E , F . Spermatocytes were immunolabeled against beta - catenin ( green ) , SYCP3 ( red ) , PCNT ( magenta ) , and stained with DAPI ( blue ) . Middle columns emphasize beta - catenin signal in diplonema and diakinesis spermatocytes . Scale bars = 5 μm . G . Quantification of beta - catenin positive centrosomes per spermatocyte at diakinesis . Immunolabeling was performed on three biological replicates , with ≥ 20 spermatocytes quantified per replicate . The total number of cells quantified for control , Aurka cKO , Plk1 cHet , and Plk1 cKO mice were 89 , 106 , 122 , and 93 , respectively .

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E, F. Spermatocytes were immunolabeled against beta-catenin (green), SYCP3 (red), PCNT (magenta), and stained with DAPI (blue). Middle columns emphasize beta-catenin signal in diplonema and diakinesis spermatocytes. Scale bars = 5 μm. G. Quantification of beta-catenin positive centrosomes per spermatocyte at diakinesis. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 89, 106, 122, and 93, respectively.


A . Spermatocytes were immunolabeled against PCNT ( red ) , CETN3 ( green ) , and SYCP3 ( blue ) , and stained with DAPI ( upper right inset ) . Zoomed images of the centrosome are shown in the bottom left inset . Scale bars = 5 μm .

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A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.


B . Quantification of the number of CETN3 foci per spermatocyte ( n = 30 ) at each meiotic prophase substage .

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B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.


C . Centrosome ( PCNT ) separation was assessed in Aurka ; Plk1 dKO spermatocytes . Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation

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C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications


( E ) Fold increase of Oct4 - GFP + colony number over control under forced expression of individual factors . n ≥ 5 , where each dot indicates an independent experiment . Box - plots show median , 1st and 3rd quartile values . One - sample Wilcoxon test p - values are as indicated , with p values < 0 . 05 shown in

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(E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red


( B ) Heatmap of average gene expression normalised to β - actin over an EpiSC resetting time course in 2i + LIF . Each row is coloured according to the unique minimum and maximum for that gene . The values shown are average expression of four independent

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(B) Heatmap of average gene expression normalised to β-actin over an EpiSC resetting time course in 2i+LIF. Each row is coloured according to the unique minimum and maximum for that gene. The values shown are average expression of four independent experiments


( C ) Gene expression for Stat3 , Klf2 , Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs . β - actin serves as an internal control . Mean + / - SEM , n = 4 independent experiments . * = p < 0 . 05 Student " s

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(C) Gene expression for Stat3, Klf2, Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs. β-actin serves as an internal control. Mean +/- SEM, n=4 independent experiments. * = p<0.05 Student"s t


( E ) Representative confocal images ( top ) and quantification ( bottom ) of Oct4 - GFP reporter mean intensity . The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6 . Mean + / - SD of 9 technical replicates . One representative experiment of two is shown . Scale bars =

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(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm


( F ) Representative alkaline phosphatase ( AP ) staining images ( left ) and quantification ( right ) of AP + colonies after clonal replating , as described in

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(F) Representative alkaline phosphatase (AP) staining images (left) and quantification (right) of AP+ colonies after clonal replating, as described in panel


( A ) Top : Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 ( + E / T ) . Bottom : Gene expression of EpiSCs harbouring empty vector ( grey ) or Esrrb / Tbx3 ( orange ) , capture at D0 ( F / A ) , D2 and D4 ( as described in Fig 3D ) . Dashed black line : expression levels in ESCs maintained in 2i + LIF . Data normalised to empty vector cultures in F / A . Gapdh serves as an internal control . Mean + / - SEM , n = 3 independent experiments . ( B ) As for panel A , comparison of control with dual expression of Esrrb and Klf4 ( + E / K , green in bottom plot ) . Mean + / - SEM , n = 3 independent experiments for bottom qRT - PCR

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(A) Top: Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 (+E/T). Bottom: Gene expression of EpiSCs harbouring empty vector (grey) or Esrrb/Tbx3 (orange), capture at D0 (F/A), D2 and D4 (as described in Fig 3D). Dashed black line: expression levels in ESCs maintained in 2i+LIF. Data normalised to empty vector cultures in F/A. Gapdh serves as an internal control. Mean +/- SEM, n=3 independent experiments. (B) As for panel A, comparison of control with dual expression of Esrrb and Klf4 (+E/K, green in bottom plot). Mean +/- SEM, n=3 independent experiments for bottom qRT-PCR panels


( C ) Left : Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb - T2A - Klf4 construct and cultured in 2i + LIF with DOX for 2 and 4 days , with the indicated fraction of cells sorted for colony formation assay . Since the Venus reporter is under the control of a DOX responsive element , and the emission spectra of Venus and GFP fluorescence overlap , Oct4 - GFP reporter could not be fully distinguished from Venus expression . Right : Number of AP + colonies formed from 250 sorted cells from indicated fractions . Data points represent two technical replicates of one out of two independent

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(C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments


( D ) Heatmap of single - cell expression measured by qRT - PCR of major ESC and EpiSC markers in un - induced EpiSCs ( black ) , established ESCs ( red ) , Day 2 High / Low ( dark and light blue ) , and Day 4 High cells ( green )

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(D) Heatmap of single-cell expression measured by qRT-PCR of major ESC and EpiSC markers in un-induced EpiSCs (black), established ESCs (red), Day 2 High/Low (dark and light blue), and Day 4 High cells (green)


( E ) Scatterplots of the relative expression of pluripotency and transitional markers . Red bar corresponds to median gene expression , and each dot represents a single

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(E) Scatterplots of the relative expression of pluripotency and transitional markers. Red bar corresponds to median gene expression, and each dot represents a single cell


Left : 0 . 782 cABN predictions of the sequence of gene activation between gene pairs ( white , OFF ; blue , ON ) along the resetting trajectory , compared to single cell gene expression measured by RT - qPCR . Each table summarises the percentage of single cells at the indicated stage of resetting ( columns ) that have the indicated expression state ( rows ) . Right : Scatterplots showing single cell coordinates based on the expression of the gene

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

Left: 0.782 cABN predictions of the sequence of gene activation between gene pairs (white, OFF; blue, ON) along the resetting trajectory, compared to single cell gene expression measured by RT-qPCR. Each table summarises the percentage of single cells at the indicated stage of resetting (columns) that have the indicated expression state (rows). Right: Scatterplots showing single cell coordinates based on the expression of the gene pair


( A ) 0 . 782 cABN predictions of essential or dispensable factors for EpiSC resetting , compared with the experiment results shown in panel ( B ) . ( B ) siRNA knockdown effects measured by Oct4 - GFP + colony formation at Day 6 of resetting . n = 4 independent

(A) 0.782 cABN predictions of essential or dispensable factors for EpiSC resetting, compared with the experiment results shown in panel (B). (B) siRNA knockdown effects measured by Oct4-GFP+ colony formation at Day 6 of resetting. n=4 independent experiments


( C ) Left , resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4 - GFP + colony formation at Day 6 of resetting . Data points represent two independent experiments . Right , representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i +

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(C) Left, resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4-GFP+ colony formation at Day 6 of resetting. Data points represent two independent experiments. Right, representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i+LIF


( D ) Expression of naïve pluripotency , transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i + LIF . Expression is normalised to wild type EpiSCs in A / F , and β - actin was used as internal control . The values shown correspond to the average expression of three technical replicates from one representative experiment out of

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of naïve pluripotency, transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i+LIF. Expression is normalised to wild type EpiSCs in A/F, and β-actin was used as internal control. The values shown correspond to the average expression of three technical replicates from one representative experiment out of two


( E ) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4 - GFP + colony formation at Day 6 of resetting in 2i + LIF . Data points represent two independent

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(E) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4-GFP+ colony formation at Day 6 of resetting in 2i+LIF. Data points represent two independent experiments


( F ) Comparison between the effect of single factor knockdowns on ESC maintenance ( n = 4 independent experiments ) and EpiSC resetting using experimental

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(F) Comparison between the effect of single factor knockdowns on ESC maintenance (n=4 independent experiments) and EpiSC resetting using experimental results


( G ) EpiSC resetting in 2i + LIF measured by Oct4 - GFP + colony formation after Stat3 siRNA in wild type EpiSCs ( left ) , or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs . n = 3 independent experiments in wild type cells ( box plots indicate min , median , max ) , n = 2 for Klf4 - / - cells ( box plots indicate min , mean , max )

