C ) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC / ΔC mice ( P21 ) . We denoted the antibody as ' SLC6A20 ' here , not ' SLC6A20A ' , because this antibody was generated using a synthetic peptide sequence ( YNEPSNNCQKHAI ) that is commonly present in SLC6A20A and SLC6A20B , being a pan - SLC6A20 antibody ( ThermoFisher ) . ( n = 3 mice for WT and ∆ C , * P < 0 . 05 , Student ' s t - test ) . The error bars represent SEM .

O O O O O O O O B-MEASURED_VAR 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 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

C) Immunoblot validation of the increase in SLC6A20 protein levels in hippocampal lysates of PtenΔC/ΔC mice (P21). We denoted the antibody as 'SLC6A20' here, not 'SLC6A20A', because this antibody was generated using a synthetic peptide sequence (YNEPSNNCQKHAI) that is commonly present in SLC6A20A and SLC6A20B, being a pan-SLC6A20 antibody (ThermoFisher). (n = 3 mice for WT and ∆C, *P < 0.05, Student's t-test). The error bars represent SEM.


( D , E ) Increased phosphorylation of β - catenin at Ser - 675 but not at Ser - 552 in the nucleus - enriched P1 fraction but not in whole - brain lysates of PtenΔC / ΔC mice ( 3 months ) . Note that total levels of β - catenin were not changed in the P1 fraction or whole - brain lysates . ( n = 4 mice for WT - WB / P1 and ΔC - WB / P1 , * P < 0 . 05 , ns , not significant , Student ' s t - test ) . The error bars represent SEM .

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

(D,E) Increased phosphorylation of β-catenin at Ser-675 but not at Ser-552 in the nucleus-enriched P1 fraction but not in whole-brain lysates of PtenΔC/ΔC mice (3 months). Note that total levels of β-catenin were not changed in the P1 fraction or whole-brain lysates. (n = 4 mice for WT-WB/P1 and ΔC-WB/P1, *P < 0.05, ns, not significant, Student's t-test). The error bars represent SEM.


( B , C ) Decreased extracellular levels of proline and glycine in the brains of PtenΔC / ΔC mice ( 2 - 4 month ) , as determined by microdialysis in the hippocampus ( see Materials and Methods for details ) . ( proline , n = 13 mice for WT and 8 for ΔC , * * P < 0 . 01 , Student ' s t - test ; glycine , n = 9 for WT and 5 for ∆ C , * * P < 0 . 01 , Student ' s t - test , Mann - Whitney U test ) . The error bars represent SEM .

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

(B,C) Decreased extracellular levels of proline and glycine in the brains of PtenΔC/ΔC mice (2-4 month), as determined by microdialysis in the hippocampus (see Materials and Methods for details). (proline, n = 13 mice for WT and 8 for ΔC, **P < 0.01, Student's t-test; glycine, n = 9 for WT and 5 for ∆C, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM.


( B ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 weeks ) by DCS treatment ( 20 μM ) , as shown by fEPSP slopes . ( n = 15 slices from 9 mice for WT - V / vehicle , 11 ( 5 ) for WT - D / DCS , 16 ( 10 ) for ΔC - V , 13 ( 7 ) for ΔC - D , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s test ) . The grey traces represent the baseline fEPSP prior to LTP induction . The error bars represent SEM .

O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by DCS treatment (20 μM), as shown by fEPSP slopes. (n = 15 slices from 9 mice for WT-V/vehicle, 11 (5) for WT-D/DCS, 16 (10) for ΔC-V, 13 (7) for ΔC-D, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.


( F ) Normalization of NMDAR - dependent HFS - LTP at synapses of PtenΔC / ΔC mice ( 4 - 5 weeks ) by sarcosine treatment ( 750 µM ) , as shown by fEPSP slopes . Note that the data for WT - V and ΔC - V are identical to those shown in Fig . 4B because the whole experiments were performed together ; we generated independent figures for DCS and sarcosine results for the clear presentation of the data . ( n = 15 slices from 9 mice for WT - V / vehicle , 9 ( 4 ) for WT - Sarc / Sarcosine , 16 ( 10 ) for ΔC - V / Vehicle , 18 ( 4 ) for ΔC - S , * P < 0 . 05 , * * * P < 0 . 001 , ns , not significant , two - way ANOVA with Tukey ' s test ) . The grey traces represent the baseline fEPSP prior to LTP induction . The error bars represent SEM .

O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normalization of NMDAR-dependent HFS-LTP at synapses of PtenΔC/ΔC mice (4-5 weeks) by sarcosine treatment (750 µM), as shown by fEPSP slopes. Note that the data for WT-V and ΔC-V are identical to those shown in Fig. 4B because the whole experiments were performed together; we generated independent figures for DCS and sarcosine results for the clear presentation of the data. (n = 15 slices from 9 mice for WT-V/vehicle, 9 (4) for WT-Sarc/Sarcosine, 16 (10) for ΔC-V/Vehicle, 18 (4) for ΔC-S, *P < 0.05, ***P < 0.001, ns, not significant, two-way ANOVA with Tukey's test). The grey traces represent the baseline fEPSP prior to LTP induction. The error bars represent SEM.


( D ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display partially normalized levels of whole - brain glycine , as shown by the lack of difference between saline - treated WT and ASO - treated PtenΔC / ΔC mice . Note that the glycine levels observed here are ~ 4 times lower than those measured in Fig . 3A , which could be attributable to different mouse ages ( P21 in Fig . 3A vs . 2 - 4 months in Fig . 5D ) , absence and presence of ASO injection , or lot - to - lot variation of the ELISA kits . ( n = 8 mice for WT - saline , 5 for WT - ASO , 7 for ΔC - saline , and 8 for ΔC - ASO , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Tukey ' s test ) . The error bars represent SEM .

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

(D) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display partially normalized levels of whole-brain glycine, as shown by the lack of difference between saline-treated WT and ASO-treated PtenΔC/ΔC mice. Note that the glycine levels observed here are ~4 times lower than those measured in Fig. 3A, which could be attributable to different mouse ages (P21 in Fig. 3A vs. 2-4 months in Fig. 5D), absence and presence of ASO injection, or lot-to-lot variation of the ELISA kits. (n = 8 mice for WT-saline, 5 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, **P < 0.01, ns, not significant, two-way ANOVA with Tukey's test). The error bars represent SEM.


( E ) PtenΔC / ΔC mice ( P20 - 37 ) treated with Slc6a20a - ASO display an NMDA / AMPA ratio at hippocampal SC - CA1 synapses that is comparable to that in ASO - treated WT mice . ( n = 11 neurons from 5 mice for WT - ASO , 10 ( 4 ) for ΔC - ASO , ns , not significant , Mann - Whitney ' s U test ) . The error bars represent SEM .

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

(E) PtenΔC/ΔC mice (P20-37) treated with Slc6a20a-ASO display an NMDA/AMPA ratio at hippocampal SC-CA1 synapses that is comparable to that in ASO-treated WT mice. (n = 11 neurons from 5 mice for WT-ASO, 10 (4) for ΔC-ASO, ns, not significant, Mann-Whitney's U test). The error bars represent SEM.


( G ) PtenΔC / ΔC mice ( 2 - 4 months ) treated with Slc6a20a - ASO display normal levels of climbing in the Laboras test . n = 6 mice for WT - saline , 7 for WT - ASO , 7 for ΔC - saline , and 8 for ΔC - ASO , * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , two - way ANOVA with Bonferroni ' s test . The error bars represent SEM .

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

(G) PtenΔC/ΔC mice (2-4 months) treated with Slc6a20a-ASO display normal levels of climbing in the Laboras test. n = 6 mice for WT-saline, 7 for WT-ASO, 7 for ΔC-saline, and 8 for ΔC-ASO, *P < 0.05, **P <0.01, ns, not significant, two-way ANOVA with Bonferroni's test. The error bars represent SEM.


( D ) Increased extracellular levels of glycine and proline in the brain of Slc6a20a + / - and Slc6a20a - / - mice , as shown by microdialysis analyses . Note that glycine levels are increased in both Slc6a20a + / - and Slc6a20a - / - mice , whereas proline levels are increased only in Slc6a20a - / - mice . ( glycine , n = 19 mice for WT , 11 for HT , 8 for KO , * * P < 0 . 01 , Mann - Whitney U test ; proline , n = 15 mice for WT , 11 for HT , 14 for KO , * * * P < 0 . 001 , ns , not significant , Mann - Whitney U test ) . The error bars represent SEM .

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

(D) Increased extracellular levels of glycine and proline in the brain of Slc6a20a+/- and Slc6a20a-/- mice, as shown by microdialysis analyses. Note that glycine levels are increased in both Slc6a20a+/- and Slc6a20a-/- mice, whereas proline levels are increased only in Slc6a20a-/- mice. (glycine, n = 19 mice for WT, 11 for HT, 8 for KO, **P < 0.01, Mann-Whitney U test; proline, n = 15 mice for WT, 11 for HT, 14 for KO, ***P < 0.001, ns, not significant, Mann-Whitney U test). The error bars represent SEM.


