( A ) Representative show hippocampal vGlut2 Homer1 immunostaining . box outlines region of interest yellow box shows regions where confocal were taken .

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(A) Representative images show hippocampal vGlut2 and Homer1 immunostaining. White box outlines the region of interest and yellow box shows the regions where confocal images were taken.


( B ) Confocal of synaptic immunostaining in CA1 striatum lacunosum - moleculare ( str . lac / mol ) at P10 . Arrows pointing to enlarged synapse in insets . ( C , D ) of synapse density in CA1 str . lac / mol in iCKO versus control at P10 ( C ) , CKO versus control at P21 ( D ) . At P10 , N ( Ctrl ) = 4 , N ( iCKO ) = 3 , p = 0 . 02 . At P21 , N = 8 , p = 0 . 005 .

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(B) Confocal images of synaptic immunostaining in CA1 striatum lacunosum-moleculare (str. lac/mol) at P10. Arrows pointing to the enlarged synapse in the insets. (C, D) Quantification of synapse density in CA1 str. lac/mol in iCKO versus control at P10 (C), and CKO versus control at P21 (D). At P10, N(Ctrl) = 4, N(iCKO) = 3, p = 0.02. At P21, N = 8, p = 0.005.


( E ) Representative show hippocampal vGlut1 Homer1 immunostaining .

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(E) Representative images show hippocampal vGlut1 and Homer1 immunostaining.


( F ) Confocal of synaptic immunostaining in CA1 striatum radiatum at P10 . ( G , H ) of synapse density in CA1 striatum radiatum in iCKO versus control at P10 ( G ) , CKO versus control at P21 ( H ) . At P10 , N = 5 , p = 0 . 83 . At P21 , N = 5 , p = 0 . 89 .

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(F) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10. (G, H) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 (G), and CKO versus control at P21 (H). At P10, N = 5, p = 0.83. At P21, N = 5, p = 0.89.


( B ) CTB - labeled dLGN shows reduced eye - segregation at P30 in CKO mice . left column shows dLGN labeled with CTB488 ( green ) , middle one is dLGN with CTB647 ( magenta ) . right column represents dLGN pseudocolored according to R - value for each pixel ( R = log ( Fipsi / Fcontra ) ) . ( C ) A histogram distribution chart of R - value for pixels within dLGN represents degree of eye - specific segregation . When R is 0 , it means equal at a pixel . A greater R - value means a bigger difference of ipsi - to - contraleteral . narrower distribution of CKO in inset indicates reduced segregation . ( D ) variance of R distributions in P30 control CKO mice . N = 4 ( Ctrl ) , N = 6 ( CKO ) , p = 0 . 004 , mean ± SD .

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(B) CTB-labeled dLGN shows reduced eye-segregation at P30 in CKO mice. The left column shows contralateral dLGN labeled with CTB488 (green), and the middle one is ipsilateral dLGN with CTB647 (magenta). The right column represents the dLGN pseudocolored according to the R-value for each pixel (R = log(Fipsi/Fcontra)). (C) A histogram distribution chart of R-value for all pixels within dLGN represents the degree of eye-specific segregation. When R is 0, it means equal ipsilateral and contralateral fluorescence intensity at a pixel. A greater R-value means a bigger difference of ipsi-to-contraleteral fluorescence intensity. The narrower distribution of CKO in the inset indicates reduced segregation. (D) The variance of R distributions in P30 control and CKO mice. N = 4 (Ctrl), N = 6 (CKO), p = 0.004, mean ± SD.


( F ) Maximal NMDAR mediated currents measured in dLGN of P28 ~ P34 mice , p = 0 . 03 , N = 14 ( Ctrl ) , 23 ( CKO ) from 5 , 7 mice , respectively . Mean ± .

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(F) Maximal NMDAR mediated currents measured in the dLGN of P28~P34 mice, p = 0.03, N = 14 (Ctrl), 23 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.


( G ) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice . ( H ) of NMDAR1 expression in CKO mice control .

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(G) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice. (H) Quantification of NMDAR1 expression in CKO mice and control.


( I ) Maximal AMPAR mediated currents measured in dLGN of P28 ~ P34 mice . P = 0 . 302 , N = 13 ( Ctrl ) , 17 ( CKO ) from 5 , 7 mice , respectively . Mean ± .

(I) Maximal AMPAR mediated currents measured in the dLGN of P28~P34 mice. P=0.302, N = 13 (Ctrl), 17 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.


( J ) Western blot of GluR1 with microdissected P30 dLGN tissue . ( K ) of GluR1 expression in CKO mice controls .

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(J) Western blot of GluR1 with microdissected P30 dLGN tissue. (K) Quantification of GluR1 expression in CKO mice and controls.


( A ) Colocalized RGC inputs PSVue signals in P6 control CKO . circles indicate PS + RGC inputs . Arrows pointing to RGC inputs enlarged in insets . ( B ) of PS + RGC inputs in total inputs in CKO controls .

(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls.


( C ) Representative of engulfed PS + PS - RGC inputs by microglia in control CKO . ( D ) of engulfed PS + RGC inputs by microglia , which is calculated as : Volume of engulfed PS + RGC inputs / Volume of microglia . ( E ) of engulfed PS - RGC inputs in control CKO .

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(C) Representative images of engulfed PS+ and PS- RGC inputs by microglia in control and CKO. (D) Quantification of engulfed PS+ RGC inputs by microglia, which is calculated as: Volume of engulfed PS+ RGC inputs / Volume of microglia cell. (E) Quantification of engulfed PS- RGC inputs in control and CKO.


A HeLa were transfected with constructs for a non - targeting shRNA ( NT ) , USP17 shRNA1 , or USP17 shRNA2 . were stained using an anti - LAMP1 antibody ( green ) nuclei counterstained with DAPI ( blue ) . Lower panels are enlarged of indicated in top panels is marked by dotted . B HeLa MDA - MB - 231 were transfected as in A distribution of LAMP1 positive vesicles was plotted as vesicle relative position ( see C ) .

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A HeLa cells were transfected with constructs for a non-targeting shRNA (NT), USP17 shRNA1, or USP17 shRNA2. Cells were stained using an anti-LAMP1 antibody (green) and the nuclei counterstained with DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. B HeLa and MDA-MB-231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position (see C). C


D HeLa transfected as in A , in conjunction with a construct coding GFP tagged CD63 . lower panels are enlarged of indicated in top panels is marked by dotted . E HeLa transfected as in A , in conjunction with constructs for either GFP tagged LAMP1 or CD63 . distribution of GFP positive vesicles plotted as vesicle relative position .

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D HeLa cells transfected as in A, in conjunction with a construct coding GFP tagged CD63. The lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. E HeLa cells transfected as in A, in conjunction with constructs for either GFP tagged LAMP1 or CD63. The distribution of GFP positive vesicles plotted as vesicle relative position. F


A HeLa were transfected with constructs for a non - targeting ( NT ) shRNA , USP17 shRNA1 or USP17 shRNA2 . 48 hours post transfection were either serum starved ( upper panels ) , or treated with serum free medium containing 100ug / ml EGF ( lower panels ) for 16 hours prior to for LAMP1 ( green ) DAPI ( blue ) . Right panels are enlarged of indicated in left panels is marked by dotted . Scale bar represents 25μm . B distribution of at least 300 LAMP1 positive vesicles from a number of ( n ) from a series of confocal across three separate experiments was plotted as vesicle relative position ( mean value is red bar ) . Error bars represent standard error , ns indicates not significant , * * * * indicates a p - values less than 0 . 0001 . One way ANOVA used to determine if statistically significant differences between groups .

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A HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 48 hours post transfection the cells were either serum starved (upper panels), or treated with serum free medium containing 100ug/ml EGF (lower panels) for 16 hours prior to staining for LAMP1 (green) and DAPI (blue). Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line. Scale bar represents 25μm. B The distribution of at least 300 LAMP1 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicates not significant, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.


A 48 hours post transfection were washed with Ca2 + Mg2 + free ice cold HBSS . different populations were split into three aliquots . One aliquot was placed into fresh medium ( Left panels ) . SLO was added to other two aliquots then toxin - containing medium was removed aliquots were either placed into Ca2 + Mg2 + free HBSS ( Middle panels ) , or EBSS containing Ca2 + Mg2 + ( Right panels ) prior to with propidium iodide by flow cytometry .

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A 48 hours post transfection the cells were washed with Ca2+ and Mg2+ free ice cold HBSS. The different cell populations were split into three aliquots. One aliquot was placed into fresh medium (Left panels). SLO was added to other two aliquots and then the toxin-containing medium was removed and the aliquots were either placed into Ca2+ and Mg2+ free HBSS (Middle panels), or EBSS containing Ca2+ and Mg2+ (Right panels) prior to staining with propidium iodide and analysis by flow cytometry.


