F Measurements of mitochondrial respiration with complex II substrates ( succinate and glycerol - 3 - phosphate ) did not show significant differences between Phb2 - deficient and control podocytes ( n = 6 ) .

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F Measurements of mitochondrial respiration with complex II substrates (succinate and glycerol-3-phosphate) did not show significant differences between Phb2-deficient and control podocytes (n = 6).


G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2 - deficient podocytes compared to control podocytes ( n = 3 ) .

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G Quantification of mtDNA as a measure for mitochondrial mass showed similar levels of mtDNA in Phb2-deficient podocytes compared to control podocytes (n = 3).


H FACS analysis of MitoTracker - and MitoSOX - stained Phb2 - deficient podocytes and control podocytes . I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2 - deficient podocytes compared to control podocytes ( n = 3 , P = 0 . 7782 for MitoTracker , P = 0 . 1088 for MitoSOX ) .

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H FACS analysis of MitoTracker- and MitoSOX-stained Phb2-deficient podocytes and control podocytes.I Quantification of mean fluorescence intensity of MitoTracker and MitoSOX by FACS analysis revealed the same mitochondrial mass and the same ROS levels in Phb2-deficient podocytes compared to control podocytes (n = 3, P = 0.7782 for MitoTracker, P = 0.1088 for MitoSOX).


A Phb2 - deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes , which could be blocked by treatment with rapamycin ( n = 5 , bars represent mean ± SEM , Student ' s t - test , * * P = 0 . 0036 ) . pS6RP , phosphorylated S6 ribosomal protein ; S6RP , S6 ribosomal protein .

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A Phb2-deficient mouse podocytes showed higher levels of phosphorylated S6 ribosomal protein than control podocytes, which could be blocked by treatment with rapamycin (n = 5, bars represent mean ± SEM, Student's t-test, **P = 0.0036). pS6RP, phosphorylated S6 ribosomal protein; S6RP, S6 ribosomal protein.


B Immunohistochemistry for phosphorylated S6 ribosomal protein ( pS6RP ) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl / fl mice ( arrows point to cells showing a signal for pS6RP ; scale bar : 20 μm ) .

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B Immunohistochemistry for phosphorylated S6 ribosomal protein (pS6RP) on kidney sections revealed increased levels of pS6RP in glomeruli of Phb2pko compared to Phb2fl/fl mice (arrows point to cells showing a signal for pS6RP; scale bar: 20 μm).


C Kaplan - Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle - treated Phb2pko mice ( n = 11 for Phb2fl / fl + rapamycin , n = 15 for Phb2pko + vehicle , n = 16 for Phb2pko + rapamycin ) . D Statistical analysis comparing rapamycin - treated Phb2pko mice with vehicle - treated Phb2pko mice .

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C Kaplan-Meier survival curve revealed that treatment of Phb2pko mice with rapamycin prolonged survival for several days compared to vehicle-treated Phb2pko mice (n = 11 for Phb2fl/fl + rapamycin, n = 15 for Phb2pko + vehicle, n = 16 for Phb2pko + rapamycin).D Statistical analysis comparing rapamycin-treated Phb2pko mice with vehicle-treated Phb2pko mice.


( C ) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells ( upper panel ) . The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel ( n = 3 independent experiments ) .

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(C) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells (upper panel). The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel (n = 3 independent experiments).


( C ) Survival curves of mice transplanted with CD4 + T cells expressing the indicated proteins . The number of animals used is indicated in the graph . Note : the mouse transplanted with EGFP - transduced cells that has died in these experiments did not show any sign of tumor development according to anatomopathological

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(C) Survival curves of mice transplanted with CD4+ T cells expressing the indicated proteins. The number of animals used is indicated in the graph. Note: the mouse transplanted with EGFP-transduced cells that has died in these experiments did not show any sign of tumor development according to anatomopathological analyses


( F ) Quantification of CD4 + versus CD8 + T cell ratio in peripheral blood from mice transplanted with CD4 + T cells expressing the indicated proteins ( bottom ) . Each point represents the measurement of an individual mouse . n as in B . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(F) Quantification of CD4+ versus CD8+ T cell ratio in peripheral blood from mice transplanted with CD4+ T cells expressing the indicated proteins (bottom). Each point represents the measurement of an individual mouse. n as in B. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( J - L ) Representative flow cytometry analysis ( J ) and quantification of the surface levels of ICOS ( K ) and CD69 ( L ) in TFH cells from mice transplanted with CD4 + T cells expressing the indicated proteins . In J , numbers indicate the relative percentage ( % ) of the cell population that has been interrogated ( boxed ) . In K and L , each point represents the values obtained with a single experimental mouse . n as in H . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(J-L) Representative flow cytometry analysis (J) and quantification of the surface levels of ICOS (K) and CD69 (L) in TFH cells from mice transplanted with CD4+ T cells expressing the indicated proteins. In J, numbers indicate the relative percentage (%) of the cell population that has been interrogated (boxed). In K and L, each point represents the values obtained with a single experimental mouse. n as in H. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( M ) Abundance of indicated transcripts ( bottom ) in splenic cells from control ( EGFP + ) , tumor - bearing mice ( expressing EGFP and Vav1 ∆ C ) and mice transplanted with cells transduced with the catalytically dead version of Vav1 ∆ C ( expressing EGFP and Vav1 ∆ C + E201A ) . Values are given relative to the expression of each transcript in samples obtained from EGFP controls ( which was given an arbitrary value of 1 ) . n = 4 animals per class analyzed . a . u . , arbitrary unit . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(M) Abundance of indicated transcripts (bottom) in splenic cells from control (EGFP+), tumor-bearing mice (expressing EGFP and Vav1∆C) and mice transplanted with cells transduced with the catalytically dead version of Vav1∆C (expressing EGFP and Vav1∆C+E201A). Values are given relative to the expression of each transcript in samples obtained from EGFP controls (which was given an arbitrary value of 1). n = 4 animals per class analyzed. a.u., arbitrary unit. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( C ) Representative immunoblot showing the abundance of the ectopic Vav1 proteins , EGFP and the endogenous tubulin α ( loading control ) in CD4 + T - cells transduced with retrovirus particles used in these experiments ( top ) .

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(C) Representative immunoblot showing the abundance of the ectopic Vav1 proteins, EGFP and the endogenous tubulin α (loading control) in CD4+ T-cells transduced with retrovirus particles used in these experiments (top).


( E , F ) Representative FACS plots ( E ) and quantification ( F ) of the proliferation of EGFP + CD4 + T cells expressing the indicated proteins using the Cell Trace Violet method . In E , the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4 + T cells before stimulation and retroviral transduction . In F , each point represents the values obtained with a single experimental mouse . n = 6 per each experimental condition , except in the case of Vav1 ∆ N ( n = 3 ) . Data information : values are shown as means ± SEM from three independent experiments .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD 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) Representative FACS plots (E) and quantification (F) of the proliferation of EGFP+ CD4+ T cells expressing the indicated proteins using the Cell Trace Violet method. In E, the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4+ T cells before stimulation and retroviral transduction. In F, each point represents the values obtained with a single experimental mouse. n = 6 per each experimental condition, except in the case of Vav1∆N (n = 3). Data information: values are shown as means ± SEM from three independent experiments.


( G - J ) Flow cytometry determination of Tox ( G ) , ICOS ( H ) , CD25 ( I ) and ICN1 ( J ) expression in EGFP + CD4 + T cells expressing the indicated Vav1 proteins . NSt , Non stimulated cells ( CD4 + T cells before stimulation and retroviral transduction ) . In all panels , each point represents the values obtained for a single experimental condition . f . i . , mean fluorescence intensity relative to the isotype - matched control antibody . n = 6 per each experimental condition , except for the Vav1 ∆ N and NSt conditions ( n = 3 ) . Data information : values are shown as means ± SEM from three independent experiments .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O 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-J) Flow cytometry determination of Tox (G), ICOS (H), CD25 (I) and ICN1 (J) expression in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. NSt, Non stimulated cells (CD4+ T cells before stimulation and retroviral transduction). In all panels, each point represents the values obtained for a single experimental condition. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. n = 6 per each experimental condition, except for the Vav1∆N and NSt conditions (n = 3). Data information: values are shown as means ± SEM from three independent experiments.


