( a ) PCFtrypanosomes ( 5 × 107 ) were added to reaction buffer ( 2 . 45 ml ) containing 2 mM succinate , 5 μM safranine , reaction was started with digitonin ( 50 μM ) . ADP ( 10 μM ) , oligomycin ( Oligo , 2 μg ml − 1 ) , CaCl2 ( 40 μM ) , EGTA ( 200 μM ) FCCP ( 10 μM ) were added where indicated . ( b ) Changes in safranine after addition of ADP or Ca2 + to − Tet + Tet PCFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - ) .

O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-ORGANISM O 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) PCFtrypanosomes (5 × 107 cells) were added to the reaction buffer (2.45 ml) containing 2 mM succinate, and 5 μM safranine, and the reaction was started with digitonin (50 μM). ADP (10 μM), oligomycin (Oligo, 2 μg ml−1), CaCl2 (40 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (b) Changes in safranine fluorescence after addition of ADP or Ca2+ to −Tet and +Tet PCFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).


( c ) BSFtrypanosomes ( 2 × 108 ) were added to reaction buffer ( 2 . 45 ml ) containing EGTA ( 20 μM ) , ATP ( 1 mM ) , sodium orthovanadate ( 500 μM ) 12 . 5 μM safranine . reaction was started with digitonin ( 40 μM ) . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) FCCP ( 10 μM ) were added where indicated . ( d , e ) Changes in safranine after addition of Ca2 + ( d ) or FCCP ( e ) to − Tet + Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - ) .

O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-ORGANISM O 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) BSFtrypanosomes (2 × 108 cells) were added to the reaction buffer (2.45 ml) containing EGTA (20 μM), ATP (1 mM), sodium orthovanadate (500 μM) and 12.5 μM safranine. The reaction was started with digitonin (40 μM). CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (d,e) Changes in safranine fluorescence after addition of Ca2+ (d) or FCCP (e) to −Tet and +Tet BSFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).


( a ) Comparison of AMP / ATP ratios between PCF trypanosomes grown in absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi , with or without glucose ( Gl ) , expressed as fold increase ( means ± s . d . , n = 3 ; * P0 . 05 ; * * P0 . 01 , Student ' s t - ) .

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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) Comparison of AMP/ATP ratios between PCF trypanosomes grown in the absence (−Tet) or presence (+Tet) of tetracycline to induce RNAi, and with or without glucose (Gl), expressed as fold increase (means±s.d., n=3; *P0.05; **P0.01, Student's t-test).


( b ) Comparison of AMP / ATP ratios between ( − Tet ) ( + Tet ) PCF trypanosomes grown in a glucose - depleted medium ( SDM - 80 ) containing 5 . 2 mM L - proline for 2 days after 2 - day growth in SDM - 79 ± tetracycline , expressed as fold increase ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - ) .

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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) Comparison of AMP/ATP ratios between (−Tet) and (+Tet) PCF trypanosomes grown in a glucose-depleted medium (SDM-80) containing 5.2 mM L-proline for 2 days after 2-day growth in SDM-79±tetracycline, expressed as fold increase (means±s.d., n=3, **P0.01, Student's t-test).


( c ) microscopic of TbATG8 . 2 in PCF trypanosomes cultured in SDM - 79 in absence ( WT ) or presence ( RNAi ) of tetracycline to induce RNAi ; scale bars , 10 μm . ( d ) of autophagosome formation in RNAi PCF trypanosomes grown in SDM - 79 or SDM - 80 medium in presence ( + Tet ) or absence ( − Tet ) of tetracycline . Over 500 from three experiments with 20 random fields / experiment were analysed ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - ) .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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) Fluorescence microscopic images of TbATG8.2 in PCF trypanosomes cultured in SDM-79 in the absence (WT) or presence (RNAi) of tetracycline to induce RNAi; scale bars, 10 μm. (d) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM-79 or SDM-80 medium in the presence (+Tet) or absence (−Tet) of tetracycline. Over 500 cells from three experiments with 20 random fields/experiment were analysed (means±s.d., n=3, **P0.001, Student's t-test).


( e ) Western blot of ATG8 . 2 or tubulin in PCFtrypanosomes cultured in SDM - 79 or SDM - 80 in absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi . lysates ( 30 μg ) were fractionated in 12 % urea - SDS - PAGE gel . Immunoblots were labelled with anti - ATG8 . 2 or anti - tubulin antibody . ( f ) of ATG8 . 2 - II / ( ATG8 . 2 - II + ATG8 . 2 - I ) presented as mean ratios ± s . d . ( n = 3 , * * P0 . 01 , Student ' s t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O 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) Western blot analysis of ATG8.2 or tubulin in PCFtrypanosomes cultured in SDM-79 or SDM-80 in the absence (−Tet) or presence (+ Tet) of tetracycline to induce RNAi. Cell lysates (30 μg) were fractionated in 12% urea-SDS-PAGE gel. Immunoblots were labelled with anti-ATG8.2 or anti-tubulin antibody. (f) Quantification of ATG8.2-II/(ATG8.2-II+ATG8.2-I) presented as mean ratios±s.d. (n=3, **P0.01, Student's t-test).


( g ) Groups of five mice were infected with WT T . brucei ( black ) or trypanosomes transfected with construct for RNAi of TbMCU ( red ) . Doxycycline ( green ; 200 μg ml − 1 ) was given in drinking water throughout a 30 - day period .

O O O O O O B-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

(g) Groups of five mice were infected with WT T. brucei (black line) or trypanosomes transfected with the construct for RNAi of TbMCU (red line). Doxycycline (green line; 200 μg ml−1) was given in the drinking water throughout a 30-day period.


( h ) Parasitemia in surviving mice were monitored at 2 - day intervals after infection . results are means ± s . d . ( n = 2 ) .

O O O O O O B-ORGANISM 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) Parasitemia levels in surviving mice were monitored at 2-day intervals after infection. The results are means±s.d. (n=2).


( a ) Growth of TbMCU - KO BSF in absence ( black ) or presence ( red ) of 1 μg ml − 1 tetracycline for indicated number of days . Addition of 10 mM threonine to medium ( green ) partially rescued mutant BSF trypanosomes but had no effect on + Tet ( yellow ; means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - ) .

O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O B-SMALL_MOLECULE O O O 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) Growth of TbMCU-KO BSF in the absence (black line) or presence (red line) of 1 μg ml−1 tetracycline for the indicated number of days. Addition of 10 mM threonine to the medium (green line) partially rescued the mutant BSF trypanosomes but had no effect on +Tet cells (yellow line; means±s.d., n=3, **P0.001, Student's t-test).


( b ) Southern blot of BSF conditional KOs . Genomic from T . brucei SM parental ( + / + ) , single - ( + / − ) or double - allele ( − / − ) KO .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O 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) Southern blot analysis of the BSF conditional KOs. Genomic DNA from the T. brucei SM parental strain (+/+), single- (+/−) or double-allele (−/−) KO cells.


( c ) Northern blot of wild - type ( WT ) TbMCU - KO BSF cultured in presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . Tubulin is shown as a loading control ( bottom panel ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O

(c) Northern blot analysis of wild-type (WT) and TbMCU-KO BSF cultured in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. Tubulin is shown as a loading control (bottom panel).


( d ) Western blot of total lysates . from BSF trypanosomes cultured in presence ( + Tet ) or absence ( − Tet ) of tetracycline were detected using anti - HA antibody . TbVDAC was used as a loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM I-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O

(d) Western blot analysis of total cell lysates. Proteins from BSF trypanosomes cultured in the presence (+Tet) or absence (−Tet) of tetracycline were detected using anti-HA antibody. TbVDAC was used as a loading control.


