Figure 2A Western blot analysis of autophagy markers , Beclin1 , ATG3 , ATG7 , ATG12 - 5 , and LC3I / II , and the mitophagy - related proteins Parkin and PINK1 in HC ( # 1 - 3 ) and DM platelets ( # 1 - 8 ) . B Quantification of autophagy markers and mitophagy - related proteins from HC ( # 1 - 5 ) and DM ( # 1 - 18 ) individuals . ( Beclin1 ; * * p = 0 . 0003 , ATG12 ; * p = 0 . 0388 , ATG3 ; * p = 0 . 0213 , ATG7 ; p = 0 . 4051 , LC3 ; * p = 0 . 0298 , PINK1 ; * p = 0 . 0155 , Parkin ; * p = 0 . 0103 vs . HC ) . GAPDH served as a loading control . C The three panels represent autophagy stages found in DM platelets . Autophagosome structures containing / enclosing organelles are indicated by the black box . Shown for each panel is an enlargement of the autophagosome ( DM1 and 2 ) or the autolysosome ( DM3 ) . Dashed structures are an outline of the autophagosome membranes . D LC3 immuno - EM analysis of HC and DM platelets . Arrows indicate immunogold - labeled LC3 clusters . No clusters were found in HC platelets . Representative areas of clusters of gold labeling in DM patients ( DM1 - 3 ) are presented . Shown in the insets are enlargements of the gold clusters adjacent to mitochondria - like structures . E Double staining for MitoTracker ( mitochondria membrane potential ) ( 50nM for 15 min ) and LC3 ( autophagy ) in HC and DM platelets . Boxed platelets are enlarged in Panel F . F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3 . G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV ( mitochondria ) and LC3 ( autophagy marker ) . The y - axis indicates percentage of LC3 positive cells in total cells ( * * p = 0 . 006 vs . HC ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Western blot analysis of autophagy markers, Beclin1, ATG3, ATG7, ATG12-5, and LC3I/II, and the mitophagy-related proteins Parkin and PINK1 in HC (#1-3) and DM platelets (#1-8).B Quantification of autophagy markers and mitophagy-related proteins from HC (#1-5) and DM (#1-18) individuals. (Beclin1; **p=0.0003, ATG12; *p=0.0388, ATG3; *p=0.0213, ATG7; p=0.4051, LC3; *p=0.0298, PINK1; *p=0.0155, Parkin; *p=0.0103 vs. HC). GAPDH served as a loading control.C The three panels represent autophagy stages found in DM platelets. Autophagosome structures containing/enclosing organelles are indicated by the black box. Shown for each panel is an enlargement of the autophagosome (DM1 and 2) or the autolysosome (DM3). Dashed structures are an outline of the autophagosome membranes.D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures.E Double staining for MitoTracker (mitochondria membrane potential) (50nM for 15 min) and LC3 (autophagy) in HC and DM platelets. Boxed platelets are enlarged in Panel F.F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3.G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV (mitochondria) and LC3 (autophagy marker). The y-axis indicates percentage of LC3 positive cells in total cells (**p=0.006 vs. HC).


Figure 3A Representative western blot analysis of recognized autophagy signaling molecules ( pJNK , JNK , pAKT , and AKT , pmTOR , and mTOR ) in HC ( # 1 - 3 ) and DM ( # 1 - 8 ) patient platelets . Quantification analysis of autophagy upstream signaling molecules on HC ( # 1 - 5 ) and DM ( # 1 - 18 ) individuals . ( pAKT / AKT ; p = 0 . 0747 , pJNK / JNK ; * p = 0 . 001 , pmTOR / mTOR ; p = 0 . 3356 vs . HC ) . GAPDH was used as the loading control . B Representative western blot analysis using HC and HG platelets with or without H2O2 . And assessing for LC3I / II . GAPDH was used as the loading control . C Platelet suspensions were incubated in HG for 2 hours and with H2O2 ( 1mM ) . ROS levels were evaluated using a detection kit ( Enzo Life Science ) for 60 mins at 37 ºC . D Western blot analysis of p53 , JNK and LC3I / II in HC platelets treated with H2O2 ( 1mM for 1 hour ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as the loading control . E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker , CoxIV , and LC3 in HC or H2O2 . The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2 - treated platelets . F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 ( JNK inhibitor ) at 3 concentrations ( 1 , 5 and 10 µM ) to assess for inhibition of autophagy . This Western blot is representative of three independent experiments . Quantification of the three independent experiments is provided at right . ( pJNK / JNK in H2O2 / SP600125 ; * * p = 0 . 0286 vs . H2O2 group , LC3II in H2O2 ; * p = 0 . 0241 vs . HC group , LC3II in H2O2 / SP600125 ; * * p = 0 . 0044 vs . H2O2 group , n = 3 for each group ) . G Triple staining of CoxIV , LC3 , and LAMP1 in HC and H2O2 with / without SP600125 - treated platelets using confocal microscopy . Arrow indicates localization of LC3 and LAMP1 . Graph indicates colocalization between LC3 , LAMP1 and CoxIV signal . The y - axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area . Signal intensity of each group was converted to fold and compared with HC values ( H2O2 ; * p = 0 . 023 vs . HC , H2O2 / SP600125 ; * p = 0 . 04 vs . H2O2 group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control.B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control.C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC.D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control.E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets.F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group).G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).


Figure 4A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53 , p53 , and LAMP1 in HC platelets . Platelets were treated with high glucose ( 25mM ) , with or without CCCP ( 10µM ) . This Western blot is representative of three independent experiments . B Quantification analysis of phosphorylated p53 and LAMP1 ( HG ; * p = 0 . 018 vs DMSO group , HG / CCCP ; * * p = 0 . 001 vs HG group , n = 4 for each group ) . C The recognized autophagy inhibitor ( 2mM 3 - Methyadenine ; 3MA for 1h ) was used to treat DM platelets compared to HC assessing for phosphorylated p53 , p53 , and LC3I / IID Quantification analysis of phosphorylated p53 / p53 was performed . Each value indicated the average band density from a total of three independent samples ( DM / 3MA ; * p = 0 . 026 vs . DM group ) . E Western blot analysis of PLPs transfected with control or ATG3 siRNA , with or without treatment of 1 mM H2O2 . Each lane displayed a different transfected group . F Quantification analysis of ATG3 , phosphorylated p53 ( ser15 ) , p53 , and GAPDH in each group . ( ATG3 in ATG3 siRNA ; * * p = 0 . 0003 vs control group , pp53 in ATG3 siRNA / H2O2 ; * p = 0 . 0145 vs . H2O2 group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53, p53, and LAMP1 in HC platelets. Platelets were treated with high glucose (25mM), with or without CCCP (10µM). This Western blot is representative of three independent experiments.B Quantification analysis of phosphorylated p53 and LAMP1 (HG; *p=0.018 vs DMSO group, HG/CCCP; **p=0.001 vs HG group, n=4 for each group).C The recognized autophagy inhibitor (2mM 3-Methyadenine; 3MA for 1h) was used to treat DM platelets compared to HC assessing for phosphorylated p53, p53, and LC3I/IID Quantification analysis of phosphorylated p53/p53 was performed. Each value indicated the average band density from a total of three independent samples (DM/3MA; *p=0.026 vs. DM group).E Western blot analysis of PLPs transfected with control or ATG3 siRNA, with or without treatment of 1 mM H2O2. Each lane displayed a different transfected group.F Quantification analysis of ATG3, phosphorylated p53 (ser15), p53, and GAPDH in each group. (ATG3 in ATG3 siRNA; **p=0.0003 vs control group, pp53 in ATG3 siRNA/ H2O2; *p=0.0145 vs. H2O2 group).


