C Density plot of recovered mRNAs in CLIP - UPF1LL or CLIP - UPF1SL affinity purifications . mRNAs were binned according to 3 ' UTR . Statistical significance was determined by K - S .

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C Density plot of recovered mRNAs in CLIP-UPF1LL or CLIP-UPF1SL affinity purifications. mRNAs were binned according to 3'UTR length. Statistical significance was determined by K-S test.


D Density plot of recovered mRNAs in CLIP - UPF1LL affinity purifications relative to that of CLIP - UPF1SL . mRNAs were subdivided by PTBP1 / or hnRNP L motif density within 3 ' UTR , as indicated by gradient triangle . Statistical significance was determined by K - W , with Dunn ' s correction for multiple comparisons .

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D Density plot of recovered mRNAs in CLIP-UPF1LL affinity purifications relative to that of CLIP-UPF1SL. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the 3'UTR, as indicated by the gradient triangle. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


E RT - qPCR of indicated transcripts from CLIP - UPF1 overexpression - seq experiments . Relative fold changes are in reference to GFP expressing control . Significance of CLIP - UPF1SL or CLIP - UPF1LL overexpression was compared to GFP expressing control . Asterisk ( * ) indicates P < 0 . 05 , as determined by two - way ANOVA . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . For protected mRNAs , motif density of PTBP1 / hnRNP L within 3 ' UTR is indicated . PTC + indicates use of primers specific to transcript isoforms with validated poison

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

E RT-qPCR analysis of indicated transcripts from CLIP-UPF1 overexpression RNA-seq experiments. Relative fold changes are in reference to the GFP expressing control line. Significance of CLIP-UPF1SL or CLIP-UPF1LL overexpression was compared to the GFP expressing control line. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. For protected mRNAs, the motif density of PTBP1/hnRNP L within the 3'UTR is indicated. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


A Semiquantitative RT - PCR of UPF1SL or UPF1LL transcript following transfection of HEK - 293 with indicated siRNAs .

A Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with the indicated siRNAs.


B RT - qPCR of indicated transcripts following transfection of a NT siRNA or SRSF1 - specific siRNA under conditions of CLIP - UPF1SL or CLIP - UPF1LL overexpression . Relative fold changes are in reference to GFP expressing control treated with a NT siRNA . Asterisk ( * ) indicates P < 0 . 05 , as determined by unpaired Student ' s t - . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) .

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B RT-qPCR analysis of indicated transcripts following transfection of a NT siRNA or SRSF1-specific siRNA under conditions of CLIP-UPF1SL or CLIP-UPF1LL overexpression. Relative fold changes are in reference to the GFP expressing control line treated with a NT siRNA. Asterisk (*) indicates P < 0.05, as determined by unpaired Student's t-test. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates).


C Density plot of changes in relative mRNA abundance as determined by - seq following SRSF1 overexpression Genes were categorized as up - regulated by siUPF1total only , siUPF1LL only , or both siUPF1total siUPF1LL . Statistical significance was determined by K - W , with Dunn ' s correction for multiple comparisons .

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

C Density plot of changes in relative mRNA abundance as determined by RNA-seq following SRSF1 overexpression Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


D Density plot as in ( C ) , with genes binned according to enrichment in CLIP - UPF1LL or CLIP - UPF1SL affinity purifications .

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D Density plot as in (C), with genes binned according to enrichment in the CLIP-UPF1LL or CLIP-UPF1SL affinity purifications.


B UPF1SLΔCH translocation along an substrate harboring a high - affinity PTBP1 binding site in absence presence of PTBP1 . Time to 50 % unwound relative total % unwound at end - of - ( 1200 sec ) are indicated . Results of four technical replicates are shown for each dataset represent at least three independent experiments . Shaded region indicates SD . C Results as in ( B ) but with UPF1LLΔCH .

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B UPF1SLΔCH translocation along an RNA substrate harboring a high-affinity PTBP1 binding site in the absence and presence of PTBP1. Time to 50% unwound and relative total % unwound at end-of-assay (1200 sec) are indicated. Results of four technical replicates are shown for each dataset and represent at least three independent experiments. Shaded region indicates SD. C Results as in (B) but with UPF1LLΔCH.


B Density plots of changes in relative mRNA abundance as determined by - seq following treatment of HEK - 293 with 1 µM thapsigargin for 6hr ( left ) or 9hr ( right ) . Genes were categorized as up - regulated by siUPF1total only or siUPF1LL only under basal conditions . Statistical significance was determined by K - W , with Dunn ' s correction for multiple comparisons .

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

B Density plots of changes in relative mRNA abundance as determined by RNA-seq following treatment of HEK-293 cells with 1 µM thapsigargin for 6hr (left) or 9hr (right). Genes were categorized as up-regulated by siUPF1total only or siUPF1LL only under basal conditions. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


D RT - qPCR of indicated transcripts following transfection of HEK - 293 with indicated siRNAs treatment with 1 µM thapsigargin for 6hr . Relative fold changes are in reference to vehicle - treated , NT siRNA . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . PTC + indicates use of primers specific to transcript isoforms with validated poison

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

D RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 1 µM thapsigargin for 6hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


C RT - qPCR of indicated transcripts following transfection of HEK - 293 with indicated siRNAs treatment with 50 µg / mL puromycin for 4hr . Relative fold changes are in reference to vehicle - treated , NT siRNA . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . PTC + indicates use of primers specific to transcript isoforms with validated poison

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

C RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


A mRNA decay measurement using Roadblock - qPCR was isolated from HEK - 293 at indicated timepoints following transfection with indicated siRNAs treatment with 400 µM 4sU 50 µg / mL puromycin . mRNA half - lives were estimated by fitting data to a single - phase exponential decay model . Puromycin treatment was compared to vehicle control siUPF1LL was compared to siNT in absence presence of puromycin treatment using extra - sum - of - squares F . Error bars indicate mean ± SD ( n = 4 biological replicates ) .

O O O O O O O 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 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

A mRNA decay measurement using Roadblock-qPCR RNA was isolated from HEK-293 cells at indicated timepoints following transfection with indicated siRNAs and treatment with 400 µM 4sU and 50 µg/mL puromycin. mRNA half-lives were estimated by fitting the data to a single-phase exponential decay model. Puromycin treatment was compared to the vehicle control and siUPF1LL was compared to the siNT in the absence and presence of puromycin treatment using the extra-sum-of-squares F test. Error bars indicate mean±SD (n = 4 biological replicates).


B RT - qPCR of indicated transcripts following transfection of HEK - 293 with indicated siRNAs treatment with 50 µg / mL puromycin for 4hr . Relative fold changes are in reference to vehicle - treated , NT siRNA . Black dots represent data points error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . PTC + indicates use of primers specific to transcript isoforms with validated poison

B RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


C Density plot of changes in relative mRNA abundance as determined from - seq following treatment of HEK - 293 with 50 µg / mL of puromycin for 4hr . mRNAs were subdivided by PTBP1 / or hnRNP L motif density within first 400 nt of 3 ' UTR . Statistical significance was determined by K - W , with Dunn ' s correction for multiple comparisons . D Density plot as in ( C ) , following UPF1LL - specific knockdown .

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

C Density plot of changes in relative mRNA abundance as determined from RNA-seq following treatment of HEK-293 cells with 50 µg/mL of puromycin for 4hr. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the first 400 nt of 3'UTR. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons. D Density plot as in (C), following UPF1LL-specific knockdown.


( a - f ) Atg8 was co - incubated with Atg19 or Atg34 ( wild type mutants ) at concentrations of 40 μM 800 μM , respectively , run on a exclusion column . Aliquots of fractions were run on SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) gels Coomassie stained . box indicates N - terminal domain , grey box coiled - coil domain , grey box Ams1 - binding domain purple box C - terminal domain .

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(a-f) Atg8 was co-incubated with Atg19 or Atg34 (wild type and mutants) at concentrations of 40 μM and 800 μM, respectively, and run on a size exclusion column. Aliquots of individual fractions were run on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and Coomassie stained. The white box indicates the N-terminal domain, the light grey box the coiled-coil domain, the dark grey box the Ams1-binding domain and the purple box the C-terminal domain.


( g - i ) Atg8 was co - incubated with indicated Atg19 domains at 40 μM run on a exclusion column . elution profiles are shown above ; Coomassie stained SDS - PAGE gels below . Columns : ( a - f , i ) S200 3 . 2 / 30 ; ( g , h ) S75 3 . 2 / 30 . M : marker . experiments shown have been carried out twice .