A HeLa were transfected with negative control siRNA RNF26 siRNA as well as constructs coding a non - targeting ( NT ) shRNA , USP17 shRNA1 or USP17 shRNA2 , as indicated . 48 hours post transfection were stained for LAMP - 1 ( green ) DAPI ( blue ) . Lower panels are enlarged of indicated in top panels is marked by dotted . Scale bar represents 25μm .

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A HeLa cells were transfected with negative control siRNA and RNF26 siRNA as well as constructs coding a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2, as indicated. 48 hours post transfection cells were stained for LAMP-1 (green) and DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. Scale bar represents 25μm.


C HeLa were transfected with expression constructs for HA - tagged p62 FLAG - tagged ubiquitin as well as empty vector , USP17 or USP17CS . After 48hrs lysates were prepared HA tagged were pulled down using anti - HA agarose . Pull downs , lysates , were immunoblotted with anti - HA , anti - FLAG , anti - USP17 antibodies , as indicated , to confirm presence of ubiquitinated p62 .

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C HeLa cells were transfected with expression constructs for HA-tagged p62 and FLAG-tagged ubiquitin as well as empty vector, USP17 or USP17CS. After 48hrs cell lysates were prepared and HA tagged proteins were pulled down using anti-HA agarose. Pull downs, and lysates, were immunoblotted with anti-HA, anti-FLAG, and anti-USP17 antibodies, as indicated, to confirm the presence of ubiquitinated p62.


A HeLa were transfected with constructs for EEA1 - GFP RAB5 - GFP in addition to non - targeting scrambled ( NT ) shRNA , USP17 shRNA1 or USP17 shRNA2 . 72 hours post transfection were stained with DAPI . scale bar represents 25μm . B distribution of at least 400 EEA1 or RAB5 positive vesicles from a number of ( n ) from a series of confocal across three separate experiments was plotted as vesicle relative position ( mean value is red bar ) . Error bars represent standard error * * * * indicates a p - values less than 0 . 0001 . One way ANOVA used to determine if statistically significant differences between groups .

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A HeLa cells were transfected with constructs for EEA1-GFP and RAB5-GFP in addition to non-targeting scrambled (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 72 hours post transfection the cells were stained with DAPI. The scale bar represents 25μm. B The distribution of at least 400 EEA1 or RAB5 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups. E


qRT - PCR of mGPDH , myogenin myosin heavy chain ( MyHC ) during C2C12 myocyte

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qRT-PCR of mGPDH, myogenin and myosin heavy chain (MyHC) levels during C2C12 myocyte differentiation


immunoblot of mGPDH , myogenin myosin heavy chain ( MyHC ) during C2C12 myocyte differentiation . represents of indicated normalized to β -

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immunoblot of mGPDH, myogenin and myosin heavy chain (MyHC) levels during C2C12 myocyte differentiation. Quantification represents the levels of indicated protein normalized to β-actin


Immunoblot of mGPDH , voltage - dependent anion channel ( VDAC ) cytochrome c ( Cyt C ) in mitochondrial lysate during C2C12 myocyte differentiation . represents of indicated normalized to COX

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Immunoblot of mGPDH, voltage-dependent anion channel (VDAC) and cytochrome c (Cyt C) levels in mitochondrial lysate during C2C12 myocyte differentiation. Quantification represents the levels of indicated protein normalized to COX IV


Activity of mGPDH at days 0 7 after C2C12 myocyte

Activity assay of mGPDH at days 0 and 7 after C2C12 myocyte differentiation


Representative of MyHC immunofluorescence ( E ) of C2C12 myocyte transfected with siRNA or overexpression plasmid for

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Representative images of MyHC immunofluorescence (E) of C2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPD


C2C12 myocyte transfected with siRNA or overexpression plasmid for mGPDH ; fusion index ( F ) distribution of nuclei per myotube ( G ) were calculated at day 5 after differentiation .

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C2C12 myocyte transfected with the siRNA or the overexpression plasmid for mGPDH; the fusion index (F) and the distribution of nuclei per myotube (G) were calculated at day 5 after differentiation.


Immunoblot of mGPDH , myogenin MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH . Quantificatio represents of indicated normalized to β - actin at indicated day after

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Immunoblot of mGPDH, myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantificatio represents the levels of indicated protein normalized to β-actin at indicated day after differentiation


myogenin MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH . Quantificatio represents of indicated normalized to β - actin at indicated day after

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myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantificatio represents the levels of indicated protein normalized to β-actin at indicated day after differentiation


qRT - PCR of mGPDH , myogenin MyHC in C2C12 myocytes transfected with siRN for mGPDH at day 4 after

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qRT-PCR analysis of mGPDH, myogenin and MyHC in C2C12 myocytes transfected with the siRN for mGPDH at day 4 after differentiation


qRT - PCR of mGPDH , myogenin MyHC in C2C12 myocytes transfected wit overexpression plasmid for mGPDH at day 4 after

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qRT-PCR analysis of mGPDH, myogenin and MyHC in C2C12 myocytes transfected wit overexpression plasmid for mGPDH at day 4 after differentiation


qRT - PC of mGPDH , myogenin developmental myosin heavy chain ( myh8 , myl4 myh3 ) in gastrocnemius ( GA ) muscle from C57BL / 6J mice at indicated day after CTX intramuscular

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qRT-PC of mGPDH, myogenin and developmental myosin heavy chain (myh8, myl4 and myh3) in gastrocnemius (GA) muscle from C57BL/6J mice at indicated day after CTX intramuscular injection


immunoblo ( B ) of mGPDH , myogeni in gastrocnemius ( GA ) muscle from C57BL / 6J mice at indicated day after CTX intramuscular

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immunoblo (B) of mGPDH, myogeni in gastrocnemius (GA) muscle from C57BL/6J mice at indicated day after CTX intramuscular injection


Activity of mGPDH in GA muscle from C57BL / 6J mice at day 0 7 after CTX

Activity assay of mGPDH in GA muscle from C57BL/6J mice at day 0 and 7 after CTX injection


Representative of H & E ( arrowhead , necrotic myofibers ; asterisks , regenerating ) ( D ) , distribution of cross - section ( CSA ) ( E ) , of myofibers with central nuclei ( F ) in GA muscle from WT mGPDH - / - mice at day 7 post CTX

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Representative images of the H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (D), distribution of the fibers cross-section area (CSA) (E), percentage of myofibers with central nuclei (F) in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection


immunofluorescence of desmin ( green ) ( G ) in GA muscle from WT mGPDH - / - mice at day 7 post CTX

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immunofluorescence staining of desmin (green) (G) in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection


Muscle ( H in GA muscle from WT mGPDH - / - mice at day 14 post CTX

Muscle weight (H in GA muscle from WT and mGPDH-/- mice at day 14 post CTX injection


trichrome ( I ) in GA muscle from WT mGPDH - / - mice at day 14 post CTX injection . represents fibrotic

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trichrome staining (I) in GA muscle from WT and mGPDH-/- mice at day 14 post CTX injection. Quantification represents the fibrotic areas


qRT - PCR ( J for mGPDH , myogenin myh3 in GA muscle from WT mGPDH - / - mice at day 7 post CTX

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qRT-PCR (J for mGPDH, myogenin and myh3 in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection


immunoblot ( K ) for mGPDH , myogeni in GA muscle from WT mGPDH - / - mice at day 7 post CTX

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immunoblot (K) for mGPDH, myogeni in GA muscle from WT and mGPDH-/- mice at day 7 post CTX injection


qRT - PCR for mGPDH , myogenin myh3 ( L ) in GA muscle from mdx mice 4 after AAV - mGPDH intramuscular

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qRT-PCR for mGPDH, myogenin and myh3 (L) in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection


H & E ( M ) , distribution of CSA ( N in GA muscle from mdx mice 4 after AAV - mGPDH intramuscular

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H&E staining (M), distribution of the fibers CSA (N in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection


) , qRT - PCR ( O in GA muscle from mdx mice 4 after AAV - mGPDH intramuscular

), qRT-PCR (O in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection


immunofluorescence ( P ) for utrophi in GA muscle from mdx mice 4 after AAV - mGPDH intramuscular

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immunofluorescence staining (P) for utrophi in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection


trichrome ( Q ) in GA muscle from mdx mice 4 after AAV - mGPDH intramuscular

trichrome staining (Q) in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection


Exercise capacity of mdx mice 6 after AAV - mGPDH tail vein injection .

Exercise capacity of mdx mice 6 weeks after AAV-mGPDH tail vein injection.