Figure 5A Western blot analysis of phosphorylated p53 ( ser15 ) , p53 , LC3I / II , and GAPDH in WT mouse platelets . Mouse platelets were treated high glucose ( 25mM ) with or without CCCP ( 10µM ) . B Quantification analysis of phosphorylated p53 ( ser15 ) and LC3 II ( pp53 in HG ; * * p = 0 . 009 vs WT group , pp53 in HG / CCCP ; * p = 0 . 022 vs HG group , LC3 II in HG / CCCP ; * p = 0 . 028 vs HG group , n = 3 for each group ) . C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT _ H2O2 or WT _ H2O2 / 3MA in mouse platelets . ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin - V ( PS externalization ) . Representative of n = 3 . D Graph indicates the percentage of TMRM positive cells in total cell population ( H2O2 ; * * p = 0 . 002 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 008 vs . not treated group , NS means no significance , n = 3 ) . E Graph indicates the percentage of Annexin V positive cell in total cell population ( H2O2 ; NS , p = 0 . 058 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 004 vs . not treated group , * * p = 0 . 008 vs . H2O2 group , NS means no significance , n = 3 ) . F Representative western blot analysis of LC3 I / II in analysis in WT or WT / H2O2 or WT / H2O2 / 3MA in mouse platelets ( n = 3 ) . G Representative western blot analysis of phosphorylated p53 , and Parkin in Parkin - / - mice compared to WT . Quantification analysis of phosphorylated p53 and Parkin ( Parkin ; * * p = 0 . 0001 vs . WT , pp53 ; * p = 0 . 019 vs . WT , n = 4 ) . H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT _ H2O2 and Parkin - / - or Parkin - / - _ H2O2 mouse platelets . ΔΨm was detected using TMRM ( WT _ H2O2 ; * * p = 0 . 002 vs . WT group , Parkin - / - _ H2O2 ; * * p = 1 . 61525E - 06 vs . Parkin - / - group , n = 3 ) I Apoptosis level was assessed with Annexin - V ( PS externalization ) . Graph indicates the percentage of Annexin V positive cell in total cell population ( Parkin - / - : * p = 0 . 010 vs . WT group , Parkin - / - _ H2O2 ; * * p = 0 . 008 vs . WT _ H2O2 ; group , Parkin - / - _ H2O2 ; * * p = 0 . 001 vs . Parkin - / - group , NS means no significance n = 3 ) J Western blot analysis in WT and Parkin - / - demonstrating the inability to induce LC3 despite the addition of H2O2 . Shown graphically are the LC3II to LC3I ratios ( WT _ H2O2 ; * p = 0 . 028 vs . WT group , n = 3 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Western blot analysis of phosphorylated p53 (ser15), p53, LC3I/II, and GAPDH in WT mouse platelets. Mouse platelets were treated high glucose (25mM) with or without CCCP (10µM).B Quantification analysis of phosphorylated p53 (ser15) and LC3 II (pp53 in HG; **p=0.009 vs WT group, pp53 in HG/CCCP ; *p=0.022 vs HG group, LC3 II in HG/CCCP ; *p=0.028 vs HG group, n=3 for each group).C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 or WT_ H2O2/3MA in mouse platelets. ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin-V (PS externalization). Representative of n=3.D Graph indicates the percentage of TMRM positive cells in total cell population (H2O2; **p=0.002 vs. not treated group, H2O2/3MA; **p=0.008 vs. not treated group, NS means no significance, n=3).E Graph indicates the percentage of Annexin V positive cell in total cell population (H2O2; NS, p=0.058 vs. not treated group, H2O2/3MA; **p=0.004 vs. not treated group, **p=0.008 vs. H2O2 group, NS means no significance, n=3).F Representative western blot analysis of LC3 I/II in analysis in WT or WT/H2O2 or WT/H2O2/3MA in mouse platelets (n=3).G Representative western blot analysis of phosphorylated p53, and Parkin in Parkin -/- mice compared to WT. Quantification analysis of phosphorylated p53 and Parkin (Parkin; **p=0.0001 vs. WT, pp53; *p=0.019 vs. WT, n=4).H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 and Parkin -/- or Parkin -/-_H2O2 mouse platelets. ΔΨm was detected using TMRM (WT_ H2O2; **p= 0.002 vs. WT group, Parkin-/-_ H2O2; **p= 1.61525E-06 vs. Parkin-/- group, n=3)I Apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of Annexin V positive cell in total cell population (Parkin-/- : *p=0.010 vs. WT group, Parkin-/-_ H2O2 ;**p= 0.008 vs. WT_ H2O2; group, Parkin-/-_ H2O2; **p=0.001 vs. Parkin-/- group, NS means no significance n=3)J Western blot analysis in WT and Parkin -/- demonstrating the inability to induce LC3 despite the addition of H2O2. Shown graphically are the LC3II to LC3I ratios (WT_ H2O2;*p=0.028 vs. WT group, n=3)


Figure 6A Representative western blot analysis of pp53 in WT ( n = 6 ) , DM ( n = 6 ) and PINK1 - / - DM mice ( n = 3 ) . GAPDH served as the loading control ( DM ; * * p = 0 . 006 vs . WT group , PINK1 - / - DM ; * * p = 0 . 004 vs . DM group ) . B Measurement of p - selectin translocation to the cell membrane by flow cytometry analysis in WT ( n = 3 ) , DM ( n = 3 ) and PINK1 - / - DM ( n = 3 ) miceplatelets . Graphs indicate mean fluorescence intensity ( MFI ) in each group . ( * p < 0 . 05 vs . WT or DM ) . C Quantification of vessel occlusion time after carotid arterial flow measurement ( WT and DM ; and PINK1 - / - : * * p < 0 . 01 vs . WT or DM ) . D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury , indicative of occlusive thrombosis . Flow was plotted as percentage of baseline flow before injury ( DM : n = 3 , PINK1 - / - DM ; n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Representative western blot analysis of pp53 in WT (n=6), DM (n=6) and PINK1-/-DM mice (n=3). GAPDH served as the loading control (DM ; **p=0.006 vs. WT group, PINK1-/-DM; **p=0.004 vs. DM group).B Measurement of p-selectin translocation to the cell membrane by flow cytometry analysis in WT (n=3), DM (n=3) and PINK1 -/- DM (n=3) miceplatelets. Graphs indicate mean fluorescence intensity (MFI) in each group. (*p<0.05 vs. WT or DM).C Quantification of vessel occlusion time after carotid arterial flow measurement (WT and DM; and PINK1 -/- : **p<0.01 vs. WT or DM).D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury, indicative of occlusive thrombosis. Flow was plotted as percentage of baseline flow before injury (DM: n=3, PINK1-/- DM; n=3).


