Figure 5 ( A ) Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle ( 0 . 1 % DMSO ) , 1 µM GSM III Data are presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 . ( B ) WT and mutant GSEC processivity estimated by the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio and presented as % of WT . Data presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 . Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle ( 0 . 1 % DMSO ) , 1 µM GSM III or ( C ) 1 µM GSM II . Data are presented as mean ± SD , N ≥ 3 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * P < 0 . 01 , * * P < 0 . 0001 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 3 , 45 ) = 56 . 9 . Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole - based GSM III . Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle ( DMSO ) or 1 µM GSM III were analysed by ELISA ( D ) ELISA - based Aβ profiles are presented as % of total Aβ ( Aβ37 + Aβ38 + Aβ40 + Aβ42 ) ( mean ± SD , N ≥ 3 ) ; One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ; * * * * P < 0 . 0001 . F ( DFn , DFd ) : F ( 4 , 17 ) = 71 . 68 . Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole - based GSM III . Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle ( DMSO ) or 1 µM GSM III were analysed by mass spectrometry ( MS ) . ( E ) APP mutation - driven effects on GSEC processivity according to the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio are presented as % of the WT condition ( mean ± SD ) . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ; * * * * P < 0 . 0001 . F ( DFn , DFd ) : F ( 4 , 17 ) = 71 . 68 . ( F ) MS - based profiles show the proportion of individual Aβ peptides on the total immunoprecipitated pool . Data shown as mean of 3 independent experiments .

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 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 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 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 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 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

Figure 5(A) Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.(B) WT and mutant GSEC processivity estimated by the Aβ (37+38)/(40+42) ratio and presented as % of WT. Data presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.Aβ profiles generated by WT and indicated mutant MEF lines in the presence of vehicle (0.1% DMSO), 1 µM GSM III or (C) 1 µM GSM II. Data are presented as mean ± SD, N ≥ 3 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). *P < 0.01, **P < 0.0001, ***P < 0.001, ****P < 0.0001 (F(DFn, DFd): F (3, 45) = 56.9.Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole-based GSM III. Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by ELISA (D) ELISA-based Aβ profiles are presented as % of total Aβ (Aβ37+ Aβ38+ Aβ40+ Aβ42) (mean ± SD, N ≥ 3); One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.Analysis of mutant APP substrates with regards to their effects on the response of GSEC to the imidazole-based GSM III. Aβ profiles secreted by HEK293T cells transiently expressing WT or mutant APPC99 substrates in presence of vehicle (DMSO) or 1 µM GSM III were analysed by mass spectrometry (MS). (E) APP mutation-driven effects on GSEC processivity according to the Aβ(37+38)/(40+42) ratio are presented as % of the WT condition (mean ± SD). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance; ****P < 0.0001. F(DFn, DFd): F (4, 17) = 71.68.(F) MS-based profiles show the proportion of individual Aβ peptides on the total immunoprecipitated pool. Data shown as mean of 3 independent experiments.


Figure 6 ( B ) Aβ profiles generated by WT or mutant PSEN1 cell lines transduced with APPC99 . Dotted lines indicate the levels of Aβ peptides ( % ) in WT profiles . Data presented as mean ± SD , N ≥ 3 independent experiments . ( C ) Pocket filling PSEN1 V236W and Y106W - V236W substitutions lead to increased Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio , indicating the activation of the sequential GSEC - mediated cleavage of APPC99 . Data presented as mean ± SD , N ≥ 3 independent experiments . ( D ) Aβ profile analysis in dose - response experiments for GSM III revealed no modulation of the PSEN1 V236W mutant cell line . Data are presented as mean ± SD , N = 2 independent experiments . For comparison purposes , WT and V236W mutant Aβ profiles are presented in panel 6B . ( E ) GSEC activity assay using purified GSEC and an APP - based fluorogenic peptide as substrate Upper panel : In the non - cleaved state the Dpn ( 2 , 4 - dinitrophenyl ) quencher group suppresses fluorescent emission from the Nma ( N - methyl - o - aminobenzoic acid ) fluorophore through Förster resonance energy transfer ( FRET ) . Upon GSEC cleavage , the Nma group emits at λ = 430 nm a fluorescent signal upon excitation at λ = 355 nm . Lower panel : Specific activities for the purified WT or mutant GSECs ( bearing the indicated substitutions in PSEN1 ) treated with DMSO ( vehicle ) , 10 µM GSMIII or GSM II . Data are normalized to the WT GSEC activity in DMSO ( vehicle ) . Data are shown as mean ± SD , N ≥ 3 independent experiments . Significance is shown for meaningful comparisons ; two - way ANOVA with comparison to WT was used to determine statistical significance ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant . ( F ) Transition state analogue inhibitor L - 685 , 458 ( Inhibitor X ) treatment of WT , V236W or Y240W mutant PSEN1 cell lines . Inhibitory profiles and derived IC50 values ( table ) indicate that the PSEN1 - V236W significantly increases the affinity for the inhibitor . Dotted line indicates 50 % inhibition . Data presented as mean ± SD , N = 3 independent experiments .

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 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 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 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

Figure 6(B) Aβ profiles generated by WT or mutant PSEN1 cell lines transduced with APPC99. Dotted lines indicate the levels of Aβ peptides (%) in WT profiles. Data presented as mean ± SD, N ≥ 3 independent experiments.(C) Pocket filling PSEN1 V236W and Y106W-V236W substitutions lead to increased Aβ(37+38)/(40+42) ratio, indicating the activation of the sequential GSEC-mediated cleavage of APPC99. Data presented as mean ± SD, N ≥ 3 independent experiments.(D) Aβ profile analysis in dose-response experiments for GSM III revealed no modulation of the PSEN1 V236W mutant cell line. Data are presented as mean ± SD, N = 2 independent experiments. For comparison purposes, WT and V236W mutant Aβ profiles are presented in panel 6B.(E) GSEC activity assay using purified GSEC and an APP-based fluorogenic peptide as substrate Upper panel: In the non-cleaved state the Dpn (2,4-dinitrophenyl) quencher group suppresses fluorescent emission from the Nma (N-methyl-o-aminobenzoic acid) fluorophore through Förster resonance energy transfer (FRET). Upon GSEC cleavage, the Nma group emits at λ = 430 nm a fluorescent signal upon excitation at λ = 355 nm. Lower panel: Specific activities for the purified WT or mutant GSECs (bearing the indicated substitutions in PSEN1) treated with DMSO (vehicle), 10 µM GSMIII or GSM II. Data are normalized to the WT GSEC activity in DMSO (vehicle). Data are shown as mean ± SD, N ≥ 3 independent experiments. Significance is shown for meaningful comparisons; two-way ANOVA with comparison to WT was used to determine statistical significance; **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.(F) Transition state analogue inhibitor L-685,458 (Inhibitor X) treatment of WT, V236W or Y240W mutant PSEN1 cell lines. Inhibitory profiles and derived IC50 values (table) indicate that the PSEN1-V236W significantly increases the affinity for the inhibitor. Dotted line indicates 50% inhibition. Data presented as mean ± SD, N = 3 independent experiments.


Figure 1C The binding of His - SOS2 to different PA species . The amount of each PA spot is 5 nmol . PA species : natural PA from soy , dipalmitoyl PA ( di16 : 0 PA ) , distearoyl PA ( di18 : 0 PA ) , dioleoyl PA ( di18 : 1 PA ) , dilinoleoyl PA ( di18 : 2 PA ) , palmitoyl - oleoyl PA ( 16 : 0 - 18 : 1 PA ) , palmitoyl - linoleoyl PA ( 16 : 0 - 18 : 2 PA ) , stearoyl - oleoyl PA ( 18 : 0 - 18 : 1 PA ) , stearoyl - linoleoyl PA ( 18 : 0 - 18 : 2 PA ) . For Mock control , no PA was incubated with the proteins . D The His - SOS2 binding to the liposome containing PA . Liposomes were made up of 16 : 0 - 18 : 2 PC only or the mixtures of 16 : 0 - 18 : 2 PC + 16 : 0 - 18 : 2 PA . After incubation with the recombinant proteins , liposomes were pelleted by centrifugation . The liposome - conjugated proteins in pellet and proteins remaining in supernatant were detected by immunoblot using anti - His antibody . For the Mock control , no liposomes were incubated with the proteins . E Microscale thermophoresis ( MST ) assay between His - SOS2 and PA . His - SOS2 was labeled and kept at a constant concentration ( 1 . 0 μM ) . His - RbohD1 - 100 , as the negative control , was labeled and kept at a constant concentration ( 4 . 5 μM ) . PA was hydrated in TBST buffer ( 0 . 005 % tween 20 ) , and diluted 16 times in a 1 : 1 ratio .

