Figure 4 ( A - B ) Mouse primary peritoneal macrophages ( MPMs ) isolated from DCLK1f / f and DCLK1MCKO mice were challenged with oxLDL ( 50 μg / mL ) for 24 h . Protein levels of TNF - α ( A ) and IL - 6 ( B ) were analyzed using ELISA ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( C - D ) MPMs isolated from DCLK1f / f and DCLK1MCKO mice were challenged with oxLDL ( 50 μg / mL ) for 6 h . mRNA levels of Tnf - α ( C ) and Il - 6 ( D ) were determined via RT - qPCR ( n = 3 biological replicates ) . The values were normalized to β - actin . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( J , K ) Western blot analysis ( J ) and densitometric quantification ( K ) of p - p65 and IκBα . GAPDH and p65 were used as loading controls ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( L - M ) MPMs were treated as described in panel ( J ) . Western blot analysis ( L ) and densitometric quantification ( M ) of p65 in nucleus and cytoplasm . GAPDH was used as the loading control for cytosolic fractions . Lamin B1 was used as the loading control for nuclear fractions ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( N - O ) MPMs were treated as described in panel ( J ) . Representative immunofluorescence staining images ( N ) and quantification ( O ) of NF - κB p65 ( red ) translocating into nucleus in MPMs . Cells were counterstained with DAPI ( blue ) . Scale bar = 25 μm , n = 3 biological replicates . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( P - Q ) Representative immunofluorescence staining images ( P ) and quantification ( Q ) of p - p65 ( red ) in aortic roots . Tissues were counterstained with DAPI ( blue ) . Scale bar = 100 μm , n = 10 biological replicates . p - p65 area was quantified by the proportion of p - p65 positive area to plaque area . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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

Figure 4(A-B) Mouse primary peritoneal macrophages (MPMs) isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/mL) for 24 h. Protein levels of TNF-α (A) and IL-6 (B) were analyzed using ELISA (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(C-D) MPMs isolated from DCLK1f/f and DCLK1MCKO mice were challenged with oxLDL (50 μg/mL) for 6 h. mRNA levels of Tnf-α (C) and Il-6 (D) were determined via RT-qPCR (n=3 biological replicates). The values were normalized to β-actin. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(J, K) Western blot analysis (J) and densitometric quantification (K) of p-p65 and IκBα. GAPDH and p65 were used as loading controls (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(L-M) MPMs were treated as described in panel (J). Western blot analysis (L) and densitometric quantification (M) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(N-O) MPMs were treated as described in panel (J). Representative immunofluorescence staining images (N) and quantification (O) of NF-κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(P-Q) Representative immunofluorescence staining images (P) and quantification (Q) of p-p65 (red) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar=100 μm, n=10 biological replicates. p-p65 area was quantified by the proportion of p-p65 positive area to plaque area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


Figure 5 ( B ) 293T cells were transfected with Flag - DCLK1 plasmid for 24 h . Control cells were transfected with empty vector ( EV ) . Levels of Flag were measured by Western blot ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( D ) Co - immunoprecipitation of DCLK1 and IKKβ in 293T cells transfected with Flag - DCLK1 . Flag - DCLK1 was immunoprecipitated by anti - Flag antibody . IgG , immunoglobulin G . ( E ) Co - immunoprecipitation of DCLK1 and IKKβ in MPMs challenged with oxLDL ( 50 μg / mL ) for 1 h . DCLK1 was immunoprecipitated by anti - DCLK1 antibody . 293T cells were co - transfected with Flag - DCLK1 and si - IKKβ for 24 h . Western blot analysis ( H ) of IκBα , p - IKKβ and p - p65 . GAPDH , IKKβ and p65 were used as loading controls ( n = 3 biological replicates ) .