Figure 1A , SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 ( SB ) or LGK974 ( LGK ) for 16 h . Total cell extracts were analyzed by WB . Protein quantification was analyzed by densitometry of three different experiments and represented B , SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB . Quantification of the WBs from three biological replicates are presented in the right panel . Values correspond to fold variation in shRor2 versus shCtl cells ( mean ± SD ) . C , RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT - PCR . Values correspond to fold variation in shRor2 versus shCtl cells . Results are the mean ± SD of at least three biological replicates performed in triplicate . D , SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB . Quantification of the WBs are presented in Appendix Fig S3D as in B .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 (SB) or LGK974 (LGK) for 16 h. Total cell extracts were analyzed by WB. Protein quantification was analyzed by densitometry of three different experiments and represented B, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs from three biological replicates are presented in the right panel. Values correspond to fold variation in shRor2 versus shCtl cells (mean ± SD).C, RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT-PCR. Values correspond to fold variation in shRor2 versus shCtl cells. Results are the mean ± SD of at least three biological replicates performed in triplicate.D, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs are presented in Appendix Fig S3D as in B.


Figure 2E , HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a - producing cells for 16 h and expression of the indicated genes assessed by qRT - PCR ( E ) ( mean ± SD of three biological replicates performed in triplicate in E ) . G , WB analysis of HT29 M6 and SW480 overexpressing Snail1 . Quantification is presented in Appendix Fig S5I . I , SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT - PCR ( I , mean ± SD of three biological replicates performed in triplicate ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2E, HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a-producing cells for 16 h and expression of the indicated genes assessed by qRT-PCR (E) (mean ± SD of three biological replicates performed in triplicate in E).G, WB analysis of HT29 M6 and SW480 overexpressing Snail1. Quantification is presented in Appendix Fig S5I.I, SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT-PCR (I, mean ± SD of three biological replicates performed in triplicate).


Figure 3A , Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells . The quantification is presented in the right panel ( mean ± SD or three biological replicates ) . D , MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D ) for the indicated times . TGFβ receptor inhibitor SB505124 ( SB , 5 μM ) was added when indicated in D . Cells were lysed and proteins analyzed by WB . Quantification of the protein levels from D is represented at the lower panel ( mean ± SD of three biological replicates ) E , GFP - labelled MSC were seeded in Matrigel - coated transwells with SW620 and HCT116 cells , and supplemented with SB when indicated . After 48 h non - invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed . GFP - labelled cells were counted in five different fields per filter by ImageJ software and represented ( bottom ) . A representative picture of SW620 cells in each condition is shown ( top ) . The bar corresponds to 500 μm . The figure shows the mean ± SD of three experiments .

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Figure 3A, Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells. The quantification is presented in the right panel (mean ± SD or three biological replicates).D, MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D) for the indicated times. TGFβ receptor inhibitor SB505124 (SB, 5 μM) was added when indicated in D. Cells were lysed and proteins analyzed by WB. Quantification of the protein levels from D is represented at the lower panel (mean ± SD of three biological replicates)E, GFP-labelled MSC were seeded in Matrigel-coated transwells with SW620 and HCT116 cells, and supplemented with SB when indicated. After 48 h non-invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed. GFP-labelled cells were counted in five different fields per filter by ImageJ software and represented (bottom). A representative picture of SW620 cells in each condition is shown (top). The bar corresponds to 500 μm. The figure shows the mean ± SD of three experiments.


Figure 4C , Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft - agar colony formation ( 21 days ) ( left ) . Representative micrographs of SW620 cells are shown in Appendix Fig S7B . Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments ( right ) . Cells were seeded for tumor - sphere formation in tumor organoid complete medium . In E , SW620 cells were supplemented with LGK ( 10 μM ) , starting the day of seeding . After four days , the wells were stained ( top ) and the number of colonies counted and represented ( bottom ) ( mean ± SD of three biological replicates ) . A more detailed micrograph of the colonies is shown in Appendix Fig S7D . F , RNA was isolated from SW620 control , stably - depleted of Ror2 , treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5 , ASCL2 , SMOC2 and the differentiation marker KRT20 was assessed by qRT - PCR . The figure shows the mean ± SD of at least three biological replicates ( performed in triplicate ) represented with respect to the corresponding control ( dashed line ) .

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Figure 4C, Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft-agar colony formation (21 days) (left). Representative micrographs of SW620 cells are shown in Appendix Fig S7B. Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments (right).Cells were seeded for tumor-sphere formation in tumor organoid complete medium. In E, SW620 cells were supplemented with LGK (10 μM), starting the day of seeding. After four days, the wells were stained (top) and the number of colonies counted and represented (bottom) (mean ± SD of three biological replicates). A more detailed micrograph of the colonies is shown in Appendix Fig S7D.F, RNA was isolated from SW620 control, stably-depleted of Ror2, treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5, ASCL2, SMOC2 and the differentiation marker KRT20 was assessed by qRT-PCR. The figure shows the mean ± SD of at least three biological replicates (performed in triplicate) represented with respect to the corresponding control (dashed line).


Figure 5B , C , Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin - embedded tumors obtained when tumors were initially detected ( days 18 or 22 ) . Staining with an anti - human antibody is also enclosed . The scale bar corresponds to 10 μm ( B ) . Fibronectin and Vimentin staining were quantified as described in Methods ( six tumors , three areas per tumor ) and represented in C ( mean ± SD ) . D , Representative images of the invading front of the tumors . The scale bar corresponds to 4 μM . E , Percentage of tumors showing muscle invasion .

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Figure 5B, C, Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin-embedded tumors obtained when tumors were initially detected (days 18 or 22). Staining with an anti-human antibody is also enclosed. The scale bar corresponds to 10 μm (B). Fibronectin and Vimentin staining were quantified as described in Methods (six tumors, three areas per tumor) and represented in C (mean ± SD).D, Representative images of the invading front of the tumors. The scale bar corresponds to 4 μM. E, Percentage of tumors showing muscle invasion.


Figure 6F , G , In vivo tumor growth of MTO cells subcutaneously injected in NOD / SCID mice ( F ) ( 5x105 cells / flank ; eight tumors per condition ) and treated with WntC59 ( 10 mg / kg / day in the drinking water ) or vehicle . Tumor weight of NOD / SCID mice at day 26 is presented in G . In F and G , the mean ± SD of the eight tumors is shown . I , J , WB ( I ) and RNA analysis ( J ) of MTO cells infected with shRor2 or shCtl . Quantification of the WB is also presented ( I , right ) . Results are the mean ± SD of three biological replicates ( performed in triplicated in J ) . N , O , C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle . In N , kinetics of in vivo luminescence ( mean ± SD of the values in five mice ) ; in O , the percentage of mice showing in vivo luminescence .

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Figure 6F, G, In vivo tumor growth of MTO cells subcutaneously injected in NOD/SCID mice (F) (5x105 cells/flank; eight tumors per condition) and treated with WntC59 (10 mg/kg/day in the drinking water) or vehicle. Tumor weight of NOD/SCID mice at day 26 is presented in G. In F and G, the mean ± SD of the eight tumors is shown.I, J, WB (I) and RNA analysis (J) of MTO cells infected with shRor2 or shCtl. Quantification of the WB is also presented (I, right). Results are the mean ± SD of three biological replicates (performed in triplicated in J).N, O, C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle. In N, kinetics of in vivo luminescence (mean ± SD of the values in five mice); in O, the percentage of mice showing in vivo luminescence.