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 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

Figure 1C The binding of His-SOS2 to different PA species. The amount of each PA spot is 5 nmol. PA species: natural PA from soy, dipalmitoyl PA (di16:0 PA), distearoyl PA (di18:0 PA), dioleoyl PA (di18:1 PA), dilinoleoyl PA (di18:2 PA), palmitoyl-oleoyl PA (16:0-18:1 PA), palmitoyl-linoleoyl PA (16:0-18:2 PA), stearoyl-oleoyl PA (18:0-18:1 PA), stearoyl-linoleoyl PA (18:0-18:2 PA). For Mock control, no PA was incubated with the proteins.D The His-SOS2 binding to the liposome containing PA. Liposomes were made up of 16:0-18:2 PC only or the mixtures of 16:0-18:2 PC+16:0-18:2 PA. After incubation with the recombinant proteins, liposomes were pelleted by centrifugation. The liposome-conjugated proteins in pellet and proteins remaining in supernatant were detected by immunoblot using anti-His antibody. For the Mock control, no liposomes were incubated with the proteins.E Microscale thermophoresis (MST) assay between His-SOS2 and PA. His-SOS2 was labeled and kept at a constant concentration (1.0 μM). His-RbohD1-100, as the negative control, was labeled and kept at a constant concentration (4.5 μM). PA was hydrated in TBST buffer (0.005% tween 20), and diluted 16 times in a 1:1 ratio.


Figure 2A The PA synthesis defective mutants pldα1 pldδ - 1 and pldα1 pldδ - 2 are salt - sensitive . Six - d - old seedlings were grown in half - strength Murashige and Skoog ( MS ) , and then transferred to half - strength MS medium with or without 100 mM NaCl for 7 d . G , H Analysis of the average values of the net Na + ( G ) and K + ( H ) fluxes of Col - 0 , sos2 , C - 1 and C - 2 . Six - d - old seedlings were treated with or without 50 mM NaCl for 24 h . Error bars represent SD ( n ≥ 3 ) . I - N The contents of shoot Na + ( I ) , shoot K + ( J ) , root Na + ( K ) and root K + ( L ) as well as the relative ratios of shoot Na + / K + ( M ) and root Na + / K + ( N ) of Col - 0 and pldα1 pldδ mutants . Six - d - old seedlings were transferred to half - strength MS medium with or without 100 mM NaCl treatment for 7 d . Error bars represent SEM of three independent biological repeats . O - T The contents of shoot Na + ( O ) , shoot K + ( P ) , root Na + ( Q ) and root K + ( R ) as well as the relative ratios of shoot Na + / K + ( S ) and root Na + / K + ( T ) of Col - 0 , sos2 , C - 1 and C - 2 mutants . Six - d - old seedlings were transferred to half - strength MS medium with or without 50 mM NaCl treatment for 7 d . Error bars represent SEM of three independent biological repeats .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The PA synthesis defective mutants pldα1 pldδ-1 and pldα1 pldδ-2 are salt-sensitive. Six-d-old seedlings were grown in half-strength Murashige and Skoog (MS), and then transferred to half-strength MS medium with or without 100 mM NaCl for 7 d.G, H Analysis of the average values of the net Na+ (G) and K+ (H) fluxes of Col-0, sos2, C-1 and C-2. Six-d-old seedlings were treated with or without 50 mM NaCl for 24 h. Error bars represent SD (n ≥ 3).I-N The contents of shoot Na+ (I), shoot K+ (J), root Na+ (K) and root K+ (L) as well as the relative ratios of shoot Na+/K+ (M) and root Na+/K+ (N) of Col-0 and pldα1 pldδ mutants. Six-d-old seedlings were transferred to half-strength MS medium with or without 100 mM NaCl treatment for 7 d. Error bars represent SEM of three independent biological repeats. O-T The contents of shoot Na+ (O), shoot K+ (P), root Na+ (Q) and root K+ (R) as well as the relative ratios of shoot Na+/K+ (S) and root Na+/K+ (T) of Col-0, sos2, C-1 and C-2 mutants. Six-d-old seedlings were transferred to half-strength MS medium with or without 50 mM NaCl treatment for 7 d. Error bars represent SEM of three independent biological repeats. D


Figure 3A PA promotes the phosphorylation of SCaBP8 by SOS2 . The phosphorylation activity of His - SOS2 on His - SCaBP8 was detected with or without 1 or 10 μM PA . The solvent of methanol was used as mock control . Coomassie brilliant blue - stained ( CBB ) gel is shown in the upper panel , and autoradiograph ( autorad ) results are shown in the middle and lower panels . The numbers indicate the relative ratios of the signal intensity of autoradiograph . Statistical analysis of the relative ratios is shown B PA promotes the phosphorylation of SOS1 C300 by SOS2 . CBB gel is shown in the upper panel , and autorad result is shown in the lower panel . The numbers indicate the relative ratios of the signal intensity of autoradiograph . Statistical analysis of the relative ratios is shownC PA promotes the activity of SOS2 under salt stress . Ten - d - old transgenic plants expressing 35S : 6 × Myc - SOS2 in Col - 0 or pldα1 pldδ - 1 background were treated with or without 100 mM NaCl or 50 μM PA for 12 h . The total protein was extracted and immunoprecipitated with anti - Myc antibody - conjugated agarose , and then the purified Myc - SOS2 proteins at similar protein levels were incubated with His - SCaBP8 in the kinase assays . His - SCaBP8 was used as a specific substrate of SOS2 . The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc - SOS2 bands ( Autorad / Myc ) . Statistical analysis of the relative ratios is shown D PA promotes the PM localization of SOS2 under salt stress . Ten - d - old seedlings were treated as described in Fig 3C . GHR1 was used as a plasma membrane marker . PEPC was used as a cytoplasm marker . The numbers represent the relative ratios of the signal intensity between Myc - SOS2 and GHR1 bands ( Myc / GHR1 ) . Statistical analysis of the relative ratios is

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

Figure 3A PA promotes the phosphorylation of SCaBP8 by SOS2. The phosphorylation activity of His-SOS2 on His-SCaBP8 was detected with or without 1 or 10 μM PA. The solvent of methanol was used as mock control. Coomassie brilliant blue-stained (CBB) gel is shown in the upper panel, and autoradiograph (autorad) results are shown in the middle and lower panels. The numbers indicate the relative ratios of the signal intensity of autoradiograph. Statistical analysis of the relative ratios is shown B PA promotes the phosphorylation of SOS1 C300 by SOS2. CBB gel is shown in the upper panel, and autorad result is shown in the lower panel. The numbers indicate the relative ratios of the signal intensity of autoradiograph. Statistical analysis of the relative ratios is shownC PA promotes the activity of SOS2 under salt stress. Ten-d-old transgenic plants expressing 35S:6×Myc-SOS2 in Col-0 or pldα1 pldδ-1 background were treated with or without 100 mM NaCl or 50 μM PA for 12 h. The total protein was extracted and immunoprecipitated with anti-Myc antibody-conjugated agarose, and then the purified Myc-SOS2 proteins at similar protein levels were incubated with His-SCaBP8 in the kinase assays. His-SCaBP8 was used as a specific substrate of SOS2. The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc-SOS2 bands (Autorad/Myc). Statistical analysis of the relative ratios is shown D PA promotes the PM localization of SOS2 under salt stress. Ten-d-old seedlings were treated as described in Fig 3C. GHR1 was used as a plasma membrane marker. PEPC was used as a cytoplasm marker. The numbers represent the relative ratios of the signal intensity between Myc-SOS2 and GHR1 bands (Myc/GHR1). Statistical analysis of the relative ratios is shown


Figure 4B MST assays show that the site K57 is critical for SOS2 binding to PA . His - SOS2 with indicated point mutations were labeled and kept at a constant concentration ( 0 . 5 μM ) . PA was hydrated in TBST buffer and diluted 15 times in a 1 : 1 ratio . D SOS2K57G has weak activity and cannot be activated by PA under salt stress . Ten - d - old seedlings expressing 35S : 6 × Myc - SOS2 or 35S : 6 × Myc - SOS2K57G in Col - 0 or pldα1 pldδ - 1 background were treated with or without 100 mM NaCl for 12 h . The brightness and contrast ( B & C ) of the autoradiograph was adjusted to clearly show the activity of SOS2K57G . The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc - SOS2 bands ( Autorad / Myc ) . Statistical analysis of the relative ratios is shownE The site K57 is important for the PM localization of SOS2 under salt stress . Ten - d - old seedlings were treated with or without 100 mM NaCl for 12 h . The numbers represent the relative ratios of the signal intensity between Myc - SOS2 and GHR1 bands ( Myc / GHR1 ) . The asterisk points to nonspecific band . Statistical analysis of the relative ratios is

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 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 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