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(G) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation after Stat3 siRNA in wild type EpiSCs (left), or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs. n=3 independent experiments in wild type cells (box plots indicate min, median, max), n=2 for Klf4-/- cells (box plots indicate min, mean, max)


( H ) EpiSC resetting in 2i + LIF measured by Oct4 - GFP + colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1 , Gbx2 and Klf4 . n = 4 independent experiments : Student " s t - test , p - value indicated on plot . Box plots indicate min , median , max . See also Appendix

(H) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1, Gbx2 and Klf4. n=4 independent experiments: Student"s t-test, p-value indicated on plot. Box plots indicate min, median, max. See also Appendix Fi


( B ) Comparison of predictions ( left ) and experimental outcome ( right ) for the potency of additional network factors in OSKM - driven MEF reprogramming in LIF containing medium n = 4 independent experiments , except Klf2 and Nanog where n = 2 independent experiments . * = p < 0 . 05 Student " s t - test . n . s . = not significant . Red dashed lines indicate empty vector + OSKM ( C ) Comparison of predictions ( left ) and experimental outcome ( right ) for the potency of additional network factors in OSKM - driven MEF reprogramming in 2i + LIF . n = 4 independent experiments , except Klf2 and Nanog where n = 2 independent experiments . * = p < 0 . 05 Student " s t - test . n . s . = not significant . Empty vector + OSKM reprogramming in LIF ( ' control no 2i ' ) was included as a control for the effect of 2i addition . Red dashed lines indicate empty vector + OSKM control level . Box plots indicate min ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in LIF containing medium n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Red dashed lines indicate empty vector + OSKM (C) Comparison of predictions (left) and experimental outcome (right) for the potency of additional network factors in OSKM-driven MEF reprogramming in 2i+LIF. n=4 independent experiments, except Klf2 and Nanog where n=2 independent experiments. * = p<0.05 Student"s t-test. n.s. = not significant. Empty vector + OSKM reprogramming in LIF ('control no 2i') was included as a control for the effect of 2i addition. Red dashed lines indicate empty vector + OSKM control level. Box plots indicate min, median


( D ) Recapitulation of the gene activation kinetics during MEF reprogramming . Top , the number of regulation steps required for permanent activation of the indicated gene . Tfcp2l1 and Sall4 are found to activate earlier than Nanog and Esrrb . Bottom , gene expression measured from sorted populations of reprogramming intermediates from O " Malley et al . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Recapitulation of the gene activation kinetics during MEF reprogramming. Top, the number of regulation steps required for permanent activation of the indicated gene. Tfcp2l1 and Sall4 are found to activate earlier than Nanog and Esrrb. Bottom, gene expression measured from sorted populations of reprogramming intermediates from O"Malley et al. (


Coomassie stained SDS - PAGE of McjD - C547 reconstituted in liposomes . The amount reconstituted was evaluated against detergent purified

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Coomassie stained SDS-PAGE of McjD-C547 reconstituted in liposomes. The amount reconstituted was evaluated against detergent purified protein


Chromatogram of McjD C547 ( black ) labelled with Alexa 555 ( blue ) and Alexa 647 ( red ) maleimide fluorophore using a Superdex 200 size - exclusion column . The fraction containing the highest degree of labeling is marked by a

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

Chromatogram of McjD C547 (black) labelled with Alexa 555 (blue) and Alexa 647 (red) maleimide fluorophore using a Superdex 200 size-exclusion column. The fraction containing the highest degree of labeling is marked by a square


The ATPase activity of all labelled variants C547 , Y64C , L67C was determined in liposomes and compared to wild - type , wt . All labelled McjD variants show basal - and ligand - induced ATPase activities in liposomes comparable to that of wild type protein , indicating that labelling does not interfere with their activity . Error bars were calculated from two replicates from two independent reconstitutions ( mean ± standard deviation , n = 2 ) . The ruler character of FRET using the fluorophore pair Alexa555 / Cy5 was supported by data of dsDNA in Figure EV1B , where different base - pair distances between both dyes correlate directly with the apparent FRET

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

The ATPase activity of all labelled variants C547, Y64C, L67C was determined in liposomes and compared to wild-type, wt. All labelled McjD variants show basal- and ligand-induced ATPase activities in liposomes comparable to that of wild type protein, indicating that labelling does not interfere with their activity. Error bars were calculated from two replicates from two independent reconstitutions (mean ± standard deviation, n=2). The ruler character of FRET using the fluorophore pair Alexa555/Cy5 was supported by data of dsDNA in Figure EV1B, where different base-pair distances between both dyes correlate directly with the apparent FRET efficiency


Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicated


Apparent ligand binding affinity values of McjD for AMPPNP ( top ) , ATP ( middle ) , and ADP - vanadate ( bottom ) . Data points were obtained from the areas of fits from the E * histograms considering the ratio ligand - free / ( ligand - free + ligand - bound ) at the indicated substrate concentrations . Extraction of Kd values from a fit to the data is described in the Material and Methods section . Data for detergent - solubilized McjD for additional biochemical conditions is shown in Figure

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

Apparent ligand binding affinity values of McjD for AMPPNP (top), ATP (middle), and ADP-vanadate (bottom). Data points were obtained from the areas of fits from the E* histograms considering the ratio ligand-free/(ligand-free+ligand-bound) at the indicated substrate concentrations. Extraction of Kd values from a fit to the data is described in the Material and Methods section. Data for detergent-solubilized McjD for additional biochemical conditions is shown in Figure EV2


Representative fluorescence time traces ( blue , donor signal ; red , acceptor signal ; black , FRET signal ; yellow , fit ) of McjD labeled with Alexa555 and Alexa647 - maleimide with indicated concentrations of corresponding substrate : ( B ) McjD L67C with 10 mM ATP and 200 µM MccJ25 , McjD Y64C with 10 mM ATP and 200 µM MccJ25 , and McjD C547 with 10 mM ATP . The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant . We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted . Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to

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

Representative fluorescence time traces (blue, donor signal; red, acceptor signal; black, FRET signal; yellow, fit) of McjD labeled with Alexa555 and Alexa647-maleimide with indicated concentrations of corresponding substrate: (B) McjD L67C with 10 mM ATP and 200 µM MccJ25, McjD Y64C with 10 mM ATP and 200 µM MccJ25, and McjD C547 with 10 mM ATP. The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant. We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted. Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to detergent


Representative FRET time traces ( black , FRET signal ; yellow , fit ) of McjD NBD C547 labeled with Alexa 555 and Alexa 647 - maleimide under apo conditions at 10 ms time resolution . Approximately ~ 85 % of all apo - McjD traces show no significant fluctuations beyond shot - noise . Apo - McjD is predominantly in the low FRET and thus open state with E * values < 0 . 3 Within the complete data set of apo - McjD ( N = 80 traces ) comprising donor and acceptor , ~ 15 % , show infrequent fluctuations to a higher FRET efficiency state with a lifetime of ( C ) 82 ± 25 ms where E * values > 0 . 5 are observed during short dwells . The data set represents a total recording time of 4 . 6 mins with a temporal resolution of 10 ms . To exclude unwanted influence on McjD due to surface - immobilization , experimental verification is provided that ATP - induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure

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

Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide under apo conditions at 10 ms time resolution. Approximately ~85% of all apo-McjD traces show no significant fluctuations beyond shot-noise. Apo-McjD is predominantly in the low FRET and thus open state with E* values <0.3 Within the complete data set of apo-McjD (N = 80 traces) comprising donor and acceptor, ~15%, show infrequent fluctuations to a higher FRET efficiency state with a lifetime of (C) 82 ± 25 ms where E* values > 0.5 are observed during short dwells. The data set represents a total recording time of 4.6 mins with a temporal resolution of 10 ms. To exclude unwanted influence on McjD due to surface-immobilization, experimental verification is provided that ATP-induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure EV4


Representative FRET time traces ( black , FRET signal ; yellow , fit ) of McjD NBD C547 labeled with Alexa 555 and Alexa 647 - maleimide in apo conditions at 10 ms time resolution . This panel shows stable ATP - loaded ( left ) and ATP - free ( right ) McjD Representative FRET time traces that show switching between ATP free McjD ( E * ~ 0 . 3 ) and ATP - loaded ( high FRET > 0 . 5 ) at ~ 4s s ( left ) and ~ 1 . 4 s ( right ) . The traces show photobleaching events of the acceptor after ~ 10 s ( left ) and ~ 3 . 3 s ( right ) Representative FRET time traces that show switching between ATP - loaded ( high FRET > 0 . 5 ) and ATP free McjD ( E * ~ 0 . 3 ) at 2 s ( left ) and 5 s ( right ) . The traces show photobleaching events after ca . 40 s ( left , donor - bleaching ) and 70 s ( right , acceptor - bleaching )

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

Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide in apo conditions at 10 ms time resolution. This panel shows stable ATP-loaded (left) and ATP-free (right) McjD Representative FRET time traces that show switching between ATP free McjD (E* ~ 0.3) and ATP-loaded (high FRET >0.5) at ~4s s (left) and ~1.4 s (right). The traces show photobleaching events of the acceptor after ~10 s (left) and ~3.3 s (right) Representative FRET time traces that show switching between ATP-loaded (high FRET >0.5) and ATP free McjD (E* ~ 0.3) at 2 s (left) and 5 s (right). The traces show photobleaching events after ca. 40 s (left, donor-bleaching) and 70 s (right, acceptor-bleaching)


TRF1 immunofluorescence of the lungs . Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ / Δ mice , respectively .

