Apg9p does not comigrate with prAPI in a strain that accumulates Cvt / autophagic vesicles . Strain TK415 ( ypt7Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , centrifuged at 100 , 000 g for 1 h , and the pellet fraction resuspended and centrifuged a second time . The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 16 h at 174 , 000 g as described in Materials and Methods . 16 fractions were collected starting from the top and subjected to SDS - PAGE and immunoblot with antisera against Apg9p and API . The graph represents an average of two experiments .

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

Apg9p does not comigrate with prAPI in a strain that accumulates Cvt/autophagic vesicles. Strain TK415 (ypt7Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, centrifuged at 100,000 g for 1 h, and the pellet fraction resuspended and centrifuged a second time. The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 16 h at 174,000 g as described in Materials and Methods. 16 fractions were collected starting from the top and subjected to SDS-PAGE and immunoblot with antisera against Apg9p and API. The graph represents an average of two experiments.


Apg9p localizes to large perivacuolar punctate structures . Immunofluorescence microscopy of strains CTD1 ( apg9Δ ) , KSL12C ( vps4Δ ) , and SKD6 - 1D ( apg6Δ ) transformed with the multicopy plasmid 3 × HA APG9 . Cells were grown in YPD to log phase ( vegetative ) , and incubated further in SD ( − N ) medium with 1 mM PMSF for 3 h ( starvation ) . The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods . Anti - HA and FITC - conjugated antibodies mark Apg9p ( left ) , and the vacuole can be visualized by Nomarski optics ( middle ) . An overlay is shown in the right panels .

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

Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.


In vivo examination of the Apg9pGFP fusion protein . Wild - type ( SEY6210 ) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4 - 64 to stain the vacuoles as described in Materials and Methods . The labeled cells were viewed directly with a Leica DM IRB confocal microscope . Large , punctate Apg9pGFP structures appear juxtaposed to the FM 4 - 64 - labeled vacuole membranes . In addition , smaller punctate structures appear throughout the cytoplasm .

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

In vivo examination of the Apg9pGFP fusion protein. Wild-type (SEY6210) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4-64 to stain the vacuoles as described in Materials and Methods. The labeled cells were viewed directly with a Leica DM IRB confocal microscope. Large, punctate Apg9pGFP structures appear juxtaposed to the FM 4-64-labeled vacuole membranes. In addition, smaller punctate structures appear throughout the cytoplasm.


Ultrastructural localization of Apg9p for immunoelectron microscopy . Strain STY1 ( pep4Δ ) expressing 3 × HA Apg9p was grown in YPD to log phase and transferred to SD ( − N ) medium for 2 h . The cells were fixed and stained with anti - HA antibody and 5 - nm colloidal gold - conjugated goat anti - mouse IgG as described in Materials and Methods . A , Section showing autophagic bodies in the vacuole lumen . B , Section showing a cytosolic autophagosome . Apg9p is seen in patches near the vacuole , but not on autophagic bodies or autophagosomes . AB , autophagic body ; AP , autophagosome ; N , nucleus ; V , vacuole ; VM , vacuolar membrane . Arrows point to areas of concentrated Apg9p .

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

Ultrastructural localization of Apg9p for immunoelectron microscopy. Strain STY1 (pep4Δ) expressing 3×HA Apg9p was grown in YPD to log phase and transferred to SD(−N) medium for 2 h. The cells were fixed and stained with anti-HA antibody and 5-nm colloidal gold-conjugated goat anti-mouse IgG as described in Materials and Methods. A, Section showing autophagic bodies in the vacuole lumen. B, Section showing a cytosolic autophagosome. Apg9p is seen in patches near the vacuole, but not on autophagic bodies or autophagosomes. AB, autophagic body; AP, autophagosome; N, nucleus; V, vacuole; VM, vacuolar membrane. Arrows point to areas of concentrated Apg9p.


Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant . A , Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway . Wild - type ( YW5 - 1B ) and apg9Δ ( CTD1 ) cells were grown in YPD ( vegetative ) or transferred to SD ( − N ) ( starvation ) for 3 h in the presence of PMSF ( starvation ) and examined by DIC ( Nomarski ) microscopy ( Zeiss Axioplan ) . PMSF inhibits the degradation of autophagic bodies . Under starvation conditions , wild - type cells accumulate autophagic bodies when vesicle breakdown is blocked . However , under the same conditions , apg9Δ cells do not accumulate autophagic bodies , indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen . The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild - type ( data not shown ) . B , The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature . Wild - type ( SEY6210 ) and apg9Δ ( JKY007 ) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38 ° C for 5 min , pulse - labeled for 10 min , and then subjected to nonradioactive chase reactions for the indicated times . Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS - PAGE as described in Materials and Methods . API - immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph . The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point .

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

Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.


Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step . A , The nature of prAPI binding and pelleting in the apg9ts strain . Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38 ° C . The spheroplasts were osmotically lysed in a buffer containing no salt ( 20 mM Pipes , pH 6 . 8 ) or a physiological concentration of salt ( 100 mM KOAc , 50 mM KCl , 5 mM MgCl2 , 20 mM Pipes , pH 6 . 8 ) and pelleted at 5 , 000 g . All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK . apg9ts retains the salt dependent binding and pelleting of prAPI . B , prAPI in the apg9ts strain accumulates in both a membrane - associated state and a large pelletable complex . Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into supernatant ( S ) and pellet ( P ) fractions by centrifugation at 5 , 000 g for 5 min . An aliquot was removed for the total lysate control ( T ) . The pellet fraction ( P ) was resuspended in 15 % Ficoll - 400 in gradient buffer ( 20 mM Pipes , 5 mM MgCl2 , complete EDTA - free protease inhibitor cocktail ) in the presence or absence of Triton X - 100 and overlaid with 13 and 2 % Ficoll - 400 in gradient buffer . The step gradients were centrifuged at 13 , 000 g for 10 min . Membrane - containing float ( F ) , nonfloat ( NF ) , and pellet ( P2 ) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods . The position of prAPI is indicated . C , prAPI in the apg9ts strain is protease accessible . Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse - labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into low - speed supernatant ( S ) and pellet ( P ) fractions after a 5 , 000 g centrifugation step . The pellet fractions were subjected to protease treatment in the absence or presence of 0 . 2 % Triton X - 100 as described in Materials and Methods . The resulting samples were immunoprecipitated with antiserum to API and PGK . The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager . The percent protease - protected prAPI was determined by dividing the value for prAPI in the protease - treated sample by the value for total prAPI before protease treatment . The percent PGK was determined by dividing the supernatant ( S ) or pellet ( P ) value over the sum of the S and P values .