( e ) Immunofluorescence of control ( upper panels ) TbMCU - KO ( lower panels ) trypanosomes . TbMCU co - localized with MitoTracker ( MT ) in control ( + Tet ; PCC = 0 . 808 ) . Scale bar , 10 μm .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(e) Immunofluorescence analysis of control (upper panels) and TbMCU-KO (lower panels) trypanosomes. TbMCU co-localized with MitoTracker (MT) in the control parasites (+Tet; PCC=0.808). Scale bar, 10 μm.


( f ) Conditional TbMCU - KO BSF trypanosomes were grown in presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . BSF trypanosomes ( 2 × 108 ) were incubated as in Fig . 2e . Multiple pulses of CaCl2 ( arrowheads ) were added to a final of 20 μM . A representative trace ( red or black ) of Ca2 + uptake from one of three independent experiments is shown .

O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

(f) Conditional TbMCU-KO BSF trypanosomes were grown in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. BSF trypanosomes (2 × 108 cells) were incubated as in Fig. 2e. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 20 μM. A representative trace (red or black) of Ca2+ uptake from one of three independent experiments is shown.


( g ) Representative traces of mitochondrial potential of digitonin - permeabilized BSF trypanosomes grown in presence ( + Tet ) or absence ( − Tet ) of tetracycline . reactions were incubated as in Fig . 3c . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) FCCP ( 10 μM ) were added where indicated .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(g) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSF trypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated.


( h , i ) Representative traces of mitochondrial potential of digitonin - permeabilized BSFtrypanosomes grown in presence ( + Tet ) or absence ( − Tet ) of tetracycline . reactions were incubated as in Fig . 3c . CaCl2 25 , 50 100 μM CaCl2 ) or FCCP ( i ) to + Tet − Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - ) .

O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-ORGANISM O 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) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSFtrypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 25, 50 and 100 μM CaCl2) or FCCP (i) to +Tet and −Tet BSFtrypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


( a ) Rhod - 2 in control or overexpressing TbMCU ( TbMCU - OE ) PCF . Scale bar , 10 μm . ( b , c ) Rhod - 2 ( mitochondrial ) ( b ) Fluo - 4 ( ) ( c ) in control ( − Tet ) overexpressing TbMCU ( + Tet ) PCF ( means ± s . d . , n = 3 , * * P0 . 05 , Student ' s t - ) ; NS , not significant .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O 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) Rhod-2 fluorescence in control or overexpressing TbMCU (TbMCU-OE) PCF. Scale bar, 10 μm. (b,c) Rhod-2 (mitochondrial) (b) and Fluo-4 (cytosolic) (c) fluorescence in control (−Tet) and overexpressing TbMCU (+Tet) PCF (means±s.d., n=3, **P0.05, Student's t-test); NS, not significant.


( d ) Ca2 + uptake by digitonin - permeabilized PCF overexpressing TbMCU . Trypanosomes were incubated as in Fig . 2c . Multiple pulses of CaCl2 ( arrowheads ) were added to a final of 8 μM . ( e ) Ca2 + uptake in TbMCU - OE PCF ( + Tet ) 200 s after addition of Ca2 + , as compared with uninduced trypanosomes considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O 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) Ca2+ uptake by digitonin-permeabilized PCF overexpressing TbMCU. Trypanosomes were incubated as in Fig. 2c. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 8 μM. (e) Ca2+ uptake in TbMCU-OE PCF (+Tet) 200 s after addition of Ca2+, as compared with uninduced trypanosomes considered as 1 (−Tet) (means±s.d., n=3, **P0.01, Student's t-test).


( f ) Mitochondrial potential of digitonin - permeabilized control ( − Tet ) TbMCU - OE ( + Tet ) PCF . reactions were incubated as in Fig . 3a .

O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O

(f) Mitochondrial membrane potential of digitonin-permeabilized control (−Tet) and TbMCU-OE (+Tet) PCF. The reactions were incubated as in Fig. 3a.


( g ) Changes in safranine after addition of ADP , Ca2 + or FCCP to - Tet + Tet PCF trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - ) .

O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-ORGANISM I-ORGANISM O 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) Changes in safranine fluorescence after addition of ADP, Ca2+ or FCCP to -Tet and +Tet PCF trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


( h ) Mitochondrial oxidative stress as measured with MitoSOX Red in control ( C ) TbMCU - overexpressing ( OE ) PCF permeabilized with 50 μM digitonin in presence of 0 . 4 mM Ca2 + . Controls were treated with 2 μM antimycin A ( C + AA ) , or were trypanosomes exposed to 0 . 4 mM Ca2 + in absence of digitonin ( − DIG + Ca2 + ) , trypanosomes permeabilized with digitonin in absence of Ca2 + ( C , yellow ) . ( i ) of change at 900 s . Results are expressed as mean ± s . d . ( n = 3 , * * P0 . 001 , Student ' s t - ) .

O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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) Mitochondrial oxidative stress as measured with MitoSOX Red in control (C) and TbMCU-overexpressing (OE) PCF permeabilized with 50 μM digitonin in the presence of 0.4 mM Ca2+. Controls were treated with 2 μM antimycin A (C+AA), or were trypanosomes exposed to 0.4 mM Ca2+ in the absence of digitonin (−DIG+Ca2+), and trypanosomes permeabilized with digitonin in the absence of Ca2+ (C, yellow line). (i) Quantification of change at 900 s. Results are expressed as mean fluorescence±s.d. (n=3, **P0.001, Student's t-test).


( j ) Growth of trypanosomes in absence ( control , black lines ) or presence ( TbMCU - OE , red lines ) of 1 μg ml − 1 tetracycline ( means ± s . d . , n = 3 ) . ( k ) Dead in representative fields of control ( − Tet ) TbMCU - OE ( + Tet ) PCF are indicated ( arrows ) . Scale bar , 10 μm .

O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O O O O

(j) Growth of trypanosomes in the absence (control, black lines) or presence (TbMCU-OE, red lines) of 1 μg ml−1 tetracycline (means±s.d., n=3). (k) Dead cells in representative fields of control (−Tet) and TbMCU-OE (+Tet) PCF are indicated (white arrows). Scale bar, 10 μm.


( l ) viability on apoptotic challenge of control ( − Tet ) or TbMCU - OE ( + Tet ) PCF trypanosomes with H2O2 C2 - ceramide . of was obtained from means ± s . d . by analysing > 60 fields ( including > 400 ) ( n = 3 ) . * * P0 . 01 , Student ' s t - .

O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O 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) Cell viability on apoptotic challenge of control (−Tet) or TbMCU-OE (+Tet) PCF trypanosomes with H2O2 and C2-ceramide. The percentage of cell death was obtained from means±s.d. by analysing >60 fields (including >400 cells) (n=3). **P0.01, Student's t-test.


Chl - HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection in N . benthamiana epidermal . Ratiometric ( F488 / 405 nm ) of excitation at 488 405 nm show oxidized state of chloroplast - targeted HyPer2 , respectively . false - blue color indicates chloroplasts . Scale bars , 30 μm . Data information : In ( A ) , data are representative of at least three independent experiments .

B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

Chl-HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection in N. benthamiana epidermal cells. Ratiometric images (F488/405 nm) of fluorescence excitation at 488 and 405 nm show the oxidized state of chloroplast-targeted HyPer2, respectively. The false-blue color indicates the chloroplasts. Scale bars, 30 μm. Data information: In (A), data are representative of at least three independent experiments.


of BSMV - dependent changes in Chl - HyPer2 at 0 to 6 dpi . of ROS - positive chloroplasts ( ) among chloroplasts ( blue ) per visual field . Data information : In ( B ) , error bars indicate mean ± ( n = 5 ) .

