Figure 2A Effects of γb palmitoylation on BSMV replication . Movement - deficient BSMV ( RNAα + RNAγ / RNAγ3CS ) was agroinfiltrated into N . benthamiana leaves . At 3 dpi , total protein and RNA were extracted for Western and Northern blot analyses . Antibodies and probes used for molecular analyses are indicated on the right of each panel . Actin was used to monitor equal protein loading , and methylene blue - stained rRNAs served as RNA loading controls . Data information : representative data are shown and three biological replicates had similar results . B Western blot analyses of chloroplast localization of γb and γb3CS . Transiently expressed αa - GFP in intact chloroplasts isolated from BSMVγb - 3xFlag - or BSMV3CS - 3xFlag - infected N . benthamiana leaves at 4 dpi . Detection of γb - 3xFlag and γb3CS - 3xFlag with an anti - Flag antibody ; TGB1 analysis with an anti - TGB1 antibody ; αa - GFP detection with an anti - GFP antibody . NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase ( PEPC ) was used to assess cytosolic contamination of isolated chloroplasts . Data information : , representative data are shown and three biological replicates had similar results . C Subcellular localization of γb - GFP and γb3CS - GFP in N . benthamiana epidermal leaf cells . The first and second rows show subcellular localization of γb - GFP and γb3CS - GFP transient expression in leaves . The third and fourth rows show localization of γb - GFP and γb3CS - GFP in BSMVγb - GFP - and BSMV3CS - GFP - infected leaves at 3 dpi . Figures on the right indicate the normalized fluorescence intensities of the GFP ( green ) or the chloroplast autofluorescence ( red ) channels along the dashed white lines shown in the merged images of the left panel . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results . D Subcellular localization of γb - GFP or γb3CS - GFP in isolated N . benthamiana protoplasts harvested at 3 dpi . The fifth and sixth columns show γb localization in BSMVγb - GFP - ( movement - deficient ) N . benthamiana protoplasts after treatment with 200 μM 2 - BP inhibitor . The released protoplasts were collected after 48 h incubation in DMSO or 2 - BP followed by confocal microscopy visualization . Figures at the bottom indicate the normalized fluorescence intensities of the GFP ( green ) and the chloroplast autofluorescence ( red ) channels along the dashed white lines in the merged images above . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results .

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

Figure 2A Effects of γb palmitoylation on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ/RNAγ3CS) was agroinfiltrated into N. benthamiana leaves. At 3 dpi, total protein and RNA were extracted for Western and Northern blot analyses. Antibodies and probes used for molecular analyses are indicated on the right of each panel. Actin was used to monitor equal protein loading, and methylene blue-stained rRNAs served as RNA loading controls. Data information: representative data are shown and three biological replicates had similar results.B Western blot analyses of chloroplast localization of γb and γb3CS. Transiently expressed αa-GFP in intact chloroplasts isolated from BSMVγb-3xFlag- or BSMV3CS-3xFlag-infected N. benthamiana leaves at 4 dpi. Detection of γb-3xFlag and γb3CS-3xFlag with an anti-Flag antibody; TGB1 analysis with an anti-TGB1 antibody; αa-GFP detection with an anti-GFP antibody. NTRC was a loading control for intact chloroplasts Phosphoenolpyruvate carboxylase (PEPC) was used to assess cytosolic contamination of isolated chloroplasts. Data information: , representative data are shown and three biological replicates had similar results.C Subcellular localization of γb-GFP and γb3CS-GFP in N. benthamiana epidermal leaf cells. The first and second rows show subcellular localization of γb-GFP and γb3CS-GFP transient expression in leaves. The third and fourth rows show localization of γb-GFP and γb3CS-GFP in BSMVγb-GFP- and BSMV3CS-GFP-infected leaves at 3 dpi. Figures on the right indicate the normalized fluorescence intensities of the GFP (green) or the chloroplast autofluorescence (red) channels along the dashed white lines shown in the merged images of the left panel. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.D Subcellular localization of γb-GFP or γb3CS-GFP in isolated N. benthamiana protoplasts harvested at 3 dpi. The fifth and sixth columns show γb localization in BSMVγb-GFP- (movement-deficient) N. benthamiana protoplasts after treatment with 200 μM 2-BP inhibitor. The released protoplasts were collected after 48 h incubation in DMSO or 2-BP followed by confocal microscopy visualization. Figures at the bottom indicate the normalized fluorescence intensities of the GFP (green) and the chloroplast autofluorescence (red) channels along the dashed white lines in the merged images above. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.


Figure 3A Effects of γb palmitoylation on virus cell - to - cell movement at 3 dpi with the BSMV duplex fluorescence ( dfBSMV ) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci , whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci . Fluorescence was evaluated by confocal microscopy . Scale bars , 100 μm . B Quantification of virus movement in Panel A . To determine the movement capacity of the viruses depicted in each panel , the mCherry and GFP fluorescent intensities were first determined by ImageJ software . Then , the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci ( n = 10 ) . The Y - axis indicates the relative sizes of the green areas in comparison to that of the red - colored areas . Data information : In ( A , representative data are shown and three biological replicates had similar results . In ( B ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) . D Biotin - switch assay to access palmitoylation of γb in BSMV - infected N . benthamiana leaves at different timepoints . BSMVγb - 3xFlag was treated with the hydroxylamine ( Hyd + ) thioester cleavage reagent . Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti - Flag antibody . Data information : In representative data are shown and three biological replicates had similar results . E Subcellular localization of BSMVγb - GFP and BSMV3CS - GFP in N . benthamiana leaf epidermal cells . The mCherry - HDEL and mTalin - RFP reporters were used as markers to indicate the ER and actin localization Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above . Scale bars , 10 μm . Data information : In E ) , representative data are shown and three biological replicates had similar results . F , G Western blot analysis of subcellular fractions extracted from N . benthamiana leaf tissues at 3 dpi . S3 indicates the supernatant separated by centrifugation at 3 , 000 g ; and S30 is the 30 , 000 g supernatant ; P30 is the 30 , 000 g pellet . The association of γb with membranes was determined by 1 % Triton X - 100 extraction .

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

Figure 3A Effects of γb palmitoylation on virus cell-to-cell movement at 3 dpi with the BSMV duplex fluorescence (dfBSMV) reporter system The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci, whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci. Fluorescence was evaluated by confocal microscopy. Scale bars, 100 μm. B Quantification of virus movement in Panel A. To determine the movement capacity of the viruses depicted in each panel, the mCherry and GFP fluorescent intensities were first determined by ImageJ software. Then, the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci (n = 10). The Y-axis indicates the relative sizes of the green areas in comparison to that of the red-colored areas. Data information: In (A, representative data are shown and three biological replicates had similar results. In (B), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).D Biotin-switch assay to access palmitoylation of γb in BSMV-infected N. benthamiana leaves at different timepoints. BSMVγb-3xFlag was treated with the hydroxylamine (Hyd+) thioester cleavage reagent. Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti-Flag antibody. Data information: In representative data are shown and three biological replicates had similar results.E Subcellular localization of BSMVγb-GFP and BSMV3CS-GFP in N. benthamiana leaf epidermal cells. The mCherry-HDEL and mTalin-RFP reporters were used as markers to indicate the ER and actin localization Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above. Scale bars, 10 μm. Data information: In E), representative data are shown and three biological replicates had similar results.F, G Western blot analysis of subcellular fractions extracted from N. benthamiana leaf tissues at 3 dpi. S3 indicates the supernatant separated by centrifugation at 3,000 g; and S30 is the 30,000 g supernatant; P30 is the 30,000 g pellet. The association of γb with membranes was determined by 1% Triton X-100 extraction.