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

Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step. A, The nature of prAPI binding and pelleting in the apg9ts strain. Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38°C. The spheroplasts were osmotically lysed in a buffer containing no salt (20 mM Pipes, pH 6.8) or a physiological concentration of salt (100 mM KOAc, 50 mM KCl, 5 mM MgCl2, 20 mM Pipes, pH 6.8) and pelleted at 5,000 g. All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK. apg9ts retains the salt dependent binding and pelleting of prAPI. B, prAPI in the apg9ts strain accumulates in both a membrane-associated state and a large pelletable complex. Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 5,000 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 in gradient buffer (20 mM Pipes, 5 mM MgCl2, complete EDTA-free protease inhibitor cocktail) in the presence or absence of Triton X-100 and overlaid with 13 and 2% Ficoll-400 in gradient buffer. The step gradients were centrifuged at 13,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods. The position of prAPI is indicated. C, prAPI in the apg9ts strain is protease accessible. Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 5,000 g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. The resulting samples were immunoprecipitated with antiserum to API and PGK. The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager. The percent protease-protected prAPI was determined by dividing the value for prAPI in the protease-treated sample by the value for total prAPI before protease treatment. The percent PGK was determined by dividing the supernatant (S) or pellet (P) value over the sum of the S and P values.


A Macroscopic seedling phenotype of 12 - day - old b16b18CRISPR , quadriCRISPR F33 # 1 / # 6 , pentaCRISPR and amiR - 2572 , compared to WT ( Col - 0 ) .

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A Macroscopic seedling phenotype of 12-day-old b16b18CRISPR, quadriCRISPR F33#1/#6, pentaCRISPR and amiR-2572, compared to WT (Col-0).


E Macroscopic seedling phenotype of 12 - day - old quadriCRISPR B64 compared to WT ( Col - 0 ) and amiR - 2572 . Scale bars = 1 cm . F , G Boxplots showing the quantification of primary root length ( F ) and LR density ( G ) in seedlings depicted in E . n = 12 ( Col - 0 ) , 13 ( quadriCRISPR B64 ) , 11 ( amiR - 2572 ) .

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E Macroscopic seedling phenotype of 12-day-old quadriCRISPR B64 compared to WT (Col-0) and amiR-2572. Scale bars = 1 cm. F, G Boxplots showing the quantification of primary root length (F) and LR density (G) in seedlings depicted in E. n = 12 (Col-0), 13 (quadriCRISPR B64), 11 (amiR-2572). D


C Confocal images of root tips in 5 - day - old Col - 0 , amiR - 2572 and syn - tasi - 1522A # 1 seedlings . LRC cells were indicated by white asterisks . The distal LRC cells were indicated by white

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C Confocal images of root tips in 5-day-old Col-0, amiR-2572 and syn-tasi-1522A#1 seedlings. LRC cells were indicated by white asterisks. The distal LRC cells were indicated by white arrows


G Macroview stereo microscopic view of DR5 : VENUS expression in root tips of 3 - day - old WT , amiR - 2572 and syn - tasi - 1522A # 1 seedlings , red arrows indicate DR5 stripes in the lateral root cap .

G Macroview stereo microscopic view of DR5:VENUS expression in root tips of 3-day-old WT, amiR-2572 and syn-tasi-1522A#1 seedlings, red arrows indicate DR5 stripes in the lateral root cap.


A Co - localisation of YFP - ABCB15 with Propidium Iodide ( PI ) in root epidermal cells of 3 - day - old seedlings . A plot of the fluorescence intensity of YFP and PI along the dashed lines shows the colocalization of the YFP and PI intensity peaks .

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A Co-localisation of YFP-ABCB15 with Propidium Iodide (PI) in root epidermal cells of 3-day-old seedlings. A plot of the fluorescence intensity of YFP and PI along the dashed lines shows the colocalization of the YFP and PI intensity peaks.


C IAA export assay . Export of [ 3H ] - IAA , assayed in parallel from tobacco mesophyll protoplasts expressing ABCB1 , ABCB15 - 22 and ABCB17P980G against vector control . mean ± SE ; n = 26 ( vector control ) , 44 ( ABCB1 ) , 5 ( ABCB15 ) , 4 ( ABCB16 ) , 9 ( ABCB17 ) , 5 ( ABCB18 ) , 9 ( ABCB22 ) and 8 ( ABCB17P980G ) , transport experiments generated from independent tobacco transfections .

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C IAA export assay. Export of [3H]-IAA, assayed in parallel from tobacco mesophyll protoplasts expressing ABCB1, ABCB15-22 and ABCB17P980G against vector control. mean ± SE; n = 26 (vector control), 44 (ABCB1), 5 (ABCB15), 4 (ABCB16), 9 (ABCB17), 5 (ABCB18), 9 (ABCB22) and 8 (ABCB17P980G),transport experiments generated from independent tobacco transfections.


B Analysis of DR5 : : VENUS expression in the root elongation zone of 4 - day - old pWOX5 : XVE > > YUC1 - 2A - TAA1 , in Control and amiR - 2572 treated with β - estradiol ( 5 µM ) for 0 , 7 . 5 and 9h . Images are composed of several tiles generated in a single snap with automatic assembly , PI in gray . The zoomed images of the yellow squares are presented below each root showing the accumulation of the DR5 : : VENUS signal in the elongation zone .

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B Analysis of DR5::VENUS expression in the root elongation zone of 4-day-old pWOX5:XVE>>YUC1-2A-TAA1, in Control and amiR-2572 treated with β-estradiol (5 µM) for 0, 7.5 and 9h. Images are composed of several tiles generated in a single snap with automatic assembly, PI in gray. The zoomed images of the yellow squares are presented below each root showing the accumulation of the DR5::VENUS signal in the elongation zone.


A , B Expression pattern of proABCB15 : NLS - GFP - GUS , proABCB16 : NLS - GFP - GUS , proABCB17 : NLS - GFP - GUS , proABCB18 : NLS - GFP - GUS , and proABCB22 : NLS - GFP - GUS expression in roots of 3 - day - old seedlings , using confocal microscopy showing longitudinal overview pictures ( Propidium iodide in magenta ) , and images zoomed on the epidermis and LRC corresponding to yellow squares ( A ) ; cytological sections of GUS stained roots of 3 - day - old seedlings , counterstained with ruthenium red ( B ) . Scale bars represent 20 µm for both graphs .