( K - N ) Example of a flow cytometry analysis ( K and M ) and final quantification of p - Akt ( L ) and p - Erk1 / 2 ( N ) levels in EGFP + CD4 + T cells expressing the indicated Vav1 proteins in primed ( blue bars ) and restimulated ( red bars ) cells . In K and M , the grey shaded histograms represent the fluorescence obtained with the isotype - matched control antibody . In L and N , NSt , nonstimulated , St , stimulated . n = 3 per each experimental condition . Data information : values are shown as means ± SEM from three independent experiments . In panels L and N , P values are given relative to nonstimulated ( blue asterisks ) and stimulated ( red asterisks ) cells expressing Vav1WT . We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t test ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-N) Example of a flow cytometry analysis (K and M) and final quantification of p-Akt (L) and p-Erk1/2 (N) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins in primed (blue bars) and restimulated (red bars) cells. In K and M, the grey shaded histograms represent the fluorescence obtained with the isotype-matched control antibody. In L and N, NSt, nonstimulated, St, stimulated. n = 3 per each experimental condition. Data information: values are shown as means ± SEM from three independent experiments. In panels L and N, P values are given relative to nonstimulated (blue asterisks) and stimulated (red asterisks) cells expressing Vav1WT. We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Student's t test).


B ) quantification of the EGFP + CD4 + T cell proliferation in the different experimental groups n = 3 independent experiments . Data information : values are shown as means ± SEM from three independent experiments . P values are given relative to nontreated ( light blue asterisks ) and treated ( dark blue asterisks ) EGFP + cells . We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Mann - Whitney U test ) .

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B) quantification of the EGFP+ CD4+ T cell proliferation in the different experimental groups n = 3 independent experiments. Data information: values are shown as means ± SEM from three independent experiments. P values are given relative to nontreated (light blue asterisks) and treated (dark blue asterisks) EGFP+ cells. We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney U test).


( C - E ) Flow cytometry determination of intracellular Tox ( C ) , intracellular ICN1 ( D ) and surface ICOS ( E ) levels in EGFP + CD4 + T cells expressing the indicated Vav1 proteins . f . i . , mean fluorescence intensity relative to the isotype - matched control antibody . n = 3 independent experiments . Data information : values are shown as means ± SEM from three independent experiments . P values are given relative to nontreated ( light blue asterisks ) and treated ( dark blue asterisks ) EGFP + cells . We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Mann - Whitney U test ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Flow cytometry determination of intracellular Tox (C), intracellular ICN1 (D) and surface ICOS (E) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. n = 3 independent experiments. Data information: values are shown as means ± SEM from three independent experiments. P values are given relative to nontreated (light blue asterisks) and treated (dark blue asterisks) EGFP+ cells. We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney U test).


Figure 1 : Growth Charts and Photographs of Affected Subjects • A Family 1 - Subject II . 1 . • B Family 1 - Subject II . 3 . • C Family 2 - Subject II . 3 . • D Family 2 - Subject II . 4 . • E Family 2 - Subject II . 5 . • F Top Row - Representative X - rays showing narrow fibulae and elongated digits in Subject II . 1 of Family 1 . Bottom Row - Lower extremities and foot from Subject II . 5 and hand from Subject II . 4 , both from Family 2 .

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Figure 1: Growth Charts and Photographs of Affected Subjects•A Family 1-Subject II.1.•B Family 1-Subject II.3.•C Family 2-Subject II.3.•D Family 2-Subject II.4.•E Family 2-Subject II.5.•F Top Row -Representative X-rays showing narrow fibulae and elongated digits in Subject II.1 of Family 1. Bottom Row - Lower extremities and foot from Subject II.5 and hand from Subject II.4, both from Family 2.


• B Pedigree and representative Sanger sequencing trace of the mutation identified in family 1 . • C Pedigree and representative Sanger sequencing trace of the mutation identified in family 2 .

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• B Pedigree and representative Sanger sequencing trace of the mutation identified in family 1.•C Pedigree and representative Sanger sequencing trace of the mutation identified in family 2.


• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP - A2 , PAPP - A2 ( D643fs ) ( family 1 ) , or PAPP - A2 ( A1033V ) ( family 2 ) . Radiolabeled substrates tested include IGFBP - 3 , IGFBP - 4 , and IGFBP - 5 . Cleavage was visualized by autoradiography following SDS - PAGE . Positions of intact IGFBPs ( i ) and cleavage products ( c ) are indicated .

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• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP-A2, PAPP-A2(D643fs) (family 1), or PAPP-A2(A1033V) (family 2). Radiolabeled substrates tested include IGFBP-3, IGFBP-4, and IGFBP-5. Cleavage was visualized by autoradiography following SDS-PAGE. Positions of intact IGFBPs (i) and cleavage products (c) are indicated.


B Western blots following nonreducing or reducing SDS - PAGE of media from transfected cells , as indicated . Polyclonal antibodies raised against the N - terminal portion of PAPP - A2 were used for detection .

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B Western blots following nonreducing or reducing SDS-PAGE of media from transfected cells, as indicated. Polyclonal antibodies raised against the N-terminal portion of PAPP-A2 were used for detection.


• C Comparison of proteolytic activity similar to the experiment of panel A , except that equimolar concentrations of PAPP - A2 and PAPP - A2 ( A1033V ) were used . The variant from Family 1 , carrying a frameshift mutation N - terminal to the proteolytic domain of PAPP - A2 , did not show any detectable expression . All gels and blots are representative of three independent experiments .

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•C Comparison of proteolytic activity similar to the experiment of panel A, except that equimolar concentrations of PAPP-A2 and PAPP-A2 (A1033V) were used. The variant from Family 1, carrying a frameshift mutation N-terminal to the proteolytic domain of PAPP-A2, did not show any detectable expression. All gels and blots are representative of three independent experiments.


• A Concentrations of endogenous insulin - like growth factor ( IGF ) - I in the different fractions obtained by neutral chromatography on a Superdex 200 column . Serum samples from the four siblings of family 1 were analyzed and compared to a non - related prepubertal control , as all siblings were prepubertal . The amount of IGF - I detected in the fractions corresponding to the ternary complex ( TC ) was markedly greater in the homozygous affected subjects ( II · 1 and II · 3 ) compared to the unaffected siblings ( II . 2 and II . 4 ) and the prepubertal control . No IGF - I was detected in the fractions corresponding to the binary complex ( BC ) or free IGF - I in any of the subjects . This is most likely due to the fact that under physiological conditions 80 - 85 % of IGF - I circulates in the TC and after the separation procedure , the amount of IGF - I in the BC or free was below the level of detection of our IGF - I assay . These results clearly demonstrate that in the affected patients the amount of IGF - I circulating in the TC is markedly elevated .

O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-ORGANISM O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

• A Concentrations of endogenous insulin-like growth factor (IGF)-I in the different fractions obtained by neutral chromatography on a Superdex 200 column. Serum samples from the four siblings of family 1 were analyzed and compared to a non-related prepubertal control, as all siblings were prepubertal. The amount of IGF-I detected in the fractions corresponding to the ternary complex (TC) was markedly greater in the homozygous affected subjects (II·1 and II·3) compared to the unaffected siblings (II.2 and II.4) and the prepubertal control. No IGF-I was detected in the fractions corresponding to the binary complex (BC) or free IGF-I in any of the subjects. This is most likely due to the fact that under physiological conditions 80-85% of IGF-I circulates in the TC and after the separation procedure, the amount of IGF-I in the BC or free was below the level of detection of our IGF-I assay. These results clearly demonstrate that in the affected patients the amount of IGF-I circulating in the TC is markedly elevated.


