A . N - linked glycans of ΔPrPs are not - type . Immunoblot comparing EndoH - non - digested lysates of ΔPrP159 Δ159 - 175 transfected . Left panel was probed with mAb 3F4 mAb , detecting transiently expressed ΔPrPs . On right panel , was re - probed with mAb 4H11 , detecting total PrP . Arrowheads depict deglycosylated ΔPrPs .

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A. N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159-175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs.


B . additional band of Δ159 - 167 represents a third N - glycan sensitive to EndoH . Upon EndoH digestion , bands converge into a non - glycoform band ( lane 3 4 ) . In Δ159 - 167 ( 169 ) third glycosylation site is not present anymore .

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B. The additional band of Δ159-167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159-167(169) the third glycosylation site is not present anymore.


C . ΔPrPs are mainly distributed in intracellular compartments rather than on surface . Confocal microscopy of N2a transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , either permeabilized ( right ) or non - permeabilized ( left ) with 0 . 2 % TX100 after fixation . were immuno - labeled with mAb 3F4 . ( 3F4 ) MoPrP - expressing show bright immunopositivity on surface , whereas ΔPrPs are not observed on surface . Also intracellular differs strongly between ( 3F4 ) MoPrP ΔPrP expressing Scale bars , 25 µm .

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C. ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.


A . ΔPrPs are insoluble in detergent solution . Immunoblots showing partition of Δ159 Δ159 - 175 in supernatant ( Sup ) pellet fraction after ultracentrifugation of lysates supplemented with 4 % sarcosyl . Upper panel is mAb 3F4 , lower panel is re - probed with mAb 4H11 detecting also endogenous wild - type PrP besides ΔPrPs . majority of ΔPrPs is found in insoluble fraction in both infected non - infected ( upper panel , lanes 3 , 4 , 7 8 ) . Wild - type PrP almost completely partitions into soluble fraction ( Sup ) in non - infected N2a with present conditions . PrP in pellet fractions of non - infected N2a is from ΔPrPs as can be seen from banding pattern ( Lane 7 8 , square bracket ) . PrP in lanes 3 4 ( lower panel ) is PrP27 - 30 .

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A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159-175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non-infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30.


B . ΔPrPs are moderately resistant to chymotrypsin digestion . Immunoblots showing chymotrypsin - resistant fragments of Δ159 Δ159 - 175 from transiently transfected N2a lysates digested with indicated concentrations of chymotrypsin ( Chy ) . Note that full - diglycoform of ΔPrP still remains at 8 µg / ml Chy ( lanes 5 10 , upper panel , closed arrowhead ) , whereas that of endogenous wild - type PrP is completely digested ( lanes 5 10 , lower panel , open arrowhead ) only small amounts of truncated fragments remained ( curly bracket ) . Other smaller fragments in lane 5 10 ( square - brackets ) represent ΔPrPs .

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B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159-175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.


A . ΔPrPs are substantially soluble in detergent without GdnHCl denaturation . Immunoblots probed with mAbs 3F4 ( left ) or 4H11 ( right , re - probed ) showing ΔPrPs endogenous PrP in detergent aqueous phases of TX114 lysates prepared from N2a transfected with Δ159 or Δ159 - 175 . Det denotes detergent phase , Aq aqueous phase .

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A. ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159-175. Det denotes detergent phase, Aq aqueous phase.


B . ΔPrPs are highly hydrophobic after denaturation with GdnHCl . Representative immunoblot of TX114 - treated lysates after denaturation with 3 M GdnHCl developed with mAb 3F4 showing Δ159 Δ159 - 175 almost exclusively in detergent phase ( lanes 1 - 4 ) . PIPLC treatment ( + , even lanes ) was done after denaturation with GdnHCl or not ( − , uneven lanes ) . PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions .

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B. ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159-175 almost exclusively in detergent phase (lanes 1-4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions.


C . Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion . After further diluting out GdnHCl by phase - separation removal of aqueous phase containing GdnHCl , PIPLC was able to digest ΔPrPs . Left two panels ( upper mAb 3F4 , lower 4H11 ) are shorter expositions , right panels longer expositions to show also difference in PrP in detergent phase . Under these conditions a substantial reduction of ΔPrPs in detergent phase was achieved by PIPLC digestion ( e . g . upper - left panel , lanes 1 , 3 vs . 2 , 4 ) . Migration of ΔPrPs in aqueous phase of PIPLC - digested samples was slightly slower than that of PIPLC - undigested samples ( − ) ( upper - right panel , lane 9 vs . 10 ) .

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C. Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).


