( C ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( D ) WT PEMs were incubated with the orgA : : tet ; spiA : : kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs ( ΔSPI1ΔSPI2 ) for 0 - 5 hours before immunoblotting with the indicated antibodies .

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

(D) WT PEMs were incubated with the orgA::tet; spiA::kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs (ΔSPI1ΔSPI2) for 0-5 hours before immunoblotting with the indicated antibodies.


( E ) WT PEMs expressing GFP - FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence . Cells were co - stained with antibodies recognizing LAMP1 ( red ) . DAPI was used to visualize nuclei and bacteria ( blue ) . Bars represent 10 µm or 2 µm in enlarged inset .

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

(E) WT PEMs expressing GFP-FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). DAPI was used to visualize nuclei and bacteria (blue). Bars represent 10 µm or 2 µm in enlarged inset.


( A ) WT and FAK − / − PEMs were incubated for 0 - 5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies .

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

(A) WT and FAK−/− PEMs were incubated for 0-5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies.


( B ) WT PEMs were pretreated for 1 hour with the FAK - kinase inhibitor PF - 228 ( 0 . 5 µm ) before incubation with ΔinvG Salmonella for 5 hours . Lysates were immunoblotted with the indicated antibodies .

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

(B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies.


( C - D ) WT or FAK − / − macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain ( C ) or LPS - coated beads ( D ) for 0 - 5 hours before immunoblotting with the indicated antibodies . In panels C and D , WT PEMs were also incubated with ΔinvG ( 5 h ) for comparison of Akt activation . Vertical white lines in panel D indicate non - contiguous lanes generated from a single exposure .

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

(C-D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0-5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure.


E ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( F ) WT PEMs expressing HA - Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours . Lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies before immunoblotting for FAK and HA . In parallel , lysates were immunoblotted for FAK , HA and ERK1 / 2 to control for the level of expression .

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

(F) WT PEMs expressing HA-Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies before immunoblotting for FAK and HA. In parallel, lysates were immunoblotted for FAK, HA and ERK1/2 to control for the level of expression.


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 .

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

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

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

(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 strain ΔinvG for 5 hours . Lysates were immunoblotted with the indicated antibodies . (

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

(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 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 .

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

. (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 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 .

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

(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 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 .

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

(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 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 .

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

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 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 .

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

(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 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 .

O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O 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) 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 the indicated antibodies .

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

(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 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 .

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

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

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

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 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 .

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

(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 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 .

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

(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 . Bacterial survival was then assayed as described in ( A ) . Values are means ± SEM , N = 3 , * p < 0 . 05 vs WT siControl .

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

(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 ) 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 .

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

. (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 and eosin ( H & amp ; 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 .

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

(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 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 .

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

. (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 the spleens of infected mice . * p < 0 . 05 .

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

(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 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 .

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

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 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 .

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

(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 and PSVue in the dLGN at P6 . Circles indicate colocalized vGlut2 and PSVue .

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

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


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

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

(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 .

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

(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 ) . 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 .

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

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

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

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


( 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 ) .

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

(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 proteins to PS and other phospholipids . HFc was used as a negative control , and GAS6 as a positive control .

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

(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 levels in isolated microglia from Gpr56 S4 mice .

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

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


( 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 .

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

(D) Representative images of 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 protein ( MBP ) staining of corpus callosum in P28 controls , Gpr56 null and Gpr56 S4 mice .

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

(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 .

(H) Quantification of MBP intensity in corpus callosum.


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

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

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


( 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 .

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

(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 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 ) .

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

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 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 .

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

(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 , 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 .

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

(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 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 .

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

(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 and controls .

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

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


( 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 .

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

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

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

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


( 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 .

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

(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 and Homer1 staining in dLGN at P10 . The yellow outline indicates the dLGN core , and the 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 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 .

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

(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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images show hippocampal vGlut1 and Homer1 immunostaining .

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

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


( 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the dLGN of P28 ~ P34 mice , p = 0 . 03 , N = 14 ( Ctrl ) , 23 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Quantification of NMDAR1 expression in CKO mice and control .

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

(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 the dLGN of P28 ~ P34 mice . P = 0 . 302 , N = 13 ( Ctrl ) , 17 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM .

(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 ) Quantification of GluR1 expression in CKO mice and controls .

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

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


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

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

(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 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 .

O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-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 O B-MEASURED_VAR O 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 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 ) .

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

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

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

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

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 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 .

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

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 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 .

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

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and myosin heavy chain ( MyHC ) levels during C2C12 myocyte

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

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


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

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

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 ) and cytochrome c ( Cyt C ) levels in mitochondrial lysate during C2C12 myocyte differentiation . Quantification represents the levels of indicated protein normalized to COX

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

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


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

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

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


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 .

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

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 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

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

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 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

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

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

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

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


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

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 and developmental myosin heavy chain ( myh8 , myl4 and myh3 ) in gastrocnemius ( GA ) muscle from C57BL / 6J mice at indicated day after CTX intramuscular

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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 assay of mGPDH in GA muscle from C57BL / 6J mice at day 0 and 7 after CTX

O O 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 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

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

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

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


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

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

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 and myh3 in GA muscle from WT and mGPDH - / - mice at day 7 post CTX

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

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 and mGPDH - / - mice at day 7 post CTX

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

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


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

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

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


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

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 weeks after AAV - mGPDH intramuscular

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


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

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

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


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

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


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

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

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


nuclear - 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 analysis ( 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 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

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

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

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 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

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 DNA ( H in C2C12 myocytes transfected by mGPDH plasmid with the AMPK inhibitor compound C ( CC ) 24 h after

O B-MEASURED_VAR 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


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

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O 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 ratio ( 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 O

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


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

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

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 and myogenin ( L in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h ( L , M after

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

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 and myogenin protein levels ( M ) in C2C12 myocytes transfected with mGPDH plasmid with the AMPK inhibitor CC at 24 h ( L ,

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

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 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

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

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 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

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

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 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 .

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

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 and myogenin in GA muscles fro indicated mic


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

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

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 and myh3 ( bottom of E in GA muscle from AAV - mGPDH - treated HFD - fed mice at days 7 post CTX intramuscular

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

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 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

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

), H&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


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

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 and myh3 ( bottom of I ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post CTX intramuscular

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

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 staining ( arrowhead , necrotic myofibers ; asterisks , regenerating fibers ) ( J ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at day post 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 O O O O

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 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

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

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 and muscle weight ( bottom of M ) in GA muscle from AAV - mGPDH - treated STZ - treated mice at da 14 ( M ) post 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 O O

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 and myogenin for the experiment described in E .

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

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 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 .

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

(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 ) 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 .

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

(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 ) 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 .

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

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

(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 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 .

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

(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 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 .

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

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

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

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 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 .

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

Exosomes were isolated from the 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 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 .

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

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 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 .

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

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 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 .

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

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

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


( C ) SGC7901 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 .

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 B-MEASURED_VAR O B-MEASURED_VAR O O O

(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 ) 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 .

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

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

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

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


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 .

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

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

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

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

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

(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 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 ) .

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

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

(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 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 .

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