( j , k ) Negative shRNA ( j ) and MCUR1 - overexpressing ( k ) HEK293T cells were permeabilized with digitonin in intracellular - like medium containing thapsigargin and bath [ Ca2 + ] indicator Fura2FF , and then pulsed with 10 μM Ca2 + . After mitochondrial clearance of bath Ca2 + , Ru360 caused an elevation of bath [ Ca2 + ] , indicating that steady - state bath [ Ca2 + ] after the pulse was maintained by a balance of MCU - mediated Ca2 + uptake and CGP37157 ( 10 μM ) - sensitive Na + - Ca2 + exchanger - mediated extrusion . CCCP was added as indicated . The solid line is the mean ; shaded areas are ± s . e . m . ( n = 3 ) .

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(j,k) Negative shRNA (j) and MCUR1-overexpressing (k) HEK293T cells were permeabilized with digitonin in intracellular-like medium containing thapsigargin and bath [Ca2+] indicator Fura2FF, and then pulsed with 10 μM Ca2+. After mitochondrial clearance of bath Ca2+, Ru360 caused an elevation of bath [Ca2+], indicating that steady-state bath [Ca2+] after the pulse was maintained by a balance of MCU-mediated Ca2+ uptake and CGP37157 (10 μM)-sensitive Na+-Ca2+ exchanger-mediated extrusion. CCCP was added as indicated. The solid line is the mean; shaded areas are ±s.e.m. (n = 3).


( l ) [ Ca2 + ] m efflux rate derived from j and k during the initial 60 s following Ru360 addition ( NS ; not significant ; n = 3 ) .

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(l) [Ca2+]m efflux rate derived from j and k during the initial 60 s following Ru360 addition (NS; not significant; n = 3).


Digitonin - permeabilized HeLa cells bathed in intracellular - like solution containing thapsigargin ( Tg ) were loaded with the ΔΨm indicator JC - 1 and the Ca2 + indicator Fura2FF , to which pulses of 10 μM Ca2 + were added before the addition of the mitochondrial uncoupler CCCP ( carbonyl cyanide m - chloro phenyl hydrazone ) . ( a - e ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] and ΔΨm in cells expressing negative shRNA ( Neg shRNA ; a ) , clone shHe1 ( b ) , clone shHe2 ( c ) , clone shHe2 re - expressing MCUR1 ( d ) and HeLa cells stably overexpressing MCUR1 ( e ) . Under similar conditions , 1 μM Ru360 was added before 10 μM Ca2 + pulses until the addition of CCCP .

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Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca2+ indicator Fura2FF, to which pulses of 10 μM Ca2+ were added before the addition of the mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). (a-e) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in cells expressing negative shRNA (Neg shRNA; a), clone shHe1 (b), clone shHe2 (c), clone shHe2 re-expressing MCUR1 (d) and HeLa cells stably overexpressing MCUR1 (e). Under similar conditions, 1 μM Ru360 was added before 10 μM Ca2+ pulses until the addition of CCCP.


( a , b ) Confocal images of HeLa cells transiently co - transfected for 48 h with GFP - tagged MCUR1 and DsRed - Mito plasmids ( a ) or mRFP - tagged MCUR1 and EYFP - Mito ( b ) . Scale bar , 20 μm .

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(a,b) Confocal images of HeLa cells transiently co-transfected for 48 h with GFP-tagged MCUR1 and DsRed-Mito plasmids (a) or mRFP-tagged MCUR1 and EYFP-Mito (b). Scale bar, 20 μm.


( c ) Immunoblot analysis of Flag - tagged MCUR1 in the HeLa cell crude mitochondrial fraction , outer mitochondrial membrane ( OMM ) and mitoplasts , using antibodies against VDAC1 ( outer mitochondrial membrane protein ) , COII ( integral membrane marker ) and Flag .

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(c) Immunoblot analysis of Flag-tagged MCUR1 in the HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (outer mitochondrial membrane protein), COII (integral membrane marker) and Flag.


( d ) Immunoblot analyses of mitochondria - containing pellet and cytosolic fractions from plasma - membrane - permeabilized HeLa cells . Permeabilized cells were treated with or without tBid ( 50 nM ) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified . Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min . These samples were probed using antibodies against HSP60 , OXA1 , Flag and MCUR1 .

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(d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma-membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane permeabilization and the appearance of cytosolic cytochrome c was verified. Intact and outer mitochondrial membrane permeabilized samples were exposed to proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1.


( e ) Reciprocal co - immunoprecipitation ( IP ) of MCU - GFP and MCUR1 - Flag transiently expressed in COS7 cells . Representative of four independent experiments . WB , western blot .

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(e) Reciprocal co-immunoprecipitation (IP) of MCU-GFP and MCUR1-Flag transiently expressed in COS7 cells. Representative of four independent experiments. WB, western blot.


( f ) Co - immunoprecipitation of MICU1 - Flag with MCU - GFP but not with MCUR1 - GFP transiently expressed in COS7 cells . Representative of four independent experiments .

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(f) Co-immunoprecipitation of MICU1-Flag with MCU-GFP but not with MCUR1-GFP transiently expressed in COS7 cells. Representative of four independent experiments.


( g ) Immunoprecipitation of LETM1 - GFP with anti - LETM1 failed to pull down MCUR1 - Flag in transiently transfected COS7 cells .

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(g) Immunoprecipitation of LETM1-GFP with anti-LETM1 failed to pull down MCUR1-Flag in transiently transfected COS7 cells.


( h ) Immunoprecipitation with Flag antibody pulled down LETM1 - Flag or MCU - Flag transiently expressed in COS7 cells ( immunoprecipitation lanes 2 and 3 , lower panel ) , but only co - immunoprecipitated MCUR1 - V5 in the MCU - Flag - expressing cells ( immunoprecipitation lanes 4 versus 5 , upper panel ) , despite lower expression of MCU - Flag in MCUR1 - co - transfected cells ( compare MCU - Flag and LETM1 - Flag expression in MCUR1 - co - transfected cells in lysate lanes 4 and 5 , bottom panel ) . Representative of three independent experiments . Uncropped images of blots / gels are shown in Supplementary Fig . S6 .

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(h) Immunoprecipitation with Flag antibody pulled down LETM1-Flag or MCU-Flag transiently expressed in COS7 cells (immunoprecipitation lanes 2 and 3, lower panel), but only co-immunoprecipitated MCUR1-V5 in the MCU-Flag-expressing cells (immunoprecipitation lanes 4 versus 5, upper panel), despite lower expression of MCU-Flag in MCUR1-co-transfected cells (compare MCU-Flag and LETM1-Flag expression in MCUR1-co-transfected cells in lysate lanes 4 and 5, bottom panel). Representative of three independent experiments. Uncropped images of blots/gels are shown in Supplementary Fig. S6.


( a ) qrtPCR of MCU mRNA from wild - type and stable MCUR1 - overexpressing HeLa cells that were transiently transfected with scrambled siRNA or siRNA against MCU . * * * P0 . 001 ( mean ± s . e . m . ) .

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(a) qrtPCR of MCU mRNA from wild-type and stable MCUR1-overexpressing HeLa cells that were transiently transfected with scrambled siRNA or siRNA against MCU. ***P0.001 (mean±s.e.m.).


( b ) [ Ca2 + ] m responses to histamine ( 100 μM ) in HeLa cells stably overexpressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA , and in stable MCUR1 - overexpressing HeLa cells transfected with MCU siRNA . After 48 h of siRNA transfection , cells were loaded with rhod - 2 and [ Ca2 + ] m responses were visualized by confocal microscopy . Solid lines are mean ; shaded regions are ± s . e . m . ; n = 3 . ( c ) Quantification of peak rhod - 2 fluorescence following histamine stimulation . * P0 . 05 , * * * P0 . 001 ( mean ± s . e . m . ; NS , not significant ; n = 3 ) .

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(b) [Ca2+]m responses to histamine (100 μM) in HeLa cells stably overexpressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA, and in stable MCUR1-overexpressing HeLa cells transfected with MCU siRNA. After 48 h of siRNA transfection, cells were loaded with rhod-2 and [Ca2+]m responses were visualized by confocal microscopy. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (c) Quantification of peak rhod-2 fluorescence following histamine stimulation. *P0.05, ***P0.001 (mean±s.e.m.; NS, not significant; n = 3).


( d ) qrtPCR of MCU mRNA from wild - type and MCUR1 - knockdown HeLa cells that were transiently transfected with MCU cDNA . * * P0 . 01 , * * * P0 . 001 ( mean ± s . e . m . ; n = 3 ) .

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(d) qrtPCR of MCU mRNA from wild-type and MCUR1-knockdown HeLa cells that were transiently transfected with MCU cDNA. **P0.01, ***P0.001 (mean±s.e.m.; n = 3).


