G ) WT FAK − / − macrophages expressing HA - Akt were stained with anti - HA anti - LAMP1 before by confocal microscopy . boxes show enlarged regions in lower panel . Arrows indicate HA + LAMP + Salmonella evident in WT PEMs . ( H ) of ΔΣΠΙ1ΔΣΠΙ2 ( first grouping ) or ΔinvG ( second grouping ) Salmonella co - localizing with HA LAMP - 1 5 h post - infection . At least 100 were counted per condition . Values are means ± , N = 3 , * p < 0 . 05 .

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G) WT and FAK−/− macrophages expressing HA-Akt were stained with anti-HA and anti-LAMP1 before analysis by confocal microscopy. White boxes show enlarged regions in lower panel. Arrows indicate HA+LAMP+ Salmonella evident in WT PEMs. (H) The percentage of ΔΣΠΙ1ΔΣΠΙ2 (first grouping) or ΔinvG (second grouping) Salmonella co-localizing with HA and LAMP-1 5 h post-infection. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05.


( A ) WT FAK − / − PEMs were incubated with S . typhimurium ΔinvG for 0 - 24 hours before immunoblotting with indicated antibodies .

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(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-24 hours before immunoblotting with the indicated antibodies.


( B ) WT PEMs were incubated with Akt inhibitor AKTV / triciribine ( 10 µm ) for 30 minutes before infection with S . typhimurium ΔinvG for 5 hours . Lysates were immunoblotted with indicated antibodies . (

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(B) WT PEMs were incubated with Akt inhibitor AKTV/triciribine (10 µm) for 30 minutes before infection with S. typhimurium strain ΔinvG for 5 hours. Lysates were immunoblotted with the indicated antibodies. (


. ( C ) WT FAK − / − PEMs were incubated with S . typhimurium ΔinvG for a total of 5 hours before with antibodies recognizing LC3 . DAPI was used to visualize nuclei . Bars represent 10 µm . boxes show enlarged regions in inset panels , bars represent 2 µm . arrowheads indicate LC3 - negative , yellow arrow heads indicate LC3 - positive ( panels c , f ) . Yellow arrowhead in panel f denotes an LC3 + cluster of . ( D ) of ΔinvG ( first grouping ) or ΔΣΠΙ1ΔΣΠΙ2 ( second grouping ) Salmonella co - localizing with LC3 at indicated time points is displayed . At least 100 were counted per condition per time point . Values are means ± , N = 3 , * p < 0 . 05 .

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. (C) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei and bacteria. Bars represent 10 µm. White boxes show enlarged regions in inset panels, bars represent 2 µm. White arrowheads indicate LC3-negative bacteria, yellow arrow heads indicate LC3-positive bacteria (panels c, f). Yellow arrowhead in panel f denotes an LC3+ cluster of bacteria. (D) The percentage of ΔinvG (first grouping) or ΔΣΠΙ1ΔΣΠΙ2 (second grouping) Salmonella co-localizing with LC3 at the indicated time points is displayed. At least 100 bacteria were counted per condition per time point. Values are means ± SEM, N = 3, *p<0.05.


( E ) WT FAK − / − PEMs were incubated with S . typhimurium ΔinvG for 5 h with or without Bafilomycin A1 ( 300 ng ) before immunoblotting with indicated antibodies . Vertical indicate non - contiguous lanes generated from a single exposure .

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(E) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 (300 ng) before immunoblotting with the indicated antibodies. Vertical white line indicate non-contiguous lanes generated from a single exposure.


( F ) FAK − / − PEMs were incubated with DQ - BSA - red for 1 hour before infection with S . typhimurium ΔinvG for 5 h . were co - stained for LC3 DAPI was used to visualize nuclei . boxes show enlarged regions in inset panel .

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(F) FAK−/− PEMs were incubated with DQ-BSA-red for 1 hour before infection with S. typhimurium strain ΔinvG for 5 h. Cells were co-stained for LC3 and DAPI was used to visualize nuclei and bacteria. White boxes show enlarged regions in inset panel.


G ) WT PEMs were pretreated with mTORC1 inhibitor rapamycin ( 4 µm ) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours . were lysed before immunoblotting with indicated antibodies .

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G) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin (4 µm) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours. Cells were lysed before immunoblotting with the indicated antibodies.


