( 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 ) .

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 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 O O O O O O O O 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 amino acid at the MAPK site . ( - ) , no co - injection of wt or mutated Smad4 .

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

(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 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 .

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 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

(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 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 .

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

(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 ( 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 ) .

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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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 , 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 ) .

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 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 B-CONTROLLED_VAR 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

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 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 . )

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

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 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 .

O O O O O O O O O O O O O O O O O O O O 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 O B-CONTROLLED_VAR 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 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 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 , 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 . )

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 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 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

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 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 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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 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 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 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 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 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 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 O B-MEASURED_VAR O 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 , 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 .

O O O O O O O O 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 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 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 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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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 ) .

B-CONTROLLED_VAR 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O 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 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 ) .

B-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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) 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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O 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 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 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 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 .

O 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 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 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

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 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 ) .

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 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 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 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 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 ) .

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 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 B-MEASURED_VAR O 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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 the 33 amino acids generated by human CCND1 intron 4 . Lamin B serves as a control . Arrow indicates the 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 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 ) .

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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 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 ) .

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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 : 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 ; 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 .

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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 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 .

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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.


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 ) .

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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 weeks after detectable tumor formation , fixed in formalin , and stained with H & amp ; 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 .

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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 , individual lines were assessed for the induction of cyclin D1a in the + / + line and 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 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 .

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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 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 .

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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 . 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 .

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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.


The indicated cell lines were grown in complete media and 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.


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 .

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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 the flanks of nude mice as in ( A ) , and tumor incidence is plotted as % of tumor - 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 , 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 .

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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 + / + 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 + / + 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 .

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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.


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 .

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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 + / + 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 .

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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 + / + 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 .

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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.


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 .

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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 ) and harvested 48 h post - transfection . Cells were analyzed for total PAR levels via immunoblot . cyclin D1 levels 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 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 .

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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 , and probed for total PAR levels 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 + / + 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 ) .

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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).


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 ) .

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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 + 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the 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 ) 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 .

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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 the majority of CRH + nerve endings in the median eminence ( ME ) . Open rectangle denotes the 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 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 .

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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 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 .

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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 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 .

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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 ) and decreased CRH content in the 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 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 .

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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 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 .

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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 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 .

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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 and 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 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 .

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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 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

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

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 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 ) .

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

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 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 .

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

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 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 ) .

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

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 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 .

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

(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.


( B ) In vitro degradation of UbLRad23 - GFP - 95 by purified S . cerevisiae proteasome . The graph plots the amount of substrate over time , estimated by fluorescence intensity monitored by plate - reader ( green ) or electronic autoradiography of SDS - PAGE gel bands ( red ) , normalized to the initial substrate amount as described in Methods . ( C ) Michaelis - Menten analysis of UbLRad23 - GFP - 95 degradation by different concentrations of purified S . cerevisiae proteasome ( green , 10 nM ; red , 40 nM ) . ( D ) UbLRad23 inhibits UbLRad23 - GFP - 95 degradation . The initial degradation rates of UbLRad23 - GFP - 95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition . ( E ) The UbLDsk2 domain is a competitive inhibitor of UbLRad23 - GFP - 95 degradation . Lineweaver - Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain . ( C - E ) Proteasomal degradation of UbLRad23 - GFP - 95 monitored by fluorescence intensity in a Tecan plate reader as described in Methods .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro degradation of UbLRad23-GFP-95 by purified S. cerevisiae proteasome. The graph plots the amount of substrate over time, estimated by fluorescence intensity monitored by plate-reader (green) or electronic autoradiography of SDS-PAGE gel bands (red), normalized to the initial substrate amount as described in Methods. (C) Michaelis-Menten analysis of UbLRad23-GFP-95 degradation by different concentrations of purified S. cerevisiae proteasome (green, 10 nM; red, 40 nM). (D) UbLRad23 inhibits UbLRad23-GFP-95 degradation. The initial degradation rates of UbLRad23-GFP-95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition. (E) The UbLDsk2 domain is a competitive inhibitor of UbLRad23-GFP-95 degradation. Lineweaver-Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain. (C-E) Proteasomal degradation of UbLRad23-GFP-95 monitored by fluorescence intensity in a Tecan plate reader as described in Methods.


