( 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 . ) .

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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 ) .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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.


( A ) Schematic representation of the SILAC - based quantitative proteomics experiment in B . subtilis . Cells overexpressing active YqgP ( BS50 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BS51 ) , both auxotrophic for lysine , were grown in parallel in " heavy " ( containing 13C615N2 - Lysine isotope ) or " light " ( containing stable 12C614N2 - Lysine ) M9 minimal medium , respectively . After mixing the cell cultures in the 1 : 1 ratio ( based on OD600 ) , cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC - MS / MS experiment . Using bioinformatic analysis of the MS data , highest - scoring candidate substrates were further evaluated .

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

(A) Schematic representation of the SILAC-based quantitative proteomics experiment in B. subtilis. Cells overexpressing active YqgP (BS50, Table EV2) or its catalytically dead mutant YqgP.S288A (BS51), both auxotrophic for lysine, were grown in parallel in "heavy" (containing 13C615N2- Lysine isotope) or "light" (containing stable 12C614N2- Lysine) M9 minimal medium, respectively. After mixing the cell cultures in the 1:1 ratio (based on OD600), cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC-MS/MS experiment. Using bioinformatic analysis of the MS data, highest-scoring candidate substrates were further evaluated.


( E ) Results of MS analyses of affinity co - purification experiments in wild - type B . subtilis control ( strain BTM2 , Table EV2 ) and B . subtilis deficient in endogenous YqgP expressing the wild - type YqgP - sfGFP bait ( strain BTM84 , Table EV2 ) or the proteolytically inactive YqgP . S288A - sfGFP bait ( strain BBM1 , Table EV2 ) . Proteins were considered as potential interactors of YqgP if they were identified only in both positive co - purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts . The protein highlighted in red was the only overlapping hit between the two proteomics approaches .

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

(E) Results of MS analyses of affinity co-purification experiments in wild-type B. subtilis control (strain BTM2, Table EV2) and B. subtilis deficient in endogenous YqgP expressing the wild-type YqgP-sfGFP bait (strain BTM84, Table EV2) or the proteolytically inactive YqgP.S288A-sfGFP bait (strain BBM1, Table EV2). Proteins were considered as potential interactors of YqgP if they were identified only in both positive co-purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts. The protein highlighted in red was the only overlapping hit between the two proteomics approaches.


( A ) Steady - state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B . subtilis ( BTM2 and BTM501 , respectively , Table EV2 ) , in minimal medium at low magnesium concentration ( 10 µM ) . Strain lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) was used as a control . Proteins were detected by immunoblotting with chemiluminescence detection . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE )

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

(A) Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B. subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 µM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE)


( B ) The cleavage is efficiently inhibited by 1 μM STS736 , a specific peptidyl ketoamide rhomboid inhibitor Ticha et al . , 2017b ( ) . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies .

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

(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b(). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( C ) To map the cleavage site region within endogenous MgtE ( second lane from the left , from strain BTM501 ) , MgtE - derived reference fragments encoding first 300 , 315 , 330 amino acids as well as full - length MgtE ( black arrow ) , were in vitro transcribed and translated . Mobility of the N - terminal cleavage product ( red arrow ) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies .

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

(C) To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE-derived reference fragments encoding first 300, 315, 330 amino acids as well as full-length MgtE (black arrow), were in vitro transcribed and translated. Mobility of the N-terminal cleavage product (red arrow) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( A ) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B . subtilis cells ( BS72 , Table EV2 ) depending on the concentrations of magnesium and manganese ions . Cells were cultivated in glucose M9 minimal medium with limiting ( 0 . 01 mM ) or high ( 1 mM ) concentration of MgSO4 , in the presence or absence of 100 μM MnCl2 , and analysed by western blotting with near - infrared detection ( upper panel ) . Black arrow denotes full - length MgtE , and red arrow denotes its N - terminal cleavage product formed by YqgP . The corresponding fluorescence signals were quantified by densitometry , and are displayed as relative specific activity , which is substrate conversion normalized to enzyme expression level ( lower panel ) .

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

(A) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B. subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalized to enzyme expression level (lower panel).


