Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the complement cascade ( C ) ( n = 4 WT , 4 ECko , multiple t - test with Holm - Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the complement cascade (C) (n= 4 WT, 4 ECko, multiple t-test with Holm- Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the mucosal healing ( D ) ( n = 4 WT , 4 ECko , multiple t - test with Holm - Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

Graphs revealing that significantly upregulated cytokines in the proteome profiler in Casp8ECko mice are associated to the mucosal healing (D) (n= 4 WT, 4 ECko, multiple t-test with Holm- Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


Representative images ( E ; area between dotted lines shows crypts ) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice ( n = 5 WT , 6 ECko Scale bars : 100µm .

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

Representative images (E; area between dotted lines shows crypts) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice (n= 5 WT, 6 ECko Scale bars: 100µm.


quantification ( F ) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice ( n = 5 WT , 6 ECko two tailed unpaired Student ' s t - test ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

quantification (F) of Ki67 staining in small intestine at an early disease stage in Casp8WT and Casp8ECko mice (n= 5 WT, 6 ECko two tailed unpaired Student's t-test). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


G . QPCR analysis of the proliferation markers Ki67 and c - myc in ileal lysates from late disease stage Casp8WT and Casp8ECko mice ( n = 4 WT , 3 - 4ECko , two - way ANOVA with Sidak method ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

G. QPCR analysis of the proliferation markers Ki67 and c-myc in ileal lysates from late disease stage Casp8WT and Casp8ECko mice (n= 4 WT, 3-4ECko, two-way ANOVA with Sidak method). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.


H . Representative images of E - Cadherin staining , showing intact epithelial barrier integrity in Casp8ECko mice in healthy areas ( h ' ) , and disrupted barrier integrity in intestinal lesions ( h ' ' ) at late disease stages compared to Casp8WT mice ( n = 4 WT , 4 ECko ) . Scale bars : 100µm .

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

H. Representative images of E-Cadherin staining, showing intact epithelial barrier integrity in Casp8ECko mice in healthy areas (h'), and disrupted barrier integrity in intestinal lesions (h'') at late disease stages compared to Casp8WT mice (n= 4 WT, 4 ECko). Scale bars: 100µm.


I . Representative pictures of CD45 and NIMPR14 staining at late disease stages ( n = 5 WT , 5 ECko ) , showing accumulation of CD45 + and NIMPR14 + cells ( black arrowheads ) in Casp8ECko intestine at sites of impaired epithelial barrier integrity and tissue necrosis ( red asterisks ) . Scale bars : 100µm .

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

I. Representative pictures of CD45 and NIMPR14 staining at late disease stages (n= 5 WT, 5 ECko), showing accumulation of CD45+ and NIMPR14+ cells (black arrowheads) in Casp8ECko intestine at sites of impaired epithelial barrier integrity and tissue necrosis (red asterisks). Scale bars: 100µm.


J . Representative images of CD4 , F4 / 80 and MPO stainings in small intestines at a late disease stage showing accumulation of these cells at the crypt base in Casp8ECko mice ( arrowheads ) ( n = 4 WT , 4 ECko ) . Scale bars : 50µm .

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

J. Representative images of CD4, F4/80 and MPO stainings in small intestines at a late disease stage showing accumulation of these cells at the crypt base in Casp8ECko mice (arrowheads) (n= 4 WT, 4 ECko). Scale bars: 50µm.


A . Representative pictures showing Peyer ´ s Patches in Casp8WT mice , and different severities of hemorrhage formation in Casp8ECko mice at an early disease stage . Scale bars : 1mm .

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

A. Representative pictures showing Peyer´s Patches in Casp8WT mice, and different severities of hemorrhage formation in Casp8ECko mice at an early disease stage. Scale bars: 1mm.


Quantification of lymphatic vessel density ( B ) in Peyer ' s Patches in Casp8WT and Casp8ECko mice at an early disease stage ( n = 9 WT , 7 - 10 ECko Peyer ' s Patches are pooled from 4 - 6 mice per genotype ; one - way ANOVA with tukey ' s multiple comparison ) . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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

Quantification of lymphatic vessel density (B) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype; one-way ANOVA with tukey's multiple comparison). Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


representative pictures of CD31 ( pan EC ) and Lyve1 ( lymphatic ) staining ( C ) in Peyer ' s Patches in Casp8WT and Casp8ECko mice at an early disease stage ( n = 9 WT , 7 - 10 ECko Peyer ' s Patches are pooled from 4 - 6 mice per genotype Scale bars : 200µm .

