( Top ) PCR detection of the recombined BrafLox − V600E allele ( Lox - V600E ) in BVE mouse tissues at 8 weeks of age without tamoxifen induction . Substantial recombination is observed in the lung , while weaker recombination is also detected in the liver . No recombination was detected in hematopoietic tissues ( bone marrow and spleen ) even after 40 cycles of amplification ( right ) . ( Bottom ) H & amp ; amp ; E staining of lung sections from wild - type ( WT , 10 weeks of age ) , BVE ( 1 - 10 weeks of age ) and Braf + / LSL − V600E mouse 8 weeks after nasal delivery of AdCre is indicated below . V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development . Scale bars , 100 μm .

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(Top) PCR detection of the recombined BrafLox−V600E allele (Lox-V600E) in BVE mouse tissues at 8 weeks of age without tamoxifen induction. Substantial recombination is observed in the lung, while weaker recombination is also detected in the liver. No recombination was detected in hematopoietic tissues (bone marrow and spleen) even after 40 cycles of amplification (right). (Bottom) H&amp;E staining of lung sections from wild-type (WT, 10 weeks of age), BVE (1-10 weeks of age) and Braf+/LSL−V600E mouse 8 weeks after nasal delivery of AdCre is indicated below. V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development. Scale bars, 100 μm.


Ki67 immunostaining of lung sections from BVE mice at 3 - 10 weeks of age . Scale bars , 25 μm . The right bar graph summarises % Ki67 + cells in tumours at different ages post - partum . Four to six mice were analysed for each age group as indicated , and more than 3 , 500 tumour cells per mouse were evaluated . The data represent mean ± SD .

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Ki67 immunostaining of lung sections from BVE mice at 3-10 weeks of age. Scale bars, 25 μm. The right bar graph summarises %Ki67+ cells in tumours at different ages post-partum. Four to six mice were analysed for each age group as indicated, and more than 3,500 tumour cells per mouse were evaluated. The data represent mean ± SD.


Representative H & amp ; amp ; E staining and Mac2 immunohistochemistry of serial lung sections from 10 - week - old BVE mice at low ( upper panels ) and high ( lower panels ) magnifications . Scale bars , 100 μm ( upper panels ) or 25 μm ( lower panels ) .

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Representative H&amp;E staining and Mac2 immunohistochemistry of serial lung sections from 10-week-old BVE mice at low (upper panels) and high (lower panels) magnifications. Scale bars, 100 μm (upper panels) or 25 μm (lower panels).


Giemsa staining of isolated stroma IMCs . These cells show round or oval - shaped nuclei that lack indentations , and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes , suggestive of immature myeloid - lineage cells that are morphologically distinct from monocytes / DCs . Scale bar , 25 μm .

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Giemsa staining of isolated stroma IMCs. These cells show round or oval-shaped nuclei that lack indentations, and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes, suggestive of immature myeloid-lineage cells that are morphologically distinct from monocytes/DCs. Scale bar, 25 μm.


Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b , which is expressed at low levels ( overlaid with isotype control histograms shown in grey ) .

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Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b, which is expressed at low levels (overlaid with isotype control histograms shown in grey).


Cell surface marker analysis ( dot plots ) of IMCs demonstrating CD11c + IMCs do not express macrophage ( F4 / 80 ) , DC ( CD24 ) or M1 - polarisation ( CD86 ) markers , but weakly express the M2 marker CD206 . Insets show isotype control staining .

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Cell surface marker analysis (dot plots) of IMCs demonstrating CD11c+ IMCs do not express macrophage (F4/80), DC (CD24) or M1-polarisation (CD86) markers, but weakly express the M2 marker CD206. Insets show isotype control staining.


Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines . The cells grown on culture plates were imaged by phase - contrast microscopy ( left ) . Then , the cells were trypsinised and smeared on glass slides for Giemsa staining ( right ) . These cells are intermediate to large sized , round or amoeboid cells with cytoplasmic vacuolation , consistent with a macrophage morphology . Scale bars , 50 μm .

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Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm.


Cell surface marker analysis of cultured IMCs showing differentiation into F4 / 80 + macrophages with CD11bhighCD11c + CD86 + CD206low surface phenotype ( overlaid with isotype control histograms shown in light grey ) .

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Cell surface marker analysis of cultured IMCs showing differentiation into F4/80+ macrophages with CD11bhighCD11c+CD86+CD206low surface phenotype (overlaid with isotype control histograms shown in light grey).


PCR detection of Braf recombination in purified IMCs , AT2 cells and tumour cell aggregates .

PCR detection of Braf recombination in purified IMCs, AT2 cells and tumour cell aggregates.


CMT64 cells ( 1 × 104 / well in 12 - well plates ) were co - cultured with 1 . 5 - 3 × 105 IMCs for 4 days . Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate . Harvested cells were subjected to counting ( left ) . Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells . The bar graph ( left ) shows CMT64 growth ( cell counting ) data pooled from two independent experiments . Phase - contrast images of CMT64 cells cultured without IMCs ( CMT64 only ) or co - cultured with IMCs ( CMT64 / IMC co - culture ) are also indicated in the middle and right . A higher magnification image of co - cultured CMT64 cells ( right ) highlights IMCs migrating into CMT64 cells ( arrow heads ) . Scale bars are 200 μm , except for the high magnification image ( right ) in which the scale bar is 100 μm .

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CMT64 cells (1 × 104/well in 12-well plates) were co-cultured with 1.5-3 × 105 IMCs for 4 days. Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate. Harvested cells were subjected to counting (left). Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells. The bar graph (left) shows CMT64 growth (cell counting) data pooled from two independent experiments. Phase-contrast images of CMT64 cells cultured without IMCs (CMT64 only) or co-cultured with IMCs (CMT64/IMC co-culture) are also indicated in the middle and right. A higher magnification image of co-cultured CMT64 cells (right) highlights IMCs migrating into CMT64 cells (arrow heads). Scale bars are 200 μm, except for the high magnification image (right) in which the scale bar is 100 μm.


( Left ) Growth and EMT - like morphological changes of CMT64 cells cultured with 25 % IMC - CM . CMT64 cells cultured for 4 days at low - density in DMEM / 10 % FCS without IMC - CM ( - IMC - CM ) or with 25 % IMC - CM ( + IMC - CM ) were counted and normalised to the average of control as 100 % . The data in the bar chart represent mean + SD of five cultures utilising IMC - CM obtained from three independent sources . ( Middle ) Phase - contrast images to indicate representative morphologies of CMT64 cells maintained without IMC - CM ( control ) or cultured with 25 % IMC - CM ( + IMC - CM ) for 2 weeks . ( Right ) E - cadherin immunofluorescence of CMT64 cells cultured as above . Scale bars are 200 μm ( phase contrast ) and 10 μm ( immunofluorescence ) .

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(Left) Growth and EMT-like morphological changes of CMT64 cells cultured with 25% IMC-CM. CMT64 cells cultured for 4 days at low-density in DMEM/10%FCS without IMC-CM (-IMC-CM) or with 25% IMC-CM (+IMC-CM) were counted and normalised to the average of control as 100%. The data in the bar chart represent mean + SD of five cultures utilising IMC-CM obtained from three independent sources. (Middle) Phase-contrast images to indicate representative morphologies of CMT64 cells maintained without IMC-CM (control) or cultured with 25% IMC-CM (+IMC-CM) for 2 weeks. (Right) E-cadherin immunofluorescence of CMT64 cells cultured as above. Scale bars are 200 μm (phase contrast) and 10 μm (immunofluorescence).