( B ) Growth curves of wild - type ( BTM843 , Table EV2 ) , yqgP deficient ( BTM844 , Table EV2 ) and rescue ( BTM845 , Table EV2 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese stress elicited by adding 75 µM MnSO4 in mid - exponential phase ( stress - phase denoted by blueish background ) . All strains further contain a deletion in the putative manganese efflux pump MntP ( ΔywlD , Table EV2 ) . Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain , and that reintroduction of YqgP rescues fitness during manganese stress to above - wild - type level . Top panel shows no difference between the strains in the absence of manganese stress . Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time - point , which illustrates the reproducibility of the assay .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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) Growth curves of wild-type (BTM843, Table EV2), yqgP deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 µM MnSO4 in mid-exponential phase (stress-phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain, and that reintroduction of YqgP rescues fitness during manganese stress to above-wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time-point, which illustrates the reproducibility of the assay.


( C ) Top panel : growth curves of wild - type ( BTM843 ) , yqgP deficient ( BTM844 ) and rescue ( BTM845 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese shock elicited by adding 75 µM MnSO4 in mid - exponential phase . Bottom panel : manganese toxicity is prevented by further adding 5 mM magnesium ( MgSO4 ) in otherwise identical conditions .

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(C) Top panel: growth curves of wild-type (BTM843), yqgP deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 µM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions.


( D ) Inhibition of YqgP by a rhomboid - specific peptidyl ketoamide inhibitor ( STS736 , i . e . compound 9 from Ticha et al . , 2017b ( ) ) abolishes the YqgP induced fitness of B . subtilis under manganese stress , in a dose - dependent manner , both with endogenous YqgP ( top panel ) and overexpressed YqgP ( bottom panel ) . Media and growth conditions were identical to those used in panel ( B ) .

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(D) Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al., 2017b()) abolishes the YqgP induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B).


( E ) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain ( BTM610 , Table EV2 ) in rich LB medium supplemented with 75 μM MnSO4 . Cell fitness is improved by overexpression of YqgP ( BTM611 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BTM612 , Table EV2 ) . For clarity , for panels C - E , representative experiments of 2 - 3 independent biological replicates are shown .

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(E) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels C - E, representative experiments of 2-3 independent biological replicates are shown.


( B ) Detection ( left panel ) and quantification ( right panel ) of steady - state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al . , 2007Ticha et al . , 2017aTicha et al . , 2017b ( , , ) ( schemes in the middle ) by ectopically expressed YqgP and its N - and C - terminally truncated variants ( strains BS184 - 187 , Table EV2 ) in living B . subtilis grown in rich LB medium . Black arrows indicate full - length substrates and red arrows indicate cleavage products .

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

(B) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al., 2007Ticha et al., 2017aTicha et al., 2017b(, , ) (schemes in the middle) by ectopically expressed YqgP and its N- and C-terminally truncated variants (strains BS184-187, Table EV2) in living B. subtilis grown in rich LB medium. Black arrows indicate full-length substrates and red arrows indicate cleavage products.


( C ) Similar analysis of the same strains but grown in minimal M9 medium ( with 10 µM MgSO4 ) supplemented with increasing concentrations of MnCl 2 . Black arrows denote full - length substrate ( MgtE ) , red arrows denote the cleavage products generated by YqgP . ( D ) Western blots of four independent experiments shown in panel ( C ) were quantified by near - infrared fluorescence detection , quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl 2 relative to 1 µM MnCl 2 activity . Average values and all fours datapoints are plotted for each indicated YqgP variant .

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(C) Similar analysis of the same strains but grown in minimal M9 medium (with 10 µM MgSO4) supplemented with increasing concentrations of MnCl2. Black arrows denote full-length substrate (MgtE), red arrows denote the cleavage products generated by YqgP. (D) Western blots of four independent experiments shown in panel (C) were quantified by near-infrared fluorescence detection, quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl2 relative to 1 µM MnCl2 activity. Average values and all fours datapoints are plotted for each indicated YqgP variant.


( A ) Detection ( left panel ) and quantification ( right panel ) of steady - state conversion of endogenous MgtE by YqgP ( strain BS72 , Table EV2 ) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 and CaCl2 ( reference conditions ) with the additions of 10 µM or 100 μM of a given divalent cation salt solution ( MnCl2 , ZnCl2 , CoCl2 , or NiCl2 ) , or 50 µM and 500 μM for CaCl2 . Western blots were quantified by near - infrared fluorescence detection ( left ) , quantified by densitometry and displayed as relative specific activity ( graph on the right ) , which is substrate conversion normalized to enzyme expression level . Black arrow denotes full - length substrate ( MgtE ) , and red arrow denotes the N - terminal cleavage product ( s ) generated by YqgP . All bands originate from the same western blot and identical treatment series .