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A, B Expression pattern of proABCB15:NLS-GFP-GUS, proABCB16:NLS-GFP-GUS, proABCB17:NLS-GFP-GUS, proABCB18:NLS-GFP-GUS, and proABCB22:NLS-GFP-GUS expression in roots of 3-day-old seedlings, using confocal microscopy showing longitudinal overview pictures (Propidium iodide in magenta), and images zoomed on the epidermis and LRC corresponding to yellow squares (A); cytological sections of GUS stained roots of 3-day-old seedlings, counterstained with ruthenium red (B). Scale bars represent 20 µm for both graphs.


A Fluorescence of YFP - ABCB17 in the root meristem and leaf in 3 - day - old F1 crosses with WT , syn - tasi - 1522A # 1 and amiR - 2572 , propidium iodide ( PI ) in magenta . Red arrows highlight the position of the epidermis . Scale bars = 50 µm . B Schematic representation of root geometry with the indication of regions of interest for YFP - ABCB17 signal quantification . Red ( Stele ) , and black ( epidermis / LRC ) . C , D Quantification of YFP - ABCB17 fluorescence intensity in the stele ( C ) and epidermis / LRC ( D ) of A , measured at regions of interest corresponding

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A Fluorescence of YFP-ABCB17 in the root meristem and leaf in 3-day-old F1 crosses with WT, syn-tasi-1522A#1 and amiR-2572, propidium iodide (PI) in magenta. Red arrows highlight the position of the epidermis . Scale bars = 50 µm. B Schematic representation of root geometry with the indication of regions of interest for YFP-ABCB17 signal quantification. Red (Stele), and black (epidermis/LRC). C, D Quantification of YFP-ABCB17 fluorescence intensity in the stele (C) and epidermis/LRC (D) of A, measured at regions of interest corresponding t


G , H Quantification of the oscillation period ( G ) and amplitude ( H ) of DR5 : : LUC in 3 - day - old WT and syn - tasi - 1522A # 1 , n = 27 ( WT ) and 23 ( syn - tasi - 1522A # 1 ) .

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G,H Quantification of the oscillation period (G) and amplitude (H) of DR5::LUC in 3-day-old WT and syn-tasi-1522A#1, n= 27 (WT) and 23 (syn-tasi-1522A#1).


( b ) N2a cells coexpressing YFP - PrP and HLA - A2 - CFP were subjected to 6 mM trypsin for 100 s . Images were taken before and after trypsin treatment as indicated . Note that the membrane signal of YFP - PrP was destroyed by the protease , whereas HLA - A2 - CFP ' s membrane signal remained unaffected .

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(b) N2a cells coexpressing YFP-PrP and HLA-A2-CFP were subjected to 6 mM trypsin for 100 s. Images were taken before and after trypsin treatment as indicated. Note that the membrane signal of YFP-PrP was destroyed by the protease, whereas HLA-A2-CFP's membrane signal remained unaffected.


( d ) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP . N2a cells expressing GFP were treated with 20 μM digitonin . Images were taken before and after digitonin application at the indicated time points . Scale bars , 10 μm .

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(d) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP. N2a cells expressing GFP were treated with 20 μM digitonin. Images were taken before and after digitonin application at the indicated time points. Scale bars, 10 μm.


( b ) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ - CFP . Images were taken before and after 1 - min treatment with 20 μM digitonin and 4 mM trypsin as indicated . Scale bar , 10 μm .

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(b) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ-CFP. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.


( b ) NRK cells expressing CD3δ - CFP and YFP - CD3δ were subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 4 mM trypsin as indicated . Scale bar , 10 μm .

(b) NRK cells expressing CD3δ-CFP and YFP-CD3δ were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.


c ) Biochemical protease protection assay of NRK cells expressing YFP - CD3δ , CD3δ - CFP and GFP . Transfected cells were subjected to SDS - PAGE and Western blot analysis before ( lane 1 ) and after separation of the cytosolic ( lane 2 ) and membrane ( lane 3 ) fractions by ultracentrifugation . Equal amounts of the membrane fraction were treated with trypsin in the absence ( lane 4 ) or presence ( lane 5 ) of Triton X - 100 . Protein extracts were analyzed by immunoblotting with monoclonal anti - GFP antibody JL - 8 .

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

c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.


( a ) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT - YFP ( red ) and ER - bound CD3δ - CFP ( green ) , and the position of the fluorescent protein tag relative to its respective membrane . ( b ) N2a cells coexpressing GalT - YFP and CD3δ - CFP were subjected to the FPP assay , as 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

(a) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT-YFP (red) and ER-bound CD3δ-CFP (green), and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing GalT-YFP and CD3δ-CFP were subjected to the FPP assay, as in Figure 2b


c ) Cartoon illustrating localization of mitochondrial EYFP - Mito ( red ) and Cd3δ - CFP ( green ) . ( d ) N2a cells coexpressing EYFP - Mito and CD3δ - CFP were subjected to the FPP assay , as in b . Scale bars , 10 μm .

c) Cartoon illustrating localization of mitochondrial EYFP-Mito (red) and Cd3δ-CFP (green). (d) N2a cells coexpressing EYFP-Mito and CD3δ-CFP were subjected to the FPP assay, as in b. Scale bars, 10 μm.


( a ) Cartoon of the FPP assay illustrating the cellular localization of RFP - SKL ( red ) and Pex3p - GFP ( green ) and the position of the fluorescent protein tag relative to its respective membrane . ( b ) N2a cells coexpressing RFP - SKL and Pex3p - GFP were subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 100 - s treatment with 4 mM trypsin as indicated . Scale bar , 10 μm .

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

(a) Cartoon of the FPP assay illustrating the cellular localization of RFP-SKL (red) and Pex3p-GFP (green) and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing RFP-SKL and Pex3p-GFP were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin as indicated. Scale bar, 10 μm.


( a ) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane . GAPDH - RFP is shown in red and CFP - LC3 is shown in green . ( b ) NRK cells stably expressing CFP - LC3 were transiently transfected to express GAPDH - RFP and subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 100 - s treatment with 4 mM trypsin . The inset shows the overlap of the GAPDH - RFP and CFP - LC3 signals ( 4 × magnification of the central area ( outlined by a box ) of the micrographs . Scale bar , 10 μm .