Co - culture of AT2 cells and autologous IMCs from BVE mice ( 8 - to 10 - week - old ) for 48 h using the Transwell ® culture system . In vitro BrdU incorporation ( flow cytometry , left and middle ) and EMT marker expression ( RT - PCR , right ) in co - cultured AT2 cells are indicated . Representative flow cytometry plots are indicated on the left , and the bar chart in the middle indicates % BrdU + AT2 cell numbers normalised to control cultures without IMCs ( n = 4 , mean + SD ) . Gapdh serves as a loading control for the RT - PCR on the right .

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Co-culture of AT2 cells and autologous IMCs from BVE mice (8- to 10-week-old) for 48 h using the Transwell® culture system. In vitro BrdU incorporation (flow cytometry, left and middle) and EMT marker expression (RT-PCR, right) in co-cultured AT2 cells are indicated. Representative flow cytometry plots are indicated on the left, and the bar chart in the middle indicates % BrdU+ AT2 cell numbers normalised to control cultures without IMCs (n = 4, mean + SD). Gapdh serves as a loading control for the RT-PCR on the right.


Morphological alterations and intracellular signalling in AT2 cells cultured with IMC - CM . On the left and middle , primary AT2 cells from 3 - week - old BVE mice were cultured for 48 h and then incubated in serum - free IMC - CM ( AT2 with IMC - CM ) or DMEM ( AT2 w / o IMC - CM , serving as a control ) for another 48 h , followed by immunostaining for E - cadherin ( red ) and vimentin ( green ) and confocal laser scanning microscopy ( CLSM ) imaging . The 3 × zoomed image in the middle right highlights dividing vimentin + cells that show internalised E - cadherin ( arrows ) . Scale bars , 50 μm . On the right , primary AT2 cells from 10 - week - old BVE mice were cultured for 48 h , serum starved for 5 h and treated with or without serum - free IMC - CM for 30 min . Phosphorylation of AKT , SMAD3 , MEK and ERK were analysed by immunoblotting .

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Morphological alterations and intracellular signalling in AT2 cells cultured with IMC-CM. On the left and middle, primary AT2 cells from 3-week-old BVE mice were cultured for 48 h and then incubated in serum-free IMC-CM (AT2 with IMC-CM) or DMEM (AT2 w/o IMC-CM, serving as a control) for another 48 h, followed by immunostaining for E-cadherin (red) and vimentin (green) and confocal laser scanning microscopy (CLSM) imaging. The 3× zoomed image in the middle right highlights dividing vimentin+ cells that show internalised E-cadherin (arrows). Scale bars, 50 μm. On the right, primary AT2 cells from 10-week-old BVE mice were cultured for 48 h, serum starved for 5 h and treated with or without serum-free IMC-CM for 30 min. Phosphorylation of AKT, SMAD3, MEK and ERK were analysed by immunoblotting.


CCR1 immunofluorescence of primary IMCs ( left , co - stained with Mac2 ) and AT2 cells ( right , co - stained with E - cadherin ) detected by CLSM . Scale bars , 10 μm . CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs , but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 ( inset in the left microphotograph ) .

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CCR1 immunofluorescence of primary IMCs (left, co-stained with Mac2) and AT2 cells (right, co-stained with E-cadherin) detected by CLSM. Scale bars, 10 μm. CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs, but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 (inset in the left microphotograph).


CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs . Scale bar , 50 μm .

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CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs. Scale bar, 50 μm.


CT imaging of lungs of AdCre - infected Braf + / LSL − V600E mice treated with vehicle ( top ) or CCR1 inhibitor ( bottom ) . H : heart ; * indicates a tumour region accompanied by atelectasis .

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CT imaging of lungs of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (top) or CCR1 inhibitor (bottom). H: heart; * indicates a tumour region accompanied by atelectasis.


H & amp ; amp ; E staining of lung sections from vehicle ( left ) or CCR1 inhibitor ( right ) treated AdCre - infected Braf + / LSL − V600E mice . Scale bars , 250 μm ( top ) or 50 μm ( bottom ) .

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H&amp;E staining of lung sections from vehicle (left) or CCR1 inhibitor (right) treated AdCre-infected Braf+/LSL−V600E mice. Scale bars, 250 μm (top) or 50 μm (bottom).


Flow cytometry analysis of CD11c + ( top , green ) and SPC + ( bottom , red ) cells in the lung of AdCre - infected Braf + / LSL − V600Emice treated with vehicle ( left ) or CCR1 inhibitor ( right ) . LungCD11c + ( left ) and SPC + ( right ) cell numbers ( per left lobe ) were quantitated in AdCre - infected Braf + / LSL − V600Emice treated with CCR1 inhibitor or vehicle ( V ) ( n = 3 , mean + SD ) . % LungCD4 + ( left ) and CD8 + ( right ) T lymphocytes were quantitated in AdCre - infected Braf + / LSL − V600E mice treated with CCR1 inhibitor or vehicle ( V ) ( n = 3 , mean + SD ) .

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Flow cytometry analysis of CD11c+ (top, green) and SPC+ (bottom, red) cells in the lung of AdCre-infected Braf+/LSL−V600Emice treated with vehicle (left) or CCR1 inhibitor (right).LungCD11c+ (left) and SPC+ (right) cell numbers (per left lobe) were quantitated in AdCre-infected Braf+/LSL−V600Emice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).% LungCD4+ (left) and CD8+ (right) T lymphocytes were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).


NPC2 immunoblots of concentrated whole lung CM ( top ) and whole lung lysates ( bottom ) generated from Braf + / + ; CreER ™ ( WT ) or BVE ( VE ) mice . Whole lung lysates were obtained from 3 - to 8 - week - old mice .

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NPC2 immunoblots of concentrated whole lung CM (top) and whole lung lysates (bottom) generated from Braf+/+;CreER™ (WT) or BVE (VE) mice. Whole lung lysates were obtained from 3-to 8-week-old mice.


NPC2 immunoblots of un - concentrated CM ( containing 10 % FCS ) from fractionated IMC and AT2 cultures . Total protein staining of the membrane by Amido black ( bottom ) serves as a loading control showing serum albumin at 70 kDa , derived from FCS in the media .

NPC2 immunoblots of un-concentrated CM (containing 10% FCS) from fractionated IMC and AT2 cultures. Total protein staining of the membrane by Amido black (bottom) serves as a loading control showing serum albumin at 70 kDa, derived from FCS in the media.


Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT - PCR . Mean + SD ( n = 3 ) of Npc2 expression normalised to Gapdh is indicated .

Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT-PCR. Mean + SD (n = 3) of Npc2 expression normalised to Gapdh is indicated.


NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells .

NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells.


NPC2 immunoblots of cell lysates from freshly isolated or cultured ( 48 h in serum - free DMEM ) IMCs .

NPC2 immunoblots of cell lysates from freshly isolated or cultured (48 h in serum-free DMEM) IMCs.


NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg / ml bNPC2 for 48 h .

NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg/ml bNPC2 for 48 h.


CLSM imaging of NPC2 - Alexa488 taken up by IMCs . ( Left ) Freshly isolated IMCs were incubated for 2 h with ( + NPC2 - Alexa488 ) or without ( - NPC2 - Alexa488 ) 82 . 5 nM NPC2 - Alexa488 , chased in NPC2 - free media for additional 2 h and live imaged by CLSM . Single - colour ( top ) and DIC - merged ( bottom ) fluorescence images are indicated . Scale bar , 20 μm . ( Right ) Zoomed images ( DIC - merged and single - colour ) from different fields showing vesicular and reticular distribution of incorporated NPC2 - Alexa488 . Scale bar , 10 μm .

CLSM imaging of NPC2-Alexa488 taken up by IMCs. (Left) Freshly isolated IMCs were incubated for 2 h with (+NPC2-Alexa488) or without (-NPC2-Alexa488) 82.5 nM NPC2-Alexa488, chased in NPC2-free media for additional 2 h and live imaged by CLSM. Single-colour (top) and DIC-merged (bottom) fluorescence images are indicated. Scale bar, 20 μm. (Right) Zoomed images (DIC-merged and single-colour) from different fields showing vesicular and reticular distribution of incorporated NPC2-Alexa488. Scale bar, 10 μm.


Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg / ml bNPC2 as determined by qRT - PCR . Mean ± SD ( n = 3 ) of Npc2 expression normalised to Hprt1 is indicated .

Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg/ml bNPC2 as determined by qRT-PCR. Mean ± SD (n = 3) of Npc2 expression normalised to Hprt1 is indicated.


Shortened survival of BVE mice following reduction in Npc2 gene dosage . Genotypes of mice are as follows : + / + = Braf + / LSL − V600E ; CreER + / 0 ; Npc2 + / + , + / hypo = Braf + / LSL − V600E ; CreER + / 0 , Npc2 + / hypo , hypo / hypo = Braf + / LSL − V600E ; CreER + / 0 ; Npc2hypo / hypo . Median survival time ( MST ) for each genotype is indicated .

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Shortened survival of BVE mice following reduction in Npc2 gene dosage. Genotypes of mice are as follows: +/+ = Braf+/LSL−V600E;CreER+/0;Npc2+/+, +/hypo = Braf+/LSL−V600E;CreER+/0,Npc2+/hypo, hypo/hypo = Braf+/LSL−V600E;CreER+/0;Npc2hypo/hypo. Median survival time (MST) for each genotype is indicated.


Representative H & amp ; amp ; E staining of lung sections from 5 - week - old BVE mice on the Npc2 + / + ( + / + ) or Npc2 + / hypo ( + / hypo ) background is shown . Scale bars , 500 μm .

Representative H&amp;E staining of lung sections from 5-week-old BVE mice on the Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) background is shown. Scale bars, 500 μm.


Stroma IMC quantification per lung in Npc2 + / + ( + / + ) or Npc2 + / hypo ( + / hypo ) BVE mice at different ages ( 39 - 109 days p . p . ) .

Stroma IMC quantification per lung in Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) BVE mice at different ages (39-109 days p.p.).


Ki67 / Mac2immunofluorescence analysis of Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVElung sections at 5 weeks of age . Merged images for Ki67 ( green ) , Mac2 ( red ) and DAPI ( blue ) are indicated . Arrows indicate dual positive cells for nuclearKi67 and Mac2 . Scale bars , 50 μm . The bar chart indicates % Ki67 in Mac2 + lung stroma cells ( mean + SD ) , obtained from 4 independent Npc2 + / + and Npc2 + / hypoBVE pairs .

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Ki67/Mac2immunofluorescence analysis of Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVElung sections at 5 weeks of age. Merged images for Ki67 (green), Mac2 (red) and DAPI (blue) are indicated. Arrows indicate dual positive cells for nuclearKi67 and Mac2. Scale bars, 50 μm. The bar chart indicates % Ki67 in Mac2+lung stroma cells (mean + SD), obtained from 4 independent Npc2+/+ and Npc2+/hypoBVE pairs.


In vivo BrdU incorporation of CD11c + IMCs from Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVE mice are shown . Representative flow cytometry plots of three independent experiments are indicated .

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In vivo BrdU incorporation of CD11c+ IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice are shown. Representative flow cytometry plots of three independent experiments are indicated.


Ki67 ( green ) / Mac2 ( red ) dual immunofluorescencestaining of lung sections from 5 - week - old Npc2 + / hypoBVEmice with nuclearDAPI ( blue ) staining , focusing on intratumour Mac2 + cell clusters . Scale bar , 50 μm . Enlarged images of the boxed areas 1 and 2 highlight Ki67 + Mac2 − cells associated with Mac2 + cells ( arrows ) .

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Ki67 (green)/Mac2 (red) dual immunofluorescencestaining of lung sections from 5-week-old Npc2+/hypoBVEmice with nuclearDAPI (blue) staining, focusing on intratumour Mac2+cell clusters. Scale bar, 50 μm. Enlarged images of the boxed areas 1 and 2 highlight Ki67+Mac2− cells associated with Mac2+ cells (arrows).


Lung SPC + / CD11c + cell numbers of Braf + / LSL − V600E ; Npc2 + / + ( + / + , n = 3 ) and Braf + / LSL − V600E ; Npc2 + / hypo ( + / hypo , n = 7 ) mice at 10 weeks post - AdCre infection .

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Lung SPC+/CD11c+cell numbers of Braf+/LSL−V600E;Npc2+/+ (+/+, n = 3) and Braf+/LSL−V600E;Npc2+/hypo (+/hypo, n = 7) mice at 10 weeks post-AdCre infection.


Intracellular un - esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2 + / + and Npc2 + / hypo IMCs . Scale bars , 25 μm . Filipin - stained cells were graded from 0 to 3 + as indicated in the microphotographs ( see Materials and Methods for criteria ) , and % of each grade in IMCs from Npc2 + / + ( + / + ) and Npc2 + / hypo ( + / hypo ) BVE mice is shown in the bar chart . The data were obtained by analysing 176 Npc2 + / + and 242 Npc2 + / hypo IMCs from three independent Npc2 + / + and Npc2 + / hypo pairs .

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Intracellular un-esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2+/+ and Npc2+/hypo IMCs. Scale bars, 25 μm. Filipin-stained cells were graded from 0 to 3+ as indicated in the microphotographs (see Materials and Methods for criteria), and % of each grade in IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice is shown in the bar chart. The data were obtained by analysing 176 Npc2+/+ and 242 Npc2+/hypo IMCs from three independent Npc2+/+ and Npc2+/hypo pairs.


CLSM imaging of fresh Npc2 + / hypo IMCs stained with filipin ( cyan ) and LAMP2 immunofluorescence ( purple ) . Scale bars , 5 μm . Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2 . Single - colour greyscale images are also demonstrated in the middle ( filipin ) and right ( LAMP2 ) .

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CLSM imaging of fresh Npc2+/hypo IMCs stained with filipin (cyan) and LAMP2 immunofluorescence (purple). Scale bars, 5 μm. Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2. Single-colour greyscale images are also demonstrated in the middle (filipin) and right (LAMP2).


Intracellular un - esterified cholesterol distribution as detected by filipin staining in Npc2 + / hypo IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . Scale bars , 25 μm . Quantitative data in the bar graph were obtained by analysing 265 bNPC2 - untreated ( - bNPC2 ) and 252 bNPC2 - treated ( + bNPC2 ) cells from three independent cultures using the method described in ( A ) .

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Intracellular un-esterified cholesterol distribution as detected by filipin staining in Npc2+/hypo IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 25 μm. Quantitative data in the bar graph were obtained by analysing 265 bNPC2-untreated (-bNPC2) and 252 bNPC2-treated (+bNPC2) cells from three independent cultures using the method described in (A).


Immunoblot ( left ) and ELISA ( right , n = 3 ) analyses of CCL6 in IMC - CM from primary Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h ( immunoblots ) or 72 h ( ELISA ) . The data in the bar graph ( ELISA ) represent mean + SD .

Immunoblot (left) and ELISA (right, n = 3) analyses of CCL6 in IMC-CM from primary Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h (immunoblots) or 72 h (ELISA). The data in the bar graph (ELISA) represent mean + SD.


Quantification of secreted CCL6 into IMC - CM during the initial 24 h ( 0 - 24 h ) and subsequent 48 h ( 24 - 72 h ) of IMC culture by ELISA ( n = 3 , mean + SD ) .

Quantification of secreted CCL6 into IMC-CM during the initial 24 h (0-24 h) and subsequent 48 h (24-72 h) of IMC culture by ELISA (n = 3, mean + SD).


Immunoblot and qRT - PCR analyses of CCL6 expression in Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . The data in the bar graph ( qRT - PCR ) represent mean + SD ; n = 3 .