Figure 4A BiFC co - localization analyses of αa and TGB1 in N . benthamiana leaf cells at 3 dpi . YFPn and YFPc was fused to the TGB1 and the αa proteins , respectively . Combinations of γb , γb3CS and the TBSV P19 control are shown above the micrographs . YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false - green color . Scale bars , 30 μm . B Quantification of the numbers of YFP foci per visual field ( n = 10 ) . Data information : In ( A , , representative data are shown and three biological replicates had similar results . In ( B , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) . C Co - IP assays to determine in vivo interactions between TGB1 and γb or γb3CS . N . benthamiana leaf tissues co - agroinfiltrated with RNAα , RNAγγb - GFP or RNAγ3CS - GFP , and TGB1 - 3xFlag plasmids were harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Input and immunoprecipitated proteins ( IP ) were analyzed by Western blotting with anti - GFP or anti - Flag antibodies . D Confocal microscopy analyses showing RFP - TGB1 localization at the chloroplast periphery in N . benthamiana leaves agroinfiltrated with αa - GFP and CFP - γb plasmids . CFP - γb or CFP - γb3CS were co - expressed with RFP - TGB1 and αa - GFP in N . benthamiana leaves . Chloroplast autofluorescence was displayed in false pink color . The third and fourth rows show leaves treated with 400 μM 2 - BP inhibitor or the DMSO control at 1 dpi . Two days later , confocal microscopy observations of RFP - TGB1 , αa - GFP , and CFP - γb were performed . Figures on the right indicate the normalized fluorescence intensity of GFP , RFP , and CFP channels along the white dashed line in the merged confocal images . Scale bars , 10 μm . Data information : In , representative data are shown and three biological replicates had similar results . E Statistical analyses of the GFP and RFP co - localization in panel D . Pearson correlation coefficient ( Pearson ' s r ) of GFP and RFP at the periphery of the chloroplasts was measured with the ImageJ PSC colocalization tool ( n = 8 ) . F Relative percentages of RFP - TGB1 foci that co - localized with αa - GFP and CFP - γb at the periphery of the chloroplast images shown in panel D ( n = 8 ) . Data information : In E and F ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) . G Chloroplast extraction experiment for detection of TGB1 chloroplast localization . Plasmids harboring αa - GFP and RFP - TGB1 were co - infiltrated into N . benthamiana along with CFP - γb or CFP - γb3CS . Intact chloroplast preparations isolated from infiltrated N . benthamiana leaves at 4 dpi were subjected to Western blot analyses . RFP - TGB1 was analyzed with an anti - TGB1 antibody . The αa - GFP , CFP - γb , and CFP - γb3CS proteins were detected with an anti - GFP antibody . NTRC was the loading control for the chloroplast preparations Phosphoenolpyruvate carboxylase ( PEPC ) served as a control to assess cytosolic contamination of isolated chloroplasts . Data information : In G ) , representative data are shown and three biological replicates had similar results .

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 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 O O O O O O O O O O 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

Figure 4A BiFC co-localization analyses of αa and TGB1 in N. benthamiana leaf cells at 3 dpi. YFPn and YFPc was fused to the TGB1 and the αa proteins, respectively. Combinations of γb, γb3CS and the TBSV P19 control are shown above the micrographs. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color. Scale bars, 30 μm. B Quantification of the numbers of YFP foci per visual field (n = 10). Data information: In (A, , representative data are shown and three biological replicates had similar results. In (B, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).C Co-IP assays to determine in vivo interactions between TGB1 and γb or γb3CS. N. benthamiana leaf tissues co-agroinfiltrated with RNAα, RNAγγb-GFP or RNAγ3CS-GFP, and TGB1-3xFlag plasmids were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blotting with anti-GFP or anti-Flag antibodies.D Confocal microscopy analyses showing RFP-TGB1 localization at the chloroplast periphery in N. benthamiana leaves agroinfiltrated with αa-GFP and CFP-γb plasmids. CFP-γb or CFP-γb3CS were co-expressed with RFP-TGB1 and αa-GFP in N. benthamiana leaves. Chloroplast autofluorescence was displayed in false pink color. The third and fourth rows show leaves treated with 400 μM 2-BP inhibitor or the DMSO control at 1 dpi. Two days later, confocal microscopy observations of RFP-TGB1, αa-GFP, and CFP-γb were performed. Figures on the right indicate the normalized fluorescence intensity of GFP, RFP, and CFP channels along the white dashed line in the merged confocal images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.E Statistical analyses of the GFP and RFP co-localization in panel D. Pearson correlation coefficient (Pearson's r) of GFP and RFP at the periphery of the chloroplasts was measured with the ImageJ PSC colocalization tool (n = 8). F Relative percentages of RFP-TGB1 foci that co-localized with αa-GFP and CFP-γb at the periphery of the chloroplast images shown in panel D (n = 8). Data information: In E and F), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).G Chloroplast extraction experiment for detection of TGB1 chloroplast localization. Plasmids harboring αa-GFP and RFP-TGB1 were co-infiltrated into N. benthamiana along with CFP-γb or CFP-γb3CS. Intact chloroplast preparations isolated from infiltrated N. benthamiana leaves at 4 dpi were subjected to Western blot analyses. RFP-TGB1 was analyzed with an anti-TGB1 antibody. The αa-GFP, CFP-γb, and CFP-γb3CS proteins were detected with an anti-GFP antibody. NTRC was the loading control for the chloroplast preparations Phosphoenolpyruvate carboxylase (PEPC) served as a control to assess cytosolic contamination of isolated chloroplasts. Data information: In G), representative data are shown and three biological replicates had similar results.


Figure 5B Analysis of γb palmitoylation in NbPAT15 - or NbPAT21 - silenced N . benthamiana plants . NbPAT15 and NbPAT21 were silenced by intron - spliced hairpin RNA - mediated RNAi approaches The γb - 3xFlag plasmid was agroinfiltrated into the leaves , followed by biotin - switch assays at 3 dpi . Data information : In ( B representative data are shown and three biological replicates had similar results . C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell - to - cell movement . Infiltrated leaves were observed by confocal microscopy at 3 dpi . Red fluorescence produced from the mCherry expression cassette shows the primary infection foci , and GFP fluorescence outside the red regions shows virus spread into secondary infected cells . Scale bars , 100 μm . Data information : In C ) , representative data are shown and three biological replicates had similar results . D Quantification of BSMV movement . The green and red fluorescent areas were measured by ImageJ software ( n = 10 ) . Data information : In ( D ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( 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 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 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 5B Analysis of γb palmitoylation in NbPAT15- or NbPAT21-silenced N. benthamiana plants. NbPAT15 and NbPAT21 were silenced by intron-spliced hairpin RNA-mediated RNAi approaches The γb-3xFlag plasmid was agroinfiltrated into the leaves, followed by biotin-switch assays at 3 dpi. Data information: In (B representative data are shown and three biological replicates had similar results.C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell-to-cell movement. Infiltrated leaves were observed by confocal microscopy at 3 dpi. Red fluorescence produced from the mCherry expression cassette shows the primary infection foci, and GFP fluorescence outside the red regions shows virus spread into secondary infected cells. Scale bars, 100 μm. Data information: In C), representative data are shown and three biological replicates had similar results.D Quantification of BSMV movement. The green and red fluorescent areas were measured by ImageJ software (n = 10). Data information: In (D), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


Figure 6A , B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N . benthamiana at 3 dpi . PAT15 - YFPn and PAT21 - YFPn were co - expressed with BSMVγb - YFPc . Chloroplast autofluorescence is depicted as a false red color and mCherry - HDEL provides the ER marker . Figures on the right show the normalized fluorescence intensities of GFP , mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images . Scale bars , 20 μm . Data information : In ( A , B , representative data are shown and three biological replicates had similar results . C Co - localization analyses of αa - GFP , RFP - TGB1 , and CFP - γb in N . benthamiana leaves at 3 dpi . Mixtures of A . tumefaciens containing αa - GFP , RFP - TGB1 , and CFP - γb were infiltrated into NbPATs - silenced N . benthamiana leaves or the non - silenced TRV : GUS control . Chloroplast autofluorescence is displayed as a false pink color . Figures on the right show the normalized fluorescence intensities of GFP , RFP , CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images . Scale bars , 10 μm . Data information : In , representative data are shown and three biological replicates had similar results . D Co - IP assays of interactions between γb and TGB1 in NbPAT15 - and NbPAT21 - silenced N . benthamiana leaves , TRV : GUS and TRV : GUS combined with the 2 - BP inhibitor provided positive and negative controls , respectively . The silenced leaves were inoculated with BSMV3xFlag - TGB1 / γb - GFP mixtures ( RNAα + RNAβ3xFlag - TGB1 + RNAγγb - GFP ) and harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Input and IP proteins were analyzed by Western blotting with anti - GFP or anti - Flag antibodies . E Average amounts of RFP - TGB1 foci co - localizing with αa - GFP and CFP - γb at the chloroplast peripheries shown in panel C ( n = 12 ) . Data information : In E ) , representative data are shown and three biological replicates had similar results . In ( E ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( 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 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 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