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

Figure 5(B) 293T cells were transfected with Flag-DCLK1 plasmid for 24 h. Control cells were transfected with empty vector (EV). Levels of Flag were measured by Western blot (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(D) Co-immunoprecipitation of DCLK1 and IKKβ in 293T cells transfected with Flag-DCLK1. Flag-DCLK1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G. (E) Co-immunoprecipitation of DCLK1 and IKKβ in MPMs challenged with oxLDL (50 μg/mL) for 1 h. DCLK1 was immunoprecipitated by anti-DCLK1 antibody.293T cells were co-transfected with Flag-DCLK1 and si-IKKβ for 24 h. Western blot analysis (H) of IκBα, p-IKKβ and p-p65. GAPDH, IKKβ and p65 were used as loading controls (n=3 biological replicates).


Figure 6 ( B ) MPMs were pretreated with DCLK1 - IN - 1 ( 5 and 10 μM ) or vehicle ( DMSO , 1 ‰ ) for 1 h , followed by exposure of oxLDL ( 50 μg / mL ) for 24 h . Protein levels of TNF - α and IL - 6 were analyzed using ELISA ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test . ( D - E ) MPMs were pretreated with DCLK1 - IN - 1 ( 5 and 10 μM ) or vehicle ( DMSO , 1 ‰ ) for 1 h , followed by exposure of oxLDL ( 50 μg / mL ) for 1 h . Western blot analysis ( D ) and densitometric quantification ( E ) of IκBα , p - IKKβ and p - p65 . GAPDH , IKKβ and p65 were used as loading controls ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test . ( F - G ) MPMs were treated as described in panel ( E ) . Western blot analysis ( F ) and densitometric quantification ( G ) of p65 in nucleus and cytoplasm . GAPDH was used as the loading control for cytosolic fractions . Lamin B1 was used as the loading control for nuclear fractions ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test . ( H - I ) MPMs were treated as described in panel ( D ) . Representative immunofluorescence staining images ( H ) and quantification ( I ) of NF - κB p65 ( red ) translocating into nucleus in MPMs . Cells were counterstained with DAPI ( blue ) . Scale bar = 25 μm , n = 3 biological replicates . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test .

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

Figure 6(B) MPMs were pretreated with DCLK1-IN-1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 24 h. Protein levels of TNF-α and IL-6 were analyzed using ELISA (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.(D-E) MPMs were pretreated with DCLK1-IN-1 (5 and 10 μM) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 μg/mL) for 1 h. Western blot analysis (D) and densitometric quantification (E) of IκBα, p-IKKβ and p-p65. GAPDH, IKKβ and p65 were used as loading controls (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.(F-G) MPMs were treated as described in panel (E). Western blot analysis (F) and densitometric quantification (G) of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.(H-I) MPMs were treated as described in panel (D). Representative immunofluorescence staining images (H) and quantification (I) of NF-κB p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar=25 μm, n=3 biological replicates. Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.


Figure 7Representative images ( C ) of plaque lesion area in aortic arches ( n = 10 biological replicates ) . The plaque area was quantified by the proportion of plaque area to aortic arches area . ( K - N ) Protein ( K - L ) and mRNA ( M - N ) levels of inflammatory cytokines TNF - α and IL - 6 in serum and aortas . The values of mRNA levels were normalized to Rn18s ( n = 10 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 , two - tailed unpaired Student ' s t - test .

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

Figure 7Representative images (C) of plaque lesion area in aortic arches (n=10 biological replicates). The plaque area was quantified by the proportion of plaque area to aortic arches area.(K-N) Protein (K-L) and mRNA (M-N) levels of inflammatory cytokines TNF-α and IL-6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05, two-tailed unpaired Student's t-test.


Figure 1B Flow cytometry based on CD49f and EpCAM staining separates lineage - negative breast tissue cells into stromal ( CD49f - EpCAM - ) and epithelial cells , which includes basal ( CD49f + EpCAMlo / - ) , luminal progenitor ( LP ) ( CD49f + EpCAM + ) and mature luminal ( ML ) ( CD49f - EpCAM + ) cells . C t - SNE plot of the integrated scRNA - seq profiles of epithelial cells from 11 reduction mammoplasties . Cell colors correspond to tissue specimens . D Same t - SNE plot as ( C ) but separated by hormonal status of the donor ( 8 premenopausal and 3 postmenopausal ) .

