Figure 7analysis of the most prominent bacterial phyla ( B ; n = 4 Control , 6 - 8 Antibiotics ; multiple t - tests with Holm - Sidak method ) by qPCR analysis , In B , the cycle number of 16S rDNA expression compared to the total number of cycles is shown . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant . Scale bars : 100μm . Graphs showing the relative weight ( C ; n = 11 WT , 7 ECko , 10 ECko + antibiotics one - way ANOVA with tukey ' s multiple comparison ) upon tamoxifen treatment in the presence of an antibiotic cocktail . Graphs showing the survival of mice ( D ; n = 6 WT , 7 ECko , 7 ECko + antibiotics ; Log - Rank test ) upon tamoxifen treatment in the presence of an antibiotic cocktail . E . Representative images of the gut of Casp8WT and Casp8ECko mice with antibiotic treatment 30 days after tamoxifen . F - H . Representative images of H & E staining ( F ) , quantification of intestinal pathology ( G ) and mucosal hemorrhages ( H ) in Casp8WT and Casp8ECko mice treated with antibiotics compared to untreated Casp8ECko mice ( G ; n = 6 WT , 4 ECko + antibiotics , 5 ECko ; one - way ANOVA with tukey ' s multiple comparison , H ; n = 6 WT , 4 ECko + antibiotics , 4 ECko ; one - way ANOVA with tukey ' s multiple comparison ) . Data information : Data shown as mean ± SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant . Scale bars : 100μm .

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

Figure 7analysis of the most prominent bacterial phyla (B; n= 4 Control, 6-8 Antibiotics; multiple t-tests with Holm-Sidak method) by qPCR analysis, In B, the cycle number of 16S rDNA expression compared to the total number of cycles is shown. Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.Graphs showing the relative weight (C; n= 11 WT, 7 ECko, 10 ECko+ antibiotics one-way ANOVA with tukey's multiple comparison) upon tamoxifen treatment in the presence of an antibiotic cocktail.Graphs showing the survival of mice (D; n= 6 WT, 7 ECko, 7 ECko+ antibiotics; Log-Rank test) upon tamoxifen treatment in the presence of an antibiotic cocktail.E. Representative images of the gut of Casp8WT and Casp8ECko mice with antibiotic treatment 30 days after tamoxifen.F-H. Representative images of H&E staining (F), quantification of intestinal pathology (G) and mucosal hemorrhages (H) in Casp8WT and Casp8ECko mice treated with antibiotics compared to untreated Casp8ECko mice (G; n= 6 WT, 4 ECko+ antibiotics, 5 ECko; one-way ANOVA with tukey's multiple comparison, H; n= 6 WT, 4 ECko+ antibiotics, 4 ECko; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. ** P <0.01, *** P <0.001, **** P <0.0001; ns: not significant. Scale bars: 100μm.


Figure 1C , Demonstration of single - molecule tracking in a single cell . Cell outline is shown as an ellipse . Left , Imaged FliM - YPet fluorescence . White dots indicate the estimated motor locations . White circles illustrate a 75 nm radius around these locations . Right , Contour map of the normalized sum of probabilities of the localization events of CheY ( I95V ) - Atto647 ( from blue to red is low to high ; black is zero probability ) . White circles show the motors ' locations from the left panel . I , CheY interacts with FliM ∆ N and this interaction is sensitive to chemotactic stimuli . Each curve is the mean of two FRET measurements of cells in response to an attractant stimulus ( 0 . 1 mM serine ; in the case of FliMwt , a single measurement was performed with serine ; another measurement of FliMwt with 1mM aspartate produced similar results ) . FRET ratio is the ratio of mCherry to YPet fluorescence . CheY - mCherry and mCherry concentrations were ~ 170 µM in the case of FliM ∆ N and ~ 15 µM in the case of FliMwt Strains used : EW677 ( FliMwt , red ) , EW659 ( negative control of FliM ∆ N without CheY , purple ) , EW637 ( FliM ∆ N , blue ) , and EW636 ( FliM ∆ N ∆ cheA , yellow ) .

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

Figure 1C, Demonstration of single-molecule tracking in a single cell. Cell outline is shown as an ellipse. Left, Imaged FliM-YPet fluorescence. White dots indicate the estimated motor locations. White circles illustrate a 75 nm radius around these locations. Right, Contour map of the normalized sum of probabilities of the localization events of CheY(I95V)-Atto647 (from blue to red is low to high; black is zero probability). White circles show the motors' locations from the left panel.I, CheY interacts with FliM∆N and this interaction is sensitive to chemotactic stimuli. Each curve is the mean of two FRET measurements of cells in response to an attractant stimulus (0.1 mM serine; in the case of FliMwt, a single measurement was performed with serine; another measurement of FliMwt with 1mM aspartate produced similar results). FRET ratio is the ratio of mCherry to YPet fluorescence. CheY-mCherry and mCherry concentrations were ~170 µM in the case of FliM∆N and ~15 µM in the case of FliMwt Strains used: EW677 (FliMwt, red), EW659 (negative control of FliM∆N without CheY, purple), EW637 (FliM∆N, blue), and EW636 (FliM∆N ∆cheA, yellow).


Figure 2B , Demonstration of rotation rate measured from a single cell ; negative and positive values ( below and above the blue bar ) are counterclockwise and clockwise , respectively . The switch from negative to positive rotation rate at 3 s is shown in the montage to the right . C , The response of tethered fliM ∆ N ΔcheZ cells ( strain EW635 ) , induced for CheY expression from a plasmid ( 200 µM IPTG ) , to positive stimuli . Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The arrow marks the estimated time at which the stimulant arrived to , or left ( as indicated ) , the flow chamber . N is the number of cells . α - Methyl - DL - aspartate ( MeAsp ) and leucine were used at 1 mM , benzoate ( pH 7 . 0 ) at 50 mM . The response of tethered fliM ∆ N ΔcheZ cells ( strain EW635 ) , induced for CheY expression from a plasmid ( 200 µM IPTG ) Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The arrow marks the estimated time at which the stimulant arrived to , or left ( as indicated ) , the flow chamber . N is the number of cells . α - Methyl - DL - aspartate ( MeAsp ) and leucine were used at 1 mM , benzoate ( pH 7 . 0 ) at 50 mM . D , negative stimuli . E , Response of tethered fliM ∆ N ΔcheA cells ( strain EW634 ) to acetate ( 50 mM , pH 7 . 0 ) at different CheY concentrations . Lines and shaded regions are mean ± SEM . F , Response of tethered fliM ∆ N ΔcheZ cells ( strain EW635 ) to acetate . G , Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation . The points are the mean clockwise rotation at time segments 0 - 20 s and 160 - 180 s . The variants shown are fliM ∆ N ∆ cheZ cells expressing CheY in the absence of presence of acetate ( 10 mM , pH 7 . 0 ) ( i . e . , CheY ~ P and CheY ~ P ~ Ac ; blue and green curves ; strain EW635 ) , fliM ∆ N ∆ cheA cells expressing CheY , in the absence of presence of acetate ( CheY and CheY ~ Ac ; purple and red , respectively ; EW634 ) , and fliM ∆ N ∆ cheA cells expressing CheY ( D13K ) ( burgundy ; EW737 ) . Each data point is the average of all experiments at a given CheY concentration , weighted by the sample number of each experiment . The CheY concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used . H , FliMN further activates CheY variants to produce clockwise rotation . The variants shown are fliM ∆ N ∆ cheZ cells expressing FliMN - CheY ( i . e . , FliMN - CheY ~ P ; blue curve ; strain EW697 ) , fliM ∆ N ∆ cheA cells expressing FliMN - CheY , in the absence of presence of acetate ( 10 mM , pH 7 . 0 ) ( FliMN - CheY and FliMN - CheY ~ Ac ; purple and red , respectively ; EW696 ) , and fliM ∆ N ∆ cheA cells expressing FliMN - CheY ( D13K ) ( burgundy ; EW739 ) . I , Response of fliM ∆ N ∆ cheA cells expressing FliMN - CheY ( strain EW696 ) to acetate and benzoate ( 10 mM each ; pH 7 . 0 ) . FliMN - CheY expression was induced with 800 μM IPTG . Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The black arrow indicates the estimated time point at which the stimulus entered the flow chamber .