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Quantification of the BSMV-dependent changes in Chl-HyPer2 fluorescence at 0 to 6 dpi. The percentage of ROS-positive chloroplasts (white) among all chloroplasts (blue and white) per visual field. Data information: In (B), error bars indicate mean ± SEM (n = 5).


Western blot of 2 - Cys Prx redox status in empty vector ( EV ) BSMV inoculated leaves under non - reducing conditions at 3 dpi . Western blots were analyzed with anti - 2 - Cys Prx or anti - CP antibodies . Rubisco large subunit ( RbcL ) served as a loading control . Data information : , representative data are shown , at least three independent replicates were performed with similar results .

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Western blot analysis of 2-Cys Prx redox status in empty vector (EV) and BSMV inoculated leaves under non-reducing conditions at 3 dpi. Western blots were analyzed with anti-2-Cys Prx or anti-CP antibodies. The Rubisco large subunit (RbcL) served as a loading control. Data information: , representative data are shown, at least three independent replicates were performed with similar results.


Relative mRNA of sAPX , 2 - Cys Prx , GPXL , PrxQ genes in response to BSMV infections at 1 , 3 5 dpi . empty vector ( EV ) was used as a negative control , NbEF1α was used as an internal control . Data information : In ( D ) , error bars indicate mean ± from three independent experiments ; * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 from Student ' s t .

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Relative mRNA levels of the sAPX, 2-Cys Prx, GPXL, and PrxQ genes in response to BSMV infections at 1, 3 and 5 dpi. The empty vector (EV) was used as a negative control, NbEF1α was used as an internal control. Data information: In (D), error bars indicate mean ± SEM from three independent experiments; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001 from Student's t test.


BSMV accumulation in N . benthamiana leaves transiently expressing chloroplast ROS scavenging . Agrobacterium harboring plasmids expressing various are indicated above panels . BSMV was agroinfiltrated into same leaves at 1 . 5 dpi . Three days later , Western blot analyses were conducted with anti - TGB1 or anti - Flag antibodies . RbcL served as loading control . 2 - CP , 2 - Cys Prx . Data information : representative data are shown , at least three independent replicates were performed with similar results .

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BSMV accumulation in N. benthamiana leaves transiently expressing chloroplast ROS scavenging proteins. Agrobacterium harboring plasmids expressing various proteins are indicated above the panels. BSMV was agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blot analyses were conducted with anti-TGB1 or anti-Flag antibodies. RbcL served as the loading control. 2-CP, 2-Cys Prx. Data information: representative data are shown, at least three independent replicates were performed with similar results.


BSMV accumulation in 2 - Cys Prx or GPXL - silenced N . benthamiana using anti - TGB1 antibody . Empty vector TRV : 00 was used as negative control . Data information : representative data are shown , at least three independent replicates were performed with similar results .

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BSMV accumulation in 2-Cys Prx or GPXL-silenced N. benthamiana plants using anti-TGB1 antibody. Empty vector TRV: 00 was used as negative control. Data information: representative data are shown, at least three independent replicates were performed with similar results.


Y2H analyses of interactions between γb NbNTRC . Various Y2H combinations are indicated on . Self - interactions of γb served as a positive control , combinations containing either empty AD or BD constructs served as negative controls .

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Y2H analyses of interactions between γb and NbNTRC. Various Y2H combinations are indicated on the images. Self-interactions of γb served as a positive control, and combinations containing either empty AD or BD constructs served as negative controls.


BiFC analyses of γb NbNTRC interactions during BSMV infections . Various BiFC combinations are indicated on left . YFP signals were visualized by confocal microscopy at 3 dpi depicted as a false - green color chloroplasts were visualized by chloroplast autofluorescence as a false - red color . Scale bars , 20 μm .

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BiFC analyses of γb and NbNTRC interactions during BSMV infections. Various BiFC combinations are indicated on the left. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. Scale bars, 20 μm.


Co - IP analyses of interactions between γb NbNTRC in N . benthamiana . Leaf tissues were agroinfiltrated with various combinations indicated above panels were harvested at 3 dpi . Total were immunoprecipitated with anti - Flag beads . Total immunoprecipitated ( IP ) were analyzed by Western blotting with anti - GFP or anti - Flag antibodies . Data information : representative data are shown , three replicates had similar results .

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Co-IP analyses of interactions between γb and NbNTRC in N. benthamiana. Leaf tissues were agroinfiltrated with various combinations indicated above the panels were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Total and immunoprecipitated (IP) proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies. Data information: representative data are shown, and three replicates had similar results.


GST pull - down assays to verify interactions between γb NbNTRC in vitro . GST - tagged NTRC or GFP were incubated with γb - His or GFP - His . Input pull - down products were analyzed by Western blot analyses with anti - GST or anti - His antibodies . GFP - His GST - GFP served as negative controls . Data information : representative data are shown , three replicates had similar results .

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GST pull-down assays to verify interactions between γb and NbNTRC in vitro. GST-tagged NTRC or GFP were incubated with γb-His or GFP-His. Input and pull-down products were analyzed by Western blot analyses with anti-GST or anti-His antibodies. GFP-His and GST-GFP served as negative controls. Data information: representative data are shown, and three replicates had similar results.


Co - localization of γb NbNTRC during BSMV infection . NbNTRC - mCherry was co - expressed with BSMVγb - GFP ( Jiang et al . , 2020 ) in N . benthamiana , confocal analyses were conducted at 3 dpi , chloroplast autofluorescence is displayed as a false - blue color . Scale bars , 20 μm .

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Co-localization of γb and NbNTRC during BSMV infection. NbNTRC-mCherry was co-expressed with BSMVγb-GFP (Jiang et al., 2020) in N. benthamiana, and confocal analyses were conducted at 3 dpi, and chloroplast autofluorescence is displayed as a false-blue color. Scale bars, 20 μm.


Y2H to analyze interactions between NbNTRC γb mutants . Various Y2H combinations are indicated on . Interactions of γb NbNTRC were confirmed earlier served as a positive control , combinations containing an empty BD construct served as a negative control .

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Y2H assay to analyze interactions between NbNTRC and γb mutants. Various Y2H combinations are indicated on the images. Interactions of γb and NbNTRC were confirmed earlier and served as a positive control, combinations containing an empty BD construct served as a negative control.


BiFC of interactions between NbNTRC γb mutants during BSMV infection of N . benthamiana . Various BiFC combinations are indicated at top . YFP signals were visualized by confocal microscopy at 3 dpi depicted as a false - green color chloroplasts were visualized by chloroplast autofluorescence as a false - red color . RbcL - fused YFPc served as a negative control . Scale bars , 20 μm .

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BiFC analysis of interactions between NbNTRC and γb mutants during BSMV infection of N. benthamiana. Various BiFC combinations are indicated at the top. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. RbcL-fused YFPc served as a negative control. Scale bars, 20 μm.


Co - IP of interactions between NbNTRC γb mutants . NbNTRC - 3xFlag together with γb - GFP , γbH85A - GFP or γbH85C - GFP were co - expressed in N . benthamiana leaves harvested at 3 dpi . Total were IP with anti - Flag beads . Total IP products were detected in Western blots with anti - GFP or anti - Flag antibodies . Uncleaved NTRC with cTP NTRC without cTP are marked with black gray arrows Data information : , representative data were shown , three independent experiments showed a similar result .