( H ) WT FAK − / − PEMs were pretreated with rapamycin ( 4 µm ) , Akt inhibitor AKTV / triciribine ( 10 µm ) or left untreated ( - Tx ) or before infection with S . typhimurium ΔinvG for a further 5 hours . WT macrophages were also pretreated with FAK inhibitor PF228 ( 0 . 5 µm ) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours . were then assessed for of ΔinvG Salmonella co - localizing with LC3 . At least 100 were counted per condition . Values are means ± , N = 3 , * p < 0 . 05 . N . s . not significant .

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(H) WT and FAK−/− PEMs were pretreated with rapamycin (4 µm), Akt inhibitor AKTV/triciribine (10 µm) or left untreated (-Tx) or before infection with S. typhimurium strain ΔinvG for a further 5 hours. WT macrophages were also pretreated with the FAK inhibitor PF228 (0.5 µm) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours. Cells were then assessed for the percentage of ΔinvG Salmonella co-localizing with LC3. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05. N.s. not significant.


A ) WT FAK − / − PEMs were incubated with LPS - coated beads ( 50 ∶ 1 ; beads ∶ ) for 1 hour before with antibodies recognizing LC3 . DAPI was used to visualize nuclei . Bars represent 10 µm . of LPS - beads co - localizing with LC3 is displayed to right . At least 100 beads were counted per condition . Values are means ± , N = 3 .

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A) WT and FAK−/− PEMs were incubated with LPS-coated beads (50∶1; beads∶cell) for 1 hour before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei. Bars represent 10 µm. The percentage of LPS-beads co-localizing with LC3 is displayed to the right. At least 100 beads were counted per condition. Values are means ± SEM, N = 3.


( B ) PEMs were treated with Atg5 , ULK1 or control siRNAs for 48 hours before immunoblotting with indicated antibodies .

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(B) PEMs were treated with Atg5, ULK1 or control siRNAs for 48 hours before immunoblotting with the indicated antibodies.


. ( C - D ) FAK − / − PEMs treated with Atg5 , ULK1 or control siRNA for 48 hours were incubated with LPS - coated beads or ΔinvG Salmonella for 1 or 5 hours before with antibodies recognizing LC3 . of LPS - beads or Salmonella co - localizing with LC3 is displayed to right . At least 100 beads or were counted per condition . Values are means ± , N = 3 .

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. (C-D) FAK−/− PEMs treated with Atg5, ULK1 or control siRNA for 48 hours were incubated with LPS-coated beads or ΔinvG Salmonella for 1 or 5 hours before analysis with antibodies recognizing LC3. The percentage of LPS-beads or Salmonella co-localizing with LC3 is displayed to the right. At least 100 beads or bacteria were counted per condition. Values are means ± SEM, N = 3.


E ) WT FAK − / − PEMs were infected with ΔinvG for 5 hours before preparation examination by transmission electron microscopy ( ) . Arrowheads indicate double .

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E) WT and FAK−/− PEMs were infected with ΔinvG for 5 hours before preparation and examination by transmission electron microscopy (TEM). Arrowheads indicate double membrane.


( A ) WT FAK − / − PEMs were incubated with S . typhimurium ΔinvG for 1 . 5 , 3 5 hours . survival was then assayed using a standard gentamycin resistance described in Materials Methods . Values are means ± , N = 6 , * p < 0 . 05 .

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(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 1.5, 3 and 5 hours. Bacterial survival was then assayed using a standard gentamycin resistance assay described in the Materials and Methods. Values are means ± SEM, N = 6, *p<0.05.


( B ) WT FAK − / − PEMs were pretreated with IFN - γ ( 50 ng / ml ) , rapamycin ( 4 µm ) , or left untreated before incubation with ΔinvG Salmonella for 3 hours . survival was then assayed as described in ( A ) . Values are means ± , N = 6 , * p < 0 . 05 .

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(B) WT and FAK−/− PEMs were pretreated with IFN-γ (50 ng/ml), rapamycin (4 µm), or left untreated before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 6, *p<0.05.


( C ) PEMs were treated with control siRNA or depleted of Atg5 before incubation with ΔinvG Salmonella for 3 hours . survival was then assayed as described in ( A ) . Values are means ± , N = 3 , * p < 0 . 05 vs WT siControl .

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(C) PEMs were treated with control siRNA or depleted of Atg5 before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 3, *p<0.05 vs WT siControl.