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TRF1 immunofluorescence of the lungs. Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ/Δ mice, respectively.


Analysis of Trf1 excision by PCR . Notice the completed excision in carcinomas of Trf1lox / lox lungs .

Analysis of Trf1 excision by PCR. Notice the completed excision in carcinomas of Trf1lox/lox lungs.


Detection of β - galactosidase activity in the lungs as a surrogate marker of oncogenic K - RasG12V expression .

Detection of β-galactosidase activity in the lungs as a surrogate marker of oncogenic K-RasG12V expression.


Tumor growth curve of Trf1 + / + K - Ras + / G12Vp53 + / + and Trf1Δ / ΔK - Ras + / G12Vp53 + / + measured by computed tomography ( CT ) . Quantification of the number and size of Trf1 + / + K - Ras + / G12Vp53 + / + and Trf1Δ / ΔK - Ras + / G12Vp53 + / + carcinomas at death point .

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Tumor growth curve of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+ measured by computed tomography (CT).Quantification of the number and size of Trf1+/+K-Ras+/G12Vp53+/+ and Trf1Δ/ΔK-Ras+/G12Vp53+/+carcinomas at death point.


Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed . Post - mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox / lox K - Ras + / G12V p53 + / + ones had excised Trf1 .

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Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed. Post-mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox/lox K-Ras+/G12V p53+/+ ones had excised Trf1.


Tumor growth curve and tumor growth slope of Trf1 + / + K - Ras + / G12V p53 − / − and Trf1 − / − K - Ras + / G12V p53 − / − measured by CT .

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Tumor growth curve and tumor growth slope of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− measured by CT.


Tumor maximal section of Trf1 + / + K - Ras + / G12Vp53 − / − and Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs measured by histological analysis before death point by CT .

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Tumor maximal section of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs measured by histological analysis before death point by CT.


Maximum 18F - FDG - glucose uptake by Trf1 + / + K - Ras + / G12Vp53 − / − and Trf1Δ / ΔK - Ras + / G12Vp53 − / − tumors 22 weeks after infection by positron emission tomography ( PET ) . Representative PET - CT image of Trf1 + / + K - Ras + / G12Vp53 − / − and Trf1Δ / ΔK - Ras + / G12Vp53 − / − lungs .

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Maximum 18F-FDG-glucose uptake by Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−tumors 22 weeks after infection by positron emission tomography (PET).Representative PET-CT image of Trf1+/+K-Ras+/G12Vp53−/− and Trf1Δ/ΔK-Ras+/G12Vp53−/−lungs.


Survival curve of Trf1 + / + K - Ras + / G12V p53 − / − and Trf1 − / − K - Ras + / G12V p53 − / − mice .

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Survival curve of Trf1+/+ K-Ras+/G12V p53−/− and Trf1−/− K-Ras+/G12V p53−/− mice.


Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes ( left panel ) . Representative images of γH2AX immunohistochemistry ( right panel ) .

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Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel).


Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci ( TIFs ) ( left panel ) . Representative images of γH2AX and RAP1 double immunofluorescence ( right panel ) . Yellow arrowheads : colocalization of γH2AX and RAP1 .

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Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci (TIFs) (left panel). Representative images of γH2AX and RAP1 double immunofluorescence (right panel). Yellow arrowheads: colocalization of γH2AX and RAP1.


Percentage of active caspase - 3 ( AC3 ) - positive cells ( left panel ) . Representative images of AC3 immunohistochemistry ( right panel ) .

Percentage of active caspase-3 (AC3)-positive cells (left panel). Representative images of AC3 immunohistochemistry (right panel).


Percentage of Ki67 - positive cells in the carcinomas of the indicated genotypes ( left panel ) . Representative images of Ki67 immunohistochemistry ( right panel ) .

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Percentage of Ki67-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of Ki67 immunohistochemistry (right panel).


Percentage of pH3 - positive cells in the carcinomas of the indicated genotypes ( left panel ) . Representative images of pH3 immunohistochemistry ( right panel ) . Red arrowheads : pH3 - positive cells .

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Percentage of pH3-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of pH3 immunohistochemistry (right panel). Red arrowheads: pH3-positive cells.


Percentage of giant nuclei in the carcinomas of the indicated genotype . Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes . Representative images of giant nuclei , multilobulated nuclei , anaphase bridges , and multipolar mitoses . Red arrowheads indicate the corresponding mitotic aberrations indicated in the image .

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Percentage of giant nuclei in the carcinomas of the indicated genotype.Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes.Representative images of giant nuclei, multilobulated nuclei, anaphase bridges, and multipolar mitoses. Red arrowheads indicate the corresponding mitotic aberrations indicated in the image.


A - C The latency ( A ) , volume ( B ) , and weight ( C ) of subcutaneous tumors generated by control and Trf1 - downregulated K - RasG12V - transformed lung cells in athymic mice . D Representative images of the subcutaneous tumors .

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A-C The latency (A), volume (B), and weight (C) of subcutaneous tumors generated by control and Trf1-downregulated K-RasG12V-transformed lung cells in athymic mice.D Representative images of the subcutaneous tumors.


E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors .

E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.


F - H Number of Ki67 - positive ( F ) , number of γH2AX - positive ( G ) , and number of active caspase - 3 - positive ( H ) cells per field in the subcutaneous tumors .

F-H Number of Ki67-positive (F), number of γH2AX-positive (G), and number of active caspase-3-positive (H) cells per field in the subcutaneous tumors.


I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1 - downregulated subcutaneous tumors compared to the normal nuclei of control tumors .

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I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.


J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1 - downregulated cells .

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J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.


K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1 - downregulated cells . L Representative images of the lungs colonized by control and Trf1 - downregulated cells , respectively .

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K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.


M - O Number of Ki67 - positive ( M ) , number of γH2AX - positive ( N ) , and number of active caspase - 3 - positive ( O ) cells per field in the lung tumors .

M-O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors.


P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh - scrambled or sh - Trf1 .

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P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.


Trf1 expression levels in the indicated tissues of wild - type and Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 weeks .

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Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1 - positive cells in skin and intestine sections of wild - type and Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 weeks .

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Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1-positive cells in skin and intestine sections of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Survival curve of wild - type and Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet for 7 weeks .

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Survival curve of wild-type and Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.


Quantification of the histological alterations observed in tamoxifen - treated Trf1lox / lox hUBC - CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild - type counterparts .

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Quantification of the histological alterations observed in tamoxifen-treated Trf1lox/lox hUBC-CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild-type counterparts.


Quantification of blood cell populations in wild - type and Trf1lox / lox hUBC - CreERT2mice subjected to a tamoxifen - containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval .

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Quantification of blood cell populations in wild-type and Trf1lox/lox hUBC-CreERT2mice subjected to a tamoxifen-containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval.


Trf1 expression levels in blood , intestine , skin , and bone marrow of Trf1lox / lox hUBC - CreERT2 mice subjected to a tamoxifen - containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild - type mice .

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Trf1 expression levels in blood, intestine, skin, and bone marrow of Trf1lox/lox hUBC-CreERT2 mice subjected to a tamoxifen-containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild-type mice.


Quantification of TRF1 levels by immunofluorescence in lung tumor - derived cell line treated with DMSO , with 10 μM ETP - 47228 ( 24 h ) , and with 10 μM ETP - 47037 ( 48 h ) . Representative images are shown to the right .