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Co-IP analysis of interactions between NbNTRC and γb mutants. NbNTRC-3xFlag proteins together with γb-GFP, γbH85A-GFP or γbH85C-GFP were co-expressed in N. benthamiana leaves and harvested at 3 dpi. Total proteins were IP with anti-Flag beads. Total and IP products were detected in Western blots with anti-GFP or anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information: , representative data were shown, and three independent experiments showed a similar result.


GST pull - down assays to evaluate in vitro interactions between NbNTRC γb mutants . Purified γb - His , γbH85A - His , γbH85C - His , or GFP - His were incubated with GST - tagged NbNTRC . Input pull - down products were analyzed by Western blotting with anti - GST or anti - His antibodies . GFP - His served as a negative control . Arrowhead indicates specific band of target . Data information : representative data were shown , three independent experiments showed a similar result .

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GST pull-down assays to evaluate in vitro interactions between NbNTRC and γb mutants. Purified γb-His, γbH85A-His, γbH85C-His, or GFP-His proteins were incubated with GST-tagged NbNTRC. Input and pull-down products were analyzed by Western blotting with anti-GST or anti-His antibodies. GFP-His served as a negative control. Arrowhead indicates the specific band of the target protein. Data information: representative data were shown, and three independent experiments showed a similar result.


of VSR activities of γb mutants . Infiltrated N . benthamiana leaves co - expressing sGFP plus γbH85A - 3xFlag , γbH85C - 3xFlag or pGD empty vector ( EV ) were observed at 5 dpi under long - wave UV illumination for GFP . γb - 3xFlag served as a positive control . dashed circles indicate infiltrated zone . Western blot of GFP in co - infiltrated leaves at 5 dpi with anti - GFP antibody . RbcL was used as loading control . Data information : representative data were shown , three independent experiments showed a similar result .

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Analysis of VSR activities of γb mutants. Infiltrated N. benthamiana leaves co-expressing sGFP plus γbH85A-3xFlag, γbH85C-3xFlag or the pGD empty vector (EV) were observed at 5 dpi under long-wave UV illumination for GFP fluorescence. The γb-3xFlag protein served as a positive control. The white dashed circles indicate the infiltrated zone.   Western blot analysis of the GFP protein in co-infiltrated leaves at 5 dpi with anti-GFP antibody. RbcL was used as loading control.  Data information:  representative data were shown, and three independent experiments showed a similar result.


Effects of NbNTRC overexpression on BSMV accumulation . N . benthamiana leaves were first infiltrated with Agrobacterium containing various contructs indicated above panel . After 1 . 5 dpi , BSMV was inoculated onto leaves by agroinfiltration . Three days later , total was extracted for Western blotting with anti - TGB1 anti - Flag antibodies . uncleaved NTRC with cTP NTRC without cTP are marked with black gray arrows RbcL was a loading control . Data information : representative data are shown three eplicates similar results .

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Effects of NbNTRC overexpression on BSMV protein accumulation. N. benthamiana leaves were first infiltrated with Agrobacterium containing various contructs indicated above the panel. After 1.5 dpi, BSMV was inoculated onto the leaves by agroinfiltration. Three days later, total protein was extracted for Western blotting with anti-TGB1 and anti-Flag antibodies. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows RbcL was a loading control. Data information: representative data are shown and three eplicates similar results.


Effects of NbNTRC overexpression on BSMV replication . Movement - deficient BSMV ( RNAα + RNAγ ) was infiltrated into leaves that had been agroinfiltrated previously with various constructs indicated above panel . NbNTRC viral γb were detected by Western blotting with anti - Flag anti - γb antibodies . Uncleaved NTRC with cTP NTRC without cTP are marked with black gray arrows ( see Appendix Fig S4 ) . RT - qPCR analyses of viral accumulation in 4B . NbPP2A was used as an internal control . Data information : representative data are shown three eplicates similar results . In ( C ) , error bars indicate mean ± from three independent experiments .

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Effects of NbNTRC overexpression on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ) was infiltrated into leaves that had been agroinfiltrated previously with various constructs indicated above the panel. NbNTRC and viral γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows (see Appendix Fig S4).   RT-qPCR analyses of viral RNA accumulation in 4B. NbPP2A was used as an internal control.   Data information: representative data are shown and three eplicates similar results. In (C), error bars indicate mean ± SEM from three independent experiments.


symptoms of infection after BSMV inoculation of a different N . benthamiana at 8 dpi . Magnified of leaves ( in boxes ) are shown in lower panels . Scale bars , 2 cm . Statistical of symptom appearance in different N . benthamiana after inoculation with BSMV . Western blot of accumulation in Figure 4D with anti - TGB1 antibody . RbcL served as loading control . Data information : , representative data are shown three eplicates similar results . In ( E ) , error bars indicate mean ± SD from three independent experiments .

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The symptoms of infection after BSMV inoculation of a different N. benthamiana plant at 8 dpi. Magnified images of leaves (in white boxes) are shown in the lower panels. Scale bars, 2 cm.   Statistical analysis of symptom appearance in different N. benthamiana plants after inoculation with BSMV.   Western blot analysis of the virus accumulation in Figure 4D with anti-TGB1 antibody. RbcL served as loading control.   Data information: , representative data are shown and three eplicates similar results. In (E), error bars indicate mean ± SD from three independent experiments.


Left panel : systemic symptoms of N . benthamiana inoculated with BSMV BSMV γb mutants . were taken at 7 dpi . Right panel : schematic representation of inoculated ( ino . ) , older systemic leaves ( o - SL ) , younger systemic leaves ( y - SL ) in N . benthamiana . Scale bars , 2 cm .

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Left panel: systemic symptoms of N. benthamiana plants inoculated with BSMV and BSMV γb mutants. Photographs were taken at 7 dpi. Right panel: schematic representation of inoculated (ino.), older systemic leaves (o-SL), and younger systemic leaves (y-SL) in N. benthamiana plants. Scale bars, 2 cm.


Western blot analyses of TGB1 CP accumulation in BSMV BSMV γb mutant inoculated N . benthamiana leaves . Anti - TGB1 anti - CP were used for detection . Data information : , representative data are shown , three replicates had similar results . RbcL served as loading control .

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Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N. benthamiana leaves. Anti-TGB1 and anti-CP were used for protein detection. Data information: , representative data are shown, and three replicates had similar results. RbcL served as loading control.


Western blot detection of BSMV with anti - CP antibodies in upper N . benthamiana leaves at 7 days ( o - SL ) or 28 days ( y - SL ) after infiltration . o - SL , older systemic leaves ; y - SL , younger systemic leaves . Data information : representative data are shown , three replicates had similar results . RbcL served as loading control .

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Western blot detection of BSMV with anti-CP antibodies in upper N. benthamiana leaves at 7 days (o-SL) or 28 days (y-SL) after infiltration. o-SL, older systemic leaves; y-SL, younger systemic leaves. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Effects of NbNTRC overexpression on infection of BSMV BSMV γb mutants . A . tumefaciens harboring indicated plasmids were infiltrated into N . benthamiana leaves . Note : Agrobacterium containing NbNTRC constructs were diluted 5 - fold compared to normal use ( OD600 = 0 . 3 ) . BSMV γb mutants were then agroinfiltrated into same leaves at 1 . 5 dpi . Three days later , Western blots were conducted with anti - TGB1 anti - Flag antibodies . Uncleaved NTRC with cTP NTRC without cTP are marked with black gray arrows Data information : representative data are shown , three replicates had similar results . RbcL served as loading control .