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

Figure 2B, Demonstration of rotation rate measured from a single cell; negative and positive values (below and above the blue bar) are counterclockwise and clockwise, respectively. The switch from negative to positive rotation rate at 3 s is shown in the montage to the right.C, The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG), to positive stimuli. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM.The response of tethered fliM∆N ΔcheZ cells (strain EW635), induced for CheY expression from a plasmid (200 µM IPTG) Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrow marks the estimated time at which the stimulant arrived to, or left (as indicated), the flow chamber. N is the number of cells. α-Methyl-DL-aspartate (MeAsp) and leucine were used at 1 mM, benzoate (pH 7.0) at 50 mM. D, negative stimuli.E, Response of tethered fliM∆N ΔcheA cells (strain EW634) to acetate (50 mM, pH 7.0) at different CheY concentrations. Lines and shaded regions are mean ± SEM.F, Response of tethered fliM∆N ΔcheZ cells (strain EW635) to acetate.G, Contribution of acetylation and phosphorylation of CheY as well as its D13K mutation to clockwise rotation. The points are the mean clockwise rotation at time segments 0-20 s and 160-180 s. The variants shown are fliM∆N ∆cheZ cells expressing CheY in the absence of presence of acetate (10 mM, pH 7.0) (i.e., CheY~P and CheY~P~Ac; blue and green curves; strain EW635), fliM∆N ∆cheA cells expressing CheY, in the absence of presence of acetate (CheY and CheY~Ac; purple and red, respectively; EW634), and fliM∆N ∆cheA cells expressing CheY(D13K) (burgundy; EW737). Each data point is the average of all experiments at a given CheY concentration, weighted by the sample number of each experiment. The CheY concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used.H, FliMN further activates CheY variants to produce clockwise rotation. The variants shown are fliM∆N ∆cheZ cells expressing FliMN-CheY (i.e., FliMN-CheY~P; blue curve; strain EW697), fliM∆N ∆cheA cells expressing FliMN-CheY, in the absence of presence of acetate (10 mM, pH 7.0) (FliMN-CheY and FliMN-CheY~Ac; purple and red, respectively; EW696), and fliM∆N ∆cheA cells expressing FliMN-CheY(D13K) (burgundy; EW739). I, Response of fliM∆N ∆cheA cells expressing FliMN-CheY (strain EW696) to acetate and benzoate (10 mM each; pH 7.0). FliMN-CheY expression was induced with 800 μM IPTG. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The black arrow indicates the estimated time point at which the stimulus entered the flow chamber.


Figure 3 , Dependence of clockwise rotation on the level of FliMN - CheY ~ Ac . The red and purple curves are two experimental days in which the clockwise production by fliM ∆ N ∆ cheA cells expressing FliMN - CheY was measured as a function of FliMN - CheY concentration ( strain EW696 ; Concentrations are estimates based on the calibration curves or which a similar CheY expression system was used . ach data point is a mean ± SEM of the data or the second and third experimental days of this strain ( in the presence of acetate ) . B , Response of the strain to addition and removal of varying acetate concentrations ( pH 7 . 0 ) . FliMN - CheY concentration was estimated to be ~ 300 μM . Lines and shaded regions are the mean time spent in clockwise rotation ± SEM . The arrows indicate the estimated time points at which acetate entered or left the flow chamber . Black lines mark the linear part of the biphasic response to acetate removal . N is the number of cells . E , Average clockwise interval length , calculated as the inverse of the fast ( blue ) and slow ( red ) rate

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

Figure 3, Dependence of clockwise rotation on the level of FliMN-CheY~Ac. The red and purple curves are two experimental days in which the clockwise production by fliM∆N ∆cheA cells expressing FliMN-CheY was measured as a function of FliMN-CheY concentration (strain EW696; Concentrations are estimates based on the calibration curves or which a similar CheY expression system was used. ach data point is a mean ± SEM of the data or the second and third experimental days of this strain (in the presence of acetate).B, Response of the strain to addition and removal of varying acetate concentrations (pH 7.0). FliMN-CheY concentration was estimated to be ~300 μM. Lines and shaded regions are the mean time spent in clockwise rotation ± SEM. The arrows indicate the estimated time points at which acetate entered or left the flow chamber. Black lines mark the linear part of the biphasic response to acetate removal. N is the number of cells.E, Average clockwise interval length, calculated as the inverse of the fast (blue) and slow (red) rate constants


Figure 4A , Effects of FliMN - CheY acetylation and phosphorylation on clockwise rotation of tethered cells containing FliM ∆ N FliN ( A93D ) motors . Acetate concentration was 10 mM each ( pH 7 . 0 ) . FliMN - CheY and FliMN - CheY ~ P were produced by using ∆ cheA and ∆ cheZ backgrounds ( strains EW713 and EW714 ) , respectively . Each data point is the average of all measurements at a given FliMN - CheY concentration , weighted by the sample number of each experiment . The concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used . Concentrations larger than 500 μM were calculated by the linear extrapolation of the calibration curve . B , Distribution of clockwise interval lengths at different average clockwise levels in ∆ cheA cells containing FliM ∆ N FliN ( A93D ) motors and expressing FliMN - CheY ( strain EW713 ) , in the presence of acetate ( 10 mM , pH 7 . 0 ) . C , Average clockwise interval length , calculated as the inverse of the rate constants from mono - phasic fits of the distributions in ( B ) . D , Response of tethered ∆ cheZ cells having FliM ∆ N FliN ( A93D ) motors to acetate addition and removal ( red ; strain EW714 ; FliMN - CheY concentration estimated at ~ 300 μM ) . Similar results were obtained with a ∆ cheA strain ( EW713 ) . Tethered cells having FliM ∆ N FliNwt motors and containing FliMN - CheY ( strain EW696 ) are shown Acetate concentration was 10 mM ( pH 7 . 0 ) . The data shown are the mean ± SEM . N is the number of cells . Black lines indicate the clockwise - decay rates of FliNwt motors ' slow phase and of FliN ( A93D ) motors following acetate removal .

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

Figure 4A, Effects of FliMN-CheY acetylation and phosphorylation on clockwise rotation of tethered cells containing FliM∆N FliN(A93D) motors. Acetate concentration was 10 mM each (pH 7.0). FliMN-CheY and FliMN-CheY~P were produced by using ∆cheA and ∆cheZ backgrounds (strains EW713 and EW714), respectively. Each data point is the average of all measurements at a given FliMN-CheY concentration, weighted by the sample number of each experiment. The concentrations shown are estimates based on the calibration curves for which a similar CheY expression system was used. Concentrations larger than 500 μM were calculated by the linear extrapolation of the calibration curve. B, Distribution of clockwise interval lengths at different average clockwise levels in ∆cheA cells containing FliM∆N FliN(A93D) motors and expressing FliMN-CheY (strain EW713), in the presence of acetate (10 mM, pH 7.0). C, Average clockwise interval length, calculated as the inverse of the rate constants from mono-phasic fits of the distributions in (B).D, Response of tethered ∆cheZ cells having FliM∆N FliN(A93D) motors to acetate addition and removal (red; strain EW714; FliMN-CheY concentration estimated at ~300 μM). Similar results were obtained with a ∆cheA strain (EW713). Tethered cells having FliM∆N FliNwt motors and containing FliMN-CheY (strain EW696) are shown Acetate concentration was 10 mM (pH 7.0). The data shown are the mean ± SEM. N is the number of cells. Black lines indicate the clockwise-decay rates of FliNwt motors' slow phase and of FliN(A93D) motors following acetate removal.


Figure 5A , CheY crosslinks with FliM ∆ N - YPet in vivo . The cytoplasm of fliM ∆ N - YPet ΔcheZ cells overexpressing CheY ( strain EW694 ) or FliMN - CheY ( strain EW697 ) from a plasmid was crosslinked by glutaraldehyde and resolved by SDS - PAGE . 1 , 2 , 3 stand for three different experiments that underwent this procedure . The plus sign before ~ 14 and ~ 15 stands for + 55 kDa of FliM ∆ N - YPet . Note that gel running suffered from parabolic distortion in band positions . The annotations relate to the lowest positions of the bands . The gel was imaged for FliM ∆ N - YPet fluorescence . To see the crosslinking products , the intense fluorescence of monomeric FliM ∆ N - YPet is shown at saturation . C , Clockwise rotation of tethered fliM ( R94S ) ∆ N ∆ cheZ cells and fliM ( R94L ) ∆ N ∆ cheZ cells expressing FliMN - CheY ( strains EW731 and EW733 , respectively ) in the absence or presence of acetate ( 10 mM , pH 7 . 0 ) . FliMN - CheY concentration was estimated to be ~ 4 mM by extrapolation of the calibration curves for which a similar CheY expression system was used . No clockwise rotation was observed when benzoate substituted for acetate . Each data point is the mean of a separate experiment . Black line is the mean of all experiments . D , Clockwise rotation of tethered fliM ( E214W ) ∆ N ΔcheZ cells ( strain EW718 ) at various concentrations of FliMN - CheY . The concentrations shown are estimates based on the calibration curve , for which a similar CheY expression system was used . Each data point is the average of all measurements at similar FliMN - CheY concentrations , weighted by the sample number of each experiment .