( e ) [ Ca2 + ] m responses to histamine ( 100 μM ) in wild - type and MCUR1 ( shHe2 ) - knockdown HeLa cells overexpressing MCU . Negative shRNA ( Neg shRNA ) and MCUR1 - shHe2 cells were used as controls . Solid lines are mean ; shaded regions are ± s . e . m . ; n = 3 . ( f ) Quantification of peak rhod - 2 fluorescence following histamine stimulation . * * * P0 . 001 ( mean ± s . e . m . ; n = 3 ) .

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(e) [Ca2+]m responses to histamine (100 μM) in wild-type and MCUR1 (shHe2)-knockdown HeLa cells overexpressing MCU. Negative shRNA (Neg shRNA) and MCUR1-shHe2 cells were used as controls. Solid lines are mean; shaded regions are ±s.e.m.; n = 3. (f) Quantification of peak rhod-2 fluorescence following histamine stimulation. ***P0.001 (mean±s.e.m.; n = 3).


( a ) AMP / ATP ratios in stable HeLa cell lines stably expressing negative shRNA ( Neg shRNA ) , MCUR1 shRNA ( clone shHe2 ) or shHe2 with MCUR1 re - expressed . * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) .

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(a) AMP/ATP ratios in stable HeLa cell lines stably expressing negative shRNA (Neg shRNA), MCUR1 shRNA (clone shHe2) or shHe2 with MCUR1 re-expressed. **P0.01 (mean±s.e.m.; n = 3).


( b ) O2 consumption rates ( OCR ) in stable HeLa cells expressing irrelevant shRNA , clone shHe2 , and clone shHe2 re - expressing MCUR1 , exposed sequentially to oligomycin ( i ) , FCCP ( ii ) and rotenone plus myzothiazol ( iii ) .

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(b) O2 consumption rates (OCR) in stable HeLa cells expressing irrelevant shRNA, clone shHe2, and clone shHe2 re-expressing MCUR1, exposed sequentially to oligomycin (i), FCCP (ii) and rotenone plus myzothiazol (iii).


( c ) Basal and maximal O2 consumption rates in cells as described in b . * P0 . 05 ( mean ± s . e . m . ; n = 3 ) .

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(c) Basal and maximal O2 consumption rates in cells as described in b. *P0.05 (mean±s.e.m.; n = 3).


( d ) Western blot of phosphorylated and total AMPK ( top ) and densitometric analysis ( bottom ) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA ( clone shHe2 ) and clone shHe2 re - expressing MCUR1 . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) . ( e ) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA ( clone shHe2 ) and clone shHe2 re - expressing MCUR1 ( top ) and quantification of LC3 - II / ( LC3 - I + LC3 - II ) ( bottom ) expressed as fold increase over levels in cells expressing irrelevant shRNA . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ; n = 3 ) .

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(d) Western blot of phosphorylated and total AMPK (top) and densitometric analysis (bottom) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1. *P0.05, **P0.01 (mean±s.e.m.; n = 3). (e) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone shHe2 re-expressing MCUR1 (top) and quantification of LC3-II/(LC3-I + LC3-II) (bottom) expressed as fold increase over levels in cells expressing irrelevant shRNA. *P0.05, **P0.01 (mean±s.e.m.; n = 3).


( f , g ) , The same as in d and e . Activation of AMPK ( f ) and autophagy ( * * P0 . 001 ; mean ± s . e . m . ; n = 3 ) ( g ) in the absence and presence of InsP3R inhibitor xestospongin B ( XeB ) . * P0 . 05 , * * P0 . 001 , NS , not significant ( mean ± s . e . m . ; n = 3 ) . Uncropped images of blots / gels are shown in Supplementary Fig . S6 .

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

(f,g), The same as in d and e. Activation of AMPK (f) and autophagy (**P0.001; mean±s.e.m.; n = 3) (g) in the absence and presence of InsP3R inhibitor xestospongin B (XeB). *P0.05, **P0.001, NS, not significant (mean±s.e.m.; n = 3). Uncropped images of blots/gels are shown in Supplementary Fig. S6.


( A ) B cell development in Igh ∆ 890 / ∆ 890 mice . The relative frequencies of the indicated cell types were determined by flow cytometric analysis of bone marrow cells from Igh ∆ 890 / ∆ 890 and Igh + / + mice at the age of 3 - 4 weeks , indicating a 1 . 3 - fold increase of pro - B cells and a moderate decrease at subsequent B cell developmental stages in Igh ∆ 890 / ∆ 890 mice . Statistical data are shown as mean value with SEM . Data information : Statistical data were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Sidak correction ; A , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot ( A , corresponds to one mouse .

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(A) B cell development in Igh∆890/∆890 mice. The relative frequencies of the indicated cell types were determined by flow cytometric analysis of bone marrow cells from Igh∆890/∆890 and Igh+/+ mice at the age of 3-4 weeks, indicating a 1.3-fold increase of pro-B cells and a moderate decrease at subsequent B cell developmental stages in Igh∆890/∆890 mice. Statistical data are shown as mean value with SEM. Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; A, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (A, corresponds to one mouse.


( B ) Two - color 3D DNA - FISH analysis of ex vivo sorted pro - B cells of the indicated genotypes with the RP23 - 340K14 ( red ) and RP24 - 275L15 ( green ) BAC probes ( Fig 1A ) . Representative images are show above . Dot plots ( below ) show the distances measured between the two DNA signals in individual Igh alleles ( 2 , 123 for Rag2 - / - , 1 , 514 for Igh ∆ 890 / ∆ 890 Rag2 - / - and 502 for Vav - Cre Pax5fl / fl ( Pax5 ∆ / ∆ ) Rag2 - / - pro - B cells ) together with the mean distance ( red bar ) and SEM determined for each genotype . In total , four independent DNA - FISH experiments were performed . Data information : Statistical data were analyzed by multiple t - tests ( unpaired one - way ANOVA ( Tukey post - hoc test ; B ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(B) Two-color 3D DNA-FISH analysis of ex vivo sorted pro-B cells of the indicated genotypes with the RP23-340K14 (red) and RP24-275L15 (green) BAC probes (Fig 1A). Representative images are show above. Dot plots (below) show the distances measured between the two DNA signals in individual Igh alleles (2,123 for Rag2-/-, 1,514 for Igh∆890/∆890 Rag2-/- and 502 for Vav-Cre Pax5fl/fl (Pax5∆/∆) Rag2-/-pro-B cells) together with the mean distance (red bar) and SEM determined for each genotype. In total, four independent DNA-FISH experiments were performed. Data information: Statistical data were analyzed by multiple t-tests (unpaired one-way ANOVA (Tukey post-hoc test; B); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


( C , VH gene recombination analysis in ex vivo sorted Igh ∆ 890 / ∆ 890 and Igh + / + pro - B cells , as determined by VDJ - seq experiments ( C ) The percentages of uniquely identified DJH and VDJH sequences are shown as mean percentage with SEM . Each dot corresponds to an independent VDJ - seq experiments performed with sorted pro - B cells from one mouse . Data information : Statistical data were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Sidak correction ; ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot C , corresponds to one mouse .

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(C, VH gene recombination analysis in ex vivo sorted Igh∆890/∆890 and Igh+/+ pro-B cells, as determined by VDJ-seq experiments (C) The percentages of uniquely identified DJH and VDJH sequences are shown as mean percentage with SEM. Each dot corresponds to an independent VDJ-seq experiments performed with sorted pro-B cells from one mouse. Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot C, corresponds to one mouse.


( B ) Flow cytometric determination of the ratio of immature IgMb ( B6 ) to IgMa ( 129 ) B cells in the bone marrow of Igh ∆ 890 ( B6 ) / + ( 129 ) , IghP4V ( B6 ) / + ( 129 ) and control Igh + ( B6 ) / + ( 129 ) mice , which were generated by crossing Igh ∆ 890 / + , IghP4V / + and Igh + / + mice on the C57BL / 6 ( B6 ) background with Igh + / + mice of the 129 / Sv ( 129 ) strain . The rearranged Igh alleles of the C57BL / 6 and 129 / Sv strains give rise to the expression of IgMb and IgMa , respectively . The IgMb ( B6 ) to IgMa ( 129 ) ratio is shown as mean value with SEM ( right ) . Data information : Statistical data were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Sidak correction ; B , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot ( B , corresponds to one mouse .

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(B) Flow cytometric determination of the ratio of immature IgMb (B6) to IgMa (129) B cells in the bone marrow of Igh∆890(B6)/+(129), IghP4V (B6)/+(129) and control Igh+(B6)/+(129) mice, which were generated by crossing Igh∆890/+, IghP4V/+ and Igh+/+ mice on the C57BL/6 (B6) background with Igh+/+ mice of the 129/Sv (129) strain. The rearranged Igh alleles of the C57BL/6 and 129/Sv strains give rise to the expression of IgMb and IgMa, respectively. The IgMb (B6) to IgMa (129) ratio is shown as mean value with SEM (right). Data information: Statistical data were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Sidak correction; B, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (B, corresponds to one mouse.


