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 .

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

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 .

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

O O O O O O O O O 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, 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 .

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 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

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.


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 RARαprotein measured in the indicated clones by Western blot analysis 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 . 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 .

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

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 RARαprotein measured in the indicated clones by Western blot analysis 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.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.


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 RARαprotein 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 . 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 .

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

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 RARαprotein 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.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.


F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 and 6 days prior to the sulforhodamine assay . Each value is the mean ± SD of five replicate culture wells .

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 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

F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.


G The panel illustrates the effects exerted by ATRA ( 0 . 1 μM ) on random cell motility of the indicated MDA - MB453 and SKBR3 clones . The results are representative of two independent experiments . Each value is the mean ± SE of the motility of at least 60 cells .

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

G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.


a ) FBXL2 binds p85α and p85β . HEK293T cells were transfected with either an empty vector ( EV ) or the indicated FLAG - tagged F - box proteins ( FBPs ) . 24 h post - transfection , cells were treated with MG132 for 3 h before collection for immunoprecipitation ( IP ) and immunoblotting as indicated . WCL , whole - cell lysate .

O O B-MEASURED_VAR 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

a) FBXL2 binds p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before collection for immunoprecipitation (IP) and immunoblotting as indicated. WCL, whole-cell lysate.


( b ) FBXL2 does not bind p110α and p110β . HEK293T and NIH3T3 cells were transfected with either an empty vector ( EV ) or the indicated FLAG - tagged proteins . The experiment was performed as described in a .

O O O B-MEASURED_VAR 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

(b) FBXL2 does not bind p110α and p110β. HEK293T and NIH3T3 cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged proteins. The experiment was performed as described in a.


( c ) The CaaX motif of FBXL2 is required to bind p85α and p85β . HEK293T cells were transfected with either an empty vector ( EV ) or the indicated FLAG - tagged F - box proteins . The experiment was performed as described in a .

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

(c) The CaaX motif of FBXL2 is required to bind p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins. The experiment was performed as described in a.


a ) HEK293T cells were transfected with an empty vector ( EV ) or the indicated FLAG - tagged constructs . 24 h after transfection , cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated .

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

a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs. 24 h after transfection, cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated.


( b ) HeLa cells stably expressing FLAG - HA - tagged FBXL2 were transfected with either siRNAs targeting FBXL2 ( four different siRNAs ) or a non - silencing siRNA ( NS ) . Cells were lysed , and proteins were immunoblotted as indicated . NT , non - transfected .

O O O O O O O O O O O O B-MEASURED_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

(b) HeLa cells stably expressing FLAG-HA-tagged FBXL2 were transfected with either siRNAs targeting FBXL2 (four different siRNAs) or a non-silencing siRNA (NS). Cells were lysed, and proteins were immunoblotted as indicated. NT, non-transfected.


( c ) During a 72 - h serum starvation , NHFs were transfected with either an siRNA targeting FBXL2 ( no . 1 ) or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . The graph shows FBXL2 mRNA levels in the different samples analysed using real - time PCR in triplicate measurements . The values represent the ratios between FBXL2 and GAPDH mRNAs . SR , serum re - addition .

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

(c) During a 72-h serum starvation, NHFs were transfected with either an siRNA targeting FBXL2 (no. 1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition.


( d ) HEK293T cells were transfected with HA - tagged p85β , FLAG - tagged FBXL2 , FLAG - tagged FBXL2 ( ΔF - box ) or an empty vector ( EV ) as indicated . After immunopurification with anti - FLAG resin , in vitro ubiquitylation of p85β was performed in the presence of E1 , E2s and ubiquitin ( Ub ) . Where indicated , an excess of methylated ubiquitin ( MeUb ) was also added . Samples were analysed by immunoblotting with the indicated antibodies . The ladder of bands with a relative molecular mass of > 85 , 000 ( lane 3 ) corresponds to ubiquitylated p85β . Immunoblots of whole - cell lysates ( WCL ) are shown at the bottom . Uncropped images of blots are shown in Supplementary Fig .

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

(d) HEK293T cells were transfected with HA-tagged p85β, FLAG-tagged FBXL2, FLAG-tagged FBXL2(ΔF-box) or an empty vector (EV) as indicated. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of p85β was performed in the presence of E1, E2s and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (MeUb) was also added. Samples were analysed by immunoblotting with the indicated antibodies. The ladder of bands with a relative molecular mass of >85,000 (lane 3) corresponds to ubiquitylated p85β. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Uncropped images of blots are shown in Supplementary Fig. S8


( b ) HEK293T cells were transfected with GFP - tagged FBXL2 and the indicated FLAG - tagged p85β mutants . Whole - cell lysates ( WCL ) were immunoprecipitated ( IP ) with anti - FLAG resin , and immunocomplexes were probed with antibodies against the indicated proteins .

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

(b) HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated FLAG-tagged p85β mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-FLAG resin, and immunocomplexes were probed with antibodies against the indicated proteins.


( c ) p85β ( ΔSH2C ) is more stable than wild - type p85β . RPE1 - hTERT cells were infected with either a retrovirus expressing wild - type p85β or p85β ( ΔSH2C ) . Cells were incubated with cycloheximide ( CHX ) for the indicated times , collected and analysed by immunoblotting as indicated . In the graph , the amount of p85β ( wild - type or mutant ) is represented relative to the amount at time 0 . (

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

(c) p85β(ΔSH2C) is more stable than wild-type p85β. RPE1-hTERT cells were infected with either a retrovirus expressing wild-type p85β or p85β (ΔSH2C). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. (


( d ) p85β ( QR / AA ) is more stable than wild - type p85β . HEK293T cells were infected with either a retrovirus expressing wild - type p85β or p85β ( QR / AA ) . Cells were incubated with cycloheximide ( CHX ) for the indicated times , collected and analysed by immunoblotting as indicated . In the graph , the amount of p85β ( wild - type or mutant ) is represented relative to the amount at time 0 .

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

(d) p85β (QR/AA) is more stable than wild-type p85β. HEK293T cells were infected with either a retrovirus expressing wild-type p85β or p85β (QR/AA). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0.


