Figure 6ρ0206 _ A and ρ0206 _ B cells were stained with mitoSOX and analyzed by confocal microscopy . DIC , Differential Interference Contrast . Scale bar , 25 µm . Quantification of the MitoSOX fluorescence intensity described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by student ' s t - test , * * p < 0 . 01 . Mitochondria carrying the 3243A > G mutation in the tRNA Leu ( UUR ) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206 _ A ( 100 % wild - type mtDNA ) and ρ0206 _ B ( ~ 90 % m . 3243A > G mtDNA ) cell lines . ρ0206 _ A and ρ0206 _ B cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 . Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 . ρ0206 _ A , ρ0206 _ B , and ρ0206 - B cells pretreated with ddC ( 50µM ) plus EB ( 0 . 5µg / ml ) for 10 days , and then harvested and lysed . Cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - GAPDH antibodies . ρ0206 _ B cells were infected with control , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA from cells was isolated and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 . ρ0206 _ B cells were infected with control , ATAD3A - Flag , ATAD3B - Flag , or ATAD3B ( mLIR ) - Flag . After two weeks , the cellular ATP levels were measured . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 . MELAS patient fibroblasts were infected with control , ATAD3A - Flag , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

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

Figure 6ρ0206_A and ρ0206_B cells were stained with mitoSOX and analyzed by confocal microscopy. DIC, Differential Interference Contrast. Scale bar, 25 µm. Quantification of the MitoSOX fluorescence intensity described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by student's t-test, **p < 0.01.Mitochondria carrying the 3243A>G mutation in the tRNA Leu(UUR) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206_A (100% wild-type mtDNA) and ρ0206_B (~90% m.3243A>G mtDNA) cell lines. ρ0206_A and ρ0206_B cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.ρ0206_A, ρ0206_B, and ρ0206-B cells pretreated with ddC (50µM) plus EB (0.5µg/ml) for 10 days, and then harvested and lysed. Cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-GAPDH antibodies.ρ0206_B cells were infected with control, ATAD3B-Flag or ATAD3B (mLIR)-Flag. After two weeks, total DNA from cells was isolated and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.ρ0206_B cells were infected with control, ATAD3A-Flag, ATAD3B-Flag, or ATAD3B (mLIR)-Flag. After two weeks, the cellular ATP levels were measured. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.MELAS patient fibroblasts were infected with control, ATAD3A-Flag, ATAD3B-Flag or ATAD3B(mLIR)-Flag. After two weeks, total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, **p < 0.01.


Figure 1Comparison of bacterial colony forming unit ( CFU ) in feces from vehicle - and ABX - treated mice ( n = 5 mice / group ) . n . d . , non - detectable . Representative image and measurements of lengths for CD31 + / LYVE - 1 + lacteal , CD31 + / LYVE - 1 - capillary plexus and DAPI + villus . Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of vehicle - or ABX - treated mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Images and comparisons of the number of Prox1 + LECs within initial 100 μm portion ( F ) presented as relative fluorescence intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Images and comparisons of the VEGFR3 expression ( G ) , presented as relative fluorescence intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Comparison of bacterial colony forming unit (CFU) in feces from vehicle- and ABX-treated mice (n = 5 mice/group). n.d., non-detectable.Representative image and measurements of lengths for CD31+/LYVE-1+ lacteal, CD31+/LYVE-1- capillary plexus and DAPI+ villus.Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of vehicle- or ABX-treated mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group).Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (F) presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.Images and comparisons of the VEGFR3 expression (G), presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


Figure 2Images and comparison of VE - cadherin LEC junctions in lacteals of jejunum between vehicle - and ABX - treated mice . Each boxed portion is highly magnified for visualization of detailed distributions of VE - cadherin to distinguish button - like ( red arrowheads ) , zipper - like ( open arrowheads ) and mixed LEC junctions in lacteals ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 50 μm . Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals . Asterisks indicate the site in lamina propria where fluorescence intensity ( FI ) was measured . Relative FI divided by mean FI of controls at 16 min ( left ) and normalized FI divided by FI at 16 min in each villus ( right ) after initial loading of BODIPY - FA were quantified . For normalization of FI , villi that showed peak intensity earlier or later than 16 min were excluded ( n = 5 - 7 mice / group , 5 - 10 villi / mouse ) . Scale bars , 100 μm . Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions ( n = 4 mice / group , 4 - 6 images / mouse ) . Jejunum of vehicle - and ABX - treated mice were harvested 2 hours after oral lipid loading . Note that the junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice . CM , chylomicron ; IS , interstitium of lamina propria ; LC , large caveola ; Lu , lacteal lumen ; Ve , vesicle containing lipoproteins . Scale bars , 1 μm . quantification of frequency of open junctions ( n = 4 mice / group , 4 - 6 images / mouse ) . Note that the junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice . Comparisons of serum triglyceride ( TG ) and free fatty acids ( FFA ) at indicated time points in vehicle - and ABX - treated mice ( n = 5 - 7 mice / group ) .

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

Figure 2Images and comparison of VE-cadherin LEC junctions in lacteals of jejunum between vehicle- and ABX-treated mice. Each boxed portion is highly magnified for visualization of detailed distributions of VE-cadherin to distinguish button-like (red arrowheads), zipper-like (open arrowheads) and mixed LEC junctions in lacteals (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 50 μm.Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm.Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm.quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice.Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).