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(g-i) Atg8 was co-incubated with the indicated Atg19 domains at 40 μM and run on a size exclusion column. Individual elution profiles are shown above; Coomassie stained SDS-PAGE gels below. Columns: (a-f,i) S200 3.2/30; (g,h) S75 3.2/30. M: size marker. All experiments shown have been carried out twice.


a ) Anti - Atg8 western blot of input pellet fractions of a pulldown experiment using GST - Atg19 as bait Atg8 as prey . black vertical indicates that unrelated samples were run on same gel but in between two samples on left one on right ( Supplementary Fig . 7 ) .

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a) Anti-Atg8 western blot of the input and pellet fractions of a pulldown experiment using GST-Atg19 as bait and Atg8 as prey. The black vertical line indicates that unrelated samples were run on the same gel but in between the two samples on the left and the one on the right (Supplementary Fig. 7).


( b ) Coomassie stained gels of pulldown experiments using GST - prApe1 propeptide as bait to pull down Atg19 , Atg19 - Atg34 chimaera Atg34 , respectively . Atg19 - Atg34 chimaera contained residues 1 - 361 of Atg19 residues 348 - 412 of Atg34 .

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(b) Coomassie stained gels of pulldown experiments using the GST-prApe1 propeptide as bait to pull down Atg19, the Atg19-Atg34 chimaera and Atg34, respectively. The Atg19-Atg34 chimaera contained residues 1-361 of Atg19 and residues 348-412 of Atg34.


( c ) Yeast two - hybrid ( Y2H ) testing for interaction of Atg11 with Atg19 , Atg19 - Atg34 chimaera Atg34 , respectively .

(c) Yeast two-hybrid (Y2H) assay testing for the interaction of Atg11 with Atg19, the Atg19-Atg34 chimaera and Atg34, respectively.


( d ) Anti - Atg8 western blot using indicated GST fusion as bait to pull down Atg8 . pulldowns were conducted in presence or absence of prApe1 propeptide . See Supplementary Fig . 1 for input gel . experiments shown have been carried out twice . of uncropped western blots gels can be found in Supplementary Fig . 7 .

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(d) Anti-Atg8 western blot using the indicated GST fusion proteins as bait to pull down Atg8. The pulldowns were conducted in the presence or absence of the prApe1 propeptide. See Supplementary Fig. 1 for input gel. All experiments shown have been carried out twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.


( a , b ) Anti - Atg8 western blots using indicated GST fusion shown on left as bait to pull down Atg8 . See Supplementary Fig . 1 for input gels .

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(a,b) Anti-Atg8 western blots using the indicated GST fusion proteins shown on the left as bait to pull down Atg8. See Supplementary Fig. 1 for input gels.


( c ) Alignment of Atg19 C - terminus lacking LIR motif with region around LIR motif of human p62 ( NP _ 003891 . 1 ) . shown in magenta green indicate residues mutated in experiments shown in d - f .

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(c) Alignment of the Atg19 C-terminus lacking the LIR motif with the region around the LIR motif of human p62 (NP_003891.1). The amino acids shown in magenta and green indicate the residues mutated in the experiments shown in d-f.


( d ) Anti - Atg8 western blots with indicated GST fusion as bait to pull down Atg8 . of amount of Atg8 pulled down by indicated GST fusion . graph is on three independent experiments ( N = 3 ) , one of which is shown below graph . Shown are averages s . d . WT , wild type .

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

(d) Anti-Atg8 western blots with the indicated GST fusion proteins as bait to pull down Atg8. Quantification of the amount of Atg8 pulled down by the indicated GST fusion protein. The graph is based on three independent experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. WT, wild type.


( e ) Green ( GFP ) - trap pulldown experiment using yeast with integrated GFP - Atg8 transformed with indicated Myc - Atg19 constructs .

(e) Green fluorescent protein (GFP)-trap pulldown experiment using yeast cells with integrated GFP-Atg8 transformed with the indicated Myc-Atg19 constructs.


( f ) Pulldown experiment using GST - prApe1 propeptide ( propep . ) to pull down Atg8 through indicated Atg19 . graph is on three experiments ( N = 3 ) , one of which is shown below graph . Shown are averages s . d . See Supplementary Fig . 2 for input gel .

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(f) Pulldown experiment using the GST-prApe1 propeptide (propep.) to pull down Atg8 through the indicated Atg19 proteins. The graph is based on three experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. See Supplementary Fig. 2 for input gel.


g ) Representative profile of a exclusion chromatography run using a 10 / 300 S200 column . Atg8 was run at 400 μM Atg19 C - terminus at 40 μM . For run both were preincubated at concentrations mentioned . average number of Atg8 molecules shifted into peak per Atg19 C - terminus was 4 . 14 ( average of three experiments ) . experiments shown in a , b , d , f , g have been conducted three times ; experiment shown in e has been conducted twice . of uncropped western blots gels can be found in Supplementary Fig . 7 .

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g) Representative profile of a size exclusion chromatography run using a 10/300 S200 column. Atg8 was run at 400 μM and the Atg19 C-terminus at 40 μM. For the complex run both proteins were preincubated at the concentrations mentioned. The average number of Atg8 molecules shifted into the complex peak per Atg19 C-terminus was 4.14 (average of three experiments). The experiments shown in a,b,d,f,g have been conducted three times; the experiment shown in e has been conducted twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.


( a ) Giant unilamellar vesicles ( GUVs ) incubated with mCherry - Atg19 . was marked by incorporation of an Oregon Green labelled lipid . ( b ) GUV coated with GFP - Atg8 incubated with mCherry - Atg19 . ( c ) Schematic representation showing reaction resulting in GFP - Atg8 conjugation to GUV shown in b . ( d ) GUVs containing nickellipids incubated with GFP - Atg8 - 6 × His wild - type mCherry - Atg19 .

(a) Giant unilamellar vesicles (GUVs) incubated with mCherry-Atg19. The membrane was marked by incorporation of an Oregon Green labelled lipid. (b) GUV coated with GFP-Atg8 incubated with mCherry-Atg19. (c) Schematic representation showing the proteins and reaction resulting in GFP-Atg8 conjugation to the GUV shown in b. (d) GUVs containing nickellipids incubated with GFP-Atg8-6×His and wild-type mCherry-Atg19.


( f ) of binding by indicated mCherry - Atg19 . experimental set - up is shown in e . graph is on three experiments . Shown are averages s . d . of these three experiments ( N = 3 , on basis of 318 GUVs for Atg19 , 171 GUVs for Atg19 W412A , 166 GUVs for Atg19 F376A , F379A , W412A , 188 GUVs for enhanced GFP ( eGFP ) control ) . Scale bars , 5 μm . Abbreviations : 3 , Atg3 ; 5 , Atg5 ; 7 , Atg7 ; 8 , Atg8 ; 12 , Atg12 ; 16 , Atg16 ; 19 , Atg19 redundant . Experiments shown in a , b have been conducted twice experiment in d three times .

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(f) Quantification of membrane binding by the indicated mCherry-Atg19 proteins. The experimental set-up is shown in e. The graph is based on three experiments. Shown are the averages and s.d. of these three experiments (N = 3, on the basis of 318 GUVs for Atg19, 171 GUVs for Atg19 W412A, 166 GUVs for Atg19 F376A, F379A, W412A, 188 GUVs for the enhanced GFP (eGFP) control). Scale bars, 5 μm. Abbreviations: 3, Atg3; 5, Atg5; 7, Atg7; 8, Atg8; 12, Atg12; 16, Atg16; 19, Atg19 redundant. Experiments shown in a,b have been conducted twice and the experiment in d three times.


( a ) prApe1 propeptide recruitment to Atg8 - harbouring GUVs in presence absence of Atg19 . experimental set - up is shown on right .

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(a) prApe1 propeptide recruitment to Atg8-harbouring GUVs in the presence and absence of Atg19. The experimental set-up is shown on the right.