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

Figure 5A, CheY crosslinks with FliM∆N-YPet in vivo. The cytoplasm of fliM∆N-YPet ΔcheZ cells overexpressing CheY (strain EW694) or FliMN-CheY (strain EW697) from a plasmid was crosslinked by glutaraldehyde and resolved by SDS-PAGE. 1, 2, 3 stand for three different experiments that underwent this procedure. The plus sign before ~14 and ~15 stands for +55 kDa of FliM∆N-YPet. Note that gel running suffered from parabolic distortion in band positions. The annotations relate to the lowest positions of the bands. The gel was imaged for FliM∆N-YPet fluorescence. To see the crosslinking products, the intense fluorescence of monomeric FliM∆N-YPet is shown at saturation.C, Clockwise rotation of tethered fliM(R94S)∆N ∆cheZ cells and fliM(R94L)∆N ∆cheZ cells expressing FliMN-CheY (strains EW731 and EW733, respectively) in the absence or presence of acetate (10 mM, pH 7.0). FliMN-CheY concentration was estimated to be ~4 mM by extrapolation of the calibration curves for which a similar CheY expression system was used. No clockwise rotation was observed when benzoate substituted for acetate. Each data point is the mean of a separate experiment. Black line is the mean of all experiments.D, Clockwise rotation of tethered fliM(E214W)∆N ΔcheZ cells (strain EW718) at various concentrations of FliMN-CheY. The concentrations shown are estimates based on the calibration curve , for which a similar CheY expression system was used. Each data point is the average of all measurements at similar FliMN-CheY concentrations, weighted by the sample number of each experiment.


Figure 1 ( A ) Levels of KRAS , COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in KRASWT , KRASG12D , and KRAS - silenced KRASG12D MEF cells . ( B ) Extracellular acidification rate ( ECAR ) in KRASWT , KRAS G12D , and KRAS - or COUP - TFII - silenced KRAS G12D MEF cells . ( C ) Lactate levels in MEF cells Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in KRASWT MEF . * * * p < 0 . 001 . ( D ) Levels of COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in COUP - TFII - silenced KRASG12D MEF cells .

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

Figure 1(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRASWT, KRASG12D, and KRAS-silenced KRASG12D MEF cells.(B) Extracellular acidification rate (ECAR) in KRASWT, KRAS G12D, and KRAS- or COUP-TFII-silenced KRAS G12D MEF cells.(C) Lactate levels in MEF cells Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in KRASWT MEF. ***p<0.001.(D) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced KRASG12D MEF cells.


Figure 2 ( A ) Levels of KRAS , COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in KRAS - silenced HCT116 and DLD - 1 cells . ( B ) Levels of COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in COUP - TFII - silenced HCT116 and DLD - 1 cells . ( C ) Effect of lactate on phosphorylated S6K ( T389 ) , 4E - BP1 ( T37 / 46 ) , ULK1 ( S757 ) in COUP - TFII - silenced HCT116 cells . ( D ) Effects of COUP - TFII and the inhibition of LDHA on phosphorylated S6K ( T389 ) and Raptor ( S792 ) in KRAS - silenced HCT116 cells . Asterisk indicates exogenous HA - COUP - TFII . Arrow indicates phosphorylated Raptor ( S792 ) . ( E ) Inhibition of COUP - TFII - silenced HCT116 , DLD - 1 , and MIA PaCa - 2 cell proliferation and its recovery by treatment with lactate . Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control . * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 . ( F ) Clonogenic assay of COUP - TFII - silenced HCT116 , DLD - 1 , and MIA PaCa - 2 cells in the presence or absence of lactate .

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Figure 2(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRAS-silenced HCT116 and DLD-1 cells.(B) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced HCT116 and DLD-1 cells.(C) Effect of lactate on phosphorylated S6K (T389), 4E-BP1 (T37/46), ULK1 (S757) in COUP-TFII-silenced HCT116 cells.(D) Effects of COUP-TFII and the inhibition of LDHA on phosphorylated S6K (T389) and Raptor (S792) in KRAS-silenced HCT116 cells. Asterisk indicates exogenous HA-COUP-TFII. Arrow indicates phosphorylated Raptor (S792).(E) Inhibition of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cell proliferation and its recovery by treatment with lactate. Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control. *p<0.05, **p<0.01, and ***p<0.001.(F) Clonogenic assay of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cells in the presence or absence of lactate.


Figure 3 ( A ) Effect of lactate on the ERK / TSC2 and AKT / TSC2 signaling pathway in serum - starved HCT116 cells . ( B ) Effect of lactate on phosphorylated S6K ( T389 ) inhibited by TSC2 overexpression in HCT116 cells . ( C ) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA - Rheb - overexpressing HCT116 cells after serum deprivation overnight . ( D ) Molecular interaction between TSC2 and recombinant GDP - loaded Rheb in the presence or absence of lactate in vitro . ( E ) Effect of lactate on Rheb activation in serum - starved HCT116 cells . ( F ) Interaction between endogenous Rheb and mTOR in the presence or absence of lactate in HCT116 cells after serum deprivation overnight . ( G ) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight .

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Figure 3(A) Effect of lactate on the ERK/TSC2 and AKT/TSC2 signaling pathway in serum-starved HCT116 cells.(B) Effect of lactate on phosphorylated S6K (T389) inhibited by TSC2 overexpression in HCT116 cells.(C) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb-overexpressing HCT116 cells after serum deprivation overnight.(D) Molecular interaction between TSC2 and recombinant GDP-loaded Rheb in the presence or absence of lactate in vitro.(E) Effect of lactate on Rheb activation in serum-starved HCT116 cells.(F) Interaction between endogenous Rheb and mTOR in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.(G) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.


Figure 4 ( A ) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP - TFII - silenced HCT116 cells . ( B ) Effect of lactate on Rheb activation in COUP - TFII - silenced HCT 116 cells . ( C ) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM . * * * p < 0 . 001 . ( D ) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA - Rheb - and / or COUP - TFII - overexpressing HCT116 cells treated with 25 nM trametinib for 24 h . ( E ) Effects of lactate and COUP - TFII overexpression on Rheb activation in HCT116 cells treated with 25 nM trametinib for 24 h . ( F ) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM . * * * p < 0 . 001 .

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Figure 4(A) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP-TFII-silenced HCT116 cells.(B) Effect of lactate on Rheb activation in COUP-TFII-silenced HCT 116 cells.(C) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001.(D) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb- and/or COUP-TFII-overexpressing HCT116 cells treated with 25 nM trametinib for 24 h.(E) Effects of lactate and COUP-TFII overexpression on Rheb activation in HCT116 cells treated with 25 nM trametinib for 24 h.(F) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001.


Figure 5Effects of COUP - TFII inhibition and lactate treatment on tumor growth in vivo . ( A ) Growth curve in tumors derived from xenografted shScramble or shCOUP - TFII - HCT116 cells and treated with sodium chloride or sodium L - lactate ( 60 l x g - 1 body weight ; 150 mM ) . ( B ) tumor volume at the experimental endpoint , in tumors derived from xenografted shScramble or shCOUP - TFII - HCT116 cells and treated with sodium chloride or sodium L - lactate ( 60 l x g - 1 body weight ; 150 mM ) . Effects of COUP - TFII inhibition and lactate treatment on tumor growth in vivo . ( C ) Western blot analysis of the indicated proteins in tumors derived from xenografted shScramble or shCOUP - TFII - HCT116 cells and treated with sodium chloride or sodium L - lactate ( 60 l x g - 1 body weight ; 150 mM ) . ( D ) Effects of trametinib treatment and overexpression of COUP - TFII on tumor growth in vivo . Tumors were derived from xenografted HCT116 expressing HA - COUP - TFII or carrying a control vector and treated with trametinib ( 3mg / kg ) . ( E ) Western blot analysis of indicated proteins in tumors derived from xenografted HCT116 treated with trametinib with or without COUP - TFII overexpression . Asterisk indicates exogenous HA - COUP - TFII .

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Figure 5Effects of COUP-TFII inhibition and lactate treatment on tumor growth in vivo. (A) Growth curve in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).(B) tumor volume at the experimental endpoint, in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).Effects of COUP-TFII inhibition and lactate treatment on tumor growth in vivo. (C) Western blot analysis of the indicated proteins in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).(D) Effects of trametinib treatment and overexpression of COUP-TFII on tumor growth in vivo. Tumors were derived from xenografted HCT116 expressing HA-COUP-TFII or carrying a control vector and treated with trametinib (3mg/kg).(E) Western blot analysis of indicated proteins in tumors derived from xenografted HCT116 treated with trametinib with or without COUP-TFII overexpression. Asterisk indicates exogenous HA-COUP-TFII.