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

representative pictures of CD31 (pan EC) and Lyve1 (lymphatic) staining (C) in Peyer's Patches in Casp8WT and Casp8ECko mice at an early disease stage (n = 9 WT, 7-10 ECko Peyer's Patches are pooled from 4-6 mice per genotype Scale bars: 200µm.


D - F . Representative images ( D ) and quantifications ( E , F ) of stainings for CD31 , Lyve1 and DAPI ( nuclei ) in small intestinal wholemounts at an early disease stage ( blue arrowheads point to defective lacteals , white arrowheads indicate edema ) in Casp8ECko mice ( E ; n = 7 WT , 6 ECko . F ; n = 7 WT , 10 ECko ; two tailed unpaired Student ' s t - test ) . Scale bars : 100µm . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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

D-F. Representative images (D) and quantifications (E, F) of stainings for CD31, Lyve1 and DAPI (nuclei) in small intestinal wholemounts at an early disease stage (blue arrowheads point to defective lacteals, white arrowheads indicate edema) in Casp8ECko mice (E; n= 7 WT, 6 ECko. F; n= 7 WT, 10 ECko; two tailed unpaired Student's t-test). Scale bars: 100µm. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


G . Representative images of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran and stained for CD31 at an early disease stage . White arrowheads point to extravasated dextran in Casp8ECko mice ( n = 5 WT , 5 ECko ) . Scale bars : 50µm . H . Quantification of extravasated dextran in small intestine and brain lysates in Casp8WT and Casp8ECko mice at an early disease stage ( n = 4 WT , 4 - 5 ECko ; two - way ANOVA with tukey ' s multiple comparison ) . RFU : relative fluorescent units . Data information : Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns : not significant .

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

G. Representative images of small intestine sections of mice injected with a 70kDa fluorescently labeled dextran and stained for CD31 at an early disease stage. White arrowheads point to extravasated dextran in Casp8ECko mice (n= 5 WT, 5 ECko). Scale bars: 50µm. H. Quantification of extravasated dextran in small intestine and brain lysates in Casp8WT and Casp8ECko mice at an early disease stage (n= 4 WT, 4-5 ECko; two-way ANOVA with tukey's multiple comparison). RFU: relative fluorescent units. Data information: Data shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, ns: not significant.


I . Representative images of CD31 and VE - Cadherin staining in intestinal wholemounts at an early disease stage ( n = 5 WT , 5 ECko ) in Casp8WT and Casp8ECko mice ( red arrowheads point to VE - Cadherin ' empty ' vessel patches ) . Scale bars : 25µm .

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

I. Representative images of CD31 and VE-Cadherin staining in intestinal wholemounts at an early disease stage (n= 5 WT, 5 ECko) in Casp8WT and Casp8ECko mice (red arrowheads point to VE-Cadherin 'empty' vessel patches). Scale bars: 25µm.


A - B . Representative images ( A ) and quantification ( B ) of CD31 , TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice at an early disease stage ( red dotted lines : epithelium ; white dotted lines : endothelium ) in ( n = 5 WT , 5 ECko , multiple t - tests with Holm - Sidak method ) . Scale bars : 100µm . Data information : All data is shown as mean ± SEM . ns : not significant .

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

A-B. Representative images (A) and quantification (B) of CD31, TUNEL and DAPI staining in small intestine sections of Casp8WT and Casp8ECko mice at an early disease stage (red dotted lines: epithelium; white dotted lines: endothelium) in (n= 5 WT, 5 ECko, multiple t-tests with Holm-Sidak method). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. ns: not significant.


D - E . Graphs showing survival and relative weight of Casp8WT / MLKLko and Casp8ECko / MLKLko mice upon tamoxifen treatment . ( D ; n = 5 - 8 WT / MLKLko , 5 - 8 ECko / MLKLko ; Log - Rank test , E ; n = 7 WT , 8 ECko ; two tailed unpaired Student ' s t - test ) .

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

D-E. Graphs showing survival and relative weight of Casp8WT/MLKLko and Casp8ECko/MLKLko mice upon tamoxifen treatment. (D; n= 5-8 WT/ MLKLko, 5-8 ECko/MLKLko; Log-Rank test, E; n= 7 WT, 8 ECko; two tailed unpaired Student's t-test).


F . Representative pictures of intestines of Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment ( n = 8 WT , 8 ECko ) .

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

F. Representative pictures of intestines of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 8 WT, 8 ECko).


G . Representative images of stainings for CD31 , Lyve1 and DAPI in small intestinal wholemounts from Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment . Scale bars : 100µm .