Figure 3Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle - or ABX - treated mice at P7 , P14 , and P28 . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm . Images and comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm . Images and comparisons of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between vehicle - or ABX - treated mice at P7 , P14 , and P28 . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in right ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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 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 3Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.Images and comparisons of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in right (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Figure 4Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of 8 - week old SPF , GF or CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm . Images and comparisons of the number of Prox1 + LECs within initial 100 μm portion ( C ) and VEGFR3 expression ( D , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from SPF , GF and CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm . Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum among SPF , GF and CONV mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Figure 4Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of 8-week old SPF, GF or CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (C) and VEGFR3 expression (D, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from SPF, GF and CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum among SPF, GF and CONV mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Figure 5Images and comparison of VEGFR3 expression ( presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum between WT and VR3iΔLEC mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm . Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and VR3iΔLEC mice . Note that , in addition to the alterations in the lacteal lengths in VR3iΔLEC mice , diameters of the lacteal also shrank by VEGFR3 ablation . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm . Images and comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from WT and VR3iΔLEC mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm . Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum from WT and VR3iΔLEC mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm . Comparisons of serum triglyceride ( TG ) at indicated time points in WT and VR3iΔLEC mice ( n = 6 mice / group ) . Comparison of protein levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) . Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) .

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

Figure 5Images and comparison of VEGFR3 expression (presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum between WT and VR3iΔLEC mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and VR3iΔLEC mice. Note that, in addition to the alterations in the lacteal lengths in VR3iΔLEC mice, diameters of the lacteal also shrank by VEGFR3 ablation. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from WT and VR3iΔLEC mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum from WT and VR3iΔLEC mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).Comparison of protein levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Figure 6Comparison of mRNA levels of VEGF - C in sorted CD45 - stromal cells and CD45 + MHCII + F4 / 80 + macrophages from jejunum and ileum of vehicle - treated mice . The mean of transcription levels of VEGF - C in CD45 - stromal cells was normalized to 1 , and the relative levels in macrophages were presented as fold change ( n = 4 mice / group ) . Comparisons of VEGF - C transcription level in CD45 - stromal cells ( left ) and CD45 + MHCII + F4 / 80 + macrophages ( right ) in jejunum and ileum of vehicle - and ABX - treated mice . The mean of transcription levels of VEGF - C in vehicle - treated mice was normalized to 1 , and the relative levels in ABX - treated mice were presented as fold change ( n = 5 - 6 mice / group ) . Images and comparison of F4 / 80 + macrophages in the villi of duodenum ( DD ) , jejunum ( JJ ) , and ileum ( IL ) from vehicle - or ABX - treated mice . Each dot indicates mean value of 5 sites in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm . Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells ( E ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) . Representative flow cytometric analysis and comparison of CX3CR1 + F4 / 80 + macrophage gated on DAPI - CD45 + MHCII + cells ( F ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) .

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Figure 6Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group).Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group).Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Figure 7Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and DTR mice ( n = 5 mice / group ) . Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from WT and DTR mice ( n = 5 mice / group ) . Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm . Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm . Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and DTR mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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Figure 7Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group).Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Figure 8Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and MyD88ΔMP mice ( n = 5 mice / group ) . Comparisons of mRNA levels of VEGF - C in sorted MHCII + F4 / 80 + macrophages from jejunum and ileum of WT and MyD88ΔMP mice . The mean of transcription levels of VEGF - C in WT mice was normalized to 1 , and the relative level in MyD88ΔMP mice were presented as fold change ( n = 5 mice / group ) . Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm . Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm . Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and MyD88ΔMP mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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Figure 8Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group).Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Figure 1Representative Southern blots showing DSB repair products in WT , rad59 ∆ , rad52 - R70A and rad59 ∆ rad52 - R70A cells . DNA was digested with Bsp1286I and probed with a MAT - distal sequence ( yellow box in panel B ) .

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Figure 1Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B).


Figure 2Viability of indicated strains ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - test was used to determine the p - valueRepresentative Southern blots showing DSB repair products in WT , rad59 ∆ , and rad52 - R70A . DNA was digested with XhoI and EcoRI and probed with a Z sequence ( yellow box ) .

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Figure 2Viability of indicated strains (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-valueRepresentative Southern blots showing DSB repair products in WT, rad59∆, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box).


Figure 3Percentage of chromosome loss of indicated mutants . Data information : χ2 test is used to determine the p - value and number of colonies tested per mutant is indicated in Appendix Table S1 . Schematic of mutation analysis during allelic recombination ( left ) . Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site . Mutation rate ( right ) of WT , rad59 ∆ , rad52 - R70A was calculated as previously described ( Saini et . al . , 2013 ) . Data information : χ Mutation rates are reported as the median value , and statistical comparisons between median mutation rates were performed using the Mann - Whitney U - test .

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Figure 3Percentage of chromosome loss of indicated mutants. Data information: χ2 test is used to determine the p-value and number of colonies tested per mutant is indicated in Appendix Table S1.Schematic of mutation analysis during allelic recombination (left). Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site. Mutation rate (right) of WT, rad59∆, rad52-R70A was calculated as previously described (Saini et. al., 2013). Data information: χ Mutation rates are reported as the median value, and statistical comparisons between median mutation rates were performed using the Mann-Whitney U-test.