Figure 4B MST assays show that the site K57 is critical for SOS2 binding to PA. His-SOS2 with indicated point mutations were labeled and kept at a constant concentration (0.5 μM). PA was hydrated in TBST buffer and diluted 15 times in a 1:1 ratio.D SOS2K57G has weak activity and cannot be activated by PA under salt stress. Ten-d-old seedlings expressing 35S:6×Myc-SOS2 or 35S:6×Myc-SOS2K57G in Col-0 or pldα1 pldδ-1 background were treated with or without 100 mM NaCl for 12 h. The brightness and contrast (B&C) of the autoradiograph was adjusted to clearly show the activity of SOS2K57G. The numbers below the blots indicate the relative ratios of the signal intensity between the autoradiograph and Myc-SOS2 bands (Autorad/Myc). Statistical analysis of the relative ratios is shownE The site K57 is important for the PM localization of SOS2 under salt stress. Ten-d-old seedlings were treated with or without 100 mM NaCl for 12 h. The numbers represent the relative ratios of the signal intensity between Myc-SOS2 and GHR1 bands (Myc/GHR1). The asterisk points to nonspecific band. Statistical analysis of the relative ratios is shown


Figure 5A The loss - of - function mutants akt1 - 1 and akt1 - 2 are salt - sensitive . Six - d - old seedlings of Col - 0 ( WT ) and akt1 mutants were treated with or without 100 mM NaCl for 7 d . B Analysis for primary root length , related to Fig 5A . Data was shown in a box and whiskers ( min to max ) form ( n ≥ 10 ) . Statistical analysis was done by two - way ANOVA with Holm - Sidak ' s multiple comparisons test ( P ≤ 0 . 05 ) . CC , D Analysis of the average values of the net Na + ( C ) and K + ( D ) fluxes of Col - 0 and akt1 mutants with or without 100 mM NaCl treatment for 24 h . Error bars represent SD ( n ≥ 4 ) . K Time - and voltage - dependent AKT1 - mediated K + current recordings from oocytes . Whole - cell K + currents were recorded in oocytes that were co - injected with indicated mixture of cRNAs for AKT1 , SCaBP8 and SOS2 ( SOS2WT or SOS2K57G ) , in the presence of CIPK23 and CBL1 . 30 % SOS2 means that the cRNA injection amount of SOS2 was 30 % of SCaBP8 . The oocytes were treated with or without 50 μM PA for 1 . 5 h .

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Figure 5A The loss-of-function mutants akt1-1 and akt1-2 are salt-sensitive. Six-d-old seedlings of Col-0 (WT) and akt1 mutants were treated with or without 100 mM NaCl for 7 d. B Analysis for primary root length, related to Fig 5A. Data was shown in a box and whiskers (min to max) form (n ≥ 10). Statistical analysis was done by two-way ANOVA with Holm-Sidak's multiple comparisons test (P ≤ 0.05). CC, D Analysis of the average values of the net Na+ (C) and K+ (D) fluxes of Col-0 and akt1 mutants with or without 100 mM NaCl treatment for 24 h. Error bars represent SD (n ≥ 4).K Time- and voltage-dependent AKT1-mediated K+ current recordings from oocytes. Whole-cell K+ currents were recorded in oocytes that were co-injected with indicated mixture of cRNAs for AKT1, SCaBP8 and SOS2 (SOS2WT or SOS2K57G), in the presence of CIPK23 and CBL1. 30% SOS2 means that the cRNA injection amount of SOS2 was 30% of SCaBP8. The oocytes were treated with or without 50 μM PA for 1.5 h.


Figure 6D SOS2K40N and SOS2T168D ∆ FISL attenuate the SCaBP8 - mediated inhibition of AKT1 in oocytes . The recorded oocytes were co - injected with indicated mixture of cRNAs for AKT1 , SCaBP8 and SOS2 ( SOS2WT , SOS2T168D ∆ FISL or SOS2K40N ) in the presence of CIPK23 and CBL1 . E I - V relationship of AKT1 steady - state currents in oocytes . Error bars represent SD ( n ≥ 7 ) . F Comparison of steady - state currents elicited by - 150 mV , related to Fig 6E .

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Figure 6D SOS2K40N and SOS2T168D∆FISL attenuate the SCaBP8-mediated inhibition of AKT1 in oocytes. The recorded oocytes were co-injected with indicated mixture of cRNAs for AKT1, SCaBP8 and SOS2 (SOS2WT, SOS2T168D∆FISL or SOS2K40N) in the presence of CIPK23 and CBL1.E I-V relationship of AKT1 steady-state currents in oocytes. Error bars represent SD (n ≥ 7).F Comparison of steady-state currents elicited by -150 mV, related to Fig 6E.


Figure 1B . Interaction between PIF7 and ASF1A / ASF1B was detected using a pull - down assay . SUMO - His - fused PIF7 and GST - ASF1A / ASF1B were purified from E . coli . C . Interaction between PIF7 and ASF1A / ASF1B was detected using a semi - in vivo pull - down assay . GST - ASF1A / ASF1B were purified from E . coli . PIF7 - Flash protein from overexpressing PIF7 - Flash seedlings grown under white light or 1 h of shade treatment was detected using an anti - MYC antibody . E . Interaction between HIRA and ASF1A was detected using a pull - down assay . TF - His - HIRA and GST - ASF1A were purified from E . coli . F . Interactions between ASF1A and PIF7 / HIRA were detected in tobacco leaf cells . The PIF7 - Flash , HIRA - HA , and GFP - ASF1A ( GFP as control ) proteins were co - expressed in tobacco leaf cells . Anti - GFP Sepharose beads were used for Co - IP assay .

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Figure 1B. Interaction between PIF7 and ASF1A/ASF1B was detected using a pull-down assay. SUMO-His-fused PIF7 and GST-ASF1A/ASF1B were purified from E. coli.C. Interaction between PIF7 and ASF1A/ASF1B was detected using a semi-in vivo pull-down assay. GST-ASF1A/ASF1B were purified from E. coli. PIF7-Flash protein from overexpressing PIF7-Flash seedlings grown under white light or 1 h of shade treatment was detected using an anti-MYC antibody.E. Interaction between HIRA and ASF1A was detected using a pull-down assay. TF-His-HIRA and GST-ASF1A were purified from E. coli.F. Interactions between ASF1A and PIF7/HIRA were detected in tobacco leaf cells. The PIF7-Flash, HIRA-HA, and GFP-ASF1A (GFP as control) proteins were co-expressed in tobacco leaf cells. Anti-GFP Sepharose beads were used for Co-IP assay.


Figure 2B , Growth analysis of Col - 0 , pif7 - 1 , asf1ab , and hira - 1 seedlings treated with shade . New hypocotyl growth and real - time growth rates were recorded at 15 - min intervals after shade treatment . D . Hypocotyl lengths of Col - 0 , phyB - 9 , asf1ab , phyB - 9 * asf1ab , hira - 1 , and phyB - 9 * hira - 1 grown under white light . The scale bar represents 2 mm . Different letters indicate significant differences ( P < 0 . 01 ) calculated using one - way ANOVA with Tukey ' s HSD test . At least 20 seedlings were used for each treatment or genotype . E . Hypocotyl lengths of Col - 0 , pif7 - 1 , asf1ab , pif7 - 1 * asf1ab , hira - 1 , and pif7 - 1 * hira - 1 under white light and shade conditions . The scale bar represents 2 mm . Different letters indicate significant differences ( P < 0 . 01 ) calculated using one - way ANOVA with Tukey ' s HSD test . At least 20 seedlings were used for each treatment or genotype .

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Figure 2B, Growth analysis of Col-0, pif7-1, asf1ab, and hira-1 seedlings treated with shade. New hypocotyl growth and real-time growth rates were recorded at 15-min intervals after shade treatment.D. Hypocotyl lengths of Col-0, phyB-9, asf1ab, phyB-9 * asf1ab, hira-1, and phyB-9 * hira-1 grown under white light. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one-way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype. E. Hypocotyl lengths of Col-0, pif7-1, asf1ab, pif7-1 * asf1ab, hira-1, and pif7-1 * hira-1 under white light and shade conditions. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one-way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype.


Figure 3B . Boxplot displays the fold changes in shade - induced genes in Col - 0 , pif7 - 1 , asf1ab , and hira - 1 by comparing the transcript levels between white light and shade conditions . Different letters indicate significant differences ( P < 0 . 05 ) as determined by one - way ANOVA .

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Figure 3B. Boxplot displays the fold changes in shade-induced genes in Col-0, pif7-1, asf1ab, and hira-1 by comparing the transcript levels between white light and shade conditions. Different letters indicate significant differences (P < 0.05) as determined by one-way ANOVA.


Figure 4D . Integrative genomics viewer ( IGV ) screenshots showing the distribution of GFP - ASF1A enrichment at the ATHB2 , PIN7 , CSLC4 , BIM1 , ARL , and NPY8 loci . G . ChIP - PCR analysis of ASF1 enrichment using anti - ASF1A antibodies at the ATHB2 , PIN7 , CSLC4 , BIM1 , ARL , and NPY8 loci . Col - 0 and pif7 - 1 seedlings were grown under continuous white light or transferred to the shade for 1 h . Top panels show a schematic representation of the gene structures . The bottom panels represent the effects of shade on ASF1A enrichment . Shade - increased enrichment of ASF1A was calculated as input % SH minus input % WL . The difference at the P2 locus in Col - 0 was normalized to one for each gene . Different letters indicate statistically significant differences ( P < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , where n refers to technical replicates ) .