Figure 7A - C , Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h . In B , cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid . The figure shows the mean ± SD of the results of three experiments . D , Cells were treated with cisplatin for the indicated times . Total extracts were lysed and analyzed by WB . I , MTO cell viability was determined as above ( mean ± SD of the results of three - four experiments ) .

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Figure 7A-C, Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h. In B, cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid. The figure shows the mean ± SD of the results of three experiments.D, Cells were treated with cisplatin for the indicated times. Total extracts were lysed and analyzed by WB.I, MTO cell viability was determined as above (mean ± SD of the results of three-four experiments).


Figure 1Quantitative RT - PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius ( GC ) and Tibialis Anterior ( TA ) , but not in White Adipose Tissue ( WAT ) from 3 - month - old HSACre / + / Lpin1fEx2 - 3 / fEx2 - 3 mice ( HSA - Cre ) compared to control mice . Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice . PAP1 and PAP2 activities were respectively substantially decreased and increased in TA , of HSA - Cre mice as compared to control mice , respectively ( n = 6 ; * P < 0 . 05 ) . Measurements of muscle / body weight ratio of GC and TA of 3 - month - old HSA - Cre and control mice . Hematoxylin and Eosin ( HE ) staining of representative transverse sections of GC muscles from 3 - month - old HSA - Cre and control mice . The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration . Scale bars : 40 μmRelative force measurements after normalization of absolute tetanic force to GC muscle weight from 3 - month - old HSA - Cre and control male mice .

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Figure 1Quantitative RT-PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius (GC) and Tibialis Anterior (TA), but not in White Adipose Tissue (WAT) from 3-month-old HSACre/+/Lpin1fEx2-3/fEx2-3 mice (HSA-Cre) compared to control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.PAP1 and PAP2 activities were respectively substantially decreased and increased in TA, of HSA-Cre mice as compared to control mice, respectively (n = 6; *P<0.05).Measurements of muscle/body weight ratio of GC and TA of 3-month-old HSA-Cre and control mice.Hematoxylin and Eosin (HE) staining of representative transverse sections of GC muscles from 3-month-old HSA-Cre and control mice. The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration. Scale bars: 40 μmRelative force measurements after normalization of absolute tetanic force to GC muscle weight from 3-month-old HSA-Cre and control male mice.


Figure 2Phosphatidic Acid ( PA ) levels in HSA - Cre and control GC muscles . Phospholipidome in HSA - Cre and control GC muscles . Proportions of indicated phospholipid classes in total phospholipids are represented . PC ; Phosphatidylcholine , PE ; Phosphatidylethanolamine , PI ; Phosphatidylinositol , PS ; Phoshpatidylserine , PA ; Phosphatidic Acid , PG ; Phosphatidylglycerol . Amounts of indicated phospholipid classes normalized by protein content in HSA - Cre and control GC muscles . ( D ) Total phospholipid ( PL ) amount normalized by protein content in HSA - Cre and control GC muscles . Distribution of the total carbon number in acyl chains of PC in HSA - Cre and control GC muscles . Distribution of the number of double bonds in acyl chains of PC in HSA - Cre and control GC muscles . Distribution of the total carbon number in acyl chains of PE in HSA - Cre and control GC muscles . Distribution of the number of double bonds in acyl chains of PE in HSA - Cre and control GC muscles .

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Figure 2Phosphatidic Acid (PA) levels in HSA-Cre and control GC muscles.Phospholipidome in HSA-Cre and control GC muscles. Proportions of indicated phospholipid classes in total phospholipids are represented. PC; Phosphatidylcholine, PE; Phosphatidylethanolamine, PI; Phosphatidylinositol, PS; Phoshpatidylserine, PA; Phosphatidic Acid, PG; Phosphatidylglycerol.Amounts of indicated phospholipid classes normalized by protein content in HSA-Cre and control GC muscles. (D) Total phospholipid (PL) amount normalized by protein content in HSA-Cre and control GC muscles.Distribution of the total carbon number in acyl chains of PC in HSA-Cre and control GC muscles.Distribution of the number of double bonds in acyl chains of PC in HSA-Cre and control GC muscles.Distribution of the total carbon number in acyl chains of PE in HSA-Cre and control GC muscles.Distribution of the number of double bonds in acyl chains of PE in HSA-Cre and control GC muscles.


Figure 3HE , Oil Red O ( ORO ) , Cytochrome c Oxidase ( COX ) and Succinate Deshydrogenase ( SDH ) staining of representative frozen sections of TA muscle from 3 - month - old HSA - Cre and control mice . Quantification of triacylglycerol ( TAG ) levels in GC muscles from 3 - month - old HSA - Cre and and control mice . Content of TAG assessed by MS in HSA - Cre and control GC muscles . Cholesterol , ( E ) Cholesteryl ester ( CE ) , ( F ) DAG was assessed by MS in HSA - Cre and control GC muscles .

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Figure 3HE, Oil Red O (ORO), Cytochrome c Oxidase (COX) and Succinate Deshydrogenase (SDH) staining of representative frozen sections of TA muscle from 3-month-old HSA-Cre and control mice.Quantification of triacylglycerol (TAG) levels in GC muscles from 3-month-old HSA-Cre and and control mice.Content of TAG assessed by MS in HSA-Cre and control GC muscles.Cholesterol, (E) Cholesteryl ester (CE), (F) DAG was assessed by MS in HSA-Cre and control GC muscles.


Figure 4Quantitative RT - PCR detection of the level of expression of Fatty acid synthase ( Fasn ) , ATP citrate lyase ( Acly ) , Thyroid hormone - inducible hepatic protein 1 ( Thrsp1 ) , Angiopoietin - like protein 4 / fast - induced adipose factor ( ANGPTL4 / Fiaf ) , Insulin - induced gene 1 ( Insig1 ) and Peroxisome proliferator - activated receptor gamma ( PPARγ ) in TA from 3 - month - old HSA - Cre and control mice . Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice . Immunoblot analysis of Fasn in TA muscle of 3 - month - old HSA - Cre and control mice . Quantitative RT - PCR measurement of Carnitine palmitoyltransferase 1B ( Cpt1b ) and Medium - chain acyl - CoA dehydrogenase ( Mcad ) in TA from 3 - month - old HSA - Cre and control mice . Immunoblot analysis of full length and N - terminal SREBP1c and SREBP2 protein levels in GC muscle of 4 - month - old HSA - Cre and control mice ( n = 7 - 4 ) . The ratio of the densitometric assay of the N - term and Full length forms is presented .

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Figure 4Quantitative RT-PCR detection of the level of expression of Fatty acid synthase (Fasn), ATP citrate lyase (Acly), Thyroid hormone-inducible hepatic protein 1 (Thrsp1), Angiopoietin-like protein 4/ fast-induced adipose factor (ANGPTL4/Fiaf), Insulin-induced gene 1 (Insig1) and Peroxisome proliferator-activated receptor gamma (PPARγ) in TA from 3-month-old HSA-Cre and control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.Immunoblot analysis of Fasn in TA muscle of 3-month-old HSA-Cre and control mice.Quantitative RT-PCR measurement of Carnitine palmitoyltransferase 1B (Cpt1b) and Medium-chain acyl-CoA dehydrogenase (Mcad) in TA from 3-month-old HSA-Cre and control mice.Immunoblot analysis of full length and N-terminal SREBP1c and SREBP2 protein levels in GC muscle of 4-month-old HSA-Cre and control mice (n=7-4). The ratio of the densitometric assay of the N-term and Full length forms is presented.


