( A ) Representative phase contrast , yellow fluorescent ( indicating p53 - Venus levels ) , and red fluorescent ( indicating reporter mCherry levels ) images at the indicated time points for MDM2 promoter reporter cells exposed to the " natural dynamics " Nutlin - 3 dosing regimen .

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(A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the "natural dynamics" Nutlin-3 dosing regimen.


( B - G ) Single cell traces of mCherry expression for the " responding " ( light gray ) or " not responding " ( dark gray ) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics ( B ) , low amplitude ( D ) , high amplitude ( D ) , high frequency ( E ) , low frequency / short duration ( F ) , or long duration ( G ) Nutlin - 3 dosing regimens . The average trace for responding and not responding cells is shown in red and blue , respectively . Heat maps show alternative representation of all single cell traces below each associated time course plot .

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(B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot.


Dose - response curves representing the rate of mCherry expression from the MDM2 ( pink dots ) and CDKN1A ( purple dots ) promoters as a function of total p53 levels . Values of expression levels were averaged across all cells in the first 15 - h response to the long duration Nutlin - 3 dosing regimen . Solid black lines represent the best fit to a Hill function model of promoter activation . Dashed lines indicate the half - maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter .

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Dose-response curves representing the rate of mCherry expression from the MDM2 (pink dots) and CDKN1A (purple dots) promoters as a function of total p53 levels. Values of expression levels were averaged across all cells in the first 15-h response to the long duration Nutlin-3 dosing regimen. Solid black lines represent the best fit to a Hill function model of promoter activation. Dashed lines indicate the half-maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter.


( B - G ) Effects of p53 duration modulation on target promoter activation in terms of the mean timing ( B , E ) , magnitude ( C , F ) , and rate ( D , G ) for the MDM2 ( B - D ) and CDKN1A ( E - G ) promoters in response to short ( light green ) and long ( dark green ) p53 durations . N = at least 45 cells per condition , error bars = SEM .

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(B-G) Effects of p53 duration modulation on target promoter activation in terms of the mean timing (B, E), magnitude (C, F), and rate (D, G) for the MDM2 (B-D) and CDKN1A (E-G) promoters in response to short (light green) and long (dark green) p53 durations. N = at least 45 cells per condition, error bars = SEM.


( H ) Mean nuclear MDM2 - YFP levels in single cells at times corresponding to the first pulse ( 5 . 5 h ) or second pulse ( 11 h ) of p53 expression in response to natural and long duration Nutlin - 3 dosing regimens . N = at least 40 cells per condition , error bars = SEM .

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(H) Mean nuclear MDM2-YFP levels in single cells at times corresponding to the first pulse (5.5 h) or second pulse (11 h) of p53 expression in response to natural and long duration Nutlin-3 dosing regimens. N = at least 40 cells per condition, error bars = SEM.


( A ) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing ( A , D ) , magnitude ( B , E ) , and rate ( C , F ) for the MDM2 ( A - C ) and CDKN1A ( D - F ) promoters in response to low ( light blue ) , natural ( medium blue ) , high ( dark blue ) p53 frequencies . N = at least 45 cells per condition , error bars = SEM .

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(A) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing (A, D), magnitude (B, E), and rate (C, F) for the MDM2 (A-C) and CDKN1A (D-F) promoters in response to low (light blue), natural (medium blue), high (dark blue) p53 frequencies. N = at least 45 cells per condition, error bars = SEM.


( H ) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low , natural , and high frequency p53 pulses .

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(H) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low, natural, and high frequency p53 pulses.


( A - B ) Representative phase contrast , red fluorescence ( indicating geminin - mCherry levels ) , and yellow fluorescence ( indicating p53 - Venus levels ) images of cells in response to a low ( A ) or high ( B ) frequency Nutlin - 3 dosing regimen over 40 h .

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(A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h.


( C ) Geminin - mCherry traces in single cells treated with the low , natural , or high frequency Nutlin - 3 dosing regimens . N = at least 50 cells per condition .

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(C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition.


Percentage of cells undergoing cell division ( D ) within the 40 h imaging in response to the low , natural , and high frequency Nutlin - 3 dosing

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Percentage of cells undergoing cell division (D) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens


Percentage of cells positive for geminin - mCherry expression ( E ) within the 40 h imaging in response to the low , natural , and high frequency Nutlin - 3 dosing

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Percentage of cells positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens


B HeLa cells expressing the indicated GFP - tagged Nups were semi - permeabilized and incubated with mitotic HeLa cell extract at 30ºC . NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC - dextran . Scale bar , 30 μm .

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B HeLa cells expressing the indicated GFP-tagged Nups were semi-permeabilized and incubated with mitotic HeLa cell extract at 30ºC. NPC disassembly was followed by monitoring the fluorescence intensity of the respective Nups at the nuclear rim and influx of TRITC-dextran. Scale bar, 30 μm.


C Quantification of dextran - positive nuclei over time of the experiments in ( B ) . N = 3 biological replicates , n > 100 cells . Error bars , SEM .

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C Quantification of dextran-positive nuclei over time of the experiments in (B). N = 3 biological replicates, n > 100 cells. Error bars, SEM.


E Quantification of 3GFP - Nup133 , GFP - Nup107 and 2GFP - Nup58 intensity at the NE over time of the experiments in ( B ) . Error bars , SEM .

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E Quantification of 3GFP-Nup133, GFP-Nup107 and 2GFP-Nup58 intensity at the NE over time of the experiments in (B). Error bars, SEM.


B Input , light and dense fractions were analyzed by immunoblotting using the indicated antibodies . The samples were derived from the same experiment but processed using several gels and blots .

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B Input, light and dense fractions were analyzed by immunoblotting using the indicated antibodies. The samples were derived from the same experiment but processed using several gels and blots.


C Quantification of the percentage of Nups in the light fractions relative to the total amount of Nups over all fractions . N = 4 biological replicates , except for Nup214 ( N = 2 biological replicates ) . Error bars , SEM .

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C Quantification of the percentage of Nups in the light fractions relative to the total amount of Nups over all fractions. N = 4 biological replicates, except for Nup214 (N = 2 biological replicates). Error bars, SEM.


B Confocal images of metaphase - arrested HeLa cells expressing 3GFP - Nup133 , GFP - Nup107 , GFP - Nup53 or 2GFP - Nup58 that were either directly fixed ( untreated ) or first semi - permeabilized and then fixed . The ER was visualized by immunostaining with an antibody targeting calnexin ; DNA was visualized with Hoechst ( visible only in the merged channels ) . Representative images of 3 biological replicates . Scale bar , 10 μm and inset scale bar , 1 μm .

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B Confocal images of metaphase-arrested HeLa cells expressing 3GFP-Nup133, GFP-Nup107, GFP-Nup53 or 2GFP-Nup58 that were either directly fixed (untreated) or first semi-permeabilized and then fixed. The ER was visualized by immunostaining with an antibody targeting calnexin; DNA was visualized with Hoechst (visible only in the merged channels). Representative images of 3 biological replicates. Scale bar, 10 μm and inset scale bar, 1 μm.