Mitochondrial ( A ) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after

Mitochondrial DNA (A) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation


- encoded OXPHOS genes ( B in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after

nuclear-encoded OXPHOS genes (B in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation


respirometry ( C in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after

respirometry analysis (C in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation


immunoblots of mGPDH , phospho - Thr172 AMPK ( p - AMPK ) , total AMPK ( AMPK ) , phospho - Ser79 - ACC ( p - ACC ) , total ACC PGC1α corresponding quantifications represent mGPDH , p - AMPK , p - ACC PGC1α level in C2C12 myocytes transfected with siRN for mGPDH 24 h after

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immunoblots of mGPDH, phospho-Thr172 AMPK (p-AMPK), total AMPK (AMPK), phospho-Ser79-ACC (p-ACC), total ACC and PGC1α and corresponding quantifications represent mGPDH, p-AMPK, p-ACC and PGC1α protein level in C2C12 myocytes transfected with siRN for mGPDH 24 h after differentiation


immunoblots of mGPDH , phospho - Thr172 AMPK ( p - AMPK ) , total AMPK ( AMPK ) , phospho - Ser79 - ACC ( p - ACC ) , total ACC PGC1α corresponding quantifications represent mGPDH , p - AMPK , p - ACC PGC1α ( D - F ) in C2C12 myocytes transfecte wit plasmid for mGPDH 24 h after

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immunoblots of mGPDH, phospho-Thr172 AMPK (p-AMPK), total AMPK (AMPK), phospho-Ser79-ACC (p-ACC), total ACC and PGC1α and corresponding quantifications represent mGPDH, p-AMPK, p-ACC and PGC1α protein levels (D-F) in C2C12 myocytes transfecte wit plasmid for mGPDH 24 h after differentiation


Immunoblots of p - AMPK , p - ACC PGC1α corresponding quantifications represent p - AMPK , p - ACC PGC1α ( G in C2C12 myocytes transfected by mGPDH plasmid with AMPK inhibitor compound C ( CC ) 24 h after

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

Immunoblots of p-AMPK, p-ACC and PGC1α and corresponding quantifications represent p-AMPK, p-ACC and PGC1α protein levels (G in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation


mitochondrial ( H in C2C12 myocytes transfected by mGPDH plasmid with AMPK inhibitor compound C ( CC ) 24 h after

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mitochondrial DNA (H in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation


- encoded OXPHOS genes combined by NDUFS8 , SDHb , Uqcrc1 , COX5b ATP5a1 ( I ) in C2C12 myocytes transfected by mGPDH plasmid with AMPK inhibitor compound C ( CC ) 24 h after

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

nuclear-encoded OXPHOS genes combined by NDUFS8, SDHb, Uqcrc1, COX5b and ATP5a1 (I) in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C (CC) 24 h after differentiation


NAD + / NADH ( J in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after

NAD+/NADH ratio (J in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation


immunoprecipitation for PGC - 1α acetyl - lysine ( Ac - Lys ) level ( K ) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after

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immunoprecipitation analysis for PGC-1α acetyl-lysine (Ac-Lys) level (K) in C2C12 myocytes transfected with siRNA or plasmid for mGPDH 24 h after differentiation


Immunoblot of c - myc myogenin ( L in C2C12 myocytes transfected with mGPDH plasmid with AMPK inhibitor CC at 24 h ( L , M after

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Immunoblot of c-myc and myogenin (L in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,M after differentiation


corresponding quantifications represent c - myc myogenin ( M ) in C2C12 myocytes transfected with mGPDH plasmid with AMPK inhibitor CC at 24 h ( L ,

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corresponding quantifications represent c-myc and myogenin protein levels (M) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h (L,M


representative of MyHC immunofluorescence ( N ) , fusion index ( O ) distribution of nuclei per myotube ( P ) in C2C12 myocytes transfected with mGPDH plasmid with AMPK inhibitor CC a 72 h ( N - P ) after

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representative images of MyHC immunofluorescence (N), fusion index (O) and the distribution of nuclei per myotube (P) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC a 72 h (N-P) after differentiation


Immunoblots of p - AMPK , p - ACC , PGC1α myogenin in C2C12 myocytes transfected with mGPDH plasmid with CaMKKβ inhibitor STO - 609 at 24 h after differentiation . Quantifications represent p - AMPK , p - ACC , PGC1α myogenin

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

Immunoblots of p-AMPK, p-ACC, PGC1α and myogenin in C2C12 myocytes transfected with mGPDH plasmid with the CaMKKβ inhibitor STO-609 at 24 h after differentiation. Quantifications represent p-AMPK, p-ACC, PGC1α and myogenin protein levels


Immunoblots of p - AMPK p - ACC in C2C12 myocytes transfected with mGPDH plasmid with Ca2 + chelator BAPTA - AM at 24 h after differentiation . Quantifications represent p - AMPK p - ACC .

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Immunoblots of p-AMPK and p-ACC in C2C12 myocytes transfected with mGPDH plasmid with the Ca2+ chelator BAPTA-AM at 24 h after differentiation. Quantifications represent p-AMPK and p-ACC protein levels.


Immunoblo of mGPD in GA muscles from obese patient


IH of mGPD in GA muscles from obese patient


Immunoblo of mGPDH myogenin in GA fro indicated

Immunoblo of mGPDH and myogenin in GA muscles fro indicated mic


qRT - PCR of mGPDH in GA muscle of indicated mice at days 0 3 post CTX intramuscular

qRT-PCR of mGPDH in GA muscle of indicated mice at days 0 and 3 post CTX intramuscular injection


Experimental setup ( upper of E ) , qRT - PCR of mGPDH , myogenin myh3 ( bottom of E in GA muscle from AAV - mGPDH - treated HFD - fed mice at days 7 post CTX intramuscular

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Experimental setup (upper of E), qRT-PCR of mGPDH, myogenin and myh3 (bottom of E in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection


) , H & E ( arrowhead , necrotic myofibers ; asterisks , regenerating ) ( F ) , distribution of fiber CSA ( G ) in GA muscle from AAV - mGPDH - treated HFD - fed mice at days 7 post CTX intramuscular

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), H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (F), distribution of the fiber CSA (G) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection


of myofibers with central nuclei ( H ) in GA muscle from AAV - mGPDH - treated HFD - fed mice at days 7 post CTX intramuscular

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percentage of myofibers with central nuclei (H) in GA muscle from AAV-mGPDH-treated HFD-fed mice at days 7 post CTX intramuscular injection


Experimental setup ( upper of I qRT - PCR of mGPDH , myogenin myh3 ( bottom of I ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post CTX intramuscular

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Experimental setup (upper of I qRT-PCR of mGPDH, myogenin and myh3 (bottom of I) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injection


H & E ( arrowhead , necrotic myofibers ; asterisks , regenerating ) ( J ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post CTX intramuscular

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H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (J) in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injection


distribution of CSA ( K ) , of myofibers with central nuclei ( L in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post CTX intramuscular

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distribution of the fibers CSA (K), percentage of myofibers with central nuclei (L in GA muscle from AAV-mGPDH-treated STZ-treated mice at day post CTX intramuscular injection


Experimental setup ( uppe muscle ( bottom of M ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at da 14 ( M ) post CTX intramuscular

Experimental setup (uppe and muscle weight (bottom of M) in GA muscle from AAV-mGPDH-treated STZ-treated mice at da 14 (M) post CTX intramuscular injection


Immunoblots of mGPDH , p - AMPK , p - ACC , PGC1α myogenin for experiment described in E .

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Immunoblots of mGPDH, p-AMPK, p-ACC, PGC1α and myogenin for the experiment described in E.


( A ) LncRNA CRNDE expression level was measured by qRT - PCR in cancerous tissues adjacent normal tissues obtained from 35 cases of gastric cancer ( GC ) . Data information : Data are expressed as mean ± SD . Student ' s t - * * P < 0 . 01 .

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(A) LncRNA CRNDE expression level was measured by qRT-PCR in cancerous tissues and adjacent normal tissues obtained from 35 cases of gastric cancer (GC) patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.


( C ) LncRNA CRNDE expression level was measured by qRT - PCR in adjacent normal tissue - derived macrophages ( NTMs ) - associated macrophages ( TAMs ) obtained from 7 cases of GC . Data information : Data are expressed as mean ± SD . Student ' s t - * * P < 0 . 01 .

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(C) LncRNA CRNDE expression level was measured by qRT-PCR in adjacent normal tissue-derived macrophages (NTMs) and tumor-associated macrophages (TAMs) obtained from 7 cases of GC patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.