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(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series.


( B ) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations .

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(B) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations.


( C ) Cation toxicity assays and their relationship to YqgP activity . Wild type B . subtilis ( BTM2 , Table EV2 ) , its variant lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) , and the rescue strain ectopically expressing YqgP ( BTM501 , Table EV2 ) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 and CaCl2 ( i . e . reference conditions ) , and in mid - exponential phase were stressed by the addition of either 70 µM MnCl2 , 500 µM ZnCl2 , 25 µM CoSO4 or 400 µM NiCl2 ( pale blue area ) . YqgP activity specifically improved cell fitness during manganese and zinc stress , while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations . Representative experiments of 2 - 3 independent replicates are shown .

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(C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.


( E Detection of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 . Black arrow marks full - length MgtE , and red arrow marks its N - terminal cleavage product by YqgP .

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(E Detection of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP.


F ) quantification of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 .

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F) quantification of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2.


( A ) The proteolytically dead mutant YqgP . S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH . Pattern of C - terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP ( strain BS72 , Table EV2 ) or its inactive S288A mutant ( BS73 , Table EV2 ) in rich LB medium in the presence or absence of endogenous FtsH . Black arrow denotes the full - length MgtE substrate , and red arrows denote its cleavage products generated by YqgP . While the presence of the faster migrating YqgP - dependent products ( full red arrows ) do not depend on the presence of FtsH , the slower migrating product formed in the presence of YqgP . S288A ( open red arrow ) does , indicating that FtsH protease is responsible for the alternative cleavage of MgtE . The same arrow symbolics are valid throughout this figure .

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(A) The proteolytically dead mutant YqgP.S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH. Pattern of C-terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP (strain BS72, Table EV2) or its inactive S288A mutant (BS73, Table EV2) in rich LB medium in the presence or absence of endogenous FtsH. Black arrow denotes the full-length MgtE substrate, and red arrows denote its cleavage products generated by YqgP. While the presence of the faster migrating YqgP-dependent products (full red arrows) do not depend on the presence of FtsH, the slower migrating product formed in the presence of YqgP.S288A (open red arrow) does, indicating that FtsH protease is responsible for the alternative cleavage of MgtE. The same arrow symbolics are valid throughout this figure.


( B ) Mapping of the boundary of the FtsH generated proteolytic product of MgtE . Immunoblot comparing the SDS PAGE mobility of the N - terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP ( catalytically dead YqgP . S288A , strains BTM78 , BTM502 , Table EV2 ) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1 - 340 , - 355 and - 370 shows that YqgP . S288A acts as an FtsH adaptor , and that FtsH processing of MgtE in the presence of YqgP . S288A stops at around amino acid 355 , which is near or at the cytoplasmic end of TMH 3 of MgtE .

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(B) Mapping of the boundary of the FtsH generated proteolytic product of MgtE. Immunoblot comparing the SDS PAGE mobility of the N-terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP (catalytically dead YqgP.S288A, strains BTM78, BTM502, Table EV2) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1-340, -355 and -370 shows that YqgP.S288A acts as an FtsH adaptor, and that FtsH processing of MgtE in the presence of YqgP.S288A stops at around amino acid 355, which is near or at the cytoplasmic end of TMH 3 of MgtE.


( D ) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full - length YqgP . S288A ( strain BS73 ) , YqgPΔNTD . S288A ( BS185 , Table EV2 ) or YqgPΔCTD . S288A ( BS186 , Table EV2 ) , in rich LB medium shows that YqgPNTD is necessary for the FtsH - dependent processing of MgtE .

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(D) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full-length YqgP.S288A (strain BS73), YqgPΔNTD.S288A (BS185, Table EV2) or YqgPΔCTD.S288A (BS186, Table EV2), in rich LB medium shows that YqgPNTD is necessary for the FtsH-dependent processing of MgtE.