A . Ncl and Pus7 mRNA levels ( normalized to TBP ) at different time points after LPS stimulation in c - mycwt / wt and c - myc ∆ / ∆ cells . Data are presented as mean ± SD ; n = 3 .

O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

A. Ncl and Pus7 mRNA levels (normalized to TBP) at different time points after LPS stimulation in c-mycwt/wt and c-myc∆/∆ cells. Data are presented as mean ± SD; n = 3.


B . To quantify RNA synthesis , we exposed our cultures to a brief pulse of ethyl - uridine ( EU ) and measured its incorporation into RNA by light microscopy . The scatter plots show the nuclear Area as detected in the 2D plane ( x axis , in pixels ) versus the EU signal ( y axis , as arbitrary units , AU ) for each single cell identified by DAPI staining in c - mycwt / wt and c - myc ∆ / ∆ populations at the indicated time points after LPS treatment . One representative experiment out of 5 is shown .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

B. To quantify RNA synthesis, we exposed our cultures to a brief pulse of ethyl-uridine (EU) and measured its incorporation into RNA by light microscopy. The scatter plots show the nuclear Area as detected in the 2D plane (x axis, in pixels) versus the EU signal (y axis, as arbitrary units, AU) for each single cell identified by DAPI staining in c-mycwt/wt and c-myc∆/∆ populations at the indicated time points after LPS treatment. One representative experiment out of 5 is shown.


C . Variation of each expressed mRNA at 4h of LPS treatment ( log2FC , relative to time 0 ) for c - mycwt / wt ( x - axis ) and c - myc ∆ / ∆ cells ( y - axis ) , as measured by RNA - seq . Regulatory groups 1 - 6 and Grey Zone ( GZ ) are as defined in the text .

O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

C. Variation of each expressed mRNA at 4h of LPS treatment (log2FC, relative to time 0) for c-mycwt/wt (x-axis) and c-myc∆/∆ cells (y-axis), as measured by RNA-seq. Regulatory groups 1-6 and Grey Zone (GZ) are as defined in the text.


D . Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation .

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D. Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation.


E . Variations ( log2FC ) in mature and precursor RNAs ( as measured by RNA - seq ) and in the modeled rates ( RNA synthesis , processing and degradation ) for Myc dependent and independent genes . The grey scale represents the starting level for each parameter in unstimulated cells .

O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

E. Variations (log2FC) in mature and precursor RNAs (as measured by RNA-seq) and in the modeled rates (RNA synthesis, processing and degradation) for Myc dependent and independent genes. The grey scale represents the starting level for each parameter in unstimulated cells.


A . Overlap between Myc ChIP - seq peaks . For each time - point indicated at the bottom , the percentage of peaks overlapping over ≥ 1 bp with any other time - point ( Reference sample ) is reported . Peaks showed consistent distributions along the time - course , almost all those called in untreated cells being included in LPS - stimulated

O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O

A. Overlap between Myc ChIP-seq peaks. For each time-point indicated at the bottom, the percentage of peaks overlapping over ≥1 bp with any other time-point (Reference sample) is reported. Peaks showed consistent distributions along the time-course, almost all those called in untreated cells being included in LPS-stimulated samples


B , C . Distribution of Myc at promoters ( B ) and distal sited ( C ) in LPS - treated B - cells ( 0 , 2 , 4 8h ) and in B - cells in vivo , in either control c - mycwt / wt ( C ) , pre - tumoral Eµ - myc mice ( P ) or tumors ( T ) 4 [ ] . Each row represents a promoter - associated Myc peak within a 6 kb genomic interval ( centered on the midpoint of the peak ) . The heatmap includes every annotated promoter in chromosome 1 ( as representative of the whole genome ) that was called as Myc - associated by ChIP - seq in at least one sample . For the same intervals , we also show the distribution of H3K4me1 , H3K4me3 , and H3K27ac in the in vivo control sample , and gene annotations ( exons in red , introns in pink ; + sense , - antisense strand ) .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD 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-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O 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, C. Distribution of Myc at promoters (B) and distal sited (C) in LPS-treated B-cells (0, 2, 4 8h) and in B-cells in vivo, in either control c-mycwt/wt (C), pre-tumoral Eµ-myc mice (P) or tumors (T) 4[]. Each row represents a promoter-associated Myc peak within a 6 kb genomic interval (centered on the midpoint of the peak). The heatmap includes every annotated promoter in chromosome 1 (as representative of the whole genome) that was called as Myc-associated by ChIP-seq in at least one sample. For the same intervals, we also show the distribution of H3K4me1, H3K4me3, and H3K27ac in the in vivo control sample, and gene annotations (exons in red, introns in pink; + sense, - antisense strand).


D , E . Numbers of Myc peaks at promoters ( - 2kb , + 1kb ) ( D ) and distal sites ( E ) at different time points after LPS stimulation . The shadings mark the subsets of peaks associated with H3K4me3 ( D ) or H3K4me1 ( E ) , with or without H3K27ac .

O O O O B-EXP_ASSAY O B-GENEPROD 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-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O

D, E. Numbers of Myc peaks at promoters (-2kb, +1kb) (D) and distal sites (E) at different time points after LPS stimulation. The shadings mark the subsets of peaks associated with H3K4me3 (D) or H3K4me1 (E), with or without H3K27ac.


F . Percentage of active promoters ( positive for H3K4me3 and H3K27ac ) or distal sites ( H3K4me1 and H3K27ac ) that overlap with at least a Myc peak at the different time points after LPS stimulation ( 0 , 2 , 4 , 8h ) or in vivo ( C , P , T1 , T2 , T3 ) .

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F. Percentage of active promoters (positive for H3K4me3 and H3K27ac) or distal sites (H3K4me1 and H3K27ac) that overlap with at least a Myc peak at the different time points after LPS stimulation (0, 2, 4, 8h) or in vivo (C, P, T1, T2, T3).


G . Quantitative heatmap showing Myc signal intensities ( read counts normalized by library size ) at each bound region in chromosome 1 , ranked based on the signal in the control sample ( 0h LPS ) .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE O O

G. Quantitative heatmap showing Myc signal intensities (read counts normalized by library size) at each bound region in chromosome 1, ranked based on the signal in the control sample (0h LPS).


H . Percentage of Myc peaks at promoters ( top ) or distal sites ( bottom ) that contain either canonical ( CACGTG ) or variant E - boxes ( CACGCG , CATGCG , CACGAG , CATGTG ) 83637 [ , , ] in a 200 bp window centered on the peak summit .

O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Percentage of Myc peaks at promoters (top) or distal sites (bottom) that contain either canonical (CACGTG) or variant E-boxes (CACGCG, CATGCG, CACGAG, CATGTG) 83637[,,] in a 200 bp window centered on the peak summit.


I . Myc enrichment by ChIP - seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs . In the boxplot , the central tendency marks the median , the box ranges mark the upper and lower quartiles , and the whiskers extend to the upper and lower extreme datapoints which are no more than 1 . 5 times the interquartile range from the box . The Myc ChIP - seq time - course was performed on a unique pool of cells , obtained from > 10 mice .

O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O

I. Myc enrichment by ChIP-seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs. In the boxplot, the central tendency marks the median, the box ranges mark the upper and lower quartiles, and the whiskers extend to the upper and lower extreme datapoints which are no more than 1.5 times the interquartile range from the box. The Myc ChIP-seq time-course was performed on a unique pool of cells, obtained from > 10 mice.