( C ) ( D ) Left : representative electron micrograph of exosomes isolated from medium of BMDMs , BMDM - polarized M2 macrophages , monocytes monocyte - polarized M2 macrophages are depicted , showing typical of exosomes . Upper right : of CD63 CD81 were measured by Western blot . Lower left : Nanoparticle tracking showed that most of particles had a similar of 50 - 150 nm in diameter with a peak at around 100 nm . Lower right : LncRNA CRNDE expression in different exosomes . Data information : Scale bar = 100 nm . results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - . * * P < 0 . 01 vs . BMDMs or monocytes or BMDMs - exo or monocytes - exo .

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

(C) and (D) Left: representative electron micrograph of exosomes isolated from the medium of BMDMs, BMDM-polarized M2 macrophages, monocytes and monocyte-polarized M2 macrophages are depicted, showing the typical morphology of exosomes. Upper right: the protein levels of CD63 and CD81 were measured by Western blot. Lower left: Nanoparticle tracking analysis showed that most of the particles had a similar size of 50-150 nm in diameter with a peak at around 100 nm. Lower right: LncRNA CRNDE expression levels in different exosomes. Data information: Scale bar= 100 nm. The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. **P<0.01 vs. BMDMs or monocytes or BMDMs-exo or monocytes-exo.


( B ) BMDM - polarized M2 macrophages were transfected with Cy3 - labelled CRNDE exosomes were isolated from medium ( Cy3 - CRNDE - EXO ) . Then , MFC were incubated with Cy3 - CRNDE - EXO . Left : Confocal microscopy was used to detect Cy3 - labelled CRNDE in MFC . nuclei were stained with DAPI . Scale bar = 20 µm . Right : Flow cytometry was performed to quantitate Cy3 - positive . * * P < 0 . 01 vs . MFC incubated with control EXO . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - .

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 B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BMDM-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, MFC cells were incubated with Cy3-CRNDE-EXO. Left: Confocal microscopy was used to detect the Cy3-labelled CRNDE in MFC cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. MFC cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.


( D ) Monocyte - polarized M2 macrophages were transfected with Cy3 - labelled CRNDE exosomes were isolated from medium ( Cy3 - CRNDE - EXO ) . Then , SGC7901 were incubated with Cy3 - CRNDE - EXO . Confocal microscopy was used to detect Cy3 - labelled CRNDE in SGC7901 . nuclei were stained with DAPI . Scale bar = 20 µm . Right : Flow cytometry was performed to quantitate Cy3 - positive . * * P < 0 . 01 vs . SGC7901 incubated with control EXO . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - .

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

(D) Monocyte-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, SGC7901 cells were incubated with Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. SGC7901 cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.


( E ) SGC7901 were incubated with chlorpromazine ( CPZ , 50 μM ) , genistein ( GEN , 200 μM ) , 5 - ( n - ethyl - n - isopropyl ) - amiloride ( EIPA , 100 μM ) , or methyl - β - cyclodextrin ( MβCD , 10 mM ) , following by incubation of Cy3 - CRNDE - EXO . Confocal microscopy was used to detect Cy3 - labelled CRNDE in SGC7901 . nuclei were stained with DAPI . Right : Flow cytometry was performed to quantitate Cy3 - positive . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA .

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

(E) SGC7901 cells were incubated with chlorpromazine (CPZ, 50 μM), genistein (GEN, 200 μM), 5-(n-ethyl-n-isopropyl)-amiloride (EIPA, 100 μM), or methyl-β-cyclodextrin (MβCD, 10 mM), following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.


( F ) SGC7901 were incubated with 0 , 25 , 50 , 100 μM CPZ , following by incubation of Cy3 - CRNDE - EXO . Confocal microscopy was used to detect Cy3 - labelled CRNDE in SGC7901 . nuclei were stained with DAPI . Right : Flow cytometry was performed to quantitate Cy3 - positive . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA .

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

(F) SGC7901 cells were incubated with 0, 25, 50, 100 μM CPZ, following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.


Exosomes were isolated from media of BMDMs ( BMDMs - exo ) , BMDM - polarized M2 macrophages ( M2 - exo ) , BMDM - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , BMDM - polarized M2 macrophages transfected with negative control of si - CRNDE ( M2 - si - control - exo ) . ( A ) LncRNA CRNDE expression in indicated exosomes was detected by qRT - PCR . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . : one - way ANOVA . * * P < 0 . 01 .

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

Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). (A) LncRNA CRNDE expression in indicated exosomes was detected by qRT-PCR. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.


Exosomes were isolated from media of BMDMs ( BMDMs - exo ) , BMDM - polarized M2 macrophages ( M2 - exo ) , BMDM - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , BMDM - polarized M2 macrophages transfected with negative control of si - CRNDE ( M2 - si - control - exo ) . Then , MFC were treated with indicated exosomes cisplatin ( CDDP ; 1μg / ml ) for 48 h . ( C ) Representative of colony formation performed on MFC .

O O O O O O O O O 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 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

Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). Then, MFC cells were treated with indicated exosomes and cisplatin (CDDP; 1μg/ml) for 48 h. (C) Representative images of colony formation assay performed on MFC cells.


Exosomes were isolated from medium of monocytes ( monocytes - exo ) , monocyte - polarized M2 macrophages ( M2 - exo ) , monocyte - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , monocyte - polarized M2 macrophages transfected with si - control ( M2 - si - control - exo ) . ( E ) LncRNA CRNDE expression were measured . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 .

O O O O O O O O O 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 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

Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). (E) LncRNA CRNDE expression were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.


Exosomes were isolated from medium of monocytes ( monocytes - exo ) , monocyte - polarized M2 macrophages ( M2 - exo ) , monocyte - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , monocyte - polarized M2 macrophages transfected with si - control ( M2 - si - control - exo ) . Then , SGC7901 were treated with indicated exosomes CDDP ( 1μg / ml ) . ( G ) proliferation were measured .

O O O O O O O O O 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

Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). Then, SGC7901 cells were treated with indicated exosomes and CDDP (1μg/ml). (G) cell proliferation were measured.


3 × 105 MFC were subcutaneously injected into nude mice . Ten days after injection , BMDMs - exo , M2 - exo , M2 - si - control - exo , or M2 - si - CRNDE - exo ( 10 µg ) was injected into center of homograft tumors of mice ( n = 7 each group ) , followed by intraperitoneal injection of CDDP ( 10 mg / kg ) . Six days after CDDP treatment , mice were sacrificed homograft tumors were collected . ( A ) Growth curves of MFC subcutaneous homograft tumors ( homograft sizes were recorded from day of CDDP treatment ) . * P < 0 . 05 , * * P < 0 . 01 vs . BMDMs - exo ; # P < 0 . 05 , # # P < 0 . 01 vs . M2 - si - control - exo . Data information : Data are expressed as mean ± SD . one - way ANOVA .

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

3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (A) Growth curves of MFC subcutaneous homograft tumors (The homograft tumor sizes were recorded from the day of CDDP treatment). *P<0.05, **P<0.01 vs. BMDMs-exo; #P<0.05, ##P<0.01 vs. M2-si-control-exo. Data information: Data are expressed as mean ± SD. one-way ANOVA.


3 × 105 MFC were subcutaneously injected into nude mice . Ten days after injection , BMDMs - exo , M2 - exo , M2 - si - control - exo , or M2 - si - CRNDE - exo ( 10 µg ) was injected into center of homograft tumors of mice ( n = 7 each group ) , followed by intraperitoneal injection of CDDP ( 10 mg / kg ) . Six days after CDDP treatment , mice were sacrificed homograft tumors were collected . ( D ) LncRNA CRNDE expression in homograft tumors of mice . * * P < 0 . 01 . Data information : Data are expressed as mean ± SD . : one - way ANOVA .

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

3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (D) LncRNA CRNDE expression in homograft tumors of mice. **P<0.01. Data information: Data are expressed as mean ± SD. : one-way ANOVA.


( C ) SGC7901 were transfected with pcDNA - CRNDE or its negative control ( Mock ) before MG132 treatment ( 10 µM , 6 h ) . Co - immunoprecipitation ( Co - IP ) was performed to examine combination of PTEN NEDD4 - 1 .

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(C) SGC7901 cells were transfected with pcDNA-CRNDE or its negative control (Mock) before MG132 treatment (10 µM, 6 h). The Co-immunoprecipitation (Co-IP) assay was performed to examine the combination of PTEN and NEDD4-1.


( E ) expressions of PTEN NEDD4 - 1 were measured in SGC7901 transfected with pcDNA - CRNDE or Mock at four time - points after cycloheximide ( CHX , 100 μg / mL ) treatment . GAPDH was served as an internal control . Left : Representative bands . Right : Quantitative results . results shown are from three biological replicates are expressed as mean ± SD . Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 vs . Mock .