( e ) p85β ( Y655A ) is less stable than wild - type p85β . The experiment was performed as described in c except that p85β ( Y655A ) was used . In the graph , the amount of p85β ( wild - type or mutant ) is represented relative to the amount at time 0 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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

(e) p85β (Y655A) is less stable than wild-type p85β. The experiment was performed as described in c except that p85β(Y655A) was used. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) Lysates from cells expressing the indicated FLAG - tagged F - box proteins ( FBPs ) were used in binding reactions with beads coupled to either a peptide or a phospho - peptide ( sequences shown on top of the panel ) . Beads were washed with lysis buffer , and bound proteins were eluted and subjected to SDS - PAGE and immunoblotting . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysates from cells expressing the indicated FLAG-tagged F-box proteins (FBPs) were used in binding reactions with beads coupled to either a peptide or a phospho-peptide (sequences shown on top of the panel). Beads were washed with lysis buffer, and bound proteins were eluted and subjected to SDS-PAGE and immunoblotting. (


( b ) Denatured extracts from HEK293T cells stably expressing either FLAG - tagged wild - type p85β or FLAG - tagged p85β ( Y655A ) were immunoprecipitated ( IP ) with either an anti - FLAG resin or a 4G10 platinum resin and immunoblotted ( IB ) as indicated . Immunoblots of whole - cell lysates ( WCL ) are also shown in the last two lanes .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Denatured extracts from HEK293T cells stably expressing either FLAG-tagged wild-type p85β or FLAG-tagged p85β(Y655A) were immunoprecipitated (IP) with either an anti-FLAG resin or a 4G10 platinum resin and immunoblotted (IB) as indicated. Immunoblots of whole-cell lysates (WCL) are also shown in the last two lanes.


( c ) Denatured extracts from HEK293T cells expressing either FLAG - tagged wild - type p85β or the indicated FLAG - tagged p85β mutants were immunoprecipitated ( IP ) with an anti - FLAG resin and immunoblotted as indicated . Uncropped images of blots are shown in Supplementary Fig . S8 .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O 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) Denatured extracts from HEK293T cells expressing either FLAG-tagged wild-type p85β or the indicated FLAG-tagged p85β mutants were immunoprecipitated (IP) with an anti-FLAG resin and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) The CaaX motif of FBXL2 is not required to bind PTPL1 . HEK293T cells were transfected with either FLAG - tagged wild - type FBXL2 or FLAG - tagged FBXL2 ( CaaX / SaaX ) . 24 h post - transfection , cells were collected and whole - cell lysates ( WCL ) were immunoprecipitated ( IP ) and immunoblotted as indicated .

O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O 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) The CaaX motif of FBXL2 is not required to bind PTPL1. HEK293T cells were transfected with either FLAG-tagged wild-type FBXL2 or FLAG-tagged FBXL2(CaaX/SaaX). 24 h post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated.


( c ) PTPL1 stimulates the binding of FBXL2 to wild - type p85β , but not p85β ( Y655A ) . HEK293T cells were transfected with GFP - tagged FBXL2 and either FLAG - tagged p85β or FLAG - tagged p85β ( Y655A ) . Where indicated , HA - tagged PTPL1 or HA - tagged PTPL1 ( C / S ) were also transfected . The experiment was performed as described in a .

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

(c) PTPL1 stimulates the binding of FBXL2 to wild-type p85β, but not p85β(Y655A). HEK293T cells were transfected with GFP-tagged FBXL2 and either FLAG-tagged p85β or FLAG-tagged p85β(Y655A). Where indicated, HA-tagged PTPL1 or HA-tagged PTPL1(C/S) were also transfected. The experiment was performed as described in a.


( d ) PTPL1 silencing inhibits the FBXL2 - p85β interaction . HeLa cells stably expressing FLAG - tagged FBXL2 were transfected with either an siRNA targeting PTPL1 or a non - silencing siRNA ( NS ) . Forty - eight hours post - transfection , cells were collected and whole - cell lysates ( WCL ) were immunoprecipitated ( IP ) and immunoblotted as indicated .

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(d) PTPL1 silencing inhibits the FBXL2-p85β interaction. HeLa cells stably expressing FLAG-tagged FBXL2 were transfected with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). Forty-eight hours post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated.


( e ) During a 72 - h serum starvation , NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1 , or a non - silencing siRNA ( NS ) . Cells were subsequently re - stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition .

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(e) During a 72-h serum starvation, NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were subsequently re-stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.


( f ) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1 , or a non - silencing siRNA ( NS ) . Cells were transfected with p85β ( Y655A ) and 16 h after cells were incubated with cycloheximide ( CHX ) for the indicated times , collected and analysed by immunoblotting as indicated .

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(f) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were transfected with p85β(Y655A) and 16 h after cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated.


g ) During a 48 - h serum starvation , U2OS cells stably transfected with a doxycycline - inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non - silencing siRNA ( NS ) . During the last 16 h before collection , p85β expression was stimulated with doxycycline . Cells were subsequently re - stimulated with media containing serum and collected 30 min later . Denatured cell lysates were immunoprecipitated with an anti - FLAG resin and immunoblotted as indicated . The asterisk indicates a non - specific band . Uncropped images of blots are shown in Supplementary Fig . S8 .

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g) During a 48-h serum starvation, U2OS cells stably transfected with a doxycycline-inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently re-stimulated with media containing serum and collected 30 min later. Denatured cell lysates were immunoprecipitated with an anti-FLAG resin and immunoblotted as indicated. The asterisk indicates a non-specific band. Uncropped images of blots are shown in Supplementary Fig. S8.


a ) During a 72 - h serum starvation , NHFs were transfected with either siRNAs targeting FBXL2 , p85β , both FBXL2 and p85β , or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . The graph at the right shows FBXL2 mRNA levels in the different samples analysed using real - time PCR in triplicate measurements . The values represent the ratios between FBXL2 and GAPDH mRNAs . SR , serum re - addition .

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

a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, p85β, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph at the right shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition.


( b ) Cells were treated as in a , except that 3 h after serum re - addition , cells were lysed , immunoprecipitated ( IP ) with an anti - IRS1 antibody and immunoblotted as indicated . Uncropped images of blots are shown in Supplementary Fig . S8 .

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(b) Cells were treated as in a, except that 3 h after serum re-addition, cells were lysed, immunoprecipitated (IP) with an anti-IRS1 antibody and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) During a 72 - h serum starvation , NHFs were transfected with either siRNAs targeting FBXL2 ( no . 1 or 2 ) or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition .

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(a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2 (no. 1 or 2) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.


( b ) During a 72 - h serum starvation , NHFs were transfected with either siRNAs targeting FBXL2 , both FBXL2 and p85β , or a non - silencing siRNA ( NS ) . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition .

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(b) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.


( c ) U2OS cells stably transfected with a doxycycline - inducible p85β construct were serum starved for 48 h . During the last 16 h before collection , p85β expression was stimulated with doxycycline . Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting . SR , serum re - addition .

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(c) U2OS cells stably transfected with a doxycycline-inducible p85β construct were serum starved for 48 h. During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition.


( d ) Cells constitutively expressing GFP - LC3 were transfected with HA - tagged p85β , fixed and incubated with an anti - HA p85β antibody ( red ) . Arrows in the top panels point to the enhanced autophagic response ( LC3 - II vesicles ) in cells expressing high levels of p85β , as shown in the bottom panels . Uncropped images of blots are shown in Supplementary Fig . S8 . Scale bars , 10 μm .

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(d) Cells constitutively expressing GFP-LC3 were transfected with HA-tagged p85β, fixed and incubated with an anti-HA p85β antibody (red). Arrows in the top panels point to the enhanced autophagic response (LC3-II vesicles) in cells expressing high levels of p85β, as shown in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S8. Scale bars, 10 μm.


B . Dot blot of tau levels in media from HEK293T cells overexpressing tau and FLAG - DnaJC5 compared to empty vector and tau . Intracellular protein levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels shown below ; data are mean ± SEM , n = 8 , * * * p < 0 . 001 .

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B. Dot blot of tau levels in media from HEK293T cells overexpressing tau and FLAG-DnaJC5 compared to empty vector and tau. Intracellular protein levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=8, ***p<0.001.


C . Light / heavy ratio of tau peptides in label - free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau . Light peptides represent endogenous tau peptides . Data are mean ± SEM , n = 3 biological repeats , * * * p < 0 . 001 .

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C. Light/heavy ratio of tau peptides in label-free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau. Light peptides represent endogenous tau peptides. Data are mean ± SEM, n=3 biological repeats, ***p<0.001.


A . Western blot of endogenous tau levels from M17 neuroblastoma cells overexpressing FLAG - DnaJC5 or empty vector . Media was concentrated prior to analysis . Intracellular DnaJC5 was detected with FLAG antibody . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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A. Western blot of endogenous tau levels from M17 neuroblastoma cells overexpressing FLAG-DnaJC5 or empty vector. Media was concentrated prior to analysis. Intracellular DnaJC5 was detected with FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Western blottau levels from concentrated media from ex - vivo organotypic slice cultures of wildtype mice overexpressing GFP - AAV9 or FLAG - DnaJC5 - AAV9 . DnaJC5 is detected by FLAG antibody . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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B. Western blottau levels from concentrated media from ex-vivo organotypic slice cultures of wildtype mice overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. DnaJC5 is detected by FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


C . Western blottau levels from concentrated media from ex - vivo organotypic slice cultures of Cspa + / + or Cspa - / - mice . DnaJC5 was detected by DnaJC5 antibody ( Synaptic Systems ) . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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. Western blottau levels from concentrated media from ex-vivo organotypic slice cultures of Cspa+/+ or Cspa-/- mice. DnaJC5 was detected by DnaJC5 antibody (Synaptic Systems). Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


A . FLAG - DnaJC5 overexpression facilitates extracellular release of wildtype ( WT ) , P301L and R406W tau by dot blot in HEK293T cells . Intracellular protein levels are shown by Western blot ; DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels shown below ; data are mean ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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A. FLAG-DnaJC5 overexpression facilitates extracellular release of wildtype (WT), P301L and R406W tau by dot blot in HEK293T cells. Intracellular protein levels are shown by Western blot; DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Overexpression of FLAG - DnaJC5 induces extracellular release of wildtype ( WT ) and A53T α - synuclein in HEK293T cells . Extracellular protein levels shown by dot blot ( quantification below , mean ± SEM , n = 3 * * * p < 0 . 001 , * * p < 0 . 01 by one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis ) and intracellular proteins levels were analyzed by Western blot , DnaJC5 was detected by FLAG antibody .

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B. Overexpression of FLAG-DnaJC5 induces extracellular release of wildtype (WT) and A53T α-synuclein in HEK293T cells. Extracellular protein levels shown by dot blot (quantification below, mean ±SEM, n=3 ***p<0.001, ** p<0.01 by one-way analysis of variance with Tukey's multiple comparison post-hoc analysis) and intracellular proteins levels were analyzed by Western blot, DnaJC5 was detected by FLAG antibody.


C . DnaJC5 overexpression induces extracellular release of GFP - tagged wildtype ( WT ) , A315T and Q343R TDP - 43 in HEK293T cells . Intracellular DnaJC5 was detected by FLAG antibody . Extracellular protein levels are analyzed by dot blot ( quantification below , mean ± SEM , n = 4 ) * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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C. DnaJC5 overexpression induces extracellular release of GFP-tagged wildtype (WT), A315T and Q343R TDP-43 in HEK293T cells. Intracellular DnaJC5 was detected by FLAG antibody. Extracellular protein levels are analyzed by dot blot (quantification below, mean ±SEM, n=4) ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


A . Dot blot of tau levels in media of cells overexpressing the phospho - deficient FLAG - DnaJC5 S10A compared to FLAG - DnaJC5 WT . Intracellular levels shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of tau levels are mean ± SEM , n = 6 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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A. Dot blot of tau levels in media of cells overexpressing the phospho-deficient FLAG-DnaJC5 S10A compared to FLAG-DnaJC5 WT. Intracellular levels shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated tau release in HEK293T cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected by FLAG antibody .

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B. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated tau release in HEK293T cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.


C . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated α - synuclein release in M17 cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 by one - way ANOVA with a Tukey ' s post - hoc analysis . DnaJC5 is detected by FLAG antibody .

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C. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated α-synuclein release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 by one-way ANOVA with a Tukey's post-hoc analysis. DnaJC5 is detected by FLAG antibody.


D . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated TDP - 43 release in M17 cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected by FLAG antibody .

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

D. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated TDP-43 release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.


A . Confocal microscopy of tau ( red pseudocolor ) and Hsp70 ( green pseudocolor ) colocalization ( merged image ) levels in wild type primary neurons overexpressing GFP - AAV9 or FLAG - DnaJC5 - AAV9 . Boxed area are 5X digital zoom on a 60X lens shown below . Scale bars are 20 µm and 5 µm as indicated . Only neurons overexpressing virus were imaged . B . Quantification of tau - Hsp70 co - localization ( mean Pearson ' s coefficient ± SEM , n = 18 , * p < 0 . 05 , one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis ) .