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

G. Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts from Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment. Scale bars: 100µm.


H - K . Quantification of vessel area ( H , I ) and lacteal length ( J , K ) at two different timepoints after tamoxifen treatment in Casp8WT / MLKLko and Casp8ECko / MLKLko mice ( n = 4 - 6 WT / MLKLko , 5 ECko / MLKLko ; two tailed unpaired Student ' s t - test ) . Data information : All data is shown as mean ± SEM . ns : not significant .

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

H-K. Quantification of vessel area (H, I) and lacteal length (J, K) at two different timepoints after tamoxifen treatment in Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.


L . Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 30 days after tamoxifen treatment ( n = 7 WT / MLKLko , 6 ECko / MLKLko ; two tailed unpaired Student ' s t - test ) . Data information : All data is shown as mean ± SEM . ns : not significant .

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

L. Graph showing quantification of extravasated 70kDa Dextran from small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 30 days after tamoxifen treatment (n= 7 WT/MLKLko, 6 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.


M - N . Representative pictures ( M ) and quantification ( N ) of lymphatic vessel density in Peyer ' s Patches of Casp8WT / MLKLko and Casp8ECko / MLKLko mice at 140 days after tamoxifen treatment ( n = 8 WT , 7 ECko Peyer ' s Patches are pooled from 4 mice per genotype ; two - tailed unpaired students t - test ) . Scale bars : 200µm . Data information : All data is shown as mean ± SEM . ns : not significant .

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

M-N. Representative pictures (M) and quantification (N) of lymphatic vessel density in Peyer's Patches of Casp8WT/MLKLko and Casp8ECko/MLKLko mice at 140 days after tamoxifen treatment (n = 8 WT, 7 ECko Peyer's Patches are pooled from 4 mice per genotype; two-tailed unpaired students t-test). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. ns: not significant.


O . Representative pictures of H & E stainings of small intestine tissue of Casp8WT / MLKLko and Casp8ECko / MLKLko mice ( n = 4 - 6 WT , 4 - 5 ECko ) . Scale bars : 100µm . P - Q . Quantification of intestinal pathology in Casp8WT / MLKLko and Casp8ECko / MLKLko mice at two different timepoints after tamoxifen treatment ( enteropathy score ; n = 4 - 6 WT / MLKLko , 5 ECko / MLKLko ; two tailed unpaired Student ' s t - test ) . Data information : All data is shown as mean ± SEM . ns : not significant .

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

O. Representative pictures of H&E stainings of small intestine tissue of Casp8WT/MLKLko and Casp8ECko/MLKLko mice (n= 4-6 WT, 4-5 ECko). Scale bars: 100µm. P-Q. Quantification of intestinal pathology in Casp8WT/MLKLko and Casp8ECko/MLKLko mice at two different timepoints after tamoxifen treatment (enteropathy score; n=4-6 WT/MLKLko, 5 ECko/MLKLko; two tailed unpaired Student's t-test). Data information: All data is shown as mean ± SEM. ns: not significant.


A . Representative pictures of TUNEL staining together with CD31 and Lyve - 1 in Peyer ' s Patches of Casp8WT and Casp8ECko mice . TUNEL + LECs , but not BECs could already be seen in non - hemorrhagic Peyer ' s Patches ( a ' ) , and further accumulated in hemorrhagic Peyer ' s Patches ( a ' ' ) . White dotted areas outline location of the lymphatic plexus . White arrowheads point to TUNEL + LECs ( note that necroptotic LECs only weakly express Lyve - 1 ) . Scale bars : 50µm , insets : 10µm .

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

A. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT and Casp8ECko mice. TUNEL+ LECs, but not BECs could already be seen in non-hemorrhagic Peyer's Patches (a'), and further accumulated in hemorrhagic Peyer's Patches (a''). White dotted areas outline location of the lymphatic plexus. White arrowheads point to TUNEL+ LECs (note that necroptotic LECs only weakly express Lyve-1). Scale bars: 50µm, insets: 10µm.


C . Survival ( n = 5 WT , 6 BECko ; Log - Rank test ) of Casp8WT and Casp8ECko mice upon tamoxifen treatment .

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

C. Survival (n= 5 WT, 6 BECko; Log-Rank test) of Casp8WT and Casp8ECko mice upon tamoxifen treatment.


D . Representative pictures of intestines of Casp8WT and Casp8BECko mice .