Figure 5Metabolomics profile of GC muscle of 4 - month - old HSA - Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of compoundsFold change of phospholipid precursors in GC muscles of 4 - month - old HSA - Cre and control mice measured by liquid - chromatography ( LC ) tandem mass spectrometry ( MS / MS ) ( n = 7 - 4 , * P < 0 . 05 ) . Fold change of Lactate / Pyruvate , Pyruvate / AcetylCoA , Pyruvate / Citrate , ATP / AMP , and ATP / ADP ratios in GC muscles of 4 month - old HSA - Cre and control mice ( n = 7 - 4 , * P < 0 . 05 ) .

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Figure 5Metabolomics profile of GC muscle of 4-month-old HSA-Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of compoundsFold change of phospholipid precursors in GC muscles of 4-month-old HSA-Cre and control mice measured by liquid-chromatography (LC) tandem mass spectrometry (MS/MS) (n=7-4, *P<0.05).Fold change of Lactate/Pyruvate, Pyruvate/AcetylCoA, Pyruvate/Citrate, ATP/AMP, and ATP/ADP ratios in GC muscles of 4 month-old HSA-Cre and control mice (n=7-4, *P<0.05).


Figure 6UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4 - month - old HSA - Cre and control mice ( n = 7 - 4 ) . Quantification by densitometric analyses of Fgf21 , Bip , sXbp1 Atf6 cleaved form , p - Eif2a and Chop protein levels is presented as a graph . Data are normalized to Gapdh and expressed as a fold change relative to the control mice . Quantitative RT - PCR measurement of Fgf21 , Gdf15 , Bip / Grp78 , hsp90b1 , and Edem1 in GC muscles from 4 - month - old HSA - Cre and control mice . EM analysis of EDL fibers of HSA - Cre mice revealed the presence of areas in which the non - junctional SR is more vesiculated and fragmented compared to control ( black arrows ) . Remodeling of the SR is also accompanied by modification in the positioning of mitochondria ( white arrows ) . A representative Calcium Release Unit ( CRU ) is indicated ( empty arrow ) . Scale bars : 0 . 50 μm .

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Figure 6UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4-month-old HSA-Cre and control mice (n=7-4). Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p-Eif2a and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice.Quantitative RT-PCR measurement of Fgf21, Gdf15, Bip/Grp78, hsp90b1, and Edem1 in GC muscles from 4-month-old HSA-Cre and control mice.EM analysis of EDL fibers of HSA-Cre mice revealed the presence of areas in which the non-junctional SR is more vesiculated and fragmented compared to control (black arrows). Remodeling of the SR is also accompanied by modification in the positioning of mitochondria (white arrows). A representative Calcium Release Unit (CRU) is indicated (empty arrow). Scale bars: 0.50 μm.


Figure 7The relative mtDNA copy number was determined by the ratio of mitochondrial DNA - encoded Cytochrome B ( CytB ) and nuclear - encoded NADH dehydrogenase ( ubiquinone ) flavoprotein 1 ( Ndufv1 ) genes in GC and TA from HSA - Cre and control mice . Mitochondria - encoded gene and Nuclear - encoded gene expression levels by quantitative RT - PCR in GC muscles from 4 - month - old HSA - Cre and control mice . Electron micrographs of HSA - Cre EDL muscles of 3 - month - old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice ( black arrows ) . On the other hand , in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron - dense contact sites ( white arrows ) . Scale bars : 0 . 20 μm . Ultrastructural analysis of HSA - Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies ( autophagosomes and lysosomes , black arrows ; multilamellar bodies , white arrow ) containing amorphous material and possibly degraded mitochondria ( M ) . Scale bars : 0 . 50

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Figure 7The relative mtDNA copy number was determined by the ratio of mitochondrial DNA-encoded Cytochrome B (CytB) and nuclear-encoded NADH dehydrogenase (ubiquinone) flavoprotein 1 (Ndufv1) genes in GC and TA from HSA-Cre and control mice.Mitochondria-encoded gene and Nuclear-encoded gene expression levels by quantitative RT-PCR in GC muscles from 4-month-old HSA-Cre and control mice.Electron micrographs of HSA-Cre EDL muscles of 3-month-old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice (black arrows). On the other hand, in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron-dense contact sites (white arrows). Scale bars: 0.20 μm.Ultrastructural analysis of HSA-Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies (autophagosomes and lysosomes, black arrows; multilamellar bodies, white arrow) containing amorphous material and possibly degraded mitochondria (M). Scale bars: 0.50 μm


Figure 8Representative immunoblots from protein extract of GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice , with the indicated antibodies . Quantitative RT - PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice . Histological analysis of TA muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice by Oil Red O staining to detect the neutral lipids . Scale bars : 40 μm . TAG level measurement in GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice . In vivo force measurements performed on GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice . Representative staining of IgG positive fibers on frozen sections of TA of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice . The sections were stained with a secondary fluorescent anti - mouse IgG antibody . Quantification of the number of necrotic fibers over the total number of fibers .

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Figure 8Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies.Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.Histological analysis of TA muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm.TAG level measurement in GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.In vivo force measurements performed on GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.Representative staining of IgG positive fibers on frozen sections of TA of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.


Figure 9Representative immunoblots from protein extract of GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice , with the indicated antibodies . Quantitative RT - PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice . Histological analysis of TA muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice by Oil Red O staining to detect the neutral lipids . TAG level measurement in GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice . In vivo force measurements performed on GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice . Representative staining of IgG positive fibers on frozen sections of TA of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice . The sections were stained with a secondary fluorescent anti - mouse IgG antibody . Quantification of the number of necrotic fibers over the total number of fibers .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9Representative immunoblots from protein extract of GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice, with the indicated antibodies.Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.Histological analysis of TA muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice by Oil Red O staining to detect the neutral lipids.TAG level measurement in GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.In vivo force measurements performed on GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.Representative staining of IgG positive fibers on frozen sections of TA of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.


figf1 ( a , b ) Immunoblot analysis of LAMP - 2A in total lysates ( a ) and quantitative PCR analysis of Lamp2a mRNA ( b ) of naive CD4 + T cells and TH1 and TH2 cells following activation with anti - CD3 and anti - CD28 . β - actin ( a ) serves as a loading control throughout ; mRNA results ( b ) are presented relative to those of resting cells . ( c ) Immunofluorescence microscopy of LAMP - 2A ( green ) and nuclei ( stained with the DNA - binding dye DAPI ; blue ) in resting TH1 cells ( Rest ) and TH1 cells activated for 24 h as in a , b ( Act ) ( left ) , and quantification of lysosomes in cells as at left ( right ) . Original magnification , × 630 ; scale bar , 10 μm . * P = 0 . 013 ( t - test ) . ( d ) Electron microscopy ( left ) of resting TH1 cells and TH1 cells activated for 24 h as in a , b ( top row ) ; below , enlargement of areas outlined above , showing individual lysosomes ( arrows ) . Original magnification ( top row ) , × 12 , 000 ; scale bars ( bottom row ) , 1 μm . Right , morphometric analysis of cells as at left . * P = 0 . 037 and * * P = 0 . 034 ( t - test ) . ( e ) Distribution of KFERQ - PA - mCherry - 1 in TH1 cells transiently transfected with a CMA reporter vector , photoactivated , and left unstimulated ( Rest ) or stimulated with anti - CD3 and anti - CD28 in the presence ( 3 - MA ) or absence ( vehicle ( veh ) ) of 3 - methyladenine ( left ) ; arrows indicate mCherry + puncta . Original magnification , × 630 . Right , quantification of the results for the cells at left . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) . ( f ) Immunoblot analysis of hsc70 in lysosomal fractions of resting ( Lys - R ) and stimulated ( Lys - A ) TH1 cells and in cytosolic fractions of TH1 cells activated as in a , b ( Cyto ) ; LAMP - 2A and LAMP - 1 serve as controls . ( g ) Immunoblot analysis of LAMP - 2A in resting TH1 cells and TH1 cells activated in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine . ( h ) Quantitative PCR analysis of Lamp2a mRNA in cells as in g ( results presented as in b ) . * P = 0 . 007 ( t test ) . ( i ) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0 , 12 or 24 h with plate - bound anti - CD3 and anti - CD28 ; results are presented relative to those of resting cells ( time 0 ) . ( j ) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated ( control ( Ctrl ) ) or stimulated for 16 h with 50 μM paraquat ( Pq ) or 1 mM H2O2 ; results are presented in relative light units ( RLU ) . ( k ) Immunoblot analysis of LAMP - 2A in total lysates of resting TH1 cells ( − ) and TH1 cells activated for 24 h as in a , b ( + ) , transfected with a control nontargeting siRNA ( with a scrambled sequence ( Scr ) ) or siRNA specific for mRNA encoding Duox1 ( either of two siRNAs : Dx ( 1 ) and Dx ( 2 ) ) or RISP ( Uq ( 1 ) ) . ( l ) Immunoblot analysis of LAMP - 2A in lysates of resting or activated TH1 cells in the presence ( right half ) or absence ( left half ) of cyclosporine A ( CsA ) . ( m ) Chromatin - immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 ( negative control ) ( left ) or to Il2 ( positive control ) ( right ) in resting and activated TH1 cells ; results are presented relative to input chromatin precipitated with anti - NFAT1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a,b) Immunoblot analysis of LAMP-2A in total lysates (a) and quantitative PCR analysis of Lamp2a mRNA (b) of naive CD4+ T cells and TH1 and TH2 cells following activation with anti-CD3 and anti-CD28. β-actin (a) serves as a loading control throughout; mRNA results (b) are presented relative to those of resting cells.(c) Immunofluorescence microscopy of LAMP-2A (green) and nuclei (stained with the DNA-binding dye DAPI; blue) in resting TH1 cells (Rest) and TH1 cells activated for 24 h as in a,b (Act) (left), and quantification of lysosomes in cells as at left (right). Original magnification, ×630; scale bar, 10 μm. *P = 0.013 (t-test).(d) Electron microscopy (left) of resting TH1 cells and TH1 cells activated for 24 h as in a,b (top row); below, enlargement of areas outlined above, showing individual lysosomes (arrows). Original magnification (top row), ×12,000; scale bars (bottom row), 1 μm. Right, morphometric analysis of cells as at left. *P = 0.037 and **P = 0.034 (t-test).(e) Distribution of KFERQ-PA-mCherry-1 in TH1 cells transiently transfected with a CMA reporter vector, photoactivated, and left unstimulated (Rest) or stimulated with anti-CD3 and anti-CD28 in the presence (3-MA) or absence (vehicle (veh)) of 3-methyladenine (left); arrows indicate mCherry+ puncta. Original magnification, ×630. Right, quantification of the results for the cells at left. *P 0.05 (analysis of variance with Tukey's post-test).(f) Immunoblot analysis of hsc70 in lysosomal fractions of resting (Lys-R) and stimulated (Lys-A) TH1 cells and in cytosolic fractions of TH1 cells activated as in a,b (Cyto); LAMP-2A and LAMP-1 serve as controls.(g) Immunoblot analysis of LAMP-2A in resting TH1 cells and TH1 cells activated in the presence (NAc) or absence (Veh) of N-acetylcysteine. (h) Quantitative PCR analysis of Lamp2a mRNA in cells as in g (results presented as in b). *P = 0.007 (t test).(i) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0, 12 or 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to those of resting cells (time 0).(j) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated (control (Ctrl)) or stimulated for 16 h with 50 μM paraquat (Pq) or 1 mM H2O2; results are presented in relative light units (RLU).(k) Immunoblot analysis of LAMP-2A in total lysates of resting TH1 cells (−) and TH1 cells activated for 24 h as in a,b (+), transfected with a control nontargeting siRNA (with a scrambled sequence (Scr)) or siRNA specific for mRNA encoding Duox1 (either of two siRNAs: Dx(1) and Dx(2)) or RISP (Uq(1)).(l) Immunoblot analysis of LAMP-2A in lysates of resting or activated TH1 cells in the presence (right half) or absence (left half) of cyclosporine A (CsA).(m) Chromatin-immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 (negative control) (left) or to Il2 (positive control) (right) in resting and activated TH1 cells; results are presented relative to input chromatin precipitated with anti-NFAT1.


figf2 ( a ) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA ( Scr ) or Lamp2a mRNA - specific shRNA ( shL2A ) ( left ) , and immunoblot analysis of LAMP - 2A in sorted GFP + T cells ( right ) . Numbers above bracketed lines ( left ) indicate percent GFP + cells . ( b ) Enzyme - linked immunosorbent assay of IL - 2 and interferon - γ ( IFN - γ ) in TH1 cells transduced as in a and then left unstimulated ( Rest ) or stimulated for 24 h with anti - CD3 and anti - CD28 ( Act ) . * P = 0 . 038 and * * P = 0 . 031 ( t - test ) . ( c ) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b , assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm ( A495 ) . * P = 0 . 013 ( t - test ) . ( d ) Quantification of annexin V - positive ( AnnV + ) cells among resting or stimulated TH1 cells transduced as in a .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA-specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells.(b) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test).(c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test).(d) Quantification of annexin V-positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a.


figf3 ( a ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti - CD3 and anti - CD28 ; LAMP - 1 serves as a control . ( b ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with ( + ) or without ( − ) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin ( Lys inh ) ; LAMP - 1 serves as a control . ( c ) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine . ( d ) Immunoblot analysis of lysates of TH1 cells transfected with GFP - Itch or GFP - mu - Itch ( top ) or with Myc - RCAN1 or Myc - mu - RCAN1 ( bottom ) and cotransfected with a GFP expression plasmid ( to assess transfection efficiency ) , then activated for 24 h as in a . ( e , f ) Proliferation ( e ) and IL - 2 production ( f ) of TH1 cells transfected with plasmid expressing GFP ( Ctrl ) or plasmids as in c and left unstimulated ( Rest ) or activated for 48 h ( e ) or 24 h ( f ) as in a ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 019 and * * P = 0 . 0034 ( e ) or * P = 0 . 046 and * * P = 0 . 044 ( f ) ( t test ) . Data are representative of four ( a , b ) , two ( c ) or three ( d ) independent experiments or are from three independent experiments ( e , f ; mean and s . e . m . ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control.(b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control.(c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine.(d) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP-mu-Itch (top) or with Myc-RCAN1 or Myc-mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a.(e,f) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).


figf4 ( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells ( left ) and liver , kidneys , thymus and lungs ( right ) of L2A - cKO mice ( KO ) and their control littermates ( Ctrl ) . ( b ) Quantitative PCR analysis of Lamp2a , Lamp2b and Lamp2c mRNA in T cells from L2A - cKO mice ( right ) ; results are presented relative to those of their control littermates ( left ) , set as 1 . ( c ) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form ( LC3 - I ) and its autophagosome membrane - associated form ( LC3 - II ) in total lysates of CD4 + T cells obtained from L2A - cKO mice or their control littermates and then left unstimulated ( Rest ) or activated for 24 h with anti - CD3 and anti - CD218 ( Act ) , cultured for the final 4 h in the presence ( + ) or absence ( − ) of NH4Cl and leupeptin ( NH4Cl + Leup ) . ( d ) Spleens from an L2A - cKO mouse and its control littermate ( left ) , and flow cytometry of peripheral T cells from such mice ( right ) . Numbers in quadrants ( right ) indicate percent CD4 − CD8 + T cells ( top left ) or CD4 + CD8 − T cells ( bottom right ) . ( e , f ) Proliferation ( e ) and IL - 2 production ( f ) by CD4 + T cells isolated from the spleen of L2A - cKO mice and their control littermates and stimulated ex vivo for 48 h with anti - CD3 and anti - CD28 ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 0062 ( e ) or 0 . 006 ( f ) ( t - test ) . ( g ) Immunoblot analysis of LAMP - 2A , Itch and RCAN1 in total lysates of CD4 + T cells obtained from L2A - cKO mice ( n = 3 ( KO1 , KO2 and KO3 ) ) and their control littermates ( n = 3 ) and activated for 24 h as in e , f . * , nonspecific band . ( h ) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4 + T cells isolated from L2A - cKO mice and their control littermates and activated for 24 h as in e , f ; results are presented relative to those of resting cells . ( i , j ) Recall responses to OVA ( 323 - 339 ) in draining lymph nodes isolated from L2A - cKO mice and their control littermates after immunization with OVA ( 323 - 339 ) , assessed as proliferation ( i ) and IL - 2 production ( j ) ( as in e , f ) . * P = 0 . 0054 ( i ) or 0 . 0052 ( j ) ( t - test ) . ( k , l ) Recall responses to L . monocytogenes in CD4 + T cells isolated from L2A - cKO mice and their control littermates ( n = 6 per group ) previously uninfected ( UI ) or previously infected with L . monocytogenes ( Inf ) , assessed as IFN - γ production ( k ) and proliferation ( l ) ( as in e , f ) . * P = 0 . 032 ( k ) or 0 . 02 ( l ) ( t - test ) . ( m ) L . monocytogenes in spleen and liver of L2A - cKO mice and their control littermates ( n = 3 per genotype ) immunized with L . monocytogenes and then reinfected with a high dose of L . monocytogenes . CFU , colony - forming units . * P = 0 . 008 and * * P = 0 . 001 ( t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Immunoblot analysis of LAMP-2A in CD4+ T cells (left) and liver, kidneys, thymus and lungs (right) of L2A-cKO mice (KO) and their control littermates (Ctrl).(b) Quantitative PCR analysis of Lamp2a, Lamp2b and Lamp2c mRNA in T cells from L2A-cKO mice (right); results are presented relative to those of their control littermates (left), set as 1.(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).(d) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right).(e,f) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test).(g) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band.(h) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells.(i,j) Recall responses to OVA(323-339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323-339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test).(k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test).(m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test).


figf5 ( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells obtained from 4 - month - old or 22 - month old mice , then left resting or stimulated for 24 h with anti - CD3 plus anti - CD28 . ( b ) Immunoblot analysis of RCAN1 and Itch in CD4 + T cells obtained from 4 - month - old or 22 - month old mice ( n = 3 per group ) , then stimulated for 24 h as in a ( left ) , and quantification of protein in 22 - month old mice , presented relative to that of 4 - month - old mice , set as 100 % . * P = 0 . 044 ( t - test ) . ( c ) Immunoblot analysis of LAMP - 2A in total lysates of naive and memory CD4 + T cells left resting or activated for 24 h as in a . ( d ) Immunoblot analysis ( bottom ) of LAMP - 2A in total lysates of naive and memory CD4 + T cells obtained from 4 - or 22 - month - old mice and left resting ( R ) or activated for 24 h as in a ( A ) ; above , quantification from results below ( n = 3 mice per group ) . * P = 0 . 009 ( t - test ) . ( e ) Electrophoretic mobility - shift assay of the binding of NFAT ( N ) and free probe ( FP ) in nuclear extracts of CD4 + T cells isolated from 3 - , 12 - or 22 - month - old mice and activated for 4 h as in a . ( f ) ROS production in CD4 + T cells isolated from 4 - or 22 - month - old mice ( n = 3 per group ) and left resting or activated for 12 h as in a , assessed by flow cytometry and presented as the mean fluorescence intensity ( MFI ) of a fluorogenic probe for oxidative stress ( CellROX Green ) . ( g - i ) Immunoblot analysis of LAMP - 2A ( g ) and analysis of the proliferation ( h ) and IL - 2 production ( i ) of TH1 cells obtained from 4 - or 22 - month - old mice and transduced with retrovirus expressing GFP alone ( RV - GFP ) or human LAMP - 2A and GFP ( RV - LAMP2A ) , sorted by GFP expression , cultured for 2 weeks and then stimulated for 24 - 48 h as in a ( assessed as in Fig . 2c ( h ) or Fig . 2b ( i ) ) . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) . ( j ) Immunoblot analysis of LAMP - 2A in total lysates of CD4 + T cells obtained from two human donors ( A and B ) and left resting or activated for 24 or 48 h as in a ( above lanes ) . ( k ) Real - time PCR analysis of LAMP2A mRNA in naive and memory CD4 + T cells obtained from young human donors ( 26 ± 2 years of age ; n = 3 ) or old human donors ( 72 ± 6 years of age ; n = 6 ) and activated for 24 h as in a ; results were calculated by the change - in - cycle - threshold method ( 2 − ΔCt ) relative to those of the gene encoding β - actin . * P = 0 . 047 and * * P = 0 . 0033 ( Mann - Whitney ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.(b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test).(c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a.(d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test).(e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).(g-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test).(j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes).(k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).