Figure 1Bioluminescence imaging and quantification of brain metastases ( H ) of BALB / c nude mice xenografted with control or IFITM1 - knockout H1299 cells ( populations of knockout cells ) by intracardiac injection ( 1 × 106 cells ) . ( The n - values denote the number of mice per group , and 4 independent Western blot experiments were performed . ) ( H , quantification of brain metastasesBioluminescence imaging overall survival ( I ) of BALB / c nude mice xenografted with control or IFITM1 - knockout H1299 cells ( populations of knockout cells ) by intracardiac injection ( 1 × 106 cells ) . # , experimental endpoint ( I ) . , the log - rank test ( I , survival ( J ) Bioluminescence imaging and quantification of brain metastases and overall survivals of BALB / c nude mice xenografted with control or IFITM1 - overexpressing H460 cells by intracardiac injection ( 1 × 105 cells ) . ( The n - values denote the number of mice per group , and 4 independent Western blot experiments were performed . ) the log - rank test survival in J ( N ) Kaplan - Meier survival curves of lung cancer patients in the TCGA dataset ( n = 415 ) stratified by IFITM1 mRNA expression . HR , hazard ratio . the log - rank test survival in N )

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Figure 1Bioluminescence imaging and quantification of brain metastases (H) of BALB/c nude mice xenografted with control or IFITM1-knockout H1299 cells (populations of knockout cells) by intracardiac injection (1×106 cells). (The n-values denote the number of mice per group, and 4 independent Western blot experiments were performed.) (H, quantification of brain metastasesBioluminescence imaging overall survival (I) of BALB/c nude mice xenografted with control or IFITM1-knockout H1299 cells (populations of knockout cells) by intracardiac injection (1×106 cells). #, experimental endpoint (I). , the log-rank test (I, survival(J) Bioluminescence imaging and quantification of brain metastases and overall survivals of BALB/c nude mice xenografted with control or IFITM1-overexpressing H460 cells by intracardiac injection (1×105 cells). (The n-values denote the number of mice per group, and 4 independent Western blot experiments were performed.) the log-rank test survival in J(N) Kaplan-Meier survival curves of lung cancer patients in the TCGA dataset (n=415) stratified by IFITM1 mRNA expression. HR, hazard ratio. the log-rank test survival in N)


Figure 2 ( A ) Representative immunofluorescence images and quantification of microglial ( TMEM119 + ) or macrophage ( TMEM119 - / F4 / 80 + ) density per microscopic field in the brain on day 1 or 7 after the intracardiac injection of control or IFITM1 - overexpressing LLC - BrM cells ( tdTomato , 1 × 106 ) into C57BL / 6 mice ( 90 , 90 , and 120 fields for normal , day 1 , and day 7 , respectively ; normal , 3 mice ; day 1 , 3 mice ; day 7 , 4 mice ) . Normal brain tissue was used as the control . ( C ) Flow cytometric analysis of violet - labeled LLC - BrM cells phagocytosed by microglia in the brains of LLC - BrM cell - allografted BALB / c nude mice on day 1 after intracardiac injection ( 1 × 106 control or IFITM1 - knockout cells ) . Representative flow cytometry pseudocolor density plots and the gating strategy are shown , and the bar graph shows the percentage of violet + microglia . ( The n - values denote the number of mice per group . ) ( E ) LLC - BrM cells ( tdTomato ) were surrounded by activated microglia ( Iba1 + , green ) and IFNγ ( white ) in the brain on day 1 or 7 after intracardiac injection of control or IFITM1 - overexpressing cells ( 1 × 106 ) into C57BL / 6 mice . IFNγ intensities in the fields enclosed in the yellow dotted lines ( ROIs ) ( 90 , 90 , and 120 fields for normal , day 1 , and day 7 , respectively ) are shown . The ROI was set as the entire area of the metastatic lesion for day 7 and as the area of single metastatic cell for day 1 . ImageJ was used to quantify the immunofluorescence intensity in the ROI . Normal brain tissue was used as the control . ROI , region of interest . ( G ) Results of ELISA for mouse IFNγ in the brain on day 1 after the intracardiac injection of control or IFITM1 - knockout LLC - BrM cells ( 1 × 106 ) into BALB / c nude mice . PLX5622 ( 1 , 200 PPM ) was administered in chow from day - 3 to day 1 . BLZ945 ( 200 mg / kg ) was administered by oral gavage from day - 2 to day 1 . Minocycline ( 33 mg / kg ) was administered intraperitoneally on days - 1 , 0 , and 1 . Clodronate liposomes ( 100 μl / 10 g ) were administered intraperitoneally on day - 1 . Normal brain tissue was used as the control . ( The n - values denote the number of mice per group . )

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

Figure 2(A) Representative immunofluorescence images and quantification of microglial (TMEM119+) or macrophage (TMEM119-/F4/80+) density per microscopic field in the brain on day 1 or 7 after the intracardiac injection of control or IFITM1-overexpressing LLC-BrM cells (tdTomato, 1×106) into C57BL/6 mice (90, 90, and 120 fields for normal, day 1, and day 7, respectively; normal, 3 mice; day 1, 3 mice; day 7, 4 mice). Normal brain tissue was used as the control.(C) Flow cytometric analysis of violet-labeled LLC-BrM cells phagocytosed by microglia in the brains of LLC-BrM cell-allografted BALB/c nude mice on day 1 after intracardiac injection (1×106 control or IFITM1-knockout cells). Representative flow cytometry pseudocolor density plots and the gating strategy are shown, and the bar graph shows the percentage of violet+ microglia. (The n-values denote the number of mice per group.)(E) LLC-BrM cells (tdTomato) were surrounded by activated microglia (Iba1+, green) and IFNγ (white) in the brain on day 1 or 7 after intracardiac injection of control or IFITM1-overexpressing cells (1×106) into C57BL/6 mice. IFNγ intensities in the fields enclosed in the yellow dotted lines (ROIs) (90, 90, and 120 fields for normal, day 1, and day 7, respectively) are shown. The ROI was set as the entire area of the metastatic lesion for day 7 and as the area of single metastatic cell for day 1. ImageJ was used to quantify the immunofluorescence intensity in the ROI. Normal brain tissue was used as the control. ROI, region of interest.(G) Results of ELISA for mouse IFNγ in the brain on day 1 after the intracardiac injection of control or IFITM1-knockout LLC-BrM cells (1×106) into BALB/c nude mice. PLX5622 (1,200 PPM) was administered in chow from day -3 to day 1. BLZ945 (200 mg/kg) was administered by oral gavage from day -2 to day 1. Minocycline (33 mg/kg) was administered intraperitoneally on days -1, 0, and 1. Clodronate liposomes (100 μl/10 g) were administered intraperitoneally on day -1. Normal brain tissue was used as the control. (The n-values denote the number of mice per group.)


Figure 3 ( A ) ELISA results of mouse TNFα in supernatants obtained from the indicated coculture systems at the indicated time points . Primary mouse microglia , 6 × 104 cells from 16 BALB / c mice . H460 , 6 × 104 cells . CM , conditioned medium ( from 6 × 104 H460 cells ) ( 1 independent experiment ) . We would like to point out that TNFα , instead of IFNγ , was used as a marker of microglial activation in vitro ( E ) ELISA results of mouse IFNγ in the brain on day 1 after intracardiac injection of the indicated LLC - BrM cells ( control , C3 knockout , IFITM1 overexpression , or IFITM1 overexpression in combination with C3 knockout , 1 × 106 ) into C57BL / 6 mice . Normal brain tissue was used as the control . ( The n - values denote the number of mice per group . ) ( G ) Control and IFITM1 - overexpressing LLC - BrM cells ( 1 × 106 , tdTomato ) were surrounded by activated microglia ( left , green , Iba1 + ) and complement C3 ( left , white ) in the brain on day 1 after intracardiac injection into C57BL / 6 mice ( normal , 3 mice ; day 1 , 3 mice ; day 7 , 4 mice ) . Complement C3 intensities in the fields enclosed in the yellow dotted lines ( ROIs ) are shown ( right ; 90 , 90 , and 120 fields for normal , day 1 , and day 7 , respectively ) . The ROI was set as the area of a single metastatic cell . ImageJ was used to quantify the immunofluorescence intensity in the ROI . Normal brain tissue was used as the control . ROI , region of interest . We would like to point out that TNFα , instead of IFNγ , was used as a marker of microglial activation in vitro ( H ) Results of ELISA for mouse TNFα in the culture supernatant of primary mouse microglia ( 6 × 104 , from 16 C57BL / 6 mice ) , primary mouse BMDMs ( 6 × 104 , from 3 C57BL / 6 mice ) , or primary mouse BrM - BMDMs ( 6 × 104 , from 37 C57BL / 6 mice ) stimulated with conditioned medium from control LLC - BrM cells ( 1 × 104 ) and IFITM1 - knockout LLC - BrM cells ( 1 × 104 ) . BMDMs , primary bone marrow - derived macrophages . BrM - BMDMs , primary bone marrow - derived macrophages from the brain metastatic lesions of LLC - BrM cell - isografted C57BL / 6 mice on day 20 after the intracardiac injection of LLC - BrM cells ( 1 × 105 cells ) . CM , conditioned medium ( 3 independent experiments ) . ( L ) Flow cytometric analysis of apoptotic LLC - BrM cells ( 6 × 104 ) in contact coculture with primary mouse microglia ( 6 × 104 , from 16 C57BL / 6 mice ) , which were activated by conditioned medium from control or C3 - knockdown H460 cells ( 1 × 106 ) . CM , conditioned medium ( 3 independent experiments ) .

