HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf , followed by co - immunoprecipitation assay to analyse the interaction of NDP52 with FIP200 , ULK1 and ATG13 ( D ) . Data information : Data are mean ± s . e . m . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments D , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) .

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

HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by co-immunoprecipitation assay to analyse the interaction of NDP52 with FIP200, ULK1 and ATG13 (D). Data information: Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments D, *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).


E , MEFs stably expressing YFP - Parkin , mt - mKeima and empty , human NDP52 ( hNDP52 ) WT or hNDP52 Mut were pre - treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement . Data information : Data are mean ± s . e . m . E ) or displayed as cell popular violin plots ( E ) . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments E ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm E ) .

O O O O O O O O O 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 O O 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, MEFs stably expressing YFP-Parkin, mt-mKeima and empty, human NDP52 (hNDP52) WT or hNDP52 Mut were pre-treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement. Data information: Data are mean ± s.e.m. E) or displayed as cell popular violin plots (E). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments E). *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm E).


C . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an AU - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . D . Dissociation constants ( Kd ) of Ago2 - miRNA complexes ( containing an GC - rich supplementary region ) binding to target RNAs with bridging regions of various lengths . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an AU-rich supplementary region) binding to target RNAs with bridging regions of various lengths. D. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an GC-rich supplementary region) binding to target RNAs with bridging regions of various lengths. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


E . Fold repression of a luciferase reporter ( relative to no target site control ) plotted versus affinity ( 1 / Kd ) values measured for the same sites in vitro . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fold repression of a luciferase reporter (relative to no target site control) plotted versus affinity (1/Kd) values measured for the same sites in vitro. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


A . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed region only . Bottom : release of the seed - paired target from Ago2 - miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time . B . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed and supplementary ( g13 - g17 ) regions . Bottom : fraction target bound to the Ago2 - miR complexes as a function of time . C . Top : schematic of miR - 122 isomiRs paired with a target complementary to the seed and shifted supplementary ( g15 - g19 ) regions . . Bottom : fraction target bound to the Ago2 - miR complexes as a function of time . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed region only. Bottom: release of the seed-paired target from Ago2-miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time. B. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and supplementary (g13-g17) regions. Bottom: fraction target bound to the Ago2-miR complexes as a function of time. C. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and shifted supplementary (g15-g19) regions. . Bottom: fraction target bound to the Ago2-miR complexes as a function of time. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


E . Release half times ( t1 / 2 ) of target RNAs in panels ( A - C ) plotted against isomiR length . Data information : All plotted data are the means of at least three independent replicates . Error bars indicate SEM .

O O O O O O O O O O O O O O O O O O O O O O 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. Release half times (t1/2) of target RNAs in panels (A-C) plotted against isomiR length. Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.


C Percentage of BrdU positive cb9Δmyc cells , in the presence or absence ( 24h after removal ) of doxycycline . Data are presented as mean ± SD ; n = 3 . Two - tailed Student t - test was used to compare between two groups and expressed as p - values .

C Percentage of BrdU positive cb9Δmyc cells, in the presence or absence (24h after removal) of doxycycline. Data are presented as mean ± SD; n = 3. Two-tailed Student t-test was used to compare between two groups and expressed as p-values.


D Immunoblot analysis of c - myc - / - HO15 . 19 rat fibroblasts infected with retroviral vectors expressing the indicated Myc proteins . Parental TGR1 cells serve as control for endogenous levels of the MycWT protein .

O O O O O O O O O O O O O O O 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 Immunoblot analysis of c-myc-/- HO15.19 rat fibroblasts infected with retroviral vectors expressing the indicated Myc proteins. Parental TGR1 cells serve as control for endogenous levels of the MycWT protein.


F Cumulative cell counts for the same cells as in ( D ) . Data are presented as mean ± SD ; n = 3 ( biological replicates ) . Two - tailed Student t - test was used to compare between two groups and expressed as 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

F Cumulative cell counts for the same cells as in (D). Data are presented as mean ± SD; n = 3 (biological replicates). Two-tailed Student t-test was used to compare between two groups and expressed as p-values.


C ChIP - seq intensities for peaks ( promoters + distal ) containing the indicated motifs in an interval of ± 100 bp around their summit . For most accurate quantitation , intensities were computed within ± 50 bp from the summit . p - values were calculated using Wilcoxon ' s test . The central band in the boxplot represents the median of the data , boxes the lower and upper quartiles ( 25 % and 75 % ) and whiskers the minimum and maximum 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 O O O O O O 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 ChIP-seq intensities for peaks (promoters + distal) containing the indicated motifs in an interval of ±100 bp around their summit. For most accurate quantitation, intensities were computed within ± 50 bp from the summit. p-values were calculated using Wilcoxon's test. The central band in the boxplot represents the median of the data, boxes the lower and upper quartiles (25% and 75%) and whiskers the minimum and maximum values.


A 3T9 fibroblasts expressing the indicated MycER proteins and control cells ( EV ) were treated with OHT ( 4h ) and subjected to biochemical fractionation in three independent experiments . The quantity of MycER protein in the different fractions was quantified by western blotting ( shown in Fig . EV4A ) and the relative proportion in the various fractions plotted as average and standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3T9 fibroblasts expressing the indicated MycER proteins and control cells (EV) were treated with OHT (4h) and subjected to biochemical fractionation in three independent experiments. The quantity of MycER protein in the different fractions was quantified by western blotting (shown in Fig. EV4A) and the relative proportion in the various fractions plotted as average and standard deviation.


F Percentage of promoters with the indicated DNA motifs under the ChIP - seq peak ( ± 100 bp from the peak summit ) within each regulatory class ( no DEG , UP or DOWN ) for either MycERWT ( left ) or MycERHEA ( right ) . p - values calculated with Fisher ' s exact 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 O O O O O

F Percentage of promoters with the indicated DNA motifs under the ChIP-seq peak (±100 bp from the peak summit) within each regulatory class (no DEG, UP or DOWN) for either MycERWT (left) or MycERHEA (right). p-values calculated with Fisher's exact test.


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 ) .

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-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 O O O O O O O O O O O O O O O O O O O O

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 .

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 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

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 ) .

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

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 .

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

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 ) .

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 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

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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

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 .

O O 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

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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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 .

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

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 .

O O O 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 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 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 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 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

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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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(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 O O O O O O O O O O O O O O O O O O O O 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 .

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(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 .

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(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 .

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(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 .

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(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 . (

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(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 O O O O O O O O O O O O O O O O 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . (

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

(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 .

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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 .

(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 .

(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 .

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 .

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 .

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 .

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 .

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 .

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 .

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 .

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 .

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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

H. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.