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Figure 4D. Integrative genomics viewer (IGV) screenshots showing the distribution of GFP-ASF1A enrichment at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci.G. ChIP-PCR analysis of ASF1 enrichment using anti-ASF1A antibodies at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci. Col-0 and pif7-1 seedlings were grown under continuous white light or transferred to the shade for 1 h. Top panels show a schematic representation of the gene structures. The bottom panels represent the effects of shade on ASF1A enrichment. Shade-increased enrichment of ASF1A was calculated as input% SH minus input% WL. The difference at the P2 locus in Col-0 was normalized to one for each gene. Different letters indicate statistically significant differences (P < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, where n refers to technical replicates).


Figure 5G . IGV screenshots show the distribution of H3 . 3 and H3 . 1 enrichment levels at the ATHB2 , PIN7 , CSLC4 , BIM1 , ARL , and NPY8 loci . H . Hypocotyl lengths of Col - 0 , pif7 - 1 , and h3 . 3kd under white light and shade conditions . The scale bar represents 2 mm . Different letters indicate significant differences ( P < 0 . 01 ) calculated using one - way ANOVA with Tukey ' s HSD test . At least 20 seedlings were used for each treatment or genotype . I . Relative expression levels of ATHB2 , PIN7 , CSLC4 , BIM1 , ARL , and NPY8 in Col - 0 , pif7 - 1 , and h3 . 3kd seedlings grown under white light or shade conditions . The seedlings were grown under white light for 6 d , and then maintained under white light , or transferred to shade for 1 h . Different letters indicate statistically significant differences ( P < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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Figure 5G. IGV screenshots show the distribution of H3.3 and H3.1 enrichment levels at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci.H. Hypocotyl lengths of Col-0, pif7-1, and h3.3kd under white light and shade conditions. The scale bar represents 2 mm. Different letters indicate significant differences (P < 0.01) calculated using one-way ANOVA with Tukey's HSD test. At least 20 seedlings were used for each treatment or genotype.I. Relative expression levels of ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 in Col-0, pif7-1, and h3.3kd seedlings grown under white light or shade conditions. The seedlings were grown under white light for 6 d, and then maintained under white light, or transferred to shade for 1 h. Different letters indicate statistically significant differences (P < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


Figure 6E . ChIP - PCR analysis of HTR5 ( H3 . 3 ) levels at the ATHB2 , PIN7 , CSLC4 , BIM1 , ARL , and NPY8 loci . Top panels show a schematic representation of the gene structures . The bottom panels represent the effects of shade on H3 . 3 enrichment . Shade - increased enrichment of H3 . 3 was calculated as the input % SH minus input % WL . The difference at the P2 locus in Col - 0 was normalized to one for each gene . Different letters indicate statistically significant differences ( P < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the means ± SDs ( n = 3 , where n refers to biological replicates ) .

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Figure 6E. ChIP-PCR analysis of HTR5 (H3.3) levels at the ATHB2, PIN7, CSLC4, BIM1, ARL, and NPY8 loci. Top panels show a schematic representation of the gene structures. The bottom panels represent the effects of shade on H3.3 enrichment. Shade-increased enrichment of H3.3 was calculated as the input% SH minus input% WL. The difference at the P2 locus in Col-0 was normalized to one for each gene. Different letters indicate statistically significant differences (P < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the means ± SDs (n = 3, where n refers to biological replicates).


Figure 1A . Western blot detection of PEX13 , ATG7 , SMURF1 , and FANCC in HeLa / GFP - LC3cells transfected with the indicated siRNA . Asterisk denotes non - specific band . B . Representative images of GFP - LC3 colocalization with mCherry - capsid at 10 h after Sindbis virus ( strain AO30 ) infection of HeLa / GFP - LC3 cells treated with the indicated siRNA . siPEX13 oligo # 2 is shown ; similar results were observed with three other siPEX13oligos . Arrowheads denote representative colocalized GFP - LC3 / mCherry - capsid puncta . Scale bars , 10 μm . C . Quantification of colocalized GFP - LC3 and mCherry - capsid puncta normalized to the number of mCherry - capsid puncta per cell ( % colocalization ) in the experiment shown in ( B ) . Bars are mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in more than three independent experiments . * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons . See Fig EV1A and B for quantification of mCherry - capsid puncta per cell and GFP - LC3 puncta per cell from the same experiment . D . Western blot detection of Pex13 in MEFs of indicated genotype . Asterisks denote non - specific bands . E . Quantification of colocalized GFP - LC3 and mCherry - capsid puncta normalized to the number of mCherry - capsid puncta per cell ( % colocalization ) at 8 h after Sindbis virus ( AO28 strain ) infection in MEFs of the indicated genotype . Bars are mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in more than three independent experiments . * p < 0 . 05 ; two - tailed unpaired t - test . See Fig EV1G and H for quantification of mCherry - capsid puncta per cell and GFP - LC3 puncta per cell from the same experiment . F . Western blot detection of p62 and LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle and cultured in normal medium ( starvation " - " ) or EBSS ( starvation " + " ) for 3 h . Similar results were observed in three independent experiments . G . Quantification of GFP - LC3 puncta in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or HBSS ( starvation " + " ) for 3 h . Bars are mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . * p < 0 . 05 ; two - tailed unpaired t - test . NC , nonsilencing control .

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Figure 1A. Western blot detection of PEX13, ATG7, SMURF1, and FANCC in HeLa/GFP-LC3cells transfected with the indicated siRNA. Asterisk denotes non-specific band.B. Representative images of GFP-LC3 colocalization with mCherry-capsid at 10 h after Sindbis virus (strain AO30) infection of HeLa/GFP-LC3 cells treated with the indicated siRNA. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13oligos. Arrowheads denote representative colocalized GFP-LC3/mCherry-capsid puncta. Scale bars, 10 μm.C. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) in the experiment shown in (B). Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. ***p<0.001; one-way ANOVA with adjustment for multiple comparisons. See Fig EV1A and B for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.D. Western blot detection of Pex13 in MEFs of indicated genotype. Asterisks denote non-specific bands.E. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) at 8 h after Sindbis virus (AO28 strain) infection in MEFs of the indicated genotype. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05; two-tailed unpaired t-test. See Fig EV1G and H for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.F. Western blot detection of p62 and LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle and cultured in normal medium (starvation "-") or EBSS (starvation "+") for 3 h. Similar results were observed in three independent experiments.G. Quantification of GFP-LC3 puncta in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or HBSS (starvation "+") for 3 h. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). *p<0.05; two-tailed unpaired t-test. NC, nonsilencing control.


Figure 2A . Representative images of Parkin - mediated clearance of mitochondrial outer membrane protein TOMM20 in HeLa / Parkin cells treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control . siPEX13 oligo # 2 is shown ; similar results were observed with three other siPEX13 oligos . Scale bars , 20 μm . B . Quantification of TOMM20 clearance in the experiment shown in ( A ) . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in more than three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons . C . Representative images of Parkin - mediated clearance of mitochondrial double - stranded DNA ( mtDNA ) in HeLa / HA - Parkin cells treated with indicated siRNA 8 h after treatment with 2 . 5 μM oligomycin and 250 nM antimycin A ( OA ) or DMSO vehicle control . siPEX13 oligo # 1 is shown ; similar results were observed with three other siPEX13 oligos . Nuclear DNA staining was masked using DAPI . Scale bars , 20 μm . D . Quantification of mtDNA clearance in the experiment shown in ( C ) . Results represent box plots of ~ 300 cells analyzed per sample . Whiskers represent 5 % - 95 % range , and each outlier is represented by a dot . Similar results were observed in three independent experiments . * * * * p < 0 . 0001 ; Kruskal - Wallis H - test . E . Representative images of TOMM20 fragmentation or compaction around perinuclear region in MEFs of the indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control . White lines indicate cell borders . Scale bars , 20 μm . F . Quantification of percentage of cells in experiment shown in ( E ) with accumulation of fragmented mitochondria after CCCP treatment . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * * p < 0 . 01 ; two - tailed unpaired t - test . G . Electron microscopic analysis of E18 . 5 mouse embryonic liver , heart , and skeletal muscle . Shown are representative images from one mouse . Similar results were observed in three mice per genotype . Arrowheads indicate damaged mitochondria . Scale bars , 1 μm .