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

(a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane. GAPDH-RFP is shown in red and CFP-LC3 is shown in green. (b) NRK cells stably expressing CFP-LC3 were transiently transfected to express GAPDH-RFP and subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin. The inset shows the overlap of the GAPDH-RFP and CFP-LC3 signals (4 × magnification of the central area (outlined by a box) of the micrographs. Scale bar, 10 μm.


Monocyte derived macrophages ( MDM ) were treated with 5μM etoposide ( ETO ) for 18h . Cells were stained for γH2AX and 53BP1 nuclear foci , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired and analysed . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; paired t ‐ test ) . Scale bars , 10μm .

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

Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.


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


MDM were treated with increasing concentrations of ETO and CTH for 66h . Cells were stained to distinguish between live and dead cells using LIVE / DEAD fixable Dead cell stain protocol . Percentages of live / dead cells were determined using automated cell ‐ imaging system Hermes WiScan and ImageJ . Addition of 20 % ethanol for 10min into cells treated with ETO was used as positive control . ( n = 3 , mean ± s . e . m ) .

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

MDM were treated with increasing concentrations of ETO and CTH for 66h. Cells were stained to distinguish between live and dead cells using LIVE/DEAD fixable Dead cell stain protocol. Percentages of live/dead cells were determined using automated cell‐imaging system Hermes WiScan and ImageJ. Addition of 20% ethanol for 10min into cells treated with ETO was used as positive control. (n = 3, mean ± s.e.m).


MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV - G pseudotyped HIV - 1 GFP viruses wild type ( wt ) HIV - 1 capsid mutants ( N74D or P90A ) , HIV - 2 , SIV sooty mangabey ( SIVsm E543 ) , replication competent Adenovirus type 5 AdV ( AdV ) , Semliki Forest Virus ( SFV ) and HSV - 1 . The percentages of infected cells were determined using an automated cell ‐ imaging system Hermes WiScan and ImageJ and normalized to untreated control ~ 100 % . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 ; ( ns ) non ‐ significant , paired t ‐ test ) . Cells from a representative donor were used for immunoblotting .

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

MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP viruses wild type (wt) HIV-1 capsid mutants (N74D or P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent Adenovirus type 5 AdV (AdV), Semliki Forest Virus (SFV) and HSV-1. The percentages of infected cells were determined using an automated cell‐imaging system Hermes WiScan and ImageJ and normalized to untreated control ~100%. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; ***P‐value ≤ 0.001; (ns) non‐significant, paired t‐test). Cells from a representative donor were used for immunoblotting.


MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV - G pseudotyped HIV - 1 GFP . 104 cells were recorded and analysed for GFP expression 48h post - infection using an automated cell ‐ imaging system Hermes WiScan and ImageJ . ( n = 4 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) .

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

MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP. 104 cells were recorded and analysed for GFP expression 48h post-infection using an automated cell‐imaging system Hermes WiScan and ImageJ. (n = 4, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).


MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full - length replication competent macrophage tropic HIV - 1 virus BaL . Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) .

MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full-length replication competent macrophage tropic HIV-1 virus BaL. Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).


G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.  Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . 2LTR circles . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) .

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

G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.2LTR circles. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05, paired t‐test).


G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) .

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

G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; ***P‐value ≤ 0.001, paired t‐test).


MDM were treated with 5μM ETO for 18h and infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / cell ) . Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) .

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

MDM were treated with 5μM ETO for 18h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ** P‐value ≤ 0.01; ***P‐value ≤ 0.001, paired t‐test).


MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / cell ) and DNA isolated 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL (3, 6, 12 ffu/cell) and DNA isolated 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL and DNA isolated 4 , 6 , and 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL and DNA isolated 4, 6, and 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


MDM were transfected with control or pool of SAMHD1 ( KD ) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV ‐ G pseudotyped HIV ‐ 1 GFP 18h later . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV‐G pseudotyped HIV‐1 GFP 18h later. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) or deleted for Vpx ( SIV VLP del Vpx ) or deleted for Vpr ( SIV VLP del Vpr ) . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . ( n = 4 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP) or deleted for Vpx (SIV VLP del Vpx) or deleted for Vpr (SIV VLP del Vpr). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 4, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).


D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).


D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).


D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).


The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).


A - B . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . MDM were differentiated and cultured in Human serum instead of FCS .

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

A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in Human serum instead of FCS.


A - B . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . MDM were differentiated and cultured in FCS . A standard culture condition used in all experiments . See Materials and Methods .

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

A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in FCS. A standard culture condition used in all experiments. See Materials and Methods.


C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . Cells from a representative donor were used for immunoblotting . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

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

C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.  The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. Cells from a representative donor were used for immunoblotting. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).


C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) .

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

C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).


C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) .

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C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).


C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

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

C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).


MDM were treated with 5μM ETO for 18h , 3μg / ml cGAMP and 10ng / ml IFNβfor 18h . RNA was isolated and qPCR performed for selected genes using TaqMan assays . Expression levels of target genes were normalised to GAPDH . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; ( ns ) non ‐ significant , paired t ‐ test ) .

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

MDM were treated with 5μM ETO for 18h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalised to GAPDH. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; (ns) non‐significant, paired t‐test).


MDM were treated with 5μM ETO or cGAMP for 18h or 100ng / ml LPS for 2h . Cells were stained and analysed for IRF3 translocation into nucleus . Scale bars : 10μm .

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MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm.


Quantification of nuclei positive for IRF3 staining . ( n = 3 , mean ± s . e . m . ) .

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Quantification of nuclei positive for IRF3 staining. (n = 3, mean ± s.e.m.).


MDM were treated with increasing concentrations of ETO and CTH . Cells were stained for MCM2 expression , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired . ( n = 3 , mean ± s . e . m . ) .

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MDM were treated with increasing concentrations of ETO and CTH. Cells were stained for MCM2 expression, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired. (n = 3, mean ± s.e.m.).


MDM were treated with 5μM ETO , lysed and immunoblotting performed to detect cell cycle / cell cycle arrest and DNA damage associated proteins .

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MDM were treated with 5μM ETO, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.


MDM were treated with 5μM ETO or 0 . 01μM CTH for 18h , lysed and immunoblotting performed to detect cell cycle / cell cycle arrest and DNA damage associated proteins .

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MDM were treated with 5μM ETO or 0.01μM CTH for 18h, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.