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

Figure 3(A) ELISA results of mouse TNFα in supernatants obtained from the indicated coculture systems at the indicated time points. Primary mouse microglia, 6×104 cells from 16 BALB/c mice. H460, 6×104 cells. CM, conditioned medium (from 6×104 H460 cells) (1 independent experiment). We would like to point out that TNFα, instead of IFNγ, was used as a marker of microglial activation in vitro(E) ELISA results of mouse IFNγ in the brain on day 1 after intracardiac injection of the indicated LLC-BrM cells (control, C3 knockout, IFITM1 overexpression, or IFITM1 overexpression in combination with C3 knockout, 1×106) into C57BL/6 mice. Normal brain tissue was used as the control. (The n-values denote the number of mice per group.)(G) Control and IFITM1-overexpressing LLC-BrM cells (1×106, tdTomato) were surrounded by activated microglia (left, green, Iba1+) and complement C3 (left, white) in the brain on day 1 after intracardiac injection into C57BL/6 mice (normal, 3 mice; day 1, 3 mice; day 7, 4 mice). Complement C3 intensities in the fields enclosed in the yellow dotted lines (ROIs) are shown (right; 90, 90, and 120 fields for normal, day 1, and day 7, respectively). The ROI was set as the area of a single metastatic cell. ImageJ was used to quantify the immunofluorescence intensity in the ROI. Normal brain tissue was used as the control. ROI, region of interest.We would like to point out that TNFα, instead of IFNγ, was used as a marker of microglial activation in vitro (H) Results of ELISA for mouse TNFα in the culture supernatant of primary mouse microglia (6×104, from 16 C57BL/6 mice), primary mouse BMDMs (6×104, from 3 C57BL/6 mice), or primary mouse BrM-BMDMs (6×104, from 37 C57BL/6 mice) stimulated with conditioned medium from control LLC-BrM cells (1×104) and IFITM1-knockout LLC-BrM cells (1×104). BMDMs, primary bone marrow-derived macrophages. BrM-BMDMs, primary bone marrow-derived macrophages from the brain metastatic lesions of LLC-BrM cell-isografted C57BL/6 mice on day 20 after the intracardiac injection of LLC-BrM cells (1×105 cells). CM, conditioned medium (3 independent experiments).(L) Flow cytometric analysis of apoptotic LLC-BrM cells (6×104) in contact coculture with primary mouse microglia (6×104, from 16 C57BL/6 mice), which were activated by conditioned medium from control or C3-knockdown H460 cells (1×106). CM, conditioned medium (3 independent experiments).


Figure 4 ( A ) Schematic of efficacy of adoptive CD8 + T cell immunotherapy in suppressing brain metastasis ( top far left ) , representative western blots of IFITM1 expression in control or IFITM1 - overexpressing LLC - BrM cells ( bottom far left ) , bioluminescence imaging ( middle left ) , quantification of brain metastases ( middle right ) and overall survivals ( far right ) of BALB / c nude mice allografted with control or IFITM1 - overexpressing LLC - BrM cells by intracardiac injection ( 1 × 105 cells ) . Primary mouse naïve CD8 + T cells from 12 C57BL / 6 mice or not were injected intravenously on days - 1 , 6 , and 13 ( 1 × 104 cells at each time point ) . ( The n - values denote the number of mice per group , and 4 independent Western blot experiments were performed . ) ( B ) Schematic of efficacy of adoptive CD8 + or CD4 + T cell immunotherapy in suppressing brain metastasis ( far left ) , bioluminescence imaging ( middle left ) , quantification of brain metastases ( middle right ) and overall survivals ( far right ) of BALB / c nude mice allografted with control or IFITM1 - overexpressing LLC - BrM cells by intracardiac injection ( 1 × 105 cells ) . Primary mouse naïve CD8 + or CD4 + T cells from 12 C57BL / 6 mice were injected intravenously on days - 1 , 6 , and 13 ( 1 × 104 cells at each time point ) . ( The n - values denote the number of mice per group . ) ( quantification of brain metastases ANOVA with uncorrected Fisher ' s LSD test or the log - rank test ( survival ( G ) Bioluminescence imaging and quantification of brain metastases on day 20 in C57BL / 6 mice isografted with control or IFITM1 - overexpressing LLC - BrM cells by intracardiac injection ( 3 × 105 cells ) . Mice were systemically primed - boosted with homologous LLC - BrM cell lysates ( 1 × 106 cells for day - 21 and 1 × 105 cells for day - 7 ) before injection of cancer cells . Then , IgG or an anti - CD8α antibody ( 2 μg / mouse ) was injected intravenously on day 3 alone or in combination with minocycline treatment ( 33 mg / kg ) on day 1 . Minocycline was administered intraperitoneally . ( The n - values denote the number of mice per group . ) one - way ( G )

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

Figure 4(A) Schematic of efficacy of adoptive CD8+ T cell immunotherapy in suppressing brain metastasis (top far left), representative western blots of IFITM1 expression in control or IFITM1-overexpressing LLC-BrM cells (bottom far left), bioluminescence imaging (middle left), quantification of brain metastases (middle right) and overall survivals (far right) of BALB/c nude mice allografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (1×105 cells). Primary mouse naïve CD8+ T cells from 12 C57BL/6 mice or not were injected intravenously on days -1, 6, and 13 (1×104 cells at each time point). (The n-values denote the number of mice per group, and 4 independent Western blot experiments were performed.) (B) Schematic of efficacy of adoptive CD8+ or CD4+ T cell immunotherapy in suppressing brain metastasis (far left), bioluminescence imaging (middle left), quantification of brain metastases (middle right) and overall survivals (far right) of BALB/c nude mice allografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (1×105 cells). Primary mouse naïve CD8+ or CD4+ T cells from 12 C57BL/6 mice were injected intravenously on days -1, 6, and 13 (1×104 cells at each time point). (The n-values denote the number of mice per group.) (quantification of brain metastases ANOVA with uncorrected Fisher's LSD test or the log-rank test (survival(G) Bioluminescence imaging and quantification of brain metastases on day 20 in C57BL/6 mice isografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (3×105 cells). Mice were systemically primed-boosted with homologous LLC-BrM cell lysates (1×106 cells for day -21 and 1×105 cells for day -7) before injection of cancer cells. Then, IgG or an anti-CD8α antibody (2 μg/mouse) was injected intravenously on day 3 alone or in combination with minocycline treatment (33 mg/kg) on day 1. Minocycline was administered intraperitoneally. (The n-values denote the number of mice per group.) one-way (G)


Figure 5 ( C ) Immunofluorescence staining of H1299 cells with anti - IFITM1 and anti - MHC - I antibodies . H1299 cells ( 6 × 104 ) were stimulated with 1 , 000 U / ml IFNγ for 48 hr and were then treated with the anti - MHC - I antibody for 1 hr . The colocalization percentages of IFITM1 ( red ) and MHC - I ( green ) are shown ( 100 cells for each condition ) . : The data are presented as the mean ± s . e . m . values . P values were determined by an unpaired two - tailed Student ' s t - test with Welch ' s correction ( C ( D ) Co - IP of endogenous IFITM1 and MHC - I proteins in H1299 cells ( 1 × 106 ) after IFNγ stimulation ( 1 , 000 U / ml ) for 48 hr ( 3 independent experiments ) . ( I ) Immunoblot analysis of MHC - I expression in the cytoplasm , on the cell membrane , and in the nucleus of control and IFITM1 - overexpressing H1299 cells ( 6 × 104 ) ( 3 independent experiments ) . ( K ) qPCR analysis of MHC - I mRNA expression in control and IFITM1 - overexpressing H1299 cells ( 6 × 104 ) with or without IFNγ stimulation ( 1 , 000 U / ml ) for 24 hr ( 3 independent experiments ) . ( L ) qPCR analysis of MHC - I mRNA expression in control and IFITM1 - knockdown H1299 cells ( 6 × 104 ) with or without IFNγ stimulation ( 1 , 000 U / ml ) for 24 hr ( 3 independent experiments ) . ( M ) Cleaved caspase 3 ( green ) - positive LLC - BrM cells ( tdTomato ) were surrounded by CD8 + T cells ( white ) in the brain metastatic lesions of LLC - BrM cell - isografted C57BL / 6 mice on day 20 after intracardiac injection of control or IFITM1 - overexpressing LLC - BrM cells ( 1 × 106 cells ) . The positively stained areas of single CD8 + T cells , clustered CD8 + T cells , cleaved caspase 3 + cancer cells , and tdTomato + cancer cells in the ROI were calculated by ImageJ . The ratios of the areas between the clustered and single CD8 + T cells are shown as the numbers of CD8 + T cells in the ROI , and the ratios of the areas between cleaved caspase 3 + cancer cells and tdTomato + cancer cells are shown as the percentages of cleaved caspase 3 + cancer cells in the ROI ( 90 fields per group , 3 mice per group ) . Yellow dotted lines ( ROIs ) , brain metastatic lesions ; ROI , region of interest . ( O ) Kaplan - Meier survival curves of lung cancer patients in the TCGA dataset stratified by IFITM1 and MHC - I mRNA expression . HR , hazard ratio . ANOVA with uncorrected Fisher ' s LSD test , or the log - rank test ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 5(C) Immunofluorescence staining of H1299 cells with anti-IFITM1 and anti-MHC-I antibodies. H1299 cells (6×104) were stimulated with 1,000 U/ml IFNγ for 48 hr and were then treated with the anti-MHC-I antibody for 1 hr. The colocalization percentages of IFITM1 (red) and MHC-I (green) are shown (100 cells for each condition). : The data are presented as the mean ± s.e.m. values. P values were determined by an unpaired two-tailed Student's t-test with Welch's correction (C(D) Co-IP of endogenous IFITM1 and MHC-I proteins in H1299 cells (1×106) after IFNγ stimulation (1,000 U/ml) for 48 hr (3 independent experiments).(I) Immunoblot analysis of MHC-I expression in the cytoplasm, on the cell membrane, and in the nucleus of control and IFITM1-overexpressing H1299 cells (6×104) (3 independent experiments).(K) qPCR analysis of MHC-I mRNA expression in control and IFITM1-overexpressing H1299 cells (6×104) with or without IFNγ stimulation (1,000 U/ml) for 24 hr (3 independent experiments). (L) qPCR analysis of MHC-I mRNA expression in control and IFITM1-knockdown H1299 cells (6×104) with or without IFNγ stimulation (1,000 U/ml) for 24 hr (3 independent experiments).(M) Cleaved caspase 3 (green)-positive LLC-BrM cells (tdTomato) were surrounded by CD8+ T cells (white) in the brain metastatic lesions of LLC-BrM cell-isografted C57BL/6 mice on day 20 after intracardiac injection of control or IFITM1-overexpressing LLC-BrM cells (1×106 cells). The positively stained areas of single CD8+ T cells, clustered CD8+ T cells, cleaved caspase 3+ cancer cells, and tdTomato+ cancer cells in the ROI were calculated by ImageJ. The ratios of the areas between the clustered and single CD8+ T cells are shown as the numbers of CD8+ T cells in the ROI, and the ratios of the areas between cleaved caspase 3+ cancer cells and tdTomato+ cancer cells are shown as the percentages of cleaved caspase 3+ cancer cells in the ROI (90 fields per group, 3 mice per group). Yellow dotted lines (ROIs), brain metastatic lesions; ROI, region of interest.(O) Kaplan-Meier survival curves of lung cancer patients in the TCGA dataset stratified by IFITM1 and MHC-I mRNA expression. HR, hazard ratio. ANOVA with uncorrected Fisher's LSD test, or the log-rank test (O).