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A. Confocal microscopy of tau (red pseudocolor) and Hsp70 (green pseudocolor) colocalization (merged image) levels in wild type primary neurons overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. Boxed area are 5X digital zoom on a 60X lens shown below. Scale bars are 20 µm and 5 µm as indicated. Only neurons overexpressing virus were imaged.B. Quantification of tau-Hsp70 co-localization (mean Pearson's coefficient ± SEM, n=18, *p <0.05, one-way analysis of variance with Tukey's multiple comparison post-hoc analysis).


C . Western blot of immunoprecipitatedtau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70 . DnaJC5 was detected with FLAG antibody .

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C. Western blot of immunoprecipitatedtau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70. DnaJC5 was detected with FLAG antibody.


D . Western blot of HEK293T intracellular lysates show the DnaJC5 / Hsc70 / tau complex is disrupted by the addition of increasing amounts of YM - 01 ( 0 , 10 , 30 , 100 µM ) in a dose - dependent fashion . DnaJC5 was detected by FLAG antibody .

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D. Western blot of HEK293T intracellular lysates show the DnaJC5/Hsc70/tau complex is disrupted by the addition of increasing amounts of YM-01 (0, 10, 30, 100 µM) in a dose-dependent fashion. DnaJC5 was detected by FLAG antibody.


E . The DnaJC5 / Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant - negative point mutation , E175S . Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70 . DnaJC5 is immunopreicipitated by DnaJC5 antibody ( Synaptic Systems ) . DnaJC5 was detected on Western blot by FLAG antibody .

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E. The DnaJC5/Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant-negative point mutation, E175S. Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70. DnaJC5 is immunopreicipitated by DnaJC5 antibody (Synaptic Systems). DnaJC5 was detected on Western blot by FLAG antibody.


F . Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM - 01 . Quantification of extracellular tau levels are mean ± SEM , n = 6 , * p < 0 . 05 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . Intracellular levels shown by Western blot . DnaJC5 was detected on Western blot by FLAG antibody .

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F. Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM-01. Quantification of extracellular tau levels are mean ± SEM, n=6, *p<0.05 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. Intracellular levels shown by Western blot. DnaJC5 was detected on Western blot by FLAG antibody.


G . Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM - 01 ( 10µM ) . FLAG - DnaJC5 was transduced into neurons using AAV9 . Quantification of extracellular tau levels are mean ± SEM , n = 6 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected on Western blot by FLAG antibody .

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G. Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM-01 (10µM). FLAG-DnaJC5 was transduced into neurons using AAV9. Quantification of extracellular tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected on Western blot by FLAG antibody.


A . Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG - DnaJC5 reduces extracellular tau levels as shown by dot blot . Intracellular levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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A. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular tau levels as shown by dot blot. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG - DnaJC5 reduces extracellular α - synuclein levels as shown by dot blot . Intracellular levels are shown by Western blotDnaJC5 was detected by FLAG antibody . Quantification of extracellular synuclein levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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B. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular α-synuclein levels as shown by dot blot. Intracellular levels are shown by Western blotDnaJC5 was detected by FLAG antibody. Quantification of extracellular synuclein levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


C . Dominant - negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot . Intracellular levels are shown by Western blot . Quantification of extracellular tau levels is shown as average ± SEM , n = 4 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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C. Dominant-negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot. Intracellular levels are shown by Western blot. Quantification of extracellular tau levels is shown as average ± SEM, n=4, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


D . Knockdown of Hsc70 reduces extracellular tau release ( dot blot ) in HEK293T cells overexpressing FLAG - DnaJC5 . Intracellular levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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D. Knockdown of Hsc70 reduces extracellular tau release (dot blot) in HEK293T cells overexpressing FLAG-DnaJC5. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


E . A mutant tau that does not bind Hsc70 is not released when FLAG - DnaJC5 is overexpressed in HEK293T cells . Dot blot and quantification of extracellular tau levels are shown . Intracellular protein levels are shown with representative Western blot , DnaJC5 was detected by FLAG antibody . Data are mean ± SEM , n = 3 p < 0 . 001 . one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

E. A mutant tau that does not bind Hsc70 is not released when FLAG-DnaJC5 is overexpressed in HEK293T cells. Dot blot and quantification of extracellular tau levels are shown. Intracellular protein levels are shown with representative Western blot, DnaJC5 was detected by FLAG antibody. Data are mean ± SEM, n=3 p<0.001. one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


F . Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot . HSPA variants were detected with V5 antibody . Data are mean ± SEM , n = 3 p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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F. Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. HSPA variants were detected with V5 antibody. Data are mean ± SEM, n=3 p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


G . Dot blot of different FLAG - DnaJ overexpression effect on extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot . DnaJ levels were detected by FLAG antibody . Data are mean ± SEM , n = 3 ; * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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G. Dot blot of different FLAG-DnaJ overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. DnaJ levels were detected by FLAG antibody. Data are mean ± SEM, n=3; *** p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


H . Dot blot of increasing FLAG - DnaJC7 levels on FLAG - DnaJC5 overexpression - mediated extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot , DnaJ proteins were detected by FLAG antibody . Quantification of extracellular levels are mean ± SEM , n = 4 .

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H. Dot blot of increasing FLAG-DnaJC7 levels on FLAG-DnaJC5 overexpression-mediated extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot, DnaJ proteins were detected by FLAG antibody. Quantification of extracellular levels are mean ± SEM, n=4.


( B ) 48h post - transfection of HeLa - CIITA cells with siRNAs targeting NDP52 , T6BP , OPTN , and p62 , AR expression was analyzed using Western Blot . Tubulin was used as control . The results are representative of at least 3 independent experiments and correspond to AR expression levels of the experiment in Figure 1B , left panel .

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(B) 48h post-transfection of HeLa-CIITA cells with siRNAs targeting NDP52, T6BP, OPTN, and p62, AR expression was analyzed using Western Blot. Tubulin was used as control. The results are representative of at least 3 independent experiments and correspond to AR expression levels of the experiment in Figure 1B, left panel.