C Relative mRNA expression of Zeb1 , Acp5 , and Ctsk during the differentiation from BMDMs to osteoclasts in culture ( n = 3 ) .

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C Relative mRNA expression of Zeb1, Acp5, and Ctsk during the differentiation from BMDMs to osteoclasts in culture (n = 3).


D Western blot of Zeb1 , c - Src , and Ctsk expression in BMDMs and mature osteoclasts ( n = 3 ) .

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D Western blot of Zeb1, c-Src, and Ctsk expression in BMDMs and mature osteoclasts (n = 3).


F Zeb1 ( red ) and TRAP ( green ) immunofluorescence of a femur section including the growth plate ( GP ) and metaphysis from a wild - type mouse . The multinucleated cells associated with the surface of the bone marrow cavity ( BM cavity ) that are shown in the red box are further enlarged in the panels to the right . Scale bar , 10 μm .

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F Zeb1 (red) and TRAP (green) immunofluorescence of a femur section including the growth plate (GP) and metaphysis from a wild-type mouse. The multinucleated cells associated with the surface of the bone marrow cavity (BM cavity) that are shown in the red box are further enlarged in the panels to the right. Scale bar, 10 μm.


A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3 - month - old wild - type and Zeb1ΔM / ΔM male mice are shown . The genotype of Zeb1 + / + , Zeb1f / + , Zebf / f mice is defined as " wild - type " control . Scale bar , 500 μm . B Quantification of BV / TV , Tb . Th , Tb . N , and Tb . Sp as determined by nanoCT in 3 - month - old wild - type and Zeb1ΔM / ΔM male mice ( n = 8 ) .

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A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown. The genotype of Zeb1+/+, Zeb1f/+, Zebf/f mice is defined as "wild-type" control. Scale bar, 500 μm. B Quantification of BV/TV, Tb.Th, Tb.N, and Tb.Sp as determined by nanoCT in 3-month-old wild-type and Zeb1ΔM/ΔM male mice (n = 8). C


C H & E and TRAP staining of the distal femurs of 3 - month - old male wild - type and Zeb1ΔM / ΔM mice . Scale bar , 200 μm . D Quantification of osteoclast number per bone surface ( N . Oc / BS ) and eroded surface per bone surface ( ES / BS ) , as well as serum CTX - I and OCN levels in 3 - month - old wild - type and Zeb1ΔM / ΔM male mice are shown ( n = 6 ) .

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C H&E and TRAP staining of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice. Scale bar, 200 μm. D Quantification of osteoclast number per bone surface (N.Oc/BS) and eroded surface per bone surface (ES/BS), as well as serum CTX-I and OCN levels in 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown (n = 6). E


E Golden ' s trichrome staining illuminating howship ' s lacunae ( arrow ) of the distal femurs of 3 - month - old male wild - type and Zeb1ΔM / ΔM mice were performed along with double - calcein bone labeling and confocal imaging to assess bone formation . Scale bar , 10 μm . F Quantification of MAR and BFR as assessed in 3 - month - old male wild - type and Zeb1ΔM / ΔM mice ( n = 6 ) .

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E Golden's trichrome staining illuminating howship's lacunae (arrow) of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice were performed along with double-calcein bone labeling and confocal imaging to assess bone formation. Scale bar, 10 μm. F Quantification of MAR and BFR as assessed in 3-month-old male wild-type and Zeb1ΔM/ΔM mice (n = 6). D


B Zeb1 , NFATc1 , c - Fos , c - Src , β3 integrin , Mmp9 , Mmp14 , and Ctsk expression as assessed by Western blot in wild - type and Zeb1ΔM / ΔM BMDMs during osteoclast differentiation ( n = 3 ) .

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B Zeb1, NFATc1, c-Fos, c-Src, β3 integrin, Mmp9, Mmp14, and Ctsk expression as assessed by Western blot in wild-type and Zeb1ΔM/ΔM BMDMs during osteoclast differentiation (n = 3).


G Supernatant CTX - I determined by ELISA ( n = 6 ) .

G Supernatant CTX-I determined by ELISA (n = 6).


H , I After a 6 - day culture atop bone slice , phalloidin staining ( red ) was performed in wild - type versus Zeb1ΔM / ΔM osteoclasts ( I ) , and actin ring area per cell quantified ( H ) . Scale bar , 20 μm .

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H, I After a 6-day culture atop bone slice, phalloidin staining (red) was performed in wild-type versus Zeb1ΔM/ΔM osteoclasts (I), and actin ring area per cell quantified (H). Scale bar, 20 μm.


E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice .

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E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice.


F MtCK1 , Ckb , VDAC , and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice ( n = 3 ) .

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F MtCK1, Ckb, VDAC, and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).


H MtCK1 ( green ) immunofluorescence of wild - type and Zeb1ΔM / ΔM osteoclasts . Scale bar , 20

H MtCK1 (green) immunofluorescence of wild-type and Zeb1ΔM/ΔM osteoclasts. Scale bar, 20 μ


A Phosphocreatine ( PCr ) and creatine ( Cr ) level , as well as PCr / Cr ratio of wild - type or Zeb1ΔM / ΔM osteoclasts cultured atop plastic substrata as determined by LC / MS analysis ( n = 3 ) .

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A Phosphocreatine (PCr) and creatine (Cr) level, as well as PCr/Cr ratio of wild-type or Zeb1ΔM/ΔM osteoclasts cultured atop plastic substrata as determined by LC/MS analysis (n = 3).


B , C Oxygen consumption rate ( OCR ) profile plot ( B ) and mitochondrial function parameters ( C ) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild - type or Zeb1ΔM / ΔM mice after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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B, C Oxygen consumption rate (OCR) profile plot (B) and mitochondrial function parameters (C) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild-type or Zeb1ΔM/ΔM mice after sequential treatment of compounds modulating mitochondrial function (n = 4).


C Wild - type BMDMs were transduced with either a mock vector , wild - type human MtCK1 , or a catalytically - inactive MtCK1C316G mutant expression vector , differentiated into osteoclasts , and cell lysates collected for MtCK1 , Ckb , Tomm20 , VDAC , Zeb1 , c - Src , and Ctsk immunoblotting ( n = 3 ) .

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C Wild-type BMDMs were transduced with either a mock vector, wild-type human MtCK1, or a catalytically-inactive MtCK1C316G mutant expression vector, differentiated into osteoclasts, and cell lysates collected for MtCK1, Ckb, Tomm20, VDAC, Zeb1, c-Src, and Ctsk immunoblotting (n = 3).