( b ) Atg19 - dependent envelopment of prApe1 - coated beads with Atg8 - positive membranes . experimental set - up is shown on right . was labelled by incorporation of a rhodamine - labelled lipid . (

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(b) Atg19-dependent envelopment of prApe1-coated beads with Atg8-positive membranes. The experimental set-up is shown on the right. The membrane was labelled by incorporation of a rhodamine-labelled lipid. (


( c ) of of GUV - associated eGFP - Ape1 - coated beads showing detectable bending ( N = 3 ; 40 GUVs per condition ) . experimental set - up is shown in b . density of Atg8 - 6 × His on was titrated by different amounts of nickel lipids in GUV . graph is on at least three independent experiments . Shown are averages s . d . (

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(c) Quantification of the percentage of GUV-associated eGFP-Ape1-coated beads showing detectable membrane bending (N = 3; 40 GUVs per condition). The experimental set-up is shown in b. The density of Atg8-6×His on the membrane was titrated by different amounts of nickel lipids in the GUV membrane. The graph is based on at least three independent experiments. Shown are the averages and s.d. (


( d ) of of GUV - associated eGFP - Ape1 - coated beads showing detectable bending . is on three independent experiments for mutant Atg19 versions 16 independent experiments for wild - type Atg19 no - Atg19 control ( Atg19 , N = 16 , total GUVs = 382 ; Atg19 W412A , N = 3 , total GUVs = 55 ; Atg19 F376A , F379A , N = 3 , total 66 GUVs ; Atg19 F376A , F379A , W412A , N = 3 , total GUVs = 84 ; Atg19 F376A , F379A , P385A , E386A , W412A , N = 3 , total GUVs = 60 ; Atg19 - 34 , N = 4 , total GUVs = 110 ; no Atg19 , N = 16 , total GUVs = 382 ) . Shown are averages s . d . experimental set - up is as shown in b ; p - values in c d were calculated using a two - Student t - .

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

(d) Quantification of the percentage of GUV-associated eGFP-Ape1-coated beads showing detectable membrane bending. The quantification is based on three independent experiments for the mutant Atg19 versions and 16 independent experiments for wild-type Atg19 and the no-Atg19 control (Atg19, N = 16, total GUVs = 382; Atg19 W412A, N = 3, total GUVs = 55; Atg19 F376A, F379A, N = 3, total 66 GUVs; Atg19 F376A, F379A, W412A, N = 3, total GUVs = 84; Atg19 F376A, F379A, P385A, E386A, W412A, N = 3, total GUVs = 60; Atg19-34, N = 4, total GUVs = 110; no Atg19, N = 16, total GUVs = 382). Shown are the averages and s.d. The experimental set-up is as shown in b; the p-values in c and d were calculated using a two-tailed Student t-test.


( e ) Visualization of Atg19 during envelopment of prApe1 - coated beads with Atg8 - containing membranes . Note that Atg19 directly binds prApe1 - coated beads ( arrows ) . experimental set - up is shown on right . Scale bars , 5 μM . Abbreviations : 8 , Atg8 ; 19 , Atg19 ; DIC , differential interference contrast . experiments have been conducted at least three times .

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(e) Visualization of Atg19 during the envelopment of prApe1-coated beads with Atg8-containing membranes. Note that Atg19 directly binds the prApe1-coated beads (arrows). The experimental set-up is shown on the right. Scale bars, 5 μM. Abbreviations: 8, Atg8; 19, Atg19; DIC, differential interference contrast. All experiments have been conducted at least three times.


( a ) Anti - Ape1 western blot of atg19Δ transformed with indicated expression constructs . lower Ape1 band indicates prApe1 processing thus its delivery into vacuole . Rap . , rapamycin ; Log . ,

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(a) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Rap., rapamycin; Log.,


( b ) Representative electron of ypt7Δ , atg19Δ yeast grown under cytoplasm - to - vacuole targetting ( Cvt ) conditions expressing indicated labelled with an anti - Myc antibody . arrowheads indicate isolation . dashed indicates circumference of prApe1 oligomer . Gold particles , 10 nm . Scale bars , 200 nm . See Supplementary Fig . 5 for full .

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(b) Representative electron micrographs of ypt7Δ, atg19Δ yeast cells grown under cytoplasm-to-vacuole targetting (Cvt) conditions expressing the indicated proteins labelled with an anti-Myc antibody. The white arrowheads indicate the isolation membrane. The dashed line indicates the circumference of the prApe1 oligomer. Gold particles, 10 nm. Scale bars, 200 nm. See Supplementary Fig. 5 for full images.


( c ) of electron of yeast expressing either Myc - Atg19 or Myc - Atg19 - Atg34 co - labelled with anti - Myc ( 10 nm gold ) anti - prApe1 ( 5 nm gold ) . See also Supplementary Fig . 5 . Three independent experiments ; Myc - Atg19 , N = 48 , Myc - Atg19 - 34 , N = 24 .

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(c) Quantification of electron micrographs of yeast cells expressing either Myc-Atg19 or Myc-Atg19-Atg34 co-labelled with anti-Myc (10 nm gold) and anti-prApe1 (5 nm gold). See also Supplementary Fig. 5. Three independent experiments; Myc-Atg19, N = 48, Myc-Atg19-34, N = 24.


( d ) Anti - Ape1 western blot of atg19Δ transformed with indicated expression constructs . lower Ape1 band indicates prApe1 processing thus its delivery into vacuole . Supplementary Fig . 6 shows expression of Myc - tagged a of .

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(d) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Supplementary Fig. 6 shows the expression of the Myc-tagged proteins and a quantification of the assay.


( e ) Y2H testing for interaction of Atg11 with Atg19 indicated Atg19 mutants .

(e) Y2H assay testing for the interaction of Atg11 with Atg19 and the indicated Atg19 mutants.


( f ) Atg19Δ yeast expressing indicated Atg19 prApe1 - RFP ( red ) were labelled with vacuolar dye MDY - 64 . Scale bars : 2 μM . experiments in a - d , f have been conducted three times , experiment in e twice . of uncropped western blots gels can be found in Supplementary Fig . 7 .

(f) Atg19Δ yeast cells expressing the indicated Atg19 proteins and prApe1-RFP (red fluorescent protein) were labelled with the vacuolar membrane dye MDY-64. Scale bars: 2 μM. The experiments in a-d,f have been conducted three times, the experiment in e twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.


A . Structure of Brca1ex2 allele . * Note that exon 4 was annotated in error in original descriptions of gene structure is not drawn . RT - PCR shows a novel product from Brca1ex2 allele , corresponding to splicing of exon 1 directly to exon 3 .

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A. Structure of Brca1ex2 allele. * Note that exon 4 was annotated in error in original descriptions of the gene structure and is not drawn. RT-PCR shows a novel product from the Brca1ex2 allele, corresponding to splicing of exon 1 directly to exon 3.


B . Western blot to detect BRCA1 BARD1 in MEFs expressing WT mutant Brca1 .

B. Western blot to detect BRCA1 and BARD1 in MEFs expressing WT and mutant Brca1.


C . Immunofluorescent ( IF ) detection of BRCA1 RAD51 at IR - induced foci in expressing WT mutant forms of Brca1 . Scale bar : 10 ingD . of IF , showing proportion of with RAD51 foci that also had > 5 BRCA1 foci . N = 2 . E . of IF , showing proportion of RAD51 foci that colocalized with BRCA1 foci . N = 2 .

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C. Immunofluorescent (IF) detection of BRCA1 and RAD51 at IR-induced nuclear foci in cells expressing WT and mutant forms of Brca1. Scale bar: 10 ingD. Quantification of IF, showing proportion of cells with RAD51 foci that also had >5 BRCA1 foci. N=2.E. Quantification of IF, showing proportion of RAD51 foci that colocalized with BRCA1 foci. N=2.


F . Immunofluorescent detection of BARD1 at IR - induced foci in MEFs expressing WT mutant forms of Brca1 . Scale bar : 10 μ

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F. Immunofluorescent detection of BARD1 at IR-induced nuclear foci in MEFs expressing WT and mutant forms of Brca1. Scale bar: 10 μ S


A . Western blot showing abundance of BRCA1 BARD1 in Brca1Δ2 / Δ2 ; Trp53bp1 - / - mouseB .

A. Western blot showing abundance of BRCA1 and BARD1 protein in Brca1Δ2/Δ2;Trp53bp1-/- mouseB cells.


B . of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 Trp53bp1 . Left , metaphase spreads were prepared from B treated with olaparib . Arrows show examples of chromosome aberrations . Right , of chromosome breaks ( CSB ) , chromatid - type breaks ( CTD ) , radial chromosomes , other abnormalities in mouseB . Error bars indicate SD , N =

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

B. Analysis of chromosome aberrations in mice with targeted deletion of Brca1 exon 2 and Trp53bp1. Left, metaphase spreads were prepared from B cells treated with olaparib. Arrows show examples of chromosome aberrations. Right, quantification of chromosome breaks (CSB), chromatid-type breaks (CTD), radial chromosomes, and other abnormalities in mouseB cells. Error bars indicate SD, N=3


C . RAD51 foci in B after 10 Gy IR exposure . Chart shows mean of with > 5 foci , N = 3 . Error bars indicate SD , statistical by two - Student t .