( C ) Summary of the ratios of immature IgMb ( B6 ) to IgMa ( 129 ) B cells , determined for the indicated six mouse strains analyzed and shown as mean values with SEM . The ratio determined for control Igh + ( B6 ) / + ( 129 ) B cells was set to 1 . Data information : Statistical data were analyzed by multiple t - tests ( unpaired one - way ANOVA ( Tukey post - hoc test ; C , ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot C , corresponds to one mouse .

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

(C) Summary of the ratios of immature IgMb (B6) to IgMa (129) B cells, determined for the indicated six mouse strains analyzed and shown as mean values with SEM. The ratio determined for control Igh+(B6)/+(129) B cells was set to 1. Data information: Statistical data were analyzed by multiple t-tests (unpaired one-way ANOVA (Tukey post-hoc test; C, ; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot C, corresponds to one mouse.


( D ) Comparison of the VDJ - seq data obtained with pro - B cells of the Igh + / + ( black ) , Igh ∆ 890 / ∆ 890 ( white bars ) and IghP4V / P4V ( gray ) genotypes . The different VH genes ( horizontal axis ) are aligned according to their position in the Igh locus The usage of each VH gene ( vertical axis ) is shown as percentage of all VDJH and DJH recombination events determined for each pro - B cell type . The relative frequency of each VH gene is shown as mean value with SEM and is based on three independent VDJ - seq experiments for pro - B cells of each genotype .

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 B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of the VDJ-seq data obtained with pro-B cells of the Igh+/+ (black), Igh∆890/∆890 (white bars) and IghP4V/P4V (gray) genotypes. The different VH genes (horizontal axis) are aligned according to their position in the Igh locus The usage of each VH gene (vertical axis) is shown as percentage of all VDJH and DJH recombination events determined for each pro-B cell type. The relative frequency of each VH gene is shown as mean value with SEM and is based on three independent VDJ-seq experiments for pro-B cells of each genotype.


( A ) Presence of open chromatin and the active histone marks H3K27ac and H3K4me2 at the V8 . 8E enhancer in pro - B cells . The location of the VH8 - 8 gene , V8 . 8E enhancer ( dashed line ) and PAIR5 element are shown together with the mm9 genomic coordinates of mouse chromosome 12 . The VH8 - 8 gene with 500 bp of its upstream and downstream sequences ( indicated by violet overlay ) was inserted with the Floxin method at the deletion point of the Igh ∆ 890 allele to generate the IghV8 - 8

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

(A) Presence of open chromatin and the active histone marks H3K27ac and H3K4me2 at the V8.8E enhancer in pro-B cells. The location of the VH8-8 gene, V8.8E enhancer (dashed line) and PAIR5 element are shown together with the mm9 genomic coordinates of mouse chromosome 12. The VH8-8 gene with 500 bp of its upstream and downstream sequences (indicated by violet overlay) was inserted with the Floxin method at the deletion point of the Igh∆890 allele to generate the IghV8-8 allele


( B , Flow - cytometric analysis ( B ) of GFP expression are shown for pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghP4GV / P4GV ( dark green , n = 16 ) , IghP4GV / + ( light green , n = 12 ) and control Igh + / + ( black , n = 13 ) mice .

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

(B, Flow-cytometric analysis (B) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/P4GV (dark green, n=16), IghP4GV/+ (light green, n=12) and control Igh+/+ (black, n=13) mice.


C ) geometric mean fluorescence intensity measurements ( MFI , C ) of GFP expression are shown for pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghP4GV / P4GV ( dark green , n = 16 ) , IghP4GV / + ( light green , n = 12 ) and control Igh + / + ( black , n = 13 ) mice . Geometric mean fluorescence intensity measurements ( C ) are shown as mean values with 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 B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-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

C) geometric mean fluorescence intensity measurements (MFI, C) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/P4GV (dark green, n=16), IghP4GV/+ (light green, n=12) and control Igh+/+ (black, n=13) mice. Geometric mean fluorescence intensity measurements (C) are shown as mean values with SEM.


G ) Absence of GFP expression in DN ( CD4 - CD8 - ) and DP ( CD4 + CD8 + ) T cells , geometric mean fluorescence intensity measurements ( G ) of GFP expression in thymocytes from the indicated mice . Geometric mean fluorescence intensity measurements ( G ) are shown as mean value with SEM based on 12 ( Igh + / + ) , 11 ( IghP4GV / + ) or 12 ( IghP4GV / P4GV ) independent replicates .

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

G) Absence of GFP expression in DN (CD4-CD8-) and DP (CD4+CD8+) T cells, geometric mean fluorescence intensity measurements (G) of GFP expression in thymocytes from the indicated mice. Geometric mean fluorescence intensity measurements (G) are shown as mean value with SEM based on 12 (Igh+/+), 11 (IghP4GV/+) or 12 (IghP4GV/P4GV) independent replicates.


( A , B ) Flow - cytometric analysis ( A ) and geometric mean fluorescence intensity measurements ( MFI , B ) of GFP expression are shown for pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghP4GV / + ( green , n = 6 ) , IghP4 ∆ Pax5GV / + ( yellow , n = 5 ) , IghP4 ∆ CtcfGV / + ( brown , n = 4 ) and control Igh + / + ( black , n = 4 ) mice . Geometric mean fluorescence intensity measurements ( B ) are shown as mean value with SEM . Data information : Each dot corresponds to one mouse .

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

(A, B) Flow-cytometric analysis (A) and geometric mean fluorescence intensity measurements (MFI, B) of GFP expression are shown for pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghP4GV/+ (green, n=6), IghP4∆Pax5GV/+ (yellow, n=5), IghP4∆CtcfGV/+ (brown, n=4) and control Igh+/+ (black, n=4) mice. Geometric mean fluorescence intensity measurements (B) are shown as mean value with SEM. Data information: Each dot corresponds to one mouse.


( C , D ) Activation of the PAIR4 - V8 . 7E module in thymocytes upon loss of CTCF binding at PAIR4 , as shown by flow - cytometric analysis ( C ) and geometric mean fluorescence intensity measurements ( MFI , D ) of GFP expression in DN , DP , CD4 + and CD8 + thymocytes of IghP4 ∆ CtcfGV / + ( light brown , n = 5 ) and IghP4 ∆ CtcfGV / P4 ∆ CtcfGV ( dark brown , n = 6 ) mice compared with control Igh + / + ( black , n = 7 ) mice . Geometric mean fluorescence intensity measurements ( D ) are shown as mean value with SEM . Data information : Each dot corresponds to one mouse .

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

(C, D) Activation of the PAIR4-V8.7E module in thymocytes upon loss of CTCF binding at PAIR4, as shown by flow-cytometric analysis (C) and geometric mean fluorescence intensity measurements (MFI, D) of GFP expression in DN, DP, CD4+ and CD8+ thymocytes of IghP4∆CtcfGV/+ (light brown, n=5) and IghP4∆CtcfGV/P4∆CtcfGV (dark brown, n=6) mice compared with control Igh+/+ (black, n=7) mice. Geometric mean fluorescence intensity measurements (D) are shown as mean value with SEM. Data information: Each dot corresponds to one mouse.


( B , C ) Flow - cytometric analysis ( B ) and geometric mean fluorescence intensity measurements ( MFI , C ) of YFP expression are shown for MPPs ( LSK ) , LMPPs , ALPs and BLPs from the bone marrow of Rosa26LSL - YPF / + IghCμCre / + ( violet ) and control Rosa26LSL - YPF / + Igh + / + ( black ) mice . Geometric mean fluorescence intensity measurements ( B ) are shown as mean value with SEM and have been normalized by setting the mean value obtained with Igh + / + cells to 1 . Data information : Each dot corresponds to one mouse .

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

(B, C) Flow-cytometric analysis (B) and geometric mean fluorescence intensity measurements (MFI, C) of YFP expression are shown for MPPs (LSK), LMPPs, ALPs and BLPs from the bone marrow of Rosa26LSL-YPF/+ IghCμCre/+ (violet) and control Rosa26LSL-YPF/+ Igh+/+ (black) mice. Geometric mean fluorescence intensity measurements (B) are shown as mean value with SEM and have been normalized by setting the mean value obtained with Igh+/+ cells to 1. Data information: Each dot corresponds to one mouse.


Flow - cytometric analysis ( E ) of GFP expression are shown for MPPs ( LSK ) , LMPPs , ALPs , BLPs , pro - B , pre - B , immature B and recirculating B cells from the bone marrow of IghEμG / + ( light blue ) , IghEμG / EμG ( dark blue ) and control Igh + / + ( black ) mice .

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Flow-cytometric analysis (E) of GFP expression are shown for MPPs (LSK), LMPPs, ALPs, BLPs, pro-B, pre-B, immature B and recirculating B cells from the bone marrow of IghEμG/+ (light blue), IghEμG/EμG (dark blue) and control Igh+/+ (black) mice.


B , Same as in A , with the exception that cells expressing flag - praja2rm or praja2 deletion mutants ( praja21 - 530 , praja21 - 630 and praja2Δ530 - 630 ) were included in the analysis .

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B, Same as in A, with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21-530, praja21-630 and praja2Δ530-630) were included in the analysis.


B . MST signal ( normalized fluorescence ) of P1 ( red curve ) , P2 ( green curve ) and P3 ( cyan curve ) plotted against TBC1D31 , at increasing concentrations of peptides . The threading modelled structure of the overlapping binding segment of praja2 ( praja2550 - 570 ) is shown .

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B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550-570) is shown.


F . MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides : wild - type ( red curve ) , R948A - R951A ( violet curve ) and R957 - R959D - H960A ( orange curve ) .

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

F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve).


D . Total lysates from mouse brain were immunoprecipitated with anti - TBC1D31 antibodies or with control IgG . The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies .

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

D. Total lysates from mouse brain were immunoprecipitated with anti-TBC1D31 antibodies or with control IgG. The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies.


E . Same as in C , with the exception that cells were co - transfected with flag - OFD1 vector and with control siRNA ( siCNT ) or siRNA targeting praja2 . F . Quantitative analysis of the experiments shown in E . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 . G

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

E. Same as in C, with the exception that cells were co-transfected with flag-OFD1 vector and with control siRNA (siCNT) or siRNA targeting praja2. F. Quantitative analysis of the experiments shown in E. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05. G


C . HEK293 cells transfected with siRNAs were serum deprived , pre - treated with cycloheximide and stimulated with FSK at different time point . Lysates were immunoblotted for endogenous praja2 and OFD1 . Hsp90 was used as loading control . D . Quantitative analysis of the experiments shown in C . A mean value ± SD of three independent experiments is shown . Student ' s t test * p < 0 , 05 ; * * < 0 , 01 versus sipraja2 ( + FSK ) and basal values ( 0 ) . E

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C. HEK293 cells transfected with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control. D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0). E


H . Immunoblot analysis of flag - OFD1 or flag - E97G mutant in HEK293 cells stimulated with FSK as in C . I . Quantitative analysis of the experiments shown in H . A mean value ± SD of three independent experiments is shown . Student ' s t test , * p < 0 , 05 versus E97G mutant ( + FSK ) and basal values ( 0 ) .

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

H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with FSK as in C. I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0).


A . siRNA transfected HEK293 cells were serum deprived and stained for acetylated - tubulin , TBC1D31 and DRAQ5 . B . Quantitative analysis of the experiments shown in A . A mean value ± SD of four independent experiments is shown . Student ' s t test * p < 0 , 05 . C

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

A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5. B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05. C


C . HEK293 cells expressing flag - OFD1 or flag - S735A were serum deprived and subjected to staining analysis for acetylated α - tubulin , flag and DRAQ5 . Images were collected by super - resolution microscopy ( upper panels ) . Where indicated , flag - OFD1 or flag - S735A was co - transfected with cilia - APEX - GFP vector ( middle panels ) . NIH3T3 expressing flag - OFD1 or flag - S735A were stained with anti - ARL13B and anti - flag antibodies . D , E . Statistical analysis of the experiments shown in D . A mean value ± SD of three independent experiments is shown . For each experimental group , a minimum of 40 cilia were analyzed . Student ' s t test p * * * < 0 , 001 . F

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were stained with anti-ARL13B and anti-flag antibodies. D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001. F


F . HEK293 cells transiently transfected with flag - OFD1 or flag - S735A were serum deprived , treated with FSK ( 6 hours ) and stained for flag , acetylated - tubulin and DRAQ5 . G . Statistical analysis of the experiments shown in F . A mean value ± SD of three independent experiments is shown . Cells analysed for each experiment : 300 for NT , 80 for flag - OFD1 and 80 for flag - S735A . Student ' s t test , p * < 0 , 05 , p * * < 0 , 01 . H

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 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 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

F. HEK293 cells transiently transfected with flag-OFD1 or flag-S735A were serum deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5. G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01. H


B . Confocal images of cilia of the neural tube cells in the wild - type , Ol - TBC1D31 KD , Ol - TBC1D31 KD + hTBC1D31 , wild - type hOFD1 , Ol - TBC1D31 KD + wild - type hOFD1 , hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735A , Ol - TBC1D31 KD + hOFD1S735D and Ol - TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti - acetylated α - tubulin antibody ( green ) and DAPI ( blue ) .

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B. Confocal images of cilia of the neural tube cells in the wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti-acetylated α-tubulin antibody (green) and DAPI (blue).


A . Characteristics of IGR39 / IGR37 and WM115 / WM266 . 4 melanoma cell lines ( Upper Panel ) . Pigmentation of melanoma cells pellets ( lower panel ) .

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A. Characteristics of IGR39 /IGR37 and WM115/WM266.4 melanoma cell lines (Upper Panel). Pigmentation of melanoma cells pellets (lower panel).


B . Growth curve of primitive IGR39 and metastatic IGR37 cell line ( N = 3 biological replicates ) . Data are mean ± SD .

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B. Growth curve of primitive IGR39 and metastatic IGR37 cell line (N=3 biological replicates). Data are mean ±SD.


C . Phase - contrast images of IGR39 and IGR37 cells . Scale bar is 100µm .

C. Phase-contrast images of IGR39 and IGR37 cells. Scale bar is 100µm.


D . Analysis of IGR39 and IGR37 speed of migration by time - lapse microscopy .

D. Analysis of IGR39 and IGR37 speed of migration by time-lapse microscopy.


B . Scatter plot of identified and quantified proteins in the secretome of primitive IGR39 and metastatic IGR37 . Red dots represent proteins that were significant with FDR < 0 . 05 , blue dots represent proteins with p - value < 0 . 05 . C . Scatter plot of identified and quantified proteins fin the secretome of primitive WM115 and metastatic WM266 . 4 . Red dots represent proteins that were significant with FDR < 0 . 05 , blue dots represent proteins with p - value < 0 . 05 .

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B. Scatter plot of identified and quantified proteins in the secretome of primitive IGR39 and metastatic IGR37. Red dots represent proteins that were significant with FDR <0.05, blue dots represent proteins with p-value<0.05. C. Scatter plot of identified and quantified proteins fin the secretome of primitive WM115 and metastatic WM266.4. Red dots represent proteins that were significant with FDR <0.05, blue dots represent proteins with p-value<0.05.


D . ( Left ) Venn diagram of the significant proteins shared by both IGR and WM cell lines . ( Right ) Histograms representing the metastatic and primitive signatures H / M ratios .

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D. (Left)Venn diagram of the significant proteins shared by both IGR and WM cell lines. (Right) Histograms representing the metastatic and primitive signatures H/M ratios.


F . Confocal fluorescence images of Proteostat ( 1 : 2000 , red spots ) and DAPI staining ( blue ) , scale bar is 10µm . G . Quantitation of aggregates / cell in IGRs cell lines by immunofluorescence analysis , left panel ; fluorescence gain of soluble proteins treated with Proteostat reagent , right panel . ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) .

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F. Confocal fluorescence images of Proteostat (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. G. Quantitation of aggregates/cell in IGRs cell lines by immunofluorescence analysis, left panel; fluorescence gain of soluble proteins treated with Proteostat reagent, right panel. (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).


A . Absolute quantitation of 24 , 25 and 27 - hydroxycholesterol in primitive and metastatic melanoma cells as indicated . ( T - test analysis , * * * p - value < 0 . 001 , N = 3 biological replicates , data are mean ± SD ) .

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A. Absolute quantitation of 24, 25 and 27-hydroxycholesterol in primitive and metastatic melanoma cells as indicated. (T-test analysis, ***p-value<0.001, N=3 biological replicates, data are mean ±SD).


B . Histogram representing R square of correlation analysis between absolute quantitation of 24 , 25 and 27 - hydroxycholesterol and label free quantitation of APOE in a cohort of primitive ( A375 , IPC298 ) and metastatic ( SKMEL5 , SKMEL28 ) melanoma cell lines .

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B. Histogram representing R square of correlation analysis between absolute quantitation of 24, 25 and 27-hydroxycholesterol and label free quantitation of APOE in a cohort of primitive (A375, IPC298) and metastatic (SKMEL5, SKMEL28) melanoma cell lines.


C . Confocal fluorescence images of anti - HMB45 PMEL antibody signal ( green ) and DAPI staining ( blue ) in IGR37 and IGR39 . Scale bar is 10µm .

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C. Confocal fluorescence images of anti-HMB45 PMEL antibody signal (green) and DAPI staining (blue) in IGR37 and IGR39. Scale bar is 10µm.


D . Western blot on IGR37 and 39 cellular lysates probed with anti - HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates .

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D. Western blot on IGR37 and 39 cellular lysates probed with anti-HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates.


A - D . Immunofluorescence images with Proteostat ( red ) and DAPI ( blue ) staining on ( A ) human normal skin , ( B ) samples of primitive melanomas , ( C ) melanoma metastases in brain and ( D ) melanoma metastases in lung .

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A-D. Immunofluorescence images with Proteostat (red) and DAPI (blue) staining on (A) human normal skin, (B) samples of primitive melanomas, (C) melanoma metastases in brain and (D) melanoma metastases in lung.


E . Details of brain metastases . Scale Bar is 30µm . F . Quantitation of Proteostat positive dots in primitive vs metastatic melanoma tissues : 6 tissues from metastatic lesions and 6 from primitive melanoma lesions were analysed . For each tissue two sections were quantified . T - test analysis was applied . T - test analysis , * * = p - value < 0 . 01 ( N = 6 , data are mean ± SEM ) .

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E. Details of brain metastases. Scale Bar is 30µm. F. Quantitation of Proteostat positive dots in primitive vs metastatic melanoma tissues: 6 tissues from metastatic lesions and 6 from primitive melanoma lesions were analysed. For each tissue two sections were quantified. T-test analysis was applied. T-test analysis, ** = p-value<0.01 (N=6, data are mean ±SEM).


A . Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB - 360 .

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A. Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB-360.


B . Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB - 360 .

B. Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB-360.


D . Confocal fluorescence images of Proteostat signal ( 1 : 2000 , red spots ) and DAPI staining ( blue ) , scale bar is 10µm . E . Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software . ( T - test analysis , * * = p - value < 0 . 01 , N = 3 biological replicates , data are mean ± SD ) .

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D. Confocal fluorescence images of Proteostat signal (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm. E. Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software. (T-test analysis, ** = p-value<0.01, N=3 biological replicates, data are mean ±SD).


F . Quantitation , by immunofluorescence analysis , of YAP in different cellular compartments ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) . Images were quantified by subdividing cells into mostly cytosolic YAP ( Cytosol ) , mostly nuclear YAP ( Nuclear ) , or equal distribution ( Total ) from three biological replicates .

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F. Quantitation, by immunofluorescence analysis, of YAP in different cellular compartments (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). Images were quantified by subdividing cells into mostly cytosolic YAP (Cytosol), mostly nuclear YAP (Nuclear), or equal distribution (Total) from three biological replicates.


G . mRNA levels of CTGF measured by real - time PCR in IGR37 treated with DMSO or NB - 360 ( T - test analysis , * = p - value < 0 . 05 , N = 3 biological replicates , data are mean ± SD ) .

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G. mRNA levels of CTGF measured by real-time PCR in IGR37 treated with DMSO or NB-360 (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).


H . CTGF mRNA level measured by real - time PCR in IGR39 treated with IGR39 conditioned medium ( CTRL ) or with IGR37 conditioned medium ( CM ) , N = 4 biological replicates . T - test analysis , * * * p - value < 0 . 001 , data are mean ± SD .

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H. CTGF mRNA level measured by real-time PCR in IGR39 treated with IGR39 conditioned medium (CTRL) or with IGR37 conditioned medium (CM), N=4 biological replicates. T-test analysis, *** p-value <0.001, data are mean ±SD.


I . CTGF mRNA level measured by real - time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils ( 0 . 5µM ) , N = 3 biological replicates . T - test analysis : * * * p - value < 0 . 001 , data are mean ± SD .

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I. CTGF mRNA level measured by real-time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis: *** p-value <0.001, data are mean ±SD.


J . CTGF mRNA level measured by real - time PCR in IGR37 treated with DMSO , NB - 360 or NB - 360 plus recombinant PMEL amyloid fibrils ( 0 . 5µM ) , N = 3 biological replicates . T - test analysis * = p - value < 0 . 05 , * * = p - value < 0 . 01 . Data are mean ± SD .

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J. CTGF mRNA level measured by real-time PCR in IGR37 treated with DMSO, NB-360 or NB-360 plus recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis* = p-value<0.05, ** = p-value<0.01. Data are mean ±SD.


A . MTT assay for IGRs treated with DMSO or NB - 360 ( 25µM ) . Biological replicates N = 4 biological replicates . T - test analysis * * = p - value < 0 . 01 , * * * p - value < 0 . 001 . Data are mean ± SD . B . MTT assay for IGRs cells treated with DMSO or inhibitor 3I ( 3µM ) . Biological replicates N = 3 biological replicates . T - test analysis * * = p - value < 0 . 01 . Data are mean ± SD .

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A. MTT assay for IGRs treated with DMSO or NB-360 (25µM). Biological replicates N=4 biological replicates. T-test analysis ** = p-value<0.01, *** p-value <0.001. Data are mean ±SD. B. MTT assay for IGRs cells treated with DMSO or inhibitor 3I (3µM). Biological replicates N=3 biological replicates. T-test analysis ** = p-value<0.01. Data are mean ±SD.


C - D . MTT assay of IGRs treated with NB - 360 ( 25µM ) and different concentration of doxorubicin as indicated . Biological replicates N = 4 biological replicates . T - test analysis : * = 0 . 01 < p - value < 0 . 05 , * * = p - value < 0 . 01 . Data are mean ± SD .

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C-D. MTT assay of IGRs treated with NB-360 (25µM) and different concentration of doxorubicin as indicated. Biological replicates N=4 biological replicates. T-test analysis: * = 0.01<p-value<0.05, ** = p-value<0.01. Data are mean ±SD.


E . T - test analysis of IC50 values for doxorubicin used alone or in combination with NB - 360 ( combo ) . Data are mean ± SD . T - test analysis : * = 0 . 01 < p - value < 0 . 05 ; * * = 0 . 001 < p - value < 0 . 01 .

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E. T-test analysis of IC50 values for doxorubicin used alone or in combination with NB-360 (combo). Data are mean ±SD. T-test analysis: * = 0.01<p-value<0.05; ** = 0.001<p-value<0.01.


A . RGFP966 ( 966 ) - dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth ( 24h in culture ) compared to HDAC class II , HDAC1 / 2 and HDAC1 or 3 inhibitions via 69A , 963 and 233 respectively . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test ) .

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A. RGFP966 (966)-dependent HDAC3 inhibition resulted in significant and robust neurite outgrowth (24h in culture) compared to HDAC class II, HDAC1/2 and HDAC1 or 3 inhibitions via 69A, 963 and 233 respectively. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle ANOVA followed by Bonferroni test).


B , C . Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth ( average neurite outgrowth after 24h in culture , Tuj1 positive neurons ) on PDL - laminin or Myelin substrates . Scale bars , 100 μm . Data information : Data expressed as average neurite length ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - test )

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B, C. Specific pharmacological inhibition of HDAC3 facilitates DRG neurite outgrowth (average neurite outgrowth after 24h in culture, Tuj1 positive neurons) on PDL-laminin or Myelin substrates. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


D , E . Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle . Data information : Data expressed as mean fold change N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus vehicle Student ' s t - test )

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D, E. Immunoblotting shows significantly increased H3K9 acetylation after 966 vs vehicle. Data information: Data expressed as mean fold change N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus vehicle Student's t-test)


F , G . AAV - mediated mutant HDAC3 ( Y298H , V5 ) infection of DRG neurons induces neurite outgrowth ( Tuj - 1 / V5 double positive neurons ) after 36h in culture , which is repressed by wildtype HDAC3 overexpression . Scale bars , 100 μm . Data information : Data expressed as average neurite length ± s . e . m . N = 3 - 9 biological replicates . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 ) indicate significant difference of 966 versus V5 vs AAV - GFP ANOVA followed by Bonferroni test ) .

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F, G. AAV-mediated mutant HDAC3 (Y298H, V5) infection of DRG neurons induces neurite outgrowth (Tuj-1/V5 double positive neurons) after 36h in culture, which is repressed by wildtype HDAC3 overexpression. Scale bars, 100 μm. Data information: Data expressed as average neurite length ± s.e.m. N= 3-9 biological replicates. (*p<0.05, **p<0.01, ***p<0.005) indicate significant difference of 966 versus V5 vs AAV-GFP ANOVA followed by Bonferroni test).


A . IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased , primarily in the nucleus 24h after sciatic nerve axotomy ( SNA ) but not spinal dorsal column axotomy ( DCA ) . Scale bars , 100 μm .

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A. IHC in DRG sections for pHDAC3 revealed that HDAC3 phosphorylation is decreased, primarily in the nucleus 24h after sciatic nerve axotomy (SNA) but not spinal dorsal column axotomy (DCA). Scale bars, 100 μm.


B - C . Bar graphs show mean fold change levels ( B ) or % of pHDAC3 + cells ( C ) ( pHDAC3 + threshold was set at the minimum intensity level of sham cells ) ± s . e . m . DAPI counter staining was used to label nuclei N = 9 - 10 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham or laminectomy ( lam ) ( ANOVA followed by Bonferroni test ) .

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B-C. Bar graphs show mean fold change levels (B) or % of pHDAC3+ cells (C) (pHDAC3+ threshold was set at the minimum intensity level of sham cells) ± s.e.m. DAPI counter staining was used to label nuclei N= 9-10 biological replicates. (***p<0.005) indicate significant difference versus sham or laminectomy (lam) (ANOVA followed by Bonferroni test).


D . Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA . E . Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * * * p < 0 . 005 ) indicate significant difference versus sham ( Student ´ s t - test ) .

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D. Western blot revealed that HDAC3 phosphorylation is decreased in DRG nuclear extracts but not in the cytoplasm 24h after SNA. E. Bar graphs show intensity measurement of nuclear vs cytoplasm immunoblot. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (***p<0.005) indicate significant difference versus sham (Student´s t-test).


F . HDAC3 activity ( HDAC3 IP and activity assay ) is strongly reduced after SNA but not DCA . Data is expressed as mean ± s . e . m . N = 3 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni test ) .

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F. HDAC3 activity (HDAC3 IP and activity assay) is strongly reduced after SNA but not DCA. Data is expressed as mean ± s.e.m. N= 3 biological replicates. (*p<0.05) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


PP4 activities are induced after SNA but not DCA , and are blocked by the calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni test ) .

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PP4 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


PP2 activities are induced after SNA but not DCA , and are blocked by the calcium chelator EGTA . Data is expressed as mean ± s . e . m . N = 3 - 4 biological replicates . ( * * * p < 0 . 001 ) indicate significant difference versus sham or lam ( Two - way ANOVA followed by Bonferroni test ) .

PP2 activities are induced after SNA but not DCA, and are blocked by the calcium chelator EGTA. Data is expressed as mean ± s.e.m. N= 3-4 biological replicates. (***p<0.001) indicate significant difference versus sham or lam (Two-way ANOVA followed by Bonferroni test).


C . SNA but not DCA significantly enhances calcium levels in DRG . Data is expressed as mean ± s . e . m . N = 4 biological replicates . ( * p < 0 . 05 ) indicate significant difference versus sham or lam ( ANOVA followed by Bonferroni test ) .

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C. SNA but not DCA significantly enhances calcium levels in DRG. Data is expressed as mean ± s.e.m. N= 4 biological replicates. (*p<0.05) indicate significant difference versus sham or lam (ANOVA followed by Bonferroni test).


D - F . Immunoblotting of DRG homogenates shows that nerve injury - dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve ( Axo = Axotomy ) . Cytoplasmic pERK levels are used as control of axotomy - dependent signalling . ( E - F ) . Data is expressed as mean fold change of band intensity levels ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 , * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni test ) .

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D-F. Immunoblotting of DRG homogenates shows that nerve injury-dependent nuclear HDAC3 dephosphorylation is reduced by the calcium scavenger EGTA upon in vivo delivery on the injured sciatic nerve (Axo=Axotomy). Cytoplasmic pERK levels are used as control of axotomy-dependent signalling. (E-F). Data is expressed as mean fold change of band intensity levels ± s.e.m. N= 3 biological replicates. (**p<0.01, ***p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


Significant Pearson correlations between calcium levels ( Fluo4AM ) and H3K9ac , r2 : 0 . 7802 ( G ) in cultured DRG neurons after KCl . The Pearson correlations were disrupted by administration of Fostriecin ( calcium and H3K9ac , r2 : 0 . 0007 Data is expressed as single cell fluorescence levels . N = 50 cells per condition from 4 biological replicates .

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Significant Pearson correlations between calcium levels (Fluo4AM) and H3K9ac, r2: 0.7802 (G) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin (calcium and H3K9ac, r2: 0.0007 Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


Significant Pearson correlations between calcium levels ( Fluo4AM ) ; or pHDAC3 , r2 : 0 . 6366 ( H ) in cultured DRG neurons after KCl . The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3 , r2 : 0 . 3025 ) . Data is expressed as single cell fluorescence levels . N = 50 cells per condition from 4 biological replicates .

Significant Pearson correlations between calcium levels (Fluo4AM) ; or pHDAC3, r2: 0.6366 (H) in cultured DRG neurons after KCl. The Pearson correlations were disrupted by administration of Fostriecin calcium and pHDAC3, r2: 0.3025). Data is expressed as single cell fluorescence levels. N=50 cells per condition from 4 biological replicates.


J - L . Immunofluorescence ( J ) in DRG tissue sections 24h after injury in vivo shows that the PP2a / PP4 inhibitor Fostriecin ( F ) inhibits the injury - induced decrease in pHDAC3 ( K ) and the increase in H3K9ac ( L ) . Scale bar , 10 μm . ( K - L ) . Data is expressed as mean fluorescence intensity in arbitrary units ± s . e . m . N = 3 ( L ) or 4 ( K ) biological replicates ( * p < 0 . 05 ; * * p < 0 . 01 ) indicate significant difference versus sham ( ANOVA followed by Bonferroni test ) .

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J-L. Immunofluorescence (J) in DRG tissue sections 24h after injury in vivo shows that the PP2a/PP4 inhibitor Fostriecin (F) inhibits the injury-induced decrease in pHDAC3 (K) and the increase in H3K9ac (L). Scale bar, 10 μm. (K-L). Data is expressed as mean fluorescence intensity in arbitrary units ± s.e.m. N= 3 (L) or 4 (K) biological replicates (*p<0.05; **p<0.01) indicate significant difference versus sham (ANOVA followed by Bonferroni test).


M - N . Pharmacological inhibition of PP2a / PP4 impairs conditioning - induced DRG neurite outgrowth ex vivo ( 12h in culture ) . M . DRG neurite outgrowth ( TUJ1 positive cells ) 24h after sham or sciatic nerve axotomy ( SNA ) prior to i . t . administration of vehicle ( V ) or Fostriecin ( F ) ( 240µM ) . Scale bar , 100µm . ( N ) Data is expressed as average neurite length per neuron ± s . e . m . N = 3 biological replicates per condition , technical triplicate . * * * p < 0 . 005 indicate significant difference versus sham / vehicle ( One way ANOVA followed by Bonferroni ) .

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M-N. Pharmacological inhibition of PP2a/PP4 impairs conditioning-induced DRG neurite outgrowth ex vivo (12h in culture). M. DRG neurite outgrowth (TUJ1 positive cells) 24h after sham or sciatic nerve axotomy (SNA) prior to i.t. administration of vehicle (V) or Fostriecin (F) (240µM). Scale bar, 100µm. (N) Data is expressed as average neurite length per neuron ± s.e.m. N= 3 biological replicates per condition, technical triplicate. *** p<0.005 indicate significant difference versus sham/vehicle (One way ANOVA followed by Bonferroni).


A - E . PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl ( A - C , arrows ) , both in the nucleus and the cytoplasm ( D ) . Scale bar , 20 μm . ( C , D ) Data is expressed as mean fold change vs scrambled ( scr ) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s . e . m . N = 3 biological replicates . ( * * p < 0 . 01 ; * * * p < 0 . 005 ) indicate significant difference versus scrambled siRNA ( Student ' s t - test ) . ( E ) PCR against PP4c demonstrates silencing of PP4c mRNA . F - G . PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 ( F ) . ( G ) Data is expressed as mean fold change of immunoblot band intensity ± s . e . m . N = 3 biological replicates ( * p < 0 . 05 ; * * * * p < 0 . 001 ) indicate significant difference versus control siRNA ( Student ' s t - test ) .

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A-E. PP4c gene silencing promotes HDAC3 phosphorylation in the presence of KCl (A-C, arrows), both in the nucleus and the cytoplasm (D). Scale bar, 20 μm. (C,D) Data is expressed as mean fold change vs scrambled (scr) siRNA of fluorescent intensity of pHDAC3 or PP4c in GFP positive cells ± s.e.m. N=3 biological replicates. (**p<0.01; ***p<0.005) indicate significant difference versus scrambled siRNA (Student's t-test). (E) PCR against PP4c demonstrates silencing of PP4c mRNA. F-G. PP2a gene silencing inhibits the KCl induced dephosphorylation of HDAC3 as shown by immunoblotting of pHDAC3 (F). (G) Data is expressed as mean fold change of immunoblot band intensity ± s.e.m. N= 3 biological replicates (*p<0.05; ****p<0.001) indicate significant difference versus control siRNA (Student's t-test).


H - I . Genetic modification of activity - dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth . ( H ) DRG neurite outgrowth after transfection of control ( V5 control ) , WT HDAC3 - V5 , phospho - dead HDAC3 ( S424A ) - V5 or phospho - mimetic HDAC3 ( S424D ) - V5 and after 24h in culture . Scale bar , 50µm . ( I ) Data is expressed as mean ± s . e . m . N = 8 - 9 biological replicates , approximately 35 V5 positive cells each . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference ( One way ANOVA followed by Bonferroni ) .

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H-I. Genetic modification of activity-dependent phosphorylation of serine 424 of HDAC3 affects DRG neurite outgrowth. (H) DRG neurite outgrowth after transfection of control (V5 control), WT HDAC3-V5, phospho-dead HDAC3 (S424A)-V5 or phospho-mimetic HDAC3 (S424D)-V5 and after 24h in culture. Scale bar, 50µm. (I) Data is expressed as mean ± s.e.m. N= 8-9 biological replicates, approximately 35 V5 positive cells each. * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference (One way ANOVA followed by Bonferroni).


A . The graph shows the number of differentially expressed ( DE ) genes 24h following SNA or DCA compared to their respective controls .

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A. The graph shows the number of differentially expressed (DE) genes 24h following SNA or DCA compared to their respective controls.


B . RNAseq read counts show differential occupancy of H3K9ac on specific gene regions , including an increased occupancy close to the transcriptional start site ( TSS ) after SNA only , when compared to corresponding controls .

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B. RNAseq read counts show differential occupancy of H3K9ac on specific gene regions, including an increased occupancy close to the transcriptional start site (TSS) after SNA only, when compared to corresponding controls.


C . Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA ( RNAseq ) with an increased or decreased H3K9ac occupancy ( ChIPseq ) , respectively . D . Odds ratio analysis of the enrichment / depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in ( C ) ; the numbers in red represent the p value given by Fisher ' s exact test ( ns = not significant ) .

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C. Bar graph shows the number of genes that are upregulated or downregulated following SNA or DCA (RNAseq) with an increased or decreased H3K9ac occupancy (ChIPseq), respectively. D. Odds ratio analysis of the enrichment/depletion of genes with differential H3K9ac occupancy across each upregulated and downregulated gene cluster in (C); the numbers in red represent the p value given by Fisher's exact test (ns= not significant).


A , B . Co - immunofluorescence of anti - neuronal and anti - regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle . Shown is increased protein expression for regeneration - associated targets in NF200 positive DRG neurons ( arrows ) after 966 treatment ( 5 weeks following SCI ) . Scale bar , 50 μm . ( B ) Data is expressed as fold change of fluorescence intensity in NF200 + cells , 966 versus vehicle ± s . e . m . N = 4 - 7 biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 indicate significant difference versus vehicle ( Student ' s t - test ) .

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A, B. Co-immunofluorescence of anti-neuronal and anti-regeneration associated proteins in DRG following SCI and treatment with 966 or vehicle. Shown is increased protein expression for regeneration-associated targets in NF200 positive DRG neurons (arrows) after 966 treatment (5 weeks following SCI). Scale bar, 50 μm. (B) Data is expressed as fold change of fluorescence intensity in NF200+ cells, 966 versus vehicle ± s.e.m. N=4-7 biological replicates * p<0.05, ** p<0.01, *** p<0.005 indicate significant difference versus vehicle (Student's t-test).


A - B . AAV - HDAC3 mutant ( Y298H ; dominant negative deacetylase inactive ) promoted DRG regenerative growth after spinal cord injury vs AAV - V5 control ( injected 5 weeks prior to SCI in the sciatic nerve bilaterally ) as shown by dextran - red traced axons across and beyond the lesion site ( GFAP , green ) ( dotted lines denote the rostral and caudal margins of the scar around the lesion ) . Scale bar , ( A - B ) 200 μm .

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A-B. AAV-HDAC3 mutant (Y298H; dominant negative deacetylase inactive) promoted DRG regenerative growth after spinal cord injury vs AAV-V5 control (injected 5 weeks prior to SCI in the sciatic nerve bilaterally) as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, (A-B) 200 μm.


C - F . Immunofluorescence for H3K9ac . AAV - HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons , but not in surrounding nuclei of satellite cells . Scale bar , ( C - F ) 50 μm .

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C-F. Immunofluorescence for H3K9ac. AAV-HDAC3 mutant induced upregulation of H3K9ac in vivo in DRG neurons, but not in surrounding nuclei of satellite cells. Scale bar, (C-F) 50 μm.


G - J . New synaptic formations ( VGlut1 + ) from regrowing axons are shown beyond the lesion site after injection of AAV - HDAC3mut . ( G ) Scale bar 200 μm , ( H - J ) High magnification insets depict co - localization of Dextran and vGlut1 staining . Scale bar 50 μm .

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G-J. New synaptic formations (VGlut1+) from regrowing axons are shown beyond the lesion site after injection of AAV-HDAC3mut. (G) Scale bar 200 μm, (H-J) High magnification insets depict co-localization of Dextran and vGlut1 staining. Scale bar 50 μm.


K - L . Quantification of dextran positive axons shows that AAV - HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site . Data is expressed as percentage of dextran + axons at each distance vs dextran + axons at - 700μm from the lesion margin ( K ) or distance from the caudal margin of the lesion to the most rostral dextran + axon tip for each animal ( L ) . N = 10 animals per condition , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 indicate a significant difference ( ANOVA followed by Bonferroni test ) .

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K-L. Quantification of dextran positive axons shows that AAV-HDAC3mut promotes DRG regenerative growth across and beyond the spinal lesion site. Data is expressed as percentage of dextran+ axons at each distance vs dextran+ axons at -700μm from the lesion margin (K) or distance from the caudal margin of the lesion to the most rostral dextran+ axon tip for each animal (L). N= 10 animals per condition, ± s.e.m. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 indicate a significant difference (ANOVA followed by Bonferroni test).


A - B . Intrathecally administered RGFP966 ( 966 ) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran - red traced axons across and beyond the lesion site ( GFAP , green ) ( dotted lines denote the rostral and caudal margins of the scar around the lesion ) . Scale bar , 200 μm .

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A-B. Intrathecally administered RGFP966 (966) through osmotic minipump for 14 days promoted DRG regenerative axonal growth after spinal cord injury as shown by dextran-red traced axons across and beyond the lesion site (GFAP, green) (dotted lines denote the rostral and caudal margins of the scar around the lesion). Scale bar, 200 μm.


C - D . Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site . Data is expressed as ratio of dextran + axons vs dextran + axons at - 700 μm from the lesion site ± s . e . m ( C ) or distance from the caudal margin of the lesion to the last dextran + axon tip ( D ) . N = 6 - 10 animals per condition . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 ) indicate a significant difference ( ANOVA followed by Bonferroni test ) .

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C-D. Quantification of axonal regeneration shows dextran axonal labelling across and beyond the spinal lesion site. Data is expressed as ratio of dextran+ axons vs dextran+ axons at -700 μm from the lesion site ± s.e.m (C) or distance from the caudal margin of the lesion to the last dextran+ axon tip (D). N= 6-10 animals per condition. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001) indicate a significant difference (ANOVA followed by Bonferroni test).


E . Immunofluorescence for H3K9ac and Neurofilament 200 ( NF200 ) shows upregulated H3K9ac in vivo in DRG neurons after 966 . Scale bar , 50 µm .

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E. Immunofluorescence for H3K9ac and Neurofilament 200 (NF200) shows upregulated H3K9ac in vivo in DRG neurons after 966. Scale bar, 50 µm.


F - G . Data is expressed as fold change of fluorescence intensity mean levels ( F ) or % of H3K9ac + cells ( G ) ( H3K9ac + threshold was set at the mean intensity level of vehicle treated animals , DAPI counter staining was used to label nuclei from NF200 + or NF200 - cells ) vs veh ± s . e . m . N = 11 - 12 biological replicates . ( * * * p < 0 . 005 ) indicate a significant difference vs respective Veh ( ANOVA followed by Bonferroni test ) .

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F-G. Data is expressed as fold change of fluorescence intensity mean levels (F) or % of H3K9ac+ cells (G) (H3K9ac+ threshold was set at the mean intensity level of vehicle treated animals, DAPI counter staining was used to label nuclei from NF200+ or NF200- cells) vs veh± s.e.m. N= 11-12 biological replicates. (***p<0.005) indicate a significant difference vs respective Veh (ANOVA followed by Bonferroni test).


( D ) HeLa cells were transfected with 10 nM control ( n = 84 ) or Nup50 siRNA ( n = 171 ) , 20 nM control ( n = 101 ) or Nup50 siRNA ( n = 157 ) and 40 nM control ( n = 86 ) or Nup50 siRNA ( n = 204 ) . 72 h post transfection , cells were fixed with 4 % PFA and stained with antibodies against Nup50 and mAB414 , chromatin was labeled with DAPI . Scale bars : 50 µm . Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations . The means are indicated as diamonds , error bars show the standard deviations . P - values have been calculated from a student t - test comparing the mean between the experimental conditions .

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(D) HeLa cells were transfected with 10 nM control (n=84) or Nup50 siRNA (n=171), 20 nM control (n=101) or Nup50 siRNA (n=157) and 40 nM control (n=86) or Nup50 siRNA (n=204). 72 h post transfection, cells were fixed with 4% PFA and stained with antibodies against Nup50 and mAB414, chromatin was labeled with DAPI. Scale bars: 50 µm. Quantitation of the mAB414 rim intensity at three different Nup50 RNAi and control oligo concentrations. The means are indicated as diamonds, error bars show the standard deviations. P-values have been calculated from a student t-test comparing the mean between the experimental conditions.


( A ) Mouse 3T3 - NIH cells were transfected with 20 nM control , Nup50A , Nup50B or a combination of Nup50A and Nup50B siRNA . After 72h cells were fixed and stained with mAB414 ( green ) and antibodies against mouse Nup50 ( red ) . Chromatin is stained with DAPI ( blue ) . The merge of the three channels is shown on the left column . Scale bar : 50 µm . An insert shows a zoom on a representative nucleus for each picture . Scale bar : 10 µm .

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(A) Mouse 3T3-NIH cells were transfected with 20 nM control, Nup50A, Nup50B or a combination of Nup50A and Nup50B siRNA. After 72h cells were fixed and stained with mAB414 (green) and antibodies against mouse Nup50 (red). Chromatin is stained with DAPI (blue). The merge of the three channels is shown on the left column. Scale bar: 50 µm. An insert shows a zoom on a representative nucleus for each picture. Scale bar: 10 µm.


( B ) Quantitation of the mAB414 rim intensity from n = 4 independent experiments done as in ( A ) , each represented by a different color . The mean of each individual experiment and condition is represented by a diamond , the mean of all four experiments by a horizontal black line . P - values have been calculated from a paired ratio t - test comparing the mean between the experimental conditions . Error bars show the standard deviations of the means .

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(B) Quantitation of the mAB414 rim intensity from n=4 independent experiments done as in (A), each represented by a different color. The mean of each individual experiment and condition is represented by a diamond, the mean of all four experiments by a horizontal black line. P-values have been calculated from a paired ratio t-test comparing the mean between the experimental conditions. Error bars show the standard deviations of the means.


( A , B ) Demembranated sperm chromatin was preincubated in Xenopus egg extract , depleted for MEL28 / ELYS or Nup50 ( B ) or control treated ( Mock , A ) . After 10 min , membranes were added to the reaction . Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α - MEL28 / ELYS ( A ) , α - Nup50 ( B ) , and mAb414 ( A , B ) . Chromatin was stained with DAPI ( blue in the overlay ) . Scale bar : 10 μm .

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(A, B) Demembranated sperm chromatin was preincubated in Xenopus egg extract, depleted for MEL28/ELYS or Nup50 (B) or control treated (Mock, A). After 10 min, membranes were added to the reaction. Reactions were stopped at the indicated time points by fixation and analyzed by confocal microscopy after immunostaining with α-MEL28/ELYS (A), α-Nup50 (B), and mAb414 (A, B). Chromatin was stained with DAPI (blue in the overlay). Scale bar: 10 μm.


3 µM recombinant EGFP , EGFP - tagged MEL28 / ELYS ( aa 2290 - 2408 ) or EGFP - tagged Nup50 were incubated with empty or DNA - coated magnetic beads , which were chromatinized with Xenopus egg extracts ( right panel ) or unchromatinized DNA - beads ( left panel ) . After 3h the beads were re - isolated , washed , co - stained with DAPI and analyzed by confocal microscopy . Scale bar : 10 µm .

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3 µM recombinant EGFP, EGFP-tagged MEL28/ELYS (aa 2290-2408) or EGFP-tagged Nup50 were incubated with empty or DNA-coated magnetic beads, which were chromatinized with Xenopus egg extracts (right panel) or unchromatinized DNA-beads (left panel). After 3h the beads were re-isolated, washed, co-stained with DAPI and analyzed by confocal microscopy. Scale bar: 10 µm.


( C ) HeLa cells were transfected with EGFP - Nup50 comprising different point mutations in the minimal NPC binding region and analyzed as in ( A ) . Scale Bar : 10 µm .

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(C) HeLa cells were transfected with EGFP-Nup50 comprising different point mutations in the minimal NPC binding region and analyzed as in (A). Scale Bar: 10 µm.


( D ) GST - fusion constructs of the Xenopus Nup50 minimal NPC binding fragment ( aa 144 - 189 ) comprising no or single point mutations were incubated with Xenopus egg extracts . Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies . The quantitation shows the average MEL28 / ELYS and Nup153 bead bound signal from three independent experiments , normalized to the signal of their respective wild - type GST fusion Data points from individual experiments are indicated .

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(D) GST-fusion constructs of the Xenopus Nup50 minimal NPC binding fragment (aa 144-189) comprising no or single point mutations were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies. The quantitation shows the average MEL28/ELYS and Nup153 bead bound signal from three independent experiments, normalized to the signal of their respective wild-type GST fusion Data points from individual experiments are indicated.


( A ) Confocal microscopy images of nuclei assembled for 120 min in mock depleted , Nup50 depleted ( ∆ Nup50 ) , and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild - type Nup50 or different mutants . Nuclei were fixed in 4 % PFA and 0 . 5 % glutaraldehyde , stained for NPCs ( mAB414 ) and the chromatin ( DAPI ) . Scale bar : 10 µm . ( B ) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in ( A ) . Data points from the individual experiments are indicated .

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(A) Confocal microscopy images of nuclei assembled for 120 min in mock depleted, Nup50 depleted (∆Nup50), and Nup50 depleted Xenopus egg extracts supplemented with recombinant wild-type Nup50 or different mutants. Nuclei were fixed in 4% PFA and 0.5 % glutaraldehyde, stained for NPCs (mAB414) and the chromatin (DAPI). Scale bar: 10 µm. (B) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (A). Data points from the individual experiments are indicated.


( C ) Confocal microscopy images of nuclei assembled with N - terminal Nup50 truncations and an minimal N - terminal fragment . Samples were analyzed as in ( A ) . ( D ) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in ( C ) . Data points from the individual experiments are indicated .

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(C) Confocal microscopy images of nuclei assembled with N-terminal Nup50 truncations and an minimal N-terminal fragment. Samples were analyzed as in (A). (D) Average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of at least three independent experiments shown in (C). Data points from the individual experiments are indicated.


( B ) Confocal microscopy images of fixed nuclei assembled for 120 min in mock depleted ( mock ) and Nup50 depleted ( ∆ Nup50 ) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologues . Nuclei were stained for Nup50 ( Nup50 antibody for Xenopus protein , His6 for mouse protein ) and NPCs ( mAB414 , red ) on the chromatin ( DAPI ) . Scale bar : 10 µm . ( C ) The average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments ( performed as in B ) is shown . Data points from individual experiments are indicated .

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(B) Confocal microscopy images of fixed nuclei assembled for 120 min in mock depleted (mock) and Nup50 depleted (∆Nup50) Xenopus egg extracts supplemented with recombinant mouse Nup50 orthologues. Nuclei were stained for Nup50 (Nup50 antibody for Xenopus protein, His6 for mouse protein) and NPCs (mAB414, red) on the chromatin (DAPI). Scale bar: 10 µm. (C) The average percentage of mAB414 positive nuclei for 100 randomly chosen chromatin substrates in each of three independent experiments (performed as in B) is shown. Data points from individual experiments are indicated.


( E ) GST - fusion constructs of the Xenopus Nup53 RRM domain ( aa 162 - 267 , control ) full - length Xenopus Nup50 and the two mouse Nup50 orthologues were incubated with Xenopus egg extracts . Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies The right panel shows the average MEL28 / ELYS , Nup153 , importin β and ran bead bound signal from two independent experiments normalized to their respective Xenopus signal . Data points from individual experiments are indicated .

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

(E) GST-fusion constructs of the Xenopus Nup53 RRM domain (aa 162-267, control) full-length Xenopus Nup50 and the two mouse Nup50 orthologues were incubated with Xenopus egg extracts. Starting material as well as bound proteins were analyzed by Western blotting with indicated antibodies The right panel shows the average MEL28/ELYS, Nup153, importin β and ran bead bound signal from two independent experiments normalized to their respective Xenopus signal. Data points from individual experiments are indicated.