( A , B ) In vitro degradation of model proteins by purified S . cerevisiae proteasome . The model proteins ( UbL - DHFR - 95 ) consisted of an N - terminal UbL domain , followed by an E . coli DHFR domain and a 95 aminoacid tail derived from S . cerevisiae cytochrome b2 . UbL - DHFR - 95 proteins were degraded by proteasome ( red solid circle ) and can be stabilized by removing the UbL domain ( no UbL , black triangles ) , by removing the 95 - amino - acid tail ( no tail , blue diamonds ) or by proteasome inhibitor ( MG132 , green squares ) . ( A ) Degradation of model proteins containing the UbL domain of S . cerevisiae Rad23 .

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 B-MEASURED_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 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 B-MEASURED_VAR O

(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23.


( A , B ) In vitro degradation of model proteins by purified S . cerevisiae proteasome . The model proteins ( UbL - DHFR - 95 ) consisted of an N - terminal UbL domain , followed by an E . coli DHFR domain and a 95 aminoacid tail derived from S . cerevisiae cytochrome b2 . UbL - DHFR - 95 proteins were degraded by proteasome ( red solid circle ) and can be stabilized by removing the UbL domain ( no UbL , black triangles ) , by removing the 95 - amino - acid tail ( no tail , blue diamonds ) or by proteasome inhibitor ( MG132 , green squares ) . ( A ) Degradation of model proteins containing the UbL domain of S . cerevisiae Rad23 . ( B ) Degradation of model proteins containing other UbL domains or ubiquitin . The graphs plot the amount of substrate estimated by electronic autoradiography in SDS - PAGE gel bands over time as normalized to the initial substrate amount as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

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

(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (B) Degradation of model proteins containing other UbL domains or ubiquitin. The graphs plot the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.


( B ) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail , and affected yeast growth under selective condition ( + 3 - AT , - his ) . Replacing UbL domains with DHFR domains rescued the growth defect . Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates . Plates were incubated at 30 ˚ C for 3 daysfor imaging .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(B) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail, and affected yeast growth under selective condition (+3-AT, -his). Replacing UbL domains with DHFR domains rescued the growth defect. Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. Plates were incubated at 30 ˚C for 3 daysfor imaging.


( B ) Fluorescence profiles of cells expressing different substrates monitored by flow cytometry . UbLRad23 targeted YFP - Su9 protein for degradation . Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP protein in cells .

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 B-MEASURED_VAR O O O O O O O

(B) Fluorescence profiles of cells expressing different substrates monitored by flow cytometry. UbLRad23 targeted YFP-Su9 protein for degradation. Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP protein in cells.


( C ) UbL domains targeted YFP substrates to degradation in the presence of an initiation region . The Y - axis plots abundance of the UbL - YFP - tail proteins normalized by a translation control , which is the same UbL - YFP protein without the tail . The ratio is equal to one for domains that are not recognized by the proteasome , and smaller than one if the UbL is recognized by the proteasome and the UbL - YFP - tail protein is degraded . YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence . The median of YFP / RFP ratio for each construct was calculated from 10 , 000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UbL domains targeted YFP substrates to degradation in the presence of an initiation region. The Y-axis plots abundance of the UbL-YFP-tail proteins normalized by a translation control, which is the same UbL-YFP protein without the tail. The ratio is equal to one for domains that are not recognized by the proteasome, and smaller than one if the UbL is recognized by the proteasome and the UbL-YFP-tail protein is degraded. YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence. The median of YFP/RFP ratio for each construct was calculated from 10,000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.