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C. RAD51 foci in B cells after 10 Gy IR exposure. Chart shows mean percentage of cells with >5 foci, N=3. Error bars indicate SD, statistical analysis by two-tailed Student t test.


D . of showing > 5 RAD51 foci after 4hrs treatment with 10 μM olaparib ( OLA ) or 10 μM camptothecin ( CPT ) . N = 2 .

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D. Percentage of cells showing >5 RAD51 foci after 4hrs treatment with 10 μM olaparib (OLA) or 10 μM camptothecin (CPT). N=2.


E . Average number of γ - H2AX foci per in that were not treated ( NT ) or exposed to 2 Gy ionizing radiation . N = 2 .

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E. Average number of γ-H2AX foci per cell in cells that were not treated (NT) or exposed to 2 Gy ionizing radiation. N=2.


F . Sister chromatid exchanges in that were not treated ( NT ) or exposed to 2 μM olaparib ( OLA ) for 16 hrs . Black arrows in show SCEs in olaparib - treated . Scale bar : 5 μm . N = 2 .

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F. Sister chromatid exchanges in cells that were not treated (NT) or exposed to 2 μM olaparib (OLA) for 16 hrs. Black arrows in images show SCEs in olaparib-treated cells. Scale bar: 5 μm. N=2.


A . Experimental scheme representative of . Scale bar : 5 μm . B . of showing proportions of replication forks that showed restart after HU treatment or remained stalled . N = 2 , > 500 scored per experiment . C . Proportion of total forks showing de novo initiation after HU treatment . N = 2 , > 500 scored per experiment . D . of initial replication fork tracts ( CldU tracts ) . N = 2 , > 200 measured per experiment .

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A. Experimental scheme and representative images of DNA fibers. Scale bar: 5 μm.B. Analysis of fibers showing proportions of replication forks that showed restart after HU treatment or remained stalled. N=2, >500 fibers scored per experiment.C. Proportion of total forks showing de novo initiation after HU treatment. N=2, >500 fibers scored per experiment.D. Length of initial replication fork tracts (CldU tracts). N=2, >200 fibers measured per experiment.


A . Kaplan - Meier survival curve for Brca1ex2 / + ; Trp53bp1 - / - Brca1ex2 / ex2 ; Trp53bp1 - / - mice . N = 20 in each group . Statistical by Mantel - Cox log - rank .

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A. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.


B . Kaplan - Meier survival curve for Brca1ex2 / + ; Trp53bp1 - / - ; Trp53 + / - Brca1ex2 / ex2 ; Trp53bp1 - / - ; Trp53 + / - mice . N = 20 in each group . Statistical by Mantel - Cox log - rank .

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B. Kaplan-Meier survival curve for Brca1ex2/+;Trp53bp1-/-;Trp53+/- and Brca1ex2/ex2;Trp53bp1-/-;Trp53+/- mice. N=20 animals in each group. Statistical analysis by Mantel-Cox log-rank test.


C . of Brca1ex2 / + ; Trp53bp1 - / - Brca1ex2 / ex2 ; Trp53bp1 - / - mice showing signs of abnormal tissue at . N = 46 inspected for Brca1ex2 / + ; Trp53bp1 + / + , N = 54 inspected for Brca1ex2 / ex2 / Trp53bp1 - / - .

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C. Percentage of Brca1ex2/+;Trp53bp1-/- and Brca1ex2/ex2;Trp53bp1-/- mice showing signs of abnormal tissue morphology at death. N=46 animals inspected for Brca1ex2/+;Trp53bp1+/+, N=54 animals inspected for Brca1ex2/ex2/Trp53bp1-/-.


D . Tissues affected by abnormal growth from N = 46 Brca1ex2 / + / Trp53bp1 - / - N = 54 Brca1ex2 / ex2 / Trp53bp1 - / - mice .

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D. Tissues affected by abnormal growth from N=46 Brca1ex2/+/Trp53bp1-/- and N=54 Brca1ex2/ex2/Trp53bp1-/- mice.


A . H & amp ; amp ; E - stained sections of seminiferous tubules from testes of WT Brca1ex2 / ex2 / Trp53bp1 - / - mice . Scale bar : 100 μm .

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A. H&amp;E-stained sections of seminiferous tubules from testes of WT and Brca1ex2/ex2/Trp53bp1-/- male mice. Scale bar: 100 μm.


B . Immunofluorescence on meiotic chromosomes from WT Brca1ex2 / ex2 ; Trp53bp1 - / - spermatocytes . Spreads were stained for SYCP3 to indicate chromosome axes extent of synapsis were stained for SYCP3 to indicate chromosome axex . Scale bar : 5

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B. Immunofluorescence on meiotic chromosomes from WT and Brca1ex2/ex2;Trp53bp1-/-spermatocytes. Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body. Scale bar: 5 apsi


C . of number of γ - H2AX foci observed at pachytene stage in indicated genotypes . P value is Mann - Whitney , two - .

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C. Quantification of the number of γ-H2AX foci observed at the pachytene stage in the indicated genotypes. P value is Mann-Whitney, two-tailed.


D . G2M checkpoint in mouse B after IR treatment . Left , mitotic were identified by flow cytometry as having 4c content ( on propidium iodide ) pSer10 - H3 + . Right , of flow cytometry data , showing mitotic after IR as of that seen in untreated . N ≥ 2 .

D. G2M checkpoint analysis in mouse B cells after IR treatment. Left, mitotic cells were identified by flow cytometry as having 4c DNA content (based on propidium iodide staining) and pSer10-H3+. Right, quantification of flow cytometry data, showing mitotic cells after IR as percentage of that seen in untreated cells. N≥2.


( A ) Macrophages were exposed to increasing concentrations of - free RNase / DNase I treated iodixanol velocity gradient purified HIV for 24 h , harvested , lysed analyzed for endogenous LC3B , SQSTM1 , ACTB by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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(A) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


( B ) Macrophages were exposed to increasing concentrations of - free RNase / DNase I treated iodixanol velocity gradient purified HIV for 24 h in presence of pepstatin A , harvested , lysed analyzed for endogenous LC3B , SQSTM1 , ACTB by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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(B) Macrophages were exposed to increasing concentrations of cell-free RNase/DNase I treated iodixanol velocity gradient purified HIV for 24 h in the presence of pepstatin A, harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


( C ) Macrophages were exposed to mock , infectious , AT - 2 - inactivated , or RNase / DNase I treated AT - 2 - inactivated iodixanol velocity gradient purified HIVBa - L for 24 h . were then harvested , lysed analyzed for endogenous LC3B SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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(C) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h. Cells were then harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


A ) Macrophages were transduced with non - specific scrambled shRNA ( shNS ) , or TLR8 shRNA ( shTLR8 ) analyzed for TLR8 expression . Bottom , a representative blot is shown . Top , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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A) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( B ) Macrophages transduced with shNS or shTLR8 from ( A ) were exposed to infectious HIV ( + ) , AT - 2 - inactivated HIV ( AT ) , or RNase / DNase I treated AT - 2 - inactivated HIV ( R ) or mock infected ( - ) for 24 h , harvested , lysed analyzed for endogenous LC3B SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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(B) Macrophages transduced with shNS or shTLR8 from (A) were exposed to infectious HIV (+), AT-2-inactivated HIV (AT), or RNase/DNase I treated AT-2-inactivated HIV (R) or mock infected (-) for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( C ) Representative microscopy of HIV - infected macrophages which were fixed , permeabilized then stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) antibody to LC3B ( green ) , SQSTM1 ( magenta ) , HIV p24 p17 ( red ) . Co - localization of HIV LC3B is represented by orange pixels , co - localization of HIV , SQSTM1 , LC3B is represented by cream pixels . Scale bars indicate 10 μm .

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(C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; blue) and antibody to LC3B (green), SQSTM1 (magenta), HIV p24 and p17 (red). Co-localization of HIV and LC3B is represented by orange pixels, and co-localization of HIV, SQSTM1, and LC3B is represented by cream pixels. Scale bars indicate 10 μm.