D. Representative pictures of intestines of Casp8WT and Casp8BECko mice.


Representative pictures of H & E staining of small intestine of Casp8WT and Casp8BECko mice ( E ) , enteropathy score ( F ; n = 5 WT , 6 ECko , two tailed unpaired Student ' s t - test ) Scale bars : 100µm , insets : 50µm . Data information : Data shown as mean ± SEM . ns : not significant .

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

Representative pictures of H&E staining of small intestine of Casp8WT and Casp8BECko mice (E), enteropathy score (F; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test) Scale bars: 100µm, insets: 50µm. Data information: Data shown as mean ± SEM. ns: not significant.


mucosal hemorrhages ( G ; n = 5 WT , 6 ECko , two tailed unpaired Student ' s t - test ) . Data information : Data shown as mean ± SEM . ns : not significant .

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

mucosal hemorrhages (G; n= 5 WT, 6 ECko, two tailed unpaired Student's t-test). Data information: Data shown as mean ± SEM. ns: not significant.


Representative images of stainings for CD31 , Lyve1 and DAPI in small intestinal wholemounts ( H ) and quantification of vessel density ( I ) in Casp8BECko compared to Casp8WT mice ( I ; n = 5 WT , 6 BECko ; two tailed unpaired Student ' s t - test , Scale bars : 100µm . Data information : Data shown as mean ± SEM . ns : not significant .

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

Representative images of stainings for CD31, Lyve1 and DAPI in small intestinal wholemounts (H) and quantification of vessel density (I) in Casp8BECko compared to Casp8WT mice (I; n= 5 WT, 6 BECko; two tailed unpaired Student's t-test, Scale bars: 100µm. Data information: Data shown as mean ± SEM. ns: not significant.


quantification of lacteal length ( J ) in Casp8BECko compared to Casp8WT mice ; n = 5 WT , 5 BECko ; two tailed unpaired Student ' s t - test with Welch ' s correction ) . Data information : Data shown as mean ± SEM . ns : not significant .

quantification of lacteal length (J) in Casp8BECko compared to Casp8WT mice ; n= 5 WT, 5 BECko; two tailed unpaired Student's t-test with Welch's correction). Data information: Data shown as mean ± SEM. ns: not significant.


K - L . Representative pictures ( K ) and quantification ( L ) of lymphatic vessel density Peyer ' s Patches in Casp8WT and Casp8BECko mice ( n = 10 WT , 11 BECko Peyer ' s Patches from 5 - 6 mice per genotype ; two tailed unpaired Student ' s t - test ) . Scale bars : 200µm . Data information : Data shown as mean ± SEM . ns : not significant .

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

K-L. Representative pictures (K) and quantification (L) of lymphatic vessel density Peyer's Patches in Casp8WT and Casp8BECko mice (n = 10 WT, 11 BECko Peyer's Patches from 5-6 mice per genotype; two tailed unpaired Student's t-test). Scale bars: 200µm. Data information: Data shown as mean ± SEM. ns: not significant.


B . Survival graph after intravenous TNF - α injection of Casp8WT vs . Casp8ECko mice within the first 24h ( n = 11 WT , 11 ECko ; Log - Rank test ) .

O O O O O O O O O O O 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. Survival graph after intravenous TNF-α injection of Casp8WT vs. Casp8ECko mice within the first 24h (n=11 WT, 11 ECko; Log-Rank test).


C . Representative images of the small intestines 24h after TNF - α injection in Casp8WT and Casp8ECko mice ( blue arrowheads point to Peyer ' s Patches ) .

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

C. Representative images of the small intestines 24h after TNF-α injection in Casp8WT and Casp8ECko mice (blue arrowheads point to Peyer's Patches).


D - E . Representative pictures of Peyer ' s Patches ( D ) and quantification of lymphatic vessel density ( E ) in Casp8WT , Casp8ECko and Casp8ECko / MLKLko mice injected with TNF - α ( n = 7 WT , 5 ECko , 15 ECko / MLKLko . Peyer ' s Patches are pooled from 3 - 5 mice per genotype ) . Scale bars : 200µm . Data information : All data is shown as mean ± SEM . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant .

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

D-E. Representative pictures of Peyer's Patches (D) and quantification of lymphatic vessel density (E) in Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α (n=7 WT, 5 ECko, 15 ECko/MLKLko. Peyer's Patches are pooled from 3-5 mice per genotype). Scale bars: 200µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.