A . Scatter plots correlating the variations in Myc share at promoters ( averaged between the 2 , 4 and 8h time points ) and in RNA synthesis ( at 8h ) , relative to untreated cells , for Myc - dependent and independent genes . The Spearman correlation between the two parameters is reported .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

A. Scatter plots correlating the variations in Myc share at promoters (averaged between the 2, 4 and 8h time points) and in RNA synthesis (at 8h), relative to untreated cells, for Myc-dependent and independent genes. The Spearman correlation between the two parameters is reported.


B . The ability of the Myc share in predicting the transcriptional outcome can be represented in terms of a Receiver operating characteristic ( ROC ) curve , which represents the Sensitivity ( true positive rate ) and Specificity ( true negative rate ) of a predictor ( i . e . Myc share ) in discriminating between two classes ( i . e . up - and down - regulated genes ) , using different thresholds . The largest the area under the curve ( AUC ) , the largest is the predictive power , in a scale from 0 . 5 ( random classification : grey line ) to 1 ( perfect predictor ) . For each system , the dot corresponds to the variation of Myc at which promoters begin increasing their share of Myc binding .

O O O O O O B-GENEPROD 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-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O

B. The ability of the Myc share in predicting the transcriptional outcome can be represented in terms of a Receiver operating characteristic (ROC) curve, which represents the Sensitivity (true positive rate) and Specificity (true negative rate) of a predictor (i.e. Myc share) in discriminating between two classes (i.e. up- and down-regulated genes), using different thresholds. The largest the area under the curve (AUC), the largest is the predictive power, in a scale from 0.5 (random classification: grey line) to 1 (perfect predictor). For each system, the dot corresponds to the variation of Myc at which promoters begin increasing their share of Myc binding.


C . Fold - change in Myc levels at each bound promoter ( at 4h LPS , relative to time zero ) as a function of the initial binding intensity at time zero ( expressed as log2 of the coverage in a 200 bp window around the summit of the peak ) . Peaks containing a canonical E - box are highlighted in black .

O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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. Fold-change in Myc levels at each bound promoter (at 4h LPS, relative to time zero) as a function of the initial binding intensity at time zero (expressed as log2 of the coverage in a 200 bp window around the summit of the peak). Peaks containing a canonical E-box are highlighted in black.


D . Percentage of Myc - bound promoters within the indicated transcriptionally regulatory categories that contains a canonical or variant E - box . The overall chi - square is 207 . 7 with a p - value < 0 . 00001 . The contribution to the chi - square of each category is reported above the corresponding bar in the plot .

O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percentage of Myc-bound promoters within the indicated transcriptionally regulatory categories that contains a canonical or variant E-box. The overall chi-square is 207.7 with a p-value <0.00001. The contribution to the chi-square of each category is reported above the corresponding bar in the plot.


B , C . Boxplots reporting LPS - induced changes ( Log2FC ) for each of the four RNAPII kinetic rates in c - mycwt / wt ( wt ) and c - myc ∆ / ∆ cells for Myc - dependent ( B ) and Myc - independent ( C ) induced and repressed genes . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported . The RNAPII ChIP - seq time - course was performed on two biological replicates ( each on pools of cells from > 10 mice ) , and reads were merged after sequencing .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O

B, C. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for Myc-dependent (B) and Myc-independent (C) induced and repressed genes. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported. The RNAPII ChIP-seq time-course was performed on two biological replicates (each on pools of cells from > 10 mice), and reads were merged after sequencing.


D , E . Heatmaps showing the variations in RNAPII kinetic rates ( Log2FC ) on Myc - dependent ( D ) and Myc - independent ( E ) genes during the LPS time - course . The grey scale represents the starting level for each parameter in unstimulated cells . The first column on the left reports the changes in Myc share for the same genes . Genes were grouped using hierarchical clustering .

O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O 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-GENEPROD O O O O O O O O O O O O O

D, E. Heatmaps showing the variations in RNAPII kinetic rates (Log2FC) on Myc-dependent (D) and Myc-independent (E) genes during the LPS time-course. The grey scale represents the starting level for each parameter in unstimulated cells. The first column on the left reports the changes in Myc share for the same genes. Genes were grouped using hierarchical clustering.


F . Boxplots reporting LPS - induced changes ( Log2FC ) for each of the four RNAPII kinetic rates in c - mycwt / wt ( wt ) and c - myc ∆ / ∆ cells for genes of clusters 1 and 9 . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported .

O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

F. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for genes of clusters 1 and 9. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.


G . Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported .

O O O O B-GENEPROD O O B-SMALL_MOLECULE O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

G. Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.


( A ) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice .

O O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O B-TISSUE O O O B-GENEPROD B-ORGANISM O

(A) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice.


( B ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 0 - 5 hours before immunoblotting with the indicated antibodies .

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(B) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-5 hours before immunoblotting with the indicated antibodies.


( C , E and G ) WT PEMs expressing GFP - FAK were incubated with either ΔinvG Salmonella ( C ) , LPS - coated beads ( E ) or E . coli ( G ) for a total of 5 hours before analysis by immunofluorescence . Cells were co - stained with antibodies recognizing LAMP1 ( red ) . Dapi was used to visualize nuclei and bacteria ( blue ) . Bars represent 10 µm . Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm . ( D , F and H ) The percent of LAMP1 - positive ( gray bars ) and LAMP1 + GFP + ( black bars ) ΔinvG Salmonella ( D ) , LPS - coated beads ( F ) and E . coli ( H ) was quantified . At least 100 bacteria were counted per condition ; N = 3 .

O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O

(C, E and G) WT PEMs expressing GFP-FAK were incubated with either ΔinvG Salmonella (C), LPS-coated beads (E) or E. coli (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) ΔinvG Salmonella (D), LPS-coated beads (F) and E. coli (H) was quantified. At least 100 bacteria were counted per condition; N = 3.


A ) TLR4 − / − PEMs were incubated with ΔinvG Salmonella for 0 - 5 hours before immunoblotting with the indicated antibodies .

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A) TLR4−/− PEMs were incubated with ΔinvG Salmonella for 0-5 hours before immunoblotting with the indicated antibodies.


( B ) WT PEMs were incubated with LPS - coated beads for 0 - 5 hours before immunoblotting for the indicated antibodies .

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(B) WT PEMs were incubated with LPS-coated beads for 0-5 hours before immunoblotting for the indicated antibodies.


( C ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation .

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(C) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( D ) WT PEMs were incubated with the orgA : : tet ; spiA : : kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs ( ΔSPI1ΔSPI2 ) for 0 - 5 hours before immunoblotting with the indicated antibodies .

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(D) WT PEMs were incubated with the orgA::tet; spiA::kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs (ΔSPI1ΔSPI2) for 0-5 hours before immunoblotting with the indicated antibodies.


( E ) WT PEMs expressing GFP - FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence . Cells were co - stained with antibodies recognizing LAMP1 ( red ) . DAPI was used to visualize nuclei and bacteria ( blue ) . Bars represent 10 µm or 2 µm in enlarged inset .

O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(E) WT PEMs expressing GFP-FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). DAPI was used to visualize nuclei and bacteria (blue). Bars represent 10 µm or 2 µm in enlarged inset.


( A ) WT and FAK − / − PEMs were incubated for 0 - 5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O O O

(A) WT and FAK−/− PEMs were incubated for 0-5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies.


( B ) WT PEMs were pretreated for 1 hour with the FAK - kinase inhibitor PF - 228 ( 0 . 5 µm ) before incubation with ΔinvG Salmonella for 5 hours . Lysates were immunoblotted with the indicated antibodies .

O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O

(B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies.


( C - D ) WT or FAK − / − macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain ( C ) or LPS - coated beads ( D ) for 0 - 5 hours before immunoblotting with the indicated antibodies . In panels C and D , WT PEMs were also incubated with ΔinvG ( 5 h ) for comparison of Akt activation . Vertical white lines in panel D indicate non - contiguous lanes generated from a single exposure .

