Figure 3A . Representative confocal micrographs of HeLa cells infected or not with B . abortus 544 GFP ( Red ) for 24 h , 48 h , and 72 h then fixed and immunostained for HIF - 1α ( Alexa 568 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . B . Quantification of the percentages of cells positive for a nuclear localisation of HIF - 1α from HeLa cells infected or not ( NI ) with B . abortus 544 GFP for 24 , 48 and 72 h from micrographs shown in ( A . B . C ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using a two - way ANOVA followed by a Šidàk ' s multiple comparisons test ; asterisks indicate significant differences compared to the control ( NI ) ; * * * * : p < 0 . 0001 . C . Representative confocal micrographs of HeLa cells infected or not with B . abortus 544 GFP ( Red ) for 48 h , then fixed and immunostained for BNIP3L ( Alexa 568 - Green ) and TOMM20 ( Alexa 633 - Magenta ) . DNA was stained with Hoechst 33258 ( Blue ) . D . Relative median fluorescence intensity ( MFI ) of BNIP3L immunostaining from HeLa cells infected or not ( NI ) with B . abortus 544 GFP for 48 h as measured by flow cytometry . Data are presented as means ± SD from n = 4 ( biological replicates ) independent experiments ( 8 , 296 cells analysed in total per condition ) . Statistical analyses were performed using a one sample t - test ; * : p < 0 . 05 ( p = 0 . 0351 ) .

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 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

Figure 3A. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Red) for 24 h, 48 h, and 72 h then fixed and immunostained for HIF-1α (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). B. Quantification of the percentages of cells positive for a nuclear localisation of HIF-1α from HeLa cells infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A.B.C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ****: p <0.0001.C. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Red) for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green) and TOMM20 (Alexa 633 - Magenta). DNA was stained with Hoechst 33258 (Blue). D. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=4 (biological replicates) independent experiments (8,296 cells analysed in total per condition). Statistical analyses were performed using a one sample t-test; *: p <0.05 (p = 0.0351).


Figure 4D . Representative confocal micrographs of HeLa cells infected or not ( NI ) with B . abortus 544 GFP ( Red ) treated or not ( ctrl ) with 500 µM FeCl2 for 48 h , then fixed and immunostained for BNIP3L ( Alexa 568 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . E . Relative median fluorescence intensity ( MFI ) of BNIP3L immunostaining from HeLa cells infected or not ( NI ) with B . abortus 544 GFP treated or not ( ctrl ) with 500 µM FeCl2 for 48 h as measured by flow cytometry . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( 10 , 199 cells analysed in total per condition ) . Statistical analyses were performed using a two - way ANOVA followed by a Šidàk ' s multiple comparisons test ; asterisks indicate significant differences compared to the control ( NI ) ; ns : not significant ; * : p < 0 , 05 ; hashtags indicate significant differences compared to the infected condition without FeCl2 ; # : p < 0 . 05 . F . Representative confocal micrographs of iBMDM infected or not ( NI ) with B . abortus 544 GFP ( Red ) treated or not ( ctrl ) with 500 µM FeCl2 for 48 h , then fixed and immunostained for BNIP3L ( Alexa 568 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . G . Relative median fluorescence intensity ( MFI ) of BNIP3L immunostaining from iBMDM infected or not ( NI ) with B . abortus 544 GFP treated or not ( ctrl ) with 500 µM FeCl2 for 48 h as measured by flow cytometry . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( 14 , 152 cells analysed in total per condition ) . Statistical analyses were performed using a two - way ANOVA followed by a Šidàk ' s multiple comparisons test ; asterisks indicate significant differences compared to the control ( NI ) ; ns : not significant ; * : p < 0 , 05 ; hashtags indicate significant differences compared to the infected condition without FeCl2 ; # # # : p < 0 . 001 .

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 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

Figure 4D. Representative confocal micrographs of HeLa cells infected or not (NI) with B. abortus 544 GFP (Red) treated or not (ctrl) with 500 µM FeCl2 for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). E. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 µM FeCl2 for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=3 (biological replicates) independent experiments (10,199 cells analysed in total per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; *: p <0,05; hashtags indicate significant differences compared to the infected condition without FeCl2; #: p <0.05.F. Representative confocal micrographs of iBMDM infected or not (NI) with B. abortus 544 GFP (Red) treated or not (ctrl) with 500 µM FeCl2 for 48 h, then fixed and immunostained for BNIP3L (Alexa 568 - Green). DNA was stained with Hoechst 33258 (Blue). G. Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from iBMDM infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 µM FeCl2 for 48 h as measured by flow cytometry. Data are presented as means ± SD from n=3 (biological replicates) independent experiments (14,152 cells analysed in total per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; *: p <0,05; hashtags indicate significant differences compared to the infected condition without FeCl2; ###: p <0.001.


Figure 5A . Representative confocal micrographs of HeLa cells transfected with a non - targeting siRNA pool ( siNT - 40 nM ) or a BNIP3L siRNA SMARTpool ( siBNIP3L - 40 nM ) , infected or not ( NI ) with B . abortus 544 GFP ( Red ) for 48 h , then fixed and immunostained for BNIP3L ( Alexa Fluor 568 - Magenta ) and TOMM20 ( Alexa Fluor 633 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . B . , C . Quantification of the mitochondrial population morphology by assessing the AR ( B ) and EBR ( C ) of the mitochondria of HeLa cells from micrographs shown in ( A ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using a multiple Mann - Whitney test followed by a Holm - Šidàk ' s multiple comparisons test ; asterisks indicate significant differences compared to the control ( NI ) ; * : p < 0 . 05 ; * * : p < 0 . 01 ; hashtags indicate significant differences compared to the infected condition transfected with a siNT ; # : p < 0 . 05 . D . Representative confocal micrographs of HeLa cells transfected with a FIS1 - GFP ( Green ) - mCherry ( Magenta ) expression construct , infected or not ( NI ) with B . abortus 544 for 48 h while being transfected with a non - targeting siRNA pool ( siNT - 40 nM ) or a BNIP3L siRNA SMARTpool ( siBNIP3L - 40 nM ) , then fixed and immunostained for B . abortus LPS ( Alexa Fluor 633 - Red ) . DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate FIS1 - mCherry - positive - GFP - negative punctae . Scale bars : 20 µm . E . Quantification of the number of FIS1 - mCherry - positive - GFP - negative punctae per cell of HeLa cells from micrographs t shown in ( D ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using a two - way ANOVA followed by a Šidàk ' s multiple comparisons test ; asterisks indicate significant differences compared to the control ( NI ) ; ns : not significant ; * * : p < 0 . 01 ; hashtags indicate significant differences compared to the infected condition transfected with a siNT ; # : p < 0 . 05 .

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 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 O O O O O O O O O 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. Representative confocal micrographs of HeLa cells transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM), infected or not (NI) with B. abortus 544 GFP (Red) for 48 h, then fixed and immunostained for BNIP3L (Alexa Fluor 568 - Magenta) and TOMM20 (Alexa Fluor 633 - Green). DNA was stained with Hoechst 33258 (Blue). B.,C. Quantification of the mitochondrial population morphology by assessing the AR (B) and EBR (C) of the mitochondria of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a multiple Mann-Whitney test followed by a Holm-Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); *: p <0.05; **: p <0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; #: p <0.05.D. Representative confocal micrographs of HeLa cells transfected with a FIS1-GFP(Green)-mCherry(Magenta) expression construct, infected or not (NI) with B. abortus 544 for 48 h while being transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM), then fixed and immunostained for B. abortus LPS (Alexa Fluor 633 - Red). DNA was stained with Hoechst 33258 (Blue). Arrows indicate FIS1-mCherry-positive-GFP-negative punctae. Scale bars: 20 µm. E. Quantification of the number of FIS1-mCherry-positive-GFP-negative punctae per cell of HeLa cells from micrographs t shown in (D). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two-way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; **: p <0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; #: p <0.05.