( B ) Glycine / proline - induced currents for mouse SLC6A20A are dependent on sodium chloride , as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate . Each experimental condition was sequentially applied to HEK293T cells , as shown in Appendix Fig . S7B . The indicated currents are average values from an ensemble of multiple ( ~ 20 ) cells in a single well . ( glycine , n = 21 cells for untransfected / blank , 17 for human SLC6A20 - V1 , 25 for human SLC6A20 - V2 , 33 for mSLC6A20A , 28 for mSLC6A20B * * * P < 0 . 001 ( relative to buffer not containing glycine ) , two - way ANOVA with Bonferroni ' s test ; proline , n = 17 cells for untransfected , 23 for human SLC6A20 - V1 , 47 for SLC6A20 - V2 , 31 for mSLC6A20A , and 31 for mSLC6A20B , * * * P < 0 . 001 , two - way ANOVA with Bonferroni ' s test ) . The error bars represent SEM .

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 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 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 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

(B) Glycine/proline-induced currents for mouse SLC6A20A are dependent on sodium chloride, as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate. Each experimental condition was sequentially applied to HEK293T cells, as shown in Appendix Fig. S7B. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 21 cells for untransfected/blank, 17 for human SLC6A20-V1, 25 for human SLC6A20-V2, 33 for mSLC6A20A, 28 for mSLC6A20B ***P < 0.001 (relative to buffer not containing glycine), two-way ANOVA with Bonferroni's test; proline, n = 17 cells for untransfected, 23 for human SLC6A20-V1, 47 for SLC6A20-V2, 31 for mSLC6A20A, and 31 for mSLC6A20B, ***P < 0.001, two-way ANOVA with Bonferroni's test). The error bars represent SEM.


( A - C ) Immunoblots for indicated proteins in BAT , extensor digitorum longus ( EDL ) , epididymal white adipose tissue ( eWAT ) and heart from 10 ‐ to 12 ‐ month ( mo ) ‐ old control ( Con ) and knock out ( KO ) mice . Arrows depict LC3 ‐ I and II .

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

(A-C) Immunoblots for indicated proteins in BAT, extensor digitorum longus (EDL), epididymal white adipose tissue (eWAT) and heart from 10‐ to 12‐month (mo)‐old control (Con) and knock out (KO) mice. Arrows depict LC3‐I and II.


( D ) ATG5 and ATG7 mRNA levels in indicated tissues ( n = 4 )

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

(D) ATG5 and ATG7 mRNA levels in indicated tissues (n=4)


( E ) immunoblots for indicated proteins in spleen , liver , lung , kidney , mediobasal hypothalamus ( MBH ) , inguinal white adipose tissue ( ingWAT ) and perinephric fat ( pnWAT ) from 10 ‐ to 12 ‐ mo ‐ old Con and KO mice .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) immunoblots for indicated proteins in spleen, liver, lung, kidney, mediobasal hypothalamus (MBH), inguinal white adipose tissue (ingWAT) and perinephric fat (pnWAT) from 10‐ to 12‐mo‐old Con and KO mice.


( F ) Body weights ( wt ) of chow diet ( RD ) ‐ fed ( n = 6 - 29 ) , and ( G ) high ‐ fat diet ( HFD ) ‐ fed Con and KO mice at indicated ages ( n = 4 - 17 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body weights (wt) of chow diet (RD)‐fed (n=6-29), and (G) high‐fat diet (HFD)‐fed Con and KO mice at indicated ages (n=4-17).


( H ) Total body fat and lean mass of 4 - 7 mo RD ‐ fed Con and KO mice ( n = 8 - 12 ) .

O O O O O 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) Total body fat and lean mass of 4-7 mo RD‐fed Con and KO mice (n=8-12).


( I ) BAT wt ( n = 4 - 7 ) , ( J ) gastrocnemius ( GA ) wt ( n = 5 - 7 ) , ( K ) soleus wt ( n = 4 - 7 ) , ( L ) eWAT wt ( n = 5 - 7 ) and ( M ) visceral ( Visc ) and subcutaneous ( Subc ) body fat distribution in 10 ‐ mo ‐ old Con and KO mice ( n = 4 ) . Values are mean ± s . e . , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

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

(I) BAT wt (n=4-7), (J) gastrocnemius (GA) wt (n=5-7), (K) soleus wt (n=4-7), (L) eWAT wt (n=5-7) and (M) visceral (Visc) and subcutaneous (Subc) body fat distribution in 10‐mo‐old Con and KO mice (n=4). Values are mean±s.e., *P0.05, **P0.01, ***P0.001.


( A - B ) mRNA for indicated genes in BAT ( n = 4 )

(A-B) mRNA for indicated genes in BAT (n=4)


( c ) mRNA for the indicated genes in epididymal white adipose tissue ( eWAT ) ( n = 4 )

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

(c) mRNA for the indicated genes in epididymal white adipose tissue (eWAT) (n=4)


( D ) immunoblots for indicated proteins in BAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice . Arrows depict protein isoforms .

O O O O O O O O O O O O O O O O O O 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) immunoblots for indicated proteins in BAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( E ) immunoblots for indicated proteins in eWAT from 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice . Arrows depict protein isoforms .

(E) immunoblots for indicated proteins in eWAT from 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice. Arrows depict protein isoforms.


( F ) Electron micrographs ( × 10 , 000 magnification ) of BAT depicting mitochondria from 4 ‐ mo ‐ old Con and KO mice . m , mitochondria ; LD , lipid droplet ; n , nucleus .

O O O O O O O O O O O O O O O O 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) Electron micrographs ( × 10,000 magnification) of BAT depicting mitochondria from 4‐mo‐old Con and KO mice. m, mitochondria; LD, lipid droplet; n, nucleus.


( G ) mRNA levels ( n = 4 ) in BAT from 10 ‐ mo ‐ old Con and KO

(G) mRNA levels (n=4) in BAT from 10‐mo‐old Con and KO mice


( H ) hematoxylin and eosin ( HE ) and ( I ) Sirius Red stains in BAT from 10 ‐ mo ‐ old Con and KO mice ( n = 3 - 4 ) . Average adipocyte and LD size , and LD number in BAT are shown . Scale bar , 50 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

(H) hematoxylin and eosin (HE) and (I) Sirius Red stains in BAT from 10‐mo‐old Con and KO mice (n=3-4). Average adipocyte and LD size, and LD number in BAT are shown. Scale bar, 50 μm.


( J ) BAT mRNA levels from 4 ‐ mo ‐ old RD ‐ fed Con and KO mice cold ‐ challenged for 75 min ( n = 3 ) . Values are mean ± s . e . * * * P0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O 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) BAT mRNA levels from 4‐mo‐old RD‐fed Con and KO mice cold‐challenged for 75 min (n=3). Values are mean±s.e. ***P0.001.


( A ) Body temperature before ( Basal ) and during cold exposure ( n = 7 - 8 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO )

(A) Body temperature before (Basal) and during cold exposure (n=7-8) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice


( B ) Oxygen consumption ( VO2 ) , ( C ) carbon dioxide production ( VCO2 ) in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice ( 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

(B) Oxygen consumption (VO2), (C) carbon dioxide production (VCO2) in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4).


( D ) energy expenditure in 10 ‐ month ( mo ) ‐ old chow diet ( RD ) ‐ fed control ( Con ) and knock out ( KO ) mice ( 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

(D) energy expenditure in 10‐month (mo)‐old chow diet (RD)‐fed control (Con) and knock out (KO) mice (n=4)


( E ) VO2 , ( F ) VCO2 and ( G ) energy expenditure in 10 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con and KO mice ( n = 3 - 5 ) .

(E) VO2, (F) VCO2 and (G) energy expenditure in 10‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=3-5).


( H ) Ambulation ( AMB ) in 10 ‐ mo ‐ old RD ‐ fed ( n = 4 ) and HFD ‐ fed Con and KO mice ( n = 3 - 5 ) .

(H) Ambulation (AMB) in 10‐mo‐old RD‐fed (n=4) and HFD‐fed Con and KO mice (n=3-5).


( I ) Hematoxylin and eosin ( HE ) ‐ stained epididymal white adipose tissue ( eWAT ) from 6 ‐ mo ‐ old RD ‐ fed Con and KO mice . Quantification for cell size is shown ( n = 3 ) . Scale bar , 50 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

(I) Hematoxylin and eosin (HE)‐stained epididymal white adipose tissue (eWAT) from 6‐mo‐old RD‐fed Con and KO mice. Quantification for cell size is shown (n=3). Scale bar, 50 μm.


( J ) Adβ3 mRNA in eWAT from 10 ‐ mo ‐ old RD ‐ fed Con and KO mice ( n = 4 ) , and in ( K ) inguinal ( ingWAT ) from 4 ‐ mo ‐ oldcold ‐ challenged RD ‐ fed Con and KO mice ( n = 3 ) .