E PCr / Cr ratio of mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts cultured on plastic substrata as determined by LC / MS analysis ( n = 3 ) .

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E PCr/Cr ratio of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts cultured on plastic substrata as determined by LC/MS analysis (n = 3).


OCR profile plot ( H ) analyzed by XF Cell Mito Stress Assay in mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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OCR profile plot (H) analyzed by XF Cell Mito Stress Assay in mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).


J , K Mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type pre - osteoclasts were cultured atop bone slices for 3 days , stained with phalloidin ( red ) . Osteoclasts were removed and resorption pits visualized by WGA - DAB staining ( J ) . The actin ring area per cell and resorption pit area were quantified ( K ; n = 6 ) . Scale bar , upper 20 μm , lower 100 μm .

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J, K Mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type pre-osteoclasts were cultured atop bone slices for 3 days, stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (J). The actin ring area per cell and resorption pit area were quantified (K; n = 6). Scale bar, upper 20 μm, lower 100 μm.


A , B Human monocyte - derived macrophages ( hMDMs ) were transfected with either shCTRL or siRNA targeting ZEB1 , differentiated into osteoclasts , and relative mRNA expression of CKMT1 ( A ) and mitochondrial creatine kinase activity determined ( B ; n = 3 ) .

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A, B Human monocyte-derived macrophages (hMDMs) were transfected with either shCTRL or siRNA targeting ZEB1, differentiated into osteoclasts, and relative mRNA expression of CKMT1 (A) and mitochondrial creatine kinase activity determined (B; n = 3).


F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non - targeting siCTRL - or siZEB1 - transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non-targeting siCTRL- or siZEB1-transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).


G , H siCTRL - or siZEB1 - transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin ( red ) . Osteoclasts were removed and resorption pits visualized by WGA - DAB staining ( G ) . Scale bar , upper 20 μm , lower 100 μm . The actin ring area per cell , resorption pit area , and the number of TRAP + MNCs were quantified ( H ; n = 6 ) .

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G, H siCTRL- or siZEB1-transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (G). Scale bar, upper 20 μm, lower 100 μm. The actin ring area per cell, resorption pit area, and the number of TRAP+ MNCs were quantified (H; n = 6).


I - L Calvaria isolated from wild - type and Zeb1ΔM / ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine , and stained for phalloidin ( red ) and Vpp3 ( green ) ( I ) . Scale bar , 10 μm . The TRAP activity of whole cell lysates , actin ring area per cell , and supernatant CTX - I were quantified ( J - L ; n = 6 ) .

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I-L Calvaria isolated from wild-type and Zeb1ΔM/ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine, and stained for phalloidin (red) and Vpp3 (green) (I). Scale bar, 10 μm. The TRAP activity of whole cell lysates, actin ring area per cell, and supernatant CTX-I were quantified (J-L; n = 6).


A panel of 42 breast cancer cell lines was challenged with increasing concentrations of ATRA ( 11 nM - 10 μM ) for 3 , 6 , and 9 days , and cell growth was determined . The graphs show the growth - inhibitory effect exerted by the indicated concentrations of ATRA in SKBR3 , HCC - 1954 , and MDA - MB436 cells which are representative of lines characterized by a high , intermediate and low ATRA score , respectively . Each point is the mean ± SD of three replicate cultures . * * Significantly lower than the corresponding vehicle - treated group ( P - value < 0 . 01 , Student ' s t - test ) .

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A panel of 42 breast cancer cell lines was challenged with increasing concentrations of ATRA (11 nM-10 μM) for 3, 6, and 9 days, and cell growth was determined.The graphs show the growth-inhibitory effect exerted by the indicated concentrations of ATRA in SKBR3, HCC-1954, and MDA-MB436 cells which are representative of lines characterized by a high, intermediate and low ATRA score, respectively. Each point is the mean ± SD of three replicate cultures. **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).


Cell lines are ranked in ascending order according to the ATRA score . The plot distinguishes four separate groups of cell lines ( D - A ) with increasing ATRA scores , as indicated by the colored scale on the right . The cell lines are also grouped in tertiles , T1 - T3 , according to an ascending ATRA score . Each calculated value is representative of at least two independent experiments .

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Cell lines are ranked in ascending order according to the ATRA score. The plot distinguishes four separate groups of cell lines (D-A) with increasing ATRA scores, as indicated by the colored scale on the right. The cell lines are also grouped in tertiles, T1-T3, according to an ascending ATRA score. Each calculated value is representative of at least two independent experiments.


SCID mice were xenografted subcutaneously with 10 × 106HCC - 1599 cells on both sides . One week after transplantation 10 animals / experimental group were treated intraperitoneally with vehicle ( DMSO ) or two doses of ATRA ( 7 . 5 and 15 . 0 mg / kg ) once / day , 5 days a week for a total of 24 days . At the end of this period , treatment was discontinued until sacrifice . The size of the tumors was determined with a caliper and the volume plotted . Each point is the mean ± SE of 20 tumors . * Significantly lower than the corresponding vehicle - treated group ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly lower than the corresponding vehicle - treated group ( P - value < 0 . 01 , Student ' s t - test ) .

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SCID mice were xenografted subcutaneously with 10 × 106HCC-1599 cells on both sides. One week after transplantation 10 animals/experimental group were treated intraperitoneally with vehicle (DMSO) or two doses of ATRA (7.5 and 15.0 mg/kg) once/day, 5 days a week for a total of 24 days. At the end of this period, treatment was discontinued until sacrifice.The size of the tumors was determined with a caliper and the volume plotted. Each point is the mean ± SE of 20 tumors. *Significantly lower than the corresponding vehicle-treated group (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).


Magnetic resonance imaging ( MRI ) analysis was performed on five animals / experimental group on day 24 . The picture shows representative 2D images of tumor sections from one animal treated with vehicle and one animal treated with 15 . 0 mg / kg ATRA . The bar graph shows the volume of the tumors calculated after 3D reconstruction of the MRI images . Each point is the mean ± SE of five tumors . The P - value of the comparisons of ATRA versus vehicle is shown .

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Magnetic resonance imaging (MRI) analysis was performed on five animals/experimental group on day 24. The picture shows representative 2D images of tumor sections from one animal treated with vehicle and one animal treated with 15.0 mg/kg ATRA. The bar graph shows the volume of the tumors calculated after 3D reconstruction of the MRI images. Each point is the mean ± SE of five tumors. The P-value of the comparisons of ATRA versus vehicle is shown.