Figure 4Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching ( H - 1400 assay ) . Viability of indicated mutants are shown . ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - test was used to determine the p -

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Figure 4Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching (H-1400 assay).Viability of indicated mutants are shown. (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


Figure 5 ( A - C ) Representative Southern blots showing DSB repair products ( top ) , percentage of BIR among repair products ( bottom left ) and viability ( bottom right ) of WT and sir2 ∆ in the H - 150 assay ( A ) , H - - 150 no - gap assay ( B ) or native mating type switching H - 1400 assay ( C ) . ( mean ± SD ; n = 3 ) . Welch ' s unpaired t - test was used to determine the p - value in panels A - C .

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Figure 5(A-C) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of WT and sir2∆ in the H-150 assay (A), H--150 no-gap assay (B) or native mating type switching H-1400 assay (C). (mean ± SD; n = 3). Welch's unpaired t-test was used to determine the p-value in panels A-C.


Figure 6 ( A - B ) Representative Southern blots showing DSB repair products ( top ) , percentage of BIR among repair products ( bottom left ) and viability ( bottom right ) of indicated mutants in the H - 150 no - gap assay ( A ) or H - 150 assay ( B ) . ( mean ± SD ; n ≥ 3 ) . Welch ' s unpaired t - test was used to determine the p - value in panels A and B .

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Figure 6(A-B) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of indicated mutants in the H-150 no-gap assay (A) or H-150 assay (B). (mean ± SD; n ≥3). Welch's unpaired t-test was used to determine the p-value in panels A and B.


Figure 1HCT116 and U87 cells were treated with 20 µM LXR623 for 24h . Transcriptome and gene set enrichment analysis was performed . Shown are enrichment plots . NES : normalized enrichment score . HCT116 cells were treated with 20 µM LXR623 for 24h . Transcriptome and gene set enrichment analysis was performed . Shown are enrichment plots . NES : normalized enrichment score . U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin ( Sim ) for 48h . Thereafter , lysates were collected and analyzed for total cholesterol levels . Shown are means and SD ( n = 3 ) . Shown is a volcano plot of patients from the TCGA ( database ) with high vs . low levels of ABCA1 . A close - up of the most significantly down - regulated genes is presented ( mRNAs related to the electron transport chain and mitochondrial metabolism ) . The x - axis highlights the fold changes ( log2 scale ) ( down - regulated genes , negative values ) , whereas the y - axis indicates the level of statistical significance ( shown in - log10 format ) . GBM12 GBM cells ( short term patient - derived xenograft ) , U87 GBM cells , HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263 , LXR623 or the combination for 72h . Thereafter , cellular viability was analyzed and statistical analysis was performed .

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Figure 1HCT116 and U87 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.HCT116 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin (Sim) for 48h. Thereafter, lysates were collected and analyzed for total cholesterol levels. Shown are means and SD (n = 3).Shown is a volcano plot of patients from the TCGA (database) with high vs. low levels of ABCA1. A close-up of the most significantly down-regulated genes is presented (mRNAs related to the electron transport chain and mitochondrial metabolism). The x-axis highlights the fold changes (log2 scale) (down-regulated genes, negative values), whereas the y-axis indicates the level of statistical significance (shown in -log10 format).GBM12 GBM cells (short term patient-derived xenograft), U87 GBM cells, HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263, LXR623 or the combination for 72h. Thereafter, cellular viability was analyzed and statistical analysis was performed.


Figure 2U87 , T98G and LN229 cells were treated with 1 µM ABT263 , 20 µM LXR623 or the combination of both for 72h . Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263 , 10 µM LXR623 or the combination of both for 72h ( MeWo ) or for 48h ( HCT116 ) . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry . , Stem - like GBM cells , NCH644 , NCH421k and NCH690 were treated with 1 µM ABT263 , 20 µM LXR623 or the combination of both for 48h . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry . U87 ( 72h treatment ) and HCT116 ( 48h treatment ) were treated with 1 µM ABT263 , 20 µM ( U87 ) or 10 µM ( HCT116 ) LXR623 or the combination of both , stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential . HCT116 colonic carcinoma cells were treated with ABT263 , LXR623 or the combination of both . Whole protein lysates were collected and subjected to capillary electrophoresis for the expression / cleavage of PARP , total caspase - 9 ( CP9 ) and Vinculin . CF : cleaved fragment , FL : full length . HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3 - mimetics , WEHI - 539 ( Bcl - xL inhibitor ) , ABT199 ( Bcl - 2 inhibitor ) or A1210477 ( Mcl - 1 inhibitor ) in the presence or absence of LXR623 for 48h . Thereafter , cells were labeled with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry .

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Figure 2U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry., Stem-like GBM cells, NCH644, NCH421k and NCH690 were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 48h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.U87 (72h treatment) and HCT116 (48h treatment) were treated with 1 µM ABT263, 20 µM (U87) or 10 µM (HCT116) LXR623 or the combination of both, stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential.HCT116 colonic carcinoma cells were treated with ABT263, LXR623 or the combination of both. Whole protein lysates were collected and subjected to capillary electrophoresis for the expression/cleavage of PARP, total caspase-9 (CP9) and Vinculin. CF: cleaved fragment, FL: full length.HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3-mimetics, WEHI-539 (Bcl-xL inhibitor), ABT199 (Bcl-2 inhibitor) or A1210477 (Mcl-1 inhibitor) in the presence or absence of LXR623 for 48h. Thereafter, cells were labeled with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