( C ) Monitoring of Gag - specific T cell activation . HeLa - CIITA cells were treated as indicated above and transfected with a plasmid encoding Gag - LC3 . Left panel , a representative experiment is shown ( + / - SD of three technical replicates ) . Right panel , three biological replicates are combined and presented as mean percentage ( + / - SD ) . The y - axis represents the relative percentage of IFNγ spots reported to the secretion of IFNγ by the CD4 + T cell clones incubated with the siCTRL - treated HeLa - CIITA and set to 100 % . The mean T cell activation levels of the three experiments using siCTRL or siT6BP were ( 100 , 195 , 119 ) and ( 35 , 53 , 38 ) IFNγ + spots , respectively . 5000 T cell clones were seeded per well in technical triplicates . Data information : For all ELISPOT experiments , the background secretions of IFNγ by CD4 + T cells co - cultured with mock - treated HeLa - CIITA cells were used as negative controls and subtracted . CTRL : control . Wilcoxon ' s test ; the symbols correspond to * * p < 0 . 01 and # p > 0 . 05 comparing each experimental conditions solely with its internal control ( siCTRL ) .

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

(C) Monitoring of Gag-specific T cell activation. HeLa-CIITA cells were treated as indicated above and transfected with a plasmid encoding Gag-LC3. Left panel, a representative experiment is shown (+/- SD of three technical replicates). Right panel, three biological replicates are combined and presented as mean percentage (+/- SD). The y-axis represents the relative percentage of IFNγ spots reported to the secretion of IFNγ by the CD4+ T cell clones incubated with the siCTRL-treated HeLa-CIITA and set to 100%. The mean T cell activation levels of the three experiments using siCTRL or siT6BP were (100, 195, 119) and (35, 53, 38) IFNγ+ spots, respectively. 5000 T cell clones were seeded per well in technical triplicates. Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols correspond to ** p<0.01 and # p>0.05 comparing each experimental conditions solely with its internal control (siCTRL).


( D ) Left panel , as in Figure 1B right panel , but using DNA encoding Gag - LC3 , Gag - LC3G120A , or Gag . With Gag - LC3 , Gag - LC3 G120A or Gag antigens , the mean T cell activation levels using siCTRL or siT6BP were ( 199 , 435 , 384 ) , ( 91 , 141 , 119 ) and ( 343 , 415 , 385 ) or ( 98 , 273 , 171 ) , ( 27 , 50 , 19 ) and ( 97 , 148 , 131 ) IFNγ + spots , respectively . 5000 T cell clones were seeded per well in technical triplicates . Three biological replicates are combined and presented as mean percentage ( + / - SD ) . Right panel , influence of T6BP silencing on peptide presentation by Hela - CIITA cells . The cognate peptide was added exogenously ( gag2 , 0 , 1µg / mL ) on siRNA - treated cells ( 2h , 37 ° C ) , washed and T cell activation monitored using IFNγ - ELISPOT . The mean T cell activation levels using siCTRL or siT6BP were ( 100 , 45 , 42 ) and ( 71 , 45 , 33 ) IFNγ + spots , respectively . 1000 T cell clones were seeded per well in technical triplicates . Three biological replicates are combined and presented as mean percentage ( + / - SD ) . Data information : For all ELISPOT experiments , the background secretions of IFNγ by CD4 + T cells co - cultured with mock - treated HeLa - CIITA cells were used as negative controls and subtracted . CTRL : control . Wilcoxon ' s test ; the

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(D) Left panel, as in Figure 1B right panel, but using DNA encoding Gag-LC3, Gag-LC3G120A, or Gag. With Gag-LC3, Gag-LC3 G120A or Gag antigens, the mean T cell activation levels using siCTRL or siT6BP were (199, 435, 384), (91, 141, 119) and (343, 415, 385) or (98, 273, 171), (27, 50, 19) and (97, 148, 131) IFNγ+ spots, respectively. 5000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (+/- SD). Right panel, influence of T6BP silencing on peptide presentation by Hela-CIITA cells. The cognate peptide was added exogenously (gag2, 0,1µg/mL) on siRNA-treated cells (2h, 37°C), washed and T cell activation monitored using IFNγ-ELISPOT. The mean T cell activation levels using siCTRL or siT6BP were (100, 45, 42) and (71, 45, 33) IFNγ+ spots, respectively. 1000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (+/- SD). Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols


( E ) As in ( D ) but using cDNA encoding HCMV pp65 antigen ( left panel ) or pp65 peptide ( 0 , 5µg / mL ; right panel ) and a pp65 - specific CD4 + T cell line . Results of three independent experiments are represented . The mean T cell activation levels using siCTRL or siT6BP and pp65 DNA were ( 66 , 106 , 99 ) and ( 39 , 50 , 33 ) IFNγ + spots , respectively . For the peptide : siCTRL or siT6BP : ( 185 , 317 , 415 ) and ( 163 , 292 , 381 ) IFNγ + spots , respectively . 1000 or 2000 T cell clones were seeded per well in technical triplicates . Data information : For all ELISPOT experiments , the background secretions of IFNγ by CD4 + T cells co - cultured with mock - treated HeLa - CIITA cells were used as negative controls and subtracted . CTRL : control . Wilcoxon ' s test ; the symbols correspond to * * p < 0 . 01 and # p > 0 . 05 comparing each experimental conditions solely with its internal control ( siCTRL ) .

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

(E) As in (D) but using cDNA encoding HCMV pp65 antigen (left panel) or pp65 peptide (0,5µg/mL; right panel) and a pp65-specific CD4+ T cell line. Results of three independent experiments are represented. The mean T cell activation levels using siCTRL or siT6BP and pp65 DNA were (66, 106, 99) and (39, 50, 33) IFNγ+ spots, respectively. For the peptide: siCTRL or siT6BP: (185, 317, 415) and (163, 292, 381) IFNγ+ spots, respectively. 1000 or 2000 T cell clones were seeded per well in technical triplicates. Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols correspond to ** p<0.01 and # p>0.05 comparing each experimental conditions solely with its internal control (siCTRL).


( D ) T6BP silencing affects the formation of stable MHC - peptide complexes . HeLa - CIITA cells were transfected as above and pulse - labeled with 35S - Met / Cys for 30 min , washed and chased for 4h at 37 ° C . MHC - II molecules were then immunoprecipitated using TÜ36 antibody and analyzed on SDS - PAGE after incubation of the immunoprecipitated protein complexes with SDS at 95 ° C ( B : boiled ) or room temperature ( NB : non - boiled ) to visualize α , β and Ii chains and SDS resistant αβ dimers , respectively . The bands corresponding to α , β and Ii chains are indicated . The arrow indicates the SDS - resistant stable HLA αβ heterodimers . This gel is representative of two independent experiments .