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

(J) Adβ3 mRNA in eWAT from 10‐mo‐old RD‐fed Con and KO mice (n=4), and in (K) inguinal (ingWAT) from 4‐mo‐oldcold‐challenged RD‐fed Con and KO mice (n=3).


( L ) Serum ‐ free fatty acid ( FFA ) and glycerol ( Gly ) from 4 ‐ to 6 ‐ mo ‐ old Con and KO mice untreated ( basal ) or treated with isoproterenol ( Iso ) i . p . for 20 min ( n = 4 - 6 ) , and ( M ) from 4 ‐ mo ‐ old RD ‐ fed cold ‐ challenged ( 75 min ) mice ( n = 3 - 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Serum‐free fatty acid (FFA) and glycerol (Gly) from 4‐ to 6‐mo‐old Con and KO mice untreated (basal) or treated with isoproterenol (Iso) i.p. for 20 min (n=4-6), and (M) from 4‐mo‐old RD‐fed cold‐challenged (75 min) mice (n=3-4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A , B ) Tmem26 and tbx1 mRNA levels in epididymal eWAT , and ( C , D ) ingWAT from Atg7Flox / Floxmice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses and cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

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

(A,B) Tmem26 and tbx1 mRNA levels in epididymal eWAT, and (C,D) ingWAT from Atg7Flox/Floxmice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( E , F ) hematoxylin and eosin ( HE ) ‐ stained ingWAT section from Atg7Flox / Flox mice ( 4 months ( mo ) ) injected in BAT with empty ( Con AdV ) or Cre ‐ expressing ( Cre AdV ) adenoviruses and cold ‐ exposed or not ( basal ) for 75 min ( n = 4 - 5 ) .

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

(E,F) hematoxylin and eosin (HE)‐stained ingWAT section from Atg7Flox/Flox mice (4 months (mo)) injected in BAT with empty (Con AdV) or Cre‐expressing (Cre AdV) adenoviruses and cold‐exposed or not (basal) for 75 min (n=4-5).


( G ) mRNA for indicated genes in ingWAT from 4 ‐ mo ‐ old cold ‐ exposed Con and KO mice ( 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

(G) mRNA for indicated genes in ingWAT from 4‐mo‐old cold‐exposed Con and KO mice (n=4).


( H , I ) mRNA levels in eWAT from 10 ‐ mo ‐ old Con and KO mice ( 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

(H,I) mRNA levels in eWAT from 10‐mo‐old Con and KO mice (n=4).


( J ) β ‐ oxidation in eWAT and ingWAT from 4 ‐ mo ‐ old Con and KO mice ( 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

(J) β‐oxidation in eWAT and ingWAT from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA levels in soleus from 4 ‐ mo ‐ old cold ‐ exposed Con and KO mice ( n = 3 - 6 ) .

(K) mRNA levels in soleus from 4‐mo‐old cold‐exposed Con and KO mice (n=3-6).


( M ) BAT from 4 ‐ mo ‐ old Con and KO mice ( n = 4 ) . Values are mean ± s . e . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 .

(M) BAT from 4‐mo‐old Con and KO mice (n=4). Values are mean±s.e. *P0.05, **P0.01, ***P0.001.


( A ) Blood glucose in 6 ‐ to 10 ‐ month ( mo ) ‐ old mice ( n = 12 - 15 ) , and ( B ) serum insulin levels in 10 ‐ mo ‐ old fed control ( Con ) and KO mice ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Blood glucose in 6‐ to 10‐month (mo)‐old mice (n=12-15), and (B) serum insulin levels in 10‐mo‐old fed control (Con) and KO mice (n=6).


( C ) Glucose tolerance tests in 10 ‐ mo ‐ old chow diet ( RD ) ‐ fed ( n = 5 ) , and ( D ) in 10 ‐ to 12 ‐ mo ‐ old high ‐ fat diet ( HFD ) ‐ fed Con and KO mice ( n = 4 - 9 ) .

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

(C) Glucose tolerance tests in 10‐mo‐old chow diet (RD)‐fed (n=5), and (D) in 10‐ to 12‐mo‐old high‐fat diet (HFD)‐fed Con and KO mice (n=4-9).


( E ) Insulin tolerance test in 10 ‐ mo ‐ old RD ‐ fed Con and KO mice ( n = 5 ) .

(E) Insulin tolerance test in 10‐mo‐old RD‐fed Con and KO mice (n=5).


( F ) Immunoblots for indicated proteins in skeletal muscle from 10 ‐ mo ‐ old Con and KO mice .

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

(F) Immunoblots for indicated proteins in skeletal muscle from 10‐mo‐old Con and KO mice.


( G ) Hematoxylin and eosin ( HE ) ‐ stained gastrocnemius ( GA ) sections from 6 ‐ mo ‐ old Con and KO mice . Myofiber cross ‐ sectional area ( μm2 ) is shown ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O 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) Hematoxylin and eosin (HE)‐stained gastrocnemius (GA) sections from 6‐mo‐old Con and KO mice. Myofiber cross‐sectional area (μm2) is shown (n=3).


( H ) Expression of MuRF ‐ 1 and Atrogin ‐ 1 in GA and TA from 10 ‐ mo ‐ old Con and KO mice ( n = 4 )

O O O O O 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 O

(H) Expression of MuRF‐1 and Atrogin‐1 in GA and TA from 10‐mo‐old Con and KO mice (n=4)


( I ) myogenic genes in GA from 10 ‐ mo ‐ old Con and KO mice ( n = 6 ) .

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

(I) myogenic genes in GA from 10‐mo‐old Con and KO mice (n=6).


( J ) Immunoblots for indicated proteins in tissues from 4 ‐ mo ‐ old Con and KO mice ( n = 4 ) .

(J) Immunoblots for indicated proteins in tissues from 4‐mo‐old Con and KO mice (n=4).


( K ) mRNA for IRS1 , ( L ) IRS2 in epididymal white adipose tissue ( eWAT ) and GA from 10 ‐ mo ‐ old Con and KO mice ( n = 6 ) .

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 O O O O O O O O O O O

(K) mRNA for IRS1, (L) IRS2 in epididymal white adipose tissue (eWAT) and GA from 10‐mo‐old Con and KO mice (n=6).


( A ) Cellomics images ( top ) of GFP - LC3 ‐ HEK cells in full medium ( FM ) or after 2 h in starvation medium ( ES ) with leupeptin ( EL ) . Scale bars = 40 μm . Optimised automated analysis ( bottom left ) programme detecting hoechst ‐ labelled nuclei ( purple line ) , cell outline ( red line ) and identifies GFP - LC3 spots ( red spots ) in FM and EL . Enlarged GFP - LC3 images in FM and EL ( bottom right ) . Scale bars = 10 μm .

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 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

(A) Cellomics images (top) of GFP-LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP-LC3 spots (red spots) in FM and EL. Enlarged GFP-LC3 images in FM and EL (bottom right). Scale bars=10 μm.


( B ) SCPO , STIPO and STAPO after indicated siRNA treatment of GFP - LC3 ‐ HEK cells incubated as in ( A ) . Error bars represent s . e . m . Significance was determined using a two ‐ tailed paired t ‐ test compared with RISCfree ( RF ) in EL : SCPO siULK1 , * * * P = 0 . 0002 ; siATG7 , * * P = 0 . 0078 ; siNRBP2 , * * P = 0 . 0088 . STIPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0060 ; siNRBP2 , * * P = 0 . 0041 . STAPO siULK1 , * * * P0 . 0001 ; siATG7 , * * P = 0 . 0037 ; siNRBP2 , * * P = 0 . 0028 . The experiments were performed : RISCfree ( n = 5 ) , siULK1 ( n = 5 ) , siATG7 ( n = 3 ) , siNRBP2 ( n = 3 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP-LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3).


( D ) Scatter plot of median SCPO Z ‐ scores from siRNA pools in the primary screen that met the minimum object count ( OC ) cutoff . Dashed lines represent approximate cutoff for 500 best increasers and decreasers . RP of known autophagy genes ( ATG4C , ATG14 , RB1CC1 , ATG9A , ATG7 ) are given in Supplementary Table S2 ; AMBRA1 ( FLJ20294 ) , TBC1D25 ( OATL1 ) and SH3GLB1 ( BIF ‐ 1 ) in Supplementary Table S1 ; LAMTOR2 has a RP = 708 . 7 and RUBICON has a RP = 1126 .

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

(D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126.


( A ) SCPO , STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP - LC3 spot phenotype , above a cutoff of greater or less than 20 % of control , and passing additional criteria ( Supplementary Table 3 ) . Red boxes indicate an increase > 1 . 2 ; pink boxes an increase between 1 . 1 and 1 . 2 ; white boxes are between 0 . 9 and 1 . 1 ; light blue boxes a decrease between 0 . 9 and 0 . 8 ; dark blue boxes a decrease to 0 . 8 . Hits underlined were taken forward as three out of four4 hits , ( § ) not chosen because of restricted tissue expression , ( * ) are three out of four when alternate method of normalisation is used .