Figure 6A . Representative confocal micrographs of HeLa cells infected with B . abortus 544 GFP ( Green ) and transfected with a non - targeting siRNA pool ( siNT - 40 nM ) or a BNIP3L siRNA SMARTpool ( siBNIP3L - 40 nM ) for 48 h , then incubated under reinfection - permissive conditions for 24 h before analysis at 72 h pi . Cells were fixed and DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate reinfected cells . B . Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in ( A ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * * : p < 0 . 01 ( p = 0 . 0032 ) . C . CFU assay expressing Log ( CFU / ml ) from the supernatant of HeLa cells infected with B . abortus 544 GFP ( Green ) and transfected with a non - targeting siRNA pool ( siNT - 40 nM ) or a BNIP3L siRNA SMARTpool ( siBNIP3L - 40 nM ) for 48 h , then incubated under reinfection - permissive conditions for 24 h before collecting the supernatant at 72 h pi for analysis . Data are presented as means ± SD from n = 5 ( biological replicates ) independent experiments ; Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; ( p = 0 . 0042 ) . D . Representative confocal micrographs of HeLa cells infected with B . abortus 544 GFP ( Red ) and transfected with a non - targeting siRNA pool ( siNT - 40 nM ) or a BNIP3L siRNA SMARTpool ( siBNIP3L - 40 nM ) for 72 h pi , then fixed and immunostained for LAMP - 1 ( Alexa Fluor 568 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate LAMP - 1 - positive BCVs ( aBCVs ) . Scale bars : 20 µm . E . Quantification of the number of LAMP - 1 - positive BCVs ( aBCVs ) per infected HeLa cells from micrographs shown in ( E ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * : p < 0 . 05 ( p = 0 . 0117 ) .

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

Figure 6A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Green) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 48 h, then incubated under reinfection-permissive conditions for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. B. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; **: p <0.01 (p = 0.0032). C. CFU assay expressing Log (CFU/ml) from the supernatant of HeLa cells infected with B. abortus 544 GFP (Green) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 48 h, then incubated under reinfection-permissive conditions for 24 h before collecting the supernatant at 72 h pi for analysis. Data are presented as means ± SD from n=5 (biological replicates) independent experiments; Statistical analyses were performed using an unpaired two-tailed Student's t-test; (p = 0.0042).D. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Red) and transfected with a non-targeting siRNA pool (siNT - 40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L - 40 nM) for 72 h pi, then fixed and immunostained for LAMP-1 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LAMP-1-positive BCVs (aBCVs). Scale bars: 20 µm. E. Quantification of the number of LAMP-1-positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p <0.05 (p = 0.0117).


Figure 7A . Representative confocal micrographs of HeLa cells infected with B . abortus 544 GFP ( Green ) treated or not ( ctrl ) with FeCl2 ( 500 µM ) for 48 h , then incubated under reinfection - permissive conditions ( with or without FeCl2 ) for 24 h before analysis at 72 h pi . Cells were fixed and DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate reinfected cells . B . Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in ( A ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * * * : p < 0 . 001 ( p = 0 . 0008 ) . C . Representative confocal micrographs of HeLa cells infected or not with B . abortus 544 GFP ( Magenta ) treated or not ( ctrl ) with FeCl2 ( 500 µM ) for 72 h , then fixed and immunostained for LAMP - 1 ( Alexa Fluor 568 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate LAMP - 1 - positive BCVs ( aBCVs ) . D . Quantification of the number of LAMP - 1 - positive BCVs ( aBCVs ) per infected HeLa cells from micrographs shown in ( C ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * : p < 0 . 05 ( p = 0 . 0368 ) . E . Representative confocal micrographs of iBMDM infected with B . abortus 544 GFP ( Green ) treated or not ( ctrl ) with FeCl2 ( 500 µM ) for 48 h , then incubated under reinfection - permissive conditions ( with or without FeCl2 ) for 24 h before analysis at 72 h pi . Cells were fixed and DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate reinfected cells . F . Quantification of the percentages of reinfection foci per infected cell at 72 h pi of iBMDM from micrographs shown in ( E ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * * : p < 0 . 01 ( p = 0 . 0043 ) .

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 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 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

Figure 7A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl2 (500 µM) for 48 h, then incubated under reinfection-permissive conditions (with or without FeCl2) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. B. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0008).C. Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Magenta) treated or not (ctrl) with FeCl2 (500 µM) for 72 h, then fixed and immunostained for LAMP-1 (Alexa Fluor 568 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate LAMP-1-positive BCVs (aBCVs). D. Quantification of the number of LAMP-1-positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (C). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p <0.05 (p = 0.0368).E. Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl2 (500 µM) for 48 h, then incubated under reinfection-permissive conditions (with or without FeCl2) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (Blue). Arrows indicate reinfected cells. F. Quantification of the percentages of reinfection foci per infected cell at 72 h pi of iBMDM from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; **: p <0.01 (p = 0.0043).


Figure 8A . Representative confocal micrographs of HeLa cells infected with B . abortus 544 GFP ( Magenta ) for 48 and 72 h , then fixed and immunostained for TOMM20 ( Alexa Fluor 647 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate when B . abortus was found inside a mitochondrion ( mBCVs ) . Scale bars : 20 µ B . Quantification of the percentage of infected HeLa cells displaying TOMM20 - positive BCVs ( mBCVs ) at the indicated times , from micrographs shown in ( A ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * * * : p < 0 . 001 ( p = 0 . 0001 ) . C . Quantification of the number of TOMM20 - positive BCVs ( mBCVs ) per infected HeLa cells , at the indicated times , from micrographs shown in ( E ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * : p = 0 . 05 . D . Representative confocal micrographs of iBMDM infected with B . abortus 544 GFP ( Magenta ) for 48 and 72 h , then fixed and immunostained for TOMM20 ( Alexa Fluor 647 - Green ) . DNA was stained with Hoechst 33258 ( Blue ) . Arrows indicate when B . abortus was found inside a mitochondrion ( mBCVs ) . Scale bars : 20 µm . E . Quantification of the percentage of infected iBMDM displaying TOMM20 - positive BCVs ( mBCVs ) at the indicated times , from micrographs shown in ( D ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; * * * : p < 0 . 001 ( p = 0 . 0009 ) . F . Quantification of the number of TOMM20 - positive BCVs ( mBCVs ) per infected HeLa cells , at the indicated times , from micrographs shown in ( E ) . Data are presented as means ± SD from n = 3 ( biological replicates ) independent experiments ( the numbers indicated in the columns represent the number of cells analysed per condition ) . Statistical analyses were performed using an unpaired two - tailed Student ' s t - test ; ns : not significant ( p = 0 . 3739 ) .

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 O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 8A. Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 µ B. Quantification of the percentage of infected HeLa cells displaying TOMM20-positive BCVs (mBCVs) at the indicated times, from micrographs shown in (A). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0001). C. Quantification of the number of TOMM20-positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; *: p = 0.05.D. Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (Magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647 - Green). DNA was stained with Hoechst 33258 (Blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 µm. E. Quantification of the percentage of infected iBMDM displaying TOMM20-positive BCVs (mBCVs) at the indicated times, from micrographs shown in (D). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ***: p <0.001 (p = 0.0009). F. Quantification of the number of TOMM20-positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n=3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two-tailed Student's t-test; ns: not significant (p = 0.3739).