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

Figure 1B Flow cytometry based on CD49f and EpCAM staining separates lineage-negative breast tissue cells into stromal (CD49f-EpCAM-) and epithelial cells, which includes basal (CD49f+EpCAMlo/-), luminal progenitor (LP) (CD49f+EpCAM+) and mature luminal (ML) (CD49f-EpCAM+) cells.C t-SNE plot of the integrated scRNA-seq profiles of epithelial cells from 11 reduction mammoplasties. Cell colors correspond to tissue specimens. D Same t-SNE plot as (C) but separated by hormonal status of the donor (8 premenopausal and 3 postmenopausal).


Figure 2A t - SNE map of combined scRNA - seq transcriptomes of total tissue cells from 13 reduction mammoplasties . Cell colors correspond to tissue specimens . B Same t - SNE map as ( A ) but colored by cell cluster ( with cluster resolution 0 . 05 ) . C Ternary plot positioning each cell according the proportion of basal , LP or ML signature genes expressed by that cell . The plot shows the same cells and cell colors as for ( B ) . D Re - clustered EPCAM - negative non - epithelial cells revealed seven clusters ( resolution 0 . 05 ) representing immune and stromal cell lineages . E Multidimensional scaling plot showing expression profile distances between the pseudo - bulk samples . Each dot corresponds to aggregated expression for a cell cluster for one patient . Cluster colors are overlaid from ( D , right panel ) . Distances on the plot correspond to leading log2 - fold - change , defined as the average log2 - fold - change for the 500 most differential genes between each pair of profiles . F Heat map of pseudo - bulk samples showing marker genes for each non - epithelial cluster . The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo - bulk read counts . Color bars at the top of the plot indicate the cluster and menopausal status ( blue / yellow for pre / post menopause ) of each sample . G Same t - SNE map of non - epithelial cells as in ( D ) but colored by menopausal status . Barplot shows relative cluster sizes ( percentage of total cells ) for each status condition . Clusters 1 and 2 have significantly different sizes in post - menopause samples after allowing for inter - patient variability ( P = 0 . 040 and P = 0 . 032 by quasi - binomial F - test ) . Sizes are not significantly different for other clusters ( P > 0 . 15 ) . H Relative cluster sizes as for ( G ) but by individual patients . Cluster colors correspond to ( D ) . by cluster . A quasi - multinomial F - test was used to test for differences in cluster frequencies between pre - and post - menopausal samples ( P = 0 . 007 ) . I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre - and post - menopausal samples and the cell clusters identified in Fig 2D .

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

Figure 2A t-SNE map of combined scRNA-seq transcriptomes of total tissue cells from 13 reduction mammoplasties. Cell colors correspond to tissue specimens. B Same t-SNE map as (A) but colored by cell cluster (with cluster resolution 0.05). C Ternary plot positioning each cell according the proportion of basal, LP or ML signature genes expressed by that cell. The plot shows the same cells and cell colors as for (B).D Re-clustered EPCAM-negative non-epithelial cells revealed seven clusters (resolution 0.05) representing immune and stromal cell lineages. E Multidimensional scaling plot showing expression profile distances between the pseudo-bulk samples. Each dot corresponds to aggregated expression for a cell cluster for one patient. Cluster colors are overlaid from (D, right panel). Distances on the plot correspond to leading log2-fold-change, defined as the average log2-fold-change for the 500 most differential genes between each pair of profiles. F Heat map of pseudo-bulk samples showing marker genes for each non-epithelial cluster. The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Color bars at the top of the plot indicate the cluster and menopausal status (blue/yellow for pre/post menopause) of each sample. G Same t-SNE map of non-epithelial cells as in (D) but colored by menopausal status. Barplot shows relative cluster sizes (percentage of total cells) for each status condition. Clusters 1 and 2 have significantly different sizes in post-menopause samples after allowing for inter-patient variability (P = 0.040 and P=0.032 by quasi-binomial F-test). Sizes are not significantly different for other clusters (P > 0.15). H Relative cluster sizes as for (G) but by individual patients. Cluster colors correspond to (D). by cluster. A quasi-multinomial F-test was used to test for differences in cluster frequencies between pre- and post-menopausal samples (P = 0.007). I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre- and post-menopausal samples and the cell clusters identified in Fig 2D.