O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SCPO, STIPO and STAPO for the 51 genes validated by two out of four individual siRNA duplexes reproducing the GFP-LC3 spot phenotype, above a cutoff of greater or less than 20% of control, and passing additional criteria (Supplementary Table 3). Red boxes indicate an increase >1.2; pink boxes an increase between 1.1 and 1.2; white boxes are between 0.9 and 1.1; light blue boxes a decrease between 0.9 and 0.8; dark blue boxes a decrease to 0.8. Hits underlined were taken forward as three out of four4 hits, (§) not chosen because of restricted tissue expression, (*) are three out of four when alternate method of normalisation is used.


( B ) Table summarising results from further validation of 20 genes . mRNA levels scored as ( + ) or ( − ) indicate efficient or poor knockdown , respectively ( see Supplementary Figures S1 and S2 ) . Increasers ( left ) or decreasers ( right ) assayed for LC3 lipidation were scored ( + ) for yes , ( − ) for no , ( + / − ) subtle change and ( nd ) not done ( Supplementary Figure S3 ) .

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

(B) Table summarising results from further validation of 20 genes. mRNA levels scored as (+) or (−) indicate efficient or poor knockdown, respectively (see Supplementary Figures S1 and S2). Increasers (left) or decreasers (right) assayed for LC3 lipidation were scored (+) for yes, (−) for no, (+/−) subtle change and (nd) not done (Supplementary Figure S3).


( C - E ) STIPO results after incubation under different autophagy ‐ inducing conditions following ( C ) SCOC knockdown ( D ) SUPT5H knockdown and ( E ) WAC knockdown . siRNA ‐ treated cells were incubated for the indicated time in FL : full medium plus leupeptin ; ES ; EL : ES plus leupeptin ; Torin : 250 nM Torin1 in FL ; Resv : 128 μM Resveratrol in FL ; LiCl : 10 mM LiCl in FM .

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(C-E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM.


( B ) Endogenous SCOC shows colocalisation with Atg9 ( red ) and TGN46 ( blue ) . HEK293 cells were fixed and permeabilised in saponin . Colocalisation indicated with arrows .

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(B) Endogenous SCOC shows colocalisation with Atg9 (red) and TGN46 (blue). HEK293 cells were fixed and permeabilised in saponin. Colocalisation indicated with arrows.


( C ) Endogenous SCOC ( green top , red bottom ) partially colocalises with endogenous LC3 ( red , top ) and GFP - LC3 ( green , bottom ) in HEK293 and GFP - LC3 ‐ HEK cells , respectively , after 2 h amino ‐ acid starvation . Boxed region is at higher magnification in merge panel on right . Scale bars = 10 μm .

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 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

(C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP-LC3 (green, bottom) in HEK293 and GFP-LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm.


( A ) SCOC is required for autophagy in HEK293 cells . Anti ‐ ULK1 , ‐ Actin and ‐ LC3 blot after siRNA treatment in HEK293 cells in FM , ES or EL . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RF EL versus siSCOC ‐ 03 EL , * P = 0 . 0419 .

O O O B-CONTROLLED_VAR O 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 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

(A) SCOC is required for autophagy in HEK293 cells. Anti‐ULK1, ‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siSCOC‐03 EL, *P=0.0419.


( B ) SCOC is required for p62 degradation . p62 levels were determined by western blot ( Supplementary Figure S5 ) after incubation in FM , ES for 2 or 4 h , or ES with BafilomycinA1 ( ES + BAF ) for 2 and 4 h . Quantification of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree ES 2 and 4 h , * * * P > 0 . 0001 and * * 0 . 0014 , respectively .

O O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P>0.0001 and **0.0014, respectively.


( C ) SCOC - GFP and SCOC - myc levels are reduced by SCOC siRNA duplex ‐ 03 . Anti ‐ GFP , ‐ Myc and ‐ Actin blots of HEK293 cells after treatment with ULK1 , RISCfree or SCOC siRNA and overexpression of GFP , SCOC - myc or SCOC - GFP .

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(C) SCOC-GFP and SCOC-myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC-myc or SCOC-GFP.


( D ) Overexpression of SCOC - myc increases autophagy . Anti ‐ Actin , ‐ LC3 and ‐ Myc blots of HEK293 cells after transfection of pcDNA or SCOC - myc . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 5 ) . SCOC - myc EL versus pcDNA EL , * P = 0 . 0215 .

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(D) Overexpression of SCOC-myc increases autophagy. Anti‐Actin, ‐LC3 and ‐Myc blots of HEK293 cells after transfection of pcDNA or SCOC-myc. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=5). SCOC-myc EL versus pcDNA EL, *P=0.0215.


( E ) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells . Arrows show partial colocalisation . Boxed region is at higher magnification in merge panel on bottom right . Scale bars = 20 μm .

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(E) Endogenous SCOC colocalises with endogenous FEZ1 in HEK293 cells. Arrows show partial colocalisation. Boxed region is at higher magnification in merge panel on bottom right. Scale bars=20 μm.


( A ) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation . Anti ‐ Actin and ‐ LC3 blot after siRNA treatment in HEK293 cells . Quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RISCfree ES versus siFEZ1 ‐ 01 ES , P = 0 . 034 . Quantification of p62 / actin ( Supplementary Figure S5 ) performed as in Figure 4B ; error bars represent s . e . m . ( n = 3 ) ; RISCfree FM versus RISCfree 2 and 4 hP > 0 . 0001 and P = 0 . 0014 , respectively . qRT - PCR showing FEZ1 mRNA levels after indicated knockdown in GFP - LC3 ‐ HEK cells ( error bars represent s . d . ) .

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

(A) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation. Anti‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells. Quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RISCfree ES versus siFEZ1‐01 ES, P=0.034. Quantification of p62/actin ( Supplementary Figure S5) performed as in Figure 4B; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree 2 and 4 hP>0.0001 and P=0.0014, respectively. qRT-PCR showing FEZ1 mRNA levels after indicated knockdown in GFP-LC3‐HEK cells (error bars represent s.d.).


( B ) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation . Anti ‐ Myc , ‐ Actin and ‐ LC3 blot after transfection with pcDNA or Myc - FEZ1 in HEK293 cells . Quantification of LC3II / actin ; error bars represent s . e . m . ( n = 3 ) ; pcDNA ES versus Myc - FEZ1 ES , P = 0 . 0318 . Anti ‐ GFP , ‐ Actin and ‐ p62 blot after transfection with GFP or FEZ1 - GFP and treatment with ES or ES plus Bafilomycin ( EB ) for 4 h . Quantification of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) ; GFP FM versus GFP ES , P = 0 . 0077 ; GFP FM versus GFP EB , P = 0 . 0212 ; GFP ES versus FEZ1 - GFP ES , P = 0 . 048 .

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

(B) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation. Anti‐Myc, ‐Actin and ‐LC3 blot after transfection with pcDNA or Myc-FEZ1 in HEK293 cells. Quantification of LC3II/actin; error bars represent s.e.m. (n=3); pcDNA ES versus Myc-FEZ1 ES, P=0.0318. Anti‐GFP, ‐Actin and ‐p62 blot after transfection with GFP or FEZ1-GFP and treatment with ES or ES plus Bafilomycin (EB) for 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); GFP FM versus GFP ES, P=0.0077; GFP FM versus GFP EB, P=0.0212; GFP ES versus FEZ1-GFP ES, P=0.048.


( C ) SCOC and FEZ1 interact . Left panel shows anti ‐ SCOC and ‐ FEZ1 blots after co ‐ expression in HEK293 cells and co ‐ immunoprecipitation of SCOC - myc and FEZ1 - GFP with anti ‐ GFP antibody . Input lysate ( Inp ) , unbound supernatant ( Unb ) . Right panel shows anti ‐ FEZ1 and ‐ FLAG blots after co ‐ expression in HEK293 cells and co ‐ immunoprecipitation of FLAG - SCOC and Myc - FEZ1 with anti ‐ FLAG antibody .

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(C) SCOC and FEZ1 interact. Left panel shows anti‐SCOC and ‐FEZ1 blots after co‐expression in HEK293 cells and co‐immunoprecipitation of SCOC-myc and FEZ1-GFP with anti‐GFP antibody. Input lysate (Inp), unbound supernatant (Unb). Right panel shows anti‐FEZ1 and ‐FLAG blots after co‐expression in HEK293 cells and co‐immunoprecipitation of FLAG-SCOC and Myc-FEZ1 with anti‐FLAG antibody.


( D ) FEZ1 interacts with SCOC in vitro . GST ‐ pulldown assays using in vitro translated 35S ‐ labelled Myc - FEZ1 ( right ) or 35S ‐ labelled Myc ‐ control ( left ) and GST or GST - SCOC ( asterisk in bottom panel ) . Bound proteins were detected by autoradiography following SDS - PAGE ( top ) , protein loading by coomassie blue ( bottom ) .

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(D) FEZ1 interacts with SCOC in vitro. GST‐pulldown assays using in vitro translated 35S‐labelled Myc-FEZ1 (right) or 35S‐labelled Myc‐control (left) and GST or GST-SCOC (asterisk in bottom panel). Bound proteins were detected by autoradiography following SDS-PAGE (top), protein loading by coomassie blue (bottom).