Figure 1 ( B ) Detection of S - acylation of BSKs in plant cells . 12 BSK proteins were fused with GFP at their C terminus and expressed in Arabidopsis protoplasts for 24 hours and the cells were collected for Biotin - switch assay . Cell lysates are shown as input , and the S - acylated proteins enriched on the resin which is dependent on hydroxylamine ( NH2OH ) are indicated as pulldown samples . All images of anti - GFP immunoblots are representative of three biological independent experiments . ( D ) Effect of 2 - BP on subcellular localization of BSK1 in root cells of intact transgenic plants . Six - day - old seedlings of 35S : BSK1 - GFP were treated with 80 μM 2 - BP for 2 h ; DMSO was supplemented in the control samples . Representative GFP ( green ) and merged ( with bright field ) signals from three biologically independent experiments are shown . Bars , 10 µm . ( E ) Effect of 2 - BP on the localization of BSK1 - GFP in transgenic plants using a cell fractionation assay . Six - day - old seedlings were treated with 80 μM 2 - BP ( DMSO was used in the control sample ) for 3 h before protein extraction . Total proteins ( T ) were divided into soluble ( S ) and pellet ( P ) fractions using ultra - centrifugation . The representative immunoblot using an anti - GFP antibody is shown .

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 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 O O O O O O O O O O O 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 1(B) Detection of S-acylation of BSKs in plant cells. 12 BSK proteins were fused with GFP at their C terminus and expressed in Arabidopsis protoplasts for 24 hours and the cells were collected for Biotin-switch assay. Cell lysates are shown as input, and the S-acylated proteins enriched on the resin which is dependent on hydroxylamine (NH2OH) are indicated as pulldown samples. All images of anti-GFP immunoblots are representative of three biological independent experiments.(D) Effect of 2-BP on subcellular localization of BSK1 in root cells of intact transgenic plants. Six-day-old seedlings of 35S:BSK1-GFP were treated with 80 μM 2-BP for 2 h; DMSO was supplemented in the control samples. Representative GFP (green) and merged (with bright field) signals from three biologically independent experiments are shown. Bars, 10 µm.(E) Effect of 2-BP on the localization of BSK1-GFP in transgenic plants using a cell fractionation assay. Six-day-old seedlings were treated with 80 μM 2-BP (DMSO was used in the control sample) for 3 h before protein extraction. Total proteins (T) were divided into soluble (S) and pellet (P) fractions using ultra-centrifugation. The representative immunoblot using an anti-GFP antibody is shown.


Figure 2 ( A , B ) Identification of S - acylation sites on BSK1 and BSK3 . The wild - type ( WT ) and indicated mutant versions of BSK1 - GFP ( A ) and BSK3 - GFP ( B ) were expressed in protoplasts for biotin - switch assay . Images of anti - GFP immunoblots are representative of three biological independent experiments . ( E , F ) Effect of S - acylation site mutation on the localization of BSK1 and BSK3 in intact root cells . Six - day - old seedlings of the WT or S - acylation site mutant versions of 35S : BSK1 - GFP ( E ) and 35S : BSK3 - GFP ( F ) were subjected to localization analysis using confocal microscopy . Representative GFP ( green ) and merged ( with bright field ) signals in root cells from three biologically independent experiments are shown . Bars , 10 µm . ( G , H ) The effect of S - acylation site mutations on the localization of BSK1 and BSK3 in a cell fractionation assay . The WT and mutant versions of BSK1 - GFP ( G ) and BSK3 - GFP ( H ) were expressed in protoplasts and the total proteins ( T ) were divided into soluble ( S ) and pellet ( P ) fractions using ultra - centrifugation . Representative immunoblotting data using an anti - GFP antibody are shown .

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

Figure 2(A, B) Identification of S-acylation sites on BSK1 and BSK3. The wild-type (WT) and indicated mutant versions of BSK1-GFP (A) and BSK3-GFP (B) were expressed in protoplasts for biotin-switch assay. Images of anti-GFP immunoblots are representative of three biological independent experiments.(E, F) Effect of S-acylation site mutation on the localization of BSK1 and BSK3 in intact root cells. Six-day-old seedlings of the WT or S-acylation site mutant versions of 35S:BSK1-GFP (E) and 35S:BSK3-GFP (F) were subjected to localization analysis using confocal microscopy. Representative GFP (green) and merged (with bright field) signals in root cells from three biologically independent experiments are shown. Bars, 10 µm.(G, H) The effect of S-acylation site mutations on the localization of BSK1 and BSK3 in a cell fractionation assay. The WT and mutant versions of BSK1-GFP (G) and BSK3-GFP (H) were expressed in protoplasts and the total proteins (T) were divided into soluble (S) and pellet (P) fractions using ultra-centrifugation. Representative immunoblotting data using an anti-GFP antibody are shown.


Figure 3 ( B ) Co - IP result for measuring the function of BSK1 S - acylation in its interaction with BRI1 . Free GFP ( negative control ) or the WT / C3S version of BSK1 - GFP was co - expressed with BRI1 - MYC in plant cells for co - IP using an anti - GFP resin . The proteins in the input and IP samples were detected via immunoblots with anti - MYC or anti - GFP antibodies . ( E , F ) The effect of S - acylation on the function of BSK3 in BR responses . The WT or 3M ( C5 , 10 , 11S ) version of BSK3 driven by a native promoter was introduced into the bsk3 - 1 mutant to generate complementation lines . The phenotype of 8 - day - old seedlings with and without 100 nM of BL treatment is shown in ( E ) . Bars , 1 cm . Quantitative data of hypocotyl and root length under the control or BL treatment condition are shown in ( F ) . Data are means ± SD from 20 seedlings in four independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(B) Co-IP result for measuring the function of BSK1 S-acylation in its interaction with BRI1. Free GFP (negative control) or the WT/C3S version of BSK1-GFP was co-expressed with BRI1-MYC in plant cells for co-IP using an anti-GFP resin. The proteins in the input and IP samples were detected via immunoblots with anti-MYC or anti-GFP antibodies.(E, F) The effect of S-acylation on the function of BSK3 in BR responses. The WT or 3M (C5, 10, 11S) version of BSK3 driven by a native promoter was introduced into the bsk3-1 mutant to generate complementation lines. The phenotype of 8-day-old seedlings with and without 100 nM of BL treatment is shown in (E). Bars, 1 cm. Quantitative data of hypocotyl and root length under the control or BL treatment condition are shown in (F). Data are means ± SD from 20 seedlings in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).


Figure 4 ( C ) Detection of the regulation of BSK1 S - acylation mediated by SA . BSK1 - GFP was expressed in protoplasts with 0 - , 20 - , 40 - , or 60 - min treatment with 100 µM of SA . Cells were collected for S - acylation detection in a biotin - switch assay . Representative immunoblots are shown in the top graph . Immunoblot signals were quantified by ImageJ and the S - acylation levels were calculated from relative signals ( [ pulldown + / input + ] - [ pulldown - / input - ] ) . The relative S - acylation level of the 0 min sample was set to 1 . The data in the bottom graph are means ± SD from three biologically independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) . ( D , E ) Effect of SA on the physiological function of BSK1 . The indicated seeds were germinated on the MS medium with or without 40 µM of SA . The photographs were taken 8 days after germination . Representative images are shown in ( D ) . Bars , 1 cm . Quantitative data of fresh weight ( per 10 seedlings ) of different genotypes are shown in ( E ) . Data are means ± SD from three biologically independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) .

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 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

Figure 4(C) Detection of the regulation of BSK1 S-acylation mediated by SA. BSK1-GFP was expressed in protoplasts with 0-, 20-, 40-, or 60-min treatment with 100 µM of SA. Cells were collected for S-acylation detection in a biotin-switch assay. Representative immunoblots are shown in the top graph. Immunoblot signals were quantified by ImageJ and the S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the 0 min sample was set to 1. The data in the bottom graph are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(D, E) Effect of SA on the physiological function of BSK1. The indicated seeds were germinated on the MS medium with or without 40 µM of SA. The photographs were taken 8 days after germination. Representative images are shown in (D). Bars, 1 cm. Quantitative data of fresh weight (per 10 seedlings) of different genotypes are shown in (E). Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).