. ( D ) loads in WT ( black circles ) FAKΔmyeloid mice ( circles ) in indicated tissues 5 days after oral infection with S . typhimurium SL1344 . Each point indicates data from an mouse . * p < 0 . 05 .

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. (D) Bacterial loads in WT (black circles) and FAKΔmyeloid mice (white circles) in the indicated tissues 5 days after oral infection with S. typhimurium strain SL1344. Each point indicates data from an individual mouse. *p<0.05.


( E ) Hematoxylin eosin ( H & amp ; amp ; E ) of spleen isolated from WT FAKΔmyeloid mice 5 days post - infection with S . typhimurium SL1344 . Arrows indicate areas of leukocyte infiltration . Lesion indicated by yellow arrow is outlined in higher magnification panel to right . Bars represent 200 µm 50 µm .

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(E) Hematoxylin and eosin (H&amp;E) staining of spleen isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Arrows indicate areas of leukocyte infiltration. Lesion indicated by yellow arrow is outlined in the higher magnification panel to the right. Bars represent 200 µm and 50 µm.


. ( F ) Gr - 1 immunostaining of spleens isolated from WT FAKΔmyeloid mice 5 days post - infection with S . typhimurium SL1344 . Panels b d are enlarged sections indicated by black boxes in panels a c . Gr - 1 - positive appear red - brown .

. (F) Gr-1 immunostaining of spleens isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Panels b and d are enlarged sections indicated by black boxes in panels a and c. Gr-1-positive cells appear red-brown.


( G ) Quantitation of flow cytometric data collected from spleens of infected mice . * p < 0 . 05 .

(G) Quantitation of flow cytometric data collected from the spleens of infected mice. *p<0.05.


B ) Binding affinity of CodB for cytosine as measured using thermostability . Cytosine was titrated into detergent solubilised membranes from overexpressing CodB . Kd was estimated to be 51 ± 9 µM . measurements are average of 4 independent titrations with error bars of s . e . m .

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B) Binding affinity of CodB for cytosine as measured using the thermostability assay. Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB. The Kd was estimated to be 51 ± 9 µM. The measurements are the average of 4 independent titrations with error bars of the s.e.m.


( C ) Top panel : Representative show PS labeling in WT dLGN at P6 P13 . RGC inputs were labeled with CTB488 . Bottom panel : Enlarged of boxed region in top panels . Circles indicate PS + RGC inputs . Arrows point to enlarged PS + RGC inputs in insets . ( D ) of of PS + RGC inputs in total RGC inputs at P6 P13 .

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(C) Top panel: Representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged images of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows point to the enlarged PS+ RGC inputs in the insets. (D) Quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and P13.


( E ) Co - labeling of vGlut2 PSVue in dLGN at P6 . Circles indicate colocalized vGlut2 PSVue .

(E) Co-labeling of vGlut2 and PSVue in the dLGN at P6. Circles indicate colocalized vGlut2 and PSVue.


( F ) Co - labeling of Homer1 PSVue in dLGN at P6 . Circles indicate colocalized Homer1 PSVue .

(F) Co-labeling of Homer1 and PSVue in the dLGN at P6. Circles indicate colocalized Homer1 and PSVue.


( G ) Orthogonal sections showed a triple positive PS + / CTB488 + / CD68 + RGC inputs inside of microglia .

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(G) Orthogonal sections showed a triple positive PS+/CTB488+/CD68+ RGC inputs inside of microglia.


( H ) Orthogonal sections showed a triple positive PS + / vGlut2 + / CD68 + synapse inside of microglia .

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(H) Orthogonal sections showed a triple positive PS+/vGlut2+/CD68+ synapse inside of microglia.


( I ) A representative image of microglia ( upper - left ) is surface rendered ( bottom - left ) . arrow points to which might be apoptotic labeled by PSVue . RGC inputs PSVue outside or inside of microglia are shown in two right panels . Scare bar , 20 µm . ( J ) of of PS + PS - RGC inputs outside of microglia in total inputs . N = 4 . ( K ) of of engulfed PS + PS - RGC inputs in total engulfed inputs . N = 4 .