Figure 6 ( F ) Bioluminescence imaging , quantification of brain metastases , and overall survival of C57BL / 6 mice isografted with control or IFITM1 - overexpressing LLC - BrM cells by intracardiac injection ( 3 × 105 cells ) . Mice were systemically primed - boosted with homologous LLC - BrM cell lysates ( 1 × 106 cells for day - 21 and 1 × 105 cells for day - 7 ) before injection of cancer cells , and IgG or an anti - PD - 1 antibody ( 100 μg / mouse ) was then injected intravenously on days 3 , 6 , and 9 . ( The n - values denote the number of mice per group . ) unpaired two - way ( quantification of brain metastases in F ) ANOVA with uncorrected Fisher ' s LSD test , or the log - rank test ( survival in F ) . ( H ) Bioluminescence imaging and quantification of brain metastases in C57BL / 6 mice isografted with control or IFITM1 - knockout LLC - BrM cells by intracardiac injection ( 3 × 105 cells ) . IgG ( 30 μg / mouse , days 3 , 6 , and 9 ) , an anti - PD - 1 antibody ( 30 μg / mouse , days 3 , 6 , and 9 ) , or UV - irradiated oncolytic virus ( 106 pfu / mouse , days 0 , 2 , and 4 ) was injected intravenously alone or in combination as indicated . ( The n - values denote the number of mice per group . ) one - way ( quantification of brain metastases in H )

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

Figure 6(F) Bioluminescence imaging, quantification of brain metastases, and overall survival of C57BL/6 mice isografted with control or IFITM1-overexpressing LLC-BrM cells by intracardiac injection (3×105 cells). Mice were systemically primed-boosted with homologous LLC-BrM cell lysates (1×106 cells for day -21 and 1×105 cells for day -7) before injection of cancer cells, and IgG or an anti-PD-1 antibody (100 μg/mouse) was then injected intravenously on days 3, 6, and 9. (The n-values denote the number of mice per group.) unpaired two-way (quantification of brain metastases in F) ANOVA with uncorrected Fisher's LSD test, or the log-rank test (survival in F).(H) Bioluminescence imaging and quantification of brain metastases in C57BL/6 mice isografted with control or IFITM1-knockout LLC-BrM cells by intracardiac injection (3×105 cells). IgG (30 μg/mouse, days 3, 6, and 9), an anti-PD-1 antibody (30 μg/mouse, days 3, 6, and 9), or UV-irradiated oncolytic virus (106 pfu/mouse, days 0, 2, and 4) was injected intravenously alone or in combination as indicated. (The n-values denote the number of mice per group.) one-way (quantification of brain metastases in H)


Figure 1A , B Hypocotyl length of ChWT , ( A ) RNAi - ChHFR1 transgenic and ( B ) chfr1 mutant seedlings grown under different R : FR . Seedlings were grown for 7 days in continuous W ( R : FR > 1 . 5 ) or for 3 days in W then transferred to W supplemented with increasing amounts of FR ( W + FR ) for 4 more days , producing various R : FR . Aspect of representative 7 - day old ChWT , RNAi - HFR1 and chfr1 - 1 seedlings grown in W or W + FR ( R : FR , 0 . 02 ) , as indicated , is shown in lower panel . Data information : Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h . Asterisks mark significant differences ( Student t - test : * * p - value < 0 . 01 ; * p - value < 0 . 05 ) relative to ChWT value at the same time point . C , D Effect of W + FR exposure on the expression of PIL1 , YUC8 and XTR7 genes in seedlings of ChWT , ( C ) RNAi - HFR1 and ( D ) chfr1 mutant lines . Expression was analyzed in 7 - day old W - grown seedlings transferred to W + FR ( R : FR , 0 . 02 ) for 0 , 1 , 4 , 8 and 12 h . Transcript abundance is normalized to EF1α levels . Data information : Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h . Asterisks mark significant differences ( Student t - test : * * p - value < 0 . 01 ; * p - value < 0 . 05 ) relative to ChWT value at the same time point .

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

Figure 1A, B Hypocotyl length of ChWT, (A) RNAi-ChHFR1 transgenic and (B) chfr1 mutant seedlings grown under different R:FR. Seedlings were grown for 7 days in continuous W (R:FR>1.5) or for 3 days in W then transferred to W supplemented with increasing amounts of FR (W+FR) for 4 more days, producing various R:FR. Aspect of representative 7-day old ChWT, RNAi-HFR1 and chfr1-1 seedlings grown in W or W+FR (R:FR, 0.02), as indicated, is shown in lower panel. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point.C, D Effect of W+FR exposure on the expression of PIL1, YUC8 and XTR7 genes in seedlings of ChWT, (C) RNAi-HFR1 and (D) chfr1 mutant lines. Expression was analyzed in 7-day old W-grown seedlings transferred to W+FR (R:FR, 0.02) for 0, 1, 4, 8 and 12 h. Transcript abundance is normalized to EF1α levels. Data information: Values are the means ± SE of three independent biological replicates relative to ChWT value at 0 h. Asterisks mark significant differences (Student t-test: ** p-value <0.01; * p-value <0.05) relative to ChWT value at the same time point.


Figure 2Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under the same conditions or transferred to W + FR ( R : FR , 0 . 02 ) for the indicated times . Plant material was harvested every 24 h . Transcript abundance of HFR1 and PIF7 was normalized to three reference genes ( EF1α , SPC25 , and YLS8 ) . Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3 . Asterisks mark significant differences ( 2 - way ANOVA : * * p - value < 0 . 01 , * * * p - value < 0 . 001 ) between ChWT and AtWT when grown under W ( black asterisks ) or W + FR ( red asterisks ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under the same conditions or transferred to W+FR (R:FR, 0.02) for the indicated times. Plant material was harvested every 24 h. Transcript abundance of HFR1 and PIF7 was normalized to three reference genes (EF1α, SPC25, and YLS8). Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: ** p-value <0.01, *** p-value <0.001) between ChWT and AtWT when grown under W (black asterisks) or W+FR (red asterisks).


Figure 3B Relative expression of HFR1 in seedlings of AtWT , At hfr1 - 5 , hfr1 > pAt : ChHFR1 ( in blue ) and hfr1 > pAt : AtHFR1 ( in red ) lines grown under W + FR ( R : FR , 0 . 02 ) . Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT . Transcript abundance is normalized to UBQ10 levels . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey test , p - value < 0 . 05 ) among means . C Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W + FR ( R : FR , 0 . 02 ) . The mean hypocotyl length in W ( HypW ) and W + FR ( HypW + FR ) of at least four biological replicates was used to calculate HypW + FR - HypW . Error bars represent SE . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey test , p - value < 0 . 05 ) among means . D Relative HFR1 protein levels in seedlings of the indicated lines , normalized to actin protein levels , are the means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 line # 22 that is taken as 1 . Seedlings were grown for 7 days in continuous W ( ~ 20 µmol m - 2 s - 1 ) after which they were incubated for 3 h in high W ( ~ 100 µmol m - 2 s - 1 ) and transferred to W + FR ( R : FR , 0 . 06 ) for 3 h . Data information : Different letters denote significant differences ( one - way ANOVA with Tukey test , p - value < 0 . 05 ) among means .