( E ) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products . B . subtilis strains expressing YqgP or its S288A mutant ( strains BTM78 ; 501 and 502 , Table EV2 ) in the presence or absence of endogenous FtsH protease ( strains BTM795 , BTM796 and BTM797 , Table EV2 ) were cultured in LB medium and at early exponential phase , after having been expressing YqgP variants for 30 min , were either left grown untreated ( as a control with ongoing translation ) or treated with 20 μg / mL tetracycline to stop proteosynthesis ( translation shut - off ) . At given time intervals afterwards , all cultures were analysed by α - MgtE western blotting with near infrared detection . Equal cell number ( judged by OD600 ) was loaded into each lane . Black arrow marks full - length MgtE , red full arrows mark YqgP - dependent and red open arrows mark YqgP . S288A - dependent cleavage product of MgtE .

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(E) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products. B. subtilis strains expressing YqgP or its S288A mutant (strains BTM78; 501 and 502, Table EV2) in the presence or absence of endogenous FtsH protease (strains BTM795, BTM796 and BTM797, Table EV2) were cultured in LB medium and at early exponential phase, after having been expressing YqgP variants for 30 min, were either left grown untreated (as a control with ongoing translation) or treated with 20 μg/mL tetracycline to stop proteosynthesis (translation shut-off). At given time intervals afterwards, all cultures were analysed by α-MgtE western blotting with near infrared detection. Equal cell number (judged by OD600) was loaded into each lane. Black arrow marks full-length MgtE, red full arrows mark YqgP-dependent and red open arrows mark YqgP.S288A-dependent cleavage product of MgtE.


B . Representative flow cytometry dot plots of CD11b + and CD11b + / Cripto + cells at day 2 , 3 and 5 after injury . PE ( Phycoerythrin / free channel ) . Data are mean ± SEM ( n = 5 biological replicates ; P ≤ 0 . 00008 , Student ' s t - test ) .

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B. Representative flow cytometry dot plots of CD11b+ and CD11b+/Cripto+ cells at day 2, 3 and 5 after injury. PE (Phycoerythrin/free channel). Data are mean±SEM (n=5 biological replicates; P≤0.00008, Student's t-test).


C . Representative flow cytometry dot plots of CD11b + / F4 / 80 + / Ly6CHigh / Low and CD11b + / Cripto + / F4 / 80 + / Ly6CHigh / Low cells at day 2 , 3 and 5 after injury . D . Quantification of F4 / 80 + / Ly6CHigh / Low cells in the CD11b + and CD11b + / Cripto + cell population from injured muscles at the indicated time points . Data represent mean ± SEM ( n = 5 biological replicates ; * * * P < 0 . 001 , Student ' s t - test ) .

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C. Representative flow cytometry dot plots of CD11b+/F4/80+/Ly6CHigh/Low and CD11b+/Cripto+/F4/80+/Ly6CHigh/Low cells at day 2, 3 and 5 after injury. D. Quantification of F4/80+/Ly6CHigh/Low cells in the CD11b+ and CD11b+/Cripto+ cell population from injured muscles at the indicated time points. Data represent mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).


A . Representative pictures of immunostaining for F4 / 80 ( green ) in Control and CriptoMy - LOF TA sections at day 2 ( top panel ) and 5 ( bottom panel ) after injury . B . Quantification of F4 / 80 staining / damaged area ( μm2 ) at day 2 ( top graph ) and 5 ( bottom graph ) after injury . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 μm . Magnification of the boxes is 3 . 5 x . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) .

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A. Representative pictures of immunostaining for F4/80 (green) in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury. B. Quantification of F4/80 staining/damaged area (μm2) at day 2 (top graph) and 5 (bottom graph) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).


C . Representative pictures of immunostaining for CD206 in Control and CriptoMy - LOF TA sections at day 2 ( top panel ) and 5 ( bottom panel ) after injury , respectively . D - E . Quantification of CD206 + MPs per area ( mm2 ) at day 2 ( D ) and 5 ( E ) after injury . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 50 μm . Magnification of the boxes is 3 . 5 x . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) .

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C. Representative pictures of immunostaining for CD206 in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury, respectively. D-E. Quantification of CD206+ MPs per area (mm2) at day 2 (D) and 5 (E) after injury. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).