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Figure 2A. Representative images of Parkin-mediated clearance of mitochondrial outer membrane protein TOMM20 in HeLa/Parkin cells treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13 oligos. Scale bars, 20 μm.B. Quantification of TOMM20 clearance in the experiment shown in (A). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.C. Representative images of Parkin-mediated clearance of mitochondrial double-stranded DNA (mtDNA) in HeLa/HA-Parkin cells treated with indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA) or DMSO vehicle control. siPEX13 oligo #1 is shown; similar results were observed with three other siPEX13 oligos. Nuclear DNA staining was masked using DAPI. Scale bars, 20 μm.D. Quantification of mtDNA clearance in the experiment shown in (C). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001; Kruskal-Wallis H-test.E. Representative images of TOMM20 fragmentation or compaction around perinuclear region in MEFs of the indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control. White lines indicate cell borders. Scale bars, 20 μm.F. Quantification of percentage of cells in experiment shown in (E) with accumulation of fragmented mitochondria after CCCP treatment. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01; two-tailed unpaired t-test.G. Electron microscopic analysis of E18.5 mouse embryonic liver, heart, and skeletal muscle. Shown are representative images from one mouse. Similar results were observed in three mice per genotype. Arrowheads indicate damaged mitochondria. Scale bars, 1 μm.


Figure 3A . Western blot detection of PEX13 in HeLa / Parkin cells transfected with indicated siRNA and siRNA - resistant PEX13 plasmid . To achieve similar PEX13 protein expression levels , 0 . 75 μg PEX13 I326T and PEX13 W313G plasmids were transfected compared to 0 . 25 μg WT PEX13 . Total plasmid level was adjusted using empty vector . Asterisk denotes non - specific band . B . Representative images of Parkin - mediated clearance of TOMM20 in HeLa / Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA - resistant plasmid and then treated with CCCP ( 10 μM , 16 h ) . Scale bars , 20 μm . See Fig EV3B for representative images of mitochondrial morphology ( TOMM20 staining ) in control cells treated with DMSO . C . Quantification of experiment shown in ( B ) . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - tailed unpaired t - test . D . Western blot detection of PEX13 expression in wild - type ( WT ) and PEX13 W313G ( W313G ) mutant primary human fibroblasts . E . Representative images of Parkin - mediated clearance of TOMM20 in wild - type ( WT ) and PEX13 W313G ( W313G ) mutant primary human fibroblasts treated with Oligomycin ( 2 . 5 μM ) + Antimycin A ( 250 nM ) for 24 h . Scale bars , 20 μm . W313G cells varied in morphology ; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology ( see Fig EV3C for higher resolution imaging ) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in ( F ) . F . Quantification of experiment shown in ( E ) . Results represent mean ± SEM of 5 groups of 10 images of random fields of cells ( > 350 cells analyzed per sample ) . Similar results were observed in two independent experiments . * * * p < 0 . 0001 ; two - tailed unpaired 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 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 O O O O O O O O O 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 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

Figure 3A. Western blot detection of PEX13 in HeLa/Parkin cells transfected with indicated siRNA and siRNA-resistant PEX13 plasmid. To achieve similar PEX13 protein expression levels, 0.75 μg PEX13 I326T and PEX13 W313G plasmids were transfected compared to 0.25 μg WT PEX13. Total plasmid level was adjusted using empty vector. Asterisk denotes non-specific band.B. Representative images of Parkin-mediated clearance of TOMM20 in HeLa/Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA-resistant plasmid and then treated with CCCP (10 μM, 16 h). Scale bars, 20 μm. See Fig EV3B for representative images of mitochondrial morphology (TOMM20 staining) in control cells treated with DMSO.C. Quantification of experiment shown in (B). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01,***p<0.001; two-tailed unpaired t-test.D. Western blot detection of PEX13 expression in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts.E. Representative images of Parkin-mediated clearance of TOMM20 in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts treated with Oligomycin (2.5 μM) + Antimycin A (250 nM) for 24 h. Scale bars, 20 μm. W313G cells varied in morphology; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology (see Fig EV3C for higher resolution imaging) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in (F).F. Quantification of experiment shown in (E). Results represent mean ± SEM of 5 groups of 10 images of random fields of cells (>350 cells analyzed per sample). Similar results were observed in two independent experiments. ***p<0.0001; two-tailed unpaired t-test.


Figure 4A - C Representative images of PEX13 and PMP70 ( A ) , PEX13 and TOMM20 ( B ) , or PEX13 - Flag and WIPI2 ( C ) colocalization in HeLa / Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment . Scale bars , 20 μm ( A - B ) and 5 μm ( C ) . D Western blot detection of endogenous PEX13 in HeLa / Parkin cells treated with 10 μM CCCP for the indicated time .

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Figure 4A-C Representative images of PEX13 and PMP70 (A), PEX13 and TOMM20 (B), or PEX13-Flag and WIPI2 (C) colocalization in HeLa/Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment. Scale bars, 20 μm (A-B) and 5 μm (C).D Western blot detection of endogenous PEX13 in HeLa/Parkin cells treated with 10 μM CCCP for the indicated time.


Figure 5A . Quantitative real - time PCR detection of mRNA levels for PEX3 , PEX13 , PEX14 , and PEX19 in HeLa cells transfected with the indicated siRNA . * * * p < 0 . 001 ; two - tailed unpaired t - test . B . Quantification of Parkin - mediated TOMM20 clearance in HeLa / Parkin cells transfected with the indicated siRNA 16 h after treatment with 10 µM CCCP . Results represent mean ± SEM of triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons . C . Quantification of mtDNA clearance in HeLa / HA - Parkin cells transfected with the indicated siRNA 8 h after treatment with 2 . 5 μM oligomycin and 250 nM antimycin A ( OA ) . Results represent box plots of ~ 300 cells analyzed per sample . Whiskers represent 5 % - 95 % range , and each outlier is represented by a dot . Similar results were observed in three independent experiments . * * * * p < 0 . 0001 , NS = not significant ; Kruskal - Wallis H - test . D . Quantification of GFP - LC3 puncta in HeLa / GFP - LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media . Results represent mean ± SEM in triplicate samples ( ~ 100 cells analyzed per sample ) . Similar results were observed in three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = not significant ; one - way ANOVA with adjustment for multiple comparisons . Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs . NC siRNA within each treatment group .

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Figure 5A. Quantitative real-time PCR detection of mRNA levels for PEX3, PEX13, PEX14, and PEX19 in HeLa cells transfected with the indicated siRNA. ***p<0.001; two-tailed unpaired t-test.B. Quantification of Parkin-mediated TOMM20 clearance in HeLa/Parkin cells transfected with the indicated siRNA 16 h after treatment with 10 µM CCCP. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.C. Quantification of mtDNA clearance in HeLa/HA-Parkin cells transfected with the indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001, NS= not significant; Kruskal-Wallis H-test.D. Quantification of GFP-LC3 puncta in HeLa/GFP-LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media. Results represent mean ± SEM in triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01, ***p<0.001, NS= not significant; one-way ANOVA with adjustment for multiple comparisons. Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs. NC siRNA within each treatment group.


Figure 1A , Cell viability of NS , GBM , and astrocyte cell lines measured 72 h after irradiation ( fold vs . non - irradiated cells , ctrl ) . * : one - way ANOVA , P = 0 . 0001 . B , Cell viability of BT308NS and their pseudo - differentiated progeny ( BT308 diff ) measured 24 h after irradiation ( fold vs . non - irradiated cells , ctrl ) . * : t - test , P = 0 . 0006 . C , Flow cytometry analysis of phosphorylated histone H2AX ( H2AX ) at the indicated time points after IR ( 5 Gy , fold vs . non - irradiated cells , ctrl ) . * : one - way ANOVA , P = 0 . 03 . D , Neutral comet assay performed 24 h after IR ( 5 Gy ) . Ctrl : non - irradiated cells . Comet tail ' s length is proportional to DSB extent . E , Western blot showing phosphorylation of ATM ( pATM ) and Chk2 ( pChk2 ) , and accumulation of RAD51 after IR ( 5 Gy ) . Total ATM protein is also shown . Vinculin was used as loading control . F , qPCR of RAD51 expression measured in NS , BT308 diff , and astrocytes at the indicated time - points after IR ( 5 Gy , fold vs . non - irradiated cells , ctrl ) . Data are represented as mean ± SEM .

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Figure 1A, Cell viability of NS, GBM, and astrocyte cell lines measured 72 h after irradiation (fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.0001.B, Cell viability of BT308NS and their pseudo-differentiated progeny (BT308 diff) measured 24 h after irradiation (fold vs. non-irradiated cells, ctrl). *: t-test, P=0.0006.C, Flow cytometry analysis of phosphorylated histone H2AX (H2AX) at the indicated time points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.03.D, Neutral comet assay performed 24 h after IR (5 Gy). Ctrl: non-irradiated cells. Comet tail's length is proportional to DSB extent.E, Western blot showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 after IR (5 Gy). Total ATM protein is also shown. Vinculin was used as loading control.F, qPCR of RAD51 expression measured in NS, BT308 diff, and astrocytes at the indicated time-points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). Data are represented as mean ± SEM.