A , B 2D dSTORM images of interphase ( A ) or metaphase ( B ) U2OS cells expressing SNAP - tagged Nup96 . Cells were semi - permeabilized with digitonin and Nup96 was labelled with the BG - AF647 SNAP dye . The SMLM data was reconstructed using Thunderstorm . Left panels were rendered to black and white and enhanced for viewing purposes . Insets ( yellow dashed boxes ) are displayed to the right . Insets in ( A ) show ring - shaped structures built by Y - complexes in interphase cells . Insets in ( B ) represents mitotic clusters outside the spindle area ( class 1 , inset 1 ) and at kinetochores ( class 2 ) . Scale bar ( left panels ) , 10 μm . Representative images of 4 biological replicates . Inset scale bars , 200 nm .

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A, B 2D dSTORM images of interphase (A) or metaphase (B) U2OS cells expressing SNAP-tagged Nup96. Cells were semi-permeabilized with digitonin and Nup96 was labelled with the BG-AF647 SNAP dye. The SMLM data was reconstructed using Thunderstorm. Left panels were rendered to black and white and enhanced for viewing purposes. Insets (yellow dashed boxes) are displayed to the right. Insets in (A) show ring-shaped structures built by Y-complexes in interphase cells. Insets in (B) represents mitotic clusters outside the spindle area (class 1, inset 1) and at kinetochores (class 2). Scale bar (left panels), 10 μm. Representative images of 4 biological replicates. Inset scale bars, 200 nm.


A 2D dSTORM images of a metaphase - arrested U2OS cell expressing Nup96 with a SNAP - tag , labelled with BG - Alexa Fluor 647 ( orange ) , in combination with confocal imaging of the ER immunostained for Climp63 ( red ) and phospho - histone H3 ( cyan ) to label DNA . Inset 1 ( mitotic clusters ) and inset 2 ( kinetochores ) are indicated with white dashed boxes . SMLM data was reconstructed using Thunderstorm . Representative images of N = 4 biological replicates . Scale bars , 10 μm . Inset scale bars , 200 nm .

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A 2D dSTORM images of a metaphase-arrested U2OS cell expressing Nup96 with a SNAP-tag, labelled with BG-Alexa Fluor 647 (orange), in combination with confocal imaging of the ER immunostained for Climp63 (red) and phospho-histone H3 (cyan) to label DNA. Inset 1 (mitotic clusters) and inset 2 (kinetochores) are indicated with white dashed boxes. SMLM data was reconstructed using Thunderstorm. Representative images of N = 4 biological replicates. Scale bars, 10 μm. Inset scale bars, 200 nm.


C Quantification of the number of localizations in interphase NPCs ( NPC ) , mitotic clusters and kinetochores of the experiments in ( A ) using DBSCAN . Error bars , SD . D Quantification of the number of protein copies in interphase NPCs ( NPC ) , mitotic clusters and kinetochores of the experiments in ( A ) using DBSCAN . Error bars , SD . E Quantification of the cluster width of interphase NPCs ( NPC ) , mitotic clusters and kinetochores of the experiments in ( A ) using DBSCAN . Error bars , SD .

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C Quantification of the number of localizations in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. D Quantification of the number of protein copies in interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD. E Quantification of the cluster width of interphase NPCs (NPC), mitotic clusters and kinetochores of the experiments in (A) using DBSCAN. Error bars, SD.


A Generation of myeloid cells genetically inactivated for Elp3 . Elp3 mRNA and protein levels were assessed by Real - Time PCR with extracts isolated from peritoneal macrophages of Elp3Control and Elp3 ∆ Mye mice . mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels ( n = 3 mice ; mean + / - SD , Student T test , * * * = p < 0 . 001 ) . An anti - Elp3 western blot ( WB ) is also illustrated . The anti - Gapdh blot is shown for normalization purposes .

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A Generation of myeloid cells genetically inactivated for Elp3. Elp3 mRNA and protein levels were assessed by Real-Time PCR with extracts isolated from peritoneal macrophages of Elp3Control and Elp3∆Mye mice. mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice; mean +/- SD, Student T test, ***= p< 0.001). An anti-Elp3 western blot (WB) is also illustrated. The anti-Gapdh blot is shown for normalization purposes.


C and D Defective mTORC2 signaling upon Elp3 deficiency in myeloid cells . Peritoneal macrophages from Elp3Control and Elp3 ∆ Mye mice were isolated and cultured ex - vivo . They were untreated or stimulated with a combination of LPS ( 100 ng / ml ) and IFNγ ( 50 ng / ml ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses .

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C and D Defective mTORC2 signaling upon Elp3 deficiency in myeloid cells. Peritoneal macrophages from Elp3Control and Elp3∆Mye mice were isolated and cultured ex-vivo. They were untreated or stimulated with a combination of LPS (100 ng/ml) and IFNγ (50 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.


F mTORC2 activation by M1 polarization signals relies on Ctu2 . Control and Ctu2 - depleted bone marrow - derived macrophages ( BMDMs ) ( SiRNA - mediated depletions ) were treated or not with IFNγ ( 50 ng / ml ) and LPS ( 100 ng / ml ) and the resulting cell extracts were subjected to WB analyses .

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F mTORC2 activation by M1 polarization signals relies on Ctu2. Control and Ctu2-depleted bone marrow-derived macrophages (BMDMs) (SiRNA-mediated depletions) were treated or not with IFNγ (50 ng/ml) and LPS (100 ng/ml) and the resulting cell extracts were subjected to WB analyses.


C Elp3 deficiency in myeloid cells exacerbates the decrease of colon length upon DSS administration ( n = 7 mice ; mean + / - SD , * * = p < 0 . 01 ) .

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C Elp3 deficiency in myeloid cells exacerbates the decrease of colon length upon DSS administration (n = 7 mice; mean +/- SD, **= p< 0.01).


E More severe histological score in mice lacking Elp3 in myeloid cells upon DSS administration . Representative intestinal crypts from the indicated genotypes are illustrated . The histological score was established as described in methods ( n = 5 mice per genotype ; mean + / - SD , Student T test , * = p < 0 . 05 ) .

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E More severe histological score in mice lacking Elp3 in myeloid cells upon DSS administration. Representative intestinal crypts from the indicated genotypes are illustrated. The histological score was established as described in methods (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05).


F Increased infiltration of iNOS + / CD68 + cells upon DSS administration in Elp3 ∆ Mye mice . The percentage of iNOS + in CD68 + cells was established in both genotypes treated with DSS ( n = 7 mice per genotype ; mean + / - SD , Student T test , * * = p < 0 . 01 ) . Anti - iNOS and - CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated .

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F Increased infiltration of iNOS+/CD68+ cells upon DSS administration in Elp3∆Mye mice. The percentage of iNOS+ in CD68+ cells was established in both genotypes treated with DSS (n = 7 mice per genotype; mean +/- SD, Student T test, **= p< 0.01). Anti-iNOS and -CD68 immunofluorescence analyses carried out in intestinal crypts from both genotypes are illustrated.


G Patients suffering from ulcerative colitis ( UC ) show reduced levels of both Elp1 and Elp3 . The central band represents the mean value of relative expression in the investigated cohort . Boxes represent 75th and 25th percentile . Whiskers represent maximum and minimum values before the upper and lower fence , respectively ( * * = p < 0 . 01 , * * * = p < 0 . 001 ) ( GSE9452 - GEO DataSets , Definition of an ulcerative colitis pre - inflammatory state ) .