( D ) Macrophages were transduced with shNS or ATG13 shRNA ( shATG13 ) analyzed for ATG13 expression . Bottom , a representative blot is shown . Top , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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(D) Macrophages were transduced with shNS or ATG13 shRNA (shATG13) and analyzed for ATG13 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( E ) Macrophages transduced with shNS or shATG13 from ( D ) were exposed to infectious HIV , 5 μg / mL ssRNA40 , 5 μg / mL ssRNA41 , or 100 nmol / L sirolimus for 24 h , harvested , lysed analyzed for endogenous LC3B SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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(E) Macrophages transduced with shNS or shATG13 from (D) were exposed to infectious HIV, 5 μg/mL ssRNA40, 5 μg/mL ssRNA41, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( F ) Macrophages were pretreated with 100 nmol / L bafilomycin A1 then exposed to mock , infectious , or RNase / DNase I treated AT - 2 - inactivated purified HIV , LyoVec , 5 μg / mL ssRNA41 , 5 μg / mL ssRNA40 , or 100 nmol / L sirolimus for 24 h , harvested , lysed analyzed for endogenous LC3B , CYP27B1 VDR by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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(F) Macrophages were pretreated with 100 nmol/L bafilomycin A1 then exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV, LyoVec, 5 μg/mL ssRNA41, 5 μg/mL ssRNA40, or 100 nmol/L sirolimus for 24 h, harvested, lysed and analyzed for endogenous LC3B, CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


Macrophages were infected with HIV harvested at 1 , 3 , 5 , 7 , 10 days post - infection . ( A ) were analyzed for endogenous LC3B SQSTM1 by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 6 .

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Macrophages were infected with HIV and cells harvested at 1, 3, 5, 7, and 10 days post-infection. (A) Cells were analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.


( B ) were harvested then subjected to flow cytometry of saponin - resistant LC3B - II in macrophages . Representative histograms of displaying saponin - resistant LC3B - II are shown .

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(B) Cells were harvested then subjected to flow cytometry analysis of saponin-resistant LC3B-II in macrophages. Representative histograms of cells displaying saponin-resistant LC3B-II are shown.


( C ) were harvested , lysed then analyzed for CYP27B1 VDR by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 6 .

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(C) Cells were harvested, lysed then analyzed for CYP27B1 and VDR by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.


( A ) Macrophages were exposed to increasing concentrations of purified HIV for 24 h , harvested , lysed , fractionated for content , analyzed for TFEB , ACTB H3 histone by Western blotting . Top , a representative blot is shown . Bottom , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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(A) Macrophages were exposed to increasing concentrations of purified HIV for 24 h, harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


( B ) Macrophages were infected with HIV at 1 , 3 , 5 , 7 , 10 days post - infection were harvested , lysed fractionated for content , analyzed for TFEB , ACTB H3 histone by Western blotting . Bottom , a representative blot is shown . Top , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 6 .

(B) Macrophages were infected with HIV and at 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 6.


C ) Representative microscopy of HIV - infected macrophages which were fixed , permeabilized then stained with 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; green ) antibody to TFEB ( red ) at indicated times post - infection . Co - localization of TFEB DAPI is represented in image by orange pixels . Scale bars indicate 20 μm .

C) Representative fluorescence microscopy images of HIV-infected macrophages which were fixed, permeabilized then stained with 4',6-diamidino-2-phenylindole (DAPI; green) and antibody to TFEB (red) at the indicated times post-infection. Co-localization of TFEB and DAPI staining is represented in the image by orange pixels. Scale bars indicate 20 μm.


( D ) Macrophages were exposed to mock , infectious , AT - 2 - inactivated , or RNase / DNase I treated AT - 2 - inactivated iodixanol velocity gradient purified HIVBa - L for 24 h , harvested , lysed fractionated for content , analyzed for TFEB , ACTB H3 histone by Western blotting . Bottom , a representative blot is shown . Top , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 3 .

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

(D) Macrophages were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated iodixanol velocity gradient purified HIVBa-L for 24 h, harvested, lysed fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 3.


( E ) Macrophages were transduced with non - specific scrambled shRNA ( shNS ) , or TLR8 shRNA ( shTLR8 ) analyzed for TLR8 expression . Bottom , a representative blot is shown . Top , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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

(E) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( F ) Macrophages transduced with shNS or shTLR8 from ( E ) were exposed to mock , infectious , AT - 2 - inactivated , or RNase / DNase I treated AT - 2 - inactivated purified HIV for 24 h . were then harvested , lysed , fractionated for content , analyzed for TFEB , ACTB H3 histone by Western blotting . Top , a representative blot is shown . Bottom , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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

(F) Macrophages transduced with shNS or shTLR8 from (E) were exposed to mock, infectious, AT-2-inactivated, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were then harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Top, a representative blot is shown. Bottom, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( A ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , or TFEB shRNA ( shTFEB ) were infected with HIV for 10 days . were lysed analyzed for TFEB ACTB content by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages transduced with non-specific scrambled shRNA (shNS), or TFEB shRNA (shTFEB) were infected with HIV for 10 days. Cells were lysed and analyzed for TFEB and ACTB content by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( B ) At 1 , 3 , 5 , 7 , 10 days post - infection from ( A ) were harvested , lysed , analyzed for endogenous LC3B SQSTM1 by Western blotting . Left , representative blots are shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

(B) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed, and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( C ) Macrophages transduced with non - specific cDNA ( Control ) , or TFEB cDNA ( TFEB ) then exposed to HIV . were lysed analyzed for TFEB ACTB content by Western blotting . Bottom , a representative blot is shown . Top , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages transduced with non-specific cDNA (Control), or TFEB cDNA (TFEB) then exposed to HIV. Cells were lysed and analyzed for TFEB and ACTB content by Western blotting. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( D ) Transduced from ( C ) were exposed to purified HIV for indicated time ( minutes ) harvested , lysed , analyzed for endogenous LC3B SQSTM1 by Western blotting . Left , representative blots are shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

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

(D) Transduced cells from (C) were exposed to purified HIV for the indicated time (minutes) harvested, lysed, and analyzed for endogenous LC3B and SQSTM1 by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( A ) qRT - PCR of mRNA expression of autophagy ( UVRAG ATG9B ) lysosomal ( MCOLN1 ) genes 24 h post - exposure to mock , infectious , or RNase / DNase I treated AT - 2 - inactivated purified HIV . Data are reported as mean ± s . e . m . , n = 4 .

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

(A) qRT-PCR analysis of mRNA expression of autophagy (UVRAG and ATG9B) and lysosomal (MCOLN1) genes 24 h post-exposure to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV. Data are reported as mean ± s.e.m., n = 4.


( B ) Macrophages were transduced with non - specific scrambled shRNA ( shNS ) , or TLR8 shRNA ( shTLR8 ) analyzed for TLR8 expression . Bottom , a representative blot is shown . Top , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

(B) Macrophages were transduced with non-specific scrambled shRNA (shNS), or TLR8 shRNA (shTLR8) and analyzed for TLR8 expression. Bottom, a representative blot is shown. Top, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( C ) Macrophages from ( B ) were exposed to mock , infectious , or RNase / DNase I treated AT - 2 - inactivated purified HIV for 24 h . were harvested qRT - PCR for UVRAG , ATG9B , MCOLN1 was performed . Data were normalized to shNS mock infected control for each gene . Bar charts are reported as mean ± s . e . m . , n = 4 .

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

(C) Macrophages from (B) were exposed to mock, infectious, or RNase/DNase I treated AT-2-inactivated purified HIV for 24 h. Cells were harvested and qRT-PCR for UVRAG, ATG9B, MCOLN1 was performed. Data were normalized to the shNS mock infected control for each gene. Bar charts are reported as mean ± s.e.m., n = 4.