Figure 6A, B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N. benthamiana at 3 dpi. PAT15-YFPn and PAT21-YFPn were co-expressed with BSMVγb-YFPc. Chloroplast autofluorescence is depicted as a false red color and mCherry-HDEL provides the ER marker. Figures on the right show the normalized fluorescence intensities of GFP, mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 20 μm. Data information: In (A, B, representative data are shown and three biological replicates had similar results.C Co-localization analyses of αa-GFP, RFP-TGB1, and CFP-γb in N. benthamiana leaves at 3 dpi. Mixtures of A. tumefaciens containing αa-GFP, RFP-TGB1, and CFP-γb were infiltrated into NbPATs-silenced N. benthamiana leaves or the non-silenced TRV:GUS control. Chloroplast autofluorescence is displayed as a false pink color. Figures on the right show the normalized fluorescence intensities of GFP, RFP, CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In , representative data are shown and three biological replicates had similar results.D Co-IP assays of interactions between γb and TGB1 in NbPAT15- and NbPAT21-silenced N. benthamiana leaves, TRV:GUS and TRV:GUS combined with the 2-BP inhibitor provided positive and negative controls, respectively. The silenced leaves were inoculated with BSMV3xFlag-TGB1/γb-GFP mixtures (RNAα + RNAβ3xFlag-TGB1 + RNAγγb-GFP) and harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and IP proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies. E Average amounts of RFP-TGB1 foci co-localizing with αa-GFP and CFP-γb at the chloroplast peripheries shown in panel C (n = 12). Data information: In E), representative data are shown and three biological replicates had similar results. In (E), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


Figure 7B Fractionation assay to detect γb membrane - associations . Separation of total protein ( T ) extracted from BSMV infected leaves into soluble ( S ) and membrane ( M ) fractions by ultracentrifugation at 100 , 000 g . PEPC and BiP , respectively , provide soluble and membrane fraction markers . C Aqueous two - phase partitioning of γb PM localization . The microsomal pellet from BSMVγb - GFP - infected leaves was obtained by ultracentrifugation at 100 , 000 g , and the two - phase fractionation procedure was used to separate the plasma membrane ( upper phase ) from other cellular membranes ( lower phase ) . H + - ATPase is a PM marker . D PM associations of γb - GFP and γb3CS - GFP in N . benthamiana protoplasts . The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb - GFP and γb3CS - GFP . The third and fourth columns show γb localization in BSMVγb - GFP and BSMV3CS - GFP - infected leaves . PIP2A provided a PM marker ( Nelson et al . , 2007 ) . Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images . Scale bars , 10 μm . Data information : In D , , representative data are shown and three biological replicates had similar results . E Western blot analysis of subcellular fractions of BSMV infected N . benthamiana leaves at 3 dpi . The S3 and S30 fractions are the 3000 and 30000 g supernatants , and P30 is the 30000 g pellet . Membrane associated γb was released by 1 % Triton X - 100 treatment . F Western blot detection of BSMV accumulation in N . benthamiana plants . A . tumefaciens mixtures harboring RNAα and RNAβmTGB2 , along with RNAγ , RNAγγb - ROP , or RNAγγb - mROP , were agroinfiltrated into N . benthamiana leaves . Western blotting to detect virus accumulation at 3 dpi used anti - TGB1 or anti - CP antibodies . Actin provided loading controls . G Analysis of the effects of enhanced PM association of γb on cell - to - cell virus movement with the BSMV duplex fluorescence ( dfBSMV ) reporter system . Mixtures of A . tumefaciens containing dfBSMV , dfBSMVγb - GFP - ROP , and dfBSMVγb - GFP - mROP were infiltrated into N . benthamiana leaves . Infiltrated leaves were observed by confocal microscope at 3 dpi . Scale bars , 100 μm . ( n = 6 ) . Data information : In G ) , representative data are shown and three biological replicates had similar results . In ( G ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 7B Fractionation assay to detect γb membrane-associations. Separation of total protein (T) extracted from BSMV infected leaves into soluble (S) and membrane (M) fractions by ultracentrifugation at 100,000 g. PEPC and BiP, respectively, provide soluble and membrane fraction markers.C Aqueous two-phase partitioning of γb PM localization. The microsomal pellet from BSMVγb-GFP-infected leaves was obtained by ultracentrifugation at 100,000 g, and the two-phase fractionation procedure was used to separate the plasma membrane (upper phase) from other cellular membranes (lower phase). H+-ATPase is a PM marker.D PM associations of γb-GFP and γb3CS-GFP in N. benthamiana protoplasts. The first and second columns show localization of γb at 3 dpi after infiltrations for transient expression of γb-GFP and γb3CS-GFP. The third and fourth columns show γb localization in BSMVγb-GFP and BSMV3CS-GFP-infected leaves. PIP2A provided a PM marker (Nelson et al., 2007). Figures below the confocal images show the normalized fluorescence intensities of the GFP and mCherry proteins along the white dashed lines in the merged images. Scale bars, 10 μm. Data information: In D, , representative data are shown and three biological replicates had similar results.E Western blot analysis of subcellular fractions of BSMV infected N. benthamiana leaves at 3 dpi. The S3 and S30 fractions are the 3000 and 30000 g supernatants, and P30 is the 30000 g pellet. Membrane associated γb was released by 1% Triton X-100 treatment.F Western blot detection of BSMV accumulation in N. benthamiana plants. A. tumefaciens mixtures harboring RNAα and RNAβmTGB2, along with RNAγ, RNAγγb-ROP, or RNAγγb-mROP, were agroinfiltrated into N. benthamiana leaves. Western blotting to detect virus accumulation at 3 dpi used anti-TGB1 or anti-CP antibodies. Actin provided loading controls.G Analysis of the effects of enhanced PM association of γb on cell-to-cell virus movement with the BSMV duplex fluorescence (dfBSMV) reporter system. Mixtures of A. tumefaciens containing dfBSMV, dfBSMVγb-GFP-ROP, and dfBSMVγb-GFP-mROP were infiltrated into N. benthamiana leaves. Infiltrated leaves were observed by confocal microscope at 3 dpi. Scale bars, 100 μm. (n = 6). Data information: In G), representative data are shown and three biological replicates had similar results. In (G), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