Figure 3A Microenvironment t - SNE map separated by menopausal status and colored by expression ( red = high expression , grey = undetectable ) of selected fibroblast markers ( upper panel ) . The dotted lines indicate the pericyte subsets . Bottom panel shows percentage of cells expressing the markers for the cell clustersB Co - immunofluorescence staining of pre - versus post - menopausal tissue for E - cadherin ( cyan ) , PDGFRβ ( yellow ) and F - actin ( pink ) . DAPI is shown in grey . The arrowheads in ( v ) depict fibroblasts in direct contact with the myoepithelial layer . For 2D and 3D confocal imaging : n = 13 premenopausal and n = 10 postmenopausal specimens . Scale bars : wholemount and optical sections : 100 μm ( panels i - iv ) ; enlargements , 30 μm ( panel v ) . C t - SNE plot of the integrated scRNA - seq profiles of fibroblasts from pre - and post - menopausal tissue Cell colors correspond to tissue specimens . D Same t - SNE plot as in ( C ) showing 5 distinct clusters ( clusters 3 and 5 were specific to one patient ) . F Heat map of same cells as ( D ) showing expression of the top 20 marker genes in each cluster . Color bars at the top of the plot show cluster membership ( colors as in ( D ) ) and pre - or post - menopausal status ( blue and yellow , respectively ) .

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

Figure 3A Microenvironment t-SNE map separated by menopausal status and colored by expression (red=high expression, grey=undetectable) of selected fibroblast markers (upper panel). The dotted lines indicate the pericyte subsets. Bottom panel shows percentage of cells expressing the markers for the cell clustersB Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).C t-SNE plot of the integrated scRNA-seq profiles of fibroblasts from pre-and post-menopausal tissue Cell colors correspond to tissue specimens. D Same t-SNE plot as in (C) showing 5 distinct clusters (clusters 3 and 5 were specific to one patient).F Heat map of same cells as (D) showing expression of the top 20 marker genes in each cluster. Color bars at the top of the plot show cluster membership (colors as in (D)) and pre- or post-menopausal status (blue and yellow, respectively).


Figure 4A t - SNE map of combined scRNA - seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers ( BRCA1 ; n = 4 ) and non - BRCA premenopausal women ( n = 8 ) with no family history of breast cancer . Cell colors represent individual samples . B Same t - SNE map as in ( A ) but colored by cluster ( cluster resolution 0 . 12 ) . C Epithelial clusters were identified by EPCAM expression and the non - epithelial cells were re - clustered to reveal immune and stromal cell populations ( cluster resolution 0 . 08 ) . Lineage identity was determined by hierarchical clustering according to top marker genesD t - SNE plot showing the combined single - cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs ( n = 4 for each ) , colored according to individual patients ( B1 = preneoplastic BRCA1 + / - tissue ; TN - B1 = BRCA1 - associated TNBCs ) . E Same t - SNE map as ( D ) but colored by cluster ( cluster resolution 0 . 15 ) . F Expression of epithelial markers indicated on the combined t - SNE plot as in ( D , E ) for BRCA1 preneoplastic and BRCA1 - associated tumor cells . G Epithelial clusters were identified by EPCAM expression and non - epithelial cells are indicated by the dotted line . H Same t - SNE map as in ( D , E ) but colored according to cancerous state : preneoplastic tissue ( blue ) and BRCA1 - associated TNBCs ( yellow ) . I Box plots of signature expression scores for the 13 cell clusters in ( D , E ) according to human breast epithelial and stromal gene signatures . Cluster 1 corresponds to tumor cells , while clusters 6 , 9 and 10 represent adjacent normal LP , basal and ML cells , respectively . Box plots show quartiles , minimum and maximum .