( A ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , or BECN1 shRNA ( shBECN1 ) were infected with HIV for 10 days . were lysed analyzed for BECN1 ACTB content by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

(A) Macrophages transduced with non-specific scrambled shRNA (shNS), or BECN1 shRNA (shBECN1) were infected with HIV for 10 days. Cells were lysed and analyzed for BECN1 and ACTB content by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( B ) At 1 , 3 , 5 , 7 , 10 days post - infection from ( A ) were harvested , lysed analyzed for endogenous LC3B , SQSTM1 , ACTB by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( C ) At 1 , 3 , 5 , 7 , 10 days post - infection from ( A ) were harvested , lysed , fractionated for content , analyzed for TFEB , ACTB H3 histone by Western blotting . Left , a representative blot is shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) At 1, 3, 5, 7, and 10 days post-infection cells from (A) were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, a representative blot is shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( A ) Macrophages were infected with HIVNL ( AD8 ) or HIVNL ( AD8 ) ΔNef . At 1 , 3 , 5 , 7 , 10 days post - infection were harvested , lysed analyzed for endogenous LC3B , SQSTM1 , ACTB by Western blotting . Left , representative blots are shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed and analyzed for endogenous LC3B, SQSTM1, and ACTB by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


( B ) Macrophages were infected with HIVNL ( AD8 ) or HIVNL ( AD8 ) ΔNef . At 1 , 3 , 5 , 7 , 10 days post - infection were harvested , lysed , fractionated for content , analyzed for TFEB , ACTB H3 histone by Western blotting . Left , representative blots are shown . Right , densitometric of immunoblots from independent donors presented as means ± s . e . m . , n = 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were infected with HIVNL(AD8) or HIVNL(AD8)ΔNef. At 1, 3, 5, 7, and 10 days post-infection cells were harvested, lysed, fractionated for cytoplasmic and nuclear content, and analyzed for TFEB, ACTB and H3 histone by Western blotting. Left, representative blots are shown. Right, densitometric analysis of immunoblots from independent donors presented as means ± s.e.m., n = 4.


B Flow cytometry on CD49f EpCAM separates lineage - negative breast tissue into stromal ( CD49f - EpCAM - ) epithelial , which includes basal ( CD49f + EpCAMlo / - ) , luminal progenitor ( LP ) ( CD49f + EpCAM + ) mature luminal ( ML ) ( CD49f - EpCAM + ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry based on CD49f and EpCAM staining separates lineage-negative breast tissue cells into stromal (CD49f-EpCAM-) and epithelial cells, which includes basal (CD49f+EpCAMlo/-), luminal progenitor (LP) (CD49f+EpCAM+) and mature luminal (ML) (CD49f-EpCAM+) cells.


C t - SNE plot of integrated scRNA - seq profiles of epithelial from 11 reduction mammoplasties . colors correspond to tissue specimens . D Same t - SNE plot as ( C ) but separated by hormonal status of donor ( 8 premenopausal 3 postmenopausal ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE plot of the integrated scRNA-seq profiles of epithelial cells from 11 reduction mammoplasties. Cell colors correspond to tissue specimens. D Same t-SNE plot as (C) but separated by hormonal status of the donor (8 premenopausal and 3 postmenopausal).


A t - SNE map of combined scRNA - seq transcriptomes of total tissue from 13 reduction mammoplasties . colors correspond to tissue specimens . B Same t - SNE map as ( A ) but colored by cluster ( with cluster resolution 0 . 05 ) . C Ternary plot positioning each according proportion of basal , LP or ML signature genes expressed by that . plot shows same colors as for ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE map of combined scRNA-seq transcriptomes of total tissue cells from 13 reduction mammoplasties. Cell colors correspond to tissue specimens. B Same t-SNE map as (A) but colored by cell cluster (with cluster resolution 0.05). C Ternary plot positioning each cell according the proportion of basal, LP or ML signature genes expressed by that cell. The plot shows the same cells and cell colors as for (B).


D Re - clustered EPCAM - negative non - epithelial revealed seven clusters ( resolution 0 . 05 ) representing immune stromal lineages . E Multidimensional scaling plot showing expression profile distances between pseudo - bulk samples . Each dot corresponds to aggregated expression for a cluster for one . Cluster colors are overlaid from ( D , right panel ) . Distances on plot correspond to leading log2 - fold - change , defined as average log2 - fold - change for 500 most differential genes between each pair of profiles . F Heat map of pseudo - bulk samples showing marker genes for each non - epithelial cluster . top 20 marker genes were identified for each cluster by differential expression of pseudo - bulk read . Color bars at top of plot indicate cluster menopausal status ( blue / yellow for pre / post menopause ) of each sample . G Same t - SNE map of non - epithelial as in ( D ) but colored by menopausal status . Barplot shows relative cluster sizes ( of total ) for each status condition . Clusters 1 2 have significantly different sizes in post - menopause samples after allowing for inter - variability ( P = 0 . 040 P = 0 . 032 by quasi - binomial F - ) . Sizes are not significantly different for other clusters ( P > 0 . 15 ) . H Relative cluster sizes as for ( G ) but by . Cluster colors correspond to ( D ) . by cluster . A quasi - multinomial F - was used to for differences in cluster frequencies between pre - post - menopausal samples ( P = 0 . 007 ) . I Bar plots showing of expressing selected immune endothelial markers for pre - post - menopausal samples clusters identified in Fig 2D .

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

D Re-clustered EPCAM-negative non-epithelial cells revealed seven clusters (resolution 0.05) representing immune and stromal cell lineages. E Multidimensional scaling plot showing expression profile distances between the pseudo-bulk samples. Each dot corresponds to aggregated expression for a cell cluster for one patient. Cluster colors are overlaid from (D, right panel). Distances on the plot correspond to leading log2-fold-change, defined as the average log2-fold-change for the 500 most differential genes between each pair of profiles. F Heat map of pseudo-bulk samples showing marker genes for each non-epithelial cluster. The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Color bars at the top of the plot indicate the cluster and menopausal status (blue/yellow for pre/post menopause) of each sample. G Same t-SNE map of non-epithelial cells as in (D) but colored by menopausal status. Barplot shows relative cluster sizes (percentage of total cells) for each status condition. Clusters 1 and 2 have significantly different sizes in post-menopause samples after allowing for inter-patient variability (P = 0.040 and P=0.032 by quasi-binomial F-test). Sizes are not significantly different for other clusters (P > 0.15). H Relative cluster sizes as for (G) but by individual patients. Cluster colors correspond to (D). by cluster. A quasi-multinomial F-test was used to test for differences in cluster frequencies between pre- and post-menopausal samples (P = 0.007). I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre- and post-menopausal samples and the cell clusters identified in Fig 2D.


A Microenvironment t - SNE map separated by menopausal status colored by expression ( red = high expression , grey = undetectable ) of selected fibroblast markers ( upper panel ) . dotted lines indicate pericyte subsets . Bottom panel shows of expressing markers for

O O O O O O O O O O O O O O O O O O O O O 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 Microenvironment t-SNE map separated by menopausal status and colored by expression (red=high expression, grey=undetectable) of selected fibroblast markers (upper panel). The dotted lines indicate the pericyte subsets. Bottom panel shows percentage of cells expressing the markers for the cell clusters


B Co - immunofluorescence of pre - versus post - menopausal tissue for E - cadherin ( cyan ) , PDGFRβ ( yellow ) F - actin ( pink ) . DAPI is shown in grey . arrowheads in ( v ) depict fibroblasts in direct contact with myoepithelial layer . For 2D 3D confocal imaging : n = 13 premenopausal n = 10 postmenopausal specimens . Scale bars : wholemount optical sections : 100 μm ( panels i - iv ) ; enlargements , 30 μm ( panel v ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).


C t - SNE plot of integrated scRNA - seq profiles of fibroblasts from pre - post - menopausal tissue colors correspond to tissue specimens . D Same t - SNE plot as in ( C ) showing 5 distinct clusters ( clusters 3 5 were specific to one ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE plot of the integrated scRNA-seq profiles of fibroblasts from pre-and post-menopausal tissue Cell colors correspond to tissue specimens. D Same t-SNE plot as in (C) showing 5 distinct clusters (clusters 3 and 5 were specific to one patient).


F Heat map of same as ( D ) showing expression of top 20 marker genes in each cluster . Color bars at top of plot show cluster membership ( colors as in ( D ) ) pre - or post - menopausal status ( blue yellow , respectively ) .

F Heat map of same cells as (D) showing expression of the top 20 marker genes in each cluster. Color bars at the top of the plot show cluster membership (colors as in (D)) and pre- or post-menopausal status (blue and yellow, respectively).