Figure 8B Co - IP analyses to analyze interactions of NbREM1 with γb or γb3CS in N . benthamiana leaf tissues infiltrated with A . tumefaciens mixtures containing movement - deficient BSMV plasmids ( RNAα + RNAγγb - GFP or RNAγ3CS - GFP ) and NbREM1 - 3xFlag . Total proteins were extracted at 3 dpi and immunoprecipitated with anti - Flag beads . Input and immunoprecipitated proteins ( IP ) were analyzed by Western blot analyses with an anti - GFP or anti - Flag antibodies . Data information : In B , , representative data are shown and three biological replicates had similar results . D Analysis of the cleaved 5 ( 6 ) - carboxyfluorescein ( CF ) movement in N . benthamiana leaves . RNAα + RNAγ / RNAγ3CS were co - infiltrated into transiently overexpressed NbREM1 N . benthamiana leaves , and 1 μL CFDA was loaded onto the adaxial leaf blade surface and incubated for 5 min followed by confocal analysis at 3 dpi . The areas of dye diffusion are marked by dashed white circles . Scale bars , 100 μm . Data information : In D , representative data are shown and three biological replicates had similar results . E Quantification of CF movement . Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software ( n = 6 ) . Data information : In ( E , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) . F Aniline blue staining of callose in N . benthamiana leaf tissues . Leaves were infiltrated with mixtures of different A . tumefaciens constructs indicated beside the panels . At 3 dpi , the 0 . 1 % aniline blue fluorochrome was infiltrated into N . benthamiana leaves . After incubation at 5 min in the dark , the leaves were observed by confocal microscopy at 405 nm . Scale bars , 20 μm . Data information : representative data are shown and three biological replicates had similar results . G Quantification of callose staining intensity as shown in panel F . The relative callose staining intensity was set to 1 in the REM samples ( n = 10 ) . Data information : In , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( p < 0 . 05 ) . H Aniline blue staining of callose in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants . Scale bars , 20 μm . Data information : In H ) , representative data are shown and three biological replicates had similar results . I Quantification of callose staining intensity as shown in panel H . The relative callose staining intensity was set as 1 in the samples of EV ( n = 8 ) . Data information : In I ) , the box boundaries indicate the upper ( 25th percentile ) and lower ( 75th percentile ) quartiles , whiskers indicate minimum and maximum values , central band indicates the median . Letters in the chart denote statistically significant differences among groups according to the Duncan ' s multiple range test ( 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 8B Co-IP analyses to analyze interactions of NbREM1 with γb or γb3CS in N. benthamiana leaf tissues infiltrated with A. tumefaciens mixtures containing movement-deficient BSMV plasmids (RNAα + RNAγγb-GFP or RNAγ3CS-GFP) and NbREM1-3xFlag. Total proteins were extracted at 3 dpi and immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blot analyses with an anti-GFP or anti-Flag antibodies. Data information: In B, , representative data are shown and three biological replicates had similar results.D Analysis of the cleaved 5(6)-carboxyfluorescein (CF) movement in N. benthamiana leaves. RNAα + RNAγ/RNAγ3CS were co-infiltrated into transiently overexpressed NbREM1 N. benthamiana leaves, and 1 μL CFDA was loaded onto the adaxial leaf blade surface and incubated for 5 min followed by confocal analysis at 3 dpi. The areas of dye diffusion are marked by dashed white circles. Scale bars, 100 μm. Data information: In D, representative data are shown and three biological replicates had similar results.E Quantification of CF movement. Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software (n = 6). Data information: In (E, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).F Aniline blue staining of callose in N. benthamiana leaf tissues. Leaves were infiltrated with mixtures of different A. tumefaciens constructs indicated beside the panels. At 3 dpi, the 0.1% aniline blue fluorochrome was infiltrated into N. benthamiana leaves. After incubation at 5 min in the dark, the leaves were observed by confocal microscopy at 405 nm. Scale bars, 20 μm. Data information: representative data are shown and three biological replicates had similar results.G Quantification of callose staining intensity as shown in panel F. The relative callose staining intensity was set to 1 in the REM samples (n = 10). Data information: In , the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).H Aniline blue staining of callose in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants. Scale bars, 20 μm. Data information: In H), representative data are shown and three biological replicates had similar results.I Quantification of callose staining intensity as shown in panel H. The relative callose staining intensity was set as 1 in the samples of EV (n = 8). Data information: In I), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).


Figure 1 ( B ) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT - PCR . Ribosomal protein 49 ( rp49 ) was used as an internal control ; n = 3 . Bars indicate mean ± s . d . ( C ) Images of the larvae of denoted genotypes at 3 days ( top ) and 6 days ( middle ) after egg ‐ laying ( AEL ) , and quantification of the larvae that developed into the mid - late third instar stage of each genotype ( bottom ) ; n = 3 . Bars indicate mean ± s . d . Fifteen larvae of each genotype were examined in each experiment . ( D ) Suppression of lipid vesicle aggregation in the fat body of dTOR mutants by reduced gene dosage of DmATG1 . The fat body images of the second / early third instar larvae of denoted genotypes under fully fed conditions . ( E ) Images of the larval salivary glands of denoted genotypes . Hoechst33342 ( pseudo - coloured green ) and phalloidin - TRITC ( red ) were used to visualize the nuclei and cell boundary of larval salivary gland cells , respectively . ( F ) Quantitative analysis of cell and nuclear sizes in Fig 1E ; n = 5 . Bars indicate mean ± s . d . DmATG1 , Drosophila Autophagy ‐ specific gene 1 ; dTOR , Drosophila TOR ; qRT - PCR , quantitative real ‐ time reverse transcriptase - PCR .

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

Figure 1(B) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT-PCR. Ribosomal protein 49 (rp49) was used as an internal control; n=3. Bars indicate mean±s.d.(C) Images of the larvae of denoted genotypes at 3 days (top) and 6 days (middle) after egg‐laying (AEL), and quantification of the larvae that developed into the mid-late third instar stage of each genotype (bottom); n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment.(D) Suppression of lipid vesicle aggregation in the fat body of dTOR mutants by reduced gene dosage of DmATG1. The fat body images of the second/early third instar larvae of denoted genotypes under fully fed conditions.(E) Images of the larval salivary glands of denoted genotypes. Hoechst33342 (pseudo-coloured green) and phalloidin-TRITC (red) were used to visualize the nuclei and cell boundary of larval salivary gland cells, respectively.(F) Quantitative analysis of cell and nuclear sizes in Fig 1E; n=5. Bars indicate mean±s.d. DmATG1, Drosophila Autophagy‐specific gene 1; dTOR, Drosophila TOR; qRT-PCR, quantitative real‐time reverse transcriptase-PCR.


Figure 2 ( A ) Quantification of adults that survived after eclosion for denoted genotypes ; n = 3 . Bars indicate mean ± s . d . Fifteen larvae of each genotype were examined in each experiment . ( B , C ) Activation of dS6K in DmATG1 mutants . Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes . Tubulinimmunoblot was used as a loading control . Rheb expressing flies ( hs > Rheb ) were used as a positive control ( C ) . hs > Rheb larvae were subjected to heat shock at 37 ° C for 1 h and then incubated at 30 ° C for 3 h before sample preparation to induce Rheb expression . For quantification , the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop , and normalized to the tubulin levels . Results are expressed as a fold change compared with the wild ‐ type controls ( w1118 ) . DmATG1 , Drosophila Autophagy ‐ specific gene 1 ; dS6k , Drosophila S6 kinase .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of adults that survived after eclosion for denoted genotypes; n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment.(B,C) Activation of dS6K in DmATG1 mutants. Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes. Tubulinimmunoblot was used as a loading control. Rheb expressing flies (hs>Rheb) were used as a positive control (C). hs>Rheb larvae were subjected to heat shock at 37°C for 1 h and then incubated at 30°C for 3 h before sample preparation to induce Rheb expression. For quantification, the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop, and normalized to the tubulin levels. Results are expressed as a fold change compared with the wild‐type controls (w1118). DmATG1, Drosophila Autophagy‐specific gene 1; dS6k, Drosophila S6 kinase.


Figure 3 ( A , B ) HEK 293T cells transfected with denoted plasmids were ( A ) deprived of nutrients for 90 min , and then replenished with DMEM for 30 min or ( B ) stimulated with 50 ng / ml epidermal growth factor ( EGF ) for 30 min . The S6K immune complexes were analysed by immunoblot using appropriate antibodies ( top three panels ) . Flag ( ATG1α ) blots were completed from the same cell lysates ( bottom panel ) . ( C ) HEK 293T cells transfected with control siRNA , ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min , and then the phosphorylation and protein levels of endogenous S6K ( top two panels ) and S6 ( bottom two panels ) were determined . ( D ) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min , and then replenished with DMEM for 30 min . The phosphorylation and protein levels of endogenous S6K were examined . For quantification , the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop , and normalized to the S6K protein levels . Results are expressed as a fold change compared with the first indicated sample . ( E ) HEK293T cells were transfected with control siRNA or ATG1α siRNA with pEGFP ‐ C1 ( a transfection marker , green ) , and nutrient ‐ starved for 90 min , fixed and stained with phosphospecific S6 antibody ( red ) . ATG1 , Autophagy ‐ specific gene 1 ; HA , haemagglutinin ; KI , ATG1α Lys 46 Ile ( kinase ‐ dead ) mutant ; siRNA , short interfering RNA ; S6K , S6 kinase ; WT , wild type .