( E ) The FEZ1 - SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled ‐ coil domain of FEZ1 as demonstrated in GST ‐ pulldown assays with in vitro translated 35S ‐ labelled WT or mutant Myc ‐ FEZ1 and GST or GST ‐ SCOC . Autoradiographs of 10 % input and bound proteins ( top panels ) and Coomassie blue ‐ stained gels of bead ‐ bound GST and GST ‐ SCOC proteins used ( bottom panels ) .

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(E) The FEZ1-SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled‐coil domain of FEZ1 as demonstrated in GST‐pulldown assays with in vitro translated 35S‐labelled WT or mutant Myc‐FEZ1 and GST or GST‐SCOC. Autoradiographs of 10% input and bound proteins (top panels) and Coomassie blue‐stained gels of bead‐bound GST and GST‐SCOC proteins used (bottom panels).


( A ) FEZ1 interacts with both the kinase domain ( 1 - 278 ) and the proline - serine rich spacer of ULK1 ( 279 - 828 ) . GFP ‐ or Myc ‐ tagged ULK1 fragments were in vitro translated with 35S ‐ methionine and subjected to GST ‐ pulldown assays using GST and GST - FEZ1 purified from E . coli ( bottom ) . Bound proteins were detected by autoradiography following SDS - PAGE ( top ) .

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(A) FEZ1 interacts with both the kinase domain (1-278) and the proline-serine rich spacer of ULK1 (279-828). GFP‐ or Myc‐tagged ULK1 fragments were in vitro translated with 35S‐methionine and subjected to GST‐pulldown assays using GST and GST-FEZ1 purified from E. coli (bottom). Bound proteins were detected by autoradiography following SDS-PAGE (top).


( B ) ULK1 interacts with FEZ1 . ULK1 was immunoprecipitated from HEK293 lysates with anti ‐ ULK1 and bound ULK1 and FEZ1 were detected with anti ‐ ULK1 and anti ‐ FEZ1 antibodies . Input lysate ( Inp ) .

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(B) ULK1 interacts with FEZ1. ULK1 was immunoprecipitated from HEK293 lysates with anti‐ULK1 and bound ULK1 and FEZ1 were detected with anti‐ULK1 and anti‐FEZ1 antibodies. Input lysate (Inp).


( C ) The FEZ1 - GFP and Myc - ULK1 interaction is unaffected by amino ‐ acid starvation . Anti ‐ Myc and ‐ GFP blots after HEK293 cells were co ‐ transfected with FEZ1 - GFP and Myc - ULK1 , incubated in FM or ES for 2 h , harvested and the complex was immunoprecipitated using anti ‐ GFP antibody .

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(C) The FEZ1-GFP and Myc-ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1-GFP and Myc-ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody.


( D ) BN - PAGE reveals SCOC and FEZ1 are in a complex . FEZ1 - GFP , FLAG - SCOC and Myc - ULK1 wild type or kinase ‐ inactive ULK1K46I ( ULK1KI ) were co ‐ expressed in HEK293 cells . Anti ‐ GFP , ‐ FLAG and ‐ Myc western blots are shown for the BN - PAGE gel ( top ) or the SDS - PAGE gel ( bottom ) . In GFP and FLAG western blots , two different exposures of the same BN - PAGE blot are shown spliced together .

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(D) BN-PAGE reveals SCOC and FEZ1 are in a complex. FEZ1-GFP, FLAG-SCOC and Myc-ULK1 wild type or kinase‐inactive ULK1K46I (ULK1KI) were co‐expressed in HEK293 cells. Anti‐GFP, ‐FLAG and ‐Myc western blots are shown for the BN-PAGE gel (top) or the SDS-PAGE gel (bottom). In GFP and FLAG western blots, two different exposures of the same BN-PAGE blot are shown spliced together.


( E ) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC . RF or ULK1 siRNA ‐ treated cells were co ‐ transfected with FEZ1 - GFP and FLAG - SCOC , then incubated and immunoprecipitated as in ( C ) . Western blot of lysates for ULK1 , GFP and FLAG , and immunoprecipitates with GFP and FLAG antibodies .

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(E) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC. RF or ULK1 siRNA‐treated cells were co‐transfected with FEZ1-GFP and FLAG-SCOC, then incubated and immunoprecipitated as in (C). Western blot of lysates for ULK1, GFP and FLAG, and immunoprecipitates with GFP and FLAG antibodies.


( F ) siRNA depletion of SCOC reduces FEZ1 - ULK1 complex . HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1 - GFP . FEZ1 - GFP was detected with anti ‐ GFP in both BN - PAGE and SDS - PAGE . Endogenous ULK1 was detected with anti ‐ ULK1 antibody . Arrowheads indicate a FEZ1 - GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex > 1236 kDa also detected in ( D ) in the triple transfection . The 200 KDa complex is sensitive to loss of SCOC and ULK1 .

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(F) siRNA depletion of SCOC reduces FEZ1-ULK1 complex. HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1-GFP. FEZ1-GFP was detected with anti‐GFP in both BN-PAGE and SDS-PAGE. Endogenous ULK1 was detected with anti‐ULK1 antibody. Arrowheads indicate a FEZ1-GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex >1236 kDa also detected in (D) in the triple transfection. The 200 KDa complex is sensitive to loss of SCOC and ULK1.


( G ) SCOC can reduce the interaction of ULK1 with MBP - FEZ1 . Immobilised MBP - FEZ1 was incubated with in vitro 35S ‐ methionine ‐ labelled Myc - ULK1 . Addition of increasing amounts of in vitro translated 35S ‐ methionine SCOC reduced the amount of Myc - ULK1 bound . Top and bottom panels are as described in ( A ) .

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(G) SCOC can reduce the interaction of ULK1 with MBP-FEZ1. Immobilised MBP-FEZ1 was incubated with in vitro 35S‐methionine‐labelled Myc-ULK1. Addition of increasing amounts of in vitro translated 35S‐methionine SCOC reduced the amount of Myc-ULK1 bound. Top and bottom panels are as described in (A).


( A ) SCOC interacts with UVRAG . HEK293 cells were transfected with FLAG - SCOC and Myc - UVRAG , lysed and incubated with anti ‐ FLAG ( left ) or anti ‐ Myc ( right ) antibodies . Co ‐ immunoprecipitation of Myc - UVRAG ( left ) and FLAG - SCOC ( right ) was confirmed using anti ‐ Myc and anti ‐ FLAG antibodies for western blotting . Asterisk in ( A - C ) and ( D ) indicates non ‐ specific bands .

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(A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG-SCOC and Myc-UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc-UVRAG (left) and FLAG-SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A-C) and (D) indicates non‐specific bands.


( B ) Interaction of SCOC with UVRAG is unaffected by FEZ1 - GFP . As in ( A ) , co ‐ immunoprecipitation was preformed in HEK293 cells transfected with FLAG - SCOC , Myc - UVRAG and FEZ1 - GFP . Myc - UVRAG was detected as in ( A ) , FEZ1 - GFP was detected with anti ‐ GFP antibody .

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(B) Interaction of SCOC with UVRAG is unaffected by FEZ1-GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG-SCOC, Myc-UVRAG and FEZ1-GFP. Myc-UVRAG was detected as in (A), FEZ1-GFP was detected with anti‐GFP antibody.


( C ) The SCOC and UVRAG interaction is reduced by starvation , and this reduction is inhibited by FEZ1 - GFP . HEK293 cells were transfected as in ( B ) and 24 h later either treated with FM or ES for 2 h . FLAG - SCOC or Myc - UVRAG was immunoprecipitated with anti ‐ FLAG or anti ‐ Myc antibodies and the bound proteins were detected with anti ‐ Myc , ‐ GFP or ‐ FLAG antibodies by western blotting . Quantification of the amount of FLAG - SCOC co ‐ immunoprecipitated with Myc - UVRAG in FM or ES , without overexpressed FEZ1 - GFP ( left ) or with overexpressed FEZ1 - GFP ( right ) . Significance was determined using a two ‐ tailed paired t ‐ test ( n = 3 ) .

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(C) The SCOC and UVRAG interaction is reduced by starvation, and this reduction is inhibited by FEZ1-GFP. HEK293 cells were transfected as in (B) and 24 h later either treated with FM or ES for 2 h. FLAG-SCOC or Myc-UVRAG was immunoprecipitated with anti‐FLAG or anti‐Myc antibodies and the bound proteins were detected with anti‐Myc, ‐GFP or ‐FLAG antibodies by western blotting. Quantification of the amount of FLAG-SCOC co‐immunoprecipitated with Myc-UVRAG in FM or ES, without overexpressed FEZ1-GFP (left) or with overexpressed FEZ1-GFP (right). Significance was determined using a two‐tailed paired t‐test (n=3).


( D ) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction . HEK293 cells were transfected with FEZ1 - GFP , GFP or FLAG - SCOC alone or in combination . Top panel input , bottom panel immunoprecipitation with anti ‐ GFP or anti ‐ FLAG . Western blot for endogenous UVRAG , GFP - FEZ1 GFP or FLAG - SCOC as indicated . Arrowhead : endogenous UVRAG .