A t - SNE map of combined scRNA - seq profiles of total isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers ( BRCA1 ; n = 4 ) non - BRCA premenopausal women ( n = 8 ) with no family history of breast cancer . colors represent samples . B Same t - SNE map as in ( A ) but colored by cluster ( cluster resolution 0 . 12 ) . C Epithelial clusters were identified by EPCAM expression non - epithelial were re - clustered to reveal immune stromal populations ( cluster resolution 0 . 08 ) . Lineage identity was determined by hierarchical clustering according to top marker

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE map of combined scRNA-seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers (BRCA1; n=4) and non-BRCA premenopausal women (n=8) with no family history of breast cancer. Cell colors represent individual samples. B Same t-SNE map as in (A) but colored by cluster (cluster resolution 0.12). C Epithelial clusters were identified by EPCAM expression and the non-epithelial cells were re-clustered to reveal immune and stromal cell populations (cluster resolution 0.08). Lineage identity was determined by hierarchical clustering according to top marker genes


D t - SNE plot showing combined single - transcriptomes of total tissue from BRCA1 preneoplastic tissue TNBCs ( n = 4 for each ) , colored according to ( B1 = preneoplastic BRCA1 + / - tissue ; TN - B1 = BRCA1 - associated TNBCs ) . E Same t - SNE map as ( D ) but colored by cluster ( cluster resolution 0 . 15 ) . F Expression of epithelial markers indicated on combined t - SNE plot as in ( D , E ) for BRCA1 preneoplastic BRCA1 - associated . G Epithelial clusters were identified by EPCAM expression non - epithelial are indicated by dotted . H Same t - SNE map as in ( D , E ) but colored according to cancerous state : preneoplastic tissue ( blue ) BRCA1 - associated TNBCs ( yellow ) . I Box plots of signature expression scores for 13 clusters in ( D , E ) according to human breast epithelial stromal gene signatures . Cluster 1 corresponds to , while clusters 6 , 9 10 represent adjacent normal LP , basal ML , respectively . Box plots show quartiles , minimum maximum .

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

D t-SNE plot showing the combined single-cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs (n=4 for each), colored according to individual patients (B1 = preneoplastic BRCA1+/- tissue; TN-B1= BRCA1-associated TNBCs). E Same t-SNE map as (D) but colored by cluster (cluster resolution 0.15). F Expression of epithelial markers indicated on the combined t-SNE plot as in (D, E) for BRCA1 preneoplastic and BRCA1-associated tumor cells. G Epithelial clusters were identified by EPCAM expression and non-epithelial cells are indicated by the dotted line. H Same t-SNE map as in (D, E) but colored according to cancerous state: preneoplastic tissue (blue) and BRCA1-associated TNBCs (yellow). I Box plots of signature expression scores for the 13 cell clusters in (D, E) according to human breast epithelial and stromal gene signatures. Cluster 1 corresponds to tumor cells, while clusters 6, 9 and 10 represent adjacent normal LP, basal and ML cells, respectively. Box plots show quartiles, minimum and maximum.


A Reclustered EPCAM - negative ( excluding clusters 1 , 6 , 9 10 revealed immune / stromal in microenvironment , identified using lineage markers . B Combined t - SNE plot as in ( A ) of single - transcriptomes of immune stromal from preneoplastic tissue of BRCA1 mutation carriers ( blue ; preneo ) BRCA1 - associated TNBCs ( yellow ; ) . C t - SNE plots showing expression profiles of cardinal markers of immune stromal . D Bar plots showing of expressing typical immune ( including Treg ) genes for clusters in ( A ) by preneoplastic vs . E Left panel , same t - SNE map as in ( A ) but separated into from preneoplastic ( blue ) specimens ( yellow ) . Endothelial ( endo ) , fibroblast ( fibro ) pericytes ( peri ) clusters are marked . Right panel , proportion of clusters as in ( A ) according to tissue type . F Relative cluster sizes as in ( E ) but by .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Reclustered EPCAM-negative cells (excluding clusters 1, 6, 9 and 10 revealed immune/stromal cells in the microenvironment, identified using lineage markers. B Combined t-SNE plot as in (A) of the single-cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers (blue; preneo) and BRCA1-associated TNBCs (yellow; tumor). C t-SNE plots showing expression profiles of cardinal markers of immune and stromal cells. D Bar plots showing the percentage of cells expressing typical immune cell (including Treg) genes for clusters in (A) by preneoplastic vs tumor. E Left panel, same t-SNE map as in (A) but separated into cells from preneoplastic (blue) and tumor specimens (yellow). Endothelial cell (endo), fibroblast cell (fibro) and pericytes (peri) clusters are marked. Right panel, proportion of clusters as in (A) according to tissue type. F Relative cluster sizes as in (E) but by individual patient.


G Co - immunofluorescence of tissue stained for epithelial marker ( cytokeratin 19 ; yellow ) - associated macrophage marker CX3CR1 ( magenta ) . DAPI is shown in ( n = 2 preneoplastic samples ; n = 2 tumors ) . Scale bar , 100 μm .

G Co-immunofluorescence of tissue stained for the epithelial marker (cytokeratin 19; yellow) and the tumor-associated macrophage marker CX3CR1 (magenta). DAPI is shown in white (n=2 preneoplastic samples; n=2 tumors). Scale bar, 100 μm.


H Heat map of top differentially expressed genes in major immune / stromal clusters , identified in ( A ) . BRCA1 + / - preneoplastic , blue ; BRCA1 - associated , yellow .

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

H Heat map of top differentially expressed genes in the major immune/stromal cell clusters, identified in (A). BRCA1+/- preneoplastic cells, blue; BRCA1-associated tumor cells, yellow.


A - C t - SNE plots of combined scRNA - seq profiles of total from 8 TNBC tumors , 6 HER2 + tumors 13 ER + tumors respectively . Integration cluster sizes for same colored by cluster ( top left panels ) , EPCAM expression ( top right ) , cancer subtype marker ( bottom left ) or MKI67 expression ( bottom right ) . Dotted lines delineate epithelial ( top panels ) cycling epithelial ( bottom panels ) . Normal epithelial subsets ( normal ) are also demarcated by dotted lines in upper - right panels of B C . D InferCNV plots to map inferred copy number variation ( CNV ) for combined scRNA - seq expression data for epithelial clusters indicated in panels A - C . scRNA - seq data from normal breast epithelial ( N - 1105 - epi ) served as a reference for normalization . Each row represents a gene each column from a cluster in a single . clusters are color - coded as in A - C . Amplifications ( red ) deletions ( blue ) are inferred from gene expression . were distinguished from normal ( N ) by abundance of CNV .

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

A-C t-SNE plots of combined scRNA-seq profiles of total cells from 8 TNBC tumors, 6 HER2+ tumors and 13 ER+ tumors respectively. Integration and cluster sizes for the same cells Cells colored by cluster (top left panels), EPCAM expression (top right), cancer subtype marker (bottom left) or MKI67 expression (bottom right). Dotted lines delineate epithelial cells (top panels) and cycling epithelial cells (bottom panels). Normal epithelial subsets (normal) are also demarcated by dotted lines in the upper-right panels of B and C. D InferCNV plots to map inferred copy number variation (CNV) for the combined tumor scRNA-seq expression data for the epithelial clusters indicated in panels A-C. scRNA-seq data from normal breast epithelial cells (N-1105-epi) served as a reference for normalization. Each row represents a gene and each column cells from a cluster in a single tumor. The tumor clusters are color-coded as in A-C. Amplifications (red) and deletions (blue) are inferred from the gene expression. Tumor cells were distinguished from normal (N) cells by abundance of CNV.


H Combined t - SNE transcriptome profiles of two distinct ER + tumors isolated from same breast of a : ER - 0029 - 7C ( blue ) ER - 0029 - 9C ( yellow ) . corresponding t - SNE clusters are shown in middle panel expression of EPCAM is shown in right - panel .

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H Combined t-SNE transcriptome profiles of two distinct ER+ tumors isolated from the same breast of a patient: ER-0029-7C (blue) and ER-0029-9C (yellow). The corresponding t-SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right-hand panel.


A - C t - SNE maps of reclustered EPCAM - negative non - epithelial Cluster resolutions 0 . 136 , 0 . 1 0 . 1 respectively . major clusters within microenvironment were identified on expression of lineage - specific genes . Heat maps of pseudo - bulk samples show marker genes for each cluster . top 30 marker genes were identified for each cluster by differential expression of pseudo - bulk read . Cluster 9 in HER2 + tumors ( B ) expressed myeloid luminal epithelial markers , suggesting phagocytosis of latter by macrophages . Color bars at top of heat map indicate cluster of each sample ; top genes that mark each cluster are indicated .