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Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right.


Quantification of γH2AX levels by immunofluorescence in lung tumor - derived cell line treated with DMSO , with ETP - 47228 ( 24 h ) , and with ETP - 47037 ( 48 h ) . Representative images are shown to the right .

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Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right.


Quantification of telomere - induced foci ( TIFs ) by double immunofluorescence with anti - RAP1 and anti - γH2AX antibodies . Representative images are shown to the right . White arrowheads : colocalization of γH2AX and RAP1 .

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Quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-γH2AX antibodies. Representative images are shown to the right. White arrowheads: colocalization of γH2AX and RAP1.


Effect of different ETP - 47228 and ETP - 47037 concentrations during 24 h on proliferation in lung tumor - derived cell line relative to the growth of DMSO - treated cells .

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Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.


Tumor growth quantification in allograft model ETP - 47037 or with ETP - 47228 .

Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.


Quantification of tumor growth relative to initial tumor size . Representative CT images are shown to the right . The white arrowhead indicates a tumor within a highly inflammatory region .

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Quantification of tumor growth relative to initial tumor size. Representative CT images are shown to the right. The white arrowhead indicates a tumor within a highly inflammatory region.


Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative images are shown to the right ( n = 4 ) .

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Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP - 47037 for 10 days . Representative images are shown to the right ( n = 4 ) .

Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).


A - C Quantification of the number of ( A ) Ki67 - , ( B ) pan - nuclear p - H3 pattern - , and ( C ) foci p - H3 pattern - positive cells in untreated and ETP - 470037 - treated lungcarcinomas . The data represent the mean values obtained for three mice in each group . Error bars represent standard errors . D Representative Ki67 and p - H3 images .

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A-C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lungcarcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.D Representative Ki67 and p-H3 images.


E Representative H & amp ; amp ; E images of intestine samples corresponding to untreated and ETP - 47037 - treated animals . High - magnification images are shown to the right indicating the presence of normal mitosis , giant multinucleated and aberrant mitotic figures .

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E Representative H&amp;E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures.


F Representative H & amp ; amp ; E images of bone marrow and skin samples corresponding to untreated and ETP - 47037 - treated animals . High - magnification images are shown indicating the presence of necrosis , hemosiderosis , multinucleated cells , and giant nuclei . Bone marrow showed moderated aplasia .

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F Representative H&amp;E images of bone marrow and skin samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown indicating the presence of necrosis, hemosiderosis, multinucleated cells, and giant nuclei. Bone marrow showed moderated aplasia.


G Telomere length in untreated and ETP - 47037 - treated lung tumor samples . Representative images are shown to the right .

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G Telomere length in untreated and ETP-47037-treated lung tumor samples. Representative images are shown to the right.


Histopathological ( A - D ) and virological ( E ) examinations of the lung tissues from an ill Malayan pangolin naturally infected with Pangolin - CoV ( A and B , 200 × and 400 × , respectively ) and an uninfected Malayan pangolin ( C and D , 200 × and 400 × , respectively ) . Interstitial pneumonia with extensive infiltration of inflammatory cells is seen in the lung tissue from the infected pangolin . Viral particles are seen in double - membrane vesicles in the transmission electron microscopy image ( small scale = 50 nm ) taken from the Verno E6 cell culture inoculated with supernatant of homogenized lung ( E ) , with morphology indicative of coronavirus ( inserts at the upper right corner of E ) .

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Histopathological (A-D) and virological (E) examinations of the lung tissues from an ill Malayan pangolin naturally infected with Pangolin-CoV (A and B, 200× and 400×, respectively) and an uninfected Malayan pangolin (C and D, 200× and 400×, respectively). Interstitial pneumonia with extensive infiltration of inflammatory cells is seen in the lung tissue from the infected pangolin. Viral particles are seen in double-membrane vesicles in the transmission electron microscopy image (small scale = 50 nm) taken from the Verno E6 cell culture inoculated with supernatant of homogenized lung (E), with morphology indicative of coronavirus (inserts at the upper right corner of E).


Similarity plot of the near full - length genome sequence ( A ) and S gene sequence ( B ) of Pangolin - CoV to sequences of 2019 - nCoV _ WIV02 , Bat - CoV RaTG13 , Bat - CoV ZC45 and Bat - CoV ZXC21 . While Pangolin - CoV has a high sequence identity to 2019 - nCoV and Bat - CoV - RaTG13 in most regions of the S gene , it is more similar to Bat SARSr - CoV ZXC21 and Bat SARSr - CoV ZC45 at the 5 ′ end ( B ) . More importantly , Pangolin - CoV is highly similar to 2019 - nCoV in the functional RBD region of the S protein , with only one amino difference ( insert in B ) . Because of the presence of genetic recombination , there is a discrepancy in cluster formation among the outcomes of phylogenetic analyses of different regions of the S gene ( C ) .

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Similarity plot of the near full-length genome sequence (A) and S gene sequence (B) of Pangolin-CoV to sequences of 2019-nCoV_WIV02, Bat-CoV RaTG13, Bat-CoV ZC45 and Bat-CoV ZXC21. While Pangolin-CoV has a high sequence identity to 2019-nCoV and Bat-CoV-RaTG13 in most regions of the S gene, it is more similar to Bat SARSr-CoV ZXC21 and Bat SARSr-CoV ZC45 at the 5′ end (B). More importantly, Pangolin-CoV is highly similar to 2019-nCoV in the functional RBD region of the S protein, with only one amino difference (insert in B). Because of the presence of genetic recombination, there is a discrepancy in cluster formation among the outcomes of phylogenetic analyses of different regions of the S gene (C).


Free energy ( kcal / mol ) for the binding between S proteins of four SARSr - CoV viruses and ACE2 of four likely hosts .

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Free energy (kcal/mol) for the binding between S proteins of four SARSr-CoV viruses and ACE2 of four likely hosts.


Serial diluted recombinant S1 proteins of SARS - CoV - 2 , SARS - CoV and MERS - CoV were coated on 96 well plates , incubated with the recombinant Fc - tagged ACE2 ( ACE2 - Fc ) for binding evaluation .

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Serial diluted recombinant S1 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV were coated on 96 well plates, incubated with the recombinant Fc-tagged ACE2 (ACE2-Fc) for binding evaluation.


Recombinant S1 proteins of SARS - CoV - 2 and SARS - CoV were incubated with 293T - ACE2 cells and subjected to FACS evaluation for binding .

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Recombinant S1 proteins of SARS-CoV-2 and SARS-CoV were incubated with 293T-ACE2 cells and subjected to FACS evaluation for binding.


Binding of SARS nAbs to SARS - CoV S1 protein were tested by ELISA . Recombinant S1 protein of SARS - CoV were coated on plates , serial diluted nAbs were added for binding to recombinant S1 protein . B . Binding of SARS nAbs to SARS - CoV - 2 S1 protein were tested by ELSIA . Recombinant S1 protein of SARS - CoV - 2 were coated on plates , serial diluted nAbs were added for binding to recombinant S1 protein .

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Binding of SARS nAbs to SARS-CoV S1 protein were tested by ELISA. Recombinant S1 protein of SARS-CoV were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.B. Binding of SARS nAbs to SARS-CoV-2 S1 protein were tested by ELSIA. Recombinant S1 protein of SARS-CoV-2 were coated on plates, serial diluted nAbs were added for binding to recombinant S1 protein.


Neutralization of SARS - CoV nAbs against SARS - CoV - 2 PSV .

Neutralization of SARS-CoV nAbs against SARS-CoV-2 PSV.


Antibody competition with SARS - CoV RBD binding to ACE2 . Recombinant SARS - CoV RBD protein was coated on plates , nAbs and recombinant ACE2 were then added for RBD binding competition measurements .

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Antibody competition with SARS-CoV RBD binding to ACE2. Recombinant SARS-CoV RBD protein was coated on plates, nAbs and recombinant ACE2 were then added for RBD binding competition measurements.


D Volcano plot showing differentially expressed genes ( adj . P . Val < 0 . 05 ) between ethanol - and R5020 - treated mouse mammary organoids , n = 3 . Genes with log2 ( FC ) > 2 are highlighted in red , genes with log2FC < - 2 in blue . Names of genes with - log10 ( adj . P . Val ) > 10 and abs ( log2 ( FC ) ) > 2 are indicated .