A . Degradation of ΔPrPs by lysosomal proteasomal systems . ( Left ) N2a transiently transfected with Δ159 or Δ159 - 175 were treated for 7 hours with inhibitors either for lysosomal degradation ( bafilomycin A1 , Baf , 120 nM ) , autophagy ( 3 - methyladenine , 3MA , 10 mM ) , or proteasome ( MG132 , 10 µM ) lysates analyzed in immunoblot for expression of Δ159 Δ159 - 175 ( mAb 3F4 ) . Square bracket bracket denote diglycoforms nonglycoforms of ΔPrPs , respectively . An increment in diglycoforms is observed in Baf - 3MA - treated , whereas in MG132 - treated also non - glycoforms are increased . ( Right ) A graph showing of PrP bands by densitometric . Data from 3 independent ( one in duplicate ) experiments for Δ159 2 independent ( one in duplicate ) for Δ159 - 175 were statistically analyzed for mean standard deviation ( error bars ) .

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A. Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159-175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159-175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159-175 were statistically analyzed for mean and standard deviation (error bars).


B . ΔPrPs can be localized in LAMP1 - immunopositive vesicles . Immunofluorescence shows distribution of ΔPrP ( mAb 3F4 ) LAMP1 in with or without 6M GdnHCl antigen - retrieval treatment . N2a transfected with ΔPrP159 were fixed , permeabilized incubated without ( upper panel ) or with 6M GdnHCl ( lower panel ) . With GndHCl treatment ΔPrP is diffusely distributed ( arrowheads ) co - localizes poorly with LAMP1 . After GdnHCl treatment ( lower panel ) , bright 3F4 - immunopositive ( arrows ) are observed which partly co - localize with LAMP1 - positive structures ( merge ) . Scale bars , 10 µm .

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B. ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.


A . DNI efficiencies of ΔPrPs are inversely correlated to deletion . Representative immunoblot showing PK - resistant PrPSc moiety of ( 3F4 ) MoPrP co - expressed with indicated ΔPrPs in 22L - ScN2a . Quantitative is shown below as scheme . Relative PrPSc were calculated as proportion of PK - resistant ( 3F4 ) MoPrP co - transfected with each ΔPrP to that of co - transfected with empty vector control in same experiment . Data from 4 ( for Δ159 - 165 , Δ159 - 167 Δ159 - 167 ( 169 ) only 3 ) independent experiments were statistically analyzed for mean standard deviation ( error bars ) .

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A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4)MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4)MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159-165, Δ159-167 and Δ159-167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars).


B . DNI of ΔPrP with a deletion in C - terminal part of H1 ∼ H2 . Δ171 - 175 ( see Fig . 7A ) was transiently expressed in N2a ( left panel ) DNI efficiency evaluated in 22L - ScN2a ( right panel ) ( mAb 3F4 ) . Despite much shorter deletion , Δ171 - 175 exerted only inefficient DNI , similar to Δ159 - 175 ( right panel ) .

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B. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171-175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171-175 exerted only inefficient DNI, similar to Δ159-175 (right panel).


. B . Δ31 - 160 variants are expressed at comparable have EndoH - sensitive - resistant N - glycans . Immunoblot comparing EndoH - digested non - digested samples from N2a transiently transfected with Δ31 - 160 variants , probed with anti - PrP mAb C16 - S raised against C - terminal portion of H3 . Non - Tf , samples prepared from N2a without transfection . Arrowhead denotes deglycosylated Δ159 ( lane 11 ) , bracket deglycosylated fragments , square bracket EndoH - resistant fragments . Asterisks indicate non - specific bands . Δ31 - 160 variants are not completely deglycosylated by EndoH , resulting in EndoH - resistant fragments ( square bracket ) which are observed also in reducing conditions ( right panel ; + / − dithiothreitol ( DTT ) treatment ) . Note that endogenous wild - type PrPc was not detected under used conditions .

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. B. Δ31-160 variants are expressed at comparable levels and have EndoH-sensitive and -resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31-160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31-160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrPc was not detected under used conditions.


C . DNI of Δ31 - 160 variants is similarly inversely related to of deletion dependent on intact C - terminal H1 - H2 portion . Immunoblot probed with mAb 3F4 showing PK - resistant ( 3F4 ) MoPrP co - transfected with indicated constructs into 22L - ScN2a ( left panel ) . Δ31 - 160 was slightly more effective than Δ159 . Right panel shows of results as obtained from a triplicate experiment , using densitometry on ImageJ . Bars illustrate mean ± standard deviation . * , p < 0 . 05 .

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C. DNI of Δ31-160 variants is similarly inversely related to size of deletion and dependent on intact C-terminal H1-H2 portion. Immunoblot probed with mAb 3F4 showing PK-resistant (3F4)MoPrP co-transfected with indicated constructs into 22L-ScN2a cells (left panel). Δ31-160 was slightly more effective than Δ159. Right panel shows quantification of results as obtained from a triplicate experiment, using densitometry on ImageJ. Bars illustrate mean ± standard deviation. *, p<0.05.