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(E) The expressions of PTEN and NEDD4-1 were measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock at four time-points after cycloheximide (CHX, 100 μg/mL) treatment. GAPDH was served as an internal control. Left: Representative bands. Right: Quantitative results. The results shown are from three biological replicates and are expressed as mean ± SD. Student's t-test. *P<0.05, **P<0.01 vs. Mock.


G ) ubiquitylation of PTEN was measured in 293T transfected with indicated vectors .

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G) The ubiquitylation of PTEN was measured in 293T cells transfected with indicated vectors.


Normal , si - control - transfected , or si - PTEN - transfected SGC7901 were treated with CDDP ( 1 µg / ml ) M2 - si - control - exo or M2 - si - CRNDE - exo . Si - control , negative control of si - PTEN . ( A ) of PTEN , phosphorylated phosphatidylinositol 3 - kinase ( p - PI3K ) , phosphorylated kinase B ( p - AKT ) , p - glycoprotein ( p - gp ) , B - lymphoma 2 ( Bcl - 2 ) were measured . GAPDH was served as an internal control .

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Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. (A) The protein levels of PTEN, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), p-glycoprotein (p-gp), B-cell lymphoma 2 (Bcl-2) were measured. GAPDH was served as an internal control.


Normal , si - control - transfected , or si - PTEN - transfected SGC7901 were treated with CDDP ( 1 µg / ml ) M2 - si - control - exo or M2 - si - CRNDE - exo . Si - control , negative control of si - PTEN . apoptosis in SGC7901 . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 .

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Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. The cell apoptosis in SGC7901 cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.


SGC7901 were divided into four groups : M2 - si - control - exo , M2 - si - CRNDE - exo , M2 - si - CRNDE - exo + PTEN inhibitor ( SF1670 ; 50 μmol / l ) , M2 - si - control - exo + PTEN inhibitor . were treated with CDDP . survival rate were measured . Data information : results shown are from seven biological replicates . Data are expressed as mean ± SD . : one - way ANOVA . * * P < 0 . 01 .

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SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell survival rate were measured. Data information: The results shown are from seven biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.


SGC7901 were divided into four groups : M2 - si - control - exo , M2 - si - CRNDE - exo , M2 - si - CRNDE - exo + PTEN inhibitor ( SF1670 ; 50 μmol / l ) , M2 - si - control - exo + PTEN inhibitor . were treated with CDDP . apoptosis were measured . Data information : results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 .

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SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell apoptosis were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.


( b ) Kinematics of surface . A kymograph of surface ( φ axis ) from a Tg ( β - actin : m - GFP ) shows continuous displacement of EVL / yolk margin , which accelerates after 50 % epiboly , while yolk does not show any shift ( see Movie EV1 ) . ( c ) Theoretical kymograph from 50 % to 70 % epiboly superimposed , between red lines , on experimental kymograph ( b ) . signal is color - coded ( blue to red ) . arrowheads point to flow lines ( black ) .

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(b) Kinematics of the embryo surface. A kymograph of the embryo surface (φ axis) from a Tg (β-actin:m-GFP) embryo shows the continuous displacement of the EVL / yolk margin, which accelerates after 50% epiboly, while the yolk cell membrane does not show any shift (see Movie EV1). (c) Theoretical kymograph from 50 % to 70 % epiboly superimposed, between the red lines, on the experimental kymograph(b). Membrane signal intensity is color-coded (blue to red). White arrowheads point to flow lines (black).


( d ) AV velocities , normalized to EVL / yolk margin velocity , were measured by PIV of time - lapse recordings of surface of lectin - soaked . plots correspond to three different epiboly stages ( 30 % , blue ; 60 % , red ; 90 % , green ) . Blue red arrows point to width of E - YSL at 30 60 % epiboly respectively ( from Appendix Figure S1 ) .

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(d) AV velocities, normalized to EVL / yolk margin velocity, were measured by PIV of time-lapse recordings of the surface of lectin-soaked embryos. The plots correspond to three different epiboly stages (30 %, blue; 60 %, red; and 90 %, green). Blue and red arrows point to the width of the E-YSL at 30 and 60 % epiboly respectively (from Appendix Figure S1).


( e ) yolk is highly convoluted ( brackets ) ahead of EVL leading condenses over time . Tg ( β - actin : m - GFP ) at 30 % ( left ) 60 % ( right ) epiboly . Scale bar 25 μm . Inset shows a high magnification of wrinkled ( arrowhead ) . Scale bar 10 μm .

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(e) The yolk cell membrane is highly convoluted (white brackets) ahead of the EVL leading cells and condenses over time. Tg (β-actin:m-GFP) embryo at 30 % (left) and 60 % (right) epiboly. Scale bar 25 μm. Inset shows a high magnification of the wrinkled area (arrowhead). Scale bar 10 μm.


( f ) Actin accumulates within wrinkles at E - YSL . yolk of LifeAct - GFP injected ( actin - green ) was stained with lectin - TRITC ( red ) ( top ) . Orthogonal projection of wrinkled ( bottom ) . Wrinkles are outlined with a dotted . Scale bar 2 μm .

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(f) Actin accumulates within wrinkles at the E-YSL. The yolk cell membrane of LifeAct-GFP injected embryos (actin - green) was stained with lectin-TRITC (red) (top). Orthogonal projection of the wrinkled area (bottom). Wrinkles are outlined with a white dotted line. Scale bar 2 μm.


( a ) Yolk granules flow in stereotyped patterns as epiboly progresses . PIV of time - lapse snapshots imaged by two - photon microscopy of a Tg ( β - actin : m - GFP ) ( from Movie EV2 ) . Upon epiboly onset , yolk granules flow towards pole displaying two internal vortices near E - YSL . At 50 % , movement of yolk granules halts , but resumes afterwards until end of epiboly . ( A ) vegetal ( V ) poles are indicated . Scale bar 100 μm . ( b ) Internal yolk content speed ( μm / min ) at center of , over time ( min ) . Inset : Spherical coordinates . main axis of sphere is represented by z , from to vegetal pole . polar angle is represented by φ radial coordinate is denoted ρ . ( c ) Vortex midpoints from center ( ρ ) , over time ( min ) . ( d ) Vortex midpoints position in relation to φ axis , over time ( min ) . Black lines represent measurements . Red lines represent averages . ( e ) Correlation between vortex displacement ( blue , average of n = 3 ) vs . EVL margin displacement ( red , average of n = 4 ) along main axis ( z ) , over time ( min ) . Standard Errors are displayed . Arrow points to 50 % epiboly .

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(a) Yolk granules flow in stereotyped patterns as epiboly progresses. PIV of time-lapse snapshots imaged by two-photon microscopy of a Tg (β-actin:m-GFP) embryo (from Movie EV2). Upon epiboly onset, yolk granules flow towards the animal pole displaying two internal vortices near the E-YSL. At 50 %, the movement of the yolk granules halts, but resumes afterwards until the end of epiboly. Animal (A) and vegetal (V) poles are indicated. Scale bar 100 μm. (b) Internal yolk content speed (μm/min) at the center of the embryo, over time (min). Inset: Spherical coordinates. The main axis of the sphere is represented by z, from the animal to the vegetal pole. The polar angle is represented by φ and the radial coordinate is denoted ρ. (c) Vortex midpoints distance from the center (ρ), over time (min). (d) Vortex midpoints position in relation to the φ axis, over time (min). Black lines represent individual measurements. Red lines represent averages. (e) Correlation between vortex displacement (blue, average of n=3 embryos) vs. EVL margin displacement (red, average of n = 4 embryos) along the main axis (z), over time (min). Standard Errors are displayed. Arrow points to 50 % epiboly.


( a ) Yolk microrheology . Mean squared displacement ( MSD ) of thermal fluctuations of nanoparticles embedded in yolk of zebrafishembryos . Solid dashed black lines are Mean ± Standard Error computed from 99 particles . Red is fit of Stokes - Einstein equation . MSD of nanoparticles embedded in yolk exhibits a proportional dependence on time lag , Δt , consistent with a Newtonian liquid behavior with a viscosity of 129 mPa · s . A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila ( He et al . , 2014 ) . Interestingly , viscosity of zebrafish yolk is two orders of magnitude lower than that of C . elegansembryos ( Daniels et al . , 2006 ) of cytoplasm of several types ( Wirtz , 2009 ) .

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(a) Yolk microrheology. Mean squared displacement (MSD) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos. Solid and dashed black lines are Mean ± Standard Error computed from 99 particles. Red line is the fit of the Stokes-Einstein equation. MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag, Δt, consistent with a Newtonian liquid behavior with a viscosity of 129 mPa·s. A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila (He et al., 2014). Interestingly, the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C. elegansembryos (Daniels et al., 2006) and of the cytoplasm of several cell types (Wirtz, 2009).