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D Volcano plot showing differentially expressed genes (adj. P.Val < 0.05) between ethanol- and R5020-treated mouse mammary organoids, n=3. Genes with log2(FC) > 2 are highlighted in red, genes with log2FC < -2 in blue. Names of genes with -log10 (adj.P.Val) > 10 and abs(log2(FC)) > 2 are indicated.


H , I Bar graphs showing relative Rankl and Wnt4 transcript levels normalized to 36B4 expression in C57Bl6 - derived mammary organoids treated for 6 hours with R5020 ( H ) or LNG ( I ) and either 10 μM or 100 μM enzalutamide , n = 3 independent experiments .

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H, I Bar graphs showing relative Rankl and Wnt4 transcript levels normalized to 36B4 expression in C57Bl6-derived mammary organoids treated for 6 hours with R5020 (H) or LNG (I) and either 10 μM or 100 μM enzalutamide, n=3 independent experiments.


G Scatter plot showing the genes which are commonly altered upon R5020 stimulation ( adj . P - value < 0 . 05 ) in both mouse and human organoids relative to their CTRL samples . Color scale as a function of the averaged human and mouse logFC values . The red dashed lines represent an adj . P - value threshold of 0 . 001 .

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G Scatter plot showing the genes which are commonly altered upon R5020 stimulation (adj. P-value < 0.05) in both mouse and human organoids relative to their CTRL samples. Color scale as a function of the averaged human and mouse logFC values. The red dashed lines represent an adj. P-value threshold of 0.001.


D Stereo micrographs of whole mounted mammary glands 12 weeks after injection of HBECs . Scale bar , 2 mm . Inset , arrows point to mouse ducts distended by the presence of HBECs . Scale bar , 0 . 8 mm .

D Stereo micrographs of whole mounted mammary glands 12 weeks after injection of HBECs. Scale bar, 2 mm. Inset, arrows point to mouse ducts distended by the presence of HBECs. Scale bar, 0.8 mm.


I Representative co - IF micrographs of a cross - sectioned " humanized " mouse milk duct 5 months after intraductal injection for AR , ERα and PR , in red , and human E - CAD , in green . Scale bar , 50 μm .

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I Representative co-IF micrographs of a cross-sectioned "humanized" mouse milk duct 5 months after intraductal injection for AR, ERα and PR, in red, and human E-CAD, in green. Scale bar, 50 μm.


K Bar graphs showing the threshold cycles of semi quantitative RT - PCR with human - specific primers on RNA from virgin ( n = 4 ) and pregnant ( n = 3 ) mice xenografted with human cells .

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K Bar graphs showing the threshold cycles of semi quantitative RT-PCR with human-specific primers on RNA from virgin (n=4) and pregnant (n=3) mice xenografted with human cells.


Progestin plasma levels in treated mice ( n = 10 , 14 , 18 , 7 , 6 , 10 ) and in women ( n = 20 , 11 , 3 , 4 , 6 , 8 ) on hormonal contraceptives measured by LC - MS , shown as means ± SD , as well as values reported in the literature ( Cmax ) . The active form of DSG , 3 - ketodesogestrel was measured .

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Progestin plasma levels in treated mice (n=10, 14, 18, 7, 6, 10) and in women (n=20, 11, 3, 4, 6, 8) on hormonal contraceptives measured by LC-MS, shown as means ± SD, as well as values reported in the literature (Cmax). The active form of DSG, 3-ketodesogestrel was measured.


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E Representative micrographs showing co - IF with anti - KI67 ( red ) , and anti - hECAD ( green ) of xenografted milk ducts after 21 days treatment with vehicle or LNG . Scale bar , 50 μm . F Violin plot showing the percentage of KI67 and hECAD double + cells of total hECAD + cells , dots represent individual sectors counted in 3 different glands , control ( n = 30 ) or LNG ( n = 28 ) , median ( red ) , statistical significance was assessed by non - parametric Mann - Whitney test . G Bar plot showing relative transcript levels of MKI67 in xenografted glands from control ( n = 6 ) and LNG - treated ( n = 3 ) mice , Student ' s t - test .

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E Representative micrographs showing co-IF with anti-KI67 (red), and anti-hECAD (green) of xenografted milk ducts after 21 days treatment with vehicle or LNG. Scale bar, 50 μm. F Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells, dots represent individual sectors counted in 3 different glands, control (n=30) or LNG (n=28), median (red), statistical significance was assessed by non-parametric Mann-Whitney test. G Bar plot showing relative transcript levels of MKI67 in xenografted glands from control (n=6) and LNG-treated (n=3) mice, Student's t-test.


Representative micrographs showing co - IF with anti - KI67 ( red ) and anti - hECAD ( green ) of histological sections from glands xenografted with HBECs from mammoplasty M310 exposed to vehicle or progestins . Scale bar , 50 μm . J Violin plot showing the percentage of KI67 and hECAD double + cells of total hECAD + cells in M310 derived HBECs , dots represent individual sectors counted in different glands , median ( red ) , CTRL ( n = 180 ) , P4 ( n = 134 ) , DSG ( n = 52 ) , GSN ( n = 161 ) , LNG ( n = 32 ) , CMA ( n = 56 ) , CPA ( n = 42 ) , DSP ( n = 100 ) . Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using the CTRL group as reference .

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Representative micrographs showing co-IF with anti-KI67 (red) and anti-hECAD (green) of histological sections from glands xenografted with HBECs from mammoplasty M310 exposed to vehicle or progestins. Scale bar, 50 μm. J Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ cells in M310 derived HBECs, dots represent individual sectors counted in different glands, median (red), CTRL (n=180), P4 (n=134), DSG (n=52), GSN (n=161), LNG (n=32), CMA (n=56), CPA (n=42), DSP (n=100). Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using the CTRL group as reference.


K Violin plot showing the percentage of KI67 and hECAD double + cells of total hECAD + HBECs derived from 11 different patients upon 21 days of CTRL ( n = 360 ) , P4 ( n = 180 ) , DSG ( n = 147 ) , GSN ( n = 298 ) , LNG ( n = 284 ) , CMA ( n = 115 ) , CPA ( n = 156 ) , DSP ( n = 123 ) treatment . Red lines show medians . L Violin plot showing percentage of AR + intraductally - engrafted HBECs derived from 11 different patients upon 21 days of CTRL ( n = 258 ) , P4 ( n = 97 ) , DSG ( n = 173 ) , GSN ( n = 159 ) , LNG ( n = 168 ) , CMA ( n = 68 ) , CPA ( n = 76 ) , DSP ( n = 135 ) pellets . Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions , with batches and patients as random variables and CTRL as reference , median in red . J - L dashed line shows 0 % .

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K Violin plot showing the percentage of KI67 and hECAD double+ cells of total hECAD+ HBECs derived from 11 different patients upon 21 days of CTRL (n=360), P4 (n=180), DSG (n=147), GSN (n=298), LNG (n=284), CMA (n=115), CPA (n=156), DSP (n=123) treatment. Red lines show medians. L Violin plot showing percentage of AR+ intraductally-engrafted HBECs derived from 11 different patients upon 21 days of CTRL (n=258), P4 (n=97), DSG (n=173), GSN (n=159), LNG (n=168), CMA (n=68), CPA (n=76), DSP (n=135) pellets. Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions, with batches and patients as random variables and CTRL as reference, median in red. J-L dashed line shows 0%.


A - F Micrographs of H & E stained tissue sections from mouse mammary glands engrafted with HBECS and exposed to LNG or vehicle for 6 months . In control mice ( A , B ) most ducts have a narrow lumen and a thin wall due to limited growth of human cells ( A ) ; a few mildly dilated ducts exhibit a cellular proliferation made out of multiple small acini burgeoning at the external side of their wall ( B , arrows ) . In LNG - treated mice ( C - F ) the peripheral acinar proliferation is diffuse , marked in some foci ( C ) , and associated with a variable degree of duct dilatation from mild ( C , arrow ) up to true cysts formation ( D ) . Proliferating human epithelial cells showing apocrine changes ( e . g . abundant eosinophilic cytoplasm and central round nuclei ) partly fill the ductal lumen with a complex architecture of micropapillae ( E ) and bridges ( F , arrows ) . Scalebars , 50 μm .