O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0-5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure.


E ) Levels of phosphorylated proteins were quantified by densitometry , normalized to the amount of total protein present in each sample , and expressed relative to the basal level in uninfected cells . Values are means ± SEM , N = 3 , * p < 0 . 05 . Dashed line drawn at 1 indicates basal levels of activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation.


( F ) WT PEMs expressing HA - Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours . Lysates were subjected to immunoprecipitation ( IP ) with anti - HA antibodies before immunoblotting for FAK and HA . In parallel , lysates were immunoblotted for FAK , HA and ERK1 / 2 to control for the level of expression .

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(F) WT PEMs expressing HA-Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies before immunoblotting for FAK and HA. In parallel, lysates were immunoblotted for FAK, HA and ERK1/2 to control for the level of expression.


G ) WT and FAK − / − macrophages expressing HA - Akt were stained with anti - HA and anti - LAMP1 before analysis by confocal microscopy . White boxes show enlarged regions in lower panel . Arrows indicate HA + LAMP + Salmonella evident in WT PEMs . ( H ) The percentage of ΔΣΠΙ1ΔΣΠΙ2 ( first grouping ) or ΔinvG ( second grouping ) Salmonella co - localizing with HA and LAMP - 1 5 h post - infection . At least 100 bacteria were counted per condition . Values are means ± SEM , N = 3 , * p < 0 . 05 .

O O O O B-GENEPROD O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O 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) WT and FAK−/− macrophages expressing HA-Akt were stained with anti-HA and anti-LAMP1 before analysis by confocal microscopy. White boxes show enlarged regions in lower panel. Arrows indicate HA+LAMP+ Salmonella evident in WT PEMs. (H) The percentage of ΔΣΠΙ1ΔΣΠΙ2 (first grouping) or ΔinvG (second grouping) Salmonella co-localizing with HA and LAMP-1 5 h post-infection. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05.


( A ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 0 - 24 hours before immunoblotting with the indicated antibodies .

(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0-24 hours before immunoblotting with the indicated antibodies.


( B ) WT PEMs were incubated with Akt inhibitor AKTV / triciribine ( 10 µm ) for 30 minutes before infection with S . typhimurium strain ΔinvG for 5 hours . Lysates were immunoblotted with the indicated antibodies . (

O O O O B-CELL_TYPE O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O O

(B) WT PEMs were incubated with Akt inhibitor AKTV/triciribine (10 µm) for 30 minutes before infection with S. typhimurium strain ΔinvG for 5 hours. Lysates were immunoblotted with the indicated antibodies. (


. ( C ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3 . DAPI was used to visualize nuclei and bacteria . Bars represent 10 µm . White boxes show enlarged regions in inset panels , bars represent 2 µm . White arrowheads indicate LC3 - negative bacteria , yellow arrow heads indicate LC3 - positive bacteria ( panels c , f ) . Yellow arrowhead in panel f denotes an LC3 + cluster of bacteria . ( D ) The percentage of ΔinvG ( first grouping ) or ΔΣΠΙ1ΔΣΠΙ2 ( second grouping ) Salmonella co - localizing with LC3 at the indicated time points is displayed . At least 100 bacteria were counted per condition per time point . Values are means ± SEM , N = 3 , * p < 0 . 05 .

O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-SUBCELLULAR O 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-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O 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) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei and bacteria. Bars represent 10 µm. White boxes show enlarged regions in inset panels, bars represent 2 µm. White arrowheads indicate LC3-negative bacteria, yellow arrow heads indicate LC3-positive bacteria (panels c, f). Yellow arrowhead in panel f denotes an LC3+ cluster of bacteria. (D) The percentage of ΔinvG (first grouping) or ΔΣΠΙ1ΔΣΠΙ2 (second grouping) Salmonella co-localizing with LC3 at the indicated time points is displayed. At least 100 bacteria were counted per condition per time point. Values are means ± SEM, N = 3, *p<0.05.


( E ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 ( 300 ng ) before immunoblotting with the indicated antibodies . Vertical white line indicate non - contiguous lanes generated from a single exposure .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(E) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 (300 ng) before immunoblotting with the indicated antibodies. Vertical white line indicate non-contiguous lanes generated from a single exposure.


( F ) FAK − / − PEMs were incubated with DQ - BSA - red for 1 hour before infection with S . typhimurium strain ΔinvG for 5 h . Cells were co - stained for LC3 and DAPI was used to visualize nuclei and bacteria . White boxes show enlarged regions in inset panel .

O O O B-GENEPROD O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O

(F) FAK−/− PEMs were incubated with DQ-BSA-red for 1 hour before infection with S. typhimurium strain ΔinvG for 5 h. Cells were co-stained for LC3 and DAPI was used to visualize nuclei and bacteria. White boxes show enlarged regions in inset panel.


G ) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin ( 4 µm ) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours . Cells were lysed before immunoblotting with the indicated antibodies .

O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O O O O O

G) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin (4 µm) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours. Cells were lysed before immunoblotting with the indicated antibodies.


( H ) WT and FAK − / − PEMs were pretreated with rapamycin ( 4 µm ) , Akt inhibitor AKTV / triciribine ( 10 µm ) or left untreated ( - Tx ) or before infection with S . typhimurium strain ΔinvG for a further 5 hours . WT macrophages were also pretreated with the FAK inhibitor PF228 ( 0 . 5 µm ) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours . Cells were then assessed for the percentage of ΔinvG Salmonella co - localizing with LC3 . At least 100 bacteria were counted per condition . Values are means ± SEM , N = 3 , * p < 0 . 05 . N . s . not significant .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) WT and FAK−/− PEMs were pretreated with rapamycin (4 µm), Akt inhibitor AKTV/triciribine (10 µm) or left untreated (-Tx) or before infection with S. typhimurium strain ΔinvG for a further 5 hours. WT macrophages were also pretreated with the FAK inhibitor PF228 (0.5 µm) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours. Cells were then assessed for the percentage of ΔinvG Salmonella co-localizing with LC3. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05. N.s. not significant.


A ) WT and FAK − / − PEMs were incubated with LPS - coated beads ( 50 ∶ 1 ; beads ∶ cell ) for 1 hour before analysis with antibodies recognizing LC3 . DAPI was used to visualize nuclei . Bars represent 10 µm . The percentage of LPS - beads co - localizing with LC3 is displayed to the right . At least 100 beads were counted per condition . Values are means ± SEM , N = 3 .

O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O 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) WT and FAK−/− PEMs were incubated with LPS-coated beads (50∶1; beads∶cell) for 1 hour before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei. Bars represent 10 µm. The percentage of LPS-beads co-localizing with LC3 is displayed to the right. At least 100 beads were counted per condition. Values are means ± SEM, N = 3.


( B ) PEMs were treated with Atg5 , ULK1 or control siRNAs for 48 hours before immunoblotting with the indicated antibodies .

O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O

(B) PEMs were treated with Atg5, ULK1 or control siRNAs for 48 hours before immunoblotting with the indicated antibodies.


. ( C - D ) FAK − / − PEMs treated with Atg5 , ULK1 or control siRNA for 48 hours were incubated with LPS - coated beads or ΔinvG Salmonella for 1 or 5 hours before analysis with antibodies recognizing LC3 . The percentage of LPS - beads or Salmonella co - localizing with LC3 is displayed to the right . At least 100 beads or bacteria were counted per condition . Values are means ± SEM , N = 3 .

O O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. (C-D) FAK−/− PEMs treated with Atg5, ULK1 or control siRNA for 48 hours were incubated with LPS-coated beads or ΔinvG Salmonella for 1 or 5 hours before analysis with antibodies recognizing LC3. The percentage of LPS-beads or Salmonella co-localizing with LC3 is displayed to the right. At least 100 beads or bacteria were counted per condition. Values are means ± SEM, N = 3.