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G Patients suffering from ulcerative colitis (UC) show reduced levels of both Elp1 and Elp3. The central band represents the mean value of relative expression in the investigated cohort. Boxes represent 75th and 25th percentile. Whiskers represent maximum and minimum values before the upper and lower fence, respectively (**= p< 0.01, ***= p<0.001) (GSE9452-GEO DataSets, Definition of an ulcerative colitis pre-inflammatory state).


A IL - 4 and IL - 13 induce the expression of tRNA - modifying enzymes in macrophages . Peritoneal macrophages were untreated ( « Control » ) or stimulated with IL - 4 ( 10 ng / ml ) , IL - 13 ( 10 ng / ml ) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB analyses .

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A IL-4 and IL-13 induce the expression of tRNA-modifying enzymes in macrophages. Peritoneal macrophages were untreated (« Control ») or stimulated with IL-4 (10 ng/ml), IL-13 (10 ng/ml) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB analyses.


B The pool of thiolated tRNAs in macrophages is enhanced by IL - 4 / IL - 13 but decreased by LPS / IFNγ . On the left , Northern blot analysis assessing t : Q ( UUG ) tRNA thiolation in Thioglycollate - elicited peritoneal macrophages subjected to the indicated treatments for 24 hours . On the right , quantification of t : Q ( UUG ) tRNA thiolation , calculated as the ration of thiolated over nonthiolated t : Q ( UUG ) , in the indicated experimental conditions .

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B The pool of thiolated tRNAs in macrophages is enhanced by IL-4/IL-13 but decreased by LPS/IFNγ. On the left, Northern blot analysis assessing t:Q(UUG) tRNA thiolation in Thioglycollate-elicited peritoneal macrophages subjected to the indicated treatments for 24 hours. On the right, quantification of t:Q(UUG) tRNA thiolation, calculated as the ration of thiolated over nonthiolated t:Q(UUG), in the indicated experimental conditions.


C Elp3 induction by IL - 4 / IL - 13 occurs through a PI3K - dependent pathway . Peritoneal macrophages were pretreated with DMSO ( control vehicle ) or with Wortmannin ( 200 nM ) for one hour and subsequently left untreated or stimulated with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for the indicated periods of time . Cell extracts from the resulting cells were subjected to WB analyses .

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C Elp3 induction by IL-4/IL-13 occurs through a PI3K-dependent pathway. Peritoneal macrophages were pretreated with DMSO (control vehicle) or with Wortmannin (200 nM) for one hour and subsequently left untreated or stimulated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.


E Rictor but not Raptor deficiency impairs Elp3 expression . BMDMs were transfected with the indicated siRNAs and the resulting cells were treated or not with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for 24 hours . Cell extracts were subjected to WB analyses .

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E Rictor but not Raptor deficiency impairs Elp3 expression. BMDMs were transfected with the indicated siRNAs and the resulting cells were treated or not with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for 24 hours. Cell extracts were subjected to WB analyses.


F IL - 4 / IL - 13 rely on Stat6 to induce Elp3 expression . Peritoneal macrophages were pretreated with the control vehicle ( DMSO ) or with the Stat6 pharmacological inhibitor AS1517499 ( 5 μM ) for one hour and subsequently left unstimulated or treated with IL - 4 ( 10 ng / ml ) / IL - 13 ( 10 ng / ml ) for the indicated periods of time . Cell extracts from the resulting cells were subjected to WB analyses .

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

F IL-4/IL-13 rely on Stat6 to induce Elp3 expression. Peritoneal macrophages were pretreated with the control vehicle (DMSO) or with the Stat6 pharmacological inhibitor AS1517499 (5 μM) for one hour and subsequently left unstimulated or treated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.


Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL - 4 ( H , , IL - 13 ( H ) or by both cytokines ( H ) . Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL - 4 ( 10 ng / ml ) ( H , IL - 13 ( 10 ng / ml ) ( H ) or with both cytokines ( H ) for 8 hours ( H ) and the resulting cell extracts were subjected to WB analyses .

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

Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 (H, , IL-13 (H) or by both cytokines (H). Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) (H, IL-13 (10 ng/ml) (H) or with both cytokines (H) for 8 hours (H) and the resulting cell extracts were subjected to WB analyses.


J Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL - 4 I and J ) Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL - 4 ( 10 ng / ml ) , I and J ) , or for the indicated periods of time ( I and J ) and the resulting cell extracts were subjected to WB analyses .

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

J Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 I and J) Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) , I and J), or for the indicated periods of time (I and J) and the resulting cell extracts were subjected to WB analyses.


L Elp3 promotes M2 macrophage polarization . Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL - 4 / IL - 13 ( 10 ng / ml ) for 24 hours and the resulting cell extracts were subjected to WB analyses .

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

L Elp3 promotes M2 macrophage polarization. Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4/IL-13 (10 ng/ml) for 24 hours and the resulting cell extracts were subjected to WB analyses.


B Defective induction of Pparγ by IL - 4 / IL - 13 upon Elp3 deficiency . Cell extracts from peritoneal macrophages of Elp3Control and Elp3 ∆ Mye mice treated or not with IL - 4 / IL - 13 were subjected to WB analyses .

B Defective induction of Pparγ by IL-4/IL-13 upon Elp3 deficiency. Cell extracts from peritoneal macrophages of Elp3Control and Elp3∆Mye mice treated or not with IL-4/IL-13 were subjected to WB analyses.


A The induction of Ric8b expression by IL - 4 relies on Elp3 . BMDMs from the indicated genotypes were treated or not with IL - 4 ( 10 ng / ml ) up to 24 hours and the resulting cell extracts were subjected to WB analyses .

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

A The induction of Ric8b expression by IL-4 relies on Elp3. BMDMs from the indicated genotypes were treated or not with IL-4 (10 ng/ml) up to 24 hours and the resulting cell extracts were subjected to WB analyses.


B Elp3 promotes Ric8B mRNA translation . BMDMs from Elp3Control and Elp3 ∆ Mye mice were treated by IL - 4 for 24 hours , followed by a treatment with 10 μg / ml puromycin for 5 minutes . To detect newly synthesized Ric8b proteins , a Puro - PLA assay was conducted . Representative images detecting Ric8b ( Red ) and α - Tubulin + microtubules ( green ) in BMDMs are illustrated . On the right , the graph shows a quantification of signals for Ric8b in α - Tubulin + areas . A random of six different areas were counted ( 6 technical replicates , mean + / - SD , Student ' s t - test , * = p < 0 . 05 ) .

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

B Elp3 promotes Ric8B mRNA translation. BMDMs from Elp3Control and Elp3∆Mye mice were treated by IL-4 for 24 hours, followed by a treatment with 10 μg/ml puromycin for 5 minutes. To detect newly synthesized Ric8b proteins, a Puro-PLA assay was conducted. Representative images detecting Ric8b (Red) and α-Tubulin+ microtubules (green) in BMDMs are illustrated. On the right, the graph shows a quantification of signals for Ric8b in α-Tubulin+ areas. A random of six different areas were counted (6 technical replicates, mean +/- SD, Student's t-test, *= p< 0.05).


