( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR ( α - IFNAR Ab ) , an isotype control antibody ( IgG1 ) or no antibody ( no Ab ) . Cells were then were transfected or not ( mock ) with Cy5 - labeled dsRNA as indicated and GFP level monitored in Cy5 + cells 72 hrs post - transfection .

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(A) Mavs+/- and Mavs-/-MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+cells 72 hrs post-transfection.


( B ) Mavs - / - MEFs stably expressing d2GFP were treated with recombinant IFN ( 200 U / ml ) for 24 hrs prior to transfection or not ( mock ) with Cy5 - labeled dsRNA , as indicated . IFN treatment was maintained for 2 days post - transfection and then GFP level monitored in Cy5 + cells .

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(B) Mavs-/-MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+cells.


( A ) Northern blot analysis of Ifnar1 - / - MEFs at 24 h . p . t with the indicated dsRNA or transfection reagent alone ( mock ) using a probe specific for dsRNA - GFP ( top ) or dsRNA - RL ( bottom ) . Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG - human Dicer were loaded in parallel . Two lanes ( delimited by dashed lines ) were left empty between the Ifnar1 - / - MEFs transfected samples and the in vitro dicing assay reactions . A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17 , 21 and 25 - nt in length was run in parallel . Endogenous U6 was used as a loading control . Signals corresponding to the dsRNA - derived siRNAs are indicated with an arrow . The membrane was first probed for dsRNA - GFP ( top ) , then probed for the miRNA marker ( top left ) and subsequently stripped and reprobed for dsRNA - RL ( middle ) and finally stripped and reprobed for both U6 ( bottom ) and for the miRNA marker again ( middle left ) .

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(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left).


( B ) Western blot of Dicer in d2GFP - expressing Mavs - / - MEFs either not transduced ( NT ) or transduced to express a non - targeting shRNA control ( CTL shRNA ) or two different Dicer - specific targeting shRNAs ( DICER shRNA 1 and 2 ) . P97 served as loading control .

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(B) Western blot of Dicer in d2GFP-expressing Mavs-/- MEFs either not transduced (NT) or transduced to express a non-targeting shRNA control (CTL shRNA) or two different Dicer-specific targeting shRNAs (DICER shRNA 1 and 2). P97 served as loading control.


( C ) Cells described in ( B ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later . Please note that the biphasic pattern of d2GFP level observed in dsRNA - GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of

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(C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP


( D ) Western blots of Ago2 in parental Mavs - / - MEFs stably expressing d2GFP and three Mavs - / - Ago2 - / - clones generated by CRISPR / CAS9 - mediated genome engineering . P97 served as loading control .

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(D) Western blots of Ago2 in parental Mavs-/- MEFs stably expressing d2GFP and three Mavs-/- Ago2-/- clones generated by CRISPR/CAS9-mediated genome engineering. P97 served as loading control.


( E ) Mavs - / - ( parental line ) and Mavs - / - Ago2 - / - MEFs ( clone 5 . 1 ) stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

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(E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


( A ) Western blots for Ago2 ( top ) and HA tag ( bottom ) of parental Mavs - / - MEFs and two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ; see Fig 4A ) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA - tagged wild - type ( HA - mAgo2 WT ) or catalytic mutant ( HA - mAgo2 D597A ) versions of mouse Ago2 . β - actin served as loading control . The membrane was first analysed for Ago2 and β - actin , stripped and then analysed for HA tag .

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(A) Western blots for Ago2 (top) and HA tag (bottom) of parental Mavs-/- MEFs and two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3; see Fig 4A) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA-tagged wild-type (HA-mAgo2 WT) or catalytic mutant (HA-mAgo2 D597A) versions of mouse Ago2. β-actin served as loading control. The membrane was first analysed for Ago2 and β-actin, stripped and then analysed for HA tag.


( B ) Parental non - transduced ( NT ) Mavs - / - MEFs and complemented Mavs - / - Ago2 - / - MEFs as described in ( A ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

(B) Parental non-transduced (NT) Mavs-/- MEFs and complemented Mavs-/- Ago2-/- MEFs as described in (A) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


( A ) Ifnar1 - / - MEFs were transfected with the indicated Cy5 - labeled dsRNA and the next day were infected with SFV - Rluc at the indicated multiplicities of infection ( MOI ) . Renilla luciferase activity was measured 24 hours later . Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold - reduction in viral activity achieved with dsRNA - RL vs . dsRNA - GFP treatment .

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(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment.


( B ) As in ( a ) but SFV - Rluc virus titre was measured by plaque assay and expressed as plaque forming units ( PFU ) per ml of supernatant at 24 h post - infection ( MOI = 0 . 1 ) .

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(B) As in (a) but SFV-Rluc virus titre was measured by plaque assay and expressed as plaque forming units (PFU) per ml of supernatant at 24 h post-infection (MOI=0.1).


( C ) As in ( a ) but with Ifnar1 - / - or Mavs - / - MEFs and a single dose of SFV - Rluc ( MOI = 0 . 1 ) .

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(C) As in (a) but with Ifnar1-/- or Mavs-/- MEFs and a single dose of SFV-Rluc (MOI=0.1).


( D ) As in ( a ) but with two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ) individually complemented to express wild - type ( HA - mAgo2 WT ) or a catalytic mutant ( HA - mAgo2 D597A ) version of mAgo2 or transduced with a control vector ( as described in Fig 5 ) . SFV - Rluc was used at an MOI = 0 . 1 .

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(D) As in (a) but with two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3) individually complemented to express wild-type (HA-mAgo2 WT) or a catalytic mutant (HA-mAgo2 D597A) version of mAgo2 or transduced with a control vector (as described in Fig 5). SFV-Rluc was used at an MOI=0.1.


( C ) Relative mRNA expression from real - time PCR analysis of the GnRH transcript in GFP - positive cells , in comparison with GFP - negative cells . The GnRH transcript appears to be selectively expressed in GFP - positive cells . Mann - Whitney U test , n = 4 mice from 2 litters .