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Immunoblot and qRT-PCR analyses of CCL6 expression in Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. The data in the bar graph (qRT-PCR) represent mean + SD; n = 3.


CLSM imaging of CCL6 ( green ) / LAMP1 ( red ) immunofluorescence of Npc2 + / + IMCs cultured with or without 50 μg / ml bNPC2 for 48 h . Scale bars , 10 μm . The right bottom image highlights the boxed area of the merged image of bNPC2 - treated IMCs showing partial co - localisation of LAMP1 and CCL6 .

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CLSM imaging of CCL6 (green)/LAMP1 (red) immunofluorescence of Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 10 μm. The right bottom image highlights the boxed area of the merged image of bNPC2-treated IMCs showing partial co-localisation of LAMP1 and CCL6.


Immunoblot analysis of intracellular and secreted ( CM ) CCL6 in bafilomycin - treated Npc2 + / + IMC culture . IMCs without ( left ) or with ( right ) 50 μg / ml bNPC2 pre - loading for 30 min were chased in serum - free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h .

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Immunoblot analysis of intracellular and secreted (CM) CCL6 in bafilomycin-treated Npc2+/+ IMC culture. IMCs without (left) or with (right) 50 μg/ml bNPC2 pre-loading for 30 min were chased in serum-free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h.


Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( B ) Dose - response assay using increasing numbers of Nrg1 type I β1a - expressing PC12 cells . For each 96 - well , 40 , 000 split TEV assay cells were co - plated with increasing numbers of Nrg1 - expressing cells , and incubated for 24h . Half - maximal activation is reached at 5 , 000 Nrg1 - expressing cells . The Z ' factor is 0 . 5 indicating a large separation band for this assay .

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Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(B) Dose-response assay using increasing numbers of Nrg1 type I β1a-expressing PC12 cells. For each 96-well, 40,000 split TEV assay cells were co-plated with increasing numbers of Nrg1-expressing cells, and incubated for 24h. Half-maximal activation is reached at 5,000 Nrg1-expressing cells. The Z' factor is 0.5 indicating a large separation band for this assay.


Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( C ) Adding 10ng / ml EGFld resulted in a two - fold activation . Per 96 - well , 40 , 000 split TEV assay cells were co - plated with empty PC12 cells ( no Nrg1 expression ) , 10 , 000 Nrg1 - expressing cells , and 10 , 000 Nrg1 - expressing cells plus 10ng / ml EGFld .

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Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(C) Adding 10ng/ml EGFld resulted in a two-fold activation. Per 96-well, 40,000 split TEV assay cells were co-plated with empty PC12 cells (no Nrg1 expression), 10,000 Nrg1-expressing cells, and 10,000 Nrg1-expressing cells plus 10ng/ml EGFld.


Fig . 1 . A co - culture assay based on the split TEV technique for monitoring NRG1 - ERBB4 signaling activity . ( D ) Lapatinib antagonizes ERBB4 - PIK3R1 signaling in a dose - dependent manner . Per 96 - well , 40 , 000 split TEV assay cells were incubated with increasing amounts of lapatinib , followed by co - plating 10 , 000 Nrg1 type I β1a expressing cells . The inset depicts the IC 50 value in µM . After compound / stimulus addition , each assay was incubated for 24h . RLU , relative luciferase units ; Fluc , firefly luciferase activity ( black line ) ; Rluc , Renilla luciferase activity ( grey line ) ; n = 6 ; data are shown as mean , error bars represent SEM .

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Fig. 1. A co-culture assay based on the split TEV technique for monitoring NRG1-ERBB4 signaling activity.(D) Lapatinib antagonizes ERBB4-PIK3R1 signaling in a dose-dependent manner. Per 96-well, 40,000 split TEV assay cells were incubated with increasing amounts of lapatinib, followed by co-plating 10,000 Nrg1 type I β1a expressing cells. The inset depicts the IC 50 value in µM. After compound/stimulus addition, each assay was incubated for 24h. RLU, relative luciferase units; Fluc, firefly luciferase activity (black line); Rluc, Renilla luciferase activity (grey line); n=6; data are shown as mean, error bars represent SEM.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( B ) Graphic visualisation of the primary screen data of the NIH - NCC library set 1 . All counts ( 320 compounds and 64 controls ) from the Nrg1 - ERBB4 - PIK3R1 split TEV compound screen were plotted against the Z - score using the Mondrian programme , with pathway activators displaying high values and inhibitors low values . For the secondary analysis , we selected all candidates that were at least three standard deviations away from the mean . EGFld positive and Lapatinib / CI - 1033 negative controls are shown in red .

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Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(B) Graphic visualisation of the primary screen data of the NIH-NCC library set 1. All counts (320 compounds and 64 controls) from the Nrg1-ERBB4-PIK3R1 split TEV compound screen were plotted against the Z-score using the Mondrian programme, with pathway activators displaying high values and inhibitors low values. For the secondary analysis, we selected all candidates that were at least three standard deviations away from the mean. EGFld positive and Lapatinib/CI-1033 negative controls are shown in red.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( C and D ) Spironolactone antagonizes Nrg1 - ERBB4 - PIK3R1 signaling . In dose - response assays using ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids transfected into PC12 cells , spironolactone was administered at increasing concentrations before seeding Nrg1 type I ( C ) expressing PC12 cells .

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Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type I (C) expressing PC12 cells.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( C and D ) Spironolactone antagonizes Nrg1 - ERBB4 - PIK3R1 signaling . In dose - response assays using ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids transfected into PC12 cells , spironolactone was administered at increasing concentrations before seeding Nrg1 type III ( D ) expressing PC12 cells .

Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen. (C and D) Spironolactone antagonizes Nrg1-ERBB4-PIK3R1 signaling. In dose-response assays using ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids transfected into PC12 cells, spironolactone was administered at increasing concentrations before seeding Nrg1 type III (D) expressing PC12 cells.


Fig . 2 . Spironolactone is the primary candidate recovered from the co - culture screen . ( E ) Spironolactone inhibits ERBB4 receptor dimerization . Dose - dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4 - NTEV - tevS - GV and ERBB4 - CTEV plasmids transfected into PC12 cells . 10 ng / ml EGFld was applied as Nrg1 stimulus . Fluc , firefly luciferase activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines , indicating toxicity levels ) ; n = 6 ; data are shown as mean , error bars represent SEM . The insets depict IC 50 values in µM .

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Fig. 2. Spironolactone is the primary candidate recovered from the co-culture screen.(E) Spironolactone inhibits ERBB4 receptor dimerization. Dose-dependent dimerization of the ERBB4 receptor was analyzed using a split TEV assay encompassing ERBB4-NTEV-tevS-GV and ERBB4-CTEV plasmids transfected into PC12 cells. 10 ng/ml EGFld was applied as Nrg1 stimulus. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( A ) Spironolactone , molecular structure shown left , inhibits ERBB4 / PIK3R1 split TEV assay activity ( black line ) , with an IC 50 value of 2 . 76 µM .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(A) Spironolactone, molecular structure shown left, inhibits ERBB4/PIK3R1 split TEV assay activity (black line), with an IC 50 value of 2.76 µM.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( B ) canrenone . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (B) canrenone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( C ) 7α - thiomethyl - spironolactone . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment. (B-D) The spironolactone metabolites(C) 7α-thiomethyl-spironolactone. Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( B - D ) The spironolactone metabolites ( D ) eplerenone do not attenuate the ERBB4 / PIK3R1 assay activity ( black lines ) . Note that only spironolactone bears a thio - ketone group attached to the sterol core structure . All ERBB4 / PIK3R1 assays were run in a single - culture assay mode using 10ng / ml EGFld as functional Nrg1 stimulus , and ERBB4 - NTEV - tevS - GV and PIK3R1 - CTEV plasmids were transfected into PC12 cells ( indicated by icon ) . Fluc , firefly luciferase activity reporting ERBB4 - PIK3R1 assay activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines ) assessing viability ; data are shown as mean , error bars represent SEM ; n = 6 .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(B-D) The spironolactone metabolites (D) eplerenone do not attenuate the ERBB4/PIK3R1 assay activity (black lines). Note that only spironolactone bears a thio-ketone group attached to the sterol core structure. All ERBB4/PIK3R1 assays were run in a single-culture assay mode using 10ng/ml EGFld as functional Nrg1 stimulus, and ERBB4-NTEV-tevS-GV and PIK3R1-CTEV plasmids were transfected into PC12 cells (indicated by icon). Fluc, firefly luciferase activity reporting ERBB4-PIK3R1 assay activity (black lines); Rluc, Renilla luciferase activity (grey lines) assessing viability; data are shown as mean, error bars represent SEM; n=6.