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

Figure 3B Relative expression of HFR1 in seedlings of AtWT, At hfr1-5, hfr1>pAt:ChHFR1 (in blue) and hfr1>pAt:AtHFR1 (in red) lines grown under W+FR (R:FR, 0.02). Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT. Transcript abundance is normalized to UBQ10 levels. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.C Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W+FR (R:FR, 0.02). The mean hypocotyl length in W (HypW) and W+FR (HypW+FR) of at least four biological replicates was used to calculate HypW+FR-HypW. Error bars represent SE. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.D Relative HFR1 protein levels in seedlings of the indicated lines, normalized to actin protein levels, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and transferred to W+FR (R:FR, 0.06) for 3 h. Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.


Figure 4A Expression of HFR1 and protein levels of HFR1 - 3xHA in seedlings of hfr1 > pAt : ChHFR1 ( line # 22 ) and hfr1 > pAt : AtHFR1 ( line # 13 ) . Seedlings were grown for 7 days in continuous W ( ~ 20 µmol m - 2 s - 1 ) after which they were incubated for 3 h in high W ( ~ 100 µmol m - 2 s - 1 ) and then either kept at high W or transferred to W + FR ( R : FR , 0 . 06 ) for 3 or 6 h , as indicated in the cartoon at the top . Relative HFR1 transcript levels , normalized to UBQ10 , are the means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 # 22 grown for 3 h under W + FR . Relative protein levels , normalized to actin , are the means ± SE of three independent biological replicates relative to hfr1 > pAt : ChHFR1 # 22 . Samples were collected at data points marked in the cartoon with asterisks . C Relative HFR1 transcript levels transiently expressed in tobacco leaves , normalized to the GFP , are the means ± SE of three independent biological replicates ( left ) . Relative HFR1 protein levels , normalized to the GFP levels , are the means ± SE of four independent biological replicates ( right ) . In A and C , asterisks mark significant differences ( Student t - test : * p - value < 0 . 05 , * * p - value < 0 . 01 ) between the indicated pairs . D Degradation of ChHFR1 ( 35S : ChHFR1 ) and AtHFR1 ( 35S : AtHFR1 ) in tobacco leaf discs treated with cycloheximide ( CHX , 100 µM ) for the indicated times . Tobacco plants were kept under high W ( ~ 200 µmol m - 2 s - 1 ) for 3 days after agroinfiltration and then leaf circles were treated with W + FR ( R : FR , 0 . 2 ) and CHX . Relative HFR1 protein levels ( ChHFR1 , blue bars ; AtHFR1 , red bars ) , normalized to the GFP levels , are the means ± SE of four biological replicates relative to data point 0 , taken as 1 for each line . Asterisks mark significant differences ( 2 - way ANOVA : * p - value < 0 . 05 ) between ChHFR1 and AtHFR1 at the same time point .

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Figure 4A Expression of HFR1 and protein levels of HFR1-3xHA in seedlings of hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and then either kept at high W or transferred to W+FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 grown for 3 h under W+FR. Relative protein levels, normalized to actin, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the cartoon with asterisks.C Relative HFR1 transcript levels transiently expressed in tobacco leaves, normalized to the GFP, are the means ± SE of three independent biological replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates (right). In A and C, asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.D Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf discs treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~200 µmol m-2 s-1) for 3 days after agroinfiltration and then leaf circles were treated with W+FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2-way ANOVA: * p-value <0.05) between ChHFR1 and AtHFR1 at the same time point.


Figure 5B Table summaries of the microscale thermophoresis binding assay The sequence of the respective synthetic peptides is indicated . D Relative HFR1 protein levels , normalized to the GFP levels , are the means ± SE of four independent biological replicates . Asterisks mark significant differences ( Student t - test : * p - value < 0 . 05 , * * p - value < 0 . 01 ) between the indicated pairs .

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Figure 5B Table summaries of the microscale thermophoresis binding assay The sequence of the respective synthetic peptides is indicated.D Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.


Figure 6A Y2H growth assay showing the interaction between AtHFR1 and AtPIF7 . The BD - and the AD - derivative constructs used in the assay are shown on the left side of the panel . SD - LW or SD - HLW refer to the selective medium ( plated as drops in dilutions of 1 , 1 : 10 and 1 : 100 ) indicative of transformed cells or interaction between the hybrid proteins , respectively . Truncated forms of murine p53 ( BD - fused ) and SV40 large T - antigen ( AD - fused ) , known to interact , were used as a positive control . Empty vectors ( / ) were used as negative controls . Hypocotyl length of seedlings of AtWT , ( B ) pif7 - 1 , hfr1 - 5 , pif7 - 1 hfr1 - 5 ( top graph ) , pif7 - 2 , hfr1 - 5 and pif7 - 2 hfr1 - 5 ( bottom graph ) mutants grown under different R : FR . Seedlings were grown in W ( R : FR > 1 . 5 ) for 7 days or for 2 days in W and then transferred to two W + FR treatments ( R : FR 0 . 06 or 0 . 02 ) for 5 additional days . Values of hypocotyl length are the means ± SE of three independent biological replicates ( at least 10 seedlings per replica ) . C Hypocotyl length of seedlings of AtWT , ( C ) transgenic 35S : GFP - ΔNt - HFR1 ( 35S : ΔNt - HFR1 ) , two lines of 35S : PIF7 - CFP ( 35S : PIF7 # 1 and # 2 ) , and 35S : GFP - ΔNt - HFR1 35S : PIF7 - CFP double transgenic ( 35S : ΔNt - HFR1 x 35S : PIF7 # 1 and # 2 ) seedlings grown under different R : FR . Seedlings were grown in W ( R : FR > 1 . 5 ) for 7 days or for 2 days in W and then transferred to two W + FR treatments ( R : FR 0 . 06 or 0 . 02 ) for 5 additional days . Values of hypocotyl length are the means ± SE of three independent biological replicates ( at least 10 seedlings per replica ) . D Aspect of representative 7 - day - old W - grown seedlings shown in C . Scale bar is 1 cm .

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Figure 6A Y2H growth assay showing the interaction between AtHFR1 and AtPIF7. The BD- and the AD- derivative constructs used in the assay are shown on the left side of the panel. SD-LW or SD-HLW refer to the selective medium (plated as drops in dilutions of 1, 1:10 and 1:100) indicative of transformed cells or interaction between the hybrid proteins, respectively. Truncated forms of murine p53 (BD-fused) and SV40 large T-antigen (AD-fused), known to interact, were used as a positive control. Empty vectors (/) were used as negative controls.Hypocotyl length of seedlings of AtWT, (B) pif7-1, hfr1-5, pif7-1 hfr1-5 (top graph), pif7-2, hfr1-5 and pif7-2 hfr1-5 (bottom graph) mutants grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica).C Hypocotyl length of seedlings of AtWT, (C) transgenic 35S:GFP-ΔNt-HFR1 (35S:ΔNt-HFR1), two lines of 35S:PIF7-CFP (35S:PIF7 #1 and #2), and 35S:GFP-ΔNt-HFR1 35S:PIF7-CFP double transgenic (35S:ΔNt-HFR1 x 35S:PIF7 #1 and #2) seedlings grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica). D Aspect of representative 7-day-old W-grown seedlings shown in C. Scale bar is 1 cm.


Figure 7C Hypocotyl length of seedlings of ( left ) AtWT , pifq , pif7 - 2 , ChWT , ( middle ) 35S : GFP - ΔNt - HFR1 ( ΔNtHFR1 ) , hfr1 - 5 and ( right ) chfr1 - 1 and chfr1 - 2 lines grown at warm temperatures . Hypocotyl lengths are the means ± SE of three biological replicates . Asterisks mark significant differences ( Student t - test : * p - value < 0 . 05 , * * p - value < 0 . 01 ) relative to the same genotype grown at 22ºC ( left and right graphs , black asterisks ) , and between the indicated pairs ( middle graph , red asterisks ) . F Relative chlorophylls levels of ( left ) AtWT , pifq , ChWT , ( middle ) ΔNtHFR1 , hfr1 - 5 and ( right ) chfr1 - 1 and chfr1 - 2 lines after DIS was promoted for the indicated time . For each genotype , values are relative to pigment levels at time 0 ( 7 days in W ) . Data are the means ± SE of four independent biological replicates . G Aspect of 4 - day old dark - grown seedlings of AtWT , pifq , pif7 - 2 , hfr1 - 5 and ΔNt - HFR1 ( left panel ) , and AtWT , ChWT and chfr1 - 1 ( right panel ) .

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Figure 7C Hypocotyl length of seedlings of (left) AtWT, pifq, pif7-2, ChWT, (middle) 35S:GFP-ΔNt-HFR1 (ΔNtHFR1), hfr1-5 and (right) chfr1-1 and chfr1-2 lines grown at warm temperatures. Hypocotyl lengths are the means ± SE of three biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) relative to the same genotype grown at 22ºC (left and right graphs, black asterisks), and between the indicated pairs (middle graph, red asterisks).F Relative chlorophylls levels of (left) AtWT, pifq, ChWT, (middle) ΔNtHFR1, hfr1-5 and (right) chfr1-1 and chfr1-2 lines after DIS was promoted for the indicated time. For each genotype, values are relative to pigment levels at time 0 (7 days in W). Data are the means ± SE of four independent biological replicates.G Aspect of 4-day old dark-grown seedlings of AtWT, pifq, pif7-2, hfr1-5 and ΔNt-HFR1 (left panel), and AtWT, ChWT and chfr1-1 (right panel).