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 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 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

Figure 3(A,B) HEK 293T cells transfected with denoted plasmids were (A) deprived of nutrients for 90 min, and then replenished with DMEM for 30 min or (B) stimulated with 50 ng/ml epidermal growth factor (EGF) for 30 min. The S6K immune complexes were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel).(C) HEK 293T cells transfected with control siRNA, ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min, and then the phosphorylation and protein levels of endogenous S6K (top two panels) and S6 (bottom two panels) were determined.(D) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min, and then replenished with DMEM for 30 min. The phosphorylation and protein levels of endogenous S6K were examined. For quantification, the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop, and normalized to the S6K protein levels. Results are expressed as a fold change compared with the first indicated sample.(E) HEK293T cells were transfected with control siRNA or ATG1α siRNA with pEGFP‐C1 (a transfection marker, green), and nutrient‐starved for 90 min, fixed and stained with phosphospecific S6 antibody (red). ATG1, Autophagy‐specific gene 1; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; siRNA, short interfering RNA; S6K, S6 kinase; WT, wild type.


Figure 4 ( A , B ) HEK 293T cells were transfected with HA - Akt ( A ) or Myc - RSK ( B ) , and / or Flag - ATG1α WT or KI plasmids . The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies ( top three panels ) . Flag ( ATG1α ) blots were completed from the same cell lysates ( bottom panel ) . ( C ) HEK 293T cells were transfected with HA - S6K Thr 389 Glu mutant and / or Flag - ATG1α WT or KI . The immunoprecipitated S6K mutants were analysed by immunoblot using anti ‐ phosphospecific S6K Thr 229 ( top panel ) and HA ( S6K ) antibodies ( middle panel ) . Flag ( ATG1α ) blot was completed from the same cell lysates ( bottom panel ) . ATG1 , Autophagy ‐ specific gene 1 ; EGF , epidermal growth factor ; HA , haemagglutinin ; KI , ATG1α Lys 46 Ile ( kinase ‐ dead ) mutant ; RSK , ribosomal S6 kinase ; WT , wild type .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A,B) HEK 293T cells were transfected with HA-Akt (A) or Myc-RSK (B), and/or Flag-ATG1α WT or KI plasmids. The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel).(C) HEK 293T cells were transfected with HA-S6K Thr 389 Glu mutant and/or Flag-ATG1α WT or KI. The immunoprecipitated S6K mutants were analysed by immunoblot using anti‐phosphospecific S6K Thr 229 (top panel) and HA (S6K) antibodies (middle panel). Flag (ATG1α) blot was completed from the same cell lysates (bottom panel). ATG1, Autophagy‐specific gene 1; EGF, epidermal growth factor; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; RSK, ribosomal S6 kinase; WT, wild type.


Figure 2Mean ΔF images of Rhod - FF and iGluSnFR signals in response to a voltage ramp and a 50 - ms - long step depolarization , respectively . The synapse marked with * on the overlaid image is further analyzed in the following panels . ( P : pillar side , M : modiolar side ; Scale bar : 2 μm ) Voltage command ( top ) , corresponding whole - cell Ca2 + - influx ( middle ) and the functional fluorescence responses ( bottom ) ( bandstop filtered at 33 . 3 Hz ) from Rhod - FF and iGluSnFR , respectively . A modified Boltzmann function ( see Materials and Methods , R2 = 0 . 81 ) was fitted to a Rhod - FF fluorescence trace in response to a voltage ramp ( C - C ' ) . iGluSnFR - AUC was calculated per depolarization voltage and used for a Boltzmann fit ( D ' , R2 = 0 . 92 ) . The voltage of half - maximal activation ( V1 / 2 ) and the dynamic range ( 10 - 90 % ) of synaptic Ca2 + - influx and glutamate release were calculated from the fits and depicted as circle and bar in C ' and D ' , respectively . The obtained fits from a Ca2 + " hotspot " ( C ' ) and from glutamate release ( D ' ) were plotted against each other in a voltage range from - 57 mV to - 17 mV in 1 mV increments . A power function was fitted to the 25 % of the maximum iGluSnFR - AUC ( R2 = 0 . 99 ) to obtain the m - estimate . ( ruptured patch - clamp , 10 mM intracellular EGTA , 5 mM [ Ca2 + ] e ) ( See also Fig EV4 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Mean ΔF images of Rhod-FF and iGluSnFR signals in response to a voltage ramp and a 50-ms-long step depolarization, respectively. The synapse marked with * on the overlaid image is further analyzed in the following panels. (P: pillar side, M: modiolar side; Scale bar: 2 μm)Voltage command (top), corresponding whole-cell Ca2+-influx (middle) and the functional fluorescence responses (bottom) (bandstop filtered at 33.3 Hz) from Rhod-FF and iGluSnFR, respectively. A modified Boltzmann function (see Materials and Methods, R2 = 0.81) was fitted to a Rhod-FF fluorescence trace in response to a voltage ramp (C-C'). iGluSnFR-AUC was calculated per depolarization voltage and used for a Boltzmann fit (D', R2 = 0.92). The voltage of half-maximal activation (V1/2) and the dynamic range (10-90%) of synaptic Ca2+-influx and glutamate release were calculated from the fits and depicted as circle and bar in C' and D', respectively. The obtained fits from a Ca2+ "hotspot" (C') and from glutamate release (D') were plotted against each other in a voltage range from -57 mV to -17 mV in 1 mV increments. A power function was fitted to the 25% of the maximum iGluSnFR-AUC (R2 = 0.99) to obtain the m-estimate. (ruptured patch-clamp, 10 mM intracellular EGTA, 5 mM [Ca2+]e) (See also Fig EV4)


Figure 3A voltage ramp was applied to obtain ΔF of Rhod - FF as a proxy synaptic Ca2 + influx . The V1 / 2 of synaptic Ca2 + - influx was calculated from a modified Boltzman function ( see Materials and Methods ) fitted to ΔF / F0 . ( mean ± SD , n = 55 synapses ; individual synapses are color coded ) ( see Appendix Fig S7 for individual fits ) . Normalized iGluSnFR - AUC , in response to 50 - ms - long step depolarizations , same as A ( see Appendix Fig S8 for individual fits ) .

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

Figure 3A voltage ramp was applied to obtain ΔF of Rhod-FF as a proxy synaptic Ca2+ influx. The V1/2 of synaptic Ca2+-influx was calculated from a modified Boltzman function (see Materials and Methods) fitted to ΔF/F0. (mean ± SD, n = 55 synapses; individual synapses are color coded) (see Appendix Fig S7 for individual fits).Normalized iGluSnFR-AUC, in response to 50-ms-long step depolarizations, same as A (see Appendix Fig S8 for individual fits).


Figure 4Left panel shows the linear regression analysis ( solid lines ) of V1 / 2 of synaptic Ca2 + - influx ( B ) , glutamate release ( C ) , threshold ( D ) and dynamic range of release ( E ) as a function of position along the pillar - modiolar axis . Shaded areas depict the associated 95 % confidence intervals . Significance of correlation coefficients is reported by a two - tailed p - value . Right panel shows box and whisker plots of these properties of synapses grouped into pillar and modiolar halves of the IHCs . Box plots indicate first quartile ( 25th percentile ) , median and third quartile ( 75th percentile ) with whiskers reaching from 10 - 90 % . For comparison of pillar and modiolar synapses , either student ' s t - test ( for normally distributed data ) or Mann - Whitney - U test ( for non - normally distributed data ) was applied . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 .