(C) Relative mRNA expression from real-time PCR analysis of the GnRH transcript in GFP-positive cells, in comparison with GFP-negative cells. The GnRH transcript appears to be selectively expressed in GFP-positive cells. Mann-Whitney U test, n=4 mice from 2 litters.


( D ) Relative mRNA expression from real - time PCR analysis of Neuropilin - 1 ( Nrp1 ) , Nrp2 , Plexin A1 ( PlxnA1 ) , and PlxnA4 transcripts in FACS - isolated GFP - positive GnRH neurons from control ( Nrp1loxP / loxP ; grey ) and mutant ( Gnrh : : cre ; Nrp1loxP / loxP ; red ) P0 mice . Unpaired t - test , n = 5 to 6 mice per group from 3 litters .

(D) Relative mRNA expression from real-time PCR analysis of Neuropilin-1 (Nrp1), Nrp2, Plexin A1 (PlxnA1), and PlxnA4 transcripts in FACS-isolated GFP-positive GnRH neurons from control (Nrp1loxP/loxP; grey) and mutant (Gnrh::cre; Nrp1loxP/loxP; red) P0 mice. Unpaired t-test, n=5 to 6 mice per group from 3 litters.


( E ) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1 - immunoreactive vomeronasal / terminal nerve fibers ( empty arrowheads ) at the nose - brain junction in sagittal slices from E14 . 5 control and mutant embryos . GnRH neurons ( green ) themselves express Nrp1 ( red , white arrows ) in control Nrp1loxP / loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh : : cre ; Nrp1loxP / loxP littermates ( black arrows ) . Scale bar : 50μm .

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(E) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose-brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50μm.


( A ) Quantification of total number of GnRH neurons during embryogenesis , at E12 . 5 , E14 . 5 and E18 . 5 . More GnRH neurons were found in E14 . 5 and E18 . 5 mutant embryos . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 4 to 6 mouse embryos from at least 3 litters .

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(A) Quantification of total number of GnRH neurons during embryogenesis, at E12.5, E14.5 and E18.5. More GnRH neurons were found in E14.5 and E18.5 mutant embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 to 6 mouse embryos from at least 3 litters.


( B ) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14 . 5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose , the olfactory bulb ( OB ) and the ventral forebrain ( VFB ) in control and mutant embryos . Scale bar : 50μm . VNO , vomeronasal organ ; OE , olfactory epithelium ; OB , olfactory bulbs ; VFB , ventral forebrain .

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(B) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14.5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose, the olfactory bulb (OB) and the ventral forebrain (VFB) in control and mutant embryos. Scale bar: 50μm. VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulbs; VFB, ventral forebrain.


( C ) Quantitative analysis showing distribution of GnRH neurons in the nose , OB and VFB of mutant and control embryos . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 5 and 6 mouse embryos for mutants and controls , respectively , from at least 3 litters .

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(C) Quantitative analysis showing distribution of GnRH neurons in the nose, OB and VFB of mutant and control embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=5 and 6 mouse embryos for mutants and controls, respectively, from at least 3 litters.


( D ) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs , in a control ( left panel ) and a mutant ( right panel ) postnatal brain . Knock - out animals show an accumulation of GnRH neurons in the accessory olfactory bulb ( circle formed by the GnRH labelling , right panel ) . AOB , accessory olfactory bulb . Scale bar : 300μm .

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(D) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs, in a control (left panel) and a mutant (right panel) postnatal brain. Knock-out animals show an accumulation of GnRH neurons in the accessory olfactory bulb (circle formed by the GnRH labelling, right panel). AOB, accessory olfactory bulb. Scale bar: 300μm.


( E ) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis ( OVLT ) in the median preoptic area , from a control ( left ) and a mutant ( right ) female postnatal brain . Scale bar : 500μm . Oc , optic chiasma .

(E) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis (OVLT) in the median preoptic area, from a control (left) and a mutant (right) female postnatal brain. Scale bar: 500μm. Oc, optic chiasma.


( F ) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates . OB , olfactory bulb ; DBB , diagonal band of Broca ; 3V , periventricular region of the preoptic area ; Tot , total . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 3 adult mice from 3 litters .

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(F) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates. OB, olfactory bulb; DBB, diagonal band of Broca; 3V, periventricular region of the preoptic area; Tot, total. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 adult mice from 3 litters.


( A ) Schematic representation of the transwell assay used to assess the effect of Sema3A , which was placed in the lower chamber , on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst ( grey ) in the lower part of the transwell membrane after overnight incubation in SFM ( left panel ) , SFM with 100ng / ml Sema3A ( middle panel ) and SFM with 100ng / ml Sema3A + 1μg Nrp1Ab ( right panel ) . Scale bar : 100μm .

(A) Schematic representation of the transwell assay used to assess the effect of Sema3A, which was placed in the lower chamber, on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst (grey) in the lower part of the transwell membrane after overnight incubation in SFM (left panel), SFM with 100ng/ml Sema3A (middle panel) and SFM with 100ng/ml Sema3A + 1μg Nrp1Ab (right panel). Scale bar: 100μm.


( B ) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber ( SFM ) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber . Kruskal - Wallis test , n = 5 to 6 wells in two independent experiments .

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(B) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber (SFM) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber. Kruskal-Wallis test, n= 5 to 6 wells in two independent experiments.


( C ) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11 . 5 mouse embryos after 8 days in vitro . NE , nasal epithelium ; NMC , nasal midline cartilage . Scale bar 500μm ( 150μm in inset ) .

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(C) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11.5 mouse embryos after 8 days in vitro. NE, nasal epithelium; NMC, nasal midline cartilage. Scale bar 500μm (150μm in inset).


( D ) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos , quantified after 8 days in culture . Mann - Whitney U test , n = 3 to 5 embryos from at least 3 litters .

(D) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos, quantified after 8 days in culture. Mann-Whitney U test, n=3 to 5 embryos from at least 3 litters.