Figure 5 ( A ) Identification of the de - S - acylation enzyme of BSK1 . BSK1 - RFP was co - overexpressed with 11 GFP - fused ABAPT members in protoplasts respectively ( free GFP was used as a control ) . Cells with both RFP and GFP signals were used for analyzing the localization of BSK1 - RFP . The predominant localization patterns of BSK1 - RFP are shown . The RFP ( magenta ) , GFP ( green ) , and the merged ( with bright field in gray ) signals are included in the top graph . Bars , 5 µm . The quantitative data of the percentages of cells with mislocalization of BSK1 - GFP ( 100 cells for each sample ) are shown in the bottom graph . Data are means ± SD from three biologically independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) . ( B ) Detection of the effect of ABAPT11 on the localization of BSK members . 12 BSK - GFP fusion proteins were co - overexpressed with RFP ( control ) or ABAPT11 - RFP in protoplasts . BSK - GFP localization in cells with both RFP and GFP signals was recorded . Representative localization patterns of BSK - GFP with RFP or ABAPT11 - RFP are shown in the top graph . Bars , 5 µm . Quantitative data of the percentages of cells with localization changes of BSK - GFP ( 100 cells for each sample ) are shown in the bottom graph . Data are means ± SD from three biologically independent experiments . * * * * P < 0 . 0001 , * * * P < 0 . 001 , * P < 0 . 05 , n . s . ( no significance ) P > 0 . 05 , Student ' s t - test ( unpaired , two - tailed ) .

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 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

Figure 5(A) Identification of the de-S-acylation enzyme of BSK1. BSK1-RFP was co-overexpressed with 11 GFP-fused ABAPT members in protoplasts respectively (free GFP was used as a control). Cells with both RFP and GFP signals were used for analyzing the localization of BSK1-RFP. The predominant localization patterns of BSK1-RFP are shown. The RFP (magenta), GFP (green), and the merged (with bright field in gray) signals are included in the top graph. Bars, 5 µm. The quantitative data of the percentages of cells with mislocalization of BSK1-GFP (100 cells for each sample) are shown in the bottom graph. Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(B) Detection of the effect of ABAPT11 on the localization of BSK members. 12 BSK-GFP fusion proteins were co-overexpressed with RFP (control) or ABAPT11-RFP in protoplasts. BSK-GFP localization in cells with both RFP and GFP signals was recorded. Representative localization patterns of BSK-GFP with RFP or ABAPT11-RFP are shown in the top graph. Bars, 5 µm. Quantitative data of the percentages of cells with localization changes of BSK-GFP (100 cells for each sample) are shown in the bottom graph. Data are means ± SD from three biologically independent experiments. ****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. (no significance) P > 0.05, Student's t-test (unpaired, two-tailed).


Figure 6 ( B ) Effect of ABAPT11 overexpression on the BSK1 membrane association in a cell fractionation assay . BSK1 - GFP was expressed in protoplasts with or without ABAPT11 overexpression . Total proteins ( T ) were separated into soluble ( S ) and pellet ( P ) fractions using ultra - centrifugation . A representative immunoblot using an anti - GFP antibody is shown . The percentages of BSK1 - GFP in the soluble fraction were calculated from relative immunoblotting signals ( S / [ S + P ] ) . Data are means ± SD from three biologically independent experiments . * * P < 0 . 01 , Student ' s t - test ( unpaired , two - tailed ) . ( C , D ) Effect of ABAPT11 overexpression on S - acylation of BSK1 and BSK3 . The S - acylation level of BSK1 - GFP ( C ) and BSK3 - GFP ( D ) with or without ABAPT11 overexpression in protoplasts was detected in a biotin - switch assay . Representative immunoblotting image is shown in the top graphs . Quantification of relative S - acylation levels was included in the bottom graphs . Immunoblotting signals were quantified by ImageJ and the S - acylation levels were calculated from relative signals ( [ pulldown + / input + ] - [ pulldown - / input - ] ) . The relative S - acylation level of the control sample was set to 1 . Data are means ± SD from three biologically independent experiments . * * P < 0 . 01 , Student ' s t - test ( unpaired , two - tailed ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of ABAPT11 overexpression on the BSK1 membrane association in a cell fractionation assay. BSK1-GFP was expressed in protoplasts with or without ABAPT11 overexpression. Total proteins (T) were separated into soluble (S) and pellet (P) fractions using ultra-centrifugation. A representative immunoblot using an anti-GFP antibody is shown. The percentages of BSK1-GFP in the soluble fraction were calculated from relative immunoblotting signals (S/[S+P]). Data are means ± SD from three biologically independent experiments. **P < 0.01, Student's t-test (unpaired, two-tailed).(C, D) Effect of ABAPT11 overexpression on S-acylation of BSK1 and BSK3. The S-acylation level of BSK1-GFP (C) and BSK3-GFP (D) with or without ABAPT11 overexpression in protoplasts was detected in a biotin-switch assay. Representative immunoblotting image is shown in the top graphs. Quantification of relative S-acylation levels was included in the bottom graphs. Immunoblotting signals were quantified by ImageJ and the S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the control sample was set to 1. Data are means ± SD from three biologically independent experiments. **P < 0.01, Student's t-test (unpaired, two-tailed).


Figure 7 ( A , B ) Expression of ABAPT11 during SA treatment . ( A ) Seven - day - old wild - type ( WT ) seedlings were treated with 100 µM SA for 0 , 10 , 20 , 30 , 40 , 50 , and 60 min before collection for RNA preparation and RT - qPCR to detect the transcript level of ABAPT11 . ( B ) Transcript levels of ABAPT11 in wild - type ( WT ) and npr1 mutant 7 - day - old seedlings with or without 100 µM SA for 20 min . ACTIN2 was used as an internal control . Data are means ± SD from triplicated replications in an experiment ; the pattern was consistent in three biologically independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) . ( C ) Accumulation of ABAPT11 proteins in the SA response . Seven - day - old seedlings expressing ABAPT11 - YFP under its own promoter were treated with 100 µM SA for 0 , 20 , 40 , and 60 min before collection for immunoblotting with an anti - GFP antibody . Coomassie blue staining of total proteins was used as the loading control . Quantitative relative protein levels ( YFP / loading ) from three biologically independent experiments are shown in the bottom graph . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) . ( D ) The S - acylation level of BSK1 in the ABAPT11 mutant during SA treatment . BSK1 - GFP was expressed in WT or abapt11 - 1 mutant cells ( with or without 100 µM SA treatment for 40 min ) for S - acylation measurement in a biotin - switch assay . Representative immunoblots are shown in the top graph . The immunoblotting signals were quantified by ImageJ and the S - acylation levels were calculated from relative signals ( [ pulldown + / input + ] - [ pulldown - / input - ] ) . The relative S - acylation level of the wild - type sample without SA treatment was set to 1 . Data are means ± SD from three biologically independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) . ( G , Confirmation of the SA - induced BSK1 translocation which is mediated by ABAPT11 . BSK1 - GFP was expressed in WT or abapt11 - 1 mutant cells , with or without 100 µM SA treatment for 40 min . Total proteins ( T ) were separated into soluble ( S ) and pellet ( P ) fractions using ultra - centrifugation . Representative immunoblots using an anti - GFP antibody are shown in ( G ) .