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A-F Micrographs of H&E stained tissue sections from mouse mammary glands engrafted with HBECS and exposed to LNG or vehicle for 6 months. In control mice (A, B) most ducts have a narrow lumen and a thin wall due to limited growth of human cells (A); a few mildly dilated ducts exhibit a cellular proliferation made out of multiple small acini burgeoning at the external side of their wall (B, arrows). In LNG-treated mice (C-F) the peripheral acinar proliferation is diffuse, marked in some foci (C), and associated with a variable degree of duct dilatation from mild (C, arrow) up to true cysts formation (D). Proliferating human epithelial cells showing apocrine changes (e.g. abundant eosinophilic cytoplasm and central round nuclei) partly fill the ductal lumen with a complex architecture of micropapillae (E) and bridges (F, arrows). Scalebars, 50 μm.


A - C Graphs showing in vivo growth of HBECs from different donors as measured by radiance after treatment with LNG , ENZ , or LNG and ENZ , means of radiance in individual glands ± SEM , n = 8 - 10 per treatment , Wilcoxon matched - pairs test . Right : Dot plot showing plasma levels of progesterone ( P ) , testosterone ( T ) and LNG in individual xenografted animals .

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A-C Graphs showing in vivo growth of HBECs from different donors as measured by radiance after treatment with LNG, ENZ, or LNG and ENZ, means of radiance in individual glands ± SEM, n= 8-10 per treatment, Wilcoxon matched-pairs test. Right: Dot plot showing plasma levels of progesterone (P), testosterone (T) and LNG in individual xenografted animals.


D Graph showing in vivo growth of HBECs from a 20 - year - old patient transduced with either sh scramble or shAR and treated with LNG . Points show means of radiance in individual glands ± SEM ; n = 8 - 10 per treatment . Wilcoxon matched - pairs test .

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D Graph showing in vivo growth of HBECs from a 20-year-old patient transduced with either sh scramble or shAR and treated with LNG. Points show means of radiance in individual glands ± SEM; n= 8-10 per treatment. Wilcoxon matched-pairs test.


E Representative micrographs showing co - IF with anti - GFP ( red ) , and anti - AR ( green ) antibodies on histological sections from glands xenografted with HBECs transduced either with sh scramble or shAR - expressing lentivirus . Scale bar , 50 μm . F Violin plot showing the percentage of AR - and GFP - double + cells of total GFP + cells in sh scramble ( n = 6 ) and shAR ( n = 12 ) conditions , dots represent individual sectors counted , median ( red ) . Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using CTRL as reference . Red line shows median .

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E Representative micrographs showing co-IF with anti-GFP (red), and anti-AR (green) antibodies on histological sections from glands xenografted with HBECs transduced either with sh scramble or shAR-expressing lentivirus. Scale bar, 50 μm. F Violin plot showing the percentage of AR- and GFP-double+ cells of total GFP+ cells in sh scramble (n=6) and shAR (n=12) conditions, dots represent individual sectors counted, median (red). Statistical significance was assessed by fitting a generalized linear mixed model with gamma distributions using CTRL as reference. Red line shows median.


( A ) UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies conditioned on yeast paste had diffuse GFP - mCherry - DrAtg8a staining in midstage egg chambers . ( B ) Nondegenerating midstage egg chambers from starved flies contained autophagosomes ( yellow ) and autolysosomes ( red ) . ( C and D ) Egg chambers early in the degeneration process showed follicle cells that take up portions of the nurse cell cytoplasm ( C ) followed by condensation and fragmentation of the nurse cell nuclei and further uptake of the nurse cell cytoplasm into follicle cells ( D ) . ( E ) Late stage degenerating egg chambers lose all GFP staining and fluoresce red .

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(A) UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies conditioned on yeast paste had diffuse GFP-mCherry-DrAtg8a staining in midstage egg chambers. (B) Nondegenerating midstage egg chambers from starved flies contained autophagosomes (yellow) and autolysosomes (red). (C and D) Egg chambers early in the degeneration process showed follicle cells that take up portions of the nurse cell cytoplasm (C) followed by condensation and fragmentation of the nurse cell nuclei and further uptake of the nurse cell cytoplasm into follicle cells (D). (E) Late stage degenerating egg chambers lose all GFP staining and fluoresce red.


( F ) Fed Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed diffuse GFP - mCherry - DrAtg8a staining in the germline . ( G ) Starved Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers . ( H and I ) Fed flies overexpressing truncated Dcp - 1 ( tDcp - 1 ) in the germline showed increased autophagosomes and autolysosomes in nondegenerating midstage egg chambers ( H ) and also contained degenerating midstage egg chambers that lose all GFP fluorescence and fluoresce red ( I ) . Bars : ( main images ) 25 µm ; ( insets ) 12 . 5 µm . Insets in A - I show diffuse cytoplasmic staining of Atg8a or autophagosomes and autolysosomes . ( J and K ) The percentages of autolysosomes ( autolysosomes / total autophagic structures ) were manually calculated in at least eight egg chambers for each genotype as indicated . Error bars represent the means ± SD . Statistical testing was performed using one - way ANOVA with a Dunnet post test ( * * * * , P < 0 . 0001 ) or a two - tailed Student ' s t test ( * * , P < 0 . 005 ) .

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(F) Fed Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed diffuse GFP-mCherry-DrAtg8a staining in the germline. (G) Starved Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies showed reduced autolysosomes in degenerating midstage egg chambers. (H and I) Fed flies overexpressing truncated Dcp-1 (tDcp-1) in the germline showed increased autophagosomes and autolysosomes in nondegenerating midstage egg chambers (H) and also contained degenerating midstage egg chambers that lose all GFP fluorescence and fluoresce red (I). Bars: (main images) 25 µm; (insets) 12.5 µm. Insets in A-I show diffuse cytoplasmic staining of Atg8a or autophagosomes and autolysosomes. (J and K) The percentages of autolysosomes (autolysosomes/total autophagic structures) were manually calculated in at least eight egg chambers for each genotype as indicated. Error bars represent the means ± SD. Statistical testing was performed using one-way ANOVA with a Dunnet post test (****, P < 0.0001) or a two-tailed Student's t test (**, P < 0.005).


( A ) Drosophila S2 cells stably expressing GFP - RFP - Atg8a showed an increase in the percentage of cells containing two or more autolysosomes after starvation , which was blocked after Bafilomycin A1 ( BafA1 ) treatment . At least 50 cells were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post test ( * * , P < 0 . 01 ) . ( B ) Representative images of S2 - GFP - RFP - Atg8a cells after the indicated treatments .

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(A) Drosophila S2 cells stably expressing GFP-RFP-Atg8a showed an increase in the percentage of cells containing two or more autolysosomes after starvation, which was blocked after Bafilomycin A1 (BafA1) treatment. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01). (B) Representative images of S2-GFP-RFP-Atg8a cells after the indicated treatments.


( C ) S2 - GFP - RFP - Atg8a cells were treated with dsRNAs and subjected to fed or starvation conditions as indicated . At least 50 cells were manually quantitated in three independent experiments ( n = 3 ) . Statistical significance was determined using one - way ANOVA with a Dunnett post test ( * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 ) . ( D ) Representative images of S2 - GFP - RFP - Atg8a cells after the indicated RNAi treatments . Error bars represent the means ± SD . Bars , 10 µm .

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(C) S2-GFP-RFP-Atg8a cells were treated with dsRNAs and subjected to fed or starvation conditions as indicated. At least 50 cells were manually quantitated in three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA with a Dunnett post test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D) Representative images of S2-GFP-RFP-Atg8a cells after the indicated RNAi treatments. Error bars represent the means ± SD. Bars, 10 µm.


( A ) Nondegenerating midstage egg chambers from fed w1118flies showed Ref ( 2 ) P staining in follicle cells and nurse cells . ( B ) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref ( 2 ) P . ( D ) Starved Atg7flies showed an accumulation of Ref ( 2 ) P in the follicle cells and nurse cells of degenerating midstage egg chambers . ( E ) Starved Dcp - 1Prev1flies contained increased levels of Ref ( 2 ) P in degenerating midstage egg chambers .

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(A) Nondegenerating midstage egg chambers from fed w1118flies showed Ref(2)P staining in follicle cells and nurse cells. (B) Starved w1118flies contained degenerating midstage egg chambers with reduced Ref(2)P.(D) Starved Atg7flies showed an accumulation of Ref(2)P in the follicle cells and nurse cells of degenerating midstage egg chambers. (E) Starved Dcp-1Prev1flies contained increased levels of Ref(2)P in degenerating midstage egg chambers.