Figure 2A , Flow cytometry analysis of PKH - 26 staining in irradiated BT308NS , 24 h after the last irradiation . The percentage of PKH - 26pos cells is indicated . Ctrl : non - irradiated cells . The mean fluorescence intensity ( MFI ) was as follow : ctrl , 58 . 21 ; 1 . 5 Gy × 4 , 69 . 35 ; 2 Gy × 3 , 68 . 20 ; 5 Gy , 60 . 29 . B , qPCR of SOX2 expression measured in NS 6 h after IR ( 5 Gy , fold vs . non - irradiated cells , ctrl ) . * : t - test , p < 0 . 02 . C , LDA ( sphere forming ) measuring the enrichment of GSCs after IR ( 5 Gy ) . Ctrl : non - irradiated cells * : 2 test , p < 0 . 05 . E , Tumor incidence in the LDA performed in p3 mice , as described in D . F , Histogram showing the in vivo GSC frequency measured by LDA in p3 tumors . GSC frequency ( 95 % CI ) was as follows : NS - ctrl , 1 / 258 . 2 ( 97 . 67 - 683 . 9 ) ; NS - IR 1 / 22 . 1 ( 8 . 05 - 62 . 6 ) . * : 2 test , P = 1 . 6 × 10 - 4 . G , LDA ( sphere forming ) measuring the in vitro frequency of GSCs in cells derived from p3 tumors . * : 2 test , P = 0 . 0006 . H , LDA ( sphere forming ) measuring the in vitro frequency of GSCs in cells derived from intracranial tumors generated by BT463NS , and irradiated in vivo ( 2 Gy × 3 days ) ( n = 3 ) . Ctrl : non - irradiated tumors . * : 2 test , P = 2 . 04 × 10 - 11 . Data are represented as mean ± CI or ± SEM in B .

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Figure 2A, Flow cytometry analysis of PKH-26 staining in irradiated BT308NS, 24 h after the last irradiation. The percentage of PKH-26pos cells is indicated. Ctrl: non-irradiated cells. The mean fluorescence intensity (MFI) was as follow: ctrl, 58.21; 1.5 Gy × 4, 69.35; 2 Gy × 3, 68.20; 5 Gy, 60.29.B, qPCR of SOX2 expression measured in NS 6 h after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.02.C, LDA (sphere forming) measuring the enrichment of GSCs after IR (5 Gy). Ctrl: non-irradiated cells *: 2 test, p<0.05.E, Tumor incidence in the LDA performed in p3 mice, as described in D.F, Histogram showing the in vivo GSC frequency measured by LDA in p3 tumors. GSC frequency (95% CI) was as follows: NS-ctrl, 1/258.2 (97.67-683.9); NS-IR 1/22.1 (8.05-62.6). *: 2 test, P=1.6×10-4.G, LDA (sphere forming) measuring the in vitro frequency of GSCs in cells derived from p3 tumors. *: 2 test, P=0.0006.H, LDA (sphere forming) measuring the in vitro frequency of GSCs in cells derived from intracranial tumors generated by BT463NS, and irradiated in vivo (2 Gy × 3 days) (n = 3). Ctrl: non-irradiated tumors. *: 2 test, P=2.04×10-11. Data are represented as mean ± CI or ± SEM in B.


Figure 3B , LDA ( sphere forming ) measuring the GSC frequency after IR ( 5 Gy ) in METhigh and METneg subpopulations sorted from BT308NS . * : 2 test , P = 0 . 001 . C , Flow cytometry analysis of MET in irradiated BT308NS , 24 h after the last irradiation ( 2 Gy × 3 days or a single dose of 5 Gy ) . Dotted lines : threshold to define the percentage of MET - expressing cells . Ctrl : non - irradiated cells . The MFI was as follows : ctrl , 18 ; 2 Gy × 3 , 22 . 77 ; 5 Gy , 19 . 49 . D , Flow cytometry analysis of MET in BT308NS weekly treated with IR ( 2 Gy ) for 6 weeks . Dotted lines : threshold to define the percentage of MET - expressing cells . Ctrl : non - irradiated cells . E , Representative immunofluorescence staining of MET ( red ) on tumors generated by subcutaneous injection of BT308NS , irradiated ( 2 Gy × 3 ) and explanted 72 h after the last irradiation . Nuclei are counterstained with DAPI ( blue ) . Ctrl : non - irradiatedtumors . Scale bar , 10 µm ( 63 × magnification ) . F left , Quantification of the percentage of MET - expressing cells in tumor sections represented in ( E ) ( n = 12 HPF / group ) . HPF : high - power field . * : t - test , P = 0 . 001 . F right , Quantification of MET mean fluorescence intensity in tumor sections represented in ( E ) ( n = 12 HPF / group , fold vs . non - irradiated cells , ctrl ) . * : t - test , P = 0 . 00016 . G , representative METimmunohistochemistry staining of matched primary and recurrent tumors . Scale bar , 50 µm ( 40 × magnification ) . H , Histogram representing the number of patient displaying different percentages of MET expressing cells in their primary or recurrent tumors : negative , 0 - 5 % ; low , 6 - 29 % ; moderate , 30 - 69 % ; high , 70 - 100 % ( see also Appendix Table 4 ) . I , Dot plot representing the MET cumulative score ( MET positive cell score + MET staining intensity score ) in 19 primary and recurrent tumors ( see Appendix Table 4 ) . Wilcoxon , p < 0 . 02 . Data are represented as mean ± SEM or ± CI 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 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 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 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

Figure 3B, LDA (sphere forming) measuring the GSC frequency after IR (5 Gy) in METhigh and METneg subpopulations sorted from BT308NS. *: 2 test, P=0.001.C, Flow cytometry analysis of MET in irradiated BT308NS, 24 h after the last irradiation (2 Gy × 3 days or a single dose of 5 Gy). Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells. The MFI was as follows: ctrl, 18; 2 Gy × 3, 22.77; 5 Gy, 19.49.D, Flow cytometry analysis of MET in BT308NS weekly treated with IR (2 Gy) for 6 weeks. Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells.E, Representative immunofluorescence staining of MET (red) on tumors generated by subcutaneous injection of BT308NS, irradiated (2 Gy × 3) and explanted 72 h after the last irradiation. Nuclei are counterstained with DAPI (blue). Ctrl: non-irradiatedtumors. Scale bar, 10 µm (63× magnification). F left, Quantification of the percentage of MET-expressing cells in tumor sections represented in (E) (n = 12 HPF/group). HPF: high-power field. *: t-test, P=0.001. F right, Quantification of MET mean fluorescence intensity in tumor sections represented in (E) (n = 12 HPF/group, fold vs. non-irradiated cells, ctrl). *: t-test, P=0.00016.G, representative METimmunohistochemistry staining of matched primary and recurrent tumors. Scale bar, 50 µm (40× magnification). H, Histogram representing the number of patient displaying different percentages of MET expressing cells in their primary or recurrent tumors: negative, 0-5%; low, 6-29%; moderate, 30-69%; high, 70-100% (see also Appendix Table 4). I, Dot plot representing the MET cumulative score (MET positive cell score + MET staining intensity score) in 19 primary and recurrent tumors (see Appendix Table 4). Wilcoxon, p<0.02. Data are represented as mean ± SEM or ± CI in B.


Figure 4A , Flow - cytometry analysis of Annexin V / DAPI incorporation and MET expression in BT308NS 36 h after IR ( 5 Gy ) . Ctrl : non - irradiated cells . Blue : MET - negative cells . Red : MET - expressing cells . B , Western Blot of METneg and METhigh subpopulations sorted from BT452NS , showing activation of PARP ( cleaved PARP ) and Caspase 3 ( cleaved casp - 3 ) after IR ( 5 Gy ) . Vinculin was used as loading control . C , Cell viability of METhigh and METneg subpopulations sorted from BT308NS , measured 96 h after IR ( 1 - 10 Gy , fold vs . non - irradiated cells , ctrl ) . * : t - test , p < 0 . 001 . D , Clonogenic assay with METhigh and METneg subpopulations sorted from BT308NS and irradiated ( 1 - 10 Gy , fold vs . non - irradiated cells , ctrl ) . * : t - test , p < 0 . 05 . E , Neutral comet assay in METhigh and METneg subpopulations sorted from BT308NS , performed 24 h after IR ( 5 Gy ) . Ctrl : non - irradiated cells . Comet tail ' s length is proportional to DSB extent . F , Western Blot of METneg and METhigh subpopulations sorted from BT308NS , showing constitutive ( ctrl ) and / or IR - induced phosphorylation of ATM ( pATM ) , Chk2 ( pChk2 ) and H2AX ( H2AX ) , and accumulation of RAD51 , 24 h after IR ( 5 Gy ) . Total ATM protein is also shown . β - actin was used as loading control . G , qPCR of RAD51 expression in METhigh and METneg subpopulations sorted from BT308NS 24 h after IR ( 5 Gy , fold vs . non - irradiated control cells , ctrl ) . * : t - test , P = 0 . 02 . Data are represented as mean ± SEM .