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

Figure 7(A, B) Expression of ABAPT11 during SA treatment. (A) Seven-day-old wild-type (WT) seedlings were treated with 100 µM SA for 0, 10, 20, 30, 40, 50, and 60 min before collection for RNA preparation and RT-qPCR to detect the transcript level of ABAPT11. (B) Transcript levels of ABAPT11 in wild-type (WT) and npr1 mutant 7-day-old seedlings with or without 100 µM SA for 20 min. ACTIN2 was used as an internal control. Data are means ± SD from triplicated replications in an experiment; the pattern was consistent in three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(C) Accumulation of ABAPT11 proteins in the SA response. Seven-day-old seedlings expressing ABAPT11-YFP under its own promoter were treated with 100 µM SA for 0, 20, 40, and 60 min before collection for immunoblotting with an anti-GFP antibody. Coomassie blue staining of total proteins was used as the loading control. Quantitative relative protein levels (YFP / loading) from three biologically independent experiments are shown in the bottom graph. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(D) The S-acylation level of BSK1 in the ABAPT11 mutant during SA treatment. BSK1-GFP was expressed in WT or abapt11-1 mutant cells (with or without 100 µM SA treatment for 40 min) for S-acylation measurement in a biotin-switch assay. Representative immunoblots are shown in the top graph. The immunoblotting signals were quantified by ImageJ and the S-acylation levels were calculated from relative signals ([pulldown+/input+] - [pulldown-/ input-]). The relative S-acylation level of the wild-type sample without SA treatment was set to 1. Data are means ± SD from three biologically independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).(G, Confirmation of the SA-induced BSK1 translocation which is mediated by ABAPT11. BSK1-GFP was expressed in WT or abapt11-1 mutant cells, with or without 100 µM SA treatment for 40 min. Total proteins (T) were separated into soluble (S) and pellet (P) fractions using ultra-centrifugation. Representative immunoblots using an anti-GFP antibody are shown in (G).


Figure 8 ( A , B ) Effect of BL and SA on hypocotyl development in ABAPT11 mutants . Seeds were germinated on regular medium ( Control ) , medium containing 80 nM BL , medium containing 40 µM SA , or medium containing both 80 nM BL and 40 µM SA . Hypocotyl length was measured 8 days after germination . Representative images from four independent experiments are shown in ( A ) . Quantitative data of hypocotyl lengths are shown in ( B ) . Data are means ± SD from 20 seedlings in four independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) . D ) Effect of BL and SA on root development of ABAPT11 mutants . Four days after germination , seedlings of WT , abapt11 - 1 , and abapt11 - 2 were transferred onto a regular medium ( Control ) , medium containing 15 nM BL , medium containing 20 µM SA , or medium containing both 15 nM BL and 20 µM SA . Quantitative data of root elongation 5 days after transferring are shown in ( D ) . Data are means ± SD from 20 seedlings in four independent experiments . Significance analysis using one - way ANOVA followed by Tukey ' s multiple comparison test is shown in different lower - case letters ( P < 0 . 05 ) .

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 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

Figure 8(A, B) Effect of BL and SA on hypocotyl development in ABAPT11 mutants. Seeds were germinated on regular medium (Control), medium containing 80 nM BL, medium containing 40 µM SA, or medium containing both 80 nM BL and 40 µM SA. Hypocotyl length was measured 8 days after germination. Representative images from four independent experiments are shown in (A). Quantitative data of hypocotyl lengths are shown in (B). Data are means ± SD from 20 seedlings in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).D) Effect of BL and SA on root development of ABAPT11 mutants. Four days after germination, seedlings of WT, abapt11-1, and abapt11-2 were transferred onto a regular medium (Control), medium containing 15 nM BL, medium containing 20 µM SA, or medium containing both 15 nM BL and 20 µM SA. Quantitative data of root elongation 5 days after transferring are shown in (D). Data are means ± SD from 20 seedlings in four independent experiments. Significance analysis using one-way ANOVA followed by Tukey's multiple comparison test is shown in different lower-case letters (P < 0.05).


Figure 1RNAi injections schedule in starved conditions . Planarians are at 14dS when the amputation is performed ( indicated by the grey cross at day 14 ) . Live images show that cct3A ( RNAi ) planarians form a minimal blastema compared to controls at the time points shown . At the bottom are the number of planarians with the phenotype shown . The remaining planarians are dead by the time point of regeneration shown . dR indicates days of regeneration . RNAi injections schedule in feeding conditions . One extra feeding in respect to ( B ) is introduced one day prior to injections . Planarians are at 8dS when the amputation is performed . The live images show that most of the cct3A ( RNAi ) planarians regenerate like controls . At the bottom are the number of planarians with the phenotype shown . At 8 - 10dR , the remaining planarians are either dead ( 4 / 124 ) or have a tiny blastema ( 23 / 124 ) and either died or regenerated later . At 51 - 68dR , the remaining planarians are either dead ( 10 / 93 ) or have a tiny blastema and died later ( 5 / 93 ) .

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

Figure 1RNAi injections schedule in starved conditions. Planarians are at 14dS when the amputation is performed (indicated by the grey cross at day 14). Live images show that cct3A(RNAi) planarians form a minimal blastema compared to controls at the time points shown. At the bottom are the number of planarians with the phenotype shown. The remaining planarians are dead by the time point of regeneration shown. dR indicates days of regeneration. RNAi injections schedule in feeding conditions. One extra feeding in respect to (B) is introduced one day prior to injections. Planarians are at 8dS when the amputation is performed. The live images show that most of the cct3A(RNAi) planarians regenerate like controls. At the bottom are the number of planarians with the phenotype shown. At 8-10dR, the remaining planarians are either dead (4/124) or have a tiny blastema (23/124) and either died or regenerated later. At 51-68dR, the remaining planarians are either dead (10/93) or have a tiny blastema and died later (5/93).


Figure 2The graph shows the mitotic numbers of trunks during different time points of regeneration in the starved condition for cct3A RNAi and controls . Error bars are s . d . from the mean and asterisks indicate P < 0 . 001 ( three asterisks ) , P < 0 . 01 ( two asterisks ) and n . s . indicates not significant using two - tailed Student ' s test with equal variance . n ≥ 9 planarians per time point . Also shown are maximum projections of representative trunks labelled with anti - H3P at different time points of regeneration in the starved condition . On the bottom , the number of planarians with the phenotype shown from the total is displayed . Note that at 50dR planarians show few ( a4 ) or no mitoses ( a5 ) . The dashed line delimits the body of the planarian . Maximum projections of representative trunks after FISH for smedwi - 1 at different time points of regeneration under starved and feeding conditions . On the bottom , the number of planarians with the phenotype shown from the total is displayed . At 15dR the expression levels are similar . At 30dR some planarians have almost no expression ( c5 ) and at 50dR almost all planarians show no expression ( c10 and c11 ) in starved conditions . Under the feeding condition the expression levels at 30dS are similar . On the bottom , the number of planarians with the phenotype shown from the total is displayed . The dashed line delimits the body of the planarian .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2The graph shows the mitotic numbers of trunks during different time points of regeneration in the starved condition for cct3A RNAi and controls. Error bars are s.d. from the mean and asterisks indicate P < 0.001 (three asterisks), P < 0.01 (two asterisks) and n.s. indicates not significant using two-tailed Student's test with equal variance. n ≥ 9 planarians per time point. Also shown are maximum projections of representative trunks labelled with anti-H3P at different time points of regeneration in the starved condition. On the bottom, the number of planarians with the phenotype shown from the total is displayed. Note that at 50dR planarians show few (a4) or no mitoses (a5). The dashed line delimits the body of the planarian.Maximum projections of representative trunks after FISH for smedwi-1 at different time points of regeneration under starved and feeding conditions. On the bottom, the number of planarians with the phenotype shown from the total is displayed. At 15dR the expression levels are similar. At 30dR some planarians have almost no expression (c5) and at 50dR almost all planarians show no expression (c10 and c11) in starved conditions. Under the feeding condition the expression levels at 30dS are similar. On the bottom, the number of planarians with the phenotype shown from the total is displayed. The dashed line delimits the body of the planarian.


