( B ) , for on 48 antihypertensive drugs CCBs , calcium channel blockers ; ARBs , angiotensin II type - I receptor blockers ; ACEIs , angiotensin - converting enzyme

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are given for (A) 48 antihypertensive drugs The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (B) , for the analysis on the 48 antihypertensive drugs CCBs, calcium channel blockers; ARBs, angiotensin II type-I receptor blockers; ACEIs, angiotensin-converting enzyme inhibitors


Differential expression for ACE2 expression changes in treated vs control samples was performed with level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . ( C ) 672 clinically approved drugs that are each tested on four carcinoma lines from CMAP ( Subramanian et al , 2017 ) .

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). (C) 672 clinically approved drugs that are each tested on four carcinoma cell lines from CMAP (Subramanian et al, 2017).


Differential expression for ACE2 expression changes in treated vs control samples was performed with level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . ACE2 expression in control drug - treated groups for top significant drugs ( vorinostat , panobinostat , isotretinoin ) among 672 clinically approved drugs are shown with boxplots in ( D ) , ACE2 expression log fold - change values adjusted P values from limma , as well as number of samples per group are labeled for each drug . In boxplot of panel ( D ) , center , box edges whiskers denotes median , interquartile range of distribution in respective order , except for points that were determined to be outliers using a method that is a function of interquartile range , as in standard box plots .

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The ACE2 expression levels in control and drug-treated groups for the top significant drugs (vorinostat, panobinostat, and isotretinoin) among the 672 clinically approved drugs are shown with boxplots in (D), the ACE2 expression log fold-change values and adjusted P values from limma, as well as the number of samples per group are labeled for each drug. In the boxplot of panel (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots.


Differential expression for ACE2 expression changes in treated vs control samples was performed with level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . enrichment of positive / negative ACE2 expression regulators in different drug classes on their mechanism of action ( MOA ) was tested with GSEA method as implemented in R package fgsea ( Segushichev , 2016 ; Methods ) , enrichment significance ( negative log10 P values ) is shown in bar plots in ( E ) , for on 672 clinically approved drugs We further extended to a larger set of 989 clinically approved drugs tested on a total of 28 lines ( 16 cancer 12 normal ; this list is provided in Table EV2A with details including site , subtype donor demographics ) where each drug may have been tested on a different subset of ( Methods ) . performed enrichment using WHO ATC drug classification data ( World Health Organization , 2006 ) , top enriched drug classes are shown in ( F ) .

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (E), for the analysis on the 672 clinically approved drugs We further extended the analysis to a larger set of 989 clinically approved drugs tested on a total of 28 cell lines (16 cancer and 12 normal cells; this list is provided in Table EV2A with details including primary site, subtype and donor demographics) where each drug may have been tested on a different subset of cells (Methods). and performed enrichment analysis using the WHO ATC drug classification data (World Health Organization, 2006), and top enriched drug classes are shown in (F).


( A - D ) This Fig summarizes differential expression results for ACE2 upon treatment by clinically approved drugs in samples of lung kidney datasets mined from GEO database , spanning both cancer non - cancer datasets ( Methods ) . Volcano plots showing log fold - change ( X - axis ) uncorrected negative log10P value ( Y - axis ) of ACE2 expression changes are displayed in ( A ) for 74 datasets of or tissue samples from lung , involving 42 clinically approved drugs , in ( C ) for 35 kidney datasets involving 29 clinically approved drugs . Among these , we focused on top significant drugs that modulate ACE2 expression in non - cancerous or tissue samples from lung kidney , which are shown in ( B ) ( D ) , respectively , where ACE2 expression ( Y - axis ) difference between control treated groups ( X - axis ) are shown with boxplots . In title of each boxplot , GEO identifier of respective studies corresponding drug name sample type are provided . Different datasets involving experiments using same drug were analyzed presented separately , since sample types can be different across datasets . P values are computed from differential expression using limma ( Ritchie et al , 2015 ) ( Methods ) . In boxplots of panels ( B ) ( D ) , center , box edges whiskers denotes median , interquartile range of distribution in respective order , except for points that were determined to be outliers using a method that is a function of interquartile range , as in standard box plots . Abbreviations : HBEC , human bronchial epithelial .

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(A-D) This Fig summarizes the differential expression analysis results for ACE2 upon treatment by clinically approved drugs in samples of lung and kidney datasets mined from the GEO database, spanning both cancer and non-cancer datasets (Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are displayed in (A) for 74 datasets of cells or tissue samples from lung, involving 42 clinically approved drugs, and in (C) for 35 kidney datasets involving 29 clinically approved drugs. Among these, we focused on the top significant drugs that modulate ACE2 expression in non-cancerous cells or tissue samples from lung and kidney, which are shown in (B) and (D), respectively, where the ACE2 expression (Y-axis) difference between control and treated groups (X-axis) are shown with boxplots. In the title of each boxplot, the GEO identifier of the respective studies and the corresponding drug name and the sample type are provided. Different datasets involving experiments using the same drug were analyzed and presented separately, since the sample types can be different across datasets. All the P values are computed from differential expression analysis using limma (Ritchie et al, 2015) (Methods). In the boxplots of panels (B) and (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots. Abbreviations: HBEC, human primary bronchial epithelial cells.