Figure 2 ( A - C ) The relative levels of ( A ) canalicular membrane ( BA ) transporter Bsep , Mrp2 , Mdr1b , Mdr2 , Abcg5 and Abcg8 mRNA transcripts , ( B ) basolateral membrane BA transporter Mrp3 , Mrp4 , Ostβ , Ntcp and Oatp1a1 mRNA transcripts , and ( C ) BA synthetic enzyme Cyp7a1 , Cyp7b1 , Cyp8b1 , Cyp27a1 , and Cyp2c70 mRNA transcripts . Wild type , wild type mice , n = 6 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 7 . Data are shown as means ± SD . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test . ( D ) Western blot analysis of the relative levels of Bsep , Mrp2 , Mdr2 , Mrp3 , Mrp4 , Ostα / β , Ntcp , Cyp7a1 and Cyp8b1 protein expression in mouse livers . Contrary to the mRNA alterations , the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers , suggesting that the decreased these proteins were might be mediated by post - translational regulation in the livers of Sema7aR145W mutant mice . Wild type , wild type mice , n = 4 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 5 . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test . ( E , F ) Multiplex IF analyses of Bsep and Mrp2 expression in the livers ( E ) and primary hepatocytes in sandwich cultures ( F ) from WT and homozygous mice . Scale bar , 50 µm . ( G ) Multiplex IF analyses revealed that transfection with the SEMA7A _ WT or SEMA7A _ R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose - dependent manner ( 0 , 0 . 2 , 1 . 0 μg per well of 6 - well plate ; 1 . 5 ml culture medium per well ) . Scale bar , 50 µm . Together , the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis ( Appendix Table S16 ) . Subsequently , cholestasis triggered an adaptive response in the liver by down - regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up - regulating the expression of BA efflux transporters Mrp3 , Mrp4 and Ostα / β .

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

Figure 2(A-C) The relative levels of (A) canalicular membrane (BA) transporter Bsep, Mrp2, Mdr1b, Mdr2, Abcg5 and Abcg8 mRNA transcripts, (B) basolateral membrane BA transporter Mrp3, Mrp4, Ostβ, Ntcp and Oatp1a1 mRNA transcripts, and (C) BA synthetic enzyme Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and Cyp2c70 mRNA transcripts. Wild type, wild type mice, n=6; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=7. Data are shown as means ± SD. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.(D) Western blot analysis of the relative levels of Bsep, Mrp2, Mdr2, Mrp3, Mrp4, Ostα/β, Ntcp, Cyp7a1 and Cyp8b1 protein expression in mouse livers. Contrary to the mRNA alterations, the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers, suggesting that the decreased these proteins were might be mediated by post-translational regulation in the livers of Sema7aR145W mutant mice. Wild type, wild type mice, n=4; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=5. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.(E, F) Multiplex IF analyses of Bsep and Mrp2 expression in the livers (E) and primary hepatocytes in sandwich cultures (F) from WT and homozygous mice. Scale bar, 50 µm.(G) Multiplex IF analyses revealed that transfection with the SEMA7A_WT or SEMA7A_R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose-dependent manner (0, 0.2, 1.0 μg per well of 6-well plate; 1.5 ml culture medium per well). Scale bar, 50 µm. Together, the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis (Appendix Table S16). Subsequently, cholestasis triggered an adaptive response in the liver by down-regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up-regulating the expression of BA efflux transporters Mrp3, Mrp4 and Ostα/β.


Figure 3Treatment with UDCA ( 13 mg / kg / d ) and GSH ( 40 mg / kg / d ) for 2 weeks restored normal liver function in the child . However , when the treatment was ceased the levels of serum ALT , AST and TBA returned to abnormally high levels in the patient . Interestingly , treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST , and reduced serum TBA in the child patient .

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Figure 3Treatment with UDCA (13 mg/kg/d) and GSH (40 mg/kg/d) for 2 weeks restored normal liver function in the child. However, when the treatment was ceased the levels of serum ALT, AST and TBA returned to abnormally high levels in the patient. Interestingly, treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST, and reduced serum TBA in the child patient.


Figure 1Transposon screen validation of UBX5 . ubx5 ∆ restores the growth of tdp1wss1 . Cells were grown in YEPD and spotted on a medium supplemented with 1 mM auxin ( for tdp1 - degron degradation ) and 5 μg / mL Camptothecin ( CPT ) . Plates were incubated for 2 days at 30 ° C . Ubx5 levels are decreased in tdp1wss1 . ( E ) HA - Ubx5 protein levels at steady state are compared by immunoblotting in different mutants . Mutant strains carrying the additional ubx5 ∆ mutation were complemented with a plasmid expressing HA - tagged Ubx5 from the endogenous pUBX5 promoter . 1mM auxin was added where indicated for 6 h to degrade tdp1 - degron , when present . Pgk1 was used as a loading control .

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Figure 1Transposon screen validation of UBX5. ubx5∆ restores the growth of tdp1wss1. Cells were grown in YEPD and spotted on a medium supplemented with 1 mM auxin (for tdp1-degron degradation) and 5 μg/mL Camptothecin (CPT). Plates were incubated for 2 days at 30°C.Ubx5 levels are decreased in tdp1wss1. (E) HA-Ubx5 protein levels at steady state are compared by immunoblotting in different mutants. Mutant strains carrying the additional ubx5∆ mutation were complemented with a plasmid expressing HA-tagged Ubx5 from the endogenous pUBX5 promoter. 1mM auxin was added where indicated for 6 h to degrade tdp1-degron, when present. Pgk1 was used as a loading control.


Figure 2The UBX ∆ mutant of Ubx5 is sufficient to suppress FA and HU sensitivities of wss1 ∆ . ubx5 ∆ wss1 ∆ mutants were complemented with plasmids encoding HA - Ubx5 - WT or HA - Ubx5 mutants overexpressed from the pGAL promoter ; cells were grown in SC - trp and either treated with 40 mM FA for 15 min or spotted directly on 20 mM HU . Plates were prepared either with glucose ( no HA - Ubx5 expression ) or galactose ( HA - Ubx5 expression ) as the source of sugar . Plates were incubated for 2 . 5 days at 30 ° C . o / e , overexpression . Protein levels of overexpressed HA - Ubx5 mutants shown in ( C ) were analyzed by immunoblotting ( as in Fig 1E ) .

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Figure 2The UBX∆ mutant of Ubx5 is sufficient to suppress FA and HU sensitivities of wss1∆. ubx5∆wss1∆ mutants were complemented with plasmids encoding HA-Ubx5-WT or HA-Ubx5 mutants overexpressed from the pGAL promoter; cells were grown in SC-trp and either treated with 40 mM FA for 15 min or spotted directly on 20 mM HU. Plates were prepared either with glucose (no HA-Ubx5 expression) or galactose (HA-Ubx5 expression) as the source of sugar. Plates were incubated for 2.5 days at 30°C. o/e, overexpression.Protein levels of overexpressed HA-Ubx5 mutants shown in (C) were analyzed by immunoblotting (as in Fig 1E).


Figure 3 ( B ) The ubx5 ∆ mutation rescues growth defects caused by Flp - nick galactose - induction in the tdp1 ∆ wss1 ∆ top1 ∆ mutant . Indicated strains were grown in YEP - 2 % raffinose prior to plating on YEP - 2 % glucose or YEP - 2 % galactose plates . Plates were incubated for 3 days at 30 ° C . ( E ) Ubx5 occupancy at the Flp - bound FRT site . Levels of Ubx5 - TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in cells grown as described in ( C ) . The graph shows enrichment of qPCR signals over the unrelated intergenic region . Data are presented as means ± SDs for n = 5 independent biological replicates . p - values for + 200 bp were defined by 2 - way ANOVA using Tukey ' s multiple comparisons test ( n . s . , non - significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) . In addition to the indicated mutations , all strains are bar1 ∆ Ubx5 - TAP . No FRT strain and raffinose ( raf ) samples were used as negative controls .

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

Figure 3(B) The ubx5∆ mutation rescues growth defects caused by Flp-nick galactose-induction in the tdp1∆ wss1∆ top1∆ mutant. Indicated strains were grown in YEP-2% raffinose prior to plating on YEP-2% glucose or YEP-2% galactose plates. Plates were incubated for 3 days at 30°C.(E) Ubx5 occupancy at the Flp-bound FRT site. Levels of Ubx5-TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in cells grown as described in (C). The graph shows enrichment of qPCR signals over the unrelated intergenic region. Data are presented as means ± SDs for n = 5 independent biological replicates. p-values for +200 bp were defined by 2-way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; *p < 0.05; ** p < 0.01; **** p < 0.0001). In addition to the indicated mutations, all strains are bar1∆ Ubx5-TAP. No FRT strain and raffinose (raf) samples were used as negative controls.