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A-C t-SNE maps of reclustered EPCAM-negative non-epithelial cells Cluster resolutions 0.136, 0.1 and 0.1 respectively. The major cell clusters within the microenvironment were identified based on expression of lineage-specific genes. Heat maps of pseudo-bulk samples show marker genes for each cluster. The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Cluster 9 in HER2+ tumors (B) expressed myeloid and luminal epithelial markers, suggesting phagocytosis of the latter by macrophages. Color bars at the top of the heat map indicate the cluster of each sample; top genes that mark each cluster are indicated.


A , B Image showing number of CD8 + Ki67 + ( A ) or CD68 + Ki67 + ( B ) per mm2 of tissue from TNBC , HER2 + ER + tumors , either within region ( K8 / 18 + ) or stroma ( defined as > 5 μm from border ) . Error bars represent s . e . m . CD8 / Ki67 : n = 11 for TNBC , n = 12 for ER + , n = 5 for HER2 + . CD68 / Ki67 : n = 6 for TNBC , n = 8 for ER + , n = 5 for HER2 + .

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A, B Image quantification showing number of CD8+Ki67+ cells (A) or CD68+Ki67+ cells (B) per mm2 of tissue from TNBC, HER2+ and ER+ tumors, either within the tumor region (K8/18+) or the stroma (defined as >5 μm from tumor border). Error bars represent s.e.m. CD8/Ki67: n=11 for TNBC, n=12 for ER+, n=5 for HER2+. CD68/Ki67: n=6 for TNBC, n=8 for ER+, n=5 for HER2+.


C - F Representative confocal of ER + ( ER - 0032 ) triple negative tumors ( TN - 0066 ) immunolabeled for K8 / 18 ( yellow ) , CD68 ( green ) Ki67 ( red ) ( C , E ) or K8 / 18 ( yellow ) , CD8 ( green ) Ki67 ( red ) ( D , F ) . DAPI is shown in blue . Arrows depict proliferative T ( CD8 + Ki67 + ) or macrophages ( CD68 + Ki67 + ) . Enlargements on shown in right - panels . Scale bars , 200 µm for large tilescans ; 50 µm for enlargements smaller tilescans .

C-F Representative confocal images of ER+ (ER-0032) and triple negative tumors ( TN-0066) immunolabeled for K8/18 (yellow), CD68 (green) and Ki67 (red) (C, E) or K8/18 (yellow), CD8 (green) and Ki67 (red) (D, F). DAPI is shown in blue. Arrows depict proliferative T cells (CD8+Ki67+) or macrophages (CD68+Ki67+). Enlargements on shown in the right-hand panels. Scale bars, 200 µm for large tilescans; 50 µm for enlargements and smaller tilescans.


C Immunostaining of LN from ER - 0064 for expression of ER , PR pan - cytokeratin . Insets show sections stained with control isotype antibodies . PR , progesterone receptor . Scale bar , 100 μm .

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C Immunostaining of tumor and LN from patient ER-0064 for expression of ER, PR and pan-cytokeratin. Insets show sections stained with control isotype antibodies. PR, progesterone receptor. Scale bar, 100 μm.


C HEK293 FT were transfected with expression plasmids for indicated . were lysed subjected to immunoprecipitation with anti - Flag antibodies , then immunoprecipitates aliquots of lysate were examined by Western blotting as indicated .

C HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated.


C were prepared by in vitro translation using reticulocyte lysates analyzed by αFlag western blotting .

C Proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.


In vitro translation / biotinylation reactions using reticulocyte lysates indicated constructs ( E , Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs (E, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


In vitro translation / biotinylation reactions using reticulocyte lysates indicated constructs F , Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs F, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


In vitro translation / biotinylation reactions using reticulocyte lysates indicated constructs Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


In vitro translation / biotinylation reactions using reticulocyte lysates indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


J In vitro translation ( IVT ) / biotinylation reactions using Hela lysates indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated . Bottom , indicated were prepared by in vitro translation using Hela lysates analyzed by αFlag western blotting .

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

J In vitro translation (IVT)/biotinylation reactions using Hela cell lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein. Bottom, the indicated proteins were prepared by in vitro translation using Hela cell lysates and analyzed by αFlag western blotting.


C - F Mouse kidney was lysed , treated with or without RNase I , was isolated . was reverse transcribed with indicated primers for ( C , E ) Hifa ( D , F ) Xbp1u mRNA , then analyzed by Real Time PCR for disomes using indicated primers . ∆ CT = ( ∆ CT = CT - RNase - CT + RNase ) . Biological replicates ( from three ) , with mean ± SD are shown . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - t - .

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

C-F Mouse kidney was lysed, treated with or without RNase I, and RNA was isolated. The RNA was reverse transcribed with the indicated primers for (C,E) Hifa and (D,F) Xbp1u mRNA, and then analyzed by Real Time PCR for disomes using the indicated primers. ∆CT = (∆CT = CT-RNase - CT+RNase). Biological replicates (from three males), with mean ± SD are shown. * P < 0.05, ** P < 0.01, by unpaired two-tailed t-test.


A HEK293 FT were transfected with expression plasmids for indicated . were lysed subjected to immunoprecipitation with anti - Flag antibodies , then immunoprecipitates aliquots of lysate were examined by Western blotting as indicated . ZFmut = C36S / C42S .

O O O O O O O O O O O O O O O O O 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 HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated. ZFmut = C36S/C42S.


B In vitro translation / biotinylation reactions using reticulocyte lysates indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated substrate . Bottom , indicated were prepared by in vitro translation using reticulocyte lysates analyzed by αFlag western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated substrate. Bottom, the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.


C HEK293 FT were transfected with expression plasmids for indicated . Tib = Tibetan . were lysed subjected to immunoprecipitation with anti - Flag antibodies , then immunoprecipitates aliquots of lysate were examined by Western blotting as indicated .

O O O O O O O O O O O O O O O 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 HEK293 FT cells were transfected with expression plasmids for the indicated proteins. Tib = Tibetan. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated.


E In vitro translation / biotinylation reactions using reticulocyte lysates construct 1 ( left ) or construct 7 ( right ) . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated substrate . Bottom , indicated were prepared by in vitro translation using reticulocyte lysates analyzed by αFlag western blotting .

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

E In vitro translation/biotinylation reactions using reticulocyte lysates and construct 1 (left) or construct 7 (right). Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated substrate. Bottom, the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.


C , Olaparib ETC - 159 synergize to prevent growth of HPAF - II xenografts in mice . NSG mice with established HPAF - II subcutaneous xenografts were randomized into four groups . Mice were gavaged daily with ETC - 159 ( 10 mg / kg ) , Olaparib ( 50 mg / kg ) or a combination of ETC - 159 ( 10 mg / kg ) Olaparib ( 50 mg / kg ) . Treatment was initiated after HPAF - II tumors were established . ( C ) volumes were measured starting from day 0 during course of treatment as shown . Data points represent mean ± SD . n = 7 - 8 tumors / group . p - values indicate significant difference compared to vehicle group .

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

C, Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (C) Tumor volumes were measured starting from day 0 and during the course of treatment as shown. Data points represent the mean ± SD. n = 7-8 tumors/group. p-values indicate significant difference compared to the vehicle group.


repair genes involved in HR FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF - II orthotopic xenografts . Comparison of log2 fold change ( FDR < 10 % ) in expression of Wnt - activated genes over time across multiple repair pathways shows that genes regulating HR FA pathways have higher fold changes compared to genes regulating BER , NER or other pathways . BER : base excision repair ; HR : homologous recombination ; FA : Fanconi anemia ; MMR : mismatch repair ; NER : nucleotide excision repair ; NHEJ : non - homologous end joining ; TLS : translesion synthesis . horizontal represents median of 4 - 6 replicates with upper lower edges of box representing 75th 25th percentile of data respectively whiskers representing 1 . 5 x interquartile range .

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

DNA repair genes involved in HR and FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF-II orthotopic xenografts. Comparison of log2 fold change (FDR < 10%) in the expression of Wnt-activated genes over time across multiple DNA repair pathways shows that genes regulating HR and FA pathways have higher fold changes compared to genes regulating BER, NER or other pathways. BER: base excision repair; HR: homologous recombination; FA: Fanconi anemia; MMR: mismatch repair; NER: nucleotide excision repair; NHEJ: non-homologous end joining; TLS: translesion DNA synthesis. The horizontal line represents median of 4-6 replicates with the upper and the lower edges of the box representing the 75th and 25th percentile of the data respectively and the whiskers representing 1.5 x interquartile range.