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

Figure 4Left panel shows the linear regression analysis (solid lines) of V1/2 of synaptic Ca2+-influx (B), glutamate release (C), threshold (D) and dynamic range of release (E) as a function of position along the pillar-modiolar axis. Shaded areas depict the associated 95% confidence intervals. Significance of correlation coefficients is reported by a two-tailed p-value. Right panel shows box and whisker plots of these properties of synapses grouped into pillar and modiolar halves of the IHCs. Box plots indicate first quartile (25th percentile), median and third quartile (75th percentile) with whiskers reaching from 10-90%. For comparison of pillar and modiolar synapses, either student's t-test (for normally distributed data) or Mann-Whitney-U test (for non-normally distributed data) was applied. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001


Figure 5 Single IHC exhibits different modes of Ca2 + - control of release . The overlaid ΔF image of Rhod - FF ( red ) and iGluSnFR ( green ) shows synaptic Ca2 + - influx and glutamate release at three neighboring modiolar synapses ( individual synapses are color coded based on their clusters ) . E ' The relation between Ca2 + - influx and glutamate release of given synapses in the voltage range of - 57 to - 17 mV plotted with 1 mV increments . A power function was fitted until the 25 % of normalized iGluSnFR AUC ( ΔF / F0 ) . Synapses showed different Ca2 + - dependencies ( m = 2 . 0 , 1 . 3 , 7 . 4 ; green , purple , orange ) . Mean glutamate release ( iGluSnFR - AUC ) as a function of depolarization voltage in the three identified clusters ( mean ± SEM , Cluster 1 : n = 27 synapses , Cluster 2 : n = 22 synapses , Cluster 3 : n = 6 synapses ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Single IHC exhibits different modes of Ca2+-control of release. The overlaid ΔF image of Rhod-FF (red) and iGluSnFR (green) shows synaptic Ca2+-influx and glutamate release at three neighboring modiolar synapses (individual synapses are color coded based on their clusters). E' The relation between Ca2+-influx and glutamate release of given synapses in the voltage range of -57 to -17 mV plotted with 1 mV increments. A power function was fitted until the 25% of normalized iGluSnFR AUC(ΔF/F0). Synapses showed different Ca2+-dependencies (m = 2.0, 1.3, 7.4; green, purple, orange).  Mean glutamate release (iGluSnFR-AUC) as a function of depolarization voltage in the three identified clusters (mean ± SEM, Cluster 1: n = 27 synapses, Cluster 2: n = 22 synapses, Cluster 3: n = 6 synapses).


Figure 1B . Detection of ALDHbr cells by high content screening . The algorithm first detect nuclear region of interest ( ROI ) in the Hoechst channel ( rainbow areas ) . Then , it computes the averaged ALDH signal in the ROI to identify ALDHbr cells ( red nuclear ROIs ) and calculate their proportion . C - D . Example of candidate genes whose silencing modified the ALDHbr cell proportion without inducing a massive cell death ( cell viability > 20 % , B - score > │ 2 . 58 │ ) . Data represent mean ± SD ( n = 3 ) . E . Representative images of the high - content screening captures . ALDEFLUOR cellular staining is represented in green . ALDHbr cells are labelled in red . F . Volcano plot of the hits . Scatter points represent genes , the x - axis is the log2 fold change of the proportion of ALDHbr cells compared to the control , and the y - axis is the B - score . Genes targeted by a siRNA inducing a significant reduction ( green dots ; B - score > 2 . 58 , log2 FC < 1 ) or increase ( red dots ; B - score > 2 . 58 , log2 FC < 1 ) of the ALDHbr cell proportion are highlighted .

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

Figure 1B. Detection of ALDHbr cells by high content screening. The algorithm first detect nuclear region of interest (ROI) in the Hoechst channel (rainbow areas). Then, it computes the averaged ALDH signal in the ROI to identify ALDHbr cells (red nuclear ROIs) and calculate their proportion.C-D. Example of candidate genes whose silencing modified the ALDHbr cell proportion without inducing a massive cell death (cell viability>20%, B-score>│2.58│). Data represent mean ±SD (n=3).E. Representative images of the high-content screening captures. ALDEFLUOR cellular staining is represented in green. ALDHbr cells are labelled in red.F. Volcano plot of the hits. Scatter points represent genes, the x-axis is the log2 fold change of the proportion of ALDHbr cells compared to the control, and the y-axis is the B-score. Genes targeted by a siRNA inducing a significant reduction (green dots; B-score>2.58, log2 FC<1) or increase (red dots; B-score>2.58, log2 FC<1) of the ALDHbr cell proportion are highlighted.


Figure 3B . Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping ( right ) and tumor SFE ( left ) using the 11 siRNAs pool ( 3 siRNAs / pool ) against the selected target genes and one scrambled siRNA pool used as control . Results for each siRNA pool ( from top to bottom ) are represented as opposite bars ( n = 3 ) . Correlations are measured using Spearman rank correlation ( R ) . C - D . Examples of dose - dependent ALDHbr cells variation in response to drug treatments . ALDHbr cell proportion in response to Tazemetostat in SUM159 ( n = 10 ) ( C ) and JQ1 in SUM149 ( n = 10 ) ( D ) . The horizontal red dashed - line corresponds to a reduction of 50 % of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed - line corresponds to the target dose predicted by the K - score . On the bottom panel , corresponding flow charts for the ALDEFLUOR staining . DEAB is an ALDH inhibitor used as negative control . F . Heat map for the variation of the ALDHbr cells proportion under drug treatment , in five different cell lines . Red and blue indicate the K - score in treated conditions , respectively , above and below the K - score in the untreated condition . G - H . Primary ( n = 4 ) ( G ) and secondary ( n = 4 ) ( H ) tumorsphere - forming efficiency ( SFE ) of SUM159 cells under treated and untreated conditions . Grey dashed - line corresponds to the level of SFE in the untreated conditions . Statistical test used is Pairwise chi - square test . Data represent mean ± SD .

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 O O O 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 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

Figure 3B. Comparison of bCSC variation after gene knockdown by ALDEFLUOR phenotyping (right) and tumor SFE (left) using the 11 siRNAs pool (3 siRNAs/pool) against the selected target genes and one scrambled siRNA pool used as control. Results for each siRNA pool (from top to bottom) are represented as opposite bars (n=3). Correlations are measured using Spearman rank correlation (R).C-D. Examples of dose-dependent ALDHbr cells variation in response to drug treatments. ALDHbr cell proportion in response to Tazemetostat in SUM159 (n=10) (C) and JQ1 in SUM149 (n=10) (D). The horizontal red dashed-line corresponds to a reduction of 50% of the ALDHbr cell proportion in the treated conditions compared to the control and the vertical red dashed-line corresponds to the target dose predicted by the K-score. On the bottom panel, corresponding flow charts for the ALDEFLUOR staining. DEAB is an ALDH inhibitor used as negative control.F. Heat map for the variation of the ALDHbr cells proportion under drug treatment, in five different cell lines. Red and blue indicate the K-score in treated conditions, respectively, above and below the K-score in the untreated condition.G-H. Primary (n=4) (G) and secondary (n=4) (H) tumorsphere-forming efficiency (SFE) of SUM159 cells under treated and untreated conditions. Grey dashed-line corresponds to the level of SFE in the untreated conditions. Statistical test used is Pairwise chi-square test. Data represent mean ±SD.


Figure 4A . Interaction surface built using the estimated combination index ( CI ) for the Salinomycin and JQ1 drug combination , in four different breast cancer cell lines ( top panels ) . The CI is represented in 2D color code interaction surface . The color code spans from strong synergism ( dark blue , CI < 1 ) to strong antagonism ( red , CI > 1 ) . The concentrations of each particular drug in the combinations are denoted on each axis . On the bottom panels , neighborhood Z - core matrix estimating the significant synergistic interaction . The color code spans from a statistically significant synergism ( dark blue , CI < 1 , p < 0 . 01 ) to a statistically significant antagonism ( dark red , CI > 1 , p < 0 . 01 ) . B . Representative examples of flow chart for the ALDEFLUOR staining following different JQ1 / Salinomycin drug combinations . DEAB is an ALDH inhibitor used as negative control . Data represent mean ± SD . C - D . Primary ( n = 4 ) ( C ) and secondary ( n = 4 ) ( D ) tumorsphere - forming efficiency ( SFE ) of SUM159 cells under mono - and combined - therapy compared to untreated conditions . Statistical test used is Pairwise chi - square test . . E . Bar plots representing the dose - dependent changes in the proportion of apoptotic cells ( YO - PRO - 3 + ) in each cell subpopulations , in response to drug treatments ( right panel ) . Cisplatin ( CisPt ) is used as a positive control . On the right panel , representative fluorescence images obtained in different treatment conditions ( n = 6 ) . Nucleus are in blue ( Hoechst ) , bCSCs are in green ( ALDH ) , and apoptotic cells in red ( YO - PRO - 3 ) . G . Representation of the BFP + / RFP + cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone , or in combination . Blue bars : BFP + cells proportion , Red bars : RFP + cells proportion . H - I . Representation of the bCSC proportion in the BFP + ( H ) and RFP + ( I ) progenies in the control cells compared to the JQ1 - and Salinomycin - treated cells alone or in combination ( n = 4 ) . Statistical test used is a student ' s t - test . JQ1 Vs CTRL and Sal Vs JQ1 p = 0 . 03 ; JQ1 / Sal Vs CTRL p = 0 . 01 . Error bars represent mean ± SD .