C Ric8b is a direct target of Elp3 . BMDMs from Elp3Control and Elp3 ∆ Mye mice were infected with a lentiviral GFP construct ( negative control ) , Flag - Ric8b WT or with Flag - Ricb MUT , as indicated and the resulting protein extracts were subjected to WB analyses to assess Ric8b and Elp3 protein levels . Gapdh was used as a loading control .

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

C Ric8b is a direct target of Elp3. BMDMs from Elp3Control and Elp3∆Mye mice were infected with a lentiviral GFP construct (negative control), Flag-Ric8b WT or with Flag-Ricb MUT, as indicated and the resulting protein extracts were subjected to WB analyses to assess Ric8b and Elp3 protein levels. Gapdh was used as a loading control.


D and E Ric8b promotes IL - 4 - dependent mTORC2 activation . Control or Ric8b - depleted BMDMs were treated or not with IL - 4 ( 10 ng / ml ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses .

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

D and E Ric8b promotes IL-4-dependent mTORC2 activation. Control or Ric8b-depleted BMDMs were treated or not with IL-4 (10 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.


C Elp3 promotes mitochondrial translation . BMDMs from the indicated genotypes were treated or not with IL - 4 / IL - 13 ( 10 ng / ml ) for the indicated hours and the resulting cell extracts were subjected to WB analyses .

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

C Elp3 promotes mitochondrial translation. BMDMs from the indicated genotypes were treated or not with IL-4/IL-13 (10 ng/ml) for the indicated hours and the resulting cell extracts were subjected to WB analyses.


D Elp3 expression in myeloid cells maintains the pool of tumor - associated macrophages ( TAMs ) . The number of TAMs , defined as CD301 + / F4 / 80 + cells among F4 / 80 + cells was quantified in intestinal crypts from both Apc + / Min Elp3Control and Apc + / Min Elp3 ∆ Mye mice ( n = 5 mice per genotype ; mean + / - SD , Student T test , * = p < 0 . 05 ) . Representative immunofluorescence analyses are illustrated .

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

D Elp3 expression in myeloid cells maintains the pool of tumor-associated macrophages (TAMs). The number of TAMs, defined as CD301+/F4/80+ cells among F4/80+ cells was quantified in intestinal crypts from both Apc+/Min Elp3Control and Apc+/Min Elp3∆Mye mice (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05). Representative immunofluorescence analyses are illustrated.


B . Typical examples of stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 12 ) human iPSC - derived NSCs / neurons immunostained for γ - Tubulin and Centrin , marked by arrows . Cells were co - immunostained with Ki67 and DAPI ( day 0 ) or Trim46 and MAP2 ( day 7 and 12 ) to define their neuronal stages . Arrowheads mark the AIS . Scale bar = 10µm in overview , 2µm in zooms .

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

B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12) human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin, marked by arrows. Cells were co-immunostained with Ki67 and DAPI (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Arrowheads mark the AIS. Scale bar = 10µm in overview, 2µm in zooms.


D . 3D STED imaging of αTubulin in stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 13 ) hiPSC - derived neurons . Scale bar = 5µm in overview , 1µm in zoom .

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

D. 3D STED imaging of αTubulin in stage 1 (day 0), stage 2 (day 7) and stage 3 (day 13) hiPSC-derived neurons. Scale bar = 5µm in overview, 1µm in zoom.


F . Typical examples of centrosomes of human iPSC - derived NSCs ( day 0 ) with STED imaging of Trim46 and Centrin immunostaining , and confocal imaging of γ - Tubulin immunostaining . Scale bar = 1µm .

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

F. Typical examples of centrosomes of human iPSC-derived NSCs (day 0) with STED imaging of Trim46 and Centrin immunostaining, and confocal imaging of γ-Tubulin immunostaining. Scale bar = 1µm.


G . Typical examples of stage 2 and stage 3 human iPSC - derived neurons immunostained for Trim46 and MAP2 . Inserts represent centrosomes . Zooms on the right represent a non - polarized neurite or a developing axon in stage 2 or stage 3 neurons , respectively . Scale bar = 20µm in overview , 1µm in insert , 10µm in zoom .

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

G. Typical examples of stage 2 and stage 3 human iPSC-derived neurons immunostained for Trim46 and MAP2. Inserts represent centrosomes. Zooms on the right represent a non-polarized neurite or a developing axon in stage 2 or stage 3 neurons, respectively. Scale bar = 20µm in overview, 1µm in insert, 10µm in zoom.


A . Typical examples of Centrinone - B treated or control human iPSC - derived NSCs immunostained for Pericentrin and Centrin . Inserts represent centriole ( s ) . Scale bar = 5µm in overview , 2µm in inserts . B . Quantifications of the percentage of cells with 0 , 1 or 2 centrioles per cell after 0 ( control ) , 2 or 5 days Centrinone - B treatment and prior to neuronal induction . n = 48 - 51 cells in 2 independent experiments . C Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Bonferroni correction * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 .

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

A. Typical examples of Centrinone-B treated or control human iPSC-derived NSCs immunostained for Pericentrin and Centrin. Inserts represent centriole(s). Scale bar = 5µm in overview, 2µm in inserts. B. Quantifications of the percentage of cells with 0, 1 or 2 centrioles per cell after 0 (control), 2 or 5 days Centrinone-B treatment and prior to neuronal induction. n = 48-51 cells in 2 independent experiments. C Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05


C . Typical examples of Centrinone - B treated or control human iPSC - derived neurons ( 12 - 15 days ) immunostained for AnkG , Trim46 , MAP2 and Centrin . Arrowheads mark AIS structures , arrows mark centrosomes . Inserts represent centrosomes , zooms on the right represent AIS structures . Scale bar = 20µm in overview , 2µm in insert , 10µm in zooms .

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

C. Typical examples of Centrinone-B treated or control human iPSC-derived neurons (12-15 days) immunostained for AnkG, Trim46, MAP2 and Centrin. Arrowheads mark AIS structures, arrows mark centrosomes. Inserts represent centrosomes, zooms on the right represent AIS structures. Scale bar = 20µm in overview, 2µm in insert, 10µm in zooms.


M . AP threshold , amplitude and after - hyperpolarization recorded in Centrinone - B treated ( n = 43 cells in 3 independent experiments ) and control human iPSC - derived neuron cultures ( n = 59 cells in 4 independent experiments ) . Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Mann Whitney test after - hyperpolarization Student ' s t test ( M : threshold , AP amplitude ) . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 .

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

M. AP threshold, amplitude and after-hyperpolarization recorded in Centrinone-B treated (n = 43 cells in 3 independent experiments) and control human iPSC-derived neuron cultures (n = 59 cells in 4 independent experiments). Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Mann Whitney test after-hyperpolarization Student's t test (M: threshold, AP amplitude). *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05


D . Representative images of fan - like growth cones at day 5 of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions . Growth cone visualized by immunostaining for phalloidin . Scale bar = 5µm . E . Quantifications of the average area ( µm² ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 25 - 83 growth cones in 3 independent experiments . F Data information : Data represent mean ± SEM . One - way ANOVA including Tukey ' s post - hoc analysis * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 0 .