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 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 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 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

Figure 4A, Flow-cytometry analysis of Annexin V/DAPI incorporation and MET expression in BT308NS 36 h after IR (5 Gy). Ctrl: non-irradiated cells. Blue: MET-negative cells. Red: MET-expressing cells.B, Western Blot of METneg and METhigh subpopulations sorted from BT452NS, showing activation of PARP (cleaved PARP) and Caspase 3 (cleaved casp-3) after IR (5 Gy). Vinculin was used as loading control.C, Cell viability of METhigh and METneg subpopulations sorted from BT308NS, measured 96 h after IR (1-10 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.001.D, Clonogenic assay with METhigh and METneg subpopulations sorted from BT308NS and irradiated (1-10 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.05.E, Neutral comet assay in METhigh and METneg subpopulations sorted from BT308NS, performed 24 h after IR (5 Gy). Ctrl: non-irradiated cells. Comet tail's length is proportional to DSB extent.F, Western Blot of METneg and METhigh subpopulations sorted from BT308NS, showing constitutive (ctrl) and/or IR-induced phosphorylation of ATM (pATM), Chk2 (pChk2) and H2AX (H2AX), and accumulation of RAD51, 24 h after IR (5 Gy). Total ATM protein is also shown. β-actin was used as loading control.G, qPCR of RAD51 expression in METhigh and METneg subpopulations sorted from BT308NS 24 h after IR (5 Gy, fold vs. non-irradiated control cells, ctrl). *: t-test, P=0.02. Data are represented as mean ± SEM.


Figure 5A , Cell viability of BT308NS , measured 48 h after IR ( 2 or 5 Gy ) in the absence ( mock ) or in the presence of 10 ng / ml HGF ( fold vs . non - irradiated mock treated cells , mock ) . Ctrl : non - irradiated cells . * : t - test ( 5 Gy + HGF ) vs . ( 5 Gy ) , p < 0 . 0001 . B , Flow - cytometry analysis of Annexin V / DAPI incorporation in BT308NS irradiated ( 5 Gy ) in the absence ( mock ) or in the presence of HGF . Histograms show the percentage of Annexin V - positive cells 48 h after IR . Ctrl : non - irradiated cells . C , Cell viability of BT308NS , irradiated in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of the MET inhibitor JNJ38877605 ( 500 nM ) , and analyzed at the indicated time points . Vehicle : non - irradiated cells ( fold vs . vehicle - treated cells at time 0 , mock ) . * : t - test , p < 0 . 01 . D , Cell viability as in C , measured 5 days after IR ( 1 - 10 Gy , fold vs . vehicle - treated cells , mock ) . * : t - test , p < 0 . 03 . E , Flow - cytometry analysis of Annexin V / DAPI incorporation in BT308NS irradiated ( 5 Gy ) in the absence ( vehicle ) or in the presence ( JNJ ) of JNJ38877605 . Histograms show the percentage of Annexin V - positive cells 36 h after IR . Ctrl : non - irradiated cells . * : t - test ( 5 Gy + JNJ ) vs . ( 5 Gy ) , P = 0 . 01 . F , Western blot of BT308NS showing caspase 3 activation ( cleaved casp - 3 ) , at the indicated time points after IR in the absence ( - ) or in the presence ( + ) of JNJ38877605 . Vinculin was used as loading control . G , Clonogenic assay with METneg and METhigh subpopulations sorted from BT308NS , irradiated ( 5 Gy ) in the absence ( vehicle ) or in the presence of MET inhibitors ( JNJ38877605 and Crizotinib , 500 nM ) . Ctrl : non - irradiated cells . * : t - test , p < 0 . 0004 . H , LDA ( sphere forming assay ) with METneg and METhigh subpopulations sorted from BT308NS , measuring GSC frequency after irradiation in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated vehicle - treated cells . * : 2 test , P = 0 . 0002 . I , Flow cytometry analysis of phosphorylated histone H2AX ( H2AX ) in the whole BT308NS at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 ( fold vs . non - irradiated cells at time 0 , mock ) . * : t - test , p < 0 . 05 . J , Neutral comet assay with BT308NS performed 24 h after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated vehicle - treated cells . Comet tail ' s length is proportional to DSB extent . Data are represented as mean ± SEM or ± CI in 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 O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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

Figure 5A, Cell viability of BT308NS, measured 48 h after IR (2 or 5 Gy) in the absence (mock) or in the presence of 10 ng/ml HGF (fold vs. non-irradiated mock treated cells, mock). Ctrl: non-irradiated cells. *: t-test (5 Gy + HGF) vs. (5 Gy), p<0.0001.B, Flow-cytometry analysis of Annexin V/DAPI incorporation in BT308NS irradiated (5 Gy) in the absence (mock) or in the presence of HGF. Histograms show the percentage of Annexin V-positive cells 48 h after IR. Ctrl: non-irradiated cells.C, Cell viability of BT308NS, irradiated in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM), and analyzed at the indicated time points. Vehicle: non-irradiated cells (fold vs. vehicle-treated cells at time 0, mock). *: t-test, p<0.01.D, Cell viability as in C, measured 5 days after IR (1-10 Gy, fold vs. vehicle-treated cells, mock). *: t-test, p<0.03.E, Flow-cytometry analysis of Annexin V/DAPI incorporation in BT308NS irradiated (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Histograms show the percentage of Annexin V-positive cells 36 h after IR. Ctrl: non-irradiated cells. *: t-test (5 Gy + JNJ) vs. (5 Gy), P=0.01.F, Western blot of BT308NS showing caspase 3 activation (cleaved casp-3), at the indicated time points after IR in the absence (-) or in the presence (+) of JNJ38877605. Vinculin was used as loading control.G, Clonogenic assay with METneg and METhigh subpopulations sorted from BT308NS, irradiated (5 Gy) in the absence (vehicle) or in the presence of MET inhibitors (JNJ38877605 and Crizotinib, 500 nM). Ctrl: non-irradiated cells. *: t-test, p<0.0004.H, LDA (sphere forming assay) with METneg and METhigh subpopulations sorted from BT308NS, measuring GSC frequency after irradiation in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. *: 2 test, P=0.0002.I, Flow cytometry analysis of phosphorylated histone H2AX (H2AX) in the whole BT308NS at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605 (fold vs. non-irradiated cells at time 0, mock). *: t-test, p<0.05.J, Neutral comet assay with BT308NS performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated vehicle-treated cells. Comet tail's length is proportional to DSB extent. Data are represented as mean ± SEM or ± CI in H.


Figure 6A , Western blot of BT463NS showing phosphorylation of ATM ( pATM ) and Chk2 ( pChk2 ) , and accumulation of RAD51 at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + HGF ) of HGF ( 10 ng / ml ) . Total ATM is also shown . H3 was used as loading control . B , Western blot of BT308NS showing phosphorylation of ATM ( pATM ) and Chk2 ( pChk2 ) , and accumulation of RAD51 at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of the MET inhibitor JNJ38877605 ( 500 nM ) . Total ATM is also shown . β - actin was used as loading control . Veh : non - irradiated vehicle - treated cells . C , Densitometric analysis of ATM phosphorylation shown in ( B ) . * : t - test , p < 0 . 01 . D , Western blot of BT308NS showing phosphorylation of ATM , Chk2 , and AKT ( pAKT ) , and accumulation of RAD51 24 h after IR ( 5 Gy ) in the absence ( vehicle , veh ) or in the presence of MAPK ( PD98059 , 20 M ) , AKT ( Ly294002 , 20 M ) , MET ( JNJ38877605 ) , or ATM ( CGK733 , 10 M ) inhibitors . Total ATM and AKT are also shown . β - actin was used as loading control . Ctrl : non - irradiated cells . E , Western blot of BT463NS showing phosphorylation of ATM , Chk2 , and Aurora Kinase A ( pAurora A ) at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + Ly ) of the AKT inhibitor Ly294002 . Total ATM and Aurora Kinase A are also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated cells . F , Western blot of BT308NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Total Aurora kinase A is also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated cells . G , Western blot of BT463NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + HGF ) of HGF . Total Aurora kinase A is also shown . H3 was used as loading control ( the same control applies to both panel A and G ) . H , Western blot of BT308NS showing phosphorylation of ATM and Aurora Kinase A at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + MLN ) of the Aurora Kinase A inhibitor MLN8237 ( 20 M ) . Total ATM and Aurora kinase A are also shown . H3 was used as loading control . Veh : non - irradiated vehicle - treated cells . I , Cell viability of BT308NS , measured 72 h after IR ( 5 Gy ) in the absence ( vehicle ) or in the presence of MET ( JNJ38877605 ) , Aurora Kinase A ( MLN8237 ) , or AKT ( Ly294002 ) inhibitors ( fold vs . non - irradiated vehicle - treated cells , mock ) . * : t - test ( 5 Gy + inhibitor ) vs . ( 5 Gy + vehicle ) , p < 0 . 01 .