Figure 3 , LN229 , T98G , U87 , U87 - EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h . Thereafter , protein lysates were collected and subjected to standard western blot ( LN229 , T98G and U87 ) or capillary electrophoresis ( U87 - EGFR - vIII and HCT116 ) for the expression of Usp9X , Mcl - 1 , Bcl - 2 , Bcl - xL , Noxa , BIM and / or Actin and Vinculin . In U87 - EGFRvIII and HCT116 , Vinculin was used in lieu of Actin . LN229 , T98G , U87 , U87 - EGFRvIII and HCT116 cells were treated with LXR623 as described in A . Thereafter , protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin . LN229 were transfected with siNT , siNoxa 1 , siNoxa 2 or BAK - siRNA for 72h . Thereafter , cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h . After conclusion of the treatment , cells were harvested , fixed , stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation . LN229 were transfected as described in B . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa , BAK and Vinculin . LN229 GBM cells were treated with 10 µM LXR623 ( LXR ) , 1 µM ABT263 or the combination for 48h . Thereafter , protein lysates were prepared and immunoprecipitated with an antibody against BAK . Standard western blotting was performed ( immunoprecipitation and the corresponding inputs ) with antibody against BAK and Mcl - 1 . The arrows highlight the specific protein bands , while stars indicate the immunoglobulin light - chains .

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

Figure 3, LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h. Thereafter, protein lysates were collected and subjected to standard western blot (LN229, T98G and U87) or capillary electrophoresis (U87-EGFR-vIII and HCT116) for the expression of Usp9X, Mcl-1, Bcl-2, Bcl-xL, Noxa, BIM and/or Actin and Vinculin. In U87-EGFRvIII and HCT116, Vinculin was used in lieu of Actin.LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with LXR623 as described in A. Thereafter, protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin.LN229 were transfected with siNT, siNoxa 1, siNoxa 2 or BAK-siRNA for 72h. Thereafter, cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h. After conclusion of the treatment, cells were harvested, fixed, stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation.LN229 were transfected as described in B. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa, BAK and Vinculin.LN229 GBM cells were treated with 10 µM LXR623 (LXR), 1 µM ABT263 or the combination for 48h. Thereafter, protein lysates were prepared and immunoprecipitated with an antibody against BAK. Standard western blotting was performed (immunoprecipitation and the corresponding inputs) with antibody against BAK and Mcl-1. The arrows highlight the specific protein bands, while stars indicate the immunoglobulin light-chains.


Figure 4U87 cells were treated with 20 µM LXR623 for 7h and 24h . Thereafter , cells were stained with annexin V / propidium iodide and analyzed by multi - parametric flow cytometry . The lower left quadrant indicates the percentage of viable cells . U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay . To account for dead cells , assays were normalized to DNA - content ( cell number ) . U87 cells were treated with LXR623 as indicated for 24h . Cell lysates were prepared and subjected to LC / MS for the determination of ATP levels . U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK ( threonine 172 ) and total AMPK . U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 . U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol . Thereafter , whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK ( threonine 172 ) and total AMPK . Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin ( G ) and by the standard western blot for the expression of Noxa and Actin . U87 cells were treated with increasing concentrations of LXR623 . Thereafter , whole cell protein lysates were prepared and analyzed for classical ER - stress markers , GRP78 , ATF4 and Noxa by capillary electrophoresis . U87 cells were transfected with non - targeting siRNA ( n . t . - siRNA ) , ATF3 - siRNA , ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs . After 48h , cells were treated with LXR623 , whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3 , ATF4 , Noxa and Vinculin .

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

Figure 4U87 cells were treated with 20 µM LXR623 for 7h and 24h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. The lower left quadrant indicates the percentage of viable cells.U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay. To account for dead cells, assays were normalized to DNA-content (cell number).U87 cells were treated with LXR623 as indicated for 24h. Cell lysates were prepared and subjected to LC/MS for the determination of ATP levels.U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4.U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin (G) and by the standard western blot for the expression of Noxa and Actin.U87 cells were treated with increasing concentrations of LXR623. Thereafter, whole cell protein lysates were prepared and analyzed for classical ER-stress markers, GRP78, ATF4 and Noxa by capillary electrophoresis.U87 cells were transfected with non-targeting siRNA (n.t.-siRNA), ATF3-siRNA, ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs. After 48h, cells were treated with LXR623, whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3, ATF4, Noxa and Vinculin.


Figure 5U87 cells were treated as in A . Shown are the levels of TCA - cycle metabolites . U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non - labeled isotopologues ( m + 0 ) . U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non - labeled isotopologues ( m + 0 ) . U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of non - essential aminoacids ( glutamatic acid and aspartic acid ) . U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of non - essential aminoacids ( glutamatic acidU87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . Shown are the isotopologues of glutathione and nucleotides . U87 cells were incubated in DMEM ( devoid of phenol red , glucose , pyruvate and glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine and 1 . 5 % dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours . Cells were then harvested for LC / MS analysis . The fractions of each different isotopologue of each metabolite were calculated ( percentage of the entire pool ) . U87 cells were treated with DMSO or LXR623 20 µM for 24h . Thereafter , cells were processed for polar metabolite analysis by LC / MS . Shown are the levels of NAD and NADH2 .

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

Figure 5U87 cells were treated as in A. Shown are the levels of TCA-cycle metabolites.U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non-labeled isotopologues (m+0).U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non-labeled isotopologues (m+0).U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid and aspartic acid).U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acidU87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of glutathione and nucleotides.U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool).U87 cells were treated with DMSO or LXR623 20 µM for 24h. Thereafter, cells were processed for polar metabolite analysis by LC/MS. Shown are the levels of NAD and NADH2.