F . Representative pictures of TUNEL staining together with CD31 and Lyve - 1 in Peyer ' s Patches of Casp8WT , Casp8ECko and Casp8ECko / MLKLko mice injected with TNF - α . TUNEL + LECs , but not BECs could already be seen in Casp8ECko , but neither in Casp8WT nor in Casp8ECko / MLKLko mice . White dotted areas outline location of the lymphatic plexus . Yellow arrowheads point to TUNEL + ECs ) . Scale bars : 50µm .

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

F. Representative pictures of TUNEL staining together with CD31 and Lyve-1 in Peyer's Patches of Casp8WT, Casp8ECko and Casp8ECko/MLKLko mice injected with TNF-α. TUNEL+ LECs, but not BECs could already be seen in Casp8ECko, but neither in Casp8WT nor in Casp8ECko/MLKLko mice. White dotted areas outline location of the lymphatic plexus. Yellow arrowheads point to TUNEL+ECs). Scale bars: 50µm.


H . Survival graph of Casp8WT and Casp8ECko mice after Enbrel ® treatment ( n = 7 WT , 5 ECko + IgG , 8 ECko + Enbrel ; Log - Rank test ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

H. Survival graph of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=7 WT, 5 ECko + IgG, 8 ECko + Enbrel; Log-Rank test).


Representative images of small intestines ( I ) of Casp8ECko mice after Enbrel ® treatment Scale bars : 100µm .

Representative images of small intestines (I) of Casp8ECko mice after Enbrel® treatment Scale bars: 100µm.


Representative images of H & E staining ( J ) with histopathological scoring ( K ) of Casp8WT and Casp8ECko mice after Enbrel ® treatment ( n = 6 WT , 4 ECko + IgG ; 8 ECko + Enbrel ( from which 3 mice were diseased , 5 mice healthy ) ; one - way ANOVA with tukey ' s multiple comparison ) . Scale bars : 100µm . Data information : All data is shown as mean ± SEM . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; ns : not significant .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of H&E staining (J) with histopathological scoring (K) of Casp8WT and Casp8ECko mice after Enbrel® treatment (n=6 WT, 4 ECko + IgG; 8 ECko + Enbrel (from which 3 mice were diseased, 5 mice healthy); one-way ANOVA with tukey's multiple comparison). Scale bars: 100µm. Data information: All data is shown as mean ± SEM. * P<0.05, *** P <0.001, **** P <0.0001; ns: not significant.


analysis 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 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

analysis 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 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O 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-CONTROLLED_VAR O O

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 .

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 O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


C , 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 .

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

C, 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 O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


B , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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

C, 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 .

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

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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, 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 O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


, 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 ) .

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

, 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 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

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

E, Average clockwise interval length, calculated as the inverse of the fast (blue) and slow (red) rate constants


A , 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 ) .

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

A, 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 O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O

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.


A , 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 .

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

A, 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 .

O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

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

D, 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.


( A ) Levels of KRAS , COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in KRASWT , KRASG12D , and KRAS - silenced KRASG12D MEF cells .

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

(A) 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 .

O O O O O 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) 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 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced KRASG12D MEF cells.


( A ) Levels of KRAS , COUP - TFII , LDHA , and phosphorylated S6K ( T389 ) in KRAS - silenced HCT116 and DLD - 1 cells .

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

(A) 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 .

(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 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 ) .

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

(D) 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 .

O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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 .

O O O O O 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-CONTROLLED_VAR O

(F) Clonogenic assay of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cells in the presence or absence of lactate.


( A ) Effect of lactate on the ERK / TSC2 and AKT / TSC2 signaling pathway in serum - starved HCT116 cells .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

(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 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(G) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.


( A ) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP - TFII - silenced HCT116 cells .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

(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 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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 .

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

(C) 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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

(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 .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

(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 .

(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.


Effects 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 ) .

O O O O O O O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

Effects 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 ) .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

(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 ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O

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 ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O

(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 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(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.


Bioluminescence 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

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

Bioluminescence 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 metastases


Bioluminescence 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 ,

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

Bioluminescence 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

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

(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 )

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

(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)


( 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 .

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

(A) 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 . )

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

(C) 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 .

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

(E) 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 O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) 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.)


( 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

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

(A) 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 . )

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

(E) 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 .

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

(G) 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 ) .

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

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

(L) 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).


( 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 (

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

(A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(G) 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)


( 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 (

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

(C) 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 ) .

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

(D) 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 ) .

(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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


( 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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) 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)


A , 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 .

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

A, B 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, 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.


Seedlings 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Seedlings 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).


B 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

C 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 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.