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(D) T6BP silencing affects the formation of stable MHC-peptide complexes. HeLa-CIITA cells were transfected as above and pulse-labeled with 35S-Met/Cys for 30 min, washed and chased for 4h at 37°C. MHC-II molecules were then immunoprecipitated using TÜ36 antibody and analyzed on SDS-PAGE after incubation of the immunoprecipitated protein complexes with SDS at 95°C (B: boiled) or room temperature (NB: non-boiled) to visualize α, β and Ii chains and SDS resistant αβ dimers, respectively. The bands corresponding to α, β and Ii chains are indicated. The arrow indicates the SDS-resistant stable HLA αβ heterodimers. This gel is representative of two independent experiments.


( A ) MHC - II and T6BP expressions were assessed using confocal microscopy . HeLa - CIITA cells were transfected with control and T6BP - silencing siRNAs . 48h post - treatment , MHC - II and T6BP were detected using L243 and anti - T6BP antibodies , respectively , and revealed with species specific secondary antibodies . Nuclei were stained using DAPI . ( B ) Quantitative analysis using in - house ImageJ script displaying distance of each MHC - II + vesicles to the nucleus and number of vesicles per cell . At least 20 , 000 vesicles from 160 cells corresponding to five biological replicates were analyzed . Right panel , quantification in the siCTRL cells of the colocalization between MHC - II + and T6BP + dots using Pearson ' s coefficient where the dotted lines ( at 0 . 5 ) indicate the limit under which no significant co - localization is measured ( number of cells = 54 ) . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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(A) MHC-II and T6BP expressions were assessed using confocal microscopy. HeLa-CIITA cells were transfected with control and T6BP-silencing siRNAs. 48h post-treatment, MHC-II and T6BP were detected using L243 and anti-T6BP antibodies, respectively, and revealed with species specific secondary antibodies. Nuclei were stained using DAPI. (B) Quantitative analysis using in-house ImageJ script displaying distance of each MHC-II+ vesicles to the nucleus and number of vesicles per cell. At least 20,000 vesicles from 160 cells corresponding to five biological replicates were analyzed. Right panel, quantification in the siCTRL cells of the colocalization between MHC-II+ and T6BP+ dots using Pearson's coefficient where the dotted lines (at 0.5) indicate the limit under which no significant co-localization is measured (number of cells = 54). Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( C ) As in A , T6BP and LAMP - 1 expressions were analyzed . ( D ) The localization of LAMP - 1 + vesicles and the number of vesicles per cell were quantified as in B . At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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(C) As in A, T6BP and LAMP-1 expressions were analyzed. (D) The localization of LAMP-1+ vesicles and the number of vesicles per cell were quantified as in B. At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed. Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( E ) As in A , adding Lysotracker staining . ( F ) As in B , quantitative analysis of Lysotracker + vesicles : localization to the nucleus and number of vesicles per cell . Colocalization of Lysotracker + vesicles with MHC - II + puncta was analyzed using JACoP plugin ( scales start at 0 . 5 above which the % of co - localization is considered significant ) , number of vesicles > 2000 from at least three biological replicates were analyzed corresponding to 20 cells . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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(E) As in A, adding Lysotracker staining. (F) As in B, quantitative analysis of Lysotracker+ vesicles: localization to the nucleus and number of vesicles per cell. Colocalization of Lysotracker+ vesicles with MHC-II+ puncta was analyzed using JACoP plugin (scales start at 0.5 above which the % of co-localization is considered significant), number of vesicles > 2000 from at least three biological replicates were analyzed corresponding to 20 cells. Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( G ) As in E , CD63 and MHC - II expressions were assessed . ( H ) Quantitative analysis as represented in F . At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed . Data information : In graphs representing the number of vesicles per cells , each dot displayed corresponds to a single cell . Within the violin plots , continuous and dotted lines correspond to medians and quartiles , respectively . Scale bars : 2μm . CTRL : control . Mann - Whitney ' s test ; * p < 0 . 05 ; * * p < 0 . 002 ; * * * p < 0 . 0003 ; * * * * p < 0 . 0001 ; # p > 0 . 05 .

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(G) As in E, CD63 and MHC-II expressions were assessed. (H) Quantitative analysis as represented in F. At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed. Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.


( A ) CD74 expression was assessed using Western Blot . HeLa - CIITA cells were transfected with siCTRL and siT6BP . 48h post treatment , the Iip33 CD74 isoform , a degradation product ( Iip16 ) , T6BP and actin were detected using indicated antibodies . Left panel , a representative Western Blot experiment is shown . Right panel , expressions levels five biological replicates were quantified using ImageJ and are presented as mean of ratios ( + / - SD ) of CD74 to actin used as control housekeeping gene expression .

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(A) CD74 expression was assessed using Western Blot. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, the Iip33 CD74 isoform, a degradation product (Iip16), T6BP and actin were detected using indicated antibodies. Left panel, a representative Western Blot experiment is shown. Right panel, expressions levels five biological replicates were quantified using ImageJ and are presented as mean of ratios (+/-SD) of CD74 to actin used as control housekeeping gene expression.


( B ) As in ( A ) assessing HLA - DR and actin expression levels . Top panel , a representative Western Blot experiment is shown . Bottom panel , in three biological replicates , HLA - DR expression was quantified using ImageJ and is presented as a mean of ratios to actin ( + / - SD ) . ( C ) As in ( B ) assessing HLA - DM and actin expression levels . Bottom panel , HLA - DM expression was quantified in three biological replicates and presented as mean of ratios to actin ( + / - SD ) .

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(B) As in (A) assessing HLA-DR and actin expression levels. Top panel, a representative Western Blot experiment is shown. Bottom panel, in three biological replicates, HLA-DR expression was quantified using ImageJ and is presented as a mean of ratios to actin (+/-SD). (C) As in (B) assessing HLA-DM and actin expression levels. Bottom panel, HLA-DM expression was quantified in three biological replicates and presented as mean of ratios to actin (+/-SD).