( b ) Viscoelasticity of yolk cortex . Effective modulus ( g * ) measured by indenting surface of zebrafishembryos with AFM . Red symbols are elastic modulus ( g ' ) yellow symbols are viscous modulus ( g ' ' ) , respectively . Standard Errors are displayed . In measured frequencies cortex rheology is dominated by a solid - like behavior with viscous modulus 5 times lower that elastic modulus . No significant frequency dependence was found in elastic ( ANOVA , p = 0 . 123 ) viscous modulus ( ANOVA , p = 0 . 719 ) .

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(b) Viscoelasticity of the yolk cortex. Effective complex modulus (g*) measured by indenting the surface of zebrafishembryos with AFM. Red symbols are the elastic modulus (g') and yellow symbols are the viscous modulus (g''), respectively. Standard Errors are displayed. In all the measured frequencies cortex rheology is dominated by a solid-like behavior with the viscous modulus 5 times lower that the elastic modulus. No significant frequency dependence was found in the elastic (ANOVA, p = 0.123) and viscous modulus (ANOVA, p = 0.719).


( c ) Flow lines from snapshots of PIV measurements ( at 40 , 60 80 % epiboly ) of a two - photon video time - lapse ( from Movie EV4 ) of a Tg ( β - actin : m - GFP ) ( top ) . Fitted flow lines using HR ar shown at bottom . relative mean square errors ( RMSE ) of fitting are displayed as a at bottom left of each panel . Scale bar 100 μm .

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(c) Flow lines from snapshots of the PIV measurements (at 40, 60 and 80 % epiboly) of a two-photon video time-lapse (from Movie EV4) of a Tg (β-actin:m-GFP) embryo (top). Fitted flow lines using HR ar shown at the bottom. The relative mean square errors (RMSE) of the fitting are displayed as a percentage at the bottom left of each panel. Scale bar 100 μm.


( d ) Mechanical power maps over time obtained by HR of same as in ( c ) ( from Movie EV5 ) . RMSE of power is shown as a at bottom left of each panel . mechanical work to produce doming ( 40 % epiboly ) arises mainly at blastoderm ( left ) ; at 60 % epiboly ( middle ) DCs undergo ingression ( red ) while E - YSL resists constriction ( blue ) . At 80 % epiboly ( right ) , active work leading to active movement of EVL / yolk margin mainly maps at E - YSL ( red ) while blastoderm undergoes elastic resistance ( blue ) . arrows point to main observed events . Scale bar 100 μm .

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(d) Mechanical power maps over time obtained by HR of the same embryos as in (c) (from Movie EV5). The RMSE of the power is shown as a percentage at the bottom left of each panel. The mechanical work to produce doming (40 % epiboly) arises mainly at the blastoderm (left); at 60 % epiboly (middle) DCs undergo ingression (red) while the E-YSL resists constriction (blue). At 80 % epiboly (right), the active work leading to the active movement of the EVL / yolk margin mainly maps at the E-YSL (red) while the blastoderm undergoes elastic resistance (blue). White arrows point to the main observed events. Scale bar 100 μm.


( e ) AV ( red ) CC ( green ) stresses their differences ( blue ) normalized values in arbitrary units from regression ( Movie EV6 ) were plotted as a function of φ angle ( 0 - 1 ) along cortex from to vegetal . equator - dotted yellow - , yolk surface - purple shadow - RMSE of mechanical pressure as a for each time point are displayed ( 40 % ; 60 % ; 80 % epiboly ) . CC stress steeps up from to vegetal AV shows a more profile . Their difference changes senses around EVL / yolk margin .

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(e) AV (red) and CC (green) stresses and their differences (blue) normalized values in arbitrary units from the regression analysis (Movie EV6) were plotted as a function of the φ angle (0-1) along the cortex from animal to vegetal. The equator - dotted yellow line -, yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed (40 %; 60 %; 80 % epiboly). The CC stress steeps up from animal to vegetal and the AV shows a more complex profile. Their difference changes senses around the EVL / yolk margin.


( a ) Correlation between differential tension ( cyan ) represented by recoil velocities differences ( m / s ) between CC AV cuts their difference ( blue ) calculated by regression at E - YSL during epiboly ( % ) . difference values in normalized relative units were fitted applying a constant adjustment at time points .

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(a) Correlation between the differential tension (cyan) represented by the recoil velocities differences (m/s) between CC and AV laser cuts and their difference (blue) calculated by regression analysis at the E-YSL during epiboly (%). The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points.


( b ) AV tension ( averaged recoil velocity in m / s ) in yolk cortex at different distances from EVL ( 0 , 10 , 20 , 40 60 m ) at different epiboly stages ( 50 % , blue ; 60 - 70 % , red ; 80 % epiboly , green ) . Significance changes are highlighted Standard Errors are displayed .

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(b) AV tension (averaged recoil velocity in m/s) in the yolk cortex at different distances from the EVL (0, 10, 20, 40 and 60 m) and at different epiboly stages (50 %, blue; 60-70 %, red; 80 % epiboly, green). Significance changes are highlighted and Standard Errors are displayed.


( c ) Probing mechanical parameters of living with AFM . Force - indentation curves recorded at cantilever velocity of 10 m / s ( blue ) fitted with a " liquid balloon " model ( red ) . Inset : AFM force - indentation description .

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(c) Probing mechanical parameters of living embryos with AFM. Force-indentation curves recorded at cantilever velocity of 10 m/s (blue) fitted with a "liquid balloon" model (red). Inset: AFM force-indentation description.


( d ) Averaged values of mean surface tension ( pN / μm ) on yolk obtained with AFM at different epiboly stages ( left ) , significant difference of tension at 60 - 70 % epiboly ( p = 0 . 0245 ) between E - YSL vegetal pole ( right ) . Standard Errors are displayed .

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(d) Averaged values of mean surface tension (pN / μm) on the yolk obtained with AFM at different epiboly stages (left), and the significant difference of tension at 60-70 % epiboly (p = 0.0245) between the E-YSL and the vegetal pole (right). Standard Errors are displayed.


( a ) Kymograph showing deformation of cortex along a segment perpendicular to a cut ( red time point ) in E - YSL of a Myosin - GFP transgenic [ Tg ( β - actin : myl 12 . 1 - e - GFP ) ] . After a rapid opening , an active contraction displaces cut centroid vegetalward indicating that EVL is more easily stretched ( elastic ) than vegetal yolk cellmembrane / cortex . When gap is closed , cut center returns to its initial position elastic equilibrium is restored . Scale bar 10 μm .

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(a) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut (red line time point) in the E-YSL of a Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. After a rapid opening, an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched (elastic) than the vegetal yolk cellmembrane/cortex. When the gap is closed, the cut center returns to its initial position and the elastic equilibrium is restored. Scale bar 10 μm.


( d ) Dynamic simulation of AV CC stresses normalized values in arbitrary units ( from Movie EV9 - 40 % ; 65 % ; 80 % epiboly ) represented as in Figure 1a . AV CC stresses are equal at poles , as epiboly simulation progresses , CC stress steeps up from to vegetal difference between CC AV stresses change sense at EVL / yolk margin .

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(d) Dynamic simulation of AV and CC stresses normalized values in arbitrary units (from Movie EV9 - 40 %; 65 %; 80 % epiboly) represented as in Figure 1a. The AV and CC stresses are equal at the poles, as the epiboly simulation progresses, the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin.


( a ) Speed of EVL advancing front at different epiboly stages ( φ angle ) for two distinct simulated conditions : 1 ) a difference of elastic tension between EVL yolk associated to a two - phase reduction of contractile E - YSL width ( from 20º to 5º ) as epiboly advances ( from 50º to 155º ) ( wild type - blue ) . contractile E - YSL width decreases at a slower rate until 80º shrinks afterwards at a rate 2 . 25 times faster , 2 ) Same assumptions as in 1 but with no shrinkage of E - YSL ( constant 20º ) ( contractileless - red ) .

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(a) Speed of the EVL advancing front at different epiboly stages (φ angle) for two distinct simulated conditions: 1) a difference of elastic tension between the EVL and the yolk associated to a two-phase reduction of the contractile E-YSL width (from 20º to 5º) as epiboly advances (from 50º to 155º) (wild type - blue). The contractile E-YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2.25 times faster, and 2) Same assumptions as in 1 but with no shrinkage of the E-YSL (constant 20º) (contractileless - red).