E ) WT and FAK − / − PEMs were infected with ΔinvG for 5 hours before preparation and examination by transmission electron microscopy ( TEM ) . Arrowheads indicate double membrane .

O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

E) WT and FAK−/− PEMs were infected with ΔinvG for 5 hours before preparation and examination by transmission electron microscopy (TEM). Arrowheads indicate double membrane.


( A ) WT and FAK − / − PEMs were incubated with S . typhimurium strain ΔinvG for 1 . 5 , 3 and 5 hours . Bacterial survival was then assayed using a standard gentamycin resistance assay described in the Materials and Methods . Values are means ± SEM , N = 6 , * p < 0 . 05 .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 1.5, 3 and 5 hours. Bacterial survival was then assayed using a standard gentamycin resistance assay described in the Materials and Methods. Values are means ± SEM, N = 6, *p<0.05.


( B ) WT and FAK − / − PEMs were pretreated with IFN - γ ( 50 ng / ml ) , rapamycin ( 4 µm ) , or left untreated before incubation with ΔinvG Salmonella for 3 hours . Bacterial survival was then assayed as described in ( A ) . Values are means ± SEM , N = 6 , * p < 0 . 05 .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O 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) WT and FAK−/− PEMs were pretreated with IFN-γ (50 ng/ml), rapamycin (4 µm), or left untreated before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 6, *p<0.05.


( C ) PEMs were treated with control siRNA or depleted of Atg5 before incubation with ΔinvG Salmonella for 3 hours . Bacterial survival was then assayed as described in ( A ) . Values are means ± SEM , N = 3 , * p < 0 . 05 vs WT siControl .

O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O 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) PEMs were treated with control siRNA or depleted of Atg5 before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 3, *p<0.05 vs WT siControl.


. ( D ) Bacterial loads in WT ( black circles ) and FAKΔmyeloid mice ( white circles ) in the indicated tissues 5 days after oral infection with S . typhimurium strain SL1344 . Each point indicates data from an individual mouse . * p < 0 . 05 .

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. (D) Bacterial loads in WT (black circles) and FAKΔmyeloid mice (white circles) in the indicated tissues 5 days after oral infection with S. typhimurium strain SL1344. Each point indicates data from an individual mouse. *p<0.05.


( E ) Hematoxylin and eosin ( H & amp ; amp ; E ) staining of spleen isolated from WT and FAKΔmyeloid mice 5 days post - infection with S . typhimurium strain SL1344 . Arrows indicate areas of leukocyte infiltration . Lesion indicated by yellow arrow is outlined in the higher magnification panel to the right . Bars represent 200 µm and 50 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O O 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) Hematoxylin and eosin (H&amp;E) staining of spleen isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Arrows indicate areas of leukocyte infiltration. Lesion indicated by yellow arrow is outlined in the higher magnification panel to the right. Bars represent 200 µm and 50 µm.


. ( F ) Gr - 1 immunostaining of spleens isolated from WT and FAKΔmyeloid mice 5 days post - infection with S . typhimurium strain SL1344 . Panels b and d are enlarged sections indicated by black boxes in panels a and c . Gr - 1 - positive cells appear red - brown .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

. (F) Gr-1 immunostaining of spleens isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Panels b and d are enlarged sections indicated by black boxes in panels a and c. Gr-1-positive cells appear red-brown.


( G ) Quantitation of flow cytometric data collected from the spleens of infected mice . * p < 0 . 05 .

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(G) Quantitation of flow cytometric data collected from the spleens of infected mice. *p<0.05.


B ) Binding affinity of CodB for cytosine as measured using the thermostability assay . Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB . The Kd was estimated to be 51 ± 9 µM . The measurements are the average of 4 independent titrations with error bars of the s . e . m .

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B) Binding affinity of CodB for cytosine as measured using the thermostability assay. Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB. The Kd was estimated to be 51 ± 9 µM. The measurements are the average of 4 independent titrations with error bars of the s.e.m.


( C ) Top panel : Representative images show PS labeling in the WT dLGN at P6 and P13 . RGC inputs were labeled with CTB488 . Bottom panel : Enlarged images of the boxed region in top panels . Circles indicate PS + RGC inputs . Arrows point to the enlarged PS + RGC inputs in the insets . ( D ) Quantification of the percentage of PS + RGC inputs in total RGC inputs at P6 and P13 .

O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-TISSUE O O O O O B-CELL_TYPE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O

(C) Top panel: Representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged images of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows point to the enlarged PS+ RGC inputs in the insets. (D) Quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and P13.


( E ) Co - labeling of vGlut2 and PSVue in the dLGN at P6 . Circles indicate colocalized vGlut2 and PSVue .

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(E) Co-labeling of vGlut2 and PSVue in the dLGN at P6. Circles indicate colocalized vGlut2 and PSVue.


( F ) Co - labeling of Homer1 and PSVue in the dLGN at P6 . Circles indicate colocalized Homer1 and PSVue .

(F) Co-labeling of Homer1 and PSVue in the dLGN at P6. Circles indicate colocalized Homer1 and PSVue.


( G ) Orthogonal sections showed a triple positive PS + / CTB488 + / CD68 + RGC inputs inside of microglia .

O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_TYPE O O O B-CELL_TYPE O

(G) Orthogonal sections showed a triple positive PS+/CTB488+/CD68+ RGC inputs inside of microglia.


( H ) Orthogonal sections showed a triple positive PS + / vGlut2 + / CD68 + synapse inside of microglia .

O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O B-CELL_TYPE O

(H) Orthogonal sections showed a triple positive PS+/vGlut2+/CD68+ synapse inside of microglia.


( I ) A representative image of microglia ( upper - left ) is surface rendered ( bottom - left ) . The white arrow points to cells which might be apoptotic cells labeled by PSVue . RGC inputs and PSVue outside or inside of microglia are shown in the two right panels . Scare bar , 20 µm . ( J ) Quantification of the percentage of PS + and PS - RGC inputs outside of microglia in total inputs . N = 4 . ( K ) Quantification of the percentage of engulfed PS + and PS - RGC inputs in total engulfed inputs . N = 4 .

O O O O O B-EXP_ASSAY O B-CELL_TYPE O 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-EXP_ASSAY O B-SMALL_MOLECULE O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O

(I) A representative image of microglia (upper-left) is surface rendered (bottom-left). The white arrow points to cells which might be apoptotic cells labeled by PSVue. RGC inputs and PSVue outside or inside of microglia are shown in the two right panels. Scare bar, 20 µm.(J) Quantification of the percentage of PS+ and PS- RGC inputs outside of microglia in total inputs. N = 4. (K) Quantification of the percentage of engulfed PS+ and PS- RGC inputs in total engulfed inputs. N=4.


( D ) The binding experiment using flow cytometry show that only the GAIN domain binds to PS , similar as Annexin V binding .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O

(D) The binding experiment using flow cytometry show that only the GAIN domain binds to PS, similar as Annexin V binding.


( E ) In the competition experiment , AF647 channel shows that GAIN domain binds 60 . 1 % of the PS + Ba / F3 cells ( left ) . Correspondingly , FITC channel reveals 39 . 7 % Annexin V binding to PS + Ba / F3 cells ( right ) .

O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

(E) In the competition experiment, AF647 channel shows that GAIN domain binds 60.1% of the PS+ Ba/F3 cells (left). Correspondingly, FITC channel reveals 39.7% Annexin V binding to PS+ Ba/F3 cells (right).


( G ) Direct binding of hFc - tagged GPR56 GAIN proteins to PS and other phospholipids . HFc was used as a negative control , and GAS6 as a positive control .

O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O

(G) Direct binding of hFc-tagged GPR56 GAIN proteins to PS and other phospholipids. HFc was used as a negative control, and GAS6 as a positive control.