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

Figure 4A t-SNE map of combined scRNA-seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers (BRCA1; n=4) and non-BRCA premenopausal women (n=8) with no family history of breast cancer. Cell colors represent individual samples. B Same t-SNE map as in (A) but colored by cluster (cluster resolution 0.12). C Epithelial clusters were identified by EPCAM expression and the non-epithelial cells were re-clustered to reveal immune and stromal cell populations (cluster resolution 0.08). Lineage identity was determined by hierarchical clustering according to top marker genesD t-SNE plot showing the combined single-cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs (n=4 for each), colored according to individual patients (B1 = preneoplastic BRCA1+/- tissue; TN-B1= BRCA1-associated TNBCs). E Same t-SNE map as (D) but colored by cluster (cluster resolution 0.15). F Expression of epithelial markers indicated on the combined t-SNE plot as in (D, E) for BRCA1 preneoplastic and BRCA1-associated tumor cells. G Epithelial clusters were identified by EPCAM expression and non-epithelial cells are indicated by the dotted line. H Same t-SNE map as in (D, E) but colored according to cancerous state: preneoplastic tissue (blue) and BRCA1-associated TNBCs (yellow). I Box plots of signature expression scores for the 13 cell clusters in (D, E) according to human breast epithelial and stromal gene signatures. Cluster 1 corresponds to tumor cells, while clusters 6, 9 and 10 represent adjacent normal LP, basal and ML cells, respectively. Box plots show quartiles, minimum and maximum.


Figure 5A Reclustered EPCAM - negative cells ( excluding clusters 1 , 6 , 9 and 10 revealed immune / stromal cells in the microenvironment , identified using lineage markers . B Combined t - SNE plot as in ( A ) of the single - cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers ( blue ; preneo ) and BRCA1 - associated TNBCs ( yellow ; tumor ) . C t - SNE plots showing expression profiles of cardinal markers of immune and stromal cells . D Bar plots showing the percentage of cells expressing typical immune cell ( including Treg ) genes for clusters in ( A ) by preneoplastic vs tumor . E Left panel , same t - SNE map as in ( A ) but separated into cells from preneoplastic ( blue ) and tumor specimens ( yellow ) . Endothelial cell ( endo ) , fibroblast cell ( fibro ) and pericytes ( peri ) clusters are marked . Right panel , proportion of clusters as in ( A ) according to tissue type . F Relative cluster sizes as in ( E ) but by individual patient . G Co - immunofluorescence of tissue stained for the epithelial marker ( cytokeratin 19 ; yellow ) and the tumor - associated macrophage marker CX3CR1 ( magenta ) . DAPI is shown in white ( n = 2 preneoplastic samples ; n = 2 tumors ) . Scale bar , 100 μm . H Heat map of top differentially expressed genes in the major immune / stromal cell clusters , identified in ( A ) . BRCA1 + / - preneoplastic cells , blue ; BRCA1 - associated tumor cells , yellow .