Figure 3The cartoon represents a regenerating trunk and the squares display the region analyzed at the anterior " A " and posterior " P " blastemas . The images are TUNEL maximum projections of representative blastemas from 72 hours and 4 days regenerating trunks in starved conditions . The dashed line delimits the body of the planarian . The graph shows the number of TUNEL + cells per mm2 . The differences in cell death are not significant ( n . s . ) at 72hR ( two - tailed Student ' s test with equal sample variance ) and significant ( * * * P < 0 . 001 using two - tailed Student ' s test with equal variance ) at 4dR . n ≥ 5 planarians per condition .

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

Figure 3The cartoon represents a regenerating trunk and the squares display the region analyzed at the anterior "A" and posterior "P" blastemas. The images are TUNEL maximum projections of representative blastemas from 72 hours and 4 days regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2. The differences in cell death are not significant (n.s.) at 72hR (two-tailed Student's test with equal sample variance) and significant (***P < 0.001 using two-tailed Student's test with equal variance) at 4dR. n ≥ 5 planarians per condition.


Figure 4Volcano plot displaying DEGs in starved cct3A ( RNAi ) planarians compared to controls at 72hR ( q - value < 0 . 05 ) . Y - axis indicates the negative log10 of the false discovery rate ( FDR ) ( q - value ) . X - axis indicates the beta values ( b ) , a biased estimator of the fold change . For better visualization , 13 dots with - log10 ( q - value ) > 20 are not displayed . This includes cct3A with q = 0 . Notice that when down - regulating cct3A in starved planarians , xbp1 , atf6 , bip - 1 and a repertoire of ER chaperones are down - regulated , whereas dnajb9 , a repressor of the UPR , and other ccts are up - regulated . Live images show that xbp1 / atf6 ( RNAi ) planarians in starved conditions form a minimal blastema when compared to controls while at the feeding condition most of them can regenerate as controls . The remaining planarians in the starved condition are dead by the time point of regeneration shown . In the feeding condition , the remaining planarians at 8 - 10dR are either dead ( 13 / 145 ) or have a tiny blastema and died or regenerated later ( 40 / 145 ) while at 21 - 27dR are dead . Maximum projections of representative regenerating trunks after FISH for smedwi - 1 in starved conditions . The dashed line delimits the body of the planarian . TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions . The dashed line delimits the body of the planarian . The graph shows the number of TUNEL + cells per mm2 at the same time points in anterior ( A ) and posterior ( P ) blastemas . n ≥ 6 planarians per time point . Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1 / atf6 RNAi and controls in starved conditions after double immunostaining with anti - tyrosine - tubulin and anti - H3P ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , * P < 0 . 05 , n . s . indicates not significant using two - sided Chi - square test ) ; n ≥ 5 planarians . Representative images are shown . Arrows indicate abnormal organization or number of spindle poles . j8 displays asymmetrical distribution of chromosome content .

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 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 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 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

Figure 4Volcano plot displaying DEGs in starved cct3A(RNAi) planarians compared to controls at 72hR (q-value < 0.05). Y-axis indicates the negative log10 of the false discovery rate (FDR) (q- value). X-axis indicates the beta values (b), a biased estimator of the fold change. For better visualization, 13 dots with -log10 (q-value) > 20 are not displayed. This includes cct3A with q = 0. Notice that when down-regulating cct3A in starved planarians, xbp1, atf6, bip-1 and a repertoire of ER chaperones are down-regulated, whereas dnajb9, a repressor of the UPR, and other ccts are up-regulated.Live images show that xbp1/atf6(RNAi) planarians in starved conditions form a minimal blastema when compared to controls while at the feeding condition most of them can regenerate as controls. The remaining planarians in the starved condition are dead by the time point of regeneration shown. In the feeding condition, the remaining planarians at 8-10dR are either dead (13/145) or have a tiny blastema and died or regenerated later (40/145) while at 21-27dR are dead.Maximum projections of representative regenerating trunks after FISH for smedwi-1 in starved conditions. The dashed line delimits the body of the planarian.TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2 at the same time points in anterior (A) and posterior (P) blastemas. n ≥ 6 planarians per time point.Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1/atf6 RNAi and controls in starved conditions after double immunostaining with anti-tyrosine-tubulin and anti-H3P (****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. indicates not significant using two-sided Chi-square test); n ≥ 5 planarians. Representative images are shown. Arrows indicate abnormal organization or number of spindle poles. j8 displays asymmetrical distribution of chromosome content.


Figure 5The Kaplan - Meier curve demonstrates increased survival of cct3A RNAi animals when treated with DTT under starved conditions . Percentages indicate the number of survivals at the indicated time points . Three asterisks indicate p < 0 . 001 with log - rank ( Mantel - Cox ) test . Images are representative surviving animals for the different conditions at the displayed time point of regeneration . At the bottom are the number of planarians with the phenotype shown . The rest of planarians are dead at the displayed time point .

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

Figure 5The Kaplan-Meier curve demonstrates increased survival of cct3A RNAi animals when treated with DTT under starved conditions. Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate p < 0.001 with log-rank (Mantel-Cox) test. Images are representative surviving animals for the different conditions at the displayed time point of regeneration. At the bottom are the number of planarians with the phenotype shown. The rest of planarians are dead at the displayed time point.


Figure 6Volcano plots displaying shRNA - Cct3 - and shRNA - luciferase - infected LSK cells cultured either in normal glucose ( upper graph ) or low glucose conditions ( lower graph ) . Dots represent all the NanoString analyzed transcripts . Red dots are UPR and ER stress related genes . The UPR and ER stress genes significantly down - regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph . For better visualization two dots with q ≤ 0 . 001 have been removed from the volcano plot ( see them in Dataset EV4 ) . Y - axis indicates the false discovery rate ( FDR ) ( q - value ) , and the X - axis indicates the log2 fold changes . The FDR - based method of p - value adjustment was conducted to calculate the q - values by nSolver software using Benjamini - Hochberg methods . Significance is established by q - value < 0 . 05 and indicated by the dotted horizontal line . n = 3 mice .

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

Figure 6Volcano plots displaying shRNA-Cct3- and shRNA-luciferase-infected LSK cells cultured either in normal glucose (upper graph) or low glucose conditions (lower graph). Dots represent all the NanoString analyzed transcripts. Red dots are UPR and ER stress related genes. The UPR and ER stress genes significantly down-regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph. For better visualization two dots with q ≤ 0.001 have been removed from the volcano plot (see them in Dataset EV4). Y-axis indicates the false discovery rate (FDR) (q- value), and the X-axis indicates the log2 fold changes. The FDR-based method of p-value adjustment was conducted to calculate the q-values by nSolver software using Benjamini-Hochberg methods. Significance is established by q-value < 0.05 and indicated by the dotted horizontal line. n= 3 mice.


Figure 7The Kaplan - Meier curve demonstrates increased survival of starved cct3A ( RNAi ) animals when treated with palmitic acid / palmitoylcarnitine ( PA / PC ) . Percentages indicate the number of survivals at the indicated time points . Three asterisks indicate P < 0 . 001 with log - rank ( Mantel - Cox ) test .

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Figure 7The Kaplan-Meier curve demonstrates increased survival of starved cct3A(RNAi) animals when treated with palmitic acid/palmitoylcarnitine (PA/PC). Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate P < 0.001 with log-rank (Mantel-Cox) test.


Figure 1Survival , gain of weight and mobility plots for Armadillidium vulgare during 75 days post - injection . There were no significant differences between animals injected with wVulC , wDil or control treatment for all these life history traits : Survival ( A ) , Gain of weight ( B ) and Mobility ( C ) .

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Figure 1Survival, gain of weight and mobility plots for Armadillidium vulgare during 75 days post-injection.There were no significant differences between animals injected with wVulC, wDil or control treatment for all these life history traits: Survival (A), Gain of weight (B) and Mobility (C).