( B ) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-ORGANISM O

(B) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice.


( D ) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16 . 5 neocortex . Arrows indicate cortical ectopias that are shown in insets . ( E ) Quantification of ectopia size per section . ( F ) The distribution of ectopia from rostral to caudal cortex .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-TISSUE O O O B-DISEASE I-DISEASE O O O O O O O O O O O B-DISEASE B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O B-TISSUE O

(D) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16.5 neocortex. Arrows indicate cortical ectopias that are shown in insets. (E) Quantification of ectopia size per section. (F) The distribution of ectopia from rostral to caudal cortex.


( G ) . Myelin basic protein ( MBP ) staining of corpus callosum in P28 controls , Gpr56 null and Gpr56 S4 mice .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O B-GENEPROD O O B-GENEPROD O B-ORGANISM O

(G). Myelin basic protein (MBP) staining of corpus callosum in P28 controls, Gpr56 null and Gpr56 S4 mice.


( H ) Quantification of MBP intensity in corpus callosum .

O O O O O B-GENEPROD B-EXP_ASSAY O B-TISSUE I-TISSUE O

(H) Quantification of MBP intensity in corpus callosum.


( I ) Gpr56 transcript levels in WT microglia at P5 and P14 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O

(I) Gpr56 transcript levels in WT microglia at P5 and P14.


( J ) An overview of vGlut2 and Homer1 staining in dLGN at P10 . The yellow outline indicates the dLGN core , and the dotted boxes show where synapses are quantified .

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(J) An overview of vGlut2 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.


K ) Representative images of vGlut2 / Homer1 staining in the dLGN of control , Gpr56 null , and Gpr56 S4 brains at P10 . Arrows pointing to the enlarged synapse in the insets . L ) Relative vGlut2 / Homer1 synapse density in dLGN . N = 10 ( Ctrl ) , N = 6 ( Gpr56 null ) , N = 3 ( Gpr56 S4 ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE O O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O

K) Representative images of vGlut2/Homer1 staining in the dLGN of control, Gpr56 null, and Gpr56 S4 brains at P10. Arrows pointing to the enlarged synapse in the insets. L) Relative vGlut2/Homer1 synapse density in dLGN. N=10 (Ctrl), N=6 (Gpr56 null), N=3 (Gpr56 S4).


( N ) Representative images and surface rendered microglia ( green ) in which CTB + ( red ) RGC inputs were engulfed . ( O ) Quantification of the percentage of engulfed RGC inputs in controls , Gpr56 null and Gpr56 S4 microglia .

O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O

(N) Representative images and surface rendered microglia (green) in which CTB+(red) RGC inputs were engulfed. (O) Quantification of the percentage of engulfed RGC inputs in controls, Gpr56 null and Gpr56 S4 microglia.


( A ) Gpr56 is deleted in CKO microglia . RNAscope shows Gpr56 transcripts co - localize with microglia ( arrow ) in controls , but not in CKO , and is absent in all cell types in the global Gpr56 KO ( Gpr56 null ) . Arrowhead indicates non - microglia cells expressing Gpr56 . RNAscope was performed in the prefrontal cortex of P30 mice . Scale bar , 20 µm . ( B ) Quantification of Gpr56 fluorescence signals in ( A ) . N = 20 - 50 cells for each .

O O O B-GENEPROD O O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

(A) Gpr56 is deleted in CKO microglia. RNAscope shows Gpr56 transcripts co-localize with microglia (arrow) in controls, but not in CKO, and is absent in all cell types in the global Gpr56 KO (Gpr56 null). Arrowhead indicates non-microglia cells expressing Gpr56. RNAscope was performed in the prefrontal cortex of P30 mice. Scale bar, 20 µm. (B) Quantification of Gpr56 fluorescence signals in (A). N = 20-50 cells for each.


( C ) Confocal images of vGlut2 ( green ) labeling retinal ganglion cell ( RGC ) presynaptic terminals and Homer1 ( red ) for post - synapses in the dLGN of CKO and controls at P10 . Overlapped vGlut2 and Homer1 are quantified as synapses , indicated by white circles . Arrows pointing to the synapse enlarged in the upper right - hand inset .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O

(C) Confocal images of vGlut2 (green) labeling retinal ganglion cell (RGC) presynaptic terminals and Homer1 (red) for post-synapses in the dLGN of CKO and controls at P10. Overlapped vGlut2 and Homer1 are quantified as synapses, indicated by white circles. Arrows pointing to the synapse enlarged in the upper right-hand inset.


( D ) A time course of dLGN synapse density ( vGlut2 + / Homer1 + ) between WT and controls .

O O O O O O O B-TISSUE B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O

(D) A time course of dLGN synapse density (vGlut2+/Homer1+) between WT and controls.


( E ) Representative super - resolution images and 3D - reconstructed images of vGlut2 and Homer1 staining of P8 dLGN . Green objects represent surface rendering of vGlut2 + presynaptic terminals . Red spots are rendered Homer1 + post - synapses . Magenta spots represent Homer1 + post - synapses within a distance of 300nm from the nearest vGlut2 + surface .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O

(E) Representative super-resolution images and 3D-reconstructed images of vGlut2 and Homer1 staining of P8 dLGN. Green objects represent surface rendering of vGlut2+ presynaptic terminals. Red spots are rendered Homer1+ post-synapses. Magenta spots represent Homer1+ post-synapses within a distance of 300nm from the nearest vGlut2+ surface.


( F ) Quantification of Homer1 + spots adjacent to vGlut2 surface within 100nm , 200nm , and 300nm , respectively . N = 3 .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O

(F) Quantification of Homer1+ spots adjacent to vGlut2 surface within 100nm, 200nm, and 300nm, respectively. N = 3.


( G ) Western blot of vGlut2 and Homer1 using microdissected dLGN tissue from P8 mice . ( H , I ) Quantification of vGlut2 and Homer1 expression in CKOs and controls . N = 3 , * p = 0 . 027 . ( J ) Quantification of synapse density ( vGlut2 + / Homer1 + ) in the dLGN of both male and female iCKO mice at P10 . N = 3 for male , N = 4 for female .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O B-ORGANISM O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

(G) Western blot of vGlut2 and Homer1 using microdissected dLGN tissue from P8 mice. (H, I) Quantification of vGlut2 and Homer1 expression in CKOs and controls. N = 3, *p = 0.027. (J) Quantification of synapse density (vGlut2+/Homer1+) in the dLGN of both male and female iCKO mice at P10. N = 3 for male, N = 4 for female.


( K ) An overview of vGlut1 and Homer1 staining in dLGN at P10 . The yellow outline indicates the dLGN core , and the dotted boxes show where synapses are quantified .

(K) An overview of vGlut1 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.


( L ) Representative images of vGlut1 + ( green ) presynaptic terminals and Homer1 + ( red ) postsynapses in the dLGN of CKO and controls at P10 . Arrows pointing to the synapse enlarged in inset . ( M ) Quantification of synapse density ( vGlut1 / homer1 ) at P10 in the dLGN of CKO mice and controls . N = 3 ( WT ) , N = 5 ( CKO ) , p = 0 . 053 .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

(L) Representative images of vGlut1+ (green) presynaptic terminals and Homer1+ (red) postsynapses in the dLGN of CKO and controls at P10. Arrows pointing to the synapse enlarged in inset. (M) Quantification of synapse density (vGlut1/homer1) at P10 in the dLGN of CKO mice and controls. N=3 (WT), N=5 (CKO), p = 0.053.


( A ) Representative images show hippocampal vGlut2 and Homer1 immunostaining . White box outlines the region of interest and yellow box shows the regions where confocal images were taken .

O O O O B-EXP_ASSAY O B-TISSUE B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

(A) Representative images show hippocampal vGlut2 and Homer1 immunostaining. White box outlines the region of interest and yellow box shows the regions where confocal images were taken.