( E ) Distance travelled by GnRH neurons from the olfactory epithelia ; Cells were quantified within a radius of 1 to 6 mm . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 3 to 5 explants .

(E) Distance travelled by GnRH neurons from the olfactory epithelia; Cells were quantified within a radius of 1 to 6 mm. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 to 5 explants.


( F ) Representative immunofluorescence for GnRH - ( red ) and BrdU - ( green ) positive cells in control and mutant E14 . 5 embryos arising from progenitors born between E9 . 5 and E10 . 5 . BrdU - positive GnRH neurons ( white arrowheads ) were more abundant in mutant than in control embryos . Scale bar : 20μm .

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(F) Representative immunofluorescence for GnRH- (red) and BrdU- (green) positive cells in control and mutant E14.5 embryos arising from progenitors born between E9.5 and E10.5. BrdU-positive GnRH neurons (white arrowheads) were more abundant in mutant than in control embryos. Scale bar: 20μm.


( G ) Number of BrdU - positive GnRH neurons in the nose , olfactory bulb ( OB ) and ventral forebrain ( VFB ) at E14 . 5 and total number of cells in control and mutant embryos . Double - labeled cells were more numerous in the mutant VFB . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 4 embryos from 2 litters .

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(G) Number of BrdU-positive GnRH neurons in the nose, olfactory bulb (OB) and ventral forebrain (VFB) at E14.5 and total number of cells in control and mutant embryos. Double-labeled cells were more numerous in the mutant VFB. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 embryos from 2 litters.


( I ) Number of GnRH neurons in E14 . 5 embryos injected with neutralizing antibodies to Neuropilin - 1 ( Nrp1Ab ) compared to an isotype goat antibody , or the antiapoptotic compound ZVAD compared to its vehicle , together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age . Note that the data are represented as fold change , as raw values are not comparable with transgenic animals that did not undergo surgery . Unpaired t - test , n = 5 to 7 embryos from at least 2 litters .

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(I) Number of GnRH neurons in E14.5 embryos injected with neutralizing antibodies to Neuropilin-1 (Nrp1Ab) compared to an isotype goat antibody, or the antiapoptotic compound ZVAD compared to its vehicle, together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age. Note that the data are represented as fold change, as raw values are not comparable with transgenic animals that did not undergo surgery. Unpaired t-test, n=5 to 7 embryos from at least 2 litters.


( A ) Olfactory sex preference test in P20 ( left panel ) and adult ( right panel ) control and mutant female mice . Half of the mutant females at P20 showed a preference for males , similar to adults of both genotypes , whereas the other half showed an even more pronounced preference for females than their control littermates at P20 . One - way ANOVA , Fisher ' s LSD multiple - comparison test for P20 mice and unpaired t - test for adult mice ; n = 4 to 6 from at least 3 litters .

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(A) Olfactory sex preference test in P20 (left panel) and adult (right panel) control and mutant female mice. Half of the mutant females at P20 showed a preference for males, similar to adults of both genotypes, whereas the other half showed an even more pronounced preference for females than their control littermates at P20. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.


( B ) Time spent sniffing after the introduction of both male and female odors into the cage of control and mutant littermates . One - way ANOVA , Fisher ' s LSD multiple - comparison test for P20 mice and unpaired t - test for adult mice ; n = 4 to 6 from at least 3 litters .

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(B) Time spent sniffing after the introduction of both male and female odors into the cage of control and mutant littermates. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.


( C ) Representative traces of spontaneous firing in GnRH neurons from control ( left , black ) and mutant ( right , red ) animals at week 2 ( upper panels ) and week 4 ( lower panels ) ; lower traces show an enlargement of the upper trace .

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(C) Representative traces of spontaneous firing in GnRH neurons from control (left, black) and mutant (right, red) animals at week 2 (upper panels) and week 4 (lower panels); lower traces show an enlargement of the upper trace.


( D ) Average spontaneous firing frequency in GnRH neurons from control and mutant mice at weeks 2 , 3 and 4 . Individual males and females used for this analysis are represented by blue and grey dots , respectively . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 8 to 14 neurons from at least 3 animals from different litters . Note that individual neurons from male ( n = 3 to 8 ) and female mice ( n = 5 to 8 ) used for this analysis are represented by blue and grey dots , respectively .

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(D) Average spontaneous firing frequency in GnRH neurons from control and mutant mice at weeks 2, 3 and 4. Individual males and females used for this analysis are represented by blue and grey dots, respectively. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=8 to 14 neurons from at least 3 animals from different litters. Note that individual neurons from male (n=3 to 8) and female mice (n=5 to 8) used for this analysis are represented by blue and grey dots, respectively.


( E ) Quantification of evoked firing in GnRH neurons from control and mutant mice in response to current steps . Two - way repeated - measures ANOVA , n = 10 to 11 neurons from at least 3 mice from different litters .

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(E) Quantification of evoked firing in GnRH neurons from control and mutant mice in response to current steps. Two-way repeated-measures ANOVA, n=10 to 11 neurons from at least 3 mice from different litters.


( A ) Evolution of body weight in control and mutant female littermates from 6 to 100 days of age . Mutant females show a significantly higher body weight from P21 . The grey lines mark body weight at puberty onset . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 7 to 17 mice from at least 4 litters .

(A) Evolution of body weight in control and mutant female littermates from 6 to 100 days of age. Mutant females show a significantly higher body weight from P21. The grey lines mark body weight at puberty onset. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=7 to 17 mice from at least 4 litters.


( B ) Fat mass or volume of white adipose tissue ( WAT ) . Adult mutant females showed higher WAT volumes . Unpaired t - test , n = 8 mice .

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(B) Fat mass or volume of white adipose tissue (WAT). Adult mutant females showed higher WAT volumes. Unpaired t-test, n=8 mice.


( C ) Volume of lean tissue . Mann - Whitney U test , n = 8 mice .

(C) Volume of lean tissue. Mann-Whitney U test, n=8 mice.


( D ) Cumulative percentage of the age at vaginal opening , n = 13 mice .

(D) Cumulative percentage of the age at vaginal opening, n=13 mice.


( E ) Cumulative percentage of animals undergoing first ovulation as assessed by the occurrence of first estrus at different ages n = 13 mice .

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(E) Cumulative percentage of animals undergoing first ovulation as assessed by the occurrence of first estrus at different ages n=13 mice.


( F ) Bar graph showing body weight at P45 , which is the mean age at puberty in mutants . Unpaired t - test , n = 9 to 14 mice .

(F) Bar graph showing body weight at P45, which is the mean age at puberty in mutants. Unpaired t-test, n=9 to 14 mice.


( G ) Plasma LH levels at P45 in control and mutant mice . Mann - Whitney U test , n = 5 to 6 mice .

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(G) Plasma LH levels at P45 in control and mutant mice. Mann-Whitney U test, n=5 to 6 mice.


( H ) Mean uterine weight normalized to body weight ( mg / g BW ) . Unpaired t - test , n = 6 to 10 mice .

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(H) Mean uterine weight normalized to body weight (mg/g BW). Unpaired t-test, n=6 to 10 mice.


PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( B ) Immunohistochemistry for dystrophin expression in body - wide muscles from mdx mice treated with PMO in saline ( PMO - S ) , PMO - G ( IV ) or PMO - G ( D ) ( scale bar = 100μm ) . PMO - G ( IV ) refers to intravenous injection of PMO in combination with intravenous injection of 5 % glycine every other day for 3 weeks . PMO - G ( D ) represents intravenous injection of PMO supplemented with 1 % glycine orally .

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

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO in saline (PMO-S), PMO-G(IV) or PMO-G (D)(scale bar=100μm).PMO-G (IV) refers to intravenous injection of PMO in combination with intravenous injection of 5% glycine every other day for 3 weeks. PMO-G (D) represents intravenous injection of PMO supplemented with 1% glycine orally.


PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( C ) Western blot for dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( IV ) , PMO - G ( D ) or PMO - S . 0 . 5µg , 2 . 5 µg , 5µg , 10µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , D - diaphragm and A - abdominal muscle . ( D ) Quantitative analysis of dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( i . v . , n = 4 ) , PMO - G ( D25 and D50 , n = 3 ) and PMO - S ( n = 3 ) ( * P < 0 . 05 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (C) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (IV), PMO-G (D) or PMO-S. 0.5µg, 2.5 µg, 5µg, 10µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, D-diaphragm and A-abdominal muscle. (D) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (i.v., n=4), PMO-G (D25 and D50, n=3) and PMO-S (n=3) (*P<0.05,**P<0.001; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( A ) Muscle function was assessed to determine the physical improvement with grip strength test ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (A) Muscle function was assessed to determine the physical improvement with grip strength test (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( B ) Measurement of bodyweight changes of DKO mice treated with PMO - G or PMO - S ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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 B-CONTROLLED_VAR 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 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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (B) Measurement of bodyweight changes of DKO mice treated with PMO-G or PMO-S (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( C ) Measurement of TA muscle mass of DKO mice treated with PMO - G or PMO - S ( n = 3 , * P < 0 . 05 ; one - way ANOVA post - hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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 B-CONTROLLED_VAR 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 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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (C) Measurement of TA muscle mass of DKO mice treated with PMO-G or PMO-S (n = 3, *P < 0.05; one-way ANOVA post-hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( D ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) . TA - tibialis

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (D) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm). TA-tibialis anterior


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( E , F ) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , A - abdominal muscle and D - diaphragm . 2 . 5 µg , 5µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (E, F) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle and D-diaphragm. 2.5 µg, 5µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( B ) Measurement of Ejection Fraction ( EF ) and Fractional Shortening ( FS ) in treated DKO mouse hearts ( n = 3 ) and C57BL / 6 mice ( n = 6 ) with echocardiography ( * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (B) Measurement of Ejection Fraction (EF) and Fractional Shortening (FS) in treated DKO mouse hearts (n=3) and C57BL/6 mice (n=6) with echocardiography (*P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( C ) Morphological examination of treated DKO mouse hearts with H & E staining ( scale bar = 800μm ) . Boxed areas are magnified . ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (C) Morphological examination of treated DKO mouse hearts with H&E staining (scale bar=800μm). Boxed areas are magnified. (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test) Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . Immunohistochemistry for immunoglobin G ( IgG ) in treated DKO mouse hearts ( D , scale bar = 100 μm ) .

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 O O B-CONTROLLED_VAR 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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Immunohistochemistry for immunoglobin G (IgG) in treated DKO mouse hearts (D, scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . Muscle function was evaluated to determine the physical improvement with grip strength ( E ) or running wheel tests ( F ) for treated DKO ( n = 3 ) or C57BL / 6 mice ( n = 4 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). Muscle function was evaluated to determine the physical improvement with grip strength (E) or running wheel tests (F) for treated DKO (n=3) or C57BL/6 mice (n=4) (*P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( G ) Immunohistochemistry for dystrophin expression in body - wide muscles from treated DKO mice ( scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (G) Immunohistochemistry for dystrophin expression in body-wide muscles from treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) and metformin daily via drinking water ( PMO - GM ) . ( H ) Western blot and quantitative analysis for dystrophin protein in treated DKO mice ( n = 3 ) . 5µg , 10µg and 20µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM). (H) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3). 5µg, 10µg and 20µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Survival rate of DKO mice treated with PMO - GM ( n = 6 ) . The number of untreated DKO controls is 15 .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Survival rate of DKO mice treated with PMO-GM (n=6).The number of untreated DKO controls is 15.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Measurement of kyphosis index of DKO mice treated with PMO - GM ( scale bar = 5mm ) and wild - type C57BL / 6 mice ( scale bar = 8mm ) ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Measurement of kyphosis index of DKO mice treated with PMO-GM (scale bar=5mm) and wild-type C57BL/6 mice (scale bar=8mm)(n=3, *P<0.05; One way- ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of tidal volume in treated DKO mice with a cardio - respiratory gated technique ( n = 3 , * P < 0 . 05 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of tidal volume in treated DKO mice with a cardio-respiratory gated technique (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( E - F ) Examination of EF and FS ( E ) and stroke volume and cardiac output ( F ) in treated DKO mouse hearts ( n = 3 ) and C57BL / 6 ( n = 5 ) with echocardiography ( * P < 0 . 05 , * * P < 0 . 001 ; one - way ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (E-F) Examination of EF and FS (E) and stroke volume and cardiac output (F) in treated DKO mouse hearts (n=3) and C57BL/6(n=5) with echocardiography (*P<0.05, **P<0.001; one-way ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Masson trichrome ' s staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts ( scale bar = 800 μm ) . Boxed areas are magnified ( n = 3 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Masson trichrome's staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts (scale bar=800 μm). Boxed areas are magnified (n=3, **P<0.001;One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( H ) Measurement of serum creatine kinase - MB ( CK - MB ) levels in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (H) Measurement of serum creatine kinase-MB (CK-MB) levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( I - J ) Muscle function was assessed to determine the physical improvement with grip strength ( I ) or running wheel tests ( J ) in treated DKO ( n = 4 ) and C57BL / 6 mice ( n = 6 ) ( * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (I-J) Muscle function was assessed to determine the physical improvement with grip strength (I) or running wheel tests (J) in treated DKO (n=4) and C57BL/6 mice (n=6) (*P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( A ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (A) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm).


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( B ) Western blot and quantitative analysis for dystrophin protein in treated DKO mice ( n = 3 ) . 5µg , 10µg and 25µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (B) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3).5µg, 10µg and 25µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( C ) Re - localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture ( scale bar = 50μm ) . The arrowheads point to identical myofibers .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (C) Re-localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar=50μm). The arrowheads point to identical myofibers.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( D ) Measurement of serum CK levels in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Measurement of serum indices from treated DKO mice to reflect liver ( E ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (D) Measurement of serum CK levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Measurement of serum indices from treated DKO mice to reflect liver (E) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . Measurement of serum indices from treated DKO mice to reflect kidney ( F ) functions ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. Measurement of serum indices from treated DKO mice to reflect kidney (F) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction , followed by 50 mg / kg / month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction , in combination with metformin daily via drinking water . ( G ) Morphological examination of liver and kidney from treated DKO mice ( scale bar = 50 μm ) .

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

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water. (G) Morphological examination of liver and kidney from treated DKO mice (scale bar=50 μm).


e . Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue . As suggested by the consensus statement on the pathology of IgG4 - RD ( Deshpande et al , 2012 ) , three 40x fields with the highest number of IgG4 + and IgG + cells were selected , counted and averaged within these fields . Cell counts as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted and averaged within these fields. Cell counts as indicated.


f . C - X - C motif chemokine 13 ( CXCL13 ) concentrations measured by ELISA from inflammatory pseudotumor ( IPT ) CSF , control CSF , and MS patient - derived CSF . Individual values , mean ± SEM ; n = 4 experiment repeats with technical 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

f. C-X-C motif chemokine 13 (CXCL13) concentrations measured by ELISA from inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. Individual values, mean ± SEM; n=4 experiment repeats with technical replicates.


g . Stacked bar chart depicting mean relative expression levels of T helper cell - associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in ( a ) . TEM , T effector memory ; TCM , T central memory ; Treg , regulatory T cell .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Stacked bar chart depicting mean relative expression levels of T helper cell-associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in (a). TEM, T effector memory; TCM, T central memory; Treg, regulatory T cell.


h . Violin plot depicting relative expression levels of IL4R ( left ) and IL10RA ( right ) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF .

O O O O O O O O O O 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. Violin plot depicting relative expression levels of IL4R (left) and IL10RA (right) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF.


a . Relative abundances of cell types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS patient - derived CSF . Boxplot depicting 25th - 75th percentiles with median shown as central band and whiskers extending from minimum to maximum values .

a. Relative abundances of cell types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS patient-derived CSF. Boxplot depicting 25th-75th percentiles with median shown as central band and whiskers extending from minimum to maximum values.


A . Confocal images of live control ( top ) and U18 - treated ( bottom ) tsA201 cells expressing P4M - YFP . Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo / lysosomal compartments , respectively . B . Quantification of perinuclear P4M - YFP mean fluorescence intensity normalized to the cytoplasm . Control : n = 19 ; U18 : n = 16 .

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

A. Confocal images of live control (top) and U18-treated (bottom) tsA201 cells expressing P4M-YFP. Black and orange rectangles show expanded views of PtdIns4P in perinuclear and endo/lysosomal compartments, respectively. B. Quantification of perinuclear P4M-YFP mean fluorescence intensity normalized to the cytoplasm. Control: n=19; U18: n=16.


K . Heat map of PtdIns , PtdInsP and PtdInsP2 levels from 4 different murine brain regions . The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild - type brains ; n = 3 for both WT and NPC1I1061T animals .

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

K. Heat map of PtdIns, PtdInsP and PtdInsP2 levels from 4 different murine brain regions. The colors from green to red indicate the relative contents of lipid isoforms in different brain regions from NPC1I1061T mice relative to wild-type brains; n=3 for both WT and NPC1I1061T animals.


C . Confocal images of control and U18 - treated tsA201 cells fixed and stained for OSBP ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of OSBP and TGN46 regions . D . Quantification of OSBP signal in TGN46 regions ( OSBPTGN46 ) . Control : n = 40 ; U18 : n = 39 .

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 B-CONTROLLED_VAR O O O O O

C. Confocal images of control and U18-treated tsA201 cells fixed and stained for OSBP (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of OSBP and TGN46 regions. D. Quantification of OSBP signal in TGN46 regions (OSBPTGN46). Control: n=40; U18: n=39.


J . Confocal images of control and U18 - treated GFP11 - SAC1 HEK293 cells ( top ) and control and NPC1I1061T patient fibroblasts immunolabeled with anti - SAC1 antibody ( bottom ) . Insets show an enlarged view of SAC1 centered on a perinuclear region . K . Quantification analyses of GFP11 - SAC1 ( top ; Control : n = 10 ; U18 : n = 10 ) and anti - SAC1 ( bottom ; Control : n = 7 ; NPC1I1061T : n = 8 ) mean fluorescence intensities in Golgi normalized to the cytoplasm .

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 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

J. Confocal images of control and U18-treated GFP11-SAC1 HEK293 cells (top) and control and NPC1I1061T patient fibroblasts immunolabeled with anti-SAC1 antibody (bottom). Insets show an enlarged view of SAC1 centered on a perinuclear region. K. Quantification analyses of GFP11-SAC1 (top; Control: n=10; U18: n=10) and anti-SAC1 (bottom; Control: n=7; NPC1I1061T: n=8) mean fluorescence intensities in Golgi normalized to the cytoplasm.


C . Left : Confocal images of control ( left ) and U18 - treated ( right ) tsA201 cells expressing PI4KIIα - GFP . Bottom panels show an enlarged view of the probe centered on a perinuclear region . Right : Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm . Control : n = 12 ; U18 : n = 14 . D . Same as ( C ) only PI4KIIIβ . Control : n = 12 ; U18 : n = 15 .

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

C. Left: Confocal images of control (left) and U18-treated (right) tsA201 cells expressing PI4KIIα -GFP. Bottom panels show an enlarged view of the probe centered on a perinuclear region. Right: Quantification of perinuclear PI4KIIα intensity relative to the cytoplasm. Control: n=12; U18: n=14. D. Same as (C) only PI4KIIIβ. Control: n=12; U18: n=15.


E . Confocal images of control and U18 - treated tsA201 cells fixed and stained for PI4KIIα ( left ) , TGN46 ( middle ) , and merge ( right ) . Black rectangles show expanded views of PI4KIIα and TGN46 regions . F . Quantification of PI4KIIα signal in TGN46 regions ( PI4KIIα TGN46 ) . Control : n = 36 ; U18 : n = 36 .

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 B-CONTROLLED_VAR O O O O O

E. Confocal images of control and U18-treated tsA201 cells fixed and stained for PI4KIIα (left), TGN46 (middle), and merge (right). Black rectangles show expanded views of PI4KIIα and TGN46 regions. F. Quantification of PI4KIIα signal in TGN46 regions (PI4KIIα TGN46). Control: n=36; U18: n=36.


B , Confocal images of wild - type and PI4KIIα - depleted ( sgPI4K2A ) HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in a Golgi region . C , Quantitative analyses of P4M - YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions . Control : n = 19 ; U18 : n = 13 , sgPI4K2A : n = 19 ; sgPI4K2A + U18 : n = 17 ; sgDHHC3 : n = 11 ; sgDHHC3 + U18 : n = 11 . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups .

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

B, Confocal images of wild-type and PI4KIIα-depleted (sgPI4K2A) HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in a Golgi region. C, Quantitative analyses of P4M-YFP mean fluorescence intensity distributed in Golgi normalized to the cytoplasm under different conditions. Control: n=19; U18: n=13, sgPI4K2A: n=19; sgPI4K2A+U18: n=17; sgDHHC3: n=11; sgDHHC3+U18: n=11. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups.


H . Control or GFP - DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ . Bottom panels show enlarged views of DHHC3 , PI4KIIα , or PI4KIIIβ at Golgi regions . I . Quantification of PI4KIIα ( left ; control : n = 42 , DHHC3 : n = 24 ) , or PI4KIIIβ ( right ; control : n = 39 , DHHC3 : n = 26 ) intensity relative to cytoplasm .

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H. Control or GFP-DHHC3 expressing tsA201 cells fixed and stained for PI4KIIα or PI4KIIIβ. Bottom panels show enlarged views of DHHC3, PI4KIIα, or PI4KIIIβ at Golgi regions. I. Quantification of PI4KIIα (left; control: n=42, DHHC3: n=24), or PI4KIIIβ (right; control: n=39, DHHC3: n=26) intensity relative to cytoplasm.


M . Confocal images of control ( top row ) , NPC1I1061T ( middle row ) , and NPC1I1061T treated with sgDHHC3 ( bottom row ) patient cells expressing P4M , LAMP1 and CAS9 . Insets are zoomed regions from LAMP1 positive areas . N . Quantification of P4M changes at endomembranes . Control : n = 149 , U18 : n = 251 , sgPI4KIIa + U18 : n = 131 , sgDHHC3 + U18 : n = 271 . Each n represents a single enodolysosome .

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

M. Confocal images of control (top row), NPC1I1061T (middle row), and NPC1I1061T treated with sgDHHC3 (bottom row) patient cells expressing P4M, LAMP1 and CAS9. Insets are zoomed regions from LAMP1 positive areas. N. Quantification of P4M changes at endomembranes. Control: n=149, U18: n=251, sgPI4KIIa +U18: n=131, sgDHHC3+U18: n= 271. Each n represents a single enodolysosome.


C . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3 . Black and orange squares show expanded views of anti - ACBD3 in Golgi and endomembrane compartments , respectively . D . Analyses of ACBD3 intensity distributed in Golgi ( left graph ; Control : n = 9 ; NPC1I1061T : n = 9 ) or in endomembranes ( right graph ; Control : n = 1360 ; NPC1I1061T : n = 1645 from 15 cells ) , normalized to the cytoplasm .

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C. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for ACBD3. Black and orange squares show expanded views of anti-ACBD3 in Golgi and endomembrane compartments, respectively. D. Analyses of ACBD3 intensity distributed in Golgi (left graph; Control: n=9; NPC1I1061T: n=9) or in endomembranes (right graph; Control: n=1360; NPC1I1061T: n=1645 from 15 cells), normalized to the cytoplasm.