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Figure 5A Reclustered EPCAM-negative cells (excluding clusters 1, 6, 9 and 10 revealed immune/stromal cells in the microenvironment, identified using lineage markers. B Combined t-SNE plot as in (A) of the single-cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers (blue; preneo) and BRCA1-associated TNBCs (yellow; tumor). C t-SNE plots showing expression profiles of cardinal markers of immune and stromal cells. D Bar plots showing the percentage of cells expressing typical immune cell (including Treg) genes for clusters in (A) by preneoplastic vs tumor. E Left panel, same t-SNE map as in (A) but separated into cells from preneoplastic (blue) and tumor specimens (yellow). Endothelial cell (endo), fibroblast cell (fibro) and pericytes (peri) clusters are marked. Right panel, proportion of clusters as in (A) according to tissue type. F Relative cluster sizes as in (E) but by individual patient.G Co-immunofluorescence of tissue stained for the epithelial marker (cytokeratin 19; yellow) and the tumor-associated macrophage marker CX3CR1 (magenta). DAPI is shown in white (n=2 preneoplastic samples; n=2 tumors). Scale bar, 100 μm.H Heat map of top differentially expressed genes in the major immune/stromal cell clusters, identified in (A). BRCA1+/- preneoplastic cells, blue; BRCA1-associated tumor cells, yellow.


Figure 6A - C t - SNE plots of combined scRNA - seq profiles of total cells from 8 TNBC tumors , 6 HER2 + tumors and 13 ER + tumors respectively . Integration and cluster sizes for the same cells Cells colored by cluster ( top left panels ) , EPCAM expression ( top right ) , cancer subtype marker ( bottom left ) or MKI67 expression ( bottom right ) . Dotted lines delineate epithelial cells ( top panels ) and cycling epithelial cells ( bottom panels ) . Normal epithelial subsets ( normal ) are also demarcated by dotted lines in the upper - right panels of B and C . D InferCNV plots to map inferred copy number variation ( CNV ) for the combined tumor scRNA - seq expression data for the epithelial clusters indicated in panels A - C . scRNA - seq data from normal breast epithelial cells ( N - 1105 - epi ) served as a reference for normalization . Each row represents a gene and each column cells from a cluster in a single tumor . The tumor clusters are color - coded as in A - C . Amplifications ( red ) and deletions ( blue ) are inferred from the gene expression . Tumor cells were distinguished from normal ( N ) cells by abundance of CNV . H Combined t - SNE transcriptome profiles of two distinct ER + tumors isolated from the same breast of a patient : ER - 0029 - 7C ( blue ) and ER - 0029 - 9C ( yellow ) . The corresponding t - SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right - hand panel .

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Figure 6A-C t-SNE plots of combined scRNA-seq profiles of total cells from 8 TNBC tumors, 6 HER2+ tumors and 13 ER+ tumors respectively. Integration and cluster sizes for the same cells Cells colored by cluster (top left panels), EPCAM expression (top right), cancer subtype marker (bottom left) or MKI67 expression (bottom right). Dotted lines delineate epithelial cells (top panels) and cycling epithelial cells (bottom panels). Normal epithelial subsets (normal) are also demarcated by dotted lines in the upper-right panels of B and C. D InferCNV plots to map inferred copy number variation (CNV) for the combined tumor scRNA-seq expression data for the epithelial clusters indicated in panels A-C. scRNA-seq data from normal breast epithelial cells (N-1105-epi) served as a reference for normalization. Each row represents a gene and each column cells from a cluster in a single tumor. The tumor clusters are color-coded as in A-C. Amplifications (red) and deletions (blue) are inferred from the gene expression. Tumor cells were distinguished from normal (N) cells by abundance of CNV.H Combined t-SNE transcriptome profiles of two distinct ER+ tumors isolated from the same breast of a patient: ER-0029-7C (blue) and ER-0029-9C (yellow). The corresponding t-SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right-hand panel.


Figure 7A - C t - SNE maps of reclustered EPCAM - negative non - epithelial cells Cluster resolutions 0 . 136 , 0 . 1 and 0 . 1 respectively . The major cell clusters within the microenvironment were identified based on expression of lineage - specific genes . Heat maps of pseudo - bulk samples show marker genes for each cluster . The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo - bulk read counts . Cluster 9 in HER2 + tumors ( B ) expressed myeloid and luminal epithelial markers , suggesting phagocytosis of the latter by macrophages . Color bars at the top of the heat map indicate the cluster of each sample ; top genes that mark each cluster are indicated .

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Figure 7A-C t-SNE maps of reclustered EPCAM-negative non-epithelial cells Cluster resolutions 0.136, 0.1 and 0.1 respectively. The major cell clusters within the microenvironment were identified based on expression of lineage-specific genes. Heat maps of pseudo-bulk samples show marker genes for each cluster. The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Cluster 9 in HER2+ tumors (B) expressed myeloid and luminal epithelial markers, suggesting phagocytosis of the latter by macrophages. Color bars at the top of the heat map indicate the cluster of each sample; top genes that mark each cluster are indicated.


Figure 8A , B Image quantification showing number of CD8 + Ki67 + cells ( A ) or CD68 + Ki67 + cells ( B ) per mm2 of tissue from TNBC , HER2 + and ER + tumors , either within the tumor region ( K8 / 18 + ) or the stroma ( defined as > 5 μm from tumor border ) . Error bars represent s . e . m . CD8 / Ki67 : n = 11 for TNBC , n = 12 for ER + , n = 5 for HER2 + . CD68 / Ki67 : n = 6 for TNBC , n = 8 for ER + , n = 5 for HER2 + . C - F Representative confocal images of ER + ( ER - 0032 ) and triple negative tumors ( TN - 0066 ) immunolabeled for K8 / 18 ( yellow ) , CD68 ( green ) and Ki67 ( red ) ( C , E ) or K8 / 18 ( yellow ) , CD8 ( green ) and Ki67 ( red ) ( D , F ) . DAPI is shown in blue . Arrows depict proliferative T cells ( CD8 + Ki67 + ) or macrophages ( CD68 + Ki67 + ) . Enlargements on shown in the right - hand panels . Scale bars , 200 µm for large tilescans ; 50 µm for enlargements and smaller tilescans .

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Figure 8A, B Image quantification showing number of CD8+Ki67+ cells (A) or CD68+Ki67+ cells (B) per mm2 of tissue from TNBC, HER2+ and ER+ tumors, either within the tumor region (K8/18+) or the stroma (defined as >5 μm from tumor border). Error bars represent s.e.m. CD8/Ki67: n=11 for TNBC, n=12 for ER+, n=5 for HER2+. CD68/Ki67: n=6 for TNBC, n=8 for ER+, n=5 for HER2+.C-F Representative confocal images of ER+ (ER-0032) and triple negative tumors ( TN-0066) immunolabeled for K8/18 (yellow), CD68 (green) and Ki67 (red) (C, E) or K8/18 (yellow), CD8 (green) and Ki67 (red) (D, F). DAPI is shown in blue. Arrows depict proliferative T cells (CD8+Ki67+) or macrophages (CD68+Ki67+). Enlargements on shown in the right-hand panels. Scale bars, 200 µm for large tilescans; 50 µm for enlargements and smaller tilescans.


Figure 9C Immunostaining of tumor and LN from patient ER - 0064 for expression of ER , PR and pan - cytokeratin . Insets show sections stained with control isotype antibodies . PR , progesterone receptor . Scale bar , 100 μm .

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Figure 9C Immunostaining of tumor and LN from patient ER-0064 for expression of ER, PR and pan-cytokeratin. Insets show sections stained with control isotype antibodies. PR, progesterone receptor. Scale bar, 100 μm.


Figure 1 ( 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 . ( 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 . ( 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 . ( 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 ) . ( 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 .

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Figure 1(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.(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.(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.(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).(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.


Figure 2Cells 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Cells 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.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.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.


Figure 3 ( 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 . ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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.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.


Figure 4Cells 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 . 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Cells 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.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.(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.(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.


Figure 5 ( 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 . 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 inIndicated 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 ) . 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 .

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

Figure 5(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.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 inIndicated 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).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.


Figure 1D 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1D 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.


Figure 2B , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B, 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.


Figure 3A - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-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.


Figure 4A - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-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.