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

D. Representative images of fan-like growth cones at day 5 of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions. Growth cone visualized by immunostaining for phalloidin. Scale bar = 5µm. E. Quantifications of the average area (µm²) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 25-83 growth cones in 3 independent experiments. F Data information: Data represent mean ± SEM. One-way ANOVA including Tukey's post-hoc analysis *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05


F . Representative images of different growth cone morphological categories : fan - like , torpedo - like and bulb - like . Scale bar = 5µm . G . Quantifications of the ratios of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . H . Quantifications of the average area ( µm² ) of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 5 - 55 growth cones in 3 independent experiments . Da Data information : Chi - square - test including post - hoc analysis with Bonferroni correction ( G ) , One - way ANOVA including Sidak ' s post - hoc analysis ( H ) . * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 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 O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

F. Representative images of different growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 5-55 growth cones in 3 independent experiments. Da Data information: Chi-square-test including post-hoc analysis with Bonferroni correction (G), One-way ANOVA including Sidak's post-hoc analysis (H). *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05


A . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . The top panel is a still of a neurite in mRFP , showing neurite morphology . The other panels show moving GFP - MT + TIP comets pointing in either an anterograde direction ( green arrowheads ) or retrograde direction ( blue arrowheads ) . P indicates the proximal direction and D the distal direction of the neurite . Timestamp in minutes : seconds given on bottom right . Scale bar = 5 µm . B . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( A ) , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Scale bar = 5 µm . C

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

A. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. The top panel is a still of a neurite in mRFP, showing neurite morphology. The other panels show moving GFP-MT+TIP comets pointing in either an anterograde direction (green arrowheads) or retrograde direction (blue arrowheads). P indicates the proximal direction and D the distal direction of the neurite. Timestamp in minutes:seconds given on bottom right. Scale bar = 5 µm. B. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (A), for different time points (day 7; day 13) and conditions (control; Centrinone-B). Scale bar = 5 µm. C


J . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . Red line denotes the location of LS . Scale bar = 5 µm . K . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( J ) following LS , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Red line and red arrowhead denote location and time of LS . Scale bar = 5 µm . L

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

J. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. Red line denotes the location of LS. Scale bar = 5 µm. K. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (J) following LS, for different time points (day 7; day 13) and conditions (control; Centrinone-B). Red line and red arrowhead denote location and time of LS. Scale bar = 5 µm. L


( A ) Representative images showing the myogenic differentiation , as assessed by immunostaining for MyHC ( green ) , of human MuSCs ( hMuSCs ) isolated from muscle biopsies of DMD patients , either before the beginning of the clinical trial ( hMuSCs ( - ) ) or after 1 year of treatment with Givinostat ( hMuSCs Giv ( - ) ) . Cells were cultured alone or ( only for hMuSCs - ) in transwell co - culture with FAPs isolated from the same patients and exposed in vitro to Giv ( + ehFAPs Giv Vitro ) or control vehicle ( + ehFAPs ) . The upper panels represent myogenic differentiation of DMD - 1 patient ; bottom panels represent myogenic differentiation of DMD - 2 patient . Dose and timing of Giv treatment as described in Bettica et al . , 2016 ( 23 ) . Scale bar = 50 μm .

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(A) Representative images showing the myogenic differentiation, as assessed by immunostaining for MyHC (green), of human MuSCs (hMuSCs) isolated from muscle biopsies of DMD patients, either before the beginning of the clinical trial (hMuSCs (-)) or after 1 year of treatment with Givinostat (hMuSCs Giv (-)). Cells were cultured alone or (only for hMuSCs -) in transwell co-culture with FAPs isolated from the same patients and exposed in vitro to Giv (+ehFAPs Giv Vitro) or control vehicle (+ehFAPs). The upper panels represent myogenic differentiation of DMD-1 patient; bottom panels represent myogenic differentiation of DMD-2 patient. Dose and timing of Giv treatment as described in Bettica et al., 2016 (23). Scale bar = 50 μm.


( C ) Representative images showing the myogenic differentiation of MuSCs isolated from 1 . 5 month old mdx mice , as assessed by immunostaining for MyHC ( green ) . MuSCs were cultured alone ( - ) or in transwell co - culture with mdx FAPs , which were pretreated with DMSO or GW4869 ( GW 10mM added to FAPs 30 minutes before starting the co - culture with MuSCs ) . Scale bar = 50 μm . ( D ) Graph showing the fusion index of MuSCs in the conditions described in ( C ) , ( n = 5 , biological replicates ) . Star ( * ) indicates statistical analysis by Tukey test relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 ; Hash ( # ) indicates statistical analysis by Tukey test relative to MuSCs in co - cultured with FAPs not treated ( DMSO ) , # p < 0 . 05 ; § represents statistical analysis by 2Way Anova test ; § § § p < 0 , 001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

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(C) Representative images showing the myogenic differentiation of MuSCs isolated from 1.5 month old mdx mice, as assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with mdx FAPs, which were pretreated with DMSO or GW4869 (GW 10mM added to FAPs 30 minutes before starting the co-culture with MuSCs). Scale bar = 50 μm. (D) Graph showing the fusion index of MuSCs in the conditions described in (C), (n=5, biological replicates). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), ** p < 0.01; Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-cultured with FAPs not treated (DMSO), # p<0.05; § represents statistical analysis by 2Way Anova test; §§§ p < 0,001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.


( E ) Representative images of PKH - 67 ( green ) and DAPI ( blue ) staining in a transwell co - culture between MuSCs and FAPs isolated from 1 . 5 month old mdx mice . FAPs exposed to PKH - 67 prior to the co - culture with MuSCs . Scale bar = 50 μm . ( F ) Graph showing the percentage of PKH - 67 positive MuSCs after the co - culture with FAPs , ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test ; * * * * p < 0 . 0001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

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(E) Representative images of PKH-67 (green) and DAPI (blue) staining in a transwell co-culture between MuSCs and FAPs isolated from 1.5 month old mdx mice. FAPs exposed to PKH-67 prior to the co-culture with MuSCs. Scale bar = 50 μm. (F) Graph showing the percentage of PKH-67 positive MuSCs after the co-culture with FAPs, (n=3, biological replicates). Star (*) indicates statistical analysis by t-test;**** p < 0.0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.