Figure 6U87 cells were treated with LXR623 for 1h , 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay ( A - C ) . From this assay , coupled respiration was calculated ( D - F ) . U87 cells were treated with LXR623 , cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay ( G ) . From this assay , mitochondrial OCR and coupled respiration was calculated ( H - I ) . U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point . Thereafter , whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes ( OXPHOS ) , Noxa , and ATF4 . A star indicates an unspecific band . U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h . Thereafter , whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot . U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol . Thereafter , whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes ( OXPHOS ) by standard western blot . U87 cells transduced with non - targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h . Thereafter , extracellular flux analysis was performed in the context of a mitochondrial stress assay . From this assay , basal respiration and coupled respiration was calculated ( M - N ) . U87 cells transduced with non - targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h . RNA and protein were collected and analyzed for the expression of LXRβ mRNA ( O ) or LXRβ protein by capillary electrophoresis ( P ) . Shown are means and SD ( n = 5 ) . Statistical significance was determined by two - sided student ' s t . test .

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

Figure 6U87 cells were treated with LXR623 for 1h, 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (A-C). From this assay, coupled respiration was calculated (D-F).U87 cells were treated with LXR623, cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (G). From this assay, mitochondrial OCR and coupled respiration was calculated (H-I).U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point. Thereafter, whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes (OXPHOS), Noxa, and ATF4. A star indicates an unspecific band.U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h. Thereafter, whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot.U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes (OXPHOS) by standard western blot.U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. Thereafter, extracellular flux analysis was performed in the context of a mitochondrial stress assay. From this assay, basal respiration and coupled respiration was calculated (M-N).U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. RNA and protein were collected and analyzed for the expression of LXRβ mRNA (O) or LXRβ protein by capillary electrophoresis (P). Shown are means and SD (n = 5). Statistical significance was determined by two-sided student's t.test.


Figure 71x106 HCT116 colon carcinoma cells were implanted subcutaneously . Animals were treated intraperitoneally with vehicle , LXR623 ( 100 - 200 mg / kg ) , ABT263 ( 100 mg / kg ) or both agents ( 3 days per week for 1 . 5 weeks . Tumor growth curves show the development of tumor size for each treatment group . Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment . Shown are means and SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0063 , * * * * p < 0 . 0001 . Statistical significance was determined by one - way ANOVA . B , E , GBM43 PDXs were implanted subcutaneously . Animals were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 weeks ) . Tumor growth curves show the development of tumor size for each treatment group . Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment . Shown are means and SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0031 . Statistical significance was determined by one - way ANOVA . C , F , A375 BRAF V600E mutated melanomas were implanted subcutaneously . Animals were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 weeks ) . Tumor growth curves show the development of tumor size for each treatment group . Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment . Shown are means and SD ( n ≥ 5 ) . * p = 0 . 0109 ( GW3965 vs Combination ) , * p = 0 . 0166 ( ABT263 vs Combination ) , * * p = 0 . 0047 . Statistical significance was determined by one - way ANOVA . Related to the HCT116 colonic xenograft model , representative histopathological images ( hematoxylin and eosin stain ) are shown from each treatment group . Scale bar : 50 µm . Sections from the same treatment groups as in G were stained with TUNEL and SDHB , repectively . Displayed are representative images of the staining . Scale bar 50 µM . Sections from the experiment in 7A were quantified for apoptotic bodies , Sections from the experiment in 7A were quantified for , number of TUNEL positive cellsSections from the experiment in 7A were quantified for number of mitosis per multiple high - power fields .

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

Figure 71x106 HCT116 colon carcinoma cells were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100-200 mg/kg), ABT263 (100 mg/kg) or both agents (3 days per week for 1.5 weeks. Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0063, ****p<0.0001. Statistical significance was determined by one-way ANOVA. B, E, GBM43 PDXs were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0031. Statistical significance was determined by one-way ANOVA. C, F, A375 BRAF V600E mutated melanomas were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.0109 (GW3965 vs Combination), *p=0.0166 (ABT263 vs Combination), **p=0.0047. Statistical significance was determined by one-way ANOVA. Related to the HCT116 colonic xenograft model, representative histopathological images (hematoxylin and eosin stain) are shown from each treatment group. Scale bar: 50 µm. Sections from the same treatment groups as in G were stained with TUNEL and SDHB, repectively. Displayed are representative images of the staining. Scale bar 50 µM.Sections from the experiment in 7A were quantified for apoptotic bodies, Sections from the experiment in 7A were quantified for , number of TUNEL positive cellsSections from the experiment in 7A were quantified for number of mitosis per multiple high-power fields.