Fig . 3 . Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment . ( F ) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays . All single dose - response assays can be found in Fig EV4 ; the combination ERBB4 - ERBB4 is shown in Fig 2E . Fluc , firefly luciferase activity ( black lines ) ; Rluc , Renilla luciferase activity ( grey lines , indicating toxicity levels ) ; n = 6 ; data are shown as mean , error bars represent SEM . The insets depict IC 50 values in µM .

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Fig. 3. Multilevel profiling approach of spironolactone treatment assessing target specificities and adapter recruitment.(F) Heat map showing the IC 50 values obtained from individual ERBB dimerization split TEV assays. All single dose-response assays can be found in Fig EV4; the combination ERBB4-ERBB4 is shown in Fig 2E. Fluc, firefly luciferase activity (black lines); Rluc, Renilla luciferase activity (grey lines, indicating toxicity levels); n=6; data are shown as mean, error bars represent SEM. The insets depict IC 50 values in µM.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( A ) Spironolactone reduces ERBB4 levels . T - 47D cells were stimulated with 10 ng / ml EGFld , 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated . Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284 .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(A) Spironolactone reduces ERBB4 levels. T-47D cells were stimulated with 10 ng/ml EGFld, 10 µM lapatinib and 10 µM spironolactone for 5 min as indicated. Cell lysates were probed for ERBB4 phosphorylation levels at Tyr1056 and Tyr1284.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( B ) Quantification of band intensities for phospho - ERBB4 levels ( n = 4 per condition ) shown in ( A ) using ImageJ . Phosphorylation levels are normalized to protein levels of ERBB4 . Data shown as mean , error bars represent SD ; t - test , with * , p = 0 . 0356 for p - ERBB4 ( Y1056 ) , and * * , p = 0 . 0079 for p - ERBB4 ( Y1284 ) .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(B) Quantification of band intensities for phospho-ERBB4 levels (n=4 per condition) shown in (A) using ImageJ. Phosphorylation levels are normalized to protein levels of ERBB4. Data shown as mean, error bars represent SD; t-test, with *, p=0.0356 for p-ERBB4 (Y1056), and **, p=0.0079 for p-ERBB4 (Y1284).


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( D ) Spironolactone reduces phospho - Erbb4 levels in Nrg1 - tg mice . Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis . Lysates were probed with indicated antibodies .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(D) Spironolactone reduces phospho-Erbb4 levels in Nrg1-tg mice. Mice were treated with spironolactone for 21 days and sacrificed for western blot analysis. Lysates were probed with indicated antibodies.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( E ) Quantification of band intensities for phospho - Erbb4 and phospho - Limk1 levels shown in ( D ) using ImageJ . Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1 . Data shown as mean , error bars represent SD ; t - test , with * , p = 0 . 0330 for p - Erbb4 , and p = 0 . 0201 for p - Limk1 .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (E) Quantification of band intensities for phospho-Erbb4 and phospho-Limk1 levels shown in (D) using ImageJ. Phosphorylation levels are normalized to protein levels of Erbb4 and Limk1. Data shown as mean, error bars represent SD; t-test, with *, p=0.0330 for p-Erbb4, and p=0.0201 for p-Limk1.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( F ) Canrenone ( applied as 10 µM ) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex ( PFC ) . Representative traces of sIPSCs ( upper ) and a histogram of mean sIPSC ( lower ) are shown for both before and after addition of canrenone .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo. (F) Canrenone (applied as 10 µM) showed no effects on frequencies and amplitudes of sIPSCs in pyramidal neurons of the prefrontal cortex (PFC). Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of canrenone.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( G ) Spironolactone ( applied as 10 µM ) significantly increases frequencies ( n = 12 ; p = 0 . 0454 ) and amplitudes ( n = 12 , p = 0 . 0478 ) of sIPSCs in pyramidal neurons of PFC . Representative traces of sIPSCs ( upper ) and a histogram of mean sIPSC ( lower ) are shown for both before and after addition of spironolactone .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(G) Spironolactone (applied as 10 µM) significantly increases frequencies (n=12; p=0.0454) and amplitudes (n=12, p=0.0478) of sIPSCs in pyramidal neurons of PFC. Representative traces of sIPSCs (upper) and a histogram of mean sIPSC (lower) are shown for both before and after addition of spironolactone.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( H ) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC ( n = 15 , p = 0 . 0286 ) . Spironolactone ( 5 µM ) was applied in the presence of canrenone ( 10 µM ) . For ( H , I ) , sample recording ( upper ) and a histogram of averaged eIPSC ( lower ) is shown for both before and after drug application . For ( F - I ) , the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals . Data are shown as mean , error bars represent SEM ( n = 4 ) ; paired t - test , with * , p ≤ 0 . 05 ; Spiro , spironolactone ; Canre , canrenone ; Lap , lapatinib .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(H) Spironolactone increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0286). Spironolactone (5 µM) was applied in the presence of canrenone (10 µM).For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.


Fig . 4 . Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo . ( I ) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC ( n = 15 , p = 0 . 0124 ) . Lapatinib was applied in 5 µM . For ( H , I ) , sample recording ( upper ) and a histogram of averaged eIPSC ( lower ) is shown for both before and after drug application . For ( F - I ) , the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals . Data are shown as mean , error bars represent SEM ( n = 4 ) ; paired t - test , with * , p ≤ 0 . 05 ; Spiro , spironolactone ; Canre , canrenone ; Lap , lapatinib .

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Fig. 4. Spironolactone antagonizes ERBB4 phosphorylation both in in vitro and in vivo.(I) Lapatinib increases amplitudes of evoked IPSCs in pyramidal neurons of PFC (n=15, p=0.0124). Lapatinib was applied in 5 µM. For (H, I), sample recording (upper) and a histogram of averaged eIPSC (lower) is shown for both before and after drug application. For (F-I), the numbers displayed inside the histogram bars indicate the number of recorded slices / number of animals. Data are shown as mean, error bars represent SEM (n=4); paired t-test, with *, p≤0.05; Spiro, spironolactone; Canre, canrenone; Lap, lapatinib.


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( B ) Nrg1 - tg mice travelled longer distances in the open field arena ( effect of genotype F ( 1 , 44 ) = 10 . 53 ; p = 0 . 0022 ; 2 - way ANOVA ) . Bonferroni post - hoc analysis revealed a significant genotype - dependent difference between vehicle - treated groups ( p = 0 . 0044 ) but not in spironolactone - treated groups ( p = 0 . 3783 ) . Genotype differences were abolished upon spironolactone treatment .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(B) Nrg1-tg mice travelled longer distances in the open field arena (effect of genotype F (1,44) =10.53; p=0.0022; 2-way ANOVA). Bonferroni post-hoc analysis revealed a significant genotype-dependent difference between vehicle-treated groups (p=0.0044) but not in spironolactone-treated groups (p=0.3783). Genotype differences were abolished upon spironolactone treatment.