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

Figure 4A. Interaction surface built using the estimated combination index (CI) for the Salinomycin and JQ1 drug combination, in four different breast cancer cell lines (top panels). The CI is represented in 2D color code interaction surface. The color code spans from strong synergism (dark blue, CI<1) to strong antagonism (red, CI>1). The concentrations of each particular drug in the combinations are denoted on each axis. On the bottom panels, neighborhood Z-core matrix estimating the significant synergistic interaction. The color code spans from a statistically significant synergism (dark blue, CI<1, p<0.01) to a statistically significant antagonism (dark red, CI>1, p<0.01).B. Representative examples of flow chart for the ALDEFLUOR staining following different JQ1/Salinomycin drug combinations. DEAB is an ALDH inhibitor used as negative control. Data represent mean ±SD.C-D. Primary (n=4) (C) and secondary (n=4) (D) tumorsphere-forming efficiency (SFE) of SUM159 cells under mono- and combined-therapy compared to untreated conditions. Statistical test used is Pairwise chi-square test..E. Bar plots representing the dose-dependent changes in the proportion of apoptotic cells (YO-PRO-3+) in each cell subpopulations, in response to drug treatments (right panel). Cisplatin (CisPt) is used as a positive control. On the right panel, representative fluorescence images obtained in different treatment conditions (n=6). Nucleus are in blue (Hoechst), bCSCs are in green (ALDH), and apoptotic cells in red (YO-PRO-3).G. Representation of the BFP+/RFP+ cells proportions for increasing drug concentrations of either JQ1 or Salinomycin alone, or in combination. Blue bars: BFP+ cells proportion, Red bars: RFP+ cells proportion.H-I. Representation of the bCSC proportion in the BFP+ (H) and RFP+ (I) progenies in the control cells compared to the JQ1- and Salinomycin-treated cells alone or in combination (n=4). Statistical test used is a student's t-test. JQ1 Vs CTRL and Sal Vs JQ1 p=0.03; JQ1/Sal Vs CTRL p=0.01. Error bars represent mean ± SD.


Figure 5B . Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 ( n = 10 ) The grey area corresponds to the period of drug treatments . C - E . Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle - treated tumors , measured by flow cytometry . Statistical test used is a student t - test . Data represent mean ± SD ( n = 10 ) . Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle - treated tumors , measured by flow cytometry . Statistical test used is a student t - test . Data represent mean ± SD ( n = 10 ) . Reimplantation assay ( CRCM434 ) . Two - week treated PDXs were reimplanted , in serial dilutions , into new recipient mice and tumor growth was monitored Each curve represents the growth kinetic from one individual injection . G - I . Bar plots displays bCSC frequency calculated using an extreme limiting dilution analysis ( ELDA ) . Results are expressed as the estimated number of bCSCs for 10 , 000 tumor cells . Statistical test used is Pairwise chi - square test . J - L . Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434 . Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs ( J ) . Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal / JQ1 treated mice compared to the control mice ( K , CRCM404 , p = 0 . 04 ; L , CRCM434 , p = 0 . 02 ) . Statistical test used is a Wilcoxon test . Error bars represent mean ± SD ( n = 10 ) .

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Figure 5B. Effect of JQ1 and Salinomycin treatment on the tumor growth of CRCM434 (n=10) The grey area corresponds to the period of drug treatments.C-E. Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle-treated tumors, measured by flow cytometry. Statistical test used is a student t-test. Data represent mean ±SD (n=10).Quantification of the proportion of residual ALDHbr cells in treated PDX normalized to the proportion of ALDHbr cells in the vehicle-treated tumors, measured by flow cytometry. Statistical test used is a student t-test. Data represent mean ±SD (n=10).Reimplantation assay (CRCM434). Two-week treated PDXs were reimplanted, in serial dilutions, into new recipient mice and tumor growth was monitored Each curve represents the growth kinetic from one individual injection.G-I. Bar plots displays bCSC frequency calculated using an extreme limiting dilution analysis (ELDA). Results are expressed as the estimated number of bCSCs for 10,000 tumor cells. Statistical test used is Pairwise chi-square test.J-L. Effect of JQ1 and Salinomycin treatments on the metastasis formation of CRCM404 and CRCM434. Metastasis formation was monitored using bioluminescence imaging in the treated mice livers and lungs (J). Quantification of the normalized photon flux revealed a statistically significant decrease in metastasis formation in Sal/JQ1 treated mice compared to the control mice (K, CRCM404, p=0.04; L, CRCM434, p=0.02). Statistical test used is a Wilcoxon test. Error bars represent mean ± SD (n=10).


Figure 6C . Kaplan - Meier relapse - free survival curve according to subnetwork metagenes expression levels in breast tumors . Curves were compared with a log - rank test ( p - value ) .

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Figure 6C. Kaplan-Meier relapse-free survival curve according to subnetwork metagenes expression levels in breast tumors. Curves were compared with a log-rank test (p-value).


Figure 1D Changes ( Δ g ) in inguinal WAT ( iWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant . E Changes ( Δ g ) in epididymal WAT ( eWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test . N . S . , not significant . F Expression of Pltp mRNA in indicated tissues . Data are shown by relative Pltp mRNA levels normalized by 18s expression . n = 3 for all tissues ( except BAT and WAT n = 5 , Testis n = 2 ) . * P < 0 . 05 Pltp expression in BAT vs other tissues ( except testis and lung ) by Student ' s t - test . G Plasma PLTP activity from control ( Ppargflox / flox ) and BAT - less mice ( Pparg UCP1 - KO , Ucp1 - Cre x Ppargflox / flox ) . Following 6 - h fasting plasma was obtained from PpargUCP1 - KO mice and littermate control . n = 3 for both groups . * P < 0 . 05 relative to control mice by Student ' s t - test . H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type ( WT ) and aP2 - PRDM16 mice [ 24 ] . Peptide levels was determined by quantitative proteomics . n = 2 for both groups . I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ . ChIP - seq data for PRDM16 and PPARγ were obtained from [ 31 , 32 ] , respectively .

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 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 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 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

Figure 1D Changes (Δ g) in inguinal WAT (iWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant. E Changes (Δ g) in epididymal WAT (eWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01, ***P<0.001 relative to GFP by Student's t-test. N.S., not significant. F Expression of Pltp mRNA in indicated tissues. Data are shown by relative Pltp mRNA levels normalized by 18s expression. n=3 for all tissues (except BAT and WAT n=5, Testis n=2). *P<0.05 Pltp expression in BAT vs other tissues (except testis and lung) by Student's t-test.G Plasma PLTP activity from control (Ppargflox/flox) and BAT-less mice (Pparg UCP1-KO, Ucp1-Cre x Ppargflox/flox). Following 6-h fasting plasma was obtained from PpargUCP1-KO mice and littermate control. n=3 for both groups. *P < 0.05 relative to control mice by Student's t-test.H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type (WT) and aP2-PRDM16 mice [24]. Peptide levels was determined by quantitative proteomics. n=2 for both groups.I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ. ChIP-seq data for PRDM16 and PPARγ were obtained from [31,32], respectively.