Figure 1C ) MiNA analysis of HeLa cells stained with the membrane potential - independent dye MitoTracker ™ Green ( MTG , 100 nM ) to visualize and determine the mitochondrial area ( = mitochondrial footprint ) . Left : HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Co - localization shown in yellow . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . D ) Left : HeLa IF1 - KO cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . E ) Left : HeLa IF1 - H49K cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . Co - localization shown in yellow . Scale bars = 10 µm . F )

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 1C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm. F)


Figure 2A ) BN - PAGE and subsequent immuno - blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1 . Monomers ( M ) , dimers ( D ) and oligomers ( O ) were detected . Left panel : Coomassie - stained gel . Right panels : Immunoblotting and detection of ATP synthase and IF1 . WT : wild type HeLa cells ; IF * : cells expressing IF1 - H49K . Stars indicate oligomeric IF1 forms . I : complex I ; III : complex III . D ) Assembly of sEcGFP - tagged subunits CoxVIIIa - sEcGFP ( CIV # ) into complex IV , and assembly of subunits γ - sEcGFP ( SUγ # ) and SU e - sEcGFP ( Sue # ) into ATP synthase . Proteins were separated by BN - PAGE and immunoblotted . Lanes # 1 - # 10 were cut into three pieces for further evaluation . Lanes 1 - 2 : complex IV assembly in WT and CoxVIIIa - sEcGFP - expressing cells , detection of CIV with anti - Cox1 . Lanes 3 - 4 : complex V assembly in WT and SUγ - sEcGFP - expressing cells , detection with anti - SU γ . Lanes 8 - 10 : assembly of CIV # into CIV , SU γ - sEcGFP ( SUγ # ) into CV , and SU e - sEcGFP ( SUe # ) into CV . At lane # 6 in the Coomassie - stained gel , the positions of molecular weight markers ( M ) are shown . SC : respiratory supercomplexes ; CIV2 : complex IV dimers ; CIV : complex IV monomer ; s1 : F1 - c8 subcomplexes ; s2 : subcomplexes of CV containing subunits b , e and g He et al . , 2018 ( ) . Blue * * : dimeric F1F0 ATP synthase ; blue * : monomeric F1F0 ATP synthase . Red asterisks : respiratory

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Figure 2A) BN-PAGE and subsequent immuno-blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1. Monomers (M), dimers (D) and oligomers (O) were detected. Left panel: Coomassie-stained gel. Right panels: Immunoblotting and detection of ATP synthase and IF1. WT: wild type HeLa cells; IF*: cells expressing IF1-H49K. Stars indicate oligomeric IF1 forms. I: complex I; III: complex III.D) Assembly of sEcGFP-tagged subunits CoxVIIIa-sEcGFP (CIV#) into complex IV, and assembly of subunits γ-sEcGFP (SUγ#) and SU e-sEcGFP (Sue#) into ATP synthase. Proteins were separated by BN-PAGE and immunoblotted. Lanes #1-#10 were cut into three pieces for further evaluation. Lanes 1-2: complex IV assembly in WT and CoxVIIIa-sEcGFP-expressing cells, detection of CIV with anti-Cox1. Lanes 3-4: complex V assembly in WT and SUγ-sEcGFP-expressing cells, detection with anti-SU γ. Lanes 8-10: assembly of CIV# into CIV, SU γ-sEcGFP (SUγ#) into CV, and SU e-sEcGFP (SUe#) into CV. At lane #6 in the Coomassie-stained gel, the positions of molecular weight markers (M) are shown. SC: respiratory supercomplexes; CIV2: complex IV dimers; CIV: complex IV monomer; s1: F1-c8 subcomplexes; s2: subcomplexes of CV containing subunits b, e and g He et al., 2018(). Blue **: dimeric F1F0 ATP synthase; blue*: monomeric F1F0 ATP synthase. Red asterisks: respiratory supercomplexes


Figure 3A , A ' Trajectory maps of ATP synthase labeled at SU γ ( A ) and SU g ( A ' ) in single mitochondria . Insets : Detailed views of the diffusion of CV - SU γ ( A ) and CV - SU g ( A ' ) . Scale bars : 1 μm overview and 0 . 5 μm insets .

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Figure 3A,A' Trajectory maps of ATP synthase labeled at SU γ (A) and SU g (A') in single mitochondria. Insets: Detailed views of the diffusion of CV-SU γ (A) and CV-SU g (A'). Scale bars: 1 μm overview and 0.5 μm insets.


Figure 4B ) Diffusion coefficients for ATP synthase under control conditions ( 5 . 6 mM glucose ) , and under OXPHOS conditions . The probability density function ( PDF ) and cumulative density function ( CDF ) of - log D is plotted . Number of trajectories analyzed : 8622 ( Glc ) and 10333 ( Gal ) from mitochondria in at least 5 different cells .

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Figure 4B) Diffusion coefficients for ATP synthase under control conditions (5.6 mM glucose), and under OXPHOS conditions. The probability density function (PDF) and cumulative density function (CDF) of -log D is plotted. Number of trajectories analyzed: 8622 (Glc) and 10333 (Gal) from mitochondria in at least 5 different cells.


Figure 5C ) Images showing matrix pH equilibration with extracellular pH . Shown are ratiometric fluorescence images of MPP - sEcGFP before and after addition of the uncouplers and inhibitors FCCP , nigericin and oligomycin ( plus 0 . 002 % w / v Triton X - 100 where indicated ) . False color images ( λ511 / λ464 ) indicate ratio values . D ) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH = 8 , pH = 7 . 4 and pH = 6 . 5 . Scale bars = 10 µm .

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Figure 5C) Images showing matrix pH equilibration with extracellular pH. Shown are ratiometric fluorescence images of MPP-sEcGFP before and after addition of the uncouplers and inhibitors FCCP, nigericin and oligomycin (plus 0.002 % w/v Triton X-100 where indicated). False color images (λ511/λ464) indicate ratio values. D) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH=8, pH=7.4 and pH=6.5. Scale bars = 10 µm. E


Figure 6B ) Mitochondrial pH values at SU e , SU γ and in the matrix bulk in hyper - glycolytic cells ( high glucose : HGlc , 25 mM ) . C ) pH at SU e , SU γ and in the matrix in respiring cells ( Gal , 10 mM ) . D ) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain .

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Figure 6B) Mitochondrial pH values at SU e, SU γ and in the matrix bulk in hyper-glycolytic cells (high glucose: HGlc, 25 mM). C) pH at SU e , SU γ and in the matrix in respiring cells (Gal, 10 mM). D) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain. Da


Figure 7A ) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic ( HGlc ) and respiring ( Gal ) conditions . Where indicated , 5 µg / mL oligomycin was added to inhibit CV activity . B ) Mitochondrial pH at CV SU e in IF1 - KO cells . Conditions : glycolytic ( HGlc ) and respiring ( Gal ) with and without oligomycin . C ) Mitochondrial pH at CV SU e in IF1 - OE cells . Conditions : glycolytic ( HGlc ) and respiring ( Gal ) with and without oligomycin . D ) F ) Effect of inhibiting the respiratory chain at complex III ( CIII ) with antimycin A ( AA ) on the local pH at SU e in WT cells ( glycolytic conditions ) . G ) Local pH at SU e in IF1 - KO cells after inhibiting CIII . H ) Local pH at SU e in IF1 - OE cells after inhibiting CIII .

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Figure 7A) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions. Where indicated, 5 µg/mL oligomycin was added to inhibit CV activity. B) Mitochondrial pH at CV SU e in IF1-KO cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. C) Mitochondrial pH at CV SU e in IF1-OE cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. D)F) Effect of inhibiting the respiratory chain at complex III (CIII) with antimycin A (AA) on the local pH at SU e in WT cells (glycolytic conditions). G) Local pH at SU e in IF1-KO cells after inhibiting CIII. H) Local pH at SU e in IF1-OE cells after inhibiting CIII. Da


Figure 8A ) Mitochondrial pH at CV SU e is compared for HeLa WT , IF1 - KO and IF1 - H49K cells under glycolytic and respiring conditions . D ) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT , IF1 - KO and IF1 - H49K cells under glycolytic and respiring conditions . J ) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ . The pH was color coded according to respective emission ratio . The pH at SU γ in IF1 - H49K - expressing cells is compared with WT cells . ( Scale bar : 1 µm ) .

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Figure 8A) Mitochondrial pH at CV SU e is compared for HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.D) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.J) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ. The pH was color coded according to respective emission ratio. The pH at SU γ in IF1-H49K-expressing cells is compared with WT cells. (Scale bar: 1 µm).


Figure 9A ) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1 . Oxygen consumption rates ( OCR ) and extracellular acidification rates ( ECAR ) were determined with an automatic flux analyzer ( Seahorse / Agilent ) . After monitoring basal respiration , oligomycin ( 1 µM ) , and rotenone plus antimycin A ( Rot ; 0 . 5 µM ; AA , 0 . 5 µM ) were added sequentially to determine glycolytic and mitochondrial ATP production rates . ~ 12 technical replicates per measurement , N = 4 independent assays .

Figure 9A) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined with an automatic flux analyzer (Seahorse/Agilent). After monitoring basal respiration, oligomycin (1 µM), and rotenone plus antimycin A (Rot; 0.5 µM; AA, 0.5 µM) were added sequentially to determine glycolytic and mitochondrial ATP production rates. ~12 technical replicates per measurement, N=4 independent assays.


Figure 1 ( A ) MYB knockdown ( MiaPaCa - shMYB and Panc1 - shMYB ) and forced MYB - overexpressing ( BxPC3 - MYB ) PC cells along with their respective control cell lines ( MiaPaCa - Scr , Panc1 - Scr , and BxPC3 - Neo ) were subjected to hypoxia ( 1 % O2 ) treatment for different time intervals ( 0 - 96 h ) . Their growth was measured by viable cell counting using a trypan blue exclusion assay . ( E , F ) ECAR ( E ) and OCR ( F ) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 ( 100 μM for 4 h ) . Thereafter , ECAR was measured in PC cells with sequential injection of glucose , oligomycin , and 2 - DG , while OCR was measured with the serial addition of oligomycin , FCCP , and antimycin A / rotenone .

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Figure 1(A) MYB knockdown (MiaPaCa-shMYB and Panc1-shMYB) and forced MYB-overexpressing (BxPC3-MYB) PC cells along with their respective control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were subjected to hypoxia (1% O2) treatment for different time intervals (0-96 h). Their growth was measured by viable cell counting using a trypan blue exclusion assay.(E, F) ECAR (E) and OCR (F) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 (100 μM for 4 h). Thereafter, ECAR was measured in PC cells with sequential injection of glucose, oligomycin, and 2-DG, while OCR was measured with the serial addition of oligomycin, FCCP, and antimycin A/rotenone.


Figure 2 ( A ) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals . HIF1α accumulation was examined as a positive control . MYB protein ( C ) levels were measured in the prostate ( LNCaP ) , lung ( A549 ) , breast ( MB231 ) , and colon ( HCT116 ) cancer cells cultured under normoxia and hypoxia ( 6 h ) by immunoblotting respectively . β - actin served as an internal control . N - normoxia and H - hypoxia . ( E ) Pancreatic cancer cells were transiently transfected either with non - targeted control siRNA ( siCtrl ) or HIF1α - targeting siRNA ( siHIF1α ) for 24 h and then incubated under hypoxia for 6 h . MYB expression was analyzed at protein and mRNA levels . ( F ) Cells were treated with either vehicle or CoCl2 ( 100µM ) for 6 h , and total protein and RNA were isolated . The expression of HIF1α and MYB was examined . β - actin was used as an internal control .