( G ) Representative images of myogenic differentiation of mdx MuSCs , as assessed by immunostaining for MyHC ( green ) . MuSCs were cultured either alone ( - ) or incubated with EVs isolated from 1 . 5 month old mdx mice ( EVs FAPs ) treated with vehicle ( CTR ) or exposed to TSA in vivo ( TSA ) or in vitro ( TSA vitro ) . Scale bar = 50 μm . ( H ) Graph showing the fusion index of MuSCs in the condition described in ( G ) , ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by Tukey test on the % of n > 5 ( % of nuclei that were MyHC + inside myotubes containing more than 5 nuclei ) relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 , * * * * p < 0 , 0001 ; Hash ( # ) indicates statistical analysis by Tukey test relative to EVs FAPs CTR , # # p < 0 . 01 , # # # p < 0 , 001 ; ns = not significant ; § represents statistical analysis by 2way Anova test , § § § § p < 0 , 0001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

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(G) Representative images of myogenic differentiation of mdx MuSCs, as assessed by immunostaining for MyHC (green). MuSCs were cultured either alone (-) or incubated with EVs isolated from 1.5 month old mdx mice (EVs FAPs) treated with vehicle (CTR) or exposed to TSA in vivo (TSA) or in vitro (TSA vitro). Scale bar = 50 μm. (H) Graph showing the fusion index of MuSCs in the condition described in (G), (n=3, biological replicates). Star (*) indicates statistical analysis by Tukey test on the % of n>5 (% of nuclei that were MyHC+ inside myotubes containing more than 5 nuclei) relative to MuSCs cultured alone (-), **p <0.01, ****p<0,0001; Hash (#) indicates statistical analysis by Tukey test relative to EVs FAPs CTR, ##p <0.01, ###p<0,001; ns= not significant; § represents statistical analysis by 2way Anova test, §§§§p < 0,0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.


( A ) Graph showing the relative expression of Drosha in FAPs after Drosha down - regulation by siRNA ( n = 4 , biological replicates ) . Values correspond to the average ± SEM . Star ( * ) indicates statistical analysis by t - test relative to FAPs TSA treated and transfected with scramble ( Scr ) ; * * p < 0 . 01 .

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(A) Graph showing the relative expression of Drosha in FAPs after Drosha down-regulation by siRNA (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by t-test relative to FAPs TSA treated and transfected with scramble (Scr); **p<0.01.


( B ) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC ( green ) . MuSCs were cultured alone ( - ) or in transwell co - culture with FAPs isolated from mdx mice treated either with vehicle ( + FAPs CTR ) or TSA ( 0 . 6 mg / kg / day for 15 days by i . p . ) ( + FAPs TSA ) , and transfected with scramble ( Scr siRNA ) or Drosha siRNA ( Drosha siRNA ) prior to co - culture with MuSCs . Scale bar = 50 μm .

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(B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm.


( E ) Representative images of miR - 206 - 3p ( violet ) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages : 1 year ( n = 2 ) , 4 years ( n = 3 ) and 9 years old ( n = 2 ) . ( F ) Graph representing miR - 206 - 3p violet area quantification relative to ( E ) .

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(E) Representative images of miR-206-3p (violet) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages: 1 year (n=2), 4 years (n=3) and 9 years old (n=2). (F) Graph representing miR-206-3p violet area quantification relative to (E).


( G ) Representative images of immunofluorescence for eMyHC ( red ) and Laminin ( green ) in conditions described in ( E ) . ( H ) Graph showing the percentage of eMyHC positive fibers of condition described in ( G ) .

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(G) Representative images of immunofluorescence for eMyHC (red) and Laminin (green) in conditions described in (E). (H) Graph showing the percentage of eMyHC positive fibers of condition described in (G).


( I ) Representative images of Masson ' s Trichrome staining in conditions described in ( E ) . ( J ) Graph showing the fibrotic area quantification of conditions described in ( I ) .

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(I) Representative images of Masson's Trichrome staining in conditions described in (E). (J) Graph showing the fibrotic area quantification of conditions described in (I).


( A ) Representative images of Sca - 1 ( green ) , Laminin ( white ) , DAPI ( blue ) immunofluorescence , and miR - 206 - 3p ( violet ) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior ( TA ) of young mdx mice ( CTR ) and after TSA treatment ( TSA ) ( 0 . 6 mg / kg / day for 15 days by i . p . ) ( n = 6 ) . Scale bar = 25 μm .

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(A) Representative images of Sca-1 (green), Laminin (white), DAPI (blue) immunofluorescence, and miR-206-3p (violet) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior (TA) of young mdx mice (CTR) and after TSA treatment (TSA) (0.6 mg/kg/day for 15 days by i.p.) (n=6). Scale bar = 25 μm.


( C ) Graph representing the miR - 206 - 3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice ( CTR ) , mdx young and old mice ( control - CTR - and TSA treated ) ( n = 4 ) . Star ( * ) indicates Tukey analysis compared to wild type mice ( CTR ) , * * * * p < 0 . 0001 ; hash ( # ) indicates Tukey analysis compared to mdx young CTR , # # # # p < 0 . 0001 . § represents statistical analysis by Anova test . § § § § p < 0 , 0001 .

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(C) Graph representing the miR-206-3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice (CTR), mdx young and old mice (control -CTR- and TSA treated) (n=4). Star (*) indicates Tukey analysis compared to wild type mice (CTR), ****p < 0.0001; hash (#) indicates Tukey analysis compared to mdx young CTR, #### p<0.0001. § represents statistical analysis by Anova test. §§§§ p<0,0001.


( D ) Representative images of CD90 ( green ) , Dapi ( blue ) and miR - 206 - 3p ( violet ) immunohistochemistry in brachial biceps bioptic samples of DMD patient before ( CTR ) and after Givinostat treatment ( GIV ) . Scale bar = 50 μm .

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(D) Representative images of CD90 (green), Dapi (blue) and miR-206-3p (violet) immunohistochemistry in brachial biceps bioptic samples of DMD patient before (CTR) and after Givinostat treatment (GIV). Scale bar = 50 μm.


( F ) Graph showing the relative expression of miR - 206 - 3p in EVs isolated from a human population of muscle resident cells enriched in FAPs ( ehFAPs ) isolated from biopsies of two DMD patients ( DMD - 1 and DMD - 2 ) before ( CTR ) and after ( GIV ) Givinostat treatment , Two technical replicates are shown .

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(F) Graph showing the relative expression of miR-206-3p in EVs isolated from a human population of muscle resident cells enriched in FAPs (ehFAPs) isolated from biopsies of two DMD patients (DMD-1 and DMD-2) before (CTR) and after (GIV) Givinostat treatment, Two technical replicates are shown.


( A ) Flow cytometry analysis of mdx muscles injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA . The left panel shows the percentage of PKH - 67 positive cells in whole muscle . The right panel shows the percentage of the PKH - 67 positive MuSCs .

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(A) Flow cytometry analysis of mdx muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA. The left panel shows the percentage of PKH-67 positive cells in whole muscle. The right panel shows the percentage of the PKH-67 positive MuSCs.


( B ) Representative images of PKH - 67 ( green ) and DAPI ( blue ) staining in MuSCs freshly isolated from muscles previously injected with PKH67 - labeled EVs . Scale bar = 25 μm . Data information : Nuclei were counterstained with DAPI ( blue ) .

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(B) Representative images of PKH-67 (green) and DAPI (blue) staining in MuSCs freshly isolated from muscles previously injected with PKH67-labeled EVs. Scale bar = 25 μm. Data information: Nuclei were counterstained with DAPI (blue).


( C ) Graph showing the relative expression of miR - 206 - 3p in MuSCs isolated as described in ( A and B ) and in MuSCs from muscles injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA , and then transfected or not with Drosha siRNA ( siRNA Drosha ) ( n = 4 ) . Star ( * ) means significance relative to MuSCs that not uptake PKH67 - labled EVs , * * p < 0 . 01 . Hash ( # ) means significance compared to MuSCs that uptake EVs - FAPs TSA , # # p < 0 . 01 . § indicates Anova analysis , § § p < 0 , 01 .

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(C) Graph showing the relative expression of miR-206-3p in MuSCs isolated as described in (A and B) and in MuSCs from muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA, and then transfected or not with Drosha siRNA (siRNA Drosha) (n=4). Star (*) means significance relative to MuSCs that not uptake PKH67-labled EVs, **p < 0.01. Hash (#) means significance compared to MuSCs that uptake EVs-FAPs TSA, ##p < 0.01. § indicates Anova analysis, §§p<0,01.


( D ) Graph showing the expression levels of miR - 206 - 3p and miR - 145 - 5p in FAPs treated or not with TSA after antagomiR treatment ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test relative to FAPs - ; * p < 0 . 05 , * * p < 0 .

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(D) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05, **p<0.01


( E ) Graph showing the expression levels of miR - 206 - 3p and miR - 145 - 5p in FAPs - derived EVs treated or not with TSA after antagomiR treatment ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test relative to FAPs - ; * p < 0 . 05 .

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(E) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs-derived EVs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05.


Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( F ) Representative images of immunofluorescence for embryonic myosin heavy chain ( eMyHC - red ) and laminin ( Lam - cyan ) stainings . Scale bar = 50 μm . ( G ) Graph showing the quantification of cross - sectional area ( CSA ) . ( H ) Graph showing the quantification of muscle regeneration ( eMyHC ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (F) Representative images of immunofluorescence for embryonic myosin heavy chain (eMyHC-red) and laminin (Lam-cyan) stainings. Scale bar = 50 μm. (G) Graph showing the quantification of cross-sectional area (CSA). (H) Graph showing the quantification of muscle regeneration (eMyHC). Data information: Nuclei were counterstained with DAPI (blue).


Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( I ) Representative images of Masson ' s Trichrome staining . Scale bar = 50 μm . ( J ) Graph showing the quantifications of muscle fiber area fraction ( MFAF ) . ( K ) Graph showing the quantifications of fibrotic area . Data information : Nuclei were counterstained with DAPI ( blue ) .

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Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (I) Representative images of Masson's Trichrome staining. Scale bar = 50 μm. (J) Graph showing the quantifications of muscle fiber area fraction (MFAF). (K) Graph showing the quantifications of fibrotic area. Data information: Nuclei were counterstained with DAPI (blue).


Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( L ) Representative images of myeloperoxidase staining ( MPO - red ) . Scale bar = 50 μm . ( M ) Graph showing the quantifications of inflammation ( MPO ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (L) Representative images of myeloperoxidase staining (MPO-red). Scale bar = 50 μm. (M) Graph showing the quantifications of inflammation (MPO). Data information: Nuclei were counterstained with DAPI (blue).


A ) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC ( green ) . Nuclei were counterstained with DAPI ( blue ) . MuSCs were cultured alone ( - ) or with EVs ( EVs FAPs ) isolated from FAPs in vivo exposed or not to TSA ( CTR and TSA ) and transfected or not with Antagomir - 206 ( - and A - 206 ) . ( n = 7 for all samples , but antagomir ' s samples n = 3 , biological replicates ) . Scale bar = 50 μm .

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A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm.


( C ) Graph relative to Notch3 expression in MuSCs isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) , and in EVs derived from FAPs ( EVs - FAPs ) isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) ( n = 3 ) . Star ( * ) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice ( CTR ) ; * p < 0 , 05 ; . hash ( # ) means significance compared to EVs - FAPs CTR , # # p < 0 , 01 . § indicates significance by Anova test ; § § p > 0 , 01 .

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(C) Graph relative to Notch3 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) (n=3). Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR, ##p<0,01. § indicates significance by Anova test; §§ p>0,01.


( D ) Graph relative to Notch1 expression in MuSCs isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) , and in EVs derived from FAPs ( EVs - FAPs ) isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) n = 3 . Star ( * ) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice ( CTR ) ; * p < 0 , 05 ; . hash ( # ) means significance compared to EVs - FAPs CTR ; * p < 0 . 05 . § indicates significance by Anova test ; § § p > 0 , 01 .

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(D) Graph relative to Notch1 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) n=3. Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR; *p < 0.05. § indicates significance by Anova test; §§ p>0,01.


( E ) Representative images showing myofibers cultured alone ( Ctr ) or with extracellular

(E) Representative images showing myofibers cultured alone (Ctr) or with extracellular vesicles


( F ) From left to right : representative images of myofibers cultured for 24 hours alone ( - ) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 ( MEDIA - FAPs ) or extracellular vesicles ( EVs - FAPs ) collected from FAPs exposed or not to TSA in vivo ( CTR and TSA ) and transfected with antagomiR - 206 ( TSA A - 206 ) . Myofibers have been stained for EdU ( Cyan ) , immunofluorescence with Pax7 ( green ) , MyoD ( red ) . Nuclei were counterstained with DAPI ( blue ) . ( n = 3 ) . Scale bar = 25 μm .

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(F) From left to right: representative images of myofibers cultured for 24 hours alone (-) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 (MEDIA-FAPs) or extracellular vesicles (EVs-FAPs) collected from FAPs exposed or not to TSA in vivo (CTR and TSA) and transfected with antagomiR-206 (TSA A-206). Myofibers have been stained for EdU(Cyan), immunofluorescence with Pax7 (green), MyoD (red). Nuclei were counterstained with DAPI (blue). (n=3). Scale bar = 25 μm.


( A ) Colocalization analysis of KIF3A / B / KAP3 with APC by immunostaining of hippocampal primary neurons .

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(A) Colocalization analysis of KIF3A/B/KAP3 with APC by immunostaining of hippocampal primary neurons.


( D ) Pull - down analysis of the binding of APCARM with KIF3 / KAP3 .

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(D) Pull-down analysis of the binding of APCARM with KIF3/KAP3.


( D ) XL - MS analysis of the interaction sites between KIF3 / KAP3 and APCARM . Two crosslinked site pairs were found to correspond to the possible KIF3 / KAP3 - APCARM binding region marked by asterisks in C . Cartoon diagrams , sequence alignment and LC - MS data are shown with crosslinked sites highlighted .

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(D) XL-MS analysis of the interaction sites between KIF3/KAP3 and APCARM. Two crosslinked site pairs were found to correspond to the possible KIF3/KAP3-APCARM binding region marked by asterisks in C. Cartoon diagrams, sequence alignment and LC-MS data are shown with crosslinked sites highlighted.


( A ) HS - AFM imaging of KIF3 / KAP3 - APC particles .

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(A) HS-AFM imaging of KIF3/KAP3-APC particles.


( B ) Time lapse HS - AFM images of a representative ABK - APCARM particle with a continuous tapping force .

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(B) Time lapse HS-AFM images of a representative ABK-APCARM particle with a continuous tapping force.


( C ) Representative HS - AFM images of ABK - APCARM illustrating the dissociation dynamic . Continuous tapping of AFM triggered the dissociation of APCARM from ABK . Red arrows indicate the binding interface between ABK and ABK - APCARM .