( b ) Experimental values for EVL advancing front speeds at different epiboly stages ( φ angle - top ) at different times ( bottom ) for wild type controls ( blue ) Msn1yolk morphants with reduced E - YSL contractility ( red ) . Experimental profiles closely approach to those inferred in simulations ( compare to ( a ) ) .

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(b) Experimental values for the EVL advancing front speeds at different epiboly stages (φ angle - top) and at different times (bottom) for wild type controls (blue) and Msn1yolk morphants with reduced E-YSL contractility (red). Experimental profiles closely approach to those inferred in the simulations (compare to (a)).


( c ) Timeline showing medial sections power isolines for wild type ( top ) Msn1yolk morphants ( bottom ) along epiboly . Mechanical parameters were inferred by HR from experimental velocity fields ( Movie EV10 ) . RMSE of power is shown as a at each time point . DCs in Msn1yolk morphants undergo ingression on schedule at 50 % epiboly ( yellow arrow ) . Importantly , as in wild type controls , active work in morphants is detected at E - YSL ( red arrow ) . This power supply fades away as epiboly slows down eventually fails . Scale bar 100 μm .

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(c) Timeline showing medial sections and power isolines for wild type (top) and Msn1yolk morphants (bottom) along epiboly. Mechanical parameters were inferred by HR from experimental velocity fields (Movie EV10). The RMSE of power is shown as a percentage at each time point. DCs in the Msn1yolk morphants undergo ingression on schedule at 50 % epiboly (yellow arrow). Importantly, as in wild type controls, the active work in morphants is detected at the E-YSL (red arrow). This power supply fades away as epiboly slows down and eventually fails. Scale bar 100 μm.


( d ) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly its temporal equivalent ( 9 hours after egg laying ) for Msn1yolk morphants . characteristic profiles of longitudinal latitudinal stresses to vegetal stress gradient that are built up during epiboly are never properly developed in morphants .

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(d) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent (9 hours after egg laying) for Msn1yolk morphants. The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants.


D - E ) Immunofluorescence against CITRINE ( electroporated ) , SOX10 ( Schwann precursors Schwann ) ISL1 ( sensory sympathetic neurons ) on electroporated with either control CRISPR plasmid ( D ) or a CRISPR plasmid containing a guide - against Sox8 ( E ) . CITRINE + populate developing dorsal root ganglia ( DRG ) peripheral nerves . Examples of CITRINE + / SOX10 + shown by arrowheads . Asterisks show ventral boundary cap glia . Scale bar is 50 µm in overviews 10 µm in insets . F ) of fate distribution of CITRINE + as a % between glial ( SOX10 + ) , sensory neurons ( ISL1 + ) or neither ( SOX10 - / ISL1 - ) in DRG of control Sox8KD chick . Biological replicates - N = 3 per condition ( wild type versus Sox8 KD ) . Data represented as mean ± . Statistical significance determined using Holm - Sidak method ( alpha = 0 . 05 ) ( multiple t - , unpaired ) . SOX10 + : p = 0 . 7901 , ISL1 + : p = 0 . 9206 , SOX10 - / ISL1 - : p = 0 . 9206 . For statistical significance : non - significant - p - value ≥ 0 . 05 . G ) of ( left ) % of SOX10 + in peripheral nerves that are CITRINE + in control Sox8KD chick ( right ) % of PH3 + CITRINE + corresponding to proliferative . Biological replicates - N = 4 per condition for SOX10 + distribution N = 2 for PH3 ( wild type versus Sox8 KD ) . Data represented as mean ± . Statistical significance determined unpaired t - with two - p value . SOX10 + : p = 0 . 0044 , PH3 + : p = 0 . 4929 . For statistical significance : non - significant - p - value ≥ 0 . 05 , * * - p - value < 0 . 01 . Data information : In total , 6 were analyzed for control electroporation 7 were analyzed for Sox8 knock down electroporation data depicted in graphs correspond to mean ± per ( corresponding to biological replicates ) : 3 - 4 electroporated were analyzed per condition ( control SOX8 knock down ) with 4 - 5 sections stained analyzed per per region of interest ( DRG , DRG : dorsal root ganglia , sg : sympathetic

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D-E) Immunofluorescence against CITRINE (electroporated cells), SOX10 (Schwann cell precursors and Schwann cells) and ISL1 (sensory and sympathetic neurons) on embryos electroporated with either control CRISPR plasmid (D) or a CRISPR plasmid containing a guide-RNA against Sox8 (E). CITRINE+ cells populate the developing dorsal root ganglia (DRG) and peripheral nerves. Examples of CITRINE+/SOX10+ cells shown by arrowheads. Asterisks show ventral boundary cap glia. Scale bar is 50 µm in overviews and 10 µm in insets. F) Quantification of the fate distribution of CITRINE+ cells as a % between glial (SOX10+) cells, sensory neurons (ISL1+) or neither (SOX10-/ISL1-) in the DRG of control and Sox8KD chick embryos. Biological replicates - N=3 embryos per condition (wild type versus Sox8 KD). Data represented as mean±SEM. Statistical significance determined using the Holm-Sidak method (alpha = 0.05) (multiple t-tests, unpaired). SOX10+ cells: p=0.7901, ISL1+ cells: p=0.9206, SOX10-/ISL1- cells: p=0.9206. For statistical significance: non-significant - p-value≥0.05. G) Quantification of (left) the % of SOX10+ cells in the peripheral nerves that are CITRINE+ in control and Sox8KD chick embryos and (right) the % of PH3+ CITRINE+ cells corresponding to proliferative cells. Biological replicates - N=4 embryos per condition for SOX10+ distribution and N=2 for PH3 quantification (wild type versus Sox8 KD). Data represented as mean±SEM. Statistical significance determined unpaired t-test with two-tailed p value. SOX10+ cells: p=0.0044, PH3+ cells: p=0.4929. For statistical significance: non-significant - p-value≥0.05, ** - p-value<0.01. Data information: In total, 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean±SEM per embryo (corresponding to biological replicates): 3-4 electroporated embryos were analyzed per condition (control and SOX8 knock down) with 4-5 sections stained and analyzed per embryo per region of interest (DRG, DRG: dorsal root ganglia, sg: sympathetic ganglion


I ) Immunofluorescence against CITRINE ( electroporated ) , SOX10 ( Schwann precursors Schwann ) TH ( sympathetic neurons chromaffin ) of sympathoadrenal domain on control Sox8KD . CITRINE + / SOX10 + shown by empty arrowheads while CITRINE + / TH + are shown by filled arrowheads . Scale bar = 50 µm . J ) of fate distribution of CITRINE + as a % between glial ( SOX10 + ) , chromaffin ( TH + ) or neither ( SOX10 - / TH - ) in proximity of dorsal aorta visceral nerve of wild type Sox8 KD chick . Biological replicates - N = 4 per condition . Data represented as mean ± . Statistical significance determined using Holm - Sidak method ( alpha = 0 . 05 ) ( multiple t - , unpaired ) . SOX10 + : p = 0 . 0067 , TH + : p = 0 . 0067 , SOX10 - / TH - : p = 0 . 8819 . For statistical significance : non - significant - p - value ≥ 0 . 05 , * - p - value < 0 . 05 . Data information : In total , 6 were analyzed for control electroporation 7 were analyzed for Sox8 knock down electroporation data depicted in graphs correspond to mean ± per ( corresponding to biological replicates ) : 3 - 4 electroporated were analyzed per condition ( control SOX8 knock down ) with 4 - 5 sections stained analyzed per per region of interest Sympathoadrenal domain ) .

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I) Immunofluorescence against CITRINE (electroporated cells), SOX10 (Schwann cell precursors and Schwann cells) and TH (sympathetic neurons and chromaffin cells) of the sympathoadrenal domain on control and Sox8KD embryos. CITRINE+/SOX10+ cells shown by empty arrowheads while CITRINE+/TH+ cells are shown by filled arrowheads. Scale bar = 50 µm. J) Quantification of the fate distribution of CITRINE+ cells as a % between glial (SOX10+) cells, chromaffin cells (TH+) or neither (SOX10-/TH-) in the proximity of the dorsal aorta and visceral nerve of wild type and Sox8 KD chick embryos. Biological replicates - N=4 embryos per condition. Data represented as mean±SEM. Statistical significance determined using the Holm-Sidak method (alpha = 0.05) (multiple t-tests, unpaired). SOX10+ cells: p=0.0067, TH+ cells: p=0.0067, SOX10-/TH- cells: p=0.8819. For statistical significance: non-significant - p-value≥0.05, * - p-value<0.05. Data information: In total, 6 embryos were analyzed for the control electroporation and 7 embryos were analyzed for the Sox8 knock down electroporation and data depicted in graphs correspond to mean±SEM per embryo (corresponding to biological replicates): 3-4 electroporated embryos were analyzed per condition (control and SOX8 knock down) with 4-5 sections stained and analyzed per embryo per region of interest Sympathoadrenal domain).