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(I) A representative image of microglia (upper-left) is surface rendered (bottom-left). The white arrow points to cells which might be apoptotic cells labeled by PSVue. RGC inputs and PSVue outside or inside of microglia are shown in the two right panels. Scare bar, 20 µm.(J) Quantification of the percentage of PS+ and PS- RGC inputs outside of microglia in total inputs. N = 4. (K) Quantification of the percentage of engulfed PS+ and PS- RGC inputs in total engulfed inputs. N=4.


( D ) binding experiment using flow cytometry show that only GAIN domain binds to PS , similar as Annexin V binding .

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(D) The binding experiment using flow cytometry show that only the GAIN domain binds to PS, similar as Annexin V binding.


( E ) In competition experiment , AF647 channel shows that GAIN domain binds 60 . 1 % of PS + Ba / F3 ( left ) . Correspondingly , FITC channel reveals 39 . 7 % Annexin V binding to PS + Ba / F3 ( right ) .

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(E) In the competition experiment, AF647 channel shows that GAIN domain binds 60.1% of the PS+ Ba/F3 cells (left). Correspondingly, FITC channel reveals 39.7% Annexin V binding to PS+ Ba/F3 cells (right).


( G ) Direct binding of hFc - tagged GPR56 GAIN to PS other phospholipids . HFc was used as a negative control , GAS6 as a positive control .

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(G) Direct binding of hFc-tagged GPR56 GAIN proteins to PS and other phospholipids. HFc was used as a negative control, and GAS6 as a positive control.


( B ) Gpr56 transcript in isolated microglia from Gpr56 S4 mice .

(B) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice.


( D ) Representative of Nissl of Gpr56 null Gpr56 S4 E16 . 5 neocortex . Arrows indicate cortical ectopias that are shown in insets . ( E ) of ectopia per section . ( F ) distribution of ectopia from rostral to caudal cortex .

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(D) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16.5 neocortex. Arrows indicate cortical ectopias that are shown in insets. (E) Quantification of ectopia size per section. (F) The distribution of ectopia from rostral to caudal cortex.


( G ) . Myelin basic ( MBP ) of corpus callosum in P28 controls , Gpr56 null Gpr56 S4 mice .

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(G). Myelin basic protein (MBP) staining of corpus callosum in P28 controls, Gpr56 null and Gpr56 S4 mice.


(H) Quantification of MBP intensity in corpus callosum.


( I ) Gpr56 transcript in WT microglia at P5 P14 .

(I) Gpr56 transcript levels in WT microglia at P5 and P14.


( J ) An overview of vGlut2 Homer1 in dLGN at P10 . yellow outline indicates dLGN , dotted boxes show where synapses are quantified .

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(J) An overview of vGlut2 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.


K ) Representative of vGlut2 / Homer1 in dLGN of control , Gpr56 null , Gpr56 S4 brains at P10 . Arrows pointing to enlarged synapse in insets . L ) Relative vGlut2 / Homer1 synapse density in dLGN . N = 10 ( Ctrl ) , N = 6 ( Gpr56 null ) , N = 3 ( Gpr56 S4 ) .

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K) Representative images of vGlut2/Homer1 staining in the dLGN of control, Gpr56 null, and Gpr56 S4 brains at P10. Arrows pointing to the enlarged synapse in the insets. L) Relative vGlut2/Homer1 synapse density in dLGN. N=10 (Ctrl), N=6 (Gpr56 null), N=3 (Gpr56 S4).


( N ) Representative surface rendered microglia ( green ) in which CTB + ( red ) RGC inputs were engulfed . ( O ) of of engulfed RGC inputs in controls , Gpr56 null Gpr56 S4 microglia .

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(N) Representative images and surface rendered microglia (green) in which CTB+(red) RGC inputs were engulfed. (O) Quantification of the percentage of engulfed RGC inputs in controls, Gpr56 null and Gpr56 S4 microglia.


( A ) Gpr56 is deleted in CKO microglia . RNAscope shows Gpr56 transcripts co - localize with microglia ( arrow ) in controls , but not in CKO , is absent in types in global Gpr56 KO ( Gpr56 null ) . Arrowhead indicates non - microglia expressing Gpr56 . RNAscope was performed in prefrontal cortex of P30 mice . Scale bar , 20 µm . ( B ) of Gpr56 signals in ( A ) . N = 20 - 50 for each .