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( C ) When vehicle treated animal were analyzed in 1 min intervals , transgenic mice showed an increased activity throughout the entire test ( effect of genotype F ( 1 , 22 ) = 9 . 27 ; p = 0 . 0060 ; 2 - way ANOVA ) , most prominent in intervals 2 , 7 , and 8 ( p = 0 . 0427 , p = 0 . 0385 and p = 0 . 000036 , respectively , Bonferroni post - hoc test ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(C) When vehicle treated animal were analyzed in 1 min intervals, transgenic mice showed an increased activity throughout the entire test (effect of genotype F (1,22) =9.27; p=0.0060; 2-way ANOVA), most prominent in intervals 2, 7, and 8 (p=0.0427, p=0.0385 and p=0.000036, respectively, Bonferroni post-hoc test).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( D ) There was no significant difference between the genotypes when treated with spironolactone ( effect of genotype F ( 1 , 22 ) = 2 . 19 ; p = 0 . 1535 ; 2 - way ANOVA ) . However , the interaction of genotype and treatment was significant ( F ( 9 , 198 ) = 1 . 94 ; p = 0 . 0481 ; 2 - way ANOVA ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(D) There was no significant difference between the genotypes when treated with spironolactone (effect of genotype F (1,22) =2.19; p=0.1535; 2-way ANOVA). However, the interaction of genotype and treatment was significant (F (9,198) =1.94; p=0.0481; 2-way ANOVA).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( E ) Nrg1 - tg mice treated with spironolactone spent more time in the light compartment during the light - dark test ( interaction gene × treatment F ( 1 , 41 ) = 4 . 90 ; p = 0 . 0324 ; 2 - way ANOVA , and p = 0 . 0219 , Bonferroni post - test ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(E) Nrg1-tg mice treated with spironolactone spent more time in the light compartment during the light-dark test (interaction gene × treatment F (1,41) =4.90; p=0.0324; 2-way ANOVA, and p=0.0219, Bonferroni post-test).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( F ) In the Y - maze test , transgenic mice performed less alterations ( effect of genotype F ( 1 , 44 ) = 11 . 50 ; p = 0 . 0015 ; 2 - way ANOVA ) . The Bonferroni test confirmed this phenotype in vehicle - treated groups ( p = 0 . 0011 ) , but not in spironolactone - treated animals ( p = 0 . 5950 ) . Spironolactone treatment had a significant effect on the number of alterations ( F ( 1 , 44 ) = 4 . 12 ; p = 0 . 0484 ; 2 - way ANOVA ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice. (F) In the Y-maze test, transgenic mice performed less alterations (effect of genotype F (1,44) =11.50; p=0.0015; 2-way ANOVA). The Bonferroni test confirmed this phenotype in vehicle-treated groups (p=0.0011), but not in spironolactone-treated animals (p=0.5950). Spironolactone treatment had a significant effect on the number of alterations (F (1,44) =4.12; p=0.0484; 2-way ANOVA).


Fig . 5 . Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1 - tg mice . ( G ) Spironolactone treatment significantly enhanced PPI in Nrg1 - tg mice ( effect of treatment F ( 1 , 21 ) = 5 . 07 ; p = 0 . 0325 ; 2 - way repeated measures ANOVA ) . Data are shown as mean , error bars represent SEM . * , p < 0 . 05 , * * , p < 0 . 01 and * * * , p < 0 . 001 , Bonferroni test following 2 - way ANOVA ; Spiro , spironolactone ; Veh , vehicle . n = 12 per genotype and treatment with an exception of ( E ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 10 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) and ( G ) ( Nrg1 - tg vehicle , n = 11 ; Nrg1 - tg Spiro , n = 12 ; wt vehicle , n = 12 ; wt Spiro , n = 12 ) .

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Fig. 5. Chronic spironolactone treatment ameliorates deficits of behavioral endophenotypes in Nrg1-tg mice.(G) Spironolactone treatment significantly enhanced PPI in Nrg1-tg mice (effect of treatment F (1,21) =5.07; p=0.0325; 2-way repeated measures ANOVA). Data are shown as mean, error bars represent SEM. *, p<0.05, **, p<0.01 and ***, p<0.001, Bonferroni test following 2-way ANOVA; Spiro, spironolactone; Veh, vehicle. n=12 per genotype and treatment with an exception of (E) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=10; wt vehicle, n=12; wt Spiro, n=12) and (G) (Nrg1-tg vehicle, n=11; Nrg1-tg Spiro, n=12; wt vehicle, n=12; wt Spiro, n=12).


B . Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs .

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B. Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs.


C . qPCR validations of neutrophil genes . n = 7 DFUs and n = 3 Skin D3 wounds . * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) . Data presented as mean ± SD .

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C. qPCR validations of neutrophil genes. n=7 DFUs and n=3 Skin D3 wounds. **P < 0.01 (two-tailed unpaired Student's t-test). Data presented as mean ± SD.


A . Representative images of human neutrophils treated with the FOXM1 inhibitor , FDI - 6 . Vehicle ( DMSO ) served as a control . Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red . Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) . Data presented as mean ± SD . ( Scale bar : 100µm ) .

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A. Representative images of human neutrophils treated with the FOXM1 inhibitor, FDI-6. Vehicle (DMSO) served as a control. Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red. Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). Data presented as mean ± SD. (Scale bar: 100µm).


B . Quantification of ROS levels in human neutrophils treated with FDI - 6 or in combination with N - acetylcysteine ( NAC ) . Phorbol 12 - myristate 13 - acetate ( PMA ) treatment served as a positive control . Data presented as mean ± SD . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) . C . Quantification of NET formation in human neutrophils treated with FDI - 6 or in combination with NAC . PMA treatment served as a positive control . Neutrophils from n = 3 different blood donors were isolated , pooled and peformed in triplicate . Data presented as mean ± SD . * P < 0 . 05 ( two - way ANOVA followed by Tukey ' s post - hoc test ) .

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B. Quantification of ROS levels in human neutrophils treated with FDI-6 or in combination with N-acetylcysteine (NAC). Phorbol 12-myristate 13-acetate (PMA) treatment served as a positive control. Data presented as mean ± SD. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). C. Quantification of NET formation in human neutrophils treated with FDI-6 or in combination with NAC. PMA treatment served as a positive control. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. Data presented as mean ± SD. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).


A . Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response .

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A. Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response.


A . Representative images of wounded skin after topical treatment with either vehicle ( IgG isotype control ) or ⍺ - TREM1 activator at 0 , 2 , 4 , 6 , and 8 days after wounding . Percent of wound area at each time following vehicle or ⍺ - TREM1 activator treatment relative to the original wound area . Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺ - TREM1 treated wounds were performed with Fiji software . Data presented as mean ± SD . * * P < 0 . 01 , and * * * * P < 0 . 0001 ( two - way ANOVA followed by Tukey ' s post - hoc test ) .

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A. Representative images of wounded skin after topical treatment with either vehicle (IgG isotype control) or ⍺-TREM1 activator at 0, 2, 4, 6, and 8 days after wounding. Percent of wound area at each time following vehicle or ⍺-TREM1 activator treatment relative to the original wound area. Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺-TREM1 treated wounds were performed with Fiji software. Data presented as mean ± SD. **P < 0.01, and ****P < 0.0001 (two-way ANOVA followed by Tukey's post-hoc test).