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(C) Representative HS-AFM images of ABK-APCARM illustrating the dissociation dynamic. Continuous tapping of AFM triggered the dissociation of APCARM from ABK. Red arrows indicate the binding interface between ABK and ABK-APCARM.


( D ) Statistical analysis of the AFM - based ABK and ABK - APCARM particle conformations . 200 frames extracted from three independent AFM movies of ABK and ABK - APCARM were used for the analysis . Distance from the coiled coil distal tip to the docking domain ( length of the protrusion as shown by the dotted lines ) was calculated and shown as violin plots . Data derived from each of the three movies were indicated by circles , triangles and rectangles , respectively . Based on the distal - center distance , particle conformations were classified into three forms , L ( cargo - locked globular form ) , T ( Transitional curved form ) and D ( cargo - docked elongated form ) . Unpaired t test was used to evaluate the statistical significance . The solid and dotted lines indicate the median and quartiles , respectively . The AFM images shown here are re - used in the figure 5A .

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(D) Statistical analysis of the AFM-based ABK and ABK-APCARM particle conformations. 200 frames extracted from three independent AFM movies of ABK and ABK-APCARM were used for the analysis. Distance from the coiled coil distal tip to the docking domain (length of the protrusion as shown by the dotted lines) was calculated and shown as violin plots. Data derived from each of the three movies were indicated by circles, triangles and rectangles, respectively. Based on the distal-center distance, particle conformations were classified into three forms, L (cargo-locked globular form), T (Transitional curved form) and D (cargo-docked elongated form). Unpaired t test was used to evaluate the statistical significance. The solid and dotted lines indicate the median and quartiles, respectively. The AFM images shown here are re-used in the figure 5A.


( b ) SAR405 inhibits the function of Vps34 in the GFP - FYVE cellular assay . HeLa cells stably transfected with GFP - FYVE were incubated with DMSO ( left ) or with 1 μM SAR405 ( right ) for 2 h . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . Fluorescence was analyzed using an imaging cytometer ( X40 ) . One representative image out of three independent experiments is depicted . Scale bars , 20 μm .

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(b) SAR405 inhibits the function of Vps34 in the GFP-FYVE cellular assay. HeLa cells stably transfected with GFP-FYVE were incubated with DMSO (left) or with 1 μM SAR405 (right) for 2 h. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). Fluorescence was analyzed using an imaging cytometer (X40). One representative image out of three independent experiments is depicted. Scale bars, 20 μm.


( c ) SAR405 does not prevent phosphorylation of Akt and S6 . U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations . The effect of compounds on phosphorylated Akt ( p - Akt ) Ser473 and Thr308 residues , total Akt and on phosphorylated S6 ( Ser240 and Ser244 residues ) and total S6 protein was measured using western blotting . One representative experiment from at least two independent experiments is shown . Full - length blots are shown in Supplementary Figure 6 .

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(c) SAR405 does not prevent phosphorylation of Akt and S6. U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations. The effect of compounds on phosphorylated Akt (p-Akt) Ser473 and Thr308 residues, total Akt and on phosphorylated S6 (Ser240 and Ser244 residues) and total S6 protein was measured using western blotting. One representative experiment from at least two independent experiments is shown. Full-length blots are shown in Supplementary Figure 6.


( a ) SAR405 induces late endosome - lysosome swelling . RKO cells were treated with DMSO ( upper panels ) or with 10 μM SAR405 for 16 h ( lower panels ) . The late endosomes - lysosomes compartment was assessed using Lysotracker green ( left ) or Alexa 488 - conjugated anti - Lamp1 antibodies ( right ) , as indicated . Nuclei were stained using Hoechst 33342 ( blue ) . One representative image chosen from three independent experiments , showing fluorescence imaged using a Zeiss microscope ( X40 ) , is shown for each condition . Arrows indicate accumulation of swollen late endosome - lysosomes . Scale bars , 20 μm .

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(a) SAR405 induces late endosome-lysosome swelling. RKO cells were treated with DMSO (upper panels) or with 10 μM SAR405 for 16 h (lower panels). The late endosomes-lysosomes compartment was assessed using Lysotracker green (left) or Alexa 488-conjugated anti-Lamp1 antibodies (right), as indicated. Nuclei were stained using Hoechst 33342 (blue). One representative image chosen from three independent experiments, showing fluorescence imaged using a Zeiss microscope (X40), is shown for each condition. Arrows indicate accumulation of swollen late endosome-lysosomes. Scale bars, 20 μm.


( b ) SAR405 triggers accumulation of p62 . RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h . The effect on the p62 biomarker was analyzed by western blotting . GAPDH was used as a loading control . The results are representative of at least two independent experiments . Full - length blots are shown in Supplementary Figure 14 .

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(b) SAR405 triggers accumulation of p62. RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h. The effect on the p62 biomarker was analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 14.


( c ) SAR405 affects the maturation of cathepsin D ( cat D ) . RKO cells were treated at the indicated concentrations of SAR405 for 24 h . The different forms of cathepsin D ( PPCD , pre - pro - catD ) were analyzed by western blotting . GAPDH was used as a loading control . The results are representative of at least two independent experiments . Full - length blots are shown in Supplementary Figure 16 .

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(c) SAR405 affects the maturation of cathepsin D (cat D). RKO cells were treated at the indicated concentrations of SAR405 for 24 h. The different forms of cathepsin D (PPCD, pre-pro-catD) were analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 16.


( a ) GFP - LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . One representative image , acquired with an iCyte imaging cytometer , for three independent experiments is depicted ( X40 ) . White arrows indicate GFP - LC3 spots , which correspond to the autophagosomes . Scale bars , 10 μm . ( b ) Histograms show the percentage of positive cells and GFP intensity , measured using an imaging cytometer for different compound concentrations on GFP - LC3 HeLa cells . Black and gray histograms correspond to fed and starved conditions , respectively . For GFP intensity , the values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

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(a) GFP-LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image, acquired with an iCyte imaging cytometer, for three independent experiments is depicted (X40). White arrows indicate GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells and GFP intensity, measured using an imaging cytometer for different compound concentrations on GFP-LC3 HeLa cells. Black and gray histograms correspond to fed and starved conditions, respectively. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( c ) Wild - type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine ( HCQ ) , as indicated . Cells were treated at the same time with SAR405 at different concentrations . LC3 - II was measured by western blotting , and GAPDH was used as a loading control . One representative experiment for three independent experiments is shown . Full - length blots are shown in Supplementary Figure 18 . ( d ) Histograms show the LC3 - II / GAPDH ratio for each condition in the two cell lines . Black and gray histograms correspond to fed condition and EBSS conditions , respectively . The addition of hydroxychloroquine at 10 μM is indicated . Values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

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(c) Wild-type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine (HCQ), as indicated. Cells were treated at the same time with SAR405 at different concentrations. LC3-II was measured by western blotting, and GAPDH was used as a loading control. One representative experiment for three independent experiments is shown. Full-length blots are shown in Supplementary Figure 18. (d) Histograms show the LC3-II/GAPDH ratio for each condition in the two cell lines. Black and gray histograms correspond to fed condition and EBSS conditions, respectively. The addition of hydroxychloroquine at 10 μM is indicated. Values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.