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 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 O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 6A, Western blot of BT463NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml). Total ATM is also shown. H3 was used as loading control.B, Western blot of BT308NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM). Total ATM is also shown. β-actin was used as loading control. Veh: non-irradiated vehicle-treated cells. C, Densitometric analysis of ATM phosphorylation shown in (B). *: t-test, p<0.01.D, Western blot of BT308NS showing phosphorylation of ATM, Chk2, and AKT (pAKT), and accumulation of RAD51 24 h after IR (5 Gy) in the absence (vehicle, veh) or in the presence of MAPK (PD98059, 20 M), AKT (Ly294002, 20 M), MET (JNJ38877605), or ATM (CGK733, 10 M) inhibitors. Total ATM and AKT are also shown. β-actin was used as loading control. Ctrl: non-irradiated cells.E, Western blot of BT463NS showing phosphorylation of ATM, Chk2, and Aurora Kinase A (pAurora A) at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + Ly) of the AKT inhibitor Ly294002. Total ATM and Aurora Kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.F, Western blot of BT308NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Total Aurora kinase A is also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.G, Western blot of BT463NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF. Total Aurora kinase A is also shown. H3 was used as loading control (the same control applies to both panel A and G).H, Western blot of BT308NS showing phosphorylation of ATM and Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + MLN) of the Aurora Kinase A inhibitor MLN8237 (20 M). Total ATM and Aurora kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells.I, Cell viability of BT308NS, measured 72 h after IR (5 Gy) in the absence (vehicle) or in the presence of MET (JNJ38877605), Aurora Kinase A (MLN8237), or AKT (Ly294002) inhibitors (fold vs. non-irradiated vehicle-treated cells, mock). *: t-test (5 Gy + inhibitor) vs. (5 Gy + vehicle), p<0.01.


Figure 7A , Western blot of BT371NS showing p21 phosphorylation ( pp21Thr145 ) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + HGF ) of HGF ( 10 ng / ml ) . B , Western blot of BT463NS showing p21 phosphorylation ( pp21Thr145 ) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of the MET inhibitor JNJ38877605 ( 500 nM ) . C , Western blot of BT308NS showing p21 localization in cytoplasmic / nuclear extracts at the indicated time points after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vinculin and lamin B were used as loading controls for cytoplasmic and nuclear fractions , respectively . D , Representative immunofluorescence staining of p21 ( red ) on BT308NS cells 3 h after IR ( 5 Gy ) in the absence ( vehicle ) or in the presence ( JNJ ) of JNJ38877605 . Ctrl : non - irradiated cells . Nuclei are counterstained with DAPI ( blue ) . Scale bar , 10 µm ( 63 × magnification ) . E , Quantification of the percentage of cells showing p21 cytoplasmic or nuclear localization in BT308NS represented in ( D ) ( n = 10 HPF / group ) . HPF : high - power field . * : t - test ( 5 Gy + JNJ ) vs . ( ctrl or 5 Gy ) , p < 0 . 0001 . F , Cell cycle analysis of BT314NS , performed 24 h after IR in the absence ( 5 Gy ) or in the presence ( 5 Gy + JNJ ) of JNJ38877605 . Vehicle : non - irradiated cells . * : one - way ANOVA , P = 0 . 008 .

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 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 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 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

Figure 7A, Western blot of BT371NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml).B, Western blot of BT463NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM).C, Western blot of BT308NS showing p21 localization in cytoplasmic/nuclear extracts at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vinculin and lamin B were used as loading controls for cytoplasmic and nuclear fractions, respectively.D, Representative immunofluorescence staining of p21 (red) on BT308NS cells 3 h after IR (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Ctrl: non-irradiated cells. Nuclei are counterstained with DAPI (blue). Scale bar, 10 µm (63× magnification). E, Quantification of the percentage of cells showing p21 cytoplasmic or nuclear localization in BT308NS represented in (D) (n = 10 HPF/group). HPF: high-power field. *: t-test (5 Gy + JNJ) vs. (ctrl or 5 Gy), p<0.0001.F, Cell cycle analysis of BT314NS, performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated cells. *: one-way ANOVA, P=0.008.


Figure 8B right , Quantification of epi - fluorescent GFP signal from tumors represented in ( B , left ) ( n = 4 / condition ) . Vehicle : non - irradiated vehicle - treated tumors . * : one - way ANOVA , P = 5 . 5 × 10 - 5 . C , Growth curves of tumors , generated by subcutaneous injection of BT308NS , irradiated in the absence ( 2 Gy × 3 days ) or in the presence ( combo ) of JNJ38877605 , which was administered for 15 days as indicated ( n = 7 / condition ) . Vehicle : non - irradiated vehicle - treated tumors . * : one - way ANOVA , P = 0 . 0006 . D , Survival analysis of mice bearing tumors generated and treated as in C . Black dot : censored mouse . Log - rank ( Mantel - Cox ) test ( combo ) vs . ( 2 Gy × 3 days ) , P = 0 . 0019 . F , In vitro LDA ( sphere - forming ) measuring GSC frequencies as described in E ( n = 3 / condition ) . * : 2 test , p < 10 - 5 . G , Tumor incidence in the in vivo LDA performed on tumors generated and treated as described in E . H , Histogram showing GSC frequencies measured by in vivo LDA , as described in E . * : 2 test , P = 5 . 47 × 10 - 6 . I , Table showing GSC frequencies measured by in vivo LDA , represented in H . Data are represented as mean ± SEM or ± CI in F , H , I .

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Figure 8B right, Quantification of epi-fluorescent GFP signal from tumors represented in (B, left) (n = 4/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=5.5×10-5.C, Growth curves of tumors, generated by subcutaneous injection of BT308NS, irradiated in the absence (2 Gy × 3 days) or in the presence (combo) of JNJ38877605, which was administered for 15 days as indicated (n = 7/condition). Vehicle: non-irradiated vehicle-treated tumors. *: one-way ANOVA, P=0.0006.D, Survival analysis of mice bearing tumors generated and treated as in C. Black dot: censored mouse. Log-rank (Mantel-Cox) test (combo) vs. (2 Gy × 3 days), P=0.0019.F, In vitro LDA (sphere-forming) measuring GSC frequencies as described in E (n = 3/condition). *: 2 test, p<10-5.G, Tumor incidence in the in vivo LDA performed on tumors generated and treated as described in E.H, Histogram showing GSC frequencies measured by in vivo LDA, as described in E. *: 2 test, P=5.47×10-6. I, Table showing GSC frequencies measured by in vivo LDA, represented in H. Data are represented as mean ± SEM or ± CI in F,H,I.


Figure 1 ( D ) Blue - native gel electrophoresis of S . cerevisiae strains with indicated mutations in Cor1 .

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Figure 1(D) Blue-native gel electrophoresis of S. cerevisiae strains with indicated mutations in Cor1.


Figure 2 ( A ) Blue - native gel electrophoresis and immunoblots of cells expressing the wild type form of Cor1 ( Cor1WT ) , as well as the mutants Cor1N63A , N187A , D192A ( Cor1 * ) and Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) in wild type ( WT ) background as well as in cells lacking CRD1 ( crd1Δ ) . Blots were probed with antibodies against Cor1 to visualize Complex III ( CIII ) , as well as Cox1 , to monitor Complex IV ( CIV ) . CV : Complex V ; Cor1Cor2 : multimer consisting of Cor1 and Cor2 ( B ) Reduced - minus - oxidized difference spectra of strains described in ( A ) . Heme peaks are indicated for Cor1WT in WT background . Steady - state protein levels of strains as described in ( A ) cultivated on YP media containing glycerolSteady - state protein levels of strains as described in ( A ) cultivated on YP media containing CM media with galactose as carbon source ( D , E ) .

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 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 B-PANEL_START 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 2(A) Blue-native gel electrophoresis and immunoblots of cells expressing the wild type form of Cor1 (Cor1WT), as well as the mutants Cor1N63A, N187A, D192A (Cor1*) and Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**) in wild type (WT) background as well as in cells lacking CRD1 (crd1Δ). Blots were probed with antibodies against Cor1 to visualize Complex III (CIII), as well as Cox1, to monitor Complex IV (CIV). CV: Complex V; Cor1Cor2: multimer consisting of Cor1 and Cor2(B) Reduced-minus-oxidized difference spectra of strains described in (A). Heme peaks are indicated for Cor1WT in WT background.Steady-state protein levels of strains as described in (A) cultivated on YP media containing glycerolSteady-state protein levels of strains as described in (A) cultivated on YP media containing CM media with galactose as carbon source (D, E).