B . Representative pictures of vehicle ( IgG isotype control ) and ⍺ - TREM1 treated diabetic wounds at day 4 show basal keratin marker K5 , and neutrophil marker Ly6G , FOXM1 and citH3 . Treatment of wounds with ⍺ - TREM1 resulted in increased FOXM1 + and decreased citH3 + neutrophils compared to vehicle treated diabetic wounds . ( Scale bar : 50µm ) . Quantification of mean fluorescence intensity was performed with Fiji software . n = 7 diabetic vehicle wounds and 6 diabetic ⍺ - TREM1 wounds . Data presented as mean ± SD . * P < 0 . 05 , * P < 0 . 05 , and * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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B. Representative pictures of vehicle (IgG isotype control) and ⍺-TREM1 treated diabetic wounds at day 4 show basal keratin marker K5, and neutrophil marker Ly6G, FOXM1 and citH3. Treatment of wounds with ⍺-TREM1 resulted in increased FOXM1+ and decreased citH3+ neutrophils compared to vehicle treated diabetic wounds. (Scale bar: 50µm). Quantification of mean fluorescence intensity was performed with Fiji software. n= 7 diabetic vehicle wounds and 6 diabetic ⍺-TREM1 wounds. Data presented as mean ± SD. *P < 0.05, *P < 0.05, and **P < 0.01 (two-tailed unpaired Student's t-test).


A . qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs ( n = 5 healing and n = 6 nonhealing ) . Data presented as mean ± SD . * P < 0 . 05 . * * P < 0 . 01 ( two - tailed unpaired Student ' s t - test ) .

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A. qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs (n = 5 healing and n= 6 nonhealing). Data presented as mean ± SD. *P < 0.05. **P < 0.01 (two-tailed unpaired Student's t-test).


B . Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing ( n = 4 ) and nonhealing ( n = 4 ) is shown in the graph . Data presented as mean ± SEM . * P < 0 . 05 ( two - tailed unpaired Student ' s t - test ) . ( Scale bar : 50µm ) .

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B. Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing (n=4) and nonhealing (n=4) is shown in the graph. Data presented as mean ± SEM. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).


C . Representative images of cit - H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs , which was confirmed by corresponding quantification from healing ( n = 3 ) and nonhealing ( n = 3 ) , shown in the graph . Data presented as mean ± SD . * P < 0 . 05 ( two - tailed unpaired Student ' s t - test ) . ( Scale bar : 50µm ) .

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C. Representative images of cit-H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs, which was confirmed by corresponding quantification from healing (n=3) and nonhealing (n=3), shown in the graph. Data presented as mean ± SD. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).


( G ) GTT or ITT in 17 - week - old wild - type or JFKTG mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(G) GTT or ITT in 17-week-old wild-type or JFKTG mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( H ) H & E and Oil Red O staining of liver sections from wild - type or JFKTG mice fed on ND or HFD . Scale bar , 100 μm .

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(H) H&E and Oil Red O staining of liver sections from wild-type or JFKTG mice fed on ND or HFD. Scale bar, 100 μm.


( D ) The body weight of male wild - type or JfkKO mice fed on HFD . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) The body weight of male wild-type or JfkKO mice fed on HFD. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


( G ) H & E and Oil Red O staining of liver sections from wild - type or JfkKO mice fed on HFD . Scale bar , 100 μm .

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(G) H&E and Oil Red O staining of liver sections from wild-type or JfkKO mice fed on HFD. Scale bar, 100 μm.


( H ) Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector or JFK via tail vein . Seven days after injection , total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) . H & E and Oil Red O staining of liver sections are shown . Scale bar , 100 μm .

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(H) Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector or JFK via tail vein. Seven days after injection, total proteins prepared from liver tissues were subjected to western blotting analysis for the expression of Jfk or measurement for hepatic TG and NEFA content. Error bars represent mean ± SEM (n = 6, *p < 0.05, one-way ANOVA with Tukey's HSD test). H&E and Oil Red O staining of liver sections are shown. Scale bar, 100 μm.


( A ) HepG2 cells were stably transfected with vector or FLAG - JFK . Cellular extracts were immunopurified with anti - FLAG immunoafﬁnity resin and eluted with FLAG peptides . The eluates were resolved on SDS - PAGE and silver - stained . The proteins bands were retrieved and analyzed by mass spectrometry .

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(A) HepG2 cells were stably transfected with vector or FLAG-JFK. Cellular extracts were immunopurified with anti-FLAG immunoafﬁnity resin and eluted with FLAG peptides. The eluates were resolved on SDS-PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry.


( B ) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins .

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(B) Cellular extracts from HepG2 cells were immunoprecipitated with antibodies against ING5 followed by immunoblotting with the antibodies against the indicated proteins.


( E ) HepG2 cells were transfected with FLAG - JFK and treated with 50 μg / ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis . Quantitation was done by densitometry and expressed as signals of ING5 / β - actin .

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(E) HepG2 cells were transfected with FLAG-JFK and treated with 50 μg/ml CHX for the indicated times in the presence or absence of MG132 for western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING5/β-actin.


( H ) Bacterially - expressed GST - JFK or JFK mutants was incubated with in vitro transcribed / translated ING5 for in vitro ubiquitination assays .

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(H) Bacterially-expressed GST-JFK or JFK mutants was incubated with in vitro transcribed/translated ING5 for in vitro ubiquitination assays.


( D ) ChIP - seq track at the representative promoter of target genes . The red rectangles indicate the peak regions / binding sites of ING5 on the target genes promoter .

(D) ChIP-seq track at the representative promoter of target genes. The red rectangles indicate the peak regions/binding sites of ING5 on the target genes promoter.


( C ) HepG2 cells were treated with control siRNAs or siRNAs against JFK or / and ING5 , or transfected with vector , JFK , or / and ING5 . Cellular extracts were prepared for western blotting analysis with the indicated antibodies .

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(C) HepG2 cells were treated with control siRNAs or siRNAs against JFK or/and ING5, or transfected with vector, JFK, or/and ING5. Cellular extracts were prepared for western blotting analysis with the indicated antibodies.


( D ) HepG2 cells were transfected with vector or JFK , AMPKα1 , or / and ING5 , or treated with control siRNAs or siRNAs against JFK , AMPKα1 , or / and ING5 , Cellular extracts were prepared for AMPK activity assays by ELISA . Error bars represent mean ± SD for triplicate experiments ( * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) HepG2 cells were transfected with vector or JFK, AMPKα1, or/and ING5, or treated with control siRNAs or siRNAs against JFK, AMPKα1, or/and ING5, Cellular extracts were prepared for AMPK activity assays by ELISA. Error bars represent mean ± SD for triplicate experiments (*p < 0.05, paired two-tailed Student's t-test).


( D ) The measurement of AMPK activity and the rate of fatty acid β - oxidation of wild - type or JfkKO mice . Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA . Mitochondria were isolated from MEFs or liver tissues , and the rate of fatty acid β - oxidation was quantified by spectrophotometric detection for ferricyanide - trapped reduced products . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , paired two - tailed Student ' s t - test ) .

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(D) The measurement of AMPK activity and the rate of fatty acid β-oxidation of wild-type or JfkKO mice. Cellular extracts prepared from MEFs or liver tissues were subjected to AMPK activity assays by ELISA. Mitochondria were isolated from MEFs or liver tissues, and the rate of fatty acid β-oxidation was quantified by spectrophotometric detection for ferricyanide-trapped reduced products. Error bars represent mean ± SEM (n = 6, *p < 0.05, paired two-tailed Student's t-test).


Wild - type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally - delivered vector control , shIng5 , or shAmpkα1 via tail vein . Seven days after injection , cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content ( E ) . Asterisk ( * ) represents significant comparison to wild - type mice , octothorpe ( # ) represents significant comparison to JfkKO mice . Error bars represent mean ± SEM ( n = 6 , * p < 0 . 05 , # p < 0 . 05 , one - way ANOVA with Tukey ' s HSD test ) .