( C ) A representative Western blot of ovaries from w1118 and Atg7d77 / d14 flies subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P protein levels relative to actin . Graph represents ± SD from five independent experiments ( n = 5 ) . Statistical significance was determined using a two - tailed Student ' s t test . * , P = 0 . 004 for fed samples ; * , P = 0 . 008 for starved samples .

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(C) A representative Western blot of ovaries from w1118 and Atg7d77/d14 flies subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test. *, P = 0.004 for fed samples; *, P = 0.008 for starved samples.


( F ) A representative Western blot of ovaries from w1118 and Dcp - 1Prev1 flies that were subjected to fed or starvation conditions for 4 d . Ref ( 2 ) P was detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate Ref ( 2 ) P protein levels relative to actin . Graph represents ± SD from five independent experiments ( n = 5 )

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(F) A representative Western blot of ovaries from w1118 and Dcp-1Prev1 flies that were subjected to fed or starvation conditions for 4 d. Ref(2)P was detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate Ref(2)P protein levels relative to actin. Graph represents ± SD from five independent experiments (n = 5)


( A and B ) l ( 2 ) mbn cells were labeled with antibodies to Dcp - 1 and ATPsyn - α ( A ) or MitoTracker red ( MTR ; B ) . Merged images show colocalization between Dcp - 1 and the mitochondria . Boxes represent zoomed images . Bars : ( main images ) 5 µm ; ( zoomed images ) 1 . 25 µm .

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(A and B) l(2)mbn cells were labeled with antibodies to Dcp-1 and ATPsyn-α (A) or MitoTracker red (MTR; B). Merged images show colocalization between Dcp-1 and the mitochondria. Boxes represent zoomed images. Bars: (main images) 5 µm; (zoomed images) 1.25 µm.


( C ) Western blot from l ( 2 ) mbn cells subjected to nutrient - rich or starvation conditions for 6 h . Cells were separated into cytosolic ( C ) and mitochondrial enriched ( M ) fractions .

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(C) Western blot from l(2)mbn cells subjected to nutrient-rich or starvation conditions for 6 h. Cells were separated into cytosolic (C) and mitochondrial enriched (M) fractions.


( D ) Ovaries from fed w1118 flies were separated into cytosolic and membrane - enriched fractions and probed with antibodies to VDAC , Tubulin , ATPsyn - α , and Dcp - 1 .

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(D) Ovaries from fed w1118 flies were separated into cytosolic and membrane-enriched fractions and probed with antibodies to VDAC, Tubulin, ATPsyn-α, and Dcp-1.


( E and F ) Intact and lysed mitochondria ( E ) or intact mitochondria isolated from l ( 2 ) mbn cells ( F ) were treated with proteinase K ( PK ) . The effects of proteinase K treatment were assessed by antibodies to VDAC , ATPsyn - α , Pink1 , and Dcp - 1 .

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(E and F) Intact and lysed mitochondria (E) or intact mitochondria isolated from l(2)mbn cells (F) were treated with proteinase K (PK). The effects of proteinase K treatment were assessed by antibodies to VDAC, ATPsyn-α, Pink1, and Dcp-1.


( G ) Control and Dcp - 1 RNAi - treated cells were subjected to nutrient - rich or starvation conditions and stained with NAO . Mean fluorescence was measured by flow cytometry . Graph represents ± SEM of three independent experiments ( n = 3 ) .

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(G) Control and Dcp-1 RNAi-treated cells were subjected to nutrient-rich or starvation conditions and stained with NAO. Mean fluorescence was measured by flow cytometry. Graph represents ± SEM of three independent experiments (n = 3).


( A ) l ( 2 ) mbn cells were labeled with the ATPsyn - α , and mitochondrial morphology was scored as fragmented , normal , or elongated . ( B ) Cells were treated with control or Dcp - 1 dsRNA and subjected to nutrient - rich media or 1 h of starvation . Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined . At least 100 cells were examined manually in three independent experiments ( n = 3 ) . Error bars represent the mean ± SD . Statistical significance was determined using one - way ANOVA with a Bonferroni post test ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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(A) l(2)mbn cells were labeled with the ATPsyn-α, and mitochondrial morphology was scored as fragmented, normal, or elongated. (B) Cells were treated with control or Dcp-1 dsRNA and subjected to nutrient-rich media or 1 h of starvation. Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined. At least 100 cells were examined manually in three independent experiments (n = 3). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA with a Bonferroni post test (*, P < 0.05; **, P < 0.01).


( C ) Mitochondrial targeted GFP ( mitoGFP ) was expressed in the germline using the nosGAL4 driver . Staining shows mitoGFP , Armadillo , and DNA . ( D ) Mitochondria were scored as healthy ( H ) , clustered ( C ) , or elongated and overly connected ( E ) . All of mitochondria from fed UASp - mitoGFP / + ; nosGAL4 / + flies were scored as healthy . n = 15 egg chambers manually scored . ( E ) mitoGFP was expressed in Dcp - 1Prev1 flies using the nosGAL4 driver . ( F ) 54 % of egg chambers from UASp - mitoGFP / + ; Dcp - 1Prev1 / Dcp - 1Prev1 ; nosGAL4 / + flies contained elongated mitochondria , 39 % contained mitochondria that were scored as healthy , and 7 % contained clustered mitochondria . n = 28 egg chambers manually scored .

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(C) Mitochondrial targeted GFP (mitoGFP) was expressed in the germline using the nosGAL4 driver. Staining shows mitoGFP, Armadillo, and DNA. (D) Mitochondria were scored as healthy (H), clustered (C), or elongated and overly connected (E). All of mitochondria from fed UASp-mitoGFP/+;nosGAL4/+ flies were scored as healthy. n = 15 egg chambers manually scored. (E) mitoGFP was expressed in Dcp-1Prev1 flies using the nosGAL4 driver. (F) 54% of egg chambers from UASp-mitoGFP/+;Dcp-1Prev1/Dcp-1Prev1;nosGAL4/+ flies contained elongated mitochondria, 39% contained mitochondria that were scored as healthy, and 7% contained clustered mitochondria. n = 28 egg chambers manually scored.


( A ) Total cellular ATP levels were measured in l ( 2 ) mbn cells treated with control or Dcp - 1 dsRNA . Data represent ± SEM of three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Students t test ( * , P = 0 . 02 ) .

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(A) Total cellular ATP levels were measured in l(2)mbn cells treated with control or Dcp-1 dsRNA. Data represent ± SEM of three independent experiments (n = 3). Statistical significance was determined using a two-tailed Students t test (*, P = 0.02).


( B ) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp - 1Prev1 flies . Data represent ± SEM of five independent experiments ( n = 5 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * * , P = 0 . 014 ; * * * , P < 0 . 001 ) .

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(B) Total cellular ATP levels were measured in ovaries from fed or starved w1118 and Dcp-1Prev1 flies. Data represent ± SEM of five independent experiments (n = 5). Statistical significance was determined using a two-tailed Student's t test (**, P = 0.014; ***, P < 0.001).


( C ) Fed or starved w1118 and Dcp - 1Prev1 flies were treated with DMSO or 25 µg / ml oligomycin A , and Ref ( 2 ) P levels were assessed by immunoblot analysis . Actin served as a loading control . Densitometry was performed to quantitate protein levels relative to actin . Graph represents ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * , P = 0 . 02 ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fed or starved w1118 and Dcp-1Prev1 flies were treated with DMSO or 25 µg/ml oligomycin A, and Ref(2)P levels were assessed by immunoblot analysis. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graph represents ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P = 0.02).


( D and E ) Control UASp - GFP - mCherry - DrAtg8a / + ; nosGAL4 / + flies ( D ) and Dcp - 1Prev1 / Dcp - 1Prev1 ; UASp - GFP - mCherry - DrAtg8a / nosGAL4 flies ( E ) were subjected to starvation conditions supplemented with DMSO or 25 µg / ml oligomycin A . Bars , 25 µm . Quantifications show percentage of autolysosomes ( autolysosomes / total autophagic structures ) . At least eight egg chambers were manually quantitated per genotype per condition ( n = 8 ) . Statistical testing was determined using a two - tailed Student ' s t test . * * * , P = 0 . 0002 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E) Control UASp-GFP-mCherry-DrAtg8a/+;nosGAL4/+ flies (D) and Dcp-1Prev1/Dcp-1Prev1;UASp-GFP-mCherry-DrAtg8a/nosGAL4 flies (E) were subjected to starvation conditions supplemented with DMSO or 25 µg/ml oligomycin A. Bars, 25 µm. Quantifications show percentage of autolysosomes (autolysosomes/total autophagic structures). At least eight egg chambers were manually quantitated per genotype per condition (n = 8). Statistical testing was determined using a two-tailed Student's t test. ***, P = 0.0002.