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Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants. A. tumefaciens harboring the indicated plasmids were infiltrated into N. benthamiana leaves. Note: Agrobacterium containing NbNTRC constructs were diluted 5-fold compared to normal use (OD600 = 0.3). BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blots were conducted with anti-TGB1 and anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Left panel : Western blot detection of BSMV CP accumulation in N . benthamiana leaves inoculated with BSMV γb movement deficient mutants . Leaf samples were collected from different regions of same leaf ( right panel ) Data information : representative data are shown , three replicates had similar results . RbcL served as loading control .

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Left panel: Western blot detection of BSMV CP accumulation in N. benthamiana leaves inoculated with BSMV and γb movement deficient mutants. Leaf samples were collected from different regions of the same leaf (right panel) Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Effects of γb γb mutants on 2 - Cys Prx in N . benthamiana . Different combinantions of pGD empty vector ( EV ) or γb γb mutants are indicated above panel . NbNTRC γb were detected by Western blotting with anti - Flag anti - γb antibodies under reducing conditions , Western blot analyses of 2 - Cys Prx abundance with anti - 2 - Cys Prx antibody under non - reducing conditions . uncleaved NTRC with cTP NTRC without cTP are marked with black gray arrows , respectively . Data information : representative data are shown , three replicates had similar results . RbcL served as loading control .

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Effects of γb and γb mutants on 2-Cys Prx in N. benthamiana. Different combinantions of the pGD empty vector (EV) or γb and γb mutants are indicated above the panel. NbNTRC and γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies under reducing conditions, Western blot analyses of 2-Cys Prx abundance with anti-2-Cys Prx antibody under non-reducing conditions. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


BiFC assays to identify interactions between NbNTRC 2 - Cys Prx in N . benthamiana during BSMV infection . NbNTRC - YFPc 2 - Cys Prx - YFPn were co - expressed with BSMV γb mutant derivatives as indicated on left . EV was used as negative control . Scale bars , 30 μm . Quantificaiton of BiFC intensities shown in Figure 6A with ImageJ software . NbNTRC - 2 - Cys Prx interaction was normalized to that of EV control , which was set to 1 . 0 . Data information : Representative data were shown in three replicates had similar results . In B , error bars indicate mean ± , a b indicate statistically significant differences among different groups ( n = 5 , ANOVA , p - value < 0 . 05 ) .

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BiFC assays to identify interactions between NbNTRC and 2-Cys Prx in N. benthamiana during BSMV infection. NbNTRC-YFPc and 2-Cys Prx-YFPn were co-expressed with BSMV and γb mutant derivatives as indicated on the left. The EV was used as negative control. Scale bars, 30 μm.   Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software. The NbNTRC-2-Cys Prx interaction was normalized to that of the EV control, which was set to 1.0.   Data information: Representative data were shown in and three replicates had similar results. In B, error bars indicate the mean ± SEM, and a and b indicate statistically significant differences among different groups (n = 5, ANOVA, p-value < 0.05).


Split - LUC assays to determine NbNTRC - 2 - Cys Prx interactions in N . benthamiana during BSMV infection . NbNTRC - cLUC 2 - Cys Prx - nLUC were co - expressed in N . benthamiana leaves infected with indicated BSMV derivatives , Bioluminescence was observed at 3 dpi . dashed circles indicate infiltrated regions . of luciferase signals shown in Figure 6C as assessed by ImageJ software . Data information : Representative data were shown , three replicates had similar results . In ( D ) , error bars indicate mean ± ( n = 6 ) . c indicate statistically significant differences among different groups ( ANOVA , p - value < 0 . 05 ) .

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Split-LUC assays to determine NbNTRC-2-Cys Prx interactions in N. benthamiana during BSMV infection. NbNTRC-cLUC and 2-Cys Prx-nLUC were co-expressed in N. benthamiana leaves infected with the indicated BSMV derivatives, Bioluminescence was observed at 3 dpi. The white dashed circles indicate the infiltrated regions.   Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software.   Data information: Representative data were shown , and three replicates had similar results. In (D), error bars indicate mean ± SEM (n = 6). c indicate statistically significant differences among different groups (ANOVA, p-value < 0.05).


Competitive GST pull - down assays showing effects of γb on interactions between NbNTRC 2 - Cys Prx . GST - NbNTRC 2 - Cys Prx - His were incubated with increasing amounts ( 5 , 10 , 20 , 40 μg ) of γb its mutant derivatives . Glutathione - Sepharose beads were washed Western blot was performed using anti - GST anti - His antibodies . Data information : representative data are shown , three independent experiments had similar results .

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Competitive GST pull-down assays showing the effects of γb on interactions between NbNTRC and 2-Cys Prx. GST-NbNTRC and 2-Cys Prx-His were incubated with increasing amounts (5, 10, 20, and 40 μg) of γb and its mutant derivatives. Glutathione-Sepharose beads were washed and Western blot analysis was performed using anti-GST and anti-His antibodies. Data information: representative data are shown, and three independent experiments had similar results.


of NbNTRC - 2 - Cys Prx interactions in presence of γb . NbNTRC - 3xFlag 2 - Cys Prx - GFP were co - expressed in N . benthamiana leaves . Total were extracted at 4 dpi immunoprecipitated with anti - Flag beads , co - incubated with increasing amounts ( 20 , 40 , 60 μg ) of γb - His γb - His mutant derivatives . Input IP were analyzed by Western blotting with anti - His , anti - GFP or anti - Flag antibodies . uncleaved 2 - Cys Prx with cTP 2 - Cys Prx without cTP are marked with black gray arrows , respectively . Data information : representative data are shown , three independent experiments had similar results .

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Analysis of NbNTRC-2-Cys Prx interactions in the presence of the γb protein. NbNTRC-3xFlag and 2-Cys Prx-GFP were co-expressed in N. benthamiana leaves. Total proteins were extracted at 4 dpi and immunoprecipitated with anti-Flag beads, co-incubated with increasing amounts (20, 40, and 60 μg) of γb-His and γb-His mutant derivatives. Input and IP proteins were analyzed by Western blotting with anti-His, anti-GFP or anti-Flag antibodies. The uncleaved 2-Cys Prx with cTP and cleaved 2-Cys Prx without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three independent experiments had similar results.


Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N . benthamiana epidermal . pCB301 empty vector ( EV ) was used as a negative control . Scale bars , 30 μm . false - blue color indicates chloroplasts . of LRSV - dependent changes in Chl - HyPer2 at 3 dpi . Data information : , data are representative of at least three independent experiments . error bars represent mean ± ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t .

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Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N. benthamiana epidermal cells. The pCB301 empty vector (EV) was used as a negative control. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of LRSV-dependent changes in Chl-HyPer2 fluorescence at 3 dpi.   Data information: , data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.


Western blot analyses of 2 - Cys Prx abundance in EV LRSV inoculated leaves under non - reducing conditions at 3 dpi . RbcL served as a loading control . Data information : representative data are shown , three replicates had similar results .

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Western blot analyses of 2-Cys Prx abundance in EV and LRSV inoculated leaves under non-reducing conditions at 3 dpi. RbcL served as a loading control. Data information: representative data are shown, and three replicates had similar results.


Western blot analyses to detect LRSV γb in NbNTRC - OE NbNTRC - KO N . benthamiana challenged with LRSV at 8 dpi . RbcL was used as loading control . Data information : representative data are shown , three replicates had similar results .

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Western blot analyses to detect LRSV γb in NbNTRC-OE and NbNTRC-KO N. benthamiana plants challenged with LRSV at 8 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.


Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N . benthamiana epidermal . Scale bars , 30 μm . false - blue color indicates chloroplasts . of TYMV - dependent changes in Chl - HyPer2 at 5 dpi . Data information : data are representative of at least three independent experiments . error bars represent mean ± ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t .

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Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N. benthamiana epidermal cells. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of the TYMV-dependent changes in Chl-HyPer2 fluorescence at 5 dpi.   Data information: data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.


Western blot of 2 - Cys Prx abundance in EV or TYMV inoculated leaves under non - reducing conditions at 5 dpi . RbcL was used as loading control . Data information : representative data are shown , three replicates had similar results .

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Western blot analysis of 2-Cys Prx abundance in EV or TYMV inoculated leaves under non-reducing conditions at 5 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.


RT - qPCR of TYMV accumulation at 8 dpi . NbEF1α served as a internal control . Data information : representative data are shown , three replicates had similar results .

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RT-qPCR analysis of TYMV RNA accumulation at 8 dpi. NbEF1α served as a internal control. Data information: representative data are shown, and three replicates had similar results.


( a , b ) Dual positive are more prevalent in epithelial organs . ( a ) Proportion of ACE2 + TMPRSS2 + ( y axis ) per dataset ( dots ) from 21 tissues organs ( rows ) .

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(a,b) Dual positive cells are more prevalent in epithelial organs and cells. (a) Proportion of ACE2+TMPRSS2+ cells (y axis) per dataset (dots) from 21 tissues and organs (rows).


( e - i ) In situ validation of dual positive in lung , airways , submucosal . ( e - g ) PLISH immunostaining in human adult lung alveoli for ( e ) ACE2 ( ) , TMPRSS2 ( green ) CTSL ( red ) , ( f ) ACE2 ( ) , TMPRSS2 ( green ) HTII - 280 ( red ) , ( g ) ACE2 ( red ) Pro - SFTPC ( green ) . ( h ) PLISH immunostaining in human large airway submucosal . ACE2 ( red ) , ACTA2 ( green ) DAPI ( blue ) . ( i ) PLISH in human adult large airway . ACE2 ( green ) DAPI ( blue ) .

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(e-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).


( a , c ) Single - ATAC - seq of lung samples from carina ( 1C ) subpleural parenchyme ( RPL ) ( n = 1 , k = 3 samples , 3 , 366 from 1C , 8 , 340 from RPL ) . Uniform Manifold Approximation Projection ( UMAP ) embedding of scATAC - seq profiles ( dots ) colored by ( a , left ) types , ( a , right ) with at least 1 fragment ( indicating accessibility , open ) mapping to ACE2 gene locus ( defined as − 2kb upstream Transcription Start Site to Transcription End Site ) , or by sample

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(a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location


( g ) Low expression in pediatric samples . Mean expression level ( log CPM , y axis ) of ACE2 ( blue ) , TMPRSS2 ( orange ) , CTSL ( green ) across bins ( x axis ) in AT2 ( left ) ciliated ( right ) . Pediatric samples : 0 - 10 years . Samples from past or current smokers were removed from this plot to avoid smoking confounders . Error bars are omitted due to y - axis limitations . They are typically 10 - fold mean value ( Supplementary Table 5 ) . Multiciliated AT2 are shown as these types are present in data , show significant associations with ACE2 expression .

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(g) Low expression in pediatric samples. Mean expression level (log CPM, y axis) of ACE2 (blue), TMPRSS2 (orange), and CTSL (green) across age bins (x axis) in AT2 (left) and ciliated (right) cells. Pediatric samples: 0-10 years. Samples from past or current smokers were removed from this plot to avoid smoking confounders. Error bars are omitted due to y-axis limitations. They are typically 10-fold the mean value (Supplementary Table 5). Multiciliated and AT2 cells are shown as these cell types are present in fetal data, and show significant age associations with ACE2 expression.


( a ) Gradual increase in Ace2 expression by airway epithelial type with . Mean expression ( y axis ) of Ace2 in different airway epithelial ( x axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , error bars ( standard error of mean ( ) ) .

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(a) Gradual increase in Ace2 expression by airway epithelial cell type with age. Mean expression (y axis) of Ace2 in different airway epithelial cells (x axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (standard error of the mean (SEM)).


( b ) Increase in proportion of Ace2 + Ctsl + goblet club with . of Ace2 + Ctsl + ( x axis ) in different airway epithelial types ( y axis ) of mice of three consecutive ages ( color legend , upper right ) . Shown are replicate mice ( dots ) , mean ( bar ) , error bars ( ) .

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(b) Increase in proportion of Ace2+Ctsl+ goblet and club cells with age. Percent of Ace2+Ctsl+ cells (x axis) in different airway epithelial cell types (y axis) of mice of three consecutive ages (color legend, upper right). Shown are replicate mice (dots), mean (bar), and error bars (SEM).


( c - j ) Increase in Ace2 expression in secretory with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( c , d ) UMAP of scRNA - seq profiles ( dots ) colored by experimental group ( c ) or by Ace2 + indicated double positive ( d ) . Alveolar epithelial ( AT1 AT2 ) airway epithelial secretory ciliated are marked .

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(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (c,d) UMAP of scRNA-seq profiles (dots) colored by experimental group (c) or by Ace2+ cells and indicated double positive cells (d). Alveolar epithelial cells (AT1 and AT2) and airway epithelial secretory and ciliated cells are marked.


( c - j ) Increase in Ace2 expression in secretory with smoking . Mice were daily exposed to cigarette smoke or filtered air as control for two months after which from whole lung suspensions were analyzed by scRNA - seq ( Drop - Seq ) . ( e ) relative frequency of Ace2 + is increased by smoking in airway secretory but not AT2 . Relative proportion ( y axis ) of Ace2 + ( red ) Ace2 - ( grey ) in smoking control mice of different types ( x axis ) .

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(c-j) Increase in Ace2 expression in secretory cells with smoking. Mice were daily exposed to cigarette smoke or filtered air as control for two months after which cells from whole lung suspensions were analyzed by scRNA-seq (Drop-Seq). (e) The relative frequency of Ace2+ cells is increased by smoking in airway secretory cells but not AT2 cells. Relative proportion (y axis) of Ace2+ (red) and Ace2- (grey) cells in smoking and control mice of different cell types (x axis).


( f , g ) Expression of Ace2 is increased in airway secretory , but not in AT2 . Distribution of Ace2 expression ( y axis ) in secretory ( f ) AT2 ( g ) from control smoking mice ( x axis ) .

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(f, g) Expression of Ace2 is increased in airway secretory cells, but not in AT2 cells. Distribution of Ace2 expression (y axis) in secretory (f) and AT2 (g) cells from control and smoking mice (x axis).


( h - j ) Re - of published bulk mRNA - Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of ( h ) Ace2 , ( i ) Tmprss2 , ( j ) Ctsl after five months of chronic exposure .

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(h-j) Re-analysis of published bulk mRNA-Seq101 of lungs exposed to different daily doses of cigarette smoke show increased expression of (h) Ace2, (i) Tmprss2, and (j) Ctsl after five months of chronic exposure.