I . Confocal images of control and sgACBD3 HEK293 - CAS9 cells expressing P4M - YFP , treated with or without U18 . Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments . J . Quantitative analyses of P4M - YFP mean fluorescence intensity at Golgi ( left graph ) or endomembranes ( right graph ) normalized to the cytoplasm . * represents a significant difference ( P < 0 . 05 ) between control and other non - U18 treated groups ; # represents a significant difference ( P < 0 . 05 ) between U18 - treated and other U18 - treated sgRNA groups . For left graph ; Control : n = 19 ; U18 : n = 13 ; sgPI4K3B : n = 12 ; sgPI4K3B + U18 : n = 11 ; sgPI4K2A + sgPI4K3B : n = 12 ; sgPI4K2A + sgPI4K3B + U18 : n = 12 ; sgACBD3 : n = 15 ; sgACBD3 + U18 : n = 16 . For right graph ; control : n = 149 ; U18 : n = 251 ; sgACBD3 : n = 138 ; sgACBD3 + U18 : n = 190 . Please note that ' control ' and ' U18 ' datasets in Fig 5J are identical to those in Fig . 4C . Data for all CRISPR experiments were collected on the same days .

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

I. Confocal images of control and sgACBD3 HEK293-CAS9 cells expressing P4M-YFP, treated with or without U18. Bottom panels show enlarged views of PtdIns4P in Golgi and in endomembrane compartments. J. Quantitative analyses of P4M-YFP mean fluorescence intensity at Golgi (left graph) or endomembranes (right graph) normalized to the cytoplasm. * represents a significant difference (P<0.05) between control and other non-U18 treated groups; # represents a significant difference (P<0.05) between U18-treated and other U18-treated sgRNA groups. For left graph; Control: n=19; U18: n=13; sgPI4K3B: n=12; sgPI4K3B+U18 : n=11; sgPI4K2A + sgPI4K3B: n=12; sgPI4K2A + sgPI4K3B +U18: n= 12; sgACBD3: n=15; sgACBD3+U18: n=16. For right graph; control: n=149; U18: n=251; sgACBD3: n=138; sgACBD3+U18: n=190. Please note that 'control' and 'U18' datasets in Fig 5J are identical to those in Fig. 4C. Data for all CRISPR experiments were collected on the same days.


A . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts expressing P4M - YFP . Black and orange squares show expanded views of PtdIns4P in Golgi and endo / lysosomal compartments respectively .

O O O O 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

A. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts expressing P4M-YFP. Black and orange squares show expanded views of PtdIns4P in Golgi and endo/lysosomal compartments respectively.


C . Confocal images of control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody . Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images . White dashed lines represent the outline of each cell . D . Quantification of mTORC1 mean integrated density normalized bycell area in control , NPC1I1061T and ACBD3 - deleted NPC1I1061T patient fibroblasts . Control : n = 15 ; NPC1I1061T : n = 14 ; NPC1I1061T + sgACBD3 : n = 11 .

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C. Confocal images of control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts immunolabeled with mTORC1 antibody. Bottom images show expanded views of mTORC1 immunolabeling in the regions indicated by the dashed squares in the main images. White dashed lines represent the outline of each cell. D. Quantification of mTORC1 mean integrated density normalized bycell area in control, NPC1I1061T and ACBD3-deleted NPC1I1061T patient fibroblasts. Control: n=15; NPC1I1061T: n=14; NPC1I1061T+sgACBD3: n=11.


B . Confocal images of tsA201 cells expressing PI4KIIα - GFP . Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . C . Quantification of PI4KIIα - GFP mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . D , E . Same as ( B , C ) but in tsA - 201 cells transfected with PI4KIIIβ - GFP . Control : n = 18 , U18 : n = 22 , U18 + PF : n = 23 .

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

B. Confocal images of tsA201 cells expressing PI4KIIα-GFP. Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. C. Quantification of PI4KIIα-GFP mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. D, E. Same as (B, C) but in tsA-201 cells transfected with PI4KIIIβ-GFP. Control: n=18, U18: n=22, U18+PF: n=23.


Confocal images of tsA201 cells Cells were treated with either vehicle ( control ) , U18 , or U18 and PF for 24h prior imaging . Bottom panels show an enlarged view of the enzyme in a perinuclear area . Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm . Control : n = 12 ; U18 : n = 14 ; U18 + PF : n = 12 . F , G . Same as ( B , C ) but in tsA - 201 cells transfected with P4M - YFP . Control : n = 39 , U18 : n = 42 , U18 + PF : n = 32 . H , I . Same as ( F , G ) but in wild - type and SCAP - / - CHO cells . Control : n = 12 , U18 : n = 11 , SCAP - / - + U18 : n = 10 .

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

Confocal images of tsA201 cells Cells were treated with either vehicle (control), U18, or U18 and PF for 24h prior imaging. Bottom panels show an enlarged view of the enzyme in a perinuclear area. Quantification of mean fluorescence intensity in Golgi normalized to the cytoplasm. Control: n=12; U18: n=14; U18+PF: n=12. F, G. Same as (B, C) but in tsA-201 cells transfected with P4M-YFP. Control: n=39, U18: n=42, U18+PF: n=32. H, I. Same as (F, G) but in wild-type and SCAP-/- CHO cells. Control: n=12, U18: n=11, SCAP-/- +U18: n=10.


A . Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI - M6PR . Insets show expanded views of each immunolabeling in the Golgi region ( upper insets ) or centered on endomembranes ( bottom inset , merged signals ) . Dashed grey and white lines represent the outline of each cell . B . Analysis of the mean area of M6P and Giantin colocalizing puncta ( top graph ; Control : n = 23 ; NPC1I1061T : n = 18 ) and of their mean fluorescence intensity ( bottom graph ; Control : n = 18 ; NPC1I1061T : n = 18 ) in control and NPC1I1061T patient fibroblasts .