( A ) A representative Western blot showing SesB and ATPsyn - α levels from fed or starved l ( 2 ) mbn cells treated with control or Dcp - 1 RNAi . Actin served as a loading control . Densitometry was performed to quantitate protein levels relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * , P < 0 . 05 ) .

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(A) A representative Western blot showing SesB and ATPsyn-α levels from fed or starved l(2)mbn cells treated with control or Dcp-1 RNAi. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( B ) A representative Western blot of ovaries from fed or starved w1118 or Dcp - 1Prev1 flies . SesB and ATPsyn - α were detected by immunoblotting . Actin served as a loading control . Densitometry was performed to quantitate protein levels relative to actin . Graphs represent ± SD from three independent experiments ( n = 3 ) . Statistical significance was determined using a two - tailed Student ' s t test ( * , P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A representative Western blot of ovaries from fed or starved w1118 or Dcp-1Prev1 flies. SesB and ATPsyn-α were detected by immunoblotting. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments (n = 3). Statistical significance was determined using a two-tailed Student's t test (*, P < 0.05).


( C ) Purified catalytically active Dcp - 1FL , but not catalytically inactive Dcp - 1C < A , cleaved in vitro translated Drice . Cleavage of in vitro translated SesB was not detected .

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(C) Purified catalytically active Dcp-1FL, but not catalytically inactive Dcp-1C<A, cleaved in vitro translated Drice. Cleavage of in vitro translated SesB was not detected.


( D ) V5 - tagged Dcp - 1C < A or V5 vector only control was expressed in l ( 2 ) mbn cells and subjected to nutrient - full or starvation conditions for 2 h . After IP with anti - V5 antibodies , lysates were separated by SDS - PAGE . Proteins were visualized with colloidal Coomassie stain .

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(D) V5-tagged Dcp-1C<A or V5 vector only control was expressed in l(2)mbn cells and subjected to nutrient-full or starvation conditions for 2 h. After IP with anti-V5 antibodies, lysates were separated by SDS-PAGE. Proteins were visualized with colloidal Coomassie stain.


( E ) FLAG - SesB or a vector only control was expressed in l ( 2 ) mbn cells and was immunoprecipitated with FLAG - agarose . Immunoblots show the interaction between FLAG - SesB and endogenous pro - Dcp - 1 . The asterisk represents a nonspecific band .

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(E) FLAG-SesB or a vector only control was expressed in l(2)mbn cells and was immunoprecipitated with FLAG-agarose. Immunoblots show the interaction between FLAG-SesB and endogenous pro-Dcp-1. The asterisk represents a nonspecific band.


( A ) Ovaries from fed SesBOrg flies show reduced Ref ( 2 ) P staining in degenerating midstage egg chamber ( arrow ) .

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(A) Ovaries from fed SesBOrg flies show reduced Ref(2)P staining in degenerating midstage egg chamber (arrow).


( B ) Ovaries from fed w1118 and SesBOrg were analyzed by Western blotting using Ref ( 2 ) P . Actin served as a loading control . Results are representative of three independent experiments ( n = 3 ) .

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(B) Ovaries from fed w1118 and SesBOrg were analyzed by Western blotting using Ref(2)P. Actin served as a loading control. Results are representative of three independent experiments (n = 3).


( C ) SesBOrg flies expressing GFP - mCherry - DrAtg8a in the germline showed increased autophagosomes and autolysosomes in nondegenerating ( left ) and degenerating ( right ) midstage egg chambers . Insets show autophagosomes and autolysosomes ( left ) and autolysosomes ( right ) in SesBOrg midstage egg chambers .

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(C) SesBOrg flies expressing GFP-mCherry-DrAtg8a in the germline showed increased autophagosomes and autolysosomes in nondegenerating (left) and degenerating (right) midstage egg chambers. Insets show autophagosomes and autolysosomes (left) and autolysosomes (right) in SesBOrg midstage egg chambers.


( D ) Quantitation of percentages of autolysosomes ( autolysosomes / total autophagic structures ) in nondegenerating ovaries from fed w1118 and SesBOrg flies . 10 egg chambers were manually examined per genotype . Error bars represent the means ± SD . Statistical significance was determined using a two - tailed Student ' s t test ( * * * , P < 0 . 0001 ) .

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(D) Quantitation of percentages of autolysosomes (autolysosomes/total autophagic structures) in nondegenerating ovaries from fed w1118 and SesBOrg flies. 10 egg chambers were manually examined per genotype. Error bars represent the means ± SD. Statistical significance was determined using a two-tailed Student's t test (***, P < 0.0001).


( E ) Degenerating midstage egg chambers ( arrows ) from fed SesBOrg flies are TUNEL positive .

(E) Degenerating midstage egg chambers (arrows) from fed SesBOrg flies are TUNEL positive.


( F ) Degenerating midstage egg chambers from starved control SesBOrg / FM7a ; Dcp - 1Prev1 / Dcp - 1Prev1flies contained persisting nurse cellnuclei and increased Ref ( 2 ) P . n = 117 ovarioles examined . Box represents zoomed image . ( G ) Degenerating midstage egg chambers from fed SesBOrg ; Dcp - 1Prev1flies contained condensed and fragmented nurse cellnuclei and low levels of Ref ( 2 ) P . Box represents zoomed image . n = 156 ovarioles examined .

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(F) Degenerating midstage egg chambers from starved control SesBOrg/FM7a;Dcp-1Prev1/Dcp-1Prev1flies contained persisting nurse cellnuclei and increased Ref(2)P. n = 117 ovarioles examined. Box represents zoomed image. (G) Degenerating midstage egg chambers from fed SesBOrg;Dcp-1Prev1flies contained condensed and fragmented nurse cellnuclei and low levels of Ref(2)P. Box represents zoomed image. n = 156 ovarioles examined.


( E ) Representative bands from SDS - PAGE blots of ATPIF1 , SDHB , ATP5A1 , and Vinculin in control and mutant patient derived fibroblasts . Right panel shows mean of ATPIF1 relative intensity normalized per ATP5A1 and represented as % of control cells ( n = 2 ) . The colored histogram illustrates the mitochondrial element impacted by each mutation . Note that ATPIF1 expression levels in patient cells is lower than in cells from control subjects . For each biological replicate , technical replicates were averaged . Data represent average ± SEM . Unpaired t - test versus Ctrl or untreated conditions shows statistical differences depicted by p - value .

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(E) Representative bands from SDS-PAGE blots of ATPIF1, SDHB, ATP5A1, and Vinculin in control and mutant patient derived fibroblasts. Right panel shows mean of ATPIF1 relative intensity normalized per ATP5A1 and represented as % of control cells (n=2). The colored histogram illustrates the mitochondrial element impacted by each mutation. Note that ATPIF1 expression levels in patient cells is lower than in cells from control subjects. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Unpaired t-test versus Ctrl or untreated conditions shows statistical differences depicted by p-value.


( J ) State 3 and maximal respiration driven by pyruvate + malate in isolated mitochondria from mouse heart . EPI was added in the respiration media at the indicated concentrations ( n ≥ 3 ) . Note that EPI does not affect maximal respiration but has a small effect on ATP synthesis - dependent OCR . For each biological replicate , technical replicates were averaged . Data represent average ± SEM . Unpaired t - test versus Ctrl or untreated conditions shows statistical differences depicted by p - value .

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(J) State 3 and maximal respiration driven by pyruvate + malate in isolated mitochondria from mouse heart. EPI was added in the respiration media at the indicated concentrations (n≥3). Note that EPI does not affect maximal respiration but has a small effect on ATP synthesis-dependent OCR. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Unpaired t-test versus Ctrl or untreated conditions shows statistical differences depicted by p-value.


( C ) Representative blot ( left ) and quantification ( right ) showing CV assembly by BNGE in mouse heart mitochondria treated with EPI as in A . For each biological replicate , technical replicates were averaged . Data represent average ± SEM .

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(C) Representative blot (left) and quantification (right) showing CV assembly by BNGE in mouse heart mitochondria treated with EPI as in A. For each biological replicate, technical replicates were averaged. Data represent average ± SEM.