( k ) Expression in placenta . UMAP plots showing expression of Ace2 , Tmprss2 Ctsl in single double positive from embryonic days 9 . 5 to 18 ( left ) embryonic day 14 . 5 ( right ) of mouse placenta

O O O B-EXP_ASSAY O B-TISSUE O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD 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-ORGANISM B-TISSUE

(k) Expression in placenta. UMAP plots showing expression of Ace2, Tmprss2 and Ctsl in single and double positive cells from embryonic days 9.5 to 18 (left) and embryonic day 14.5 (right) of mouse placenta development


( a ) Multiple proteases are co - expressed with ACE2 in human lung scRNA - seq . Scatter plot of significance ( y axis , - log10 ( adjusted P value ) ) effect ( x axis ) of co - expression of each protease gene ( dot ) with ACE within each indicated epithelial type ( color ) . Dashed : significance threshold . TMPRSS2 PCSKs that significantly co - expressed with ACE2 are marked .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-ORGANISM B-TISSUE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O

(a) Multiple proteases are co-expressed with ACE2 in human lung scRNA-seq. Scatter plot of significance (y axis, -log10(adjusted P value)) and effect size (x axis) of co-expression of each protease gene (dot) with ACE within each indicated epithelial cell type (color). Dashed line: significance threshold. TMPRSS2 and PCSKs that significantly co-expressed with ACE2 are marked.


A CD4 + T from wild - type ( WT ) , CBLOST CBLBOSTmice were left unstimulated ( - ) or stimulated for 2 min with anti - CD3 anti - CD4 antibodies ( + ) . Equal amounts of lysates were subjected to affinity purification on Strep - Tactin - Sepharose beads , followed by elution of with D - biotin . Eluted were analyzed by immunoblot with antibodies specific for CBLB CBL . Equal amounts of from whole lysates ( WCL ) were also probed for CBLB CBL . Note that in case of CBLB two bands are detected likely corresponded to presence of two isoforms differing at their N - terminus ( http : / / www . uniprot . org / uniprot / Q3TTA7 ) . Also shown are loading control corresponding to same immunoblot probed with anti - CD5 , molecular masses ( kDa ) . Data are representative of at least three experiments .

O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O

A CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (-) or stimulated for 2 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads, followed by elution of proteins with D-biotin. Eluted proteins were analyzed by immunoblot with antibodies specific for CBLB and CBL. Equal amounts of proteins from whole cell lysates (WCL) were also probed for CBLB and CBL. Note that in the case of CBLB two bands are detected and likely corresponded to the presence of two isoforms differing at their N-terminus (http://www.uniprot.org/uniprot/Q3TTA7). Also shown are loading control corresponding to the same immunoblot probed with anti-CD5, and molecular masses (kDa). Data are representative of at least three experiments.


B CD4 + CD8 + thymocytes mature CD4 + T isolated from mice with specified genotypes were permeabilized stained with saturating amounts of Strep - Tactin - APC analyzed by flow cytometry . Background was deduced using from WT mouse . Data are representative of 2 experiments .

O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O

B CD4+CD8+thymocytes and mature CD4+T cells isolated from mice with the specified genotypes were permeabilized and stained with saturating amounts of Strep-Tactin-APC and analyzed by flow cytometry. Background staining was deduced using cells from WT mouse. Data are representative of 2 experiments.


CD4 + T from wild - type ( WT ) , CBLOST CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 anti - CD4 antibodies . Equal amounts of lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads subjected to MS as described in Material Methods . A Histogram comparing normalized CBL intensities resulting from AP - MS of CD4 + T from WT CBLOSTmice prior to after specified stimulation times . For each time point , distributions of normalized intensities of 9 data points ( 3 biological replicates x 3 technical replicates ) corresponding to CD4 + T from wild - type ( WT ) CBLOSTmice were compared using adjusted p - value from a one - way ANOVA ( denoted as p ( t ) ) value r ( t ) corresponding to enrichment observed in CBLOST samples as compared to WT samples . Different colors represent distinct biological replicates .

B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O 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-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.A Histogram comparing the normalized CBL protein intensities resulting from AP-MS analysis of CD4+T cells from WT and CBLOSTmice prior to and after the specified stimulation times. For each time point, the distributions of the normalized intensities of the 9 data points (3 biological replicates x 3 technical replicates) corresponding to CD4+ T from wild-type (WT) and CBLOSTmice were compared using the adjusted p-value from a one-way ANOVA test (denoted as p(t)) and the value r(t) corresponding to the enrichment observed in the CBLOST samples as compared to WT samples. Different colors represent distinct biological replicates.


CD4 + T from wild - type ( WT ) , CBLOST CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 anti - CD4 antibodies . Equal amounts of lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads subjected to MS as described in Material Methods . B are classified as CBL interactors according to their position in a volcano plot in which mean p - valueis plotted against corresponding mean enrichment for CBL ( t ) > ( t ) > OST versus WT samples . time point shown corresponds to unstimulated condition ( t = 0s ) . Mean was calculated using bootstrap resampling ( see Results Material Methods ) . that displayed an enrichment r ( t ) greater than 2 - fold a corresponding p - value p ( t ) lower than a set threshold ( see Material Methods ) in more than 90 % of bootstrap iterations were selected as specific partners ( indicated in red ) . Names of four of most significantly enriched are indicated . Dashed lines represent thresholds on p - value p * = 10 - 3 enrichment r * = 2 used to identify specific interactors of CBL .

B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.B Proteins are classified as CBL interactors according to their position in a volcano plot in which the mean p-valueis plotted against the corresponding mean enrichment for CBL(t)>(t)>OST versus WT samples. The time point shown corresponds to the unstimulated condition (t=0s). Mean was calculated using bootstrap resampling (see Results and Material and Methods). Proteins that displayed an enrichment r(t) greater than 2-fold and a corresponding p-value p(t) lower than a set threshold (see Material and Methods) in more than 90% of the bootstrap iterations were selected as specific partners (indicated in red). Names of four of the most significantly enriched proteins are indicated. Dashed lines represent thresholds on the p-value p*=10-3 and the enrichment r*=2 used to identify specific interactors of CBL.


CD4 + T from wild - type ( WT ) , CBLOST CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 anti - CD4 antibodies . Equal amounts of lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads subjected to MS as described in Material Methods . C , D List of specific interacting partners of CBLB ( C ) CBL ( D ) ranked according to their maximum mean enrichment across time points . Heat maps show , for each time point , fraction of bootstrap iterations for which corresponding were detected as specific partners ( denoted as " P ( Detection ) " ) . Red dots indicate that have been previously reported as CBLB ( C ) interactors in BioGRID database . Ubiquitin ( UB ) is highlighted with a black arrow .

B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBLB (C) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow.


CD4 + T from wild - type ( WT ) , CBLOST CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 anti - CD4 antibodies . Equal amounts of lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads subjected to MS as described in Material Methods . C , D List of specific interacting partners of CBLB ( C ) CBL ( D ) ranked according to their maximum mean enrichment across time points . Heat maps show , for each time point , fraction of bootstrap iterations for which corresponding were detected as specific partners ( denoted as " P ( Detection ) " ) . Red dots indicate that have been previously reported as CBL ( D ) interactors in BioGRID database . Ubiquitin ( UB ) is highlighted with a black arrow .

B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as "P(Detection)"). Red dots indicate the proteins that have been previously reported as CBL (D) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow.


CD4 + T from wild - type ( WT ) , CBLOST CBLBOSTmice were left unstimulated ( unstim ) or stimulated for 30 , 120 , 300 or 600 seconds with anti - CD3 anti - CD4 antibodies . Equal amounts of lysates were subjected to affinity purification using Strep - Tactin - Sepharose beads subjected to MS as described in Material Methods . E Histograms showing numbers of specific interactors binding to CBL - OST or CBLB - OST prior to after TCR stimulation for different times for which P ( Detection ) > 0 . 9 .

B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.E Histograms showing the numbers of specific interactors binding to the CBL-OST or CBLB-OST proteins prior to and after TCR stimulation for different times and for which P(Detection) >0.9.


D , E , F , G , H Experimental validation of PPIs predicted on basis of high confidence CN shown in C . CD4 + T were left unstimulated ( - ) or stimulated ( + ) with anti - CD3 anti - CD4 antibodies subsequently lysed . Equal amounts of lysates were incubated with isotype control ( Iso Ctrl ) or specified antibodies resulting immunoprecipitates ( IP ) analyzed by immunoblot with antibodies specified in left margin . validated PPIs predicted in C correspond to CSK - CBLB CSK - CD5 ( D ) , CRKL - PI3Kp85β CSK - PI3Kp85β ( E ) . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to CSK-CBLB and CSK-CD5 (D), CRKL-PI3Kp85β and CSK- PI3Kp85β (E). Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


D , E , F , G , H Experimental validation of PPIs predicted on basis of high confidence CN shown in C . CD4 + T were left unstimulated ( - ) or stimulated ( + ) with anti - CD3 anti - CD4 antibodies subsequently lysed . Equal amounts of lysates were incubated with isotype control ( Iso Ctrl ) or specified antibodies resulting immunoprecipitates ( IP ) analyzed by immunoblot with antibodies specified in left margin . validated PPIs predicted in C correspond to PI3Kp85β - PI3Kp110α , CSK - PI3Kp110α ( F ) CRKL - PI3Kp110α ( G ) . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

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-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to PI3Kp85β-PI3Kp110α, CSK-PI3Kp110α (F) CRKL-PI3Kp110α (G). Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


A CD4 + T from wild - type ( WT ) , CBLOST CBLBOSTmice were left unstimulated ( - ) or stimulated for 1 min with anti - CD3 anti - CD4 antibodies ( + ) . Equal amounts of lysates were subjected to affinity purification on Strep - Tactin - Sepharose beads resulting eluates were analyzed by immunoblotting with anti - ubiquitin ( UB ) , anti - CBLB or anti - CBL antibodies . Also shown are a loading control corresponding to equal amounts of from whole lysates ( WCL ) probed with anti - CD5 , molecular masses ( kDa ) . TCR stimulation resulted in some CBLB degradation in detection of a higher molecular CBLB form , likely due to its ubiquitylation ( marked with an asterisk ) . In contrast , no degradation of CBL was detectable upon TCR stimulation its molecular remained unchanged . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O 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-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O 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 CD4+T cells from wild-type (WT), CBLOST and CBLBOSTmice were left unstimulated (-) or stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads and the resulting protein eluates were analyzed by immunoblotting with anti-ubiquitin (UB), anti-CBLB or anti-CBL antibodies. Also shown are a loading control corresponding to equal amounts of proteins from whole cell lysates (WCL) probed with anti-CD5, and molecular masses (kDa). TCR stimulation resulted in some CBLB degradation and in the detection of a higher molecular weight CBLB form, likely due to its ubiquitylation (marked with an asterisk). In contrast, no degradation of CBL was detectable upon TCR stimulation and its molecular weight remained unchanged. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


C Immunoprecipitation of CBL from lysates of CD4 + T isolated from WT or CBLB - deficient mouse ( Cblb - / - ) kept unstimulated ( - ) or stimulated as in ( A ) . Immunoprecipitates whole lystates ( WCLs ) were analyzed by immunoblot with antibodies specific for specified in left margin . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O 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 Immunoprecipitation of CBL from cell lysates of CD4+T cells isolated from WT or CBLB-deficient mouse (Cblb-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


D Immunoprecipitation of CBLB from lysates of CD4 + T isolated from WT or CBL - deficient mouse ( Cbl - / - ) kept unstimulated ( - ) or stimulated as in ( A ) . Immunoprecipitates whole lystates ( WCLs ) were analyzed by immunoblot with antibodies specific for specified in left margin . Molecular masses are shown ( kDa ) . Data are representative of at least 3 experiments .

D Immunoprecipitation of CBLB from cell lysates of CD4+T cells isolated from WT or CBL-deficient mouse (Cbl-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 3 experiments.


A , B CD4 + T from wild - type , Cblb - / - Cd5 - / - mice were stimulated for 1 min with anti - CD3 anti - CD4 antibodies ( + ) . CBL ( A ) were immunoprecipitated from equal amounts of lysates . Level of ubiquitylation amount of CD5 associated were evaluated by immunoblot with antibodies specific for specified in left margin . Whole lystates ( WCLs ) were also analyzed with specified antibodies . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

O O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). CBL (A) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


A , B CD4 + T from wild - type , Cblb - / - Cd5 - / - mice were stimulated for 1 min with anti - CD3 anti - CD4 antibodies ( + ) . PI3KR1 ( PI3Kp85α ) ( B ) were immunoprecipitated from equal amounts of lysates . Level of ubiquitylation amount of CD5 associated were evaluated by immunoblot with antibodies specific for specified in left margin . Whole lystates ( WCLs ) were also analyzed with specified antibodies . Molecular masses are shown ( kDa ) . Data are representative of at least 2 experiments .

O O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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, B CD4+T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). PI3KR1 (PI3Kp85α) (B) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.


of differentially expressed cholesterol - related genes between normal brain tissues ( n = 28 ) glioblastomas ( n = 217 ) from Rembrandt dataset . Gene expression values are z - transformed colored red for high expression blue for low expression , as indicated in scale bar . Volcano plot showing fold - change ( log2 ) in cholesterol - related gene on GBM vs normal brain tissue . Data were obtained from Rembrandt dataset .

O O B-EXP_ASSAY B-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O B-DISEASE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-DISEASE O O B-TISSUE O O O O O O O O O

Heatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 28) and glioblastomas (n = 217) from the Rembrandt dataset. Gene expression values are z-transformed and colored red for high expression and blue for low expression, as indicated in the scale bar. Volcano plot showing the fold-change (log2) in cholesterol-related gene levels based on GBM vs normal brain tissue. Data were obtained from the Rembrandt dataset.


Representative of for CYP46A1 in normal brain different pathological grades of gliomas ( n = 64 ) . Scale bar = 30 μm . of CYP46A1 in normal brain ( n = 6 ) different pathological grades of gliomas ( n = 58 ) .

O O O B-GENEPROD O O B-TISSUE O O O O B-DISEASE O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O B-DISEASE O O O O O O

Representative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas (n = 64). Scale bar = 30 μm. Quantification of CYP46A1 IHC staining in normal brain (n = 6) and different pathological grades of gliomas (n = 58).


Representative of CYP46A1 in GBM adjacent brain tissues from one specific case . Scale bar = 30 μm .

O O B-GENEPROD O B-DISEASE O B-TISSUE O O O O O O O O O O O O

Representative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case. Scale bar = 30 μm.


CYP46A1 expression in tumours from TCGA dataset using 2016 WHO classification . Data are shown as mean ± standard error of mean ( ; n = 667 ) . * * * P < 0 . 0001 . Statistical significance was determined by one ‐ way ANOVA .

B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification. Data are shown as the mean ± the standard error of the mean (SEM; n = 667). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.


CYP46A1 expression in different molecular subtypes from Rembrandt GBM dataset . Shown are means ( n = 245 ) . * * * P < 0 . 0001 . Statistical significance was determined by one ‐ way ANOVA .

B-GENEPROD B-EXP_ASSAY O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset. Shown are means and SEM (n = 245). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.


Kaplan - Meier for OS PFS on high vs low expression of CYP46A1 in LGG GBM . Data were obtained from CGGA dataset . P - values were obtained from log - rank .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O

Kaplan-Meier analysis for patient OS and PFS based on high vs low expression of CYP46A1 in LGG and GBM. Data were obtained from the CGGA dataset. P-values were obtained from the log-rank test.