( B ) Confocal images of synaptic immunostaining in CA1 striatum lacunosum - moleculare ( str . lac / mol ) at P10 . Arrows pointing to the enlarged synapse in the insets . ( C , D ) Quantification of synapse density in CA1 str . lac / mol in iCKO versus control at P10 ( C ) , and CKO versus control at P21 ( D ) . At P10 , N ( Ctrl ) = 4 , N ( iCKO ) = 3 , p = 0 . 02 . At P21 , N = 8 , p = 0 . 005 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of synaptic immunostaining in CA1 striatum lacunosum-moleculare (str. lac/mol) at P10. Arrows pointing to the enlarged synapse in the insets. (C, D) Quantification of synapse density in CA1 str. lac/mol in iCKO versus control at P10 (C), and CKO versus control at P21 (D). At P10, N(Ctrl) = 4, N(iCKO) = 3, p = 0.02. At P21, N = 8, p = 0.005.


( E ) Representative images show hippocampal vGlut1 and Homer1 immunostaining .

O O O O B-EXP_ASSAY O B-TISSUE B-GENEPROD O B-GENEPROD B-EXP_ASSAY O

(E) Representative images show hippocampal vGlut1 and Homer1 immunostaining.


( F ) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10 . ( G , H ) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 ( G ) , and CKO versus control at P21 ( H ) . At P10 , N = 5 , p = 0 . 83 . At P21 , N = 5 , p = 0 . 89 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10. (G, H) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 (G), and CKO versus control at P21 (H). At P10, N = 5, p = 0.83. At P21, N = 5, p = 0.89.


( B ) CTB - labeled dLGN shows reduced eye - segregation at P30 in CKO mice . The left column shows contralateral dLGN labeled with CTB488 ( green ) , and the middle one is ipsilateral dLGN with CTB647 ( magenta ) . The right column represents the dLGN pseudocolored according to the R - value for each pixel ( R = log ( Fipsi / Fcontra ) ) . ( C ) A histogram distribution chart of R - value for all pixels within dLGN represents the degree of eye - specific segregation . When R is 0 , it means equal ipsilateral and contralateral fluorescence intensity at a pixel . A greater R - value means a bigger difference of ipsi - to - contraleteral fluorescence intensity . The narrower distribution of CKO in the inset indicates reduced segregation . ( D ) The variance of R distributions in P30 control and CKO mice . N = 4 ( Ctrl ) , N = 6 ( CKO ) , p = 0 . 004 , mean ± SD .

O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-TISSUE O O B-TISSUE O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O O O O O O 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-TISSUE O O O O B-TISSUE O O B-EXP_ASSAY O O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O 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) CTB-labeled dLGN shows reduced eye-segregation at P30 in CKO mice. The left column shows contralateral dLGN labeled with CTB488 (green), and the middle one is ipsilateral dLGN with CTB647 (magenta). The right column represents the dLGN pseudocolored according to the R-value for each pixel (R = log(Fipsi/Fcontra)). (C) A histogram distribution chart of R-value for all pixels within dLGN represents the degree of eye-specific segregation. When R is 0, it means equal ipsilateral and contralateral fluorescence intensity at a pixel. A greater R-value means a bigger difference of ipsi-to-contraleteral fluorescence intensity. The narrower distribution of CKO in the inset indicates reduced segregation. (D) The variance of R distributions in P30 control and CKO mice. N = 4 (Ctrl), N = 6 (CKO), p = 0.004, mean ± SD.


( F ) Maximal NMDAR mediated currents measured in the dLGN of P28 ~ P34 mice , p = 0 . 03 , N = 14 ( Ctrl ) , 23 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM .

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(F) Maximal NMDAR mediated currents measured in the dLGN of P28~P34 mice, p = 0.03, N = 14 (Ctrl), 23 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.


( G ) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice . ( H ) Quantification of NMDAR1 expression in CKO mice and control .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM O O O

(G) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice. (H) Quantification of NMDAR1 expression in CKO mice and control.


( I ) Maximal AMPAR mediated currents measured in the dLGN of P28 ~ P34 mice . P = 0 . 302 , N = 13 ( Ctrl ) , 17 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM .

(I) Maximal AMPAR mediated currents measured in the dLGN of P28~P34 mice. P=0.302, N = 13 (Ctrl), 17 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.


( J ) Western blot of GluR1 with microdissected P30 dLGN tissue . ( K ) Quantification of GluR1 expression in CKO mice and controls .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-TISSUE O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM O O O

(J) Western blot of GluR1 with microdissected P30 dLGN tissue. (K) Quantification of GluR1 expression in CKO mice and controls.


( A ) Colocalized RGC inputs and PSVue signals in P6 control and CKO . White circles indicate PS + RGC inputs . Arrows pointing to the RGC inputs enlarged in the insets . ( B ) The percentage of PS + RGC inputs in total inputs in CKO and controls .

O O O B-EXP_ASSAY B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O

(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls.


( C ) Representative images of engulfed PS + and PS - RGC inputs by microglia in control and CKO . ( D ) Quantification of engulfed PS + RGC inputs by microglia , which is calculated as : Volume of engulfed PS + RGC inputs / Volume of microglia cell . ( E ) Quantification of engulfed PS - RGC inputs in control and CKO .

O O O O B-EXP_ASSAY O B-EXP_ASSAY B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O

(C) Representative images of engulfed PS+ and PS- RGC inputs by microglia in control and CKO. (D) Quantification of engulfed PS+ RGC inputs by microglia, which is calculated as: Volume of engulfed PS+ RGC inputs / Volume of microglia cell. (E) Quantification of engulfed PS- RGC inputs in control and CKO.


A HeLa cells were transfected with constructs for a non - targeting shRNA ( NT ) , USP17 shRNA1 , or USP17 shRNA2 . Cells were stained using an anti - LAMP1 antibody ( green ) and the nuclei counterstained with DAPI ( blue ) . Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line . B HeLa and MDA - MB - 231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position ( see C ) .

O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

A HeLa cells were transfected with constructs for a non-targeting shRNA (NT), USP17 shRNA1, or USP17 shRNA2. Cells were stained using an anti-LAMP1 antibody (green) and the nuclei counterstained with DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. B HeLa and MDA-MB-231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position (see C). C


D HeLa cells transfected as in A , in conjunction with a construct coding GFP tagged CD63 . The lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line . E HeLa cells transfected as in A , in conjunction with constructs for either GFP tagged LAMP1 or CD63 . The distribution of GFP positive vesicles plotted as vesicle relative position .

O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O

D HeLa cells transfected as in A, in conjunction with a construct coding GFP tagged CD63. The lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. E HeLa cells transfected as in A, in conjunction with constructs for either GFP tagged LAMP1 or CD63. The distribution of GFP positive vesicles plotted as vesicle relative position. F


A HeLa cells were transfected with constructs for a non - targeting ( NT ) shRNA , USP17 shRNA1 or USP17 shRNA2 . 48 hours post transfection the cells were either serum starved ( upper panels ) , or treated with serum free medium containing 100ug / ml EGF ( lower panels ) for 16 hours prior to staining for LAMP1 ( green ) and DAPI ( blue ) . Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line . Scale bar represents 25μm . B The distribution of at least 300 LAMP1 positive vesicles from a number of cells ( n ) from a series of confocal images across three separate experiments was plotted as vesicle relative position ( mean value is red bar ) . Error bars represent standard error , ns indicates not significant , and * * * * indicates a p - values less than 0 . 0001 . One way ANOVA used to determine if statistically significant differences between groups .

O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 48 hours post transfection the cells were either serum starved (upper panels), or treated with serum free medium containing 100ug/ml EGF (lower panels) for 16 hours prior to staining for LAMP1 (green) and DAPI (blue). Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line. Scale bar represents 25μm. B The distribution of at least 300 LAMP1 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicates not significant, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.