Figure 2Animals were injected by pure suspension of wVulC , diluted wVulC inoculum ( only for females ) , wDil or control treatment and monitored during 75 days post - injection . ( A ) Gain of weight : Both Wolbachia strains negatively impact this parameter compared to the control treatment . However , wVulC ( i . e pure or 1 / 100 diluted inoculum ) induces a lower growth rate than the native strain wDil ( B ) Mobility : this parameter is dramatically reduced for animals injected by wVulC compared to those from other treatments . On the opposite , injection of wDil has no significant effect on the host ' s mobility . ( C ) Burying behaviour : The level 0 corresponds to the surface of the substrate , negative values correspond to the number of buried animals while positive values correspond to the number of animals found at the surface of the substrate . For control treatment animals , not a single individual rose up at the surface and for individuals injected by wDil only few animals were spotted at the surface . When inoculated by wVulC , all animals rose up to the substrate after 60 days . ( D ) Survival ( a ) data for females and ( b ) data for males : The injection of wVulC kills all the individuals . However , the males die quicker than the females . No extra - mortalities were reported for injection with wDil compared to control treatment .

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Figure 2Animals were injected by pure suspension of wVulC, diluted wVulC inoculum (only for females), wDil or control treatment and monitored during 75 days post-injection. (A) Gain of weight: Both Wolbachia strains negatively impact this parameter compared to the control treatment. However, wVulC (i.e pure or 1/100 diluted inoculum) induces a lower growth rate than the native strain wDil(B) Mobility: this parameter is dramatically reduced for animals injected by wVulC compared to those from other treatments. On the opposite, injection of wDil has no significant effect on the host's mobility.(C) Burying behaviour: The level 0 corresponds to the surface of the substrate, negative values correspond to the number of buried animals while positive values correspond to the number of animals found at the surface of the substrate. For control treatment animals, not a single individual rose up at the surface and for individuals injected by wDil only few animals were spotted at the surface. When inoculated by wVulC, all animals rose up to the substrate after 60 days.(D) Survival (a) data for females and (b) data for males: The injection of wVulC kills all the individuals. However, the males die quicker than the females. No extra-mortalities were reported for injection with wDil compared to control treatment.


Figure 3Wolbachia loads in CNS , gonads and hemocytes of Porcellio d . dilatatus and Armadillidium vulgare . ( A ) Comparison between ( i ) P . d . dilatatus in dark grey and A . vulgare in white injected with wVulC and ( ii ) naturally infected A . vulgare in light grey revealed no significant differences for the Wolbachia loads in CNS cells , gonads and hemocytes ( i . e immune cells ) , at both 30 and 60 days post - injection . ( B ) Comparison between ( i ) P . d . dilatatus in dark grey and A . vulgare in white injected with wDil and ( ii ) naturally infected P . d . dilatatus in dim grey revealed no significant differences for the Wolbachia loads in CNS cells at both 30 and 60 days PI . However , at 60 days post - injection , for gonads and hemocytes wDil loads were significantly higher in the native host P . d . dilatatus than in A . vulgare . A global comparison between wDil and wVulC loads showed that wVulC ( A ) always exhibited higher bacterial loads than wDil ( B ) in both host species .

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Figure 3Wolbachia loads in CNS, gonads and hemocytes of Porcellio d. dilatatus and Armadillidium vulgare.(A) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wVulC and (ii) naturally infected A. vulgare in light grey revealed no significant differences for the Wolbachia loads in CNS cells, gonads and hemocytes (i.e immune cells), at both 30 and 60 days post-injection. (B) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wDil and (ii) naturally infected P. d. dilatatus in dim grey revealed no significant differences for the Wolbachia loads in CNS cells at both 30 and 60 days PI. However, at 60 days post-injection, for gonads and hemocytes wDil loads were significantly higher in the native host P. d. dilatatus than in A. vulgare. A global comparison between wDil and wVulC loads showed that wVulC (A) always exhibited higher bacterial loads than wDil (B) in both host species.


Figure 4Micrograph of nerve cells of Porcellio d . dilatatus injected by wVulC . These cells are localised in the CNS ganglion . ( A ) At 30 days post - injection , some autolysosomes and bacteria are visible : autophagic process is already quite high compared to control [ N : Nucleus , V : Vacuole , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Wo : Wolbachia , * : a Wolbachia which seems to be surrounded by an autolysosome , Arrow head : magnified area ] . ( B ) At 60 days post - injection , the nerve cells are severely damaged by the accumulation of autophagic vesicles ( N : Nucleus , V : Vacuole , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Arrow head : magnified area and probably a Wolbachia in autophagic degradation ) .

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Figure 4Micrograph of nerve cells of Porcellio d. dilatatus injected by wVulC.These cells are localised in the CNS ganglion. (A) At 30 days post-injection, some autolysosomes and bacteria are visible: autophagic process is already quite high compared to control [N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, *: a Wolbachia which seems to be surrounded by an autolysosome, Arrow head: magnified area]. (B) At 60 days post-injection, the nerve cells are severely damaged by the accumulation of autophagic vesicles (N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Arrow head: magnified area and probably a Wolbachia in autophagic degradation).


Figure 5Micrograph and LC3B antibody labelling of adipocytes neighboring the nerve cells of Porcellio d . dilatatus . These adipocytes are localised around the nerve cells of the CNS and constitutes the sheathing of this organ . ( A ) At 30 days post - injection , autolysosomes , autophagosomes but also bacteria were observed : autophagic process was already higher compared to the control ( Ad : adipocyte , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified area ) . ( B ) At 60 days post - injection , the adipocytes were severely damaged by the accumulation of autophagic vesicles ( Ad : adipocyte , Ap : Autophagosomes , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified area ) . ( C ) With the antibody labelling anti - LC3B , the protein LC3B implicated in autophagic processes was detected in adipocytes of P . d . dilatatus injected with wVulC at 45 days post - injection . The highly labeled spherical structures would reflect the incorporation of the LC3B protein in the phagophores . ( D ) With adipocytes of P . d . dilatatus injected by wDil but also in other tested situations , only really few of these structures were detected by epiflorescent microscopy .

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Figure 5Micrograph and LC3B antibody labelling of adipocytes neighboring the nerve cells of Porcellio d. dilatatus.These adipocytes are localised around the nerve cells of the CNS and constitutes the sheathing of this organ. (A) At 30 days post-injection, autolysosomes, autophagosomes but also bacteria were observed: autophagic process was already higher compared to the control (Ad: adipocyte, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (B) At 60 days post-injection, the adipocytes were severely damaged by the accumulation of autophagic vesicles (Ad: adipocyte, Ap: Autophagosomes, Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (C) With the antibody labelling anti-LC3B, the protein LC3B implicated in autophagic processes was detected in adipocytes of P. d. dilatatus injected with wVulC at 45 days post-injection. The highly labeled spherical structures would reflect the incorporation of the LC3B protein in the phagophores. (D) With adipocytes of P. d. dilatatus injected by wDil but also in other tested situations, only really few of these structures were detected by epiflorescent microscopy.


Figure 1 ( A ) Confocal observation of mitochondrial potential after TMRM staining of LMP1 - negative or - positive NPC cells . Red fluorescence , which indicates normal mitochondrial potential , was converted into green fluorescence after a reduction in mitochondrial potential . Images were analyzed using Image J software ( scale bar , 10 μm ) . Data are presented as means ± S . E . M . ( paired t - test , n = 6 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) . ( C ) Extent of Ca2 + - mediated mitochondrial swelling in NPC cells . Data are presented as means ± S . E . M . ( paired t - test , n = 6 , biological replicates per group , * * p < 0 . 01 ) .

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Figure 1(A) Confocal observation of mitochondrial potential after TMRM staining of LMP1-negative or -positive NPC cells. Red fluorescence, which indicates normal mitochondrial potential, was converted into green fluorescence after a reduction in mitochondrial potential. Images were analyzed using Image J software (scale bar, 10 μm). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05, ** p < 0.01).(C) Extent of Ca2+-mediated mitochondrial swelling in NPC cells. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).