ATRA - dependent anti - tumor activity in short - term cultures of primary breast tumorsTissue slices deriving from surgical specimens were challenged with vehicle ( DMSO ) or ATRA ( 0 . 1 μM ) for 48 h . The panel illustrates examples of the immuno - histochemical data obtained in four representative cases : ( i ) Luminal - A ( Lum A ) ; ( ii ) Luminal - B ( Lum B ) ; ( iii ) Her2 + with RARA coamplification ( Her2 + / RARA + ) and ( iv ) triple negative ( TN ) .

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ATRA-dependent anti-tumor activity in short-term cultures of primary breast tumorsTissue slices deriving from surgical specimens were challenged with vehicle (DMSO) or ATRA (0.1 μM) for 48 h.The panel illustrates examples of the immuno-histochemical data obtained in four representative cases: (i) Luminal-A (Lum A); (ii) Luminal-B (Lum B); (iii) Her2+ with RARA coamplification (Her2+/RARA+) and (iv) triple negative (TN).


The percentage of Ki67 - positive tumor cells in the 45 samples considered are illustrated by the bar graphs . Each value represents the mean ± SE of at least five separate fields for each experimental sample . * Significantly lower than the corresponding vehicle - treated control ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly lower than the corresponding vehicle - treated control ( P - value < 0 . 01 , Student ' s t - test ) .

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The percentage of Ki67-positive tumor cells in the 45 samples considered are illustrated by the bar graphs. Each value represents the mean ± SE of at least five separate fields for each experimental sample. *Significantly lower than the corresponding vehicle-treated control (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated control (P-value < 0.01, Student's t-test).


Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell linesThe figure illustrates the associations between the indicated retinoid receptors / binding proteins and the Luminal versus Basal phenotype ( left panels ) , ER positivity versus ER negativity ( middle panels ) as well as HER2 positivity versus HER2 negativity ( right panels ) . The gene - expression microarray and RNA - seq data refer to 42 and 40 breast cancer cell lines , respectively . The P - values of the indicated comparisons after Student ' s t - test are shown in red . L ( red ) = Luminal cell lines ; B ( blue ) = Basal cell lines ; ER + ( red ) = ER - positive cell lines ; ER − ( red ) = ER - negative cell lines ; ER − ( blue ) = ER - negative Luminal cell lines ; H + ( red ) = HER2 - positive cell lines ; H − ( red ) = HER2 - negative cell lines ; H + ( blue ) = HER2 - positive Luminal cell lines ; H − ( blue ) = HER2 - negative Luminal cell lines . fpkm = fragments per kilobase of exon per million fragments mapped .

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Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell linesThe figure illustrates the associations between the indicated retinoid receptors/binding proteins and the Luminal versus Basal phenotype (left panels), ER positivity versus ER negativity (middle panels) as well as HER2 positivity versus HER2 negativity (right panels). The gene-expression microarray and RNA-seq data refer to 42 and 40 breast cancer cell lines, respectively. The P-values of the indicated comparisons after Student's t-test are shown in red. L (red) = Luminal cell lines; B (blue) = Basal cell lines; ER+ (red) = ER-positive cell lines; ER− (red) = ER-negative cell lines; ER− (blue) = ER-negative Luminal cell lines; H+ (red) = HER2-positive cell lines; H− (red) = HER2-negative cell lines; H+ (blue) = HER2-positive Luminal cell lines; H− (blue) = HER2-negative Luminal cell lines. fpkm = fragments per kilobase of exon per million fragments mapped.


Associations between components of the retinoid pathway and breast cancer phenotypeAssociations between the expression of the indicated members of the retinoid pathway in the TCGA gene - expression database are shown . Mammary tumors are classified into Luminal - A , Luminal - B , HER2 - like , Normal - like , and Basal according to the PAM50 fingerprint . The average expression levels and the corresponding SD values of the indicated members of the retinoid pathway are shown by the upper box plots . For each member of the retinoid pathway , significant differences between the indicated groups of tumors are shown in the lower table . Significant P - values for the indicated comparisons ( Student ' s t - test ) are shown in red .

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Associations between components of the retinoid pathway and breast cancer phenotypeAssociations between the expression of the indicated members of the retinoid pathway in the TCGA gene-expression database are shown. Mammary tumors are classified into Luminal-A, Luminal-B, HER2-like, Normal-like, and Basal according to the PAM50 fingerprint. The average expression levels and the corresponding SD values of the indicated members of the retinoid pathway are shown by the upper box plots. For each member of the retinoid pathway, significant differences between the indicated groups of tumors are shown in the lower table. Significant P-values for the indicated comparisons (Student's t-test) are shown in red.


The gene - expression microarray and RNA - seq data associated with 40 of the breast cancer cell lines were used for the analyses . The panels illustrate the associations of RARα and PPARβ / δ with ATRA sensitivity . The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups ( 13 cell lines in each of the T1 and T3 groups ) defined by ascending ATRA scores . The intermediate and right panels indicate the same results after stratification for the Luminal ( microarray and RNA - seq data = 7 cell lines in each of the T1 and T3 groups ) and the Basal ( microarray and RNA - seq data = 7 cell lines in each of the T1 and T3 groups ) phenotype , respectively . fpkm = fragments per kilobase of exon per million fragments mapped .

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

The gene-expression microarray and RNA-seq data associated with 40 of the breast cancer cell lines were used for the analyses. The panels illustrate the associations of RARα and PPARβ/δ with ATRA sensitivity. The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups (13 cell lines in each of the T1 and T3 groups) defined by ascending ATRA scores. The intermediate and right panels indicate the same results after stratification for the Luminal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) and the Basal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) phenotype, respectively. fpkm = fragments per kilobase of exon per million fragments mapped.


The basal expression levels of the indicated RAR - isoform variants were determined with the use of specific Taqman assays . The results are associated with ATRA sensitivity before ( TOTAL ) and after stratification of the cell lines for the Luminal and Basal phenotype as in ( A ) . Data information : Significant P - values ( Student ' s t - test ) are indicated in red .

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The basal expression levels of the indicated RAR-isoform variants were determined with the use of specific Taqman assays. The results are associated with ATRA sensitivity before (TOTAL) and after stratification of the cell lines for the Luminal and Basal phenotype as in (A).Data information: Significant P-values (Student's t-test) are indicated in red.