( D ) CD74 expression is partially recovered by blocking lysosomal acidification . As in ( A ) , following siRNA transfection of Hela - CIITA cells , CD74 expression was assessed . The last 16h prior to harvesting , cells were treated with chloroquine ( CQ ) or epoxomicin ( Epo ) . Left panel a representative experiment is shown , membranes were blotted using anti - T6BP , - CD74 and - actin antibodies . The different degradation fragments of CD74 are indicated ( Iip33 , Iip22 and Iip16 ) . Right panel , expression levels of Iip33 ( top ) and Iip16 ( bottom ) were quantified in three biological replicates using ImageJ and are presented as mean of ratios of each fragment to actin ( + / - SD ) . Data information : ( D ) One - way Anova statistical test combined with Bonferronis multiple comparison test was applied . Dotted lines indicate statistically significant differences between conditions .

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(D) CD74 expression is partially recovered by blocking lysosomal acidification. As in (A), following siRNA transfection of Hela-CIITA cells, CD74 expression was assessed. The last 16h prior to harvesting, cells were treated with chloroquine (CQ) or epoxomicin (Epo). Left panel a representative experiment is shown, membranes were blotted using anti-T6BP, -CD74 and -actin antibodies. The different degradation fragments of CD74 are indicated (Iip33, Iip22 and Iip16). Right panel, expression levels of Iip33 (top) and Iip16 (bottom) were quantified in three biological replicates using ImageJ and are presented as mean of ratios of each fragment to actin (+/-SD). Data information: (D) One-way Anova statistical test combined with Bonferronis multiple comparison test was applied. Dotted lines indicate statistically significant differences between conditions.


( E Analysis of CD74 proteolysis . HeLa - CIITA cells were transfected with siCTRL and siT6BP . 48h post treatment , cells were pulsed for 30 min with 35S - Met / Cys , washed and chased for 1 , 2 and 4h . HLA - DR / CD74 complexes were first immunoprecipitated using Tü36 antibody ( E ) Samples were boiled and analyzed using SDS - PAGE . The bands corresponding to CD74 isoforms ( Iip41 and Iip33 ) and the cleavage products ( Iip16 ) are indicated . ( E right panel ) Iip16 expression was quantified using ImageJ and presented as a percentage of Iip16 normalized to the highest quantity of Iip16 detected after 1h of chase in the control condition . Results are representative of two biological replicates . Ii : invariant chain ( CD74 ) ; CTRL : control .

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(E Analysis of CD74 proteolysis. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed and chased for 1, 2 and 4h. HLA-DR/CD74 complexes were first immunoprecipitated using Tü36 antibody (E) Samples were boiled and analyzed using SDS-PAGE. The bands corresponding to CD74 isoforms (Iip41 and Iip33) and the cleavage products (Iip16) are indicated. (E right panel) Iip16 expression was quantified using ImageJ and presented as a percentage of Iip16 normalized to the highest quantity of Iip16 detected after 1h of chase in the control condition. Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control.


F ) . Analysis of CD74 proteolysis . HeLa - CIITA cells were transfected with siCTRL and siT6BP . 48h post treatment , cells were pulsed for 30 min with 35S - Met / Cys , washed and chased for 1 , 2 and 4h . HLA - DR / CD74 complexes were immunoprecipitated using VICY1 antibody ( F ) . Samples were boiled and analyzed using SDS - PAGE . The bands corresponding to CD74 isoforms Iip33 ) Results are representative of two biological replicates . Ii : invariant chain ( CD74 ) ; CTRL : control .

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F). Analysis of CD74 proteolysis. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed and chased for 1, 2 and 4h. HLA-DR/CD74 complexes were immunoprecipitated using VICY1 antibody (F). Samples were boiled and analyzed using SDS-PAGE. The bands corresponding to CD74 isoforms Iip33) Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control.


( A - B ) CANX co - immunoprecipitates with T6BP in model and professional APCs . Endogenous T6BP was immunoprecipitated from HeLa - CIITA , B ( DG - 75 ) and dendritic - like ( KG - 1 , DC ) cells . The input , the flow through ( FT ) and the immunoprecipitation ( IP ) fractions were analyzed by Western blot using the indicated antibodies . Bottom panels , control IPs using the beads without antibody ( IP CTRL ) are presented for each IP . ( B ) As in ( A ) but using anti - CANX Ab for the IP . For A and B , one representative experiment out of three biological replicates is shown .

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(A-B) CANX co-immunoprecipitates with T6BP in model and professional APCs. Endogenous T6BP was immunoprecipitated from HeLa-CIITA, B (DG-75) and dendritic-like (KG-1, DC) cells. The input, the flow through (FT) and the immunoprecipitation (IP) fractions were analyzed by Western blot using the indicated antibodies. Bottom panels, control IPs using the beads without antibody (IP CTRL) are presented for each IP. (B) As in (A) but using anti-CANX Ab for the IP. For A and B, one representative experiment out of three biological replicates is shown.


( C ) CANX interacts with T6BP using proximity ligation assay ( PLA ) . 48h post - transfection with the indicated siRNAs , HeLa - CIITA cells were fixed , stained with anti - T6BP and anti - CANX antibodies and proximity revealed using PLA ( Duolink ) . Nuclei were stained using DAPI . Top panel , two representative fields are shown . Bottom panel , quantitative analysis using ImageJ displaying the number of PLA per cell . The continuous line indicates the mean ( + / - SEM ) . PLA were quantified in at least 130 cells corresponding to three biological replicates . Scale bars : 10μm . CTRL : control ; Mann - Whitney ' s test ; * * * : p < 0 . 0003 .

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(C) CANX interacts with T6BP using proximity ligation assay (PLA). 48h post-transfection with the indicated siRNAs, HeLa-CIITA cells were fixed, stained with anti-T6BP and anti-CANX antibodies and proximity revealed using PLA (Duolink). Nuclei were stained using DAPI. Top panel, two representative fields are shown. Bottom panel, quantitative analysis using ImageJ displaying the number of PLA per cell. The continuous line indicates the mean (+/-SEM). PLA were quantified in at least 130 cells corresponding to three biological replicates. Scale bars: 10μm. CTRL: control; Mann-Whitney's test; ***: p<0.0003.


( D ) Calnexin interacts with T6BP through its cytosolic tail . HeLa - CIITA cells were transfected with a plasmid encoding the GST - tagged transmembrane ( TM ) and cytosolic ( CytoTail ) domains of CANX ( GST - TM - CytoTail - CANX ) or the vector not encoding TM - CytoTail - CANX as control and immunoprecipitated with anti - GST antibodies . The input , FT , the wash and the IP fractions were analyzed by Western blot and revealed using the indicated antibodies . One representative experiment out of three biological replicates is shown .