Figure 2B Changes in body weight of mice in ( A ) during 10 weeks of HFD . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test . C Changes in food intake of mice in ( A ) during 10 weeks of HFD ( cohort 1 ) . n = 9 for GFP ( cage 1 = 5 mice ; cage 2 = 4 mice ) and n = 8 for PLTP ( cage 1 = 4 mice ; cage 2 = 4 mice ) D Body composition of mice in ( A ) at 10 weeks of HFD determined by EchoMRI . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 relative to GFP by two - way ANOVA followed by Tukey ' s test . N . S . , not significant . E Whole - body energy expenditure of mice at 3 weeks of HFD . Area under the curve ( AUC ) of VO2 is shown in the right graph . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . * P < 0 . 05 relative to GFP by Student ' s t - test . F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . N . S . , not significant by Student ' s t - testG OCR in the interscapular BAT and iWAT of control and PLTP - treated mice . One week after the treatment , BAT and iWAT were isolated from mice ( 7 weeks old ) that received adenovirus for GFP or PLTP . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant . H Gene expression of indicated thermogenic genes in ( A ) from interscapular BAT for GFP ( n = 8 ) and PLTP ( n = 8 ) groups ( cohort 1 ) except n = 7 for Ucp1 of the PLTP group . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant . I H & E staining of interscapular BAT from mice in ( A ) at 10 weeks of HFD ( cohort 1 ) . Representative images from n = 3 mice for both groups . Scale Bar , 50µm .

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

Figure 2B Changes in body weight of mice in (A) during 10 weeks of HFD. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05, **P<0.01, ***P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.C Changes in food intake of mice in (A) during 10 weeks of HFD (cohort 1). n=9 for GFP (cage 1 = 5 mice; cage 2 = 4 mice) and n=8 for PLTP (cage 1 = 4 mice; cage 2 = 4 mice)D Body composition of mice in (A) at 10 weeks of HFD determined by EchoMRI. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05 relative to GFP by two-way ANOVA followed by Tukey's test. N.S., not significant.E Whole-body energy expenditure of mice at 3 weeks of HFD. Area under the curve (AUC) of VO2 is shown in the right graph. n=7 for GFP and n=8 for PLTP (cohort 2). *P<0.05 relative to GFP by Student's t-test.F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis. n=7 for GFP and n=8 for PLTP (cohort 2). N.S., not significant by Student's t-testG OCR in the interscapular BAT and iWAT of control and PLTP-treated mice. One week after the treatment, BAT and iWAT were isolated from mice (7 weeks old) that received adenovirus for GFP or PLTP. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.H Gene expression of indicated thermogenic genes in (A) from interscapular BAT for GFP (n=8) and PLTP (n=8) groups (cohort 1) except n=7 for Ucp1 of the PLTP group. Tbp was used as an internal control. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant.I H&E staining of interscapular BAT from mice in (A) at 10 weeks of HFD (cohort 1). Representative images from n=3 mice for both groups. Scale Bar, 50µm.


Figure 3A Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 10 weeks of HFD . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 9 for GFP and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test . B Insulin tolerance test of mice in ( A ) at 10 weeks of HFD . Mice were fasted for four hours and administered insulin ( 0 . 75 U / kg body weight ) . n = 9 for GFP control and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test . C Fasting insulin levels of mice in ( A ) at 10 weeks of HFD . Blood samples were harvested after eight hours of fasting . n = 5 for GFP and n = 4 for PLTP . * P < 0 . 05 relative to GFP by Student ' s t - test . D Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 6 weeks of HFD . Note that no difference was found in body weight between the groups by two - way ANOVA repeated measures followed by Bonferroni ' s test . . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 7 for GFP control and n = 10 for PLTP . AUC of glucose is shown in the right graph . * P < 0 . 05 by Student ' s t - test . E 18F - FDG PET / CT scan images of mice that received adenovirus GFP ( control ) or PLTP at 14 weeks of HFD . Mice received CL316 , 243 at dose of 1 mg / kg of body weight 30 min before the PET / CT images . Representative images of n = 5 for both groups . Yellow arrows indicate anatomical references as bladder , heart and interscapular BAT . F Quantification of 18F - FDG uptake in indicated tissues from mice in ( E ) . After the 18F - FDG PET / CT imaging , indicated tissues were harvested and the radioactivity was measured by scintillation counter . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant .

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

Figure 3A Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 10 weeks of HFD. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=9 for GFP and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.B Insulin tolerance test of mice in (A) at 10 weeks of HFD. Mice were fasted for four hours and administered insulin (0.75 U/kg body weight). n=9 for GFP control and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test.D Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 6 weeks of HFD. Note that no difference was found in body weight between the groups by two-way ANOVA repeated measures followed by Bonferroni's test.. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=7 for GFP control and n=10 for PLTP. AUC of glucose is shown in the right graph. *P<0.05 by Student's t-test.E 18F-FDG PET/CT scan images of mice that received adenovirus GFP (control) or PLTP at 14 weeks of HFD. Mice received CL316,243 at dose of 1 mg/kg of body weight 30 min before the PET/CT images. Representative images of n=5 for both groups. Yellow arrows indicate anatomical references as bladder, heart and interscapular BAT.F Quantification of 18F-FDG uptake in indicated tissues from mice in (E). After the 18F-FDG PET/CT imaging, indicated tissues were harvested and the radioactivity was measured by scintillation counter. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.


Figure 4A Lipidomics - based quantification of indicated lipid species in mice that received tail - vein injection of adenovirus expressing PLTP or GFP ( control ) . Blood and tissues were harvested after 10 weeks of HFD . n = 4 for GFP and n = 5 for PLTP in mice on HFD . LPC = ( lyso ) phosphatidylcholine , LPE = ( lyso ) phosphatidylethalonamine , PA = phosphatidic acid , SM = sphingomyelin , TAG = triglycerides , FFA = free fatty acid . Statistical analysis is demonstrated ( P value ) relative to GFP by Student ' s t - test . B Plasma total cholesterol levels in mice that received adenovirus GFP ( control ) or PLTP . Blood and tissues were harvested seven days after adenovirus infection . n = 8 in mice on RD and n = 7 in mice on HFD for 14 weeks . * * * P < 0 . 001 relative to GFP by Student ' s t - test . C Relative mRNA levels of indicated genes in the liver of mice on HFD . Tbp was used as an internal control . n = 4 for GFP and n = 6 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . D Bile acid levels in the liver of mice in ( B ) . n = 8 in mice on RD and n = 6 in mice on HFD . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . E Bile acid levels in the plasma of mice in ( B ) . n = 8 in mice on RD and n = 7 in mice on HFD . * * * P < 0 . 001 relative to GFP by Student ' s t - test . F Bile acid levels in the feces of mice in ( B ) . Feces were collected from mice on RD and HFD after seven days of treatment . n = 7 in mice on RD and HFD . * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test . H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid ( CA ) . Cells were treated with CA at indicated concentrations for 24 h . 0 . 1 % ETOH was used as vehicle control ( Ctr ) . n = 3 . Ucp1 mRNA expression was normalized by Tbp as an internal control . * * P < 0 . 01 relative to Ctr by one - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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

Figure 4A Lipidomics-based quantification of indicated lipid species in mice that received tail-vein injection of adenovirus expressing PLTP or GFP (control). Blood and tissues were harvested after 10 weeks of HFD. n=4 for GFP and n=5 for PLTP in mice on HFD. LPC = (lyso)phosphatidylcholine, LPE = (lyso)phosphatidylethalonamine, PA = phosphatidic acid, SM = sphingomyelin, TAG = triglycerides, FFA = free fatty acid. Statistical analysis is demonstrated (P value) relative to GFP by Student's t-test.B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test.C Relative mRNA levels of indicated genes in the liver of mice on HFD. Tbp was used as an internal control. n=4 for GFP and n=6 for PLTP. *P<0.05, **P<0.01 relative to GFP by Student's t-test.D Bile acid levels in the liver of mice in (B). n=8 in mice on RD and n=6 in mice on HFD. *P<0.05, **P<0.01 relative to GFP by Student's t-test.E Bile acid levels in the plasma of mice in (B). n=8 in mice on RD and n=7 in mice on HFD. ***P<0.001 relative to GFP by Student's t-test. F Bile acid levels in the feces of mice in (B). Feces were collected from mice on RD and HFD after seven days of treatment. n=7 in mice on RD and HFD. **P<0.01, ***P<0.001 relative to GFP by Student's t-test. H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid (CA). Cells were treated with CA at indicated concentrations for 24 h. 0.1% ETOH was used as vehicle control (Ctr). n=3. Ucp1 mRNA expression was normalized by Tbp as an internal control. **P<0.01 relative to Ctr by one-way ANOVA followed by Tukey's test. N.S., not significant.