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(D) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction. HEK293 cells were transfected with FEZ1-GFP, GFP or FLAG-SCOC alone or in combination. Top panel input, bottom panel immunoprecipitation with anti‐GFP or anti‐FLAG. Western blot for endogenous UVRAG, GFP-FEZ1 GFP or FLAG-SCOC as indicated. Arrowhead: endogenous UVRAG.


( E ) UVRAG can interact with both FEZ1 and SCOC in vitro . GST , GST - FEZ1 or GST - SCOC were immobilised and incubated with 35S ‐ labelled Myc - UVRAG as indicated . Arrowheads indicate GST - FEZ1 ( left ) and GST - SCOC ( right ) .

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(E) UVRAG can interact with both FEZ1 and SCOC in vitro. GST, GST-FEZ1 or GST-SCOC were immobilised and incubated with 35S‐labelled Myc-UVRAG as indicated. Arrowheads indicate GST-FEZ1 (left) and GST-SCOC (right).


( F ) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells . Arrows show colocalisation . Scale bar = 10 μm .

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(F) Endogenous UVRAG partially colocalises with endogenous FEZ1 in HEK293 cells. Arrows show colocalisation. Scale bar=10 μm.


( A ) Depletion of endogenous WAC . Anti ‐ WAC and ‐ Actin blot and WAC mRNA levels by qRT - PCR after treatment with RF or individual WAC siRNAs ( W ‐ 01 to W ‐ 04 ) in HEK293 cells . qRT - PCR error bars represent standard deviation of triplicates ( n = 2 ) .

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(A) Depletion of endogenous WAC. Anti‐WAC and ‐Actin blot and WAC mRNA levels by qRT-PCR after treatment with RF or individual WAC siRNAs (W‐01 to W‐04) in HEK293 cells. qRT-PCR error bars represent standard deviation of triplicates (n=2).


( B ) ( B ) WAC is required for autophagy . Anti ‐ ULK1 , ‐ WAC , ‐ Actin and ‐ LC3 blots after siRNA treatment in HEK293 cells and incubation in FM , ES or EL . Representative blot ; quantification of LC3II / actin of averaged duplicates ; error bars represent s . e . m . ( n = 3 ) . Significance was determined using a two ‐ tailed paired t ‐ test : RF EL versus siWAC ‐ 03 EL , * * P = 0 . 0079 .

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(B) (B) WAC is required for autophagy. Anti‐ULK1, ‐WAC, ‐Actin and ‐LC3 blots after siRNA treatment in HEK293 cells and incubation in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siWAC‐03 EL, **P=0.0079.


( C ) p62 degradation requires WAC . Anti ‐ p62 and ‐ Actin blot after siRNA treatment of GFP - LC3 ‐ HEK cells in FM or ES for 2 h in duplicate . Bars represent p62 / actin levels normalised to RF FM ; quantification of p62 / actin ; error bars represent s . e . m . ( n = 3 ) : RF FM versus RF ES , * * P = 0 . 0057 ; RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0025 ; RF ES versus siWAC ‐ 03 ES , * P = 0 . 0386 .

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(C) p62 degradation requires WAC. Anti‐p62 and ‐Actin blot after siRNA treatment of GFP-LC3‐HEK cells in FM or ES for 2 h in duplicate. Bars represent p62/actin levels normalised to RF FM; quantification of p62/actin; error bars represent s.e.m. (n=3): RF FM versus RF ES, **P=0.0057; RF FM versus siWAC‐03 FM, **P=0.0025; RF ES versus siWAC‐03 ES, *P=0.0386.


( D ) WAC depletion accelerates degradation of a UPS reporter . Anti ‐ p62 , ‐ GFP , ‐ Actin , ‐ LC3 , ‐ WAC and ‐ Atg16 blots after siRNA treatment in UbG76V - YFP MelJuSo cells and incubation conditions FM , ES or FM with 10 μM MG132 ( MG ) for 2 h . Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC ‐ 03 siRNA treatment ( n = 3 ) and compared with ATG16 siRNA treatment ( n = 2 ) . Error bars represent s . e . m . : RF FM versus siWAC ‐ 03 FM , * * P = 0 . 0082 .

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(D) WAC depletion accelerates degradation of a UPS reporter. Anti‐p62, ‐GFP, ‐Actin, ‐LC3, ‐WAC and ‐Atg16 blots after siRNA treatment in UbG76V-YFP MelJuSo cells and incubation conditions FM, ES or FM with 10 μM MG132 (MG) for 2 h. Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC‐03 siRNA treatment (n=3) and compared with ATG16 siRNA treatment (n=2). Error bars represent s.e.m.: RF FM versus siWAC‐03 FM, **P=0.0082.


( E ) Soluble HttQ25 - CFP degradation is increased after WAC depletion . Anti ‐ WAC , ‐ Actin and ‐ GFP blots after indicated siRNA treatment ( siWAC duplexes ‐ 02 , ‐ 03 , ‐ 04 ) of HttPolyQ ‐ mutant HeLa cell lines and incubation conditions DMSO or DOX ( Doxycyline ) for 3 days . The insoluble fraction was obtained by pelleting the HttQ103 lysates ( see Materials and methods ) . Quantification of normalised average HttQ25 - CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment ; error bar represents s . e . m . ( n = 3 ) : HttQ25 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0350 ; soluble HttQ103 - CFP / actin RF DMSO versus siWAC DMSO , * P = 0 . 0380 .

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(E) Soluble HttQ25-CFP degradation is increased after WAC depletion. Anti‐WAC, ‐Actin and ‐GFP blots after indicated siRNA treatment (siWAC duplexes ‐02, ‐03, ‐04) of HttPolyQ‐mutant HeLa cell lines and incubation conditions DMSO or DOX (Doxycyline) for 3 days. The insoluble fraction was obtained by pelleting the HttQ103 lysates (see Materials and methods). Quantification of normalised average HttQ25-CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment; error bar represents s.e.m. (n=3): HttQ25-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0350; soluble HttQ103-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0380.


A . Characterization of C . perfringens β - toxin ( CPB ) constructs with different N - termini by gel electrophoretic analysis under denaturing ( A ) conditions . SDS - PAGE gel analyis of CPB samples in cholate ( 1μg per lane ) . CPB samples containing cholate were boiled for 5 min at 95 ° C ( + ) or not ( - ) .

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A. Characterization of C. perfringens β-toxin (CPB) constructs with different N-termini by gel electrophoretic analysis under denaturing (A) conditions. SDS-PAGE gel analyis of CPB samples in cholate (1μg per lane). CPB samples containing cholate were boiled for 5 min at 95°C (+) or not (-).


B . Coomassie stained Blue native PAGE gel ( 4 - 16 % ) of CPB samples ( 10ug per lane ) showing oligomer formation for all different N - termini constructs .

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B. Coomassie stained Blue native PAGE gel (4-16%) of CPB samples (10ug per lane) showing oligomer formation for all different N-termini constructs.


C . Alignment of CPB N - terminus with N - termini of different hemolysins and cryo - EM 2D classification and average of N - terminally tagged WT CPB ( 1 ) , Δ23CPB ( 2 ) , δ - toxin N - terminus Δ23CPB ( 3 ) , C - terminally tagged WT CPB ( 4 ) , Hla N - terminus Δ23CPB ( 5 ) and HlgB N - terminus Δ23CPB ( 6 ) .

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C. Alignment of CPB N-terminus with N-termini of different hemolysins and cryo-EM 2D classification and average of N-terminally tagged WT CPB (1), Δ23CPB (2), δ-toxin N-terminus Δ23CPB (3), C-terminally tagged WT CPB (4), Hla N-terminus Δ23CPB (5) and HlgB N-terminus Δ23CPB (6).


D . Viability of HEK 293FT / CD31 - GFP cells ( transduced with CD31 - GFP ) as a percentage of untreated control cells after incubation with indicated concentrations of toxins ( 24 h , 37 ° C ) . Data ( technical replicates ) are represented as means ( n = 4 ) ± SD .

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D. Viability of HEK 293FT/CD31-GFP cells (transduced with CD31-GFP) as a percentage of untreated control cells after incubation with indicated concentrations of toxins (24 h, 37°C). Data (technical replicates) are represented as means (n = 4) ± SD.