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A. Confocal images of control and NPC1I1061T patient fibroblasts immunolabeled for Giantin and CI-M6PR. Insets show expanded views of each immunolabeling in the Golgi region (upper insets) or centered on endomembranes (bottom inset, merged signals). Dashed grey and white lines represent the outline of each cell. B. Analysis of the mean area of M6P and Giantin colocalizing puncta (top graph; Control: n=23; NPC1I1061T: n=18) and of their mean fluorescence intensity (bottom graph; Control: n=18; NPC1I1061T: n=18) in control and NPC1I1061T patient fibroblasts.


C . Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1 - GFP . Insets show enlarged views of ARF1 in the Golgi area . Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows . D . Quantification of ΑRF - 1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts . Control : n = 10 ; NPC1I1061T : n = 11 .

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C. Confocal images of control and NPC1I1061T patient fibroblasts expressing ARF1-GFP. Insets show enlarged views of ARF1 in the Golgi area. Tubular structures were observed in NPC1I1061T patient fibroblasts as indicated by the black arrows. D. Quantification of ΑRF-1 fluorescence intensity in Golgi normalized to the cytoplasm in control and NPC1I1061T patient fibroblasts. Control: n=10; NPC1I1061T: n=11.


B . Results of the sequential digestion workflow applied to a synthetic 7 - protein mix , compared to using trypsin alone in four replicates and parallel digestion with trypsin , AspN , chymotrypsin and GluC . A trypsin four replicate experiment shows a large overlap of the four datasets with little gain . Parallel digestions with trypsin , AspN , chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin . Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs . C . Gains of repeated analysis ( trypsin only ) , parallel digestion and sequential digestion for the same data as shown in panel B .

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B. Results of the sequential digestion workflow applied to a synthetic 7-protein mix, compared to using trypsin alone in four replicates and parallel digestion with trypsin, AspN, chymotrypsin and GluC. A trypsin four replicate experiment shows a large overlap of the four datasets with little gain. Parallel digestions with trypsin, AspN, chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin. Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs. C. Gains of repeated analysis (trypsin only), parallel digestion and sequential digestion for the same data as shown in panel B.


D . Crosslinked peptides obtained by sequential digestion of a synthetic 7 - protein mix are smaller than their corresponding tryptic peptides .

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D. Crosslinked peptides obtained by sequential digestion of a synthetic 7-protein mix are smaller than their corresponding tryptic peptides.


B . The in vitro helicase loading assay demonstrates that an Mcm2 C - terminal deletion mutant supports complex assembly ( lanes 6 and 7 ) and blocks formation of the final helicase loading product ( lanes 8 and 9 ) .

O O O O O O O O O O O O O O O O O O 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. The in vitro helicase loading assay demonstrates that an Mcm2 C-terminal deletion mutant supports complex assembly (lanes 6 and 7) and blocks formation of the final helicase loading product (lanes 8 and 9).


C . Overexpression analysis of Mcm2 - 7ΔC2 shows that this mutant causes dominant lethality , indicating that the C - terminus of Mcm2 is essential in cell survival .

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C. Overexpression analysis of Mcm2-7ΔC2 shows that this mutant causes dominant lethality, indicating that the C-terminus of Mcm2 is essential in cell survival.


Plot showing the average Z - scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one - sample t - test of whether average Z - score ≠ 0 . Green circles represent significantly upregulated genes ( Z > 0 and P < 0 . 05 ) . Magenta circles represent significantly downregulated genes ( Z < 0 and P < 0 . 05 ) . Grey circles represent genes whose expression is not significantly changed ( P > 0 . 05 ) . All comparisons were between cancers and normal cells from the same tissue . Dashed line shows position on Y - axis that corresponds to P = 0 . 05 . Positions of MCOLN1 ( red circle ) , VAC14 ( blue circle ) , and MTM1 ( yellow circle ) are indicated . Inset , schematic showing that Mtm1 and Vac14 regulate the levels of PI ( 3 , 5 ) P2 , and thereby , influence TRPML1 activity .

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

Plot showing the average Z-scores of 210 endolysosomal genes plotted against the negative log10 of the P values from the one-sample t-test of whether average Z-score ≠ 0. Green circles represent significantly upregulated genes (Z > 0 and P < 0.05). Magenta circles represent significantly downregulated genes (Z < 0 and P < 0.05). Grey circles represent genes whose expression is not significantly changed (P > 0.05). All comparisons were between cancers and normal cells from the same tissue. Dashed line shows position on Y-axis that corresponds to P = 0.05. Positions of MCOLN1 (red circle), VAC14 (blue circle), and MTM1 (yellow circle) are indicated. Inset, schematic showing that Mtm1 and Vac14 regulate the levels of PI(3,5)P2, and thereby, influence TRPML1 activity.


Unsupervised hierarchical clustering of Pearson ' s coefficients of pairwise correlation of gene expression reveal 4 indicated clusters . Violin plots of average Z - scores of genes that belong to the four clusters derived from hierarchical clustering . The thick horizontal dashed lines in each sample set represent median ; thin dashed lines represent the quartiles ; polygons represent density estimates of data and extend to extreme values . Statistical tests employed were 1 - sample t - test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Unsupervised hierarchical clustering of Pearson's coefficients of pairwise correlation of gene expression reveal 4 indicated clusters. Violin plots of average Z-scores of genes that belong to the four clusters derived from hierarchical clustering. The thick horizontal dashed lines in each sample set represent median; thin dashed lines represent the quartiles; polygons represent density estimates of data and extend to extreme values. Statistical tests employed were 1-sample t-test or Wilcoxon signed rank test to examine significance of differences from a hypothetical value of 0.


GSEA conducted on endolysosomal genes ranked on the basis of increasing Z - scores . Positions of the CLEAR target genes are indicated .

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GSEA conducted on endolysosomal genes ranked on the basis of increasing Z-scores. Positions of the CLEAR target genes are indicated.