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(D) Calnexin interacts with T6BP through its cytosolic tail. HeLa-CIITA cells were transfected with a plasmid encoding the GST-tagged transmembrane (TM) and cytosolic (CytoTail) domains of CANX (GST-TM-CytoTail-CANX) or the vector not encoding TM-CytoTail-CANX as control and immunoprecipitated with anti-GST antibodies. The input, FT, the wash and the IP fractions were analyzed by Western blot and revealed using the indicated antibodies. One representative experiment out of three biological replicates is shown.


( E ) Silencing of T6BP does not influence CANX expression levels while silencing of CANX reduces the level of CD74 expression . HeLa - CIITA cells transfected with the indicated siRNAs and samples analyzed , 48h post transfection , by Western blot with the indicated antibodies . These western blot results are representative of at least three biological replicates .

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(E) Silencing of T6BP does not influence CANX expression levels while silencing of CANX reduces the level of CD74 expression. HeLa-CIITA cells transfected with the indicated siRNAs and samples analyzed, 48h post transfection, by Western blot with the indicated antibodies. These western blot results are representative of at least three biological replicates.


A Liver biopsies showing no ( IS0 ) , moderate ( IS3 ) , and severe ( IS6 ) fibrosis stained by PSR . Size bar , 300 μm .

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A Liver biopsies showing no (IS0), moderate (IS3), and severe (IS6) fibrosis stained by PSR. Size bar, 300 μm.


B Immunohistochemistry ( brown staining ) for αSMA and SOX9 on consecutive sections from patients with no ( IS0 ) , moderate ( IS3 ) , and severe ( IS6 ) fibrosis . Boxed areas are shown to the right at higher magnification . Bile duct ( bd ) . SOX9 detected in HSCs with elongated nuclei ( hatched line ) next to scar and in hepatocytes with rounded nuclei ( arrows ) . Size bars , 200 µm .

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B Immunohistochemistry (brown staining) for αSMA and SOX9 on consecutive sections from patients with no (IS0), moderate (IS3), and severe (IS6) fibrosis. Boxed areas are shown to the right at higher magnification. Bile duct (bd). SOX9 detected in HSCs with elongated nuclei (hatched line) next to scar and in hepatocytes with rounded nuclei (arrows). Size bars, 200 µm.


C Immunofluorescence for SOX9 ( red ) co - stained with CK19 , CK7 , α1AT , and αSMA ( green ) . Arrowheads show elongated nuclei in αSMA - positive HSCs .

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C Immunofluorescence for SOX9 (red) co-stained with CK19, CK7, α1AT, and αSMA (green). Arrowheads show elongated nuclei in αSMA-positive HSCs.


D Immunohistochemistry for SOX9 and EpCAM ( brown staining ) on consecutive sections from severe ( IS6 ) fibrosis and normal fetal liver at 18 weeks post - conception ( wpc ) . Dual immunofluorescence confirms SOX9 and EpCAM in the same cell in severe fibrosis .

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D Immunohistochemistry for SOX9 and EpCAM (brown staining) on consecutive sections from severe (IS6) fibrosis and normal fetal liver at 18 weeks post-conception (wpc). Dual immunofluorescence confirms SOX9 and EpCAM in the same cell in severe fibrosis.


E , F , G Data for SOX9 index for the biliary , HSC / Hep , and total ( biliary + HSC / Hep ) populations . In worsening categories of liver fibrosis ( E ) . In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow - up biopsy ( F ) . The lines shows the best fit by ordinal logistic regression analysis . In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow - up biopsy ( G ) .

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E, F, G Data for SOX9 index for the biliary, HSC/Hep, and total (biliary + HSC/Hep) populations. In worsening categories of liver fibrosis (E). In the initial biopsy plotted against the increment in Ishak fibrosis stage between the initial and follow-up biopsy (F). The lines shows the best fit by ordinal logistic regression analysis. In the initial biopsy categorized by whether patients did or did not progress by at least two stages on follow-up biopsy (G).


I - L Plots of SOX9 index ( total , HSC / Hep , and biliary ) and NI score marking progressors in green and non - progressors in red . The solid line indicates the mean in each graph . The broken line in NI dot plot ( L ) indicates the threshold ( score = 8 ) below which all NI is categorized as ' mild ' .

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I-L Plots of SOX9 index (total, HSC/Hep, and biliary) and NI score marking progressors in green and non-progressors in red. The solid line indicates the mean in each graph. The broken line in NI dot plot (L) indicates the threshold (score = 8) below which all NI is categorized as 'mild'.


A Immunohistochemistry for SOX9 ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis . SOX9 positive bile ducts ( bd ) and hepatocytes ( arrowheads ) indicated .

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A Immunohistochemistry for SOX9 (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis. SOX9 positive bile ducts (bd) and hepatocytes (arrowheads) indicated.


B ( Right panel ) Immunofluorescence for SOX9 ( green ) and HNF4α ( red ) in WT fibrotic mice . Arrowheads ( orange / yellow staining ) indicate SOX9 + / HNF4α + hepatocytes and star ( * ; red ) indicates SOX - / HNF4α + hepatocytes . ( Left panel ) Immunofluorescence for SOX9 ( green nucleus ) and CK19 ( red cytoplasm ) in WT fibrotic mice . Arrowheads ( green ) indicate SOX9 + hepatocytes and star ( * ) indicates SOX - hepatocytes identified by DAPI staining in Appendix Figure S1 .

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B (Right panel) Immunofluorescence for SOX9 (green) and HNF4α (red) in WT fibrotic mice. Arrowheads (orange/yellow staining) indicate SOX9+/HNF4α+ hepatocytes and star (*; red) indicates SOX-/ HNF4α+ hepatocytes. (Left panel) Immunofluorescence for SOX9 (green nucleus) and CK19 (red cytoplasm) in WT fibrotic mice. Arrowheads (green) indicate SOX9+ hepatocytes and star (*) indicates SOX- hepatocytes identified by DAPI staining in Appendix Figure S1.


C Co - localization by immunohistochemistry ( top panel ) and in situ hybridization ( lower panel ) for SOX9 ( red ) and αSMA ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis .

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C Co-localization by immunohistochemistry (top panel) and in situ hybridization (lower panel) for SOX9 (red) and αSMA (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis.


D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control .

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D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control.