- Seq of : different time points of CD4 + T - infected in vitro with HIV - 1 or mock infected Expression were normalized as Log2 fold change in infected vs matched mock - infected controls For ( A ) data were analyzed by Fisher ( number of donors = 3 biological replicates ) . Boxplots in ( A ) depict median 25 - 75 percentiles while whiskers extend from hinge to highest or lowest value that is within 1 . 5 * IQR ( inter - quartile range ) of hinge . Data beyond end of whiskers are outliers plotted as points . For each time point , dots illustrate pathway enrichment of genes up - regulated in infected vs matched mock - infected controls . Dots are color - coded on enrichment q - values their indicates fraction of differentially expressed genes ( DEGs ) in pathway ; grey dots are not statistically

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RNA-Seq analysis of: different time points of primary CD4+ T-cells infected in vitro with HIV-1 or mock infected Expression levels were normalized as Log2 fold change in infected vs matched mock-infected controls For (A) data were analyzed by Fisher test (number of donors =3 biological replicates). Boxplots in (A) depict median and 25-75 percentiles while whiskers extend from the hinge to the highest or lowest value that is within 1.5 * IQR (inter-quartile range) of the hinge. Data beyond the end of the whiskers are outliers and plotted as points. For each time point, dots illustrate the pathway enrichment analysis of genes up-regulated in infected vs matched mock-infected controls. Dots are color-coded based on the enrichment q-values and their size indicates the fraction of differentially expressed genes (DEGs) in the pathway; grey dots are not statistically significant


- Seq of PBMCs of macaques infected with SIVmac239 before after suppression of viremia with ART Expression were normalized as Log2 fold change or as enrichment score in ART - treated vs ART - naïve ( B ) . ( B ) data were analyzed by Gene Set Enrichment ( GSEA , number of = 8 ) . Symbols in GSEA enrichment plots represent position of gene set members in transcriptome ranked by differential expression between ART - naïve ART - treated time points . Red color indicates leading edge genes . analyses were conducted on genes of pathway GO response to oxidative stress ( 184 genes ; GO : 0034599 ) .

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RNA-Seq analysis of PBMCs of macaques infected with SIVmac239 before and after suppression of viremia with ART Expression levels were normalized as Log2 fold change or as enrichment score in ART-treated vs ART-naïve animals (B). (B) data were analyzed by Gene Set Enrichment Analysis (GSEA, number of animals =8). Symbols in the GSEA enrichment plots represent the position of the gene set members in the transcriptome ranked by differential expression between ART-naïve and ART-treated time points. Red color indicates leading edge genes. All analyses were conducted on the genes of the pathway GO cellular response to oxidative stress (184 genes; GO:0034599).


( A ) Total to oxidized glutathione ( GSH / GSSG ) in mock infected or HIV - 1 infected CD4 + T - ( 7 dpi , n = 4 biological replicates ; mean ± ) .

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(A) Total to oxidized glutathione ratio (GSH/GSSG) in mock infected or HIV-1 infected CD4+ T-cells (7 dpi, n=4 biological replicates; mean±SEM).


representative picture ( B ) of ROS content in HIV - 1 infected or mock infected CD4 + T - at 7dpi ( productive infection ) 14 dpi ( latent infection ) . ROS content was measured by immunofluorescence , HIV - 1 + were identified by

representative picture (B) of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH


of ROS content in HIV - 1 infected or mock infected CD4 + T - at 7dpi ( productive infection ) 14 dpi ( latent infection ) . ROS content was measured by immunofluorescence , HIV - 1 + were identified by FISH ( D ) . black dash in ( C ) indicates median . Box plots in ( D ) depict median 25 - 75 percentile , while whiskers extend from min to max . n = number of . Scale bar = 2μm .

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quantification of ROS content in HIV-1 infected or mock infected primary CD4+ T-cells at 7dpi (productive infection) and 14 dpi (latent infection). ROS content was measured by immunofluorescence, HIV-1 DNA+ cells were identified by FISH (D). The black dash in (C) indicates the median. Box plots in (D) depict median and 25-75 percentile, while whiskers extend from min to max. n= number of cells. Scale bar = 2μm.


( E ) Subcellular localization of Nrf2 in CD4 + T - infected with HIV - 1 or mock infected ( 3dpi ) as analyzed by biochemical fractionation western blot ; Whole Extract ( WCE ) , Cytoplasm ( Cyt ) , Nucleoplasm ( Nuc ) .

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(E) Subcellular localization of Nrf2 in CD4+ T-cells infected with HIV-1 or mock infected (3dpi) as analyzed by biochemical fractionation and western blot; Whole Cell Extract (WCE), Cytoplasm (Cyt), Nucleoplasm (Nuc).


( F ) localization ( left ) content ( right ) of Nrf2 in HIV - 1 + HIV - 1 - ( 7dpi ) as measured by combining immunofluorescence HIV - 1 FISH . Black dash indicates median , n = number of . Scale bar =

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(F) Nuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA+ and HIV-1 RNA- cells (7dpi) as measured by combining immunofluorescence and HIV-1 RNA FISH. Black dash indicates median, n= number of cells. Scale bar = 2μm


Time course of mRNA of main downstream antioxidant targets of Nrf2 during transition from productive ( 3 - 9 dpi ) to latent ( 14 dpi ) infection , as measured by qPCR Data in ( G ) are depicted as mean ± of 3 biological replicates . raw data were first normalized using 18S as housekeeping control then expressed as Log2 fold mRNA expression in infected vs mock infected Trx = thioredoxin ; NQO1 = NAD ( P ) H [ quinone ] dehydrogenase 1 ; HMOX - 1 = heme oxygenase 1 ; G6PD = glucose - 6 - phosphate dehydrogenase ; GCLC = glutamate - cysteine ligase ; TrxR1 = thioredoxin reductase 1 .

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Time course of the mRNA levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection, as measured by qPCR Data in (G) are depicted as mean±SEM of 3 biological replicates. raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in infected vs mock infected cells Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC= glutamate-cysteine ligase ; TrxR1= thioredoxin reductase 1.


Time course of of main downstream antioxidant targets of Nrf2 during transition from productive ( 3 - 9 dpi ) to latent ( 14 dpi ) infection , as measured by western blot GCLC HMOX - 1 in ( H ) were probed upon stripping .

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Time course of the protein levels of main downstream antioxidant targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection, as measured by western blot GCLC and HMOX-1 in (H) were probed upon membrane stripping.


( I ) Relative mRNA expression of Nrf2 downstream antioxidant targets in J - Lat 9 . 2 left untreated ( latent ) or treated for 24h with 10µM TPA ( reactivated ) . Data are depicted as mean ± of 3 biological replicates . raw data were first normalized using 18S as housekeeping control then expressed as Log2 fold mRNA expression in TPA - reactivated vs latent Trx = thioredoxin ; NQO1 = NAD ( P ) H [ quinone ] dehydrogenase 1 ; HMOX - 1 = heme oxygenase 1 ; G6PD = glucose - 6 - phosphate dehydrogenase ; GCLC =

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(I) Relative mRNA expression of Nrf2 downstream antioxidant targets in J-Lat 9.2 cells left untreated (latent) or treated for 24h with 10µM TPA (reactivated). Data are depicted as mean±SEM of 3 biological replicates. raw data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in TPA-reactivated vs latent cells Trx= thioredoxin; NQO1= NAD(P)H [quinone] dehydrogenase 1; HMOX-1= heme oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC=


( A ) gag mRNA expression ( left ) relative viability ( right ) of HIV - 1 infected Jurkat - TAg transfected with non targeting siRNA ( NC ) or siRNAs targeting HMOX - 1 gene . were infected 24hr post - transfection assayed for mRNA expression ( by qPCR ) or viability ( by MTT ) 48hr post - infection . Data ( mean ± ; n = 3 technical replicates ) were normalized using NC control analyzed by unpaired t - . P < 0 . 05 ; * * P < 0 . 01 .

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(A) gag mRNA expression (left) and relative viability (right) of HIV-1 infected Jurkat-TAg cells transfected with non targeting siRNA (NC) or siRNAs targeting the HMOX-1 gene. Cells were infected 24hr post-transfection and assayed for mRNA expression (by qPCR) or viability (by MTT assay) 48hr post-infection. Data (mean±SEM; n=3 technical replicates) were normalized using the NC control and analyzed by unpaired t-test. P<0.05; **P<0.01.