Figure 1A , B . Western blot showing the absence of HUWE1 protein in 293T ( A ) and HeLa ( B ) cells subjected to CRISPR / Cas9 - mediated HUWE1 - deletion . C , D . HUWE1 - knockout 293T ( C ) and HeLa ( D ) cells show increased DNA breaks in the absence of exogenous DNA damage treatment . Results from the Alkaline Comet Assay are shown . The " n " numbers of comet tails analyzed ( pooled from two independent experiments ) , as well as the mean , are indicated on the graphs . HUWE1 - knockout HeLa cells did not show increases breakage in the Neutral Comet assay ( Fig EV1B ) , indicating that the majority of breaks observed in cycling HUWE1 - knockout cells are not double strand breaks , but rather single strand breaks and other types of lesions . E , F . Increased S - phase arrest in HUWE1 - knockout 293T ( E ) and HeLa ( F ) cells . Cycling cells were incubated with BrdU and subjected to BrdU / PI bi - dimensional flow cytometry . Representative flow cytometry profiles are presented in Fig EV1D - F . Bars represent the fold increase in the percentage of cells with S - phase DNA content ( between 2N and 4N ) but negative for BrdU staining . Bars represent the average of three independent experiments , with error bars showing SD . The p - values are 0 . 0091 ( E ) and 0 . 0007 ( F ) . G , H . The DNA fiber assay shows reduced replication tract length in HUWE1 - knockout 293T ( G ) and HeLa ( H ) cells in the absence of exogenous DNA damage treatment . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . P - values are 9 . 8x10 - 22 ( G ) and 1 . 0x10 - 10 ( H ) .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B. Western blot showing the absence of HUWE1 protein in 293T (A) and HeLa (B) cells subjected to CRISPR/Cas9-mediated HUWE1-deletion.C, D. HUWE1-knockout 293T (C) and HeLa (D) cells show increased DNA breaks in the absence of exogenous DNA damage treatment. Results from the Alkaline Comet Assay are shown. The "n" numbers of comet tails analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs. HUWE1-knockout HeLa cells did not show increases breakage in the Neutral Comet assay (Fig EV1B), indicating that the majority of breaks observed in cycling HUWE1-knockout cells are not double strand breaks, but rather single strand breaks and other types of lesions.E, F. Increased S-phase arrest in HUWE1-knockout 293T (E) and HeLa (F) cells. Cycling cells were incubated with BrdU and subjected to BrdU/PI bi-dimensional flow cytometry. Representative flow cytometry profiles are presented in Fig EV1D-F. Bars represent the fold increase in the percentage of cells with S-phase DNA content (between 2N and 4N) but negative for BrdU staining. Bars represent the average of three independent experiments, with error bars showing SD. The p-values are 0.0091 (E) and 0.0007 (F).G, H. The DNA fiber assay shows reduced replication tract length in HUWE1-knockout 293T (G) and HeLa (H) cells in the absence of exogenous DNA damage treatment. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-values are 9.8x10-22 (G) and 1.0x10-10 (H).


Figure 2A . HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells . B . Quantification of HUWE1 expression following siRNA treatment . Band intensity was quantified using ImageJ software and normalized to Tubulin loading control . The average of three experiments is shown . Error bars indicate SD . C - E . Cell cycle analyses by flow cytometry using BrdU / PI double staining show increased replication arrest in HUWE1 - depleted 8988T cells in the absence of exogenous DNA damage treatment . C . Representative flow cytometry profiles of control and HUWE1 - knockdown cells . R1 - labeled region indicates mid and late S - phase cells ( BrdU - positive , > 2N DNA content ) , while R2 - labeled region indicates S - phase arrested cells ( BrdU - negative , DNA content between 2N and 4N ) . C - E . Cell cycle analyses by flow cytometry using BrdU / PI double staining show increased replication arrest in HUWE1 - depleted 8988T cells in the absence of exogenous DNA damage treatment . C . Representative flow cytometry profiles of control and HUWE1 - knockdown cells . R1 - labeled region indicates mid and late S - phase cells ( BrdU - positive , > 2N DNA content ) , while R2 - labeled region indicates S - phase arrested cells ( BrdU - negative , DNA content between 2N and 4N ) . D . Quantification of S - phase cells . Percentage of cells in R1 region is shown . Bars represent the average of three independent experiments . Error bars indicate SD . P - value is 0 . 0014 . E . Quantification of S - phase arrested cells . Bars represent the fold increase in the percentage of cells in R2 region , normalized to siControl - treated cells . The average of three independent experiments is shown . Error bars indicate SD . P - value is 0 . 0135 . F . DNA fiber experiment showing reduced replication tract length in HUWE1 - depleted HeLa cells in the absence of exogenous DNA damage treatment . The " n " numbers of fibers analyzed ( pooled from two independent experiments ) , as well as the mean , are indicated on the graphs . G , H . Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV ( G ) , and HU ( H ) . As controls , both cells transfected with non - targeting siRNA ( siControl ) , and non - transfected cells , were used . Note that the siHUWE1 # 2 oligonucleotide shows a stronger downregulation of HUWE1 , and confers increased sensitivity compared to siHUWE1 # 1 . The average of nine experiments is shown . Error bars represent SEM . P - values ( representing the statistical difference between the samples at the highest dose treatment ) are : 4 . 55x10 - 9 ( G ) and 1 . 8x10 - 8 ( H ) . I . DNA fiber experiments showing decreased replication tract length following UV - B treatment ( 30J / m2 ) in HUWE1 - depleted HeLa cells . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . P - value is 2 . 11x10 - 20 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells. B. Quantification of HUWE1 expression following siRNA treatment. Band intensity was quantified using ImageJ software and normalized to Tubulin loading control. The average of three experiments is shown. Error bars indicate SD.C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N).C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N). D. Quantification of S-phase cells. Percentage of cells in R1 region is shown. Bars represent the average of three independent experiments. Error bars indicate SD. P-value is 0.0014. E. Quantification of S-phase arrested cells. Bars represent the fold increase in the percentage of cells in R2 region, normalized to siControl-treated cells. The average of three independent experiments is shown. Error bars indicate SD. P-value is 0.0135.F. DNA fiber experiment showing reduced replication tract length in HUWE1-depleted HeLa cells in the absence of exogenous DNA damage treatment. The "n" numbers of fibers analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs.G, H. Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV (G), and HU (H). As controls, both cells transfected with non-targeting siRNA (siControl), and non- transfected cells, were used. Note that the siHUWE1#2 oligonucleotide shows a stronger downregulation of HUWE1, and confers increased sensitivity compared to siHUWE1#1. The average of nine experiments is shown. Error bars represent SEM. P-values (representing the statistical difference between the samples at the highest dose treatment) are: 4.55x10-9 (G) and 1.8x10-8 (H).I. DNA fiber experiments showing decreased replication tract length following UV-B treatment (30J/m2) in HUWE1-depleted HeLa cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-value is 2.11x10-20.


Figure 3A . iPOND experiment showing that HUWE1 localizes to replication forks in 293T cells . Binding to nascent DNA was increased after UV exposure . Because of the difficulties in removing all the crosslinks in high molecular weightproteins , HUWE1 is detected as a high molecular weight smear . A control experiment using the HUWE1 - knockout cells ( shown in the right side panel ) confirmed the specificity of the HUWE1 signal . B . Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU ( 2mM for 16h ) and UV ( 40J / m2 , analyzed 2h later ) . C . Co - immunofluorescence experiments showing co - localization of HUWE1 and PCNA in chromatin foci . U2OS cells were analyzed 2h after exposure to UV ( 40J / m2 ) . Quantifications of co - localization , and additional micrographs are presented in Fig EV2A - D .

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Figure 3A. iPOND experiment showing that HUWE1 localizes to replication forks in 293T cells. Binding to nascent DNA was increased after UV exposure. Because of the difficulties in removing all the crosslinks in high molecular weightproteins, HUWE1 is detected as a high molecular weight smear. A control experiment using the HUWE1-knockout cells (shown in the right side panel) confirmed the specificity of the HUWE1 signal.B. Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU (2mM for 16h) and UV (40J/m2, analyzed 2h later).C. Co-immunofluorescence experiments showing co-localization of HUWE1 and PCNA in chromatin foci. U2OS cells were analyzed 2h after exposure to UV (40J/m2). Quantifications of co-localization, and additional micrographs are presented in Fig EV2A-D.