( B ) Cells expressing fluorescently - tagged α2 - tubulin ( atb2 ) were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs recorded within the final 1 . 1 µm of the cell for each strain . Red bars signify the

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(B) Cells expressing fluorescently-tagged α2-tubulin (atb2) were imaged every 5 seconds and the dwell time of ~100 individual iMTs recorded within the final 1.1 µm of the cell for each strain. Red bars signify the mean


( C ) Kymographs showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged microtubules ( MT ) in the presence ( top panels ) and absence ( bottom panels ) of Mcp1 . Banding on MT , caused by unequal incorporation of fluorescence , illustrates the force exerted on the MT by continued growth into the cell cortex . Dashed yellow line indicates the cell

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(C) Kymographs showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged microtubules (MT) in the presence (top panels) and absence (bottom panels) of Mcp1. Banding on MT, caused by unequal incorporation of fluorescence, illustrates the force exerted on the MT by continued growth into the cell cortex. Dashed yellow line indicates the cell end


( D ) MT growth speed was calculated from kymographs from control ( n = 16 ) and ∆ mcp1 cells ( n = 11 ) , Klp5 / Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control ( n = 44 ) and ∆ mcp1 cells ( n = 32 )

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(D) MT growth speed was calculated from kymographs from control (n=16) and ∆mcp1 cells (n=11), Klp5/Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control (n=44) and ∆mcp1 cells (n=32)


( E ) Average intensity of Klp5 / Klp6 at the plus ends of iMTs from multiple kymographs of control ( n = 19 ) or ∆ mcp1 cells ( n = 14 )

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(E) Average intensity of Klp5/Klp6 at the plus ends of iMTs from multiple kymographs of control (n=19) or ∆mcp1 cells (n=14)


( F ) Mixing experiment to compare fluorescently - tagged Klp5 / Klp6 levels between cells either expressing ( blue , closed arrowheads ) or deleted ( red , open arrowhead ) for Mcp1 distinguished by the absence of fluorescently - tagged nuclear envelope protein Cut11 ( left panel ) . Scale bar , 5µm . Box plot ( right panel ) shows quantitated fluorescence values for nuclear levels of Klp5 / Klp6 in control ( n = 44 ) and ∆ mcp1 cells ( n = 45 ) and at the MT plus end in control ( n = 64 ) and ∆ mcp1 cells ( n = 35 ) prior to

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(F) Mixing experiment to compare fluorescently-tagged Klp5/Klp6 levels between cells either expressing (blue, closed arrowheads) or deleted (red, open arrowhead) for Mcp1 distinguished by the absence of fluorescently-tagged nuclear envelope protein Cut11 (left panel). Scale bar, 5µm. Box plot (right panel) shows quantitated fluorescence values for nuclear levels of Klp5/Klp6 in control (n=44) and ∆mcp1 cells (n=45) and at the MT plus end in control (n=64) and ∆mcp1 cells (n=35) prior to shrinkage


( A ) Log phase cells expressing fluorescently - tagged MTs were imaged and the levels of either fluorescently - tagged Klp5 / Klp6 or Tea2 at the plus ends of growing MTs determined . Measurements ( klp5 - mNG klp6 - mNG , n = 305 ; tea2 - GFP , n = 359 ) were plotted against microtubule ( MT ) length and third order polynomial curves fitted to the

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(A) Log phase cells expressing fluorescently-tagged MTs were imaged and the levels of either fluorescently-tagged Klp5/Klp6 or Tea2 at the plus ends of growing MTs determined. Measurements (klp5-mNG klp6-mNG, n=305; tea2-GFP, n=359) were plotted against microtubule (MT) length and third order polynomial curves fitted to the data


( B ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Tea2 kinesin in control cells ( top panels ) or in the absence of Klp6 ( middle panels ) or Mcp1 ( bottom panels ) . Dashed yellow line indicates the cell

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(B) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Tea2 kinesin in control cells (top panels) or in the absence of Klp6 (middle panels) or Mcp1 (bottom panels). Dashed yellow line indicates the cell end


( C ) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in ( B ) for control ( n = 16 ) , ∆ klp6 ( n = 13 ) or ∆ mcp1 ( n = 14 ) cells

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(C) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in (B) for control (n=16), ∆klp6 (n=13) or ∆mcp1 (n=14) cells


( D ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Klp5 / Klp6 in control ( top panels , repeated from 1C ) or ∆ tea2 cells ( bottom panels ) . Dashed yellow line indicates the cell

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(D) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Klp5/Klp6 in control (top panels, repeated from 1C) or ∆tea2 cells (bottom panels). Dashed yellow line indicates the cell end


( E ) Cells expressing fluorescently - tagged tubulin were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs for each condition recorded within the final 1 . 1 µm of the cell . Red bars signify the

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(E) Cells expressing fluorescently-tagged tubulin were imaged every 5 seconds and the dwell time of ~100 individual iMTs for each condition recorded within the final 1.1 µm of the cell. Red bars signify the mean


( A ) Kymograph ( top left panels ) showing fluorescently - tagged Tea2 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the position of the cell end . Plots ( bottom left panels ) show both the relative fluorescence intensity and position for Tea2 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar 2µm . Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 ( green ) and Tip1 ( magenta ) over time ( top right ) and the distance of these pixels from the cell end ( bottom right )

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(A) Kymograph (top left panels) showing fluorescently-tagged Tea2 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the position of the cell end. Plots (bottom left panels) show both the relative fluorescence intensity and position for Tea2 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar 2µm. Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 (green) and Tip1 (magenta) over time (top right) and the distance of these pixels from the cell end (bottom right)


( A ) Kymograph ( top left panels ) showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the cell end . Plots ( bottom left panels ) show both the fluorescence intensity and position of Klp5 / Klp6 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar , 2µm . Intensity of the maximal value pixel at the plus end ( top right panel ) and its position relative to the cell end ( bottom right panel ) for both Klp5 / Klp6 ( green ) and Tip1 ( magenta ) plotted relative to MT shrinkage for this kymograph

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(A) Kymograph (top left panels) showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the cell end. Plots (bottom left panels) show both the fluorescence intensity and position of Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2µm. Intensity of the maximal value pixel at the plus end (top right panel) and its position relative to the cell end (bottom right panel) for both Klp5/Klp6 (green) and Tip1 (magenta) plotted relative to MT shrinkage for this kymograph


A ) The absolute abundance of centrosomal proteins in whole cell lysates , prepared from asynchronous growing cells , was determined using SRM mass spectrometry combined with stable isotope dilution . Copy numbers per cell were measured in three independent experiments . Because not all peptides could be detected in all experiments , histograms show average values determined in those two measurements in which most peptides could be measured reliably ; Results presented are means ± SEM . RPE - 1 ( non - transformed ) cells were compared with various tumor - derived ( U2OS , HeLa , KE37 ) or transformed cells ( HEK293T ) . Note the different scale for γ - tubulin on the y - axis .

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A) The absolute abundance of centrosomal proteins in whole cell lysates, prepared from asynchronous growing cells, was determined using SRM mass spectrometry combined with stable isotope dilution. Copy numbers per cell were measured in three independent experiments. Because not all peptides could be detected in all experiments, histograms show average values determined in those two measurements in which most peptides could be measured reliably; Results presented are means ± SEM. RPE-1 (non-transformed) cells were compared with various tumor-derived (U2OS, HeLa, KE37) or transformed cells (HEK293T). Note the different scale for γ-tubulin on the y-axis.


B ) Representative image showing centrosome preparation stained with antibodies to NEDD1 ( green ) and CP110 ( red ) . Bar : 10 μm .

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B) Representative image showing centrosome preparation stained with antibodies to NEDD1 (green) and CP110 (red). Bar: 10 μm.


C ) Purified centrosomes from KE37 cells were analyzed by SRM with stable isotope dilution . Histogram a priori represents relative copy numbers per centrosome ( Results presented are means ± SEM . ) . To predict protein abundance in absolute numbers ( average number of copies / centrosome ) , data were normalized to 1340 γ - tubulin molecules , i . e . the average number of γ - tubulin per centrosome as deduced by combining SRM and EGFP fluorescence measurements ( Figs 4 and 5 ) . Bars for Sas - 6 and STIL are marked in red to indicate that the corresponding values should be approximately doubled to correct for the fact that some 50 % of purified centrosomes are derived from G1 phase cells that mostly lack these two proteins . Note the different scale for γ - tubulin on the y - axis .

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C) Purified centrosomes from KE37 cells were analyzed by SRM with stable isotope dilution. Histogram a priori represents relative copy numbers per centrosome (Results presented are means ± SEM.). To predict protein abundance in absolute numbers (average number of copies/centrosome), data were normalized to 1340 γ-tubulin molecules, i.e. the average number of γ-tubulin per centrosome as deduced by combining SRM and EGFP fluorescence measurements (Figs 4 and 5). Bars for Sas-6 and STIL are marked in red to indicate that the corresponding values should be approximately doubled to correct for the fact that some 50% of purified centrosomes are derived from G1 phase cells that mostly lack these two proteins. Note the different scale for γ-tubulin on the y-axis.


Centrosome preparations from KE37 cells were analyzed by shotgun proteomics and relative protein abundances determined using the iBAQ shotgun proteomics method . The iBAQ values were normalized with the trendline as obtained from the SRM measurements ( Fig EV1 , panel E ) and γ - tubulin was normalized to 1340 copies / centrosome . A subset of centrosomal proteins is shown as described in ( Azimzadeh et al , 2012 ) . Black bars represent the centrosome subset and red bars represent the values obtained by SRM . Results presented are means ± SEM .