Figure 2 ( A ) LMP1 is localized to the inner mitochondrial membrane . Isolated mitochondria were incubated with proteinase K ( 40 μM ) for 30 min before LMP1 expression was detected ( left ) ; the mitochondria were further subjected to submitochondrial fractionation . VDAC1 , TIM23 , and HSP60 were used to represent MOM , MIM , and mitochondrial matrix protein , respectively ( right ) . ( B ) The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay . HK1 - LMP1 cells were immunostained with anti - LMP1 ( green ) and anti - ANT1 ( red ) antibodies and then subjected to confocal microscopy ， and use imageJ to calculate the co - location level of both . The nuclei were stained with DAPI ( scale bar , 5 μm ) . The quantified graph on the top right shows the percentage of yellow fluorescence ( merge ) to red fluorescence ( ANT1 ) in HK1 - LMP1 cells . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) . ( G ) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells . IP / IB was used to detect the interaction between Flag - tagged LMP1 mutants and Myc - tagged ANT1 . IP of cell lysates using a Flag or Myc antibody .

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Figure 2(A) LMP1 is localized to the inner mitochondrial membrane. Isolated mitochondria were incubated with proteinase K (40 μM) for 30 min before LMP1 expression was detected (left); the mitochondria were further subjected to submitochondrial fractionation. VDAC1, TIM23, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively (right).(B) The interaction of LMP1 and endogenous ANT1 was analyzed by an immunofluorescence confocal assay. HK1-LMP1 cells were immunostained with anti-LMP1 (green) and anti-ANT1 (red) antibodies and then subjected to confocal microscopy，and use imageJ to calculate the co-location level of both. The nuclei were stained with DAPI (scale bar, 5 μm). The quantified graph on the top right shows the percentage of yellow fluorescence (merge) to red fluorescence (ANT1) in HK1-LMP1 cells. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).(G) LMP1 binds directly to ANT1 dependent on its transmembrane domain in 293T cells. IP/IB was used to detect the interaction between Flag-tagged LMP1 mutants and Myc-tagged ANT1. IP of cell lysates using a Flag or Myc antibody.


Figure 3 ( A Effect of inhibitors BKA and CATR on ANT1 - VDAC1 binding in 293T cells . B ) Effect of inhibitors BKA and CATR on ANT1 - VDAC1 binding in 293T cells . Data are presented as means ± S . E . M . ( paired t - test , n = 3 , biological replicates per group , * * p < 0 . 01 ) . ( C The effect of different amounts of EBV - LMP1 on the binding of ANT1 - VDAC1 was detected by CO - IP . D ) The effect of different amounts of EBV - LMP1 on the binding of ANT1 - VDAC1 was detected by CO - IP . Data are presented as means ± S . E . M . ( paired t - test , n = 3 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) . ( G ) 19F - NMR spectra of ANT1 - Y195 - F2Y and ANT1 - Y290 - F2Y after titration with different amounts of LMP1 . After incubation at room temperature for 30 min , the nuclei were continuously monitored at 500 MZ for 30 min . ( H ) 19F - NMR spectra of ANT1 - Y195 - F2Y and ANT1 - Y290 - F2Y after titration with different amounts of LMP1 - ΔT . After incubation at room temperature for 30 min , the nuclei were continuously monitored at 500 MZ for 30 min .

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Figure 3(A Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells.B) Effect of inhibitors BKA and CATR on ANT1-VDAC1 binding in 293T cells. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, ** p < 0.01).(C The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP.D) The effect of different amounts of EBV-LMP1 on the binding of ANT1-VDAC1 was detected by CO-IP. Data are presented as means ± S.E.M. (paired t-test, n = 3, biological replicates per group, * p < 0.05, ** p < 0.01).(G) 19F-NMR spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min. (H) 19F-NMR spectra of ANT1-Y195-F2Y and ANT1-Y290-F2Y after titration with different amounts of LMP1-ΔT. After incubation at room temperature for 30 min, the nuclei were continuously monitored at 500 MZ for 30 min.


Figure 4 ( A - B ) The ADP / ATP exchange rate in EBV - LMP1 - positive nasopharyngeal carcinoma cells is decreased . The fluorescence value of Magnesium Green ™ ( 1 μM ) was measured at 505 nm ( Ex ) / 535 nm ( Em ) by enzymography ( A ) , and the ADP / ATP exchange rate was calculated after conversion to ATP ( B ) . Data are presented as means ± S . E . M . ( paired t - test , n = 5 , biological replicates per group , * * p < 0 . 01 ) . ( C ) LMP1 - negative or - positive NPC cells were treated with BKA ( 1 μM ) or CATR ( 5 μM ) for 24 h , Mitochondrial OCR assessed with the XF24 extracellular flux analyzer ( Seahorse ) under sequential treatment with oligomycin , FCCP , and antimycin A . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) . ( D ) Oxidative phosphorylation - related metrics based on OCR datas . Data are presented as means ± S . E . M . ( paired t - test , n = 6 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) .

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Figure 4(A-B) The ADP/ATP exchange rate in EBV-LMP1-positive nasopharyngeal carcinoma cells is decreased. The fluorescence value of Magnesium Green ™ (1 μM) was measured at 505 nm (Ex)/535 nm (Em) by enzymography (A), and the ADP/ATP exchange rate was calculated after conversion to ATP (B). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, ** p < 0.01).(C) LMP1-negative or -positive NPC cells were treated with BKA (1 μM) or CATR (5 μM) for 24 h, Mitochondrial OCR assessed with the XF24 extracellular flux analyzer (Seahorse) under sequential treatment with oligomycin, FCCP, and antimycin A. Data are presented as means ± S.E.M. (n = 6, biological replicates per group). (D) Oxidative phosphorylation-related metrics based on OCR datas. Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, * p < 0.05,** p < 0.01).


Figure 5 ( A ) HK1 - LMP1 were treated with increasing concentrations of cisplatin for 24 h . Cell viability was determined by MTS assay . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) . ( D ) The IC50 value of the combination of CATR and cisplatin in CNE1 - LMP1 cells was changed . Data are presented as means ± S . E . M . ( n = 6 , biological replicates per group ) . ( G ) Flow cytometry analysis of death of NPC cells treated for 24 h with CATR ( 1 μM ) alone , cisplatin ( 20 μM ) alone , or cisplatin ( 20 μM ) combined with CATR ( 1 μM ) . Data are presented as means ± S . E . M . ( paired t - test , n = 5 , biological replicates per group , * p < 0 . 05 , * * p < 0 . 01 ) . ( H ) Trypan blue staining was used to detect death of NPC cells treated for 24 h with vehicle , cisplatin , CATR , or a combination of cisplatin and CATR ( 1 μM ) . Data are presented as means ± S . E . M . ( paired t - test , n = 6 , biological replicates per group , * * p < 0 . 01 ) .

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

Figure 5(A) HK1-LMP1 were treated with increasing concentrations of cisplatin for 24 h. Cell viability was determined by MTS assay. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).(D) The IC50 value of the combination of CATR and cisplatin in CNE1-LMP1 cells was changed. Data are presented as means ± S.E.M. (n = 6, biological replicates per group).(G) Flow cytometry analysis of death of NPC cells treated for 24 h with CATR (1 μM) alone, cisplatin (20 μM) alone, or cisplatin (20 μM) combined with CATR (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 5, biological replicates per group, * p < 0.05, ** p < 0.01).(H) Trypan blue staining was used to detect death of NPC cells treated for 24 h with vehicle, cisplatin, CATR, or a combination of cisplatin and CATR (1 μM). Data are presented as means ± S.E.M. (paired t-test, n = 6, biological replicates per group, ** p < 0.01).