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(A) Gpr56 is deleted in CKO microglia. RNAscope shows Gpr56 transcripts co-localize with microglia (arrow) in controls, but not in CKO, and is absent in all cell types in the global Gpr56 KO (Gpr56 null). Arrowhead indicates non-microglia cells expressing Gpr56. RNAscope was performed in the prefrontal cortex of P30 mice. Scale bar, 20 µm. (B) Quantification of Gpr56 fluorescence signals in (A). N = 20-50 cells for each.


( C ) Confocal of vGlut2 ( green ) labeling retinal ganglion ( RGC ) presynaptic terminals Homer1 ( red ) for post - synapses in dLGN of CKO controls at P10 . Overlapped vGlut2 Homer1 are quantified as synapses , indicated by circles . Arrows pointing to synapse enlarged in upper right - inset .

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(C) Confocal images of vGlut2 (green) labeling retinal ganglion cell (RGC) presynaptic terminals and Homer1 (red) for post-synapses in the dLGN of CKO and controls at P10. Overlapped vGlut2 and Homer1 are quantified as synapses, indicated by white circles. Arrows pointing to the synapse enlarged in the upper right-hand inset.


( D ) A time course of dLGN synapse density ( vGlut2 + / Homer1 + ) between WT controls .

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(D) A time course of dLGN synapse density (vGlut2+/Homer1+) between WT and controls.


( E ) Representative super - resolution 3D - reconstructed of vGlut2 Homer1 of P8 dLGN . Green objects represent surface rendering of vGlut2 + presynaptic terminals . Red spots are rendered Homer1 + post - synapses . Magenta spots represent Homer1 + post - synapses within a of 300nm from nearest vGlut2 + surface .

(E) Representative super-resolution images and 3D-reconstructed images of vGlut2 and Homer1 staining of P8 dLGN. Green objects represent surface rendering of vGlut2+ presynaptic terminals. Red spots are rendered Homer1+ post-synapses. Magenta spots represent Homer1+ post-synapses within a distance of 300nm from the nearest vGlut2+ surface.


( F ) of Homer1 + spots adjacent to vGlut2 surface within 100nm , 200nm , 300nm , respectively . N = 3 .

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(F) Quantification of Homer1+ spots adjacent to vGlut2 surface within 100nm, 200nm, and 300nm, respectively. N = 3.


( G ) Western blot of vGlut2 Homer1 using microdissected dLGN tissue from P8 mice . ( H , I ) of vGlut2 Homer1 expression in CKOs controls . N = 3 , * p = 0 . 027 . ( J ) of synapse density ( vGlut2 + / Homer1 + ) in dLGN of both iCKO mice at P10 . N = 3 for , N = 4 for .

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(G) Western blot of vGlut2 and Homer1 using microdissected dLGN tissue from P8 mice. (H, I) Quantification of vGlut2 and Homer1 expression in CKOs and controls. N = 3, *p = 0.027. (J) Quantification of synapse density (vGlut2+/Homer1+) in the dLGN of both male and female iCKO mice at P10. N = 3 for male, N = 4 for female.


( K ) An overview of vGlut1 Homer1 in dLGN at P10 . yellow outline indicates dLGN , dotted boxes show where synapses are quantified .

(K) An overview of vGlut1 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.


( L ) Representative of vGlut1 + ( green ) presynaptic terminals Homer1 + ( red ) postsynapses in dLGN of CKO controls at P10 . Arrows pointing to synapse enlarged in inset . ( M ) of synapse density ( vGlut1 / homer1 ) at P10 in dLGN of CKO mice controls . N = 3 ( WT ) , N = 5 ( CKO ) , p = 0 . 053 .

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(L) Representative images of vGlut1+ (green) presynaptic terminals and Homer1+ (red) postsynapses in the dLGN of CKO and controls at P10. Arrows pointing to the synapse enlarged in inset. (M) Quantification of synapse density (vGlut1/homer1) at P10 in the dLGN of CKO mice and controls. N=3 (WT), N=5 (CKO), p = 0.053.


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

(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 .

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

(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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ± .

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

(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 .

(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 .

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(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 .

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

(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 ) .

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

A 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 .

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

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

A 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 .

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

A 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 .

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-CONTROLLED_VAR O O

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 .

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

A 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

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

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

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

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 .

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

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

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

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

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

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

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

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

O O O O O O O O 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

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

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

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

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

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

O O 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 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

(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 .

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 .

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

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 .

O O O O O O O O O O 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

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 .

O O O O O O O O O O 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

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 ) ) .

(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)).