D . Δ31 - 160 forms spontaneous with moderate chymotrypsin ( Chy ) resistance . Immunoblots probed with mAbs C16 - S ( upper panel ) or 3F4 ( lower panel ) for comparing Chy - resistant fragments of Δ31 - 160 with that of Δ159 . Lysates from N2a expressing Δ159 or Δ31 - 160 were digested with indicated concentrations of Chy for 30 minutes then digested with PNGaseF . A sample from non - transfected N2a is shown in lane 5 . Asterisks indicate non - specific C16 - S bands . Arrowhead denotes deglycoform of Δ159 , bracket deglycoform of Δ31 - 160 .

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D. Δ31-160 forms spontaneous aggregates with moderate chymotrypsin (Chy) resistance. Immunoblots probed with mAbs C16-S (upper panel) or 3F4 (lower panel) for comparing Chy-resistant fragments of Δ31-160 with that of Δ159. Lysates from N2a cells expressing Δ159 or Δ31-160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF. A sample from non-transfected N2a cells is shown in the lane 5. Asterisks indicate non-specific C16-S bands. Arrowhead denotes deglycoform of Δ159, bracket deglycoform of Δ31-160.


B ( Top ) Semiquantitative RT - PCR of UPF1SL or UPF1LL transcript following transfection of HEK - 293 with a NT siRNA or a siRNA that specifically targets UPF1LL isoform . ( Bottom ) Venn diagram ( to scale ) of overlapping targets identified from - seq following total UPF1 or UPF1LL - specific knockdown . Depicted are genes that increased in abundance at least 1 . 4 - fold ( FDR < 0 . 05 ) met read cutoffs in both datasets . P value indicates enrichment of genes that increased in abundance at least 1 . 4 - fold ( FDR < 0 . 05 ) with UPF1LL - specific knockdown among those regulated by total UPF1 , as determined by Fisher ' s exact .

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B (Top) Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with a NT siRNA or a siRNA that specifically targets the UPF1LL isoform. (Bottom) Venn diagram (to scale) of overlapping targets identified from RNA-seq following total UPF1 or UPF1LL-specific knockdown. Depicted are genes that increased in abundance at least 1.4-fold (FDR < 0.05) and met read count cutoffs in both datasets. P value indicates enrichment of genes that increased in abundance at least 1.4-fold (FDR < 0.05) with UPF1LL-specific knockdown among those regulated by total UPF1, as determined by Fisher's exact test.


C Heat map of changes in relative mRNA abundance for genes encoding NMD factors , as determined from - seq following transfection of HEK - 293 with a siRNA that targets both UPF1 isoforms ( UPF1total ) or a siRNA that specifically targets UPF1LL isoform .

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C Heat map of changes in relative mRNA abundance for genes encoding NMD factors, as determined from RNA-seq following transfection of HEK-293 cells with a siRNA that targets both UPF1 isoforms (UPF1total) or a siRNA that specifically targets the UPF1LL isoform.


D Density plot of changes in relative mRNA abundance as determined by - seq in SMG7ko / SMG6kd , relative to a parental treated with control siRNAs 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 .

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D Density plot of changes in relative mRNA abundance as determined by RNA-seq in SMG7ko/SMG6kd cells, relative to a parental cell line treated with control siRNAs 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.


E RT - qPCR of indicated transcripts following transfection of HEK - 293 with indicated siRNAs . Relative fold changes are in reference to NT siRNA . 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 indicates log2 ( fold change ) of + 0 . 5 . PTC + indicates use of primers specific to transcript isoforms with validated poison

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E RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with the indicated siRNAs. Relative fold changes are in reference to NT siRNA. 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 line indicates log2(fold change) of +0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


F Gene ontology of 1621 genes that increased in expression at least 1 . 4 - fold upon UPF1LL - specific knockdown in HEK - 293 under normal conditions . Genes may map to multiple categories .

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F Gene ontology analysis of 1621 genes that increased in expression at least 1.4-fold upon UPF1LL-specific knockdown in HEK-293 cells under normal cellular conditions. Genes may map to multiple categories.


B Scatterplot of CLIP - UPF1LL vs CLIP - UPF1SL RIP - seq efficiency . mRNAs were binned according to 3 ' UTR ( short , medium , or long ) .

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B Scatterplot of CLIP-UPF1LL vs CLIP-UPF1SL RIP-seq efficiency. mRNAs were binned according to 3'UTR length (short, medium, or long).


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

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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 .

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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 .

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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

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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

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

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 .

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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 .

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(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 .

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(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 .

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(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 .

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(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 - .

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(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 .

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(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 .

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(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 .

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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 .

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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 =

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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 .

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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 .

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

(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 .

O O O O B-EXP_ASSAY O B-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM B-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

(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 .

O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

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

(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 .

B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O

(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 .

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

(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 .

O O O B-CELL_TYPE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(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 .

O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(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 .

O O O B-EXP_ASSAY O B-ORGANISM O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O

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

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

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).