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Centrosome preparations from KE37 cells were analyzed by shotgun proteomics and relative protein abundances determined using the iBAQ shotgun proteomics method. The iBAQ values were normalized with the trendline as obtained from the SRM measurements (Fig EV1, panel E) and γ-tubulin was normalized to 1340 copies/centrosome. A subset of centrosomal proteins is shown as described in (Azimzadeh et al, 2012). Black bars represent the centrosome subset and red bars represent the values obtained by SRM. Results presented are means ± SEM.


A ) Fluorescence analysis of RPE - 1 cells expressing endogenously tagged γ - tubulin - EGFP . Cells were synchronized in G2 , using RO - 3306 , to allow for a clear distinction of the two centrosomes present at this cell cycle stage . Panels illustrate sum projections of the stacks from an original picture ( left ) , a whole cell mask ( middle ) and a centrosome mask ( right ) ; both masks are based on intensity thresholding . Bar : 10 μm .

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A) Fluorescence analysis of RPE-1 cells expressing endogenously tagged γ-tubulin-EGFP. Cells were synchronized in G2, using RO-3306, to allow for a clear distinction of the two centrosomes present at this cell cycle stage. Panels illustrate sum projections of the stacks from an original picture (left), a whole cell mask (middle) and a centrosome mask (right); both masks are based on intensity thresholding. Bar: 10 μm.


B ) EGFP - tagged viral like particles ( GFP - VLP2 / 6 ) are used as a reference for quantification of γ - tubulin - EGFP . The histogram shows the distribution of the total fluorescence intensity associated with single GFP - VLP2 / 6 ; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2 / 6 particle ( Charpilienne et al , 2001 ) . The inset shows a sum projection of EGFP - VLPs fluorescence image . Bar : 10 μm .

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B) EGFP-tagged viral like particles (GFP-VLP2/6) are used as a reference for quantification of γ-tubulin-EGFP. The histogram shows the distribution of the total fluorescence intensity associated with single GFP-VLP2/6; the 2 dashed lines delimit the population used for assigning an average fluorescence intensity value to represent the 120 GFP molecules associated with each VLP2/6 particle (Charpilienne et al, 2001). The inset shows a sum projection of EGFP-VLPs fluorescence image. Bar: 10 μm.


C ) Scatter plots show the numbers of γ - tubulin molecules per centrosome determined by calibrations using either SRM measurements ( n = 46 cells ) or EGFP - VLP2 / 6 fluorescence ( n = 40 cells ) . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot .

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C) Scatter plots show the numbers of γ-tubulin molecules per centrosome determined by calibrations using either SRM measurements (n=46 cells) or EGFP-VLP2/6 fluorescence (n=40 cells). The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot.


To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing ( endogenous ) γ - tubulin - EGFP ( A ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of γ - tubulin ( A ) molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : γ - tubulin : 45 ; in late S / G2 phase : γ - tubulin : 45 ; in late G2 phase : γ - tubulin : 48 ; in early mitosis : γ - tubulin : 31 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( P > 0 . 05 ) .

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To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing (endogenous) γ-tubulin-EGFP (A) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of γ-tubulin (A) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: γ-tubulin: 45; in late S/G2 phase: γ-tubulin: 45; in late G2 phase: γ-tubulin: 48; in early mitosis: γ-tubulin: 31. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing Sas - 6 ( B ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : Sas - 6 : 43 ; in late S / G2 phase : Sas - 6 : 46 ; in late G2 phase : Sas - 6 : 48 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( P > 0 . 05 ) .

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To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing Sas-6 (B) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: Sas-6: 43; in late S/G2 phase: Sas-6: 46; in late G2 phase: Sas-6: 48. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification , endogenously tagged RPE - 1 cells were synchronized as described under Materials and Methods . ( See also Fig EV5 , panel D ) . A - C ) Top panels : RPE - 1 cells expressing STIL ( C ) at the indicted cell cycle stages . Fluorescence images of representative cells are shown as maximum projections . Bars : 10μm . Bottom panels : Fluorescence quantifications were performed using the EGFP - VLP2 / 6 method for calibration . Scatter plots represent the number of STIL ( C ) molecules per centrosome at the indicated cell cycle stages . The black lines represent mean values ; numbers ± SEM are indicated on top of each scatter plot . The following numbers of cells were analyzed in early S phase : STIL : 79 ; in late S / G2 phase : STIL : 68 ; in late G2 phase : STIL : 68 . Note that fluorescence images were recorded using identical settings . P values from t - tests are indicated by the following symbols : * * * ( P < 0 . 001 ) , * * ( P = 0 . 001 - 0 . 01 ) , * ( P = 0 . 01 - 0 . 05 ) and ns ( P > 0 . 05 ) .

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To determine the cell cycle dependence of centrosomal protein abundance by fluorescence quantification, endogenously tagged RPE-1 cells were synchronized as described under Materials and Methods. (See also Fig EV5, panel D). A-C) Top panels: RPE-1 cells expressing STIL (C) at the indicted cell cycle stages. Fluorescence images of representative cells are shown as maximum projections. Bars: 10μm. Bottom panels: Fluorescence quantifications were performed using the EGFP-VLP2/6 method for calibration. Scatter plots represent the number of STIL (C) molecules per centrosome at the indicated cell cycle stages. The black lines represent mean values; numbers ± SEM are indicated on top of each scatter plot. The following numbers of cells were analyzed in early S phase: STIL: 79; in late S/G2 phase: STIL: 68; in late G2 phase: STIL: 68. Note that fluorescence images were recorded using identical settings. P values from t-tests are indicated by the following symbols: *** (P < 0.001), ** (P = 0.001-0.01), * (P = 0.01-0.05) and ns (P > 0.05).


a , Correlative light and electron microscopy of HeLa / GFP - LC3 cell infected with SIN / mCherry . capsid virus . Top left , deconvolved image of red and green fluorescence channels ; middle , three - dimensional surface reconstruction of red and green channels ; right , yellow ( red + green ) colocalization channel . Arrows denote yellow puncta that correspond to ' 1 ' and ' 2 ' in electron microscopy images below . Bottom left , electron microscopy of identical cell ; middle and right , high magnification images of insets ' 1 ' and ' 2 ' . Scale bars , left , 10 µm ; middle and right , 200 nm .

O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Correlative light and electron microscopy of HeLa/GFP-LC3 cell infected with SIN/mCherry.capsid virus. Top left, deconvolved image of red and green fluorescence channels; middle, three-dimensional surface reconstruction of red and green channels; right, yellow (red + green) colocalization channel. Arrows denote yellow puncta that correspond to '1' and '2' in electron microscopy images below. Bottom left, electron microscopy of identical cell; middle and right, high magnification images of insets '1' and '2'. Scale bars, left, 10 µm; middle and right, 200 nm.


a , Electron microscopy analysis of wild - type ( WT ) and Smurf1 - / - ( KO ) MEFs grown in normal media or EBSS ( starvation ) for 4 h . Arrowheads , representative autophagosomes ; arrows , representative autolysosomes . Scale bars , 500 nm .

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a, Electron microscopy analysis of wild-type (WT) and Smurf1-/- (KO) MEFs grown in normal media or EBSS (starvation) for 4 h. Arrowheads, representative autophagosomes; arrows, representative autolysosomes. Scale bars, 500 nm.


b , Western blot analyses of LC3 - I / II ( non - lipidated and lipidated forms of MAP1LC3 , respectively ) and p62 levels in MEFs of indicated genotype .

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b, Western blot analyses of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in MEFs of indicated genotype.


c , Quantification of colocalization of SIN / mCherry . capsid and GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection . Data shown represent mean ± s . e . m . number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples . * P < 0 . 001 against wild - type , Students t - test .

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 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 O O O O O 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, Quantification of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection. Data shown represent mean ± s.e.m. number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples. *P<0.001 against wild-type, Students t-test.


d , Representative confocal micrographs of colocalization of SIN / mCherry . capsid and GFP - LC3 in indicated MEFs 16 h after SIN / mCherry . capsid / GFP - LC3 infection images used for quantification in c . Arrows , colocalized red and green puncta . Scale bar , 15 & amp ; amp ; mgr ; m .

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

d, Representative confocal micrographs of colocalization of SIN/mCherry.capsid and GFP-LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP-LC3 infection images used for quantification in c. Arrows, colocalized red and green puncta. Scale bar, 15 &amp;mgr;m.


e , Representative electron microscopy images of indicated MEFs infected with HSV - 1 ( strain 17termA ) . White arrowheads , partially degraded viral nucleocapsids inside autolysosomes ; black arrowheads , intact viral nucleocapsids inside viral vesicles . Scale bar , 0 . 5 µm . For a - e , similar results were obtained in 3 - 5 independent experiments .

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e, Representative electron microscopy images of indicated MEFs infected with HSV-1 (strain 17termA). White arrowheads, partially degraded viral nucleocapsids inside autolysosomes; black arrowheads, intact viral nucleocapsids inside viral vesicles. Scale bar, 0.5 µm. For a-e, similar results were obtained in 3-5 independent experiments.