Basal levels of RARα , RARβ , and RARγ mRNA splicing variants in mammary tumorsTotal RNA was extracted from the tissue slices deriving from the surgical specimens of breast cancer patients used in Fig3 before any treatment with DMSO or ATRA . RNA was subjected to RT - PCR analysis to determine the basal expression of the indicated RAR splicing variants . Each value represents the mean ± SD of two replicate measurements . The table shows the statistical significance of the indicated comparisons . * Significantly different ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly different ( P - value < 0 . 01 , Student ' s t - test ) .

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Basal levels of RARα, RARβ, and RARγ mRNA splicing variants in mammary tumorsTotal RNA was extracted from the tissue slices deriving from the surgical specimens of breast cancer patients used in Fig3 before any treatment with DMSO or ATRA. RNA was subjected to RT-PCR analysis to determine the basal expression of the indicated RAR splicing variants.Each value represents the mean ± SD of two replicate measurements. The table shows the statistical significance of the indicated comparisons. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).


The plots illustrate the average expression levels of the indicated mRNAs ( mean ± SD of two replicates ) in tumor samples classified as ATRA - sensitive ( Sens ) and ATRA - resistant ( Res ) according to the response of Ki67 . * Significantly different ( P - value < 0 . 05 , Student ' s t - test ) .

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The plots illustrate the average expression levels of the indicated mRNAs (mean ± SD of two replicates) in tumor samples classified as ATRA-sensitive (Sens) and ATRA-resistant (Res) according to the response of Ki67. *Significantly different (P-value < 0.05, Student's t-test).


RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis . The panel illustrates representative Western blots for a number of cell lines . The blots were sequentially developed with RARα - specific and control tubulin antibodies . The positions of the RARα and tubulin bands ( right ) along with the position of a relevant molecular weight marker ( left ) are indicated . To normalize the Western blot signals in different gels , the same preparation of RARα - transfected COS - 7cell extracts ( COS - 7 ) and MCF7 extracts was loaded in each gel . The blots are representative of at least two independent experiments providing similar results .

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RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.


The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores . Cell lines are grouped according to the ATRA score ( A - D groups and T1 - T3 tertiles ) .

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The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A-D groups and T1-T3 tertiles).


Left : The upper graph indicates the averages expression levels of the RARα protein in T1 - T3 cell lines . The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal ( six cell lines in each of the T1 and T3 groups ) and the Basal ( six cell lines in each of the T1 and T3 groups ) phenotype , respectively . Right : The levels of the RARα protein in Basal and Luminal cell lines ( upper graph ) , in ER + , ER − , and Luminal ER − cell lines ( middle graph ) and the indicated classes of HER2 + and HER2 − cell lines ( lower graph ) are shown . * Significantly different ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly different ( P - value < 0 . 01 , Student ' s t - test ) .

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Left: The upper graph indicates the averages expression levels of the RARα protein in T1-T3 cell lines. The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal (six cell lines in each of the T1 and T3 groups) and the Basal (six cell lines in each of the T1 and T3 groups) phenotype, respectively. Right: The levels of the RARα protein in Basal and Luminal cell lines (upper graph), in ER+, ER−, and Luminal ER−cell lines (middle graph) and the indicated classes of HER2+ and HER2−cell lines (lower graph) are shown. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).


The plots show the correlation curves between the levels of the indicated RARα - variant transcripts and the RARα protein .

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The plots show the correlation curves between the levels of the indicated RARα-variant transcripts and the RARα protein.


Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell linesThe indicated Luminal and Basal cell lines were challenged with increasing concentrations of ATRA , the RARα agonist , AM580 , the RARβ agonist , UVI2003 , and the RARγ agonist , BMS961 , for 6 days . The complement of RAR - variant transcripts expressed in each cell line is shown in the left bar graphs ( mean ± SD of two replicate measurements ) . The growth curves ( sulforhodamine assay ) of the cell lines are illustrated by the right linear plots . The results are expressed in % values relative to the corresponding control dishes treated with vehicle alone ( right graphs ) . Each result is the mean ± SD of five replicate wells . ATRA sc = ATRA score .

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Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell linesThe indicated Luminal and Basal cell lines were challenged with increasing concentrations of ATRA, the RARα agonist, AM580, the RARβ agonist, UVI2003, and the RARγ agonist, BMS961, for 6 days. The complement of RAR-variant transcripts expressed in each cell line is shown in the left bar graphs (mean ± SD of two replicate measurements). The growth curves (sulforhodamine assay) of the cell lines are illustrated by the right linear plots. The results are expressed in % values relative to the corresponding control dishes treated with vehicle alone (right graphs). Each result is the mean ± SD of five replicate wells. ATRA sc = ATRA score.


A RARα1 / 3 plasmid construct and the corresponding control void vector were stably transfected into ATRA - resistantMDA - MB453 cells . Two cell clones over - expressing RARα ( RARA - C5 andRARA - C7 ) and two appropriate control clones ( Vect - C1 andVect - C2 ) were isolated . A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA - sensitiveSKBR3 cells . After selection , two cell clones silenced for RARα3 ( RARA - sh18 andRARA - sh19 ) and two appropriate control clones ( Vect - C6 andVect - C8 ) were isolated . A , D The indicated clones and the parental cell line ( WT ) were transiently transfected with the RARE - DR5 - Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle ( DMSO ) and ATRA ( 100 nM ) , as illustrated in the upper bar graph . Each value is the mean ± SD of three replicate cultures . The levels of the RARα protein measured in the indicated clones by Western blotanalysis is shown under the bar graph . To demonstrate that similar levels of total proteins were loaded in each lane , the β - actin band signal obtained after re - blotting of the gel is shown . FI = Fluorescence intensity .

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A RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistantMDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 andRARA-C7) and two appropriate control clones (Vect-C1 andVect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitiveSKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 andRARA-sh19) and two appropriate control clones (Vect-C6 andVect-C8) were isolated.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blotanalysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.


B , E The panels illustrate the growth curves of the indicated MDA - MB453 and SKBR3 clones and the SKBR3 parental cell lines ( WT ) measured with the sulforhodamine assay .

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B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.


C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA - MB453 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 , 6 , and 9 days prior to the sulforhodamine assay . OD = optical density at 540 nm . Each value is the mean ± SD of five replicate culture wells .

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C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.