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Figure 2(A) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals. HIF1α accumulation was examined as a positive control.MYB protein (C) levels were measured in the prostate (LNCaP), lung (A549), breast (MB231), and colon (HCT116) cancer cells cultured under normoxia and hypoxia (6 h) by immunoblotting respectively. β-actin served as an internal control. N- normoxia and H- hypoxia.(E) Pancreatic cancer cells were transiently transfected either with non-targeted control siRNA (siCtrl) or HIF1α -targeting siRNA (siHIF1α) for 24 h and then incubated under hypoxia for 6 h. MYB expression was analyzed at protein and mRNA levels.(F) Cells were treated with either vehicle or CoCl2 (100µM) for 6 h, and total protein and RNA were isolated. The expression of HIF1α and MYB was examined. β-actin was used as an internal control.


Figure 3 ( B ) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h . HuR ( green ) , and nucleus ( blue ) . Scale bar , 25 μm ( lower left corner ) . Lower panels are digitally magnified views of the marked rectangles . ( C ) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting . α - Tubulin ( nuclear ) and Lamin A ( cytoplasm ) were used as loading controls . ( G ) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h , and MYB and HuR expression was determined by immunoblotting or qRT - PCR .

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Figure 3(B) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h. HuR (green), and nucleus (blue). Scale bar, 25 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles.(C) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting. α-Tubulin (nuclear) and Lamin A (cytoplasm) were used as loading controls.(G) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h, and MYB and HuR expression was determined by immunoblotting or qRT-PCR.


Figure 4 ( A ) MYB - silenced ( MiaPaCa - shMYB and Panc1 - shMYB ) and MYB - overexpressing ( BxPC3 - MYB ) PC cells , along with their control cell lines ( MiaPaCa - Scr , Panc1 - Scr , and BxPC3 - Neo ) were exposed to hypoxia for 6 h . Protein lysates were made , and the expression of MYB and HIF1α was examined by immunoblotting . β - actin served as a loading control . ( C ) The transcriptional activity of the HIF1α promoter was determined by performing the promoter - reporter assay . MYB - modulated PC cell lines were transfected with the reporter plasmid , and after 24 h of transfection , cells were cultured under hypoxia or normoxia for 24 h , and the supernatant was collected . HIF1α promoter - driven Gaussia luciferase activity was normalized with SEAP activity , and the data was presented as a bar diagram . ( E ) MYB - silenced or - overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti - MYB antibodies or IgG control followed by qPCR analysis using site - specific primers .

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Figure 4(A) MYB-silenced (MiaPaCa-shMYB and Panc1-shMYB) and MYB-overexpressing (BxPC3-MYB) PC cells, along with their control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were exposed to hypoxia for 6 h. Protein lysates were made, and the expression of MYB and HIF1α was examined by immunoblotting. β-actin served as a loading control.(C) The transcriptional activity of the HIF1α promoter was determined by performing the promoter-reporter assay. MYB-modulated PC cell lines were transfected with the reporter plasmid, and after 24 h of transfection, cells were cultured under hypoxia or normoxia for 24 h, and the supernatant was collected. HIF1α promoter-driven Gaussia luciferase activity was normalized with SEAP activity, and the data was presented as a bar diagram.(E) MYB-silenced or -overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti-MYB antibodies or IgG control followed by qPCR analysis using site-specific primers.


Figure 5 ( A ) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB - silenced ( shMYB ) and those having a forced overexpression of HIF1α ( shMYB - HIF1αOE ) along with their respective controls ( Scr and shMYB - Neo ) cultured under hypoxia . β - actin served as a loading control . ( C ) Live / Dead staining was performed in MYB - silenced MiaPaCa and Panc - 1 or MYB silenced - HIF1α overexpressed cells cultured under hypoxia for 48 h . Cells were visualized under a fluorescence microscope . Representative fluorescence micrographs are presented . The number of live and dead cells was counted from the captured random images . Scale bar , 150 μm ( lower right corner ) . ( F ) MYB - silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h . Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels . The data was normalized with the number of cells and presented as relative fold changes .

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Figure 5(A) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB-silenced (shMYB) and those having a forced overexpression of HIF1α (shMYB-HIF1αOE) along with their respective controls (Scr and shMYB-Neo) cultured under hypoxia. β-actin served as a loading control.(C) Live/Dead staining was performed in MYB-silenced MiaPaCa and Panc-1 or MYB silenced- HIF1α overexpressed cells cultured under hypoxia for 48 h. Cells were visualized under a fluorescence microscope. Representative fluorescence micrographs are presented. The number of live and dead cells was counted from the captured random images. Scale bar, 150 μm (lower right corner).(F) MYB-silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h. Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels. The data was normalized with the number of cells and presented as relative fold changes.


Figure 6 ( C ) Quantitative ChIP - qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes ( GLUT3 , HK2 , PFKL , ENO2 , and MCT4 ) using anti - MYB or IgG control antibodies . The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia . p300 ( E ) , and H3K9ac ( F ) on the promoter regions were also confirmed by ChIP - qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG .

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Figure 6(C) Quantitative ChIP-qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes (GLUT3, HK2, PFKL, ENO2, and MCT4) using anti-MYB or IgG control antibodies. The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia.p300 (E), and H3K9ac (F) on the promoter regions were also confirmed by ChIP- qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG.