( A ) Relative expression of the BBSome subunits in the indicated organs and T cells measured by qPCR . CT values of the BBS genes were normalized to the geometric mean of the CT values of reference genes Gapdh , Tubb2a , and Eef1a1 . The expression levels are normalized to those of brain ( = 1 ) . Mean + SD . Three biological replicates ( the result of each biological replicate is a median of three technical replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative expression of the BBSome subunits in the indicated organs and T cells measured by qPCR. CT values of the BBS genes were normalized to the geometric mean of the CT values of reference genes Gapdh, Tubb2a, and Eef1a1. The expression levels are normalized to those of brain (=1). Mean+SD. Three biological replicates (the result of each biological replicate is a median of three technical replicates).


( B ) Immunoblot analysis of BBS4 expression in T and B lymphocytes isolated from the lymph nodes and spleen of C57Bl / 6 mouse . β - actin staining served as a loading control .

O O O 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

(B) Immunoblot analysis of BBS4 expression in T and B lymphocytes isolated from the lymph nodes and spleen of C57Bl/6 mouse. β-actin staining served as a loading control.


( D ) Immunoblot analysis of BBS4 expression in testes and thymi lysates of Bbs4 + / + ( WT ) , Bbs4GT / + , and Bbs4GT / GT mice . β - actin staining serves as a loading control . A representative experiment out of three biological replicates in total is shown .

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

(D) Immunoblot analysis of BBS4 expression in testes and thymi lysates of Bbs4+/+ (WT), Bbs4GT/+, and Bbs4GT/GT mice. β-actin staining serves as a loading control. A representative experiment out of three biological replicates in total is shown.


( E ) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4 + / + , Bbs4GT / GT , and Bbs4KO / KO mice . β - actin staining served as a loading control . A representative experiment out of two biological replicates is shown .

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

(E) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4+/+, Bbs4GT/GT, and Bbs4KO/KO mice. β-actin staining served as a loading control. A representative experiment out of two biological replicates is shown.


( F ) Hematoxylin and eosin staining of sections of seminiferous tubules from ~ 4 weeks old Bbs4 + / + ( n = 2 mice ) and Bbs4GT / GT ( n = 4 ) males . Scale bar , 100 µm . Representative images are shown .

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

(F) Hematoxylin and eosin staining of sections of seminiferous tubules from ~4 weeks old Bbs4+/+ (n=2 mice) and Bbs4GT/GT (n=4) males. Scale bar, 100 µm. Representative images are shown.


( G ) Genotypic ratio of Bbs4 + / + , Bbs4GT / + or Bbs4KO / + ( Het ) , and Bbs4GT / GT ( GT ) or Bbs4KO / KO ( KO ) offspring at weaning from mating of Bbs4GT / + ( n = 145 pups ) or Bbs4 + / KO ( n = 168 pups ) parents . Binomial test was used for statistical comparison of the observed distribution to the expected Mendelian ratio .

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

(G) Genotypic ratio of Bbs4+/+, Bbs4GT/+ or Bbs4KO/+ (Het), and Bbs4GT/GT (GT) or Bbs4KO/KO (KO) offspring at weaning from mating of Bbs4GT/+ (n=145 pups) or Bbs4+/KO (n=168 pups) parents. Binomial test was used for statistical comparison of the observed distribution to the expected Mendelian ratio.


( H ) Bbs4 + / + and Bbs4GT / GT female littermates at 20 weeks of age . Representative litter out of seven in total . ( I ) Bbs4 + / + and Bbs4KO / KO female littermates at 20 weeks of age . Representative litter out of five in total .

O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

(H) Bbs4+/+ and Bbs4GT/GT female littermates at 20 weeks of age. Representative litter out of seven in total. (I) Bbs4+/+ and Bbs4KO/KO female littermates at 20 weeks of age. Representative litter out of five in total.


( J ) Growth curves of Bbs4 - deficient mice , mean ± SD is shown . Females : Bbs4 + / + ( n = 12 mice ) , Bbs4GT / GT ( n = 12 ) , Bbs4KO / KO ( n = 6 ) . Males : Bbs4 + / + ( n = 6 mice ) , Bbs4GT / GT ( n = 7 ) , Bbs4KO / KO ( n = 5 ) .

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

(J) Growth curves of Bbs4-deficient mice, mean ± SD is shown. Females: Bbs4+/+ (n=12 mice), Bbs4GT/GT (n=12), Bbs4KO/KO (n=6). Males: Bbs4+/+ (n=6 mice), Bbs4GT/GT (n=7), Bbs4KO/KO (n=5).


( K ) Leptin concentration in blood plasma taken from young adult ( 7 - 8 weeks ) or mid - age ( 14 - 20 weeks ) mice . Young adult mice : Bbs4 + / + ( n = 4 mice ) , Bbs4KO / KO ( n = 3 ) , analyzed in two independent experiments . Mean + SEM . Mid - age adult mice : Bbs4 + / + ( n = 7 mice ) , Bbs4KO / KO ( n = 5 ) , Bbs4GT / GT ( n = 3 ) , analyzed in 4 independent experiments . Kruskal - Wallis tests was used for the statistical analysis . Mean + SEM .

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

(K) Leptin concentration in blood plasma taken from young adult (7-8 weeks) or mid-age (14-20 weeks) mice. Young adult mice: Bbs4+/+ (n=4 mice), Bbs4KO/KO (n=3), analyzed in two independent experiments. Mean+SEM. Mid-age adult mice: Bbs4+/+ (n=7 mice), Bbs4KO/KO (n=5), Bbs4GT/GT (n=3), analyzed in 4 independent experiments. Kruskal-Wallis tests was used for the statistical analysis. Mean+SEM.


Cells isolated from the bone marrow ( BM ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( A ) Percentage of B220low positive cells in the bone marrow . Up : Bbs4 + / + n = 9 , Bbs4GT / GT n = 10 . Down : Bbs4 + / + ( n = 11 ) , Bbs4KO / KO ( n = 10 ) . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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

Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (A) Percentage of B220low positive cells in the bone marrow . Up: Bbs4+/+ n=9, Bbs4GT/GT n=10. Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the bone marrow ( BM ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( B ) Percentage of B - cell precursors ( IgM - IgD - ) in the bone marrow . Gated on viable B220 + cells . Up : Bbs4 + / + ( n = 9 ) , Bbs4GT / GT ( n = 10 ) . Down : Bbs4 + / + ( n = 11 ) , Bbs4KO / KO ( n = 10 ) . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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

Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (B) Percentage of B-cell precursors (IgM- IgD-) in the bone marrow. Gated on viable B220+ cells. Up: Bbs4+/+ (n=9), Bbs4GT/GT (n=10). Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the bone marrow ( BM ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( C ) Percentage of pre - B cells ( CD43 - CD24high ) in the bone marrow of Bbs4 + / + ( n = 11 ) and Bbs4KO / KO mice ( n = 10 ) . Gated on viable B220 + IgM - IgD - cells . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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

Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (C) Percentage of pre-B cells (CD43- CD24high) in the bone marrow of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10). Gated on viable B220+ IgM- IgD- cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the spleens ( SPL ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( D ) Percentage of late mature ( IgM - IgD + ) B cells in the spleen of Bbs4 + / + ( n = 11 ) and Bbs4KO / KO mice ( n = 10 ) Gated on viable CD19 + cells . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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

Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (D) Percentage of late mature (IgM- IgD+) B cells in the spleen of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10) Gated on viable CD19+ cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


Cells isolated from the spleens ( SPL ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( E ) An alternative analysis of the experiment shown in ( D ) . Geometric mean fluorescence intensity ( MFI ) of BV421 ( IgM ) on IgD + B cells was determined . Mean of MFI values for each genotype per experiment was quantified , and obtained values were normalized to Bbs4 + / + ( = 1 ) for each experiment . Two - tailed One Sample Wilcoxon Signed Rank Test was used for the statistical analysis .

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

Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (E) An alternative analysis of the experiment shown in (D). Geometric mean fluorescence intensity (MFI) of BV421 (IgM) on IgD+ B cells was determined. Mean of MFI values for each genotype per experiment was quantified, and obtained values were normalized to Bbs4+/+ (=1) for each experiment. Two-tailed One Sample Wilcoxon Signed Rank Test was used for the statistical analysis.


Cells isolated from the spleens ( SPL ) of 18 - 25 weeks old Bbs4 + / + ( WT ) , Bbs4GT / GT , and Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT strain and Bbs4WT / WT controls ) or eight ( Bbs4KO / KO strain and Bbs4WT / WT controls ) independent experiments were performed . ( F ) Percentage of splenic marginal zone ( MZ ) B cells ( CD23 - CD1d + ) in Bbs4 + / + controls ( n = 10 ) and Bbs4KO / KO mice ( n = 9 ) was determined . Gated on viable CD19 + , IgD - IgM + , CD138 - cells . Statistical significance was calculated using two - tailed Mann - Whitney test . Medians are shown .

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

Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (F) Percentage of splenic marginal zone (MZ) B cells (CD23- CD1d+) in Bbs4+/+ controls (n=10) and Bbs4KO/KO mice (n=9) was determined. Gated on viable CD19+, IgD- IgM+, CD138- cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


( A , B ) Splenocytes isolated from Bbs4 + / + ( n = 4 mice ) and Bbs4GT / GT ( n = 3 ) B - cell transgenic B1 - 8 littermates were activated with 4 - hydroxy - 3 - nitrophenylacetic acid succinimide ester - loaded T2 - Kb cells in three independent experiments . Percentage of activated B cells ( gated as CD69 + B220 + IgLλ + viable cells ) in the samples without T2 - Kb ( negative control ) , and in the samples with T2 - Kb added in ratios 1 : 10 or 1 : 3 was determined by flow cytometry . ( A ) Representative mice are shown . ( B ) Mean ± SEM . Statistical significance was calculated using two - tailed Mann - Whitney test .

O O O O O O O O B-CONTROLLED_VAR 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O 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) Splenocytes isolated from Bbs4+/+ (n=4 mice) and Bbs4GT/GT (n=3) B-cell transgenic B1-8 littermates were activated with 4-hydroxy-3-nitrophenylacetic acid succinimide ester-loaded T2-Kb cells in three independent experiments. Percentage of activated B cells (gated as CD69+ B220+ IgLλ+ viable cells) in the samples without T2-Kb (negative control), and in the samples with T2-Kb added in ratios 1:10 or 1:3 was determined by flow cytometry. (A) Representative mice are shown. (B) Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.


( C ) B cells from 18 - 20 weeks old Bbs4KO / KO ( red circle , n = 3 mice ) , and control mice ( Bbs4 + / + , dark grey square , n = 4 , or Bbs4 + / KO , light grey square , n = 2 ) were activated with F ( ab ' ) 2 - goat anti - mouse IgM ( Mu chain ) . Percentage of activated B cells ( gated as CD69 + CD19 + viable cells ) was determined by flow cytometry . Representative experiment out of three biological replicates is shown . Mean ± SEM . Statistical significance was calculated using two - tailed Mann - Whitney test .

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

(C) B cells from 18-20 weeks old Bbs4KO/KO (red circle, n=3 mice), and control mice (Bbs4+/+, dark grey square, n=4, or Bbs4+/KO, light grey square, n=2) were activated with F(ab')2-goat anti-mouse IgM (Mu chain). Percentage of activated B cells (gated as CD69+ CD19+ viable cells) was determined by flow cytometry. Representative experiment out of three biological replicates is shown. Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.


( D ) CFSE - loaded T cells isolated from lymph nodes of Bbs4FL / FL OT - I Rag2KO / KO ( OT - I ) and CD4 - Cre Bbs4FL / FL OT - I Rag2KO / KO ( Bbs4 cKO , OT - I ) littermates were incubated with DDAO - labeled WT splenocytes loaded with OVA peptide or with the indicated altered peptide ligands for 20 min . Percentage of T cells conjugated with the APCs was determined by flow cytometry . Four biological replicates were performed . Mean + SEM . Statistical significance was calculated using two - tailed Mann - Whitney test , p > 0 . 05 for all peptides .

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 B-CONTROLLED_VAR 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 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

(D) CFSE-loaded T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were incubated with DDAO-labeled WT splenocytes loaded with OVA peptide or with the indicated altered peptide ligands for 20 min. Percentage of T cells conjugated with the APCs was determined by flow cytometry. Four biological replicates were performed. Mean+SEM. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all peptides.


500 or 1000 T cells isolated from lymph nodes of Bbs4FL / FL OT - I Rag2KO / KO ( OT - I ) and CD4 - Cre Bbs4FL / FL OT - I Rag2KO / KO ( Bbs4 cKO , OT - I ) littermates were adoptively transferred into RIP . OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin ( LM - OVA ) on the next day . ( E ) Glucose level in the urine of mice was monitored on a daily basis . The mouse was considered diabetic when it had urine glucose level ≥ 1000 mg / dL for two consecutive days . Statistical significance was calculated by Log - rank ( Mantel - Cox ) 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 O B-CONTROLLED_VAR 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 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 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

500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (E) Glucose level in the urine of mice was monitored on a daily basis. The mouse was considered diabetic when it had urine glucose level ≥ 1000 mg/dL for two consecutive days. Statistical significance was calculated by Log-rank (Mantel-Cox) test.


500 or 1000 T cells isolated from lymph nodes of Bbs4FL / FL OT - I Rag2KO / KO ( OT - I ) and CD4 - Cre Bbs4FL / FL OT - I Rag2KO / KO ( Bbs4 cKO , OT - I ) littermates were adoptively transferred into RIP . OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin ( LM - OVA ) on the next day . ( F ) Glucose concentration in blood on day 7 post - infection . 500 OT - I ctrl ( n = 13 mice ) , 500 OT - I Bbs4 cKO ( n = 13 ) , 1000 OT - I ctrl ( n = 9 ) , 1000 OT - I Bbs4 cKO ( n = 11 ) , analyzed in four independent experiments , mean is shown . Statistical significance was calculated using two - tailed Mann - Whitney 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 O B-CONTROLLED_VAR 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 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 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 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

500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (F) Glucose concentration in blood on day 7 post-infection. 500 OT-I ctrl (n=13 mice), 500 OT-I Bbs4 cKO (n=13), 1000 OT-I ctrl (n=9), 1000 OT-I Bbs4 cKO (n=11), analyzed in four independent experiments, mean is shown. Statistical significance was calculated using two-tailed Mann-Whitney test.


Cells isolated from bone marrow of Bbs4FL / FL ( control ) and Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . ( A ) Percentage of B220low in bone marrow . Representative experiment out of four in total is shown . Bbs4FL / FL ( n = 6 mice ) , Vav - iCre Bbs4FL / FL ( n = 9 ) . Representative experiment out of six in total is shown . Statistical significance was calculated using two - tailed Mann - Whitney test , p > 0 . 05 for all tests . Median is shown .

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

Cells isolated from bone marrow of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (A) Percentage of B220low in bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


Cells isolated from bone marrow of Bbs4FL / FL ( control ) and Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . ( B ) Percentage of B - cell precursors ( B220 + , IgM - IgD - ) in the bone marrow . Representative experiment out of four in total is shown . Bbs4FL / FL ( n = 6 mice ) , Vav - iCre Bbs4FL / FL ( n = 9 ) . Representative experiment out of six in total is shown . Statistical significance was calculated using two - tailed Mann - Whitney test , p > 0 . 05 for all tests . Median is shown .

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

Cells isolated from bone marrow of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (B) Percentage of B-cell precursors (B220+, IgM- IgD-) in the bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


Cells isolated from , lymph nodes and spleen of Bbs4FL / FL ( control ) and Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . ( C ) Percentage of CD19 + B cells in spleens and lymph nodes of Vav - iCre Bbs4FL / FL ( n = 12 mice ) and Bbs4FL / FL ( n = 9 ) mice , six independent experiments . Representative experiment out of six in total is shown . Statistical significance was calculated using two - tailed Mann - Whitney test , p > 0 . 05 for all tests . Median is shown .

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

Cells isolated from , lymph nodes and spleen of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (C) Percentage of CD19+ B cells in spleens and lymph nodes of Vav-iCre Bbs4FL/FL (n=12 mice) and Bbs4FL/FL (n=9) mice, six independent experiments. Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


Cells isolated from spleen of Bbs4FL / FL ( control ) and Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . D ) Percentage of late mature B cells ( CD19 + , IgM - , IgD + ) in spleens of Vav - iCre Bbs4FL / FL ( n = 12 mice ) , and Bbs4FL / FL mice ( n = 9 ) . Representative experiment out of six in total is shown . Statistical significance was calculated using two - tailed Mann - Whitney test , p > 0 . 05 for all tests . Median is shown .

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

Cells isolated from spleen of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. D) Percentage of late mature B cells (CD19+, IgM-, IgD+) in spleens of Vav-iCre Bbs4FL/FL (n=12 mice), and Bbs4FL/FL mice (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


( E ) Expression of Cxcl12 and Il - 7 in the bone marrow from Bbs4 + / + Bbs18 + / + ( WT ) mice ( black circles , n = 7 mice ) , Bbs4 + / KO ( Het , green circle , n = 3 ) , Bbs18 + / KO ( Het , blue circle , n = 3 ) , Bbs4KO / KO ( green square , n = 5 ) , and Bbs18KO / KO ( blue square , n = 2 ) was analyzed by RT - qPCR in five independent experiments . The expression was normalized to Gapdh . The statistical significance of the difference between the BBSome - deficient mice ( Bbs4KO / KO and Bbs18KO / KO ) and controls were calculated using two - tailed Mann - Whitney test . Median is shown .

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

(E) Expression of Cxcl12 and Il-7 in the bone marrow from Bbs4+/+ Bbs18+/+ (WT) mice (black circles, n=7 mice), Bbs4+/KO (Het, green circle, n=3), Bbs18+/KO (Het, blue circle, n=3), Bbs4KO/KO (green square, n=5), and Bbs18KO/KO (blue square, n=2) was analyzed by RT-qPCR in five independent experiments. The expression was normalized to Gapdh. The statistical significance of the difference between the BBSome-deficient mice (Bbs4KO/KO and Bbs18KO/KO) and controls were calculated using two-tailed Mann-Whitney test. Median is shown.


( F ) MEF cell lines were derived from a single Bbs4 + / + ( WT ) or a single Bbs4KO / KO embryos . The expression of Cxcl12 in these cell lines was analyzed by RT - qPCR . The expression was normalized to Gapdh and to Bbs4 + / + MEFs ( = 1 ) for each experiment . Three biological replicates . One sample t test was used for the statistical analysis .

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

(F) MEF cell lines were derived from a single Bbs4+/+ (WT) or a single Bbs4KO/KO embryos. The expression of Cxcl12 in these cell lines was analyzed by RT-qPCR. The expression was normalized to Gapdh and to Bbs4+/+ MEFs (=1) for each experiment. Three biological replicates. One sample t test was used for the statistical analysis.


( G ) Expression of Cxcl12 and canonical - WNT responsive gene Alpl in untreated ( NT ) ST2 cells , or ST2 cells treated with WNT3A or WNT5A was analyzed by RT - qPCR . The expression was normalized to Gapdh and untreated ST2 sample ( = 1 ) for each experiment . Four biological replicates were performed . One sample t test was used for the statistical analysis .

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

(G) Expression of Cxcl12 and canonical-WNT responsive gene Alpl in untreated (NT) ST2 cells, or ST2 cells treated with WNT3A or WNT5A was analyzed by RT-qPCR. The expression was normalized to Gapdh and untreated ST2 sample (=1) for each experiment. Four biological replicates were performed. One sample t test was used for the statistical analysis.


( A Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . BBS patients n = 43 for parameters White blood cell count n = 41 for parameters Neutrophils , Lymphocytes , Monocytes , Eosinophils Both data sets of healthy controls were selected as 10 - fold larger than the set of BBS patients . Median is shown . Kruskal - Wallis test was used for the statistical analysis 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters White blood cell count n=41 for parameters Neutrophils, Lymphocytes, Monocytes, Eosinophils Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in (A


( B Indicated parameters were measured using the blood from 20 - 21 weeks old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , and Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis test with Dunn ' s Multiple Comparison Post - tests was used for the statistical analysis . Median is shown .

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 B-CONTROLLED_VAR 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

(B Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . In ( C ) , the percentage of patients or controls having CRP ≥ 5 were compared using Fisher ' s exact test with post - hoc Sidak correction for multiple comparisons , BBS patients n = 42 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. In (C), the percentage of patients or controls having CRP≥5 were compared using Fisher's exact test with post-hoc Sidak correction for multiple comparisons, BBS patients n=42.


Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . BBS patients n = 43 for parameters Mean cell volume , Hemoglobin n = 39 for parameters Red Blood cells , Mean cell Hemoglobin and red cell distribution width ( RDW ) . Both data sets of healthy controls were selected as 10 - fold larger than the set of BBS patients . Median is shown . Kruskal - Wallis test was used for the statistical analysis

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O 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 O O O O O O O O O

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters Mean cell volume, Hemoglobin n=39 for parameters Red Blood cells, Mean cell Hemoglobin and red cell distribution width (RDW). Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in


Indicated parameters were measured using the blood from 20 - 21 weeks old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , and Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis test with Dunn ' s Multiple Comparison Post - tests was used for the statistical analysis . Median is shown .

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 B-CONTROLLED_VAR 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

Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


Results of the blood tests of BBS patients from the Guy ' s Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank . The BMI - random controls were age - and gender - matched to the set of BBS patients , with random BMIs . The BMI - matched controls were matched for age , gender , and BMI . BBS patients n = 43 for parameters Platelets ; Both data sets of healthy controls were selected as 10 - fold larger than the set of BBS patients . Median is shown . Kruskal - Wallis test was used for the statistical analysis 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

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters Platelets; Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in F).


G ) Indicated parameters were measured using the blood from 20 - 21 weeks old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , and Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis test with Dunn ' s Multiple Comparison Post - tests was used for the statistical analysis . Median is shown .

G) Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


D Microscale thermophoresis ( MST ) curves for binding of peptides to labelled GyraseA14 . The circles indicate the experimental values and the solid lines indicate the fitted fluorescence values obtained upon analysis using the MO . Affinity Analysis software ( version 2 . 2 . 5 , NanoTemper Technologies ) . Data information : In panels D , data are obtained from n = 3 biological replicates ; wherein for each biological replicate , the experiment is performed in duplicates ( technical duplicates for each biological replicate ) . In panel D , the data are presented as mean ± SD values obtained from all the replicates .

O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Microscale thermophoresis (MST) curves for binding of peptides to labelled GyraseA14. The circles indicate the experimental values and the solid lines indicate the fitted fluorescence values obtained upon analysis using the MO.Affinity Analysis software (version 2.2.5, NanoTemper Technologies). Data information: In panels D, data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panel D, the data are presented as mean ± SD values obtained from all the replicates.


B , In vitro bactericidal action of the CP1 - WT peptide on E . coli Top10 cells . B Growth profile of E . coli Top10 in the presence of increasing concentrations of the CP1 - WT peptide . Data information : data are obtained from n = 3 biological replicates ; wherein for each biological replicate , the experiment is performed in duplicates ( technical duplicates for each biological replicate ) . In panels B data are presented as mean ± SD values obtained from all the replicates . In panel B , N . T . - No treatment ; 4 . 5 % DMSO ( solvent control ) ; CP1 - WT ( test peptide ) ; CP1 - Mut ( negative control peptide ) and Cip - 10µg / mL ciprofloxacin ( positive 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 O O O O O O O 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, In vitro bactericidal action of the CP1-WT peptide on E. coli Top10 cells. B Growth profile of E. coli Top10 in the presence of increasing concentrations of the CP1-WT peptide. Data information: data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panels B data are presented as mean ± SD values obtained from all the replicates. In panel B, N.T. - No treatment; 4.5% DMSO (solvent control); CP1-WT (test peptide); CP1-Mut (negative control peptide) and Cip - 10µg/mL ciprofloxacin (positive control).


C In vitro bactericidal action of the CP1 - WT peptide on E . coli Top10 cells . C Estimation of Minimum Inhibitory Concentration ( MIC ) , indicated in terms of OD600 and Minimum Bactericidal Dose ( MBD ) , indicated in terms of CFU / mL , show that the CP1 - WT peptide has similar MIC and MBD values of 40µM ( ~ 130µg / mL ) . Data information : data are obtained from n = 3 biological replicates ; wherein for each biological replicate , the experiment is performed in duplicates ( technical duplicates for each biological replicate ) . In panels C , data are presented as mean ± SD values obtained from all the replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro bactericidal action of the CP1-WT peptide on E. coli Top10 cells. C Estimation of Minimum Inhibitory Concentration (MIC), indicated in terms of OD600 and Minimum Bactericidal Dose (MBD), indicated in terms of CFU/mL, show that the CP1-WT peptide has similar MIC and MBD values of 40µM (~130µg/mL). Data information: data are obtained from n = 3 biological replicates; wherein for each biological replicate, the experiment is performed in duplicates (technical duplicates for each biological replicate). In panels C, data are presented as mean ± SD values obtained from all the replicates.


A - D Peptide - induced membrane damage was studied by confocal microscopy and measured by propidium iodide ( PI ) influx ( % PI uptake ) in ( A ) S . Typhimurium , ( B ) A . baumannii , ( C ) S . aureus and ( D ) P . aeruginosa . Data information : In panels A - D , n = 5 biological replicates ; scale bars : 5µm . Representative images are shown . In the bar graphs , data are presented as mean ± SD values of percentage of propidium iodide uptake ( % PI uptake ) , averaged across all the replicates . 3 % , 7 % , 8 % and 3 % DMSO , and 80µM , 50µM , 50µM and 80µM of the peptides in the respective DMSO concentrations were used to treat S . Typhimurium , A . baumannii , S . aureus and P . aeruginosa respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Peptide-induced membrane damage was studied by confocal microscopy and measured by propidium iodide (PI) influx (% PI uptake) in (A) S. Typhimurium, (B) A. baumannii, (C) S. aureus and (D) P. aeruginosa. Data information: In panels A-D, n = 5 biological replicates; scale bars: 5µm. Representative images are shown. In the bar graphs, data are presented as mean ± SD values of percentage of propidium iodide uptake (% PI uptake), averaged across all the replicates. 3%, 7%, 8% and 3% DMSO, and 80µM, 50µM, 50µM and 80µM of the peptides in the respective DMSO concentrations were used to treat S. Typhimurium, A. baumannii, S. aureus and P. aeruginosa respectively.


E - H Minimum Inhibitory Concentration ( MIC ) and Minimum Bactericidal Dose ( MBD ) estimation of CP1 - WT on stationary phase cells of ( E ) S . Typhimurium , ( F ) A . baumannii , ( G ) S . aureus and ( H ) P . aeruginosa . No bactericidal action is observed for P . aeruginosa . In each of the panels and its corresponding bar graph , the black lines and black bars represent the MIC data indicated by their OD600 values , and the red lines and red bars represent the MBD data indicated by their CFU / mL values . The bar graphs in each panel depict control reactions used for each strain where the cells were treated with DMSO , peptides and ciprofloxacin ( Cip - postive control ; 10µg / mL ) . The strain of A . baumannii used was reported to be ciprofloxacin - resistant ( Nagarajan et al , 2018 ) as is observed here ( inset in panel F ) . In panels E - H , n = 5 biological replicates . Data are presented as mean ± SD values , averaged across all the replicates . For the bar graphs in these panels , 7 % , 13 % , 8 % and 4 % DMSO , and 80µM , 100µM , 100µM and 50µM of the peptides , were used for S . Typhimurium , A . baumannii , S . aureus and P . aeruginosa respectively . N . T - No treatment ; WT - CP1 - WT peptide ; Mut - CP1 - Mut peptide ( negative control ) . P values are calculated by Student ' s unpaired t - test ; nsP > 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 Student ' s unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Dose (MBD) estimation of CP1-WT on stationary phase cells of (E) S. Typhimurium, (F) A. baumannii, (G) S. aureus and (H) P. aeruginosa. No bactericidal action is observed for P. aeruginosa. In each of the panels and its corresponding bar graph, the black lines and black bars represent the MIC data indicated by their OD600 values, and the red lines and red bars represent the MBD data indicated by their CFU/mL values. The bar graphs in each panel depict control reactions used for each strain where the cells were treated with DMSO, peptides and ciprofloxacin (Cip - postive control; 10µg/mL). The strain of A. baumannii used was reported to be ciprofloxacin-resistant (Nagarajan et al, 2018) as is observed here (inset in panel F). In panels E-H, n = 5 biological replicates. Data are presented as mean ± SD values, averaged across all the replicates. For the bar graphs in these panels, 7%, 13%, 8% and 4% DMSO, and 80µM, 100µM, 100µM and 50µM of the peptides, were used for S. Typhimurium, A. baumannii, S. aureus and P. aeruginosa respectively. N.T- No treatment; WT - CP1-WT peptide; Mut - CP1-Mut peptide (negative control). P values are calculated by Student's unpaired t-test; nsP > 0.05; *** P < 0.001; **** P < 0.0001 Student's unpaired t-test.


A - D Transmission electron micrographs of the indicated bacterial strains ( left ) depicting the loss of membrane integrity upon treatment with the CP1 - WT peptide which is time - dependent . Arrows indicate membrane blebs , generalized membrane damage and the exclusion of cytoplasmic contents out of the cell . 1h , 2h and 3h indicate different treatment times of 1 , 2 and 3 hours respectively with the CP1 - WT and CP1 - Mut peptides as indicated in the panels . Untreated cells , used as controls , are also shown . Scale bars : 2μm . Data information : In each panel , across each fluorescent channel , the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 ( immediate addition ) and 2 hours respectively . nsP > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 ; Student ' s unpaired t - test . WT and Mut indicate CP1 - WT and CP1 - Mut treatments respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Transmission electron micrographs of the indicated bacterial strains (left) depicting the loss of membrane integrity upon treatment with the CP1-WT peptide which is time-dependent. Arrows indicate membrane blebs, generalized membrane damage and the exclusion of cytoplasmic contents out of the cell. 1h, 2h and 3h indicate different treatment times of 1, 2 and 3 hours respectively with the CP1-WT and CP1-Mut peptides as indicated in the panels. Untreated cells, used as controls, are also shown. Scale bars: 2μm. Data information: In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.


E - H Mean fluorescence intensities across the three fluorescent channels ( FM 4 - 64FX , FAM and DAPI fluorophores ) were estimated from confocal microscopy experiments to understand if the peptide was bound to the membrane or internalized in the cytosol of the bacteria indicated in each of the panels ( top ) . Triple staining procedure with FM 4 - 64FX ( membrane - binding dye ; red ) , FAM - conjugated peptides ( green ) and DAPI ( nuclear staining dye ; blue ) was used and the mean fluorescence intensities across these three fluorescent channels were calculated using ZEN 2 . 3 software . The bacterial cells were treated with 2 % DMSO / FAM - labelled CP1 - WT / FAM - labelled CP1 - Mut for 0 ( 0hr ) or 2 hours ( 2hr ) , followed by staining with FM 4 - 64FX and DAPI consecutively . The 0th hour data represents the data obtained after immediate addition of DMSO or peptide . In each panel , green circles represent autofluorescence ( cells emitting light of wavelength corresponding to that observed for FAM fluorophore ) of DMSO - treated cells at 0 and 2 hours . Data information : In panels E - H , n = 5 biological replicates are used . In each of these panels , the mean ± SD intensity values ( averaged across all the replicates ) obtained across each fluorescent channel ( FM 4 - 64FX , FAM and DAPI ) are presented for a particular bacterial strain ( indicated on top of the panel ) . In each panel , across each fluorescent channel , the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 ( immediate addition ) and 2 hours respectively . nsP > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 ; Student ' s unpaired t - test . WT and Mut indicate CP1 - WT and CP1 - Mut treatments respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H Mean fluorescence intensities across the three fluorescent channels (FM 4-64FX, FAM and DAPI fluorophores) were estimated from confocal microscopy experiments to understand if the peptide was bound to the membrane or internalized in the cytosol of the bacteria indicated in each of the panels (top). Triple staining procedure with FM 4-64FX (membrane-binding dye; red), FAM-conjugated peptides (green) and DAPI (nuclear staining dye; blue) was used and the mean fluorescence intensities across these three fluorescent channels were calculated using ZEN 2.3 software. The bacterial cells were treated with 2% DMSO/ FAM-labelled CP1-WT/ FAM-labelled CP1-Mut for 0 (0hr) or 2 hours (2hr), followed by staining with FM 4-64FX and DAPI consecutively. The 0th hour data represents the data obtained after immediate addition of DMSO or peptide. In each panel, green circles represent autofluorescence (cells emitting light of wavelength corresponding to that observed for FAM fluorophore) of DMSO-treated cells at 0 and 2 hours. Data information: In panels E-H, n = 5 biological replicates are used. In each of these panels, the mean ± SD intensity values (averaged across all the replicates) obtained across each fluorescent channel (FM 4-64FX, FAM and DAPI) are presented for a particular bacterial strain (indicated on top of the panel). In each panel, across each fluorescent channel, the unstriped and striped bars indicate the data for cells treated with DMSO or peptides for 0 (immediate addition) and 2 hours respectively. nsP > 0.05, * P < 0.05, ** P < 0.01; Student's unpaired t-test. WT and Mut indicate CP1-WT and CP1-Mut treatments respectively.


In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . A Changes in body weights relative to day 0 were noted . The mean percentage changes in body weights for each cohort are plotted across all the days . Data information : n = 2 biological replicates with 5 mice per cohort were used . In panel A , across the entire experimental tenure of 14 days , data are presented as mean ± SD values for each cohort , averaged across both the replicates for all the five mice ( studied in the respective cohort ) . Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights across DMSO - treated and the respective peptide treated cohorts ( ns P > 0 . 05 ) .

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

In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. A Changes in body weights relative to day 0 were noted. The mean percentage changes in body weights for each cohort are plotted across all the days. Data information: n = 2 biological replicates with 5 mice per cohort were used. In panel A, across the entire experimental tenure of 14 days, data are presented as mean ± SD values for each cohort, averaged across both the replicates for all the five mice (studied in the respective cohort). Multiple t test with Bonferroni Dunn correction was used to compare percentage change in body weights across DMSO-treated and the respective peptide treated cohorts (ns P > 0.05).


In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . B , After 14 days post peptide treatment , blood was collected from the animals by retro - orbital puncture and analysed for ( B ) alanine transaminase ( SGPT ) , alanine aminotransferase ( SGOT ) , alkaline phosphatase ( ALP ) Relative to the values obtained for the DMSO cohort , insignificant changes in serum levels of these parameters indicate that the peptide shows no significant hepatotoxic ( as seen in panel B ) Data information : biological replicates with 5 mice per cohort were used . In the panels B representative data for only one biological replicate across all the cohorts ( five mice per cohort ) are presented as mean ± SD values . P values were calculated in comparison to the DMSO - treated cohorts using Mann - Whitney rank sum test ( ns P > 0 . 05 ) .

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

In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. B, After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (B) alanine transaminase (SGPT), alanine aminotransferase (SGOT), alkaline phosphatase (ALP) Relative to the values obtained for the DMSO cohort, insignificant changes in serum levels of these parameters indicate that the peptide shows no significant hepatotoxic (as seen in panel B) Data information: biological replicates with 5 mice per cohort were used. In the panels B representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).


In vivo toxicity determination of the CP1 - WT peptide using a murine model . BALB / c mice ( Five mice per cohort ) were intraperitoneally injected with 1 . 6 % DMSO or varying peptide doses as indicated and monitored for 14 days . C After 14 days post peptide treatment , blood was collected from the animals by retro - orbital puncture and analysed for ( C ) creatinine and blood urea nitrogen ( BUN ) levels . Relative to the values obtained for the DMSO cohort , insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects ( as seen in panel C ) . Data information : n = 2 biological replicates with 5 mice per cohort were used . In panels C , representative data for only one biological replicate across all the cohorts ( five mice per cohort ) are presented as mean ± SD values . For panels C , P values were calculated in comparison to the DMSO - treated cohorts using Mann - Whitney rank sum test ( ns P > 0 . 05 ) .

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

In vivo toxicity determination of the CP1-WT peptide using a murine model. BALB/c mice (Five mice per cohort) were intraperitoneally injected with 1.6% DMSO or varying peptide doses as indicated and monitored for 14 days. C After 14 days post peptide treatment, blood was collected from the animals by retro-orbital puncture and analysed for (C) creatinine and blood urea nitrogen (BUN) levels. Relative to the values obtained for the DMSO cohort, insignificant changes of these parameters indicate that the peptide shows no significant nephrotoxic effects (as seen in panel C). Data information: n = 2 biological replicates with 5 mice per cohort were used. In panels C, representative data for only one biological replicate across all the cohorts (five mice per cohort) are presented as mean ± SD values. For panels C, P values were calculated in comparison to the DMSO-treated cohorts using Mann-Whitney rank sum test (ns P > 0.05).


Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . ( A ) Survival for symptoms were monitored . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . In panel A , percent survival was compared between untreated cohort ( N . T ) and the DMSO / peptide / ciprofloxacin - treated cohort using a two - way ANOVA test using Bonferroni correction ( ns P > 0 . 05 ; * P < 0 . 01 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively .

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

Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. (A) Survival for symptoms were monitored. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. In panel A, percent survival was compared between untreated cohort (N.T) and the DMSO/peptide/ciprofloxacin-treated cohort using a two-way ANOVA test using Bonferroni correction (ns P > 0.05; * P < 0.01). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.


Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . C - E Bacterial loads in S . Typhimurium infected organs , namely , ( C ) liver , ( D ) kidney and ( E ) spleen , were estimated . Data are presented as mean ± SD CFU / mL values for the bacterial organ loads isolated from seven mice in each cohort . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . In panels , ( C - E ) , Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively .

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

Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. C-E Bacterial loads in S. Typhimurium infected organs, namely, (C) liver, (D) kidney and (E) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice in each cohort. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. In panels, (C-E), Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.


Action of the CP1 - WT peptide in treatment of S . Typhimurium oral infection . F - J Histopathology analyses with representative H & E - stained liver sections at different magnifications are shown for S . Typhimurium infected liver treated with various agents . K Mean pathological scoring from the tissue sections used in histopathology analyses in panels F - J . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . In panels K to assign histopathological scores , data are collected from five mice in each cohort , and the representative data for only one biological replicate are presented as mean ± SD values . Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively . For histopathology analyses in panels ( F - J ) except for uninfected and peptide - treated mice , pathology sections from infected or treated ( DMSO or ciprofloxacin ) mice showed aggregation of multiple inflammatory cells ( INF ) and edema ( E ) in liver parenchyma , central vein congestion ( C ) , distortion of hepatic portal vein ( HPV ) , hemorrhage ( H ) , necrosis ( N ) and pyknotic nuclei ( arrows in panels H , Scale bar : 50µm in panels ( F - J )

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

Action of the CP1-WT peptide in treatment of S. Typhimurium oral infection. F-J Histopathology analyses with representative H&E-stained liver sections at different magnifications are shown for S. Typhimurium infected liver treated with various agents. K Mean pathological scoring from the tissue sections used in histopathology analyses in panels F-J. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. In panels K to assign histopathological scores, data are collected from five mice in each cohort, and the representative data for only one biological replicate are presented as mean ± SD values. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively. For histopathology analyses in panels (F-J) except for uninfected and peptide-treated mice, pathology sections from infected or treated (DMSO or ciprofloxacin) mice showed aggregation of multiple inflammatory cells (INF) and edema (E) in liver parenchyma, central vein congestion (C), distortion of hepatic portal vein (HPV), hemorrhage (H), necrosis (N) and pyknotic nuclei (arrows in panels H, Scale bar: 50µm in panels (F-J)


Action of the CP1 - WT peptide in treatment of A . baumannii intraperitoneal infections . L - N Bacterial loads in A . baumannii infected organs , namely , ( L ) liver , ( M ) kidney and ( N ) spleen , were estimated . Data are presented as mean ± SD CFU / mL values for the bacterial organ loads isolated from seven mice in each cohort . Data information : For all the three infection models stated above , n = 2 biological replicates with 7 mice per cohort were used . In panels ( L - N ) representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort . Mann - Whitney rank sum test was used ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) . The notations , namely , N . T denote no treatment ; Pep and Cip denote CP1 - WT and ciprofloxacin treatments respectively .

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

Action of the CP1-WT peptide in treatment of A. baumannii intraperitoneal infections. L-N Bacterial loads in A. baumannii infected organs, namely, (L) liver, (M) kidney and (N) spleen, were estimated. Data are presented as mean ± SD CFU/mL values for the bacterial organ loads isolated from seven mice in each cohort. Data information: For all the three infection models stated above, n = 2 biological replicates with 7 mice per cohort were used. In panels (L-N) representative data for only one biological replicate are presented as mean ± SD values for the parameters obtained from seven mice in each cohort. Mann-Whitney rank sum test was used (ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001). The notations, namely, N.T denote no treatment; Pep and Cip denote CP1-WT and ciprofloxacin treatments respectively.


( F ) UMAP plot showing the expression levels of EMT markers . The magenta dotted lines indicate cluster 6 ( partial EMT ) .

O 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) UMAP plot showing the expression levels of EMT markers. The magenta dotted lines indicate cluster 6 (partial EMT).


( E ) , ( F ) Heatmap of DEGs in ROR1high and ROR1low cells in the S2 - VP10 xenograft ( E ) or PDO # 1 xenograft ( 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

(E), (F) Heatmap of DEGs in ROR1high and ROR1low cells in the S2-VP10 xenograft (E) or PDO#1 xenograft (F).


( A ) Kaplan - Meier disease - specific survival curves based on ROR1 expression levels in PDAC patients from the TCGA database ( n = 154 ) . Data information : Graphs are presented as mean ± s . e . m . , * , p < 0 . 05 ; log - rank test ( A )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier disease-specific survival curves based on ROR1 expression levels in PDAC patients from the TCGA database (n = 154) . Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; log-rank test (A)


( C ) FACS gating to sort ROR1high and ROR1low cells and the number of organoids or colonies ( n = 3 or n = 5 , biological replicates ) . Mouse cells expressing H - 2Kd / H - 2Dd were eliminated . Data information : Graphs are presented as mean ± s . e . m . , * , p < 0 . 05 ; two - sided t - test ( C )

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

(C) FACS gating to sort ROR1high and ROR1low cells and the number of organoids or colonies (n = 3 or n = 5, biological replicates). Mouse cells expressing H-2Kd/H-2Dd were eliminated. Data information: Graphs are presented as mean ± s.e.m., *, p < 0.05; two-sided t-test (C)


( G ) Representative images of tissue stained with H & E and AB / PAS , and for ROR1 in the PDO # 1 xenograft and of tumor derived from ROR1high or ROR1low cells in the PDO # 1 xenograft . Data information : Scale bars , 100 µm ( G ) .

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(G) Representative images of tissue stained with H&E and AB/PAS, and for ROR1 in the PDO#1 xenograft and of tumor derived from ROR1high or ROR1low cells in the PDO#1 xenograft. Data information: Scale bars, 100 µm (G).


( H ) The expression level of AURKB in PANC - 1 cells transfected with control or ROR1 siRNA and in ROR1high or ROR1low cells from S2 - VP10 and PDO # 1 xenografts ( n = 3 ) . Data information : Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant .

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(H) The expression level of AURKB in PANC-1 cells transfected with control or ROR1 siRNA and in ROR1high or ROR1low cells from S2-VP10 and PDO#1 xenografts (n = 3). Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.


( I ) Western blot analysis of ROR1 , AURKB , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA .

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(I) Western blot analysis of ROR1, AURKB, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.


( J ) Treatment of PDO # 1 and S2 - VP10 organoids with DMSO ( vehicle ) or Aurora kinase inhibitors ( n = 3 , biological replicates ) . Tozasertib , pan - Aurora kinase inhibitor ; Barasertib , selective Aurora B kinase inhibitor . Representative images of organoids are shown . An area of 2 , 000 μm2 and more was identified as an organoid . The area of organoids is shown in the violin plot . Black or white solid lines indicate the median value for each violin . Data information : Scale bars 1 mm ( J ) Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant .

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(J) Treatment of PDO#1 and S2-VP10 organoids with DMSO (vehicle) or Aurora kinase inhibitors (n = 3, biological replicates). Tozasertib, pan-Aurora kinase inhibitor; Barasertib, selective Aurora B kinase inhibitor. Representative images of organoids are shown. An area of 2,000 μm2 and more was identified as an organoid. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars 1 mm (J) Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.


( K ) Organoid formation assay of ROR1high or ROR1low cells derived from S2 - VP10 EGFP ( control ) or S2 - VP10 AURKB - EGFP xenografts ( n = 3 , biological replicates ) . Representative images and the number of organoids are shown . Data information : Scale bars , 1 mm ( K ) . Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 ; n . s . , not significant .

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(K) Organoid formation assay of ROR1high or ROR1low cells derived from S2-VP10 EGFP (control) or S2-VP10 AURKB-EGFP xenografts (n = 3, biological replicates). Representative images and the number of organoids are shown. Data information: Scale bars, 1 mm (K). Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05; n.s., not significant.


( L ) Western blot analysis of ROR1 , AKT , phospho - AKT ( Ser473 ) , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA .

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(L) Western blot analysis of ROR1, AKT, phospho-AKT (Ser473), and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.


( N ) Western blot analysis of ROR1 , c - Myc , phospho - RB ( Ser780 ) , E2F1 , and β - actin in S2 - VP10 and S2 - 013 cells transfected with control or ROR1 siRNA .

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(N) Western blot analysis of ROR1, c-Myc, phospho-RB (Ser780), E2F1, and β-actin in S2-VP10 and S2-013 cells transfected with control or ROR1 siRNA.


( C ) Tumor growth curve of the four groups ( control , Dox only , gemcitabine only , and Dox + gemcitabine ) ( n = 8 ) . The endpoint for each group is shown by a slash . Data information : Data are presented as mean ± s . e . m . , two - sided t - test . * , p < 0 . 05 .

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(C) Tumor growth curve of the four groups (control, Dox only, gemcitabine only, and Dox + gemcitabine) (n = 8). The endpoint for each group is shown by a slash. Data information: Data are presented as mean ± s.e.m., two-sided t-test. *, p < 0.05.


( A ) - ( C ) Representative images of ROR1 , mCherry , and aSMA staining in the primary tumor ( pancreas ) ( A ) and metastatic lesions in the lung ( B ) and mesenteric lymph node ( C ) after orthotopic transplantation of S2 - VP10 - mCherry cells into the pancreas of BRJ mice . Data information : Scale bars , 100

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(A)-(C) Representative images of ROR1, mCherry, and aSMA staining in the primary tumor (pancreas) (A) and metastatic lesions in the lung (B) and mesenteric lymph node (C) after orthotopic transplantation of S2-VP10-mCherry cells into the pancreas of BRJ mice. Data information: Scale bars, 100 µm


( D ) , ( E ) Representative images of S2 - 013 orthotopic xenografts stained with H & E and for ROR1 in the pancreas ( D ) and mesenteric lymph node ( E ) . Data information : Scale bars , 100

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(D), (E) Representative images of S2-013 orthotopic xenografts stained with H&E and for ROR1 in the pancreas (D) and mesenteric lymph node (E). Data information: Scale bars, 100 µm


( A ) , H3K4me1 , K3K4me3 , H3K27ac , and open chromatin profiles around the ROR1 gene by CUT & RUN and ATAC - sequencing in cultured S2 - VP10 cells ( A ) The ROR1 promoter ( blue - shaded boxes ) and enhancer elements ( green - shaded box ) of ROR1 are indicated .

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(A), H3K4me1, K3K4me3, H3K27ac, and open chromatin profiles around the ROR1 gene by CUT&RUN and ATAC-sequencing in cultured S2-VP10 cells (A) The ROR1 promoter (blue-shaded boxes) and enhancer elements (green-shaded box) of ROR1 are indicated.


( E ) YAP occupancy at the ROR1 enhancer as determined by ChIP - qPCR . The known YAP targets , CTGF and CYR61 promoters , were tested as positive controls , and ROR1 intron 1 and AFM promoter were tested as negative controls ( n = 4 , biological replicates ) .

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(E) YAP occupancy at the ROR1 enhancer as determined by ChIP-qPCR. The known YAP targets, CTGF and CYR61 promoters, were tested as positive controls, and ROR1 intron 1 and AFM promoter were tested as negative controls (n = 4, biological replicates).


( G ) Western blot analysis of YAP , TAZ , ROR1 , and β - actin in S2 - VP10 cells transfected with control or YAP1 / WWTR1 siRNA .

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(G) Western blot analysis of YAP, TAZ, ROR1, and β-actin in S2-VP10 cells transfected with control or YAP1/WWTR1 siRNA.


( I ) Relative expression of ROR1 and YAP target genes ( CTGF and CYR61 ) in S2 - VP10 cells treated with DMSO or verteporfin ( VP ) ( n = 3 , biological replicates ) . mRNA levels are normalized to that of RPS18 .

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(I) Relative expression of ROR1 and YAP target genes (CTGF and CYR61) in S2-VP10 cells treated with DMSO or verteporfin (VP) (n = 3, biological replicates). mRNA levels are normalized to that of RPS18.


( M ) Relative luciferase activity using the ROR1 reporter in S2 - VP10 cells treated with DMSO or BET inhibitor ( JQ1 ) ( n = 3 , biological replicates ) . Data information : Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( M )

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(M) Relative luciferase activity using the ROR1 reporter in S2-VP10 cells treated with DMSO or BET inhibitor (JQ1) (n = 3, biological replicates). Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (M)


( O ) , Treatment of PDO # 1 and S2 - VP10 organoids with DMSO or JQ1 ( n = 3 , biological replicates ) . Relative expression of ROR1 ( O ) , mRNA levels are normalized to that of RPS18 . Data information : Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( O )

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(O), Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). Relative expression of ROR1 (O), mRNA levels are normalized to that of RPS18. Data information: Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (O)


( P ) Treatment of PDO # 1 and S2 - VP10 organoids with DMSO or JQ1 ( n = 3 , biological replicates ) . representative images of organoids , and organoid area ( P ) are shown . The area of organoids is shown in the violin plot . Black or white solid lines indicate the median value for each violin . Data information : Scale bars , 1 mm ( P ) . Data are presented as mean ± s . e . m . , * , p < 0 . 05 . ; two - sided t - test ( P ) .

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(P) Treatment of PDO#1 and S2-VP10 organoids with DMSO or JQ1 (n = 3, biological replicates). representative images of organoids, and organoid area (P) are shown. The area of organoids is shown in the violin plot. Black or white solid lines indicate the median value for each violin. Data information: Scale bars, 1 mm (P). Data are presented as mean ± s.e.m., *, p < 0.05. ; two-sided t-test (P).


A BAK - 6H permeabilising activity is exacerbated by cBID . BAK - 6H ( mBAK ∆ C21 - 6H ) was loaded at the indicated concentrations onto Ni - NTA liposomes ( 5 μM ) prior to the addition of cBID . The release of carboxyfluorescein was measured over time . Results representative of at least 3 independent experiments .

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A BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 μM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least 3 independent experiments.


B BAK - 6H oligomerises on liposomes induced by cBID . Liposomes were incubated with BAK - 6H ( 150 nM ) and the indicated concentration of cBID or a BID BH3 peptide for 60 min . Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN - PAGE . In parallel , samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran , normalised to total fluorescence of detergent permeabilised liposomes . Results representative of 3 independent experiments .

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B BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of 3 independent experiments.


C BAK - 6H oligomerisation on liposomes is blocked by BCL - XL . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( WT , 90 nM ) or cBID M97A ( 90 nM ) that has reduced affinity for BCL - XL , in the presence or absence of BCL - XL for 60 mins prior to analysis of BAK oligomerisation by BN - PAGE . Results representative of 2 independent experiments . Liposome permeabilisation was monitored with the indicated combinations of cBID ( 90 nM ) , BAK - 6H ( 150 nM ) and BCL - XL ( 1 μM ) . Data is presented as mean of 2 independent experiments with percentage release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50 % of detergent - lysis maximum .

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C BAK-6H oligomerisation on liposomes is blocked by BCL-XL. Liposomes were incubated with BAK-6H (150 nM) together with cBID (WT, 90 nM) or cBID M97A (90 nM) that has reduced affinity for BCL-XL, in the presence or absence of BCL-XL for 60 mins prior to analysis of BAK oligomerisation by BN-PAGE. Results representative of 2 independent experiments. Liposome permeabilisation was monitored with the indicated combinations of cBID (90 nM), BAK-6H (150 nM) and BCL-XL (1 μM). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


D BAK - 6H oligomerisation on liposomes involves its BH3 domain . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( 90 nM ) . Antibody recognising the BAK BH3 domain ( 4B5 ) or α1 - α2 loop ( 7D10 ) , as shown on the structure of BAK ( PDB : 2IMS ) , were added ( 2 . 4 μg / μl ) either prior to ( pre ) or after ( post ) incubation with cBID for 60 mins . BAK : Ab complex is indicated ( arrowhead ) . 7D10 which recognises the activated form of mBAK ( Iyer et al . , 2016 ) , bound to and gel - shifted BAK when added either before or after activation with cBID . In contrast , 4B5 failed to appreciably gel - shift inactive BAK ( lanes 3 and 5 ) or BAK that was already activated ( lane 6 ) , but gel - shifted BAK when present during cBID activation ( lane 4 ) . Liposome permeabilisation was assessed after 20 min incubation with cBID ( 90 nM ) , BAK - 6H ( 150 nM ) and antibodies 4B5 ( 2 . 5 μg ) or 7D10 ( 2 . 5 μg ) . Data is presented as mean of 2 independent experiments with percentage release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50 % of detergent - lysis maximum .

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D BAK-6H oligomerisation on liposomes involves its BH3 domain. Liposomes were incubated with BAK-6H (150 nM) together with cBID (90 nM). Antibody recognising the BAK BH3 domain (4B5) or α1-α2 loop (7D10), as shown on the structure of BAK (PDB:2IMS), were added (2.4 μg/μl) either prior to (pre) or after (post) incubation with cBID for 60 mins. BAK:Ab complex is indicated (arrowhead). 7D10 which recognises the activated form of mBAK (Iyer et al., 2016), bound to and gel-shifted BAK when added either before or after activation with cBID. In contrast, 4B5 failed to appreciably gel-shift inactive BAK (lanes 3 and 5) or BAK that was already activated (lane 6), but gel-shifted BAK when present during cBID activation (lane 4). Liposome permeabilisation was assessed after 20 min incubation with cBID (90 nM), BAK-6H (150 nM) and antibodies 4B5 (2.5 μg) or 7D10 (2.5 μg). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


B BAK , or BAK with cysteine introduced at the indicated positions ( on a BAK ∆ Cys background ) were stably expressed in Bax - / - Bak - / - MEFs and assess for BAK expression by immunoblotting ( inset panel ; * indicates non - specific band . ) and apoptotic activity in response to etoposide treatment .

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B BAK, or BAK with cysteine introduced at the indicated positions (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and assess for BAK expression by immunoblotting (inset panel; * indicates non-specific band.) and apoptotic activity in response to etoposide treatment.


C E24 and R169 are proximal in inactive BAK on mitochondria . Mitochondria - enriched membrane fractions from cells were incubated with oxidant ( CuPhe ) and induced intramolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

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C E24 and R169 are proximal in inactive BAK on mitochondria. Mitochondria-enriched membrane fractions from cells were incubated with oxidant (CuPhe) and induced intramolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


D E24 and R169 dissociate during BAK activation . Mitochondria - enriched membrane fractions from cells were incubated with cBID ( 100 nM ) prior to oxidant ( CuPhe ) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

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D E24 and R169 dissociate during BAK activation. Mitochondria-enriched membrane fractions from cells were incubated with cBID (100 nM) prior to oxidant (CuPhe) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


C Bax - / - Bak - / - MEFs expressing the indicated BAK variants were treated with doxycycline ( Dox , 3 h ) , incubated for a further 24 h in the presence or absence of BH3 - mimetic compounds , and cell death was assessed by PI uptake .

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C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake.


D Mutation of BH4 promotes BAK activating conformation change . Cells were treated with doxycycline ( 3 h ) to induce BAK expression followed by incubation with BH3 - mimetics where indicated ( for 2 h ) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK ( G3172 ) .

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D Mutation of BH4 promotes BAK activating conformation change. Cells were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172).