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Wild-type or JfkKO mice were fed with HFD for 11 weeks prior to injection of adenovirally-delivered vector control, shIng5, or shAmpkα1 via tail vein. Seven days after injection, cellular extracts prepared from liver tissues were subjected to western blotting analysis or measurement for hepatic TG and NEFA content (E). Asterisk (*) represents significant comparison to wild-type mice, octothorpe (#) represents significant comparison to JfkKO mice. Error bars represent mean ± SEM (n = 6, *p < 0.05, #p < 0.05, one-way ANOVA with Tukey's HSD test).


( A ) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n ( number of animals per group ) = 6 .

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(A) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n (number of animals per group)=6.


( B ) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n ( number of animals per group ) = 5 .

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(B) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n (number of animals per group)=5.


( C ) Tumor volume in SCIDmice treated or not with LPS n ( number of animals per group ) = 5 .

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(C) Tumor volume in SCIDmice treated or not with LPS n (number of animals per group)=5.


( D ) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n ( number of animals per group ) = 6 .

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(D) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n (number of animals per group)=6.


( E ) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n ( number of animals per group ) ≥ 4 . NT , control ; DT , diphtheria toxin treated animals to deplete DCs ; LPS , mice treated with LPS . Error bars depict SEM . Statistical significance was determined with a two - tail t test . NT , untreated .

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(E) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n (number of animals per group) ≥ 4. NT, control; DT, diphtheria toxin treated animals to deplete DCs; LPS, mice treated with LPS. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel .

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(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel.


( B ) Detection of blood vessels by immunohistochemistry using anti - CD31 antibody ( brown staining ) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in ( A ) . Nuclei are stained with hematoxylin . Two representative sections are shown , magnification 20X . Left panel scale bars , 500m ; right panel scale bars 100 m . Quantification of vessel mean dimensions and area coverage by vessels is also shown . Three sections from three tumors have been analyzed . Error bars depict SEM . Statistical significance was determined with a two - tail t test . NT , untreated .

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(B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A , B ) Absolute numbers of IFN - γ + and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 ( 25g / mouse ) to reduce the ingress of lymphocytes in the efferent lymphatics .

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(A,B) Absolute numbers of IFN-γ+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics.


( C ) Tumor volume in mice treated or not with LPS and / or FTY720 according to the scheme .

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(C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme.


( D ) Percent of IFN - γ + NK cells within the tumor in mice treated or not with LPS and with FTY720 . n ( number of animals per group ) = 4 . Error bars depict SEM . FTY : FTY720 . Statistical significance was determined with a two - tail t test . NT , untreated .

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(D) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti - CD62L antibody to inhibit NK cel ingress in the lymph node from blood . Where indicated mice were challenged with LPS .

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(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cel ingress in the lymph node from blood. Where indicated mice were challenged with LPS.


( B ) Absolute numbers of IFN - γ + NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti - CD62L treatment .

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(B) Absolute numbers of IFN-γ+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment.


( C ) ( left panel ) Absolute numbers of endogenous and adoptively transferred ( CFSE + ) NK cells in the draining and contralateral lymph node 5 hours after LPS administration ; ( right panel ) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration .

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(C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration.


( D ) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration . Lymph node cells were stained with anti - CD3 , anti - DX5 and anti - IFN - γ antibodies and the analysis performed on gated CD3 - DX5high cells . One representative of 4 independent experiments is shown .

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(D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN-γ antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown.


( E ) Tumor volume in mice treated or not with LPS , where indicated mice received anti - CD62L antibody 12h before LPS treatment .

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(E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment.


( F ) Percent of IFN - γ + NK cells within the tumor in mice treated or not with LPS and with the anti - CD62L antibody .

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(F) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.


( A ) Representative image of an NK cell ( red ) interacting with four DCs ( green ) . ( B , C , D ) Plot of the confinement ratio ( CR , black squares ) , the normalized NK speed Vthr ( ti ) ( red circles ) and the digitalized NK - DC distance , DistD ( ti ) ( green triangles ) as a function of time for the NK cell reported in the image . The B , C and D panels correspond respectively to the interaction with the DC1 , DC2 and DC4 displayed in ( A ) . For reference also the normalized distance Dist ( ti ) is reported in the plots ( blue down triangles ) . The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots . The green up - triangles indicate the parameter TonToff defined as TonToff = - 1 if Dist ( ti ) < d = 25 µm and TonToff = - 1 if Dist ( ti ) d and indicate the putative interactions according to a more simplified algorithm based on the DC - NK cell distance alone .

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(A) Representative image of an NK cell (red) interacting with four DCs (green). (B, C, D) Plot of the confinement ratio (CR, black squares), the normalized NK speed Vthr(ti) (red circles) and the digitalized NK-DC distance, DistD(ti) (green triangles) as a function of time for the NK cell reported in the image. The B, C and D panels correspond respectively to the interaction with the DC1, DC2 and DC4 displayed in (A). For reference also the normalized distance Dist(ti) is reported in the plots (blue down triangles). The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots. The green up-triangles indicate the parameter TonToff defined as TonToff = -1 if Dist(ti) < d = 25 µm and TonToff = -1 if Dist(ti) d and indicate the putative interactions according to a more simplified algorithm based on the DC-NK cell distance alone.


( A ) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope . The sequence of images shows the interaction between an NK cell ( red ) and a DC ( green ) . In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back . In the right lower panel the stable contact DC - NK cell is clearly visible . ( B ) Time duration of the DC - NK contacts in steady state ( - LPS ) and inflammatory ( + LPS ) conditions . Bars indicate the mean of the distribution . The percent of cells showing long interaction times ( ≥ 15 min ) are shown . ( C ) NK cell 3D velocities . 3D velocities of NK cells taking contacts with DCs before ( black ) and 5 h ( green ) after LPS treatment .

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(A) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope. The sequence of images shows the interaction between an NK cell (red) and a DC (green). In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back. In the right lower panel the stable contact DC-NK cell is clearly visible. (B) Time duration of the DC-NK contacts in steady state (-LPS) and inflammatory (+LPS) conditions. Bars indicate the mean of the distribution. The percent of cells showing long interaction times (≥ 15 min) are shown. (C) NK cell 3D velocities. 3D velocities of NK cells taking contacts with DCs before (black) and 5 h (green) after LPS treatment.


( A ) Differential interference contrast images ( left panels ) and confocal microscopy analysis of IL - 18 ( right panels ) in a DC / NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells ( NT ) . Representative images from 3 experiments are shown , magnification 63X . Scale bars , 4 . 36 m .

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(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m.


( B ) IFN - γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs ( untreated ) or DCs treated O / N with LPS ( LPS ) . Where indicated IL - 18 was added to the cultures .

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(B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures.


( C ) Cell - contact dependent activation of NK cells by DCs depends on IL - 18 . Unstimulated or LPS - activated DCs and NK cells were cocultured in the same wells ( DC + NK + LPS ) or separated by a porous membrane ( ( DC + LPS ) / NK ) . Where indicated IL - 18 was added to the transwells ( ( DCs + LPS ) / ( NK + IL - 18 ) ) . NK cells alone were also cultured in the presence of the three selected cytokines ; IL - 2 and IFN - β in the upper chamber and NK cells and IL - 18 in the lower chamber . IFN - γ in the supernatant was then measured by ELISA after 8 hours of coculture . ( B , C ) . n ( number of independent experiments ) = 3 . Error bars depict SEM .

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(C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS-activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.


C Western blot analysis of ACE2 in Caco - 2 cells treated with vitamin ( Vit ) compounds ( VitB1 , 500 µM ; VitB6 , 500 µM ; VitB12 , 50 nM ; VitC , 5 mM ; VitD3 , 25 µM ; VitK1 , 0 . 5 µM ) for 24 hrs . Data are representative of three biological replicates .

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C Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. Data are representative of three biological replicates.