A . Representative flow cytometry dot plots showing the percentage of GFP + cell population in GFP - Control and GFP - CriptoMy - LOF muscles at day 2 ( top panel ) and 5 ( bottom panel ) after injury . Percentage of GFP + cells expressing F4 / 80 in GFP - Control and GFP - CriptoMy - LOF muscles is shown at day 2 after injury ( middle panel ) . Data are mean ± SEM ( n = 4 biological replicates ; P = ns ; Student ' s t - test ) .

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A. Representative flow cytometry dot plots showing the percentage of GFP+ cell population in GFP-Control and GFP-CriptoMy-LOF muscles at day 2 (top panel) and 5 (bottom panel) after injury. Percentage of GFP+ cells expressing F4/80 in GFP-Control and GFP-CriptoMy-LOF muscles is shown at day 2 after injury (middle panel). Data are mean±SEM (n=4 biological replicates; P=ns; Student's t-test).


B . qRT - PCR analysis of pro - ( Nos2 , Mcp1 and Tnfα ) and anti - ( Arg1 , Fizz1 , Il10 , Il4rα and Tgfβ ) inflammatory markers in GFP - CriptoMy - LOF and GFP - Control MPs at day 2 and 5 after injury . Data represent mean ± SEM of relative mRNA level normalized with Gapdh ( n ≥ 4 biological replicates ; * * * P < 0 . 001 , Student ' s t - test ) .

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B. qRT-PCR analysis of pro- (Nos2, Mcp1 and Tnfα) and anti- (Arg1, Fizz1, Il10, Il4rα and Tgfβ) inflammatory markers in GFP-CriptoMy-LOF and GFP-Control MPs at day 2 and 5 after injury. Data represent mean±SEM of relative mRNA level normalized with Gapdh (n≥4 biological replicates; ***P<0.001, Student's t-test).


C . Representative pictures of immunostaining for GFP ( green ) , CD206 ( red ) , pSMAD3 ( white ) in GFP - Control and GFP - CriptoMy - LOF TA sections at day 5 after injury . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x . D . Quantification of GFP + / CD206 ± / pSMAD3 ± cell distribution in TA sections from GFP - Control and GFP - CriptoMy - LOF at day 5 after injury . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Data are mean ± SEM ( n = 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) .

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C. Representative pictures of immunostaining for GFP (green), CD206 (red), pSMAD3 (white) in GFP-Control and GFP-CriptoMy-LOF TA sections at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x. D. Quantification of GFP+/CD206±/pSMAD3± cell distribution in TA sections from GFP-Control and GFP-CriptoMy-LOF at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean±SEM (n=5 biological replicates; **P<0.01, Student's t-test).


E . Representative flow cytometry dot plots of F4 / 80 + / Ly6CLow cell population gated on GFP + cells from GFP - Control and GFP - CriptoMy - LOF muscles at day 5 after injury . Data are mean ± SEM ( n = 3 biological replicates ; P = ns ; Student ' s t - test ) .

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E. Representative flow cytometry dot plots of F4/80+/Ly6CLow cell population gated on GFP+ cells from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data are mean±SEM (n=3 biological replicates; P=ns; Student's t-test).


( F ) Smear plot of RNA - seq data from GFP + / F4 / 80 + / Ly6CLow cells FACS sorted from GFP - Control and GFP - CriptoMy - LOF muscles at day 5 after injury . Data show the expression level as Log2 fold - change ( FC ) [ Log2 ( GFP - Control / GFP - CriptoMy - LOF ) ] , against Log2 ( expression ) [ Log2 ( average of gene expression across all samples ] , for each individual gene . The blue lines correspond to LogFC of 1 and - 1 . ( n = 3 biological replicates ) .

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(F) Smear plot of RNA-seq data from GFP+/F4/80+/Ly6CLow cells FACS sorted from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data show the expression level as Log2 fold-change (FC) [Log2(GFP-Control/GFP-CriptoMy-LOF)], against Log2(expression) [Log2(average of gene expression across all samples], for each individual gene. The blue lines correspond to LogFC of 1 and -1. (n=3 biological replicates).


B . Representative pictures of double immunostaining with Laminin ( green ) and embryonic myosin heavy chain ( eMHC ; red ) at day 5 ( left panel ) and 30 ( right panel ) after cardiotoxin ( CTX ) injection . Data information : Nuclei were counterstained with DAPI ( blue ) .

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B. Representative pictures of double immunostaining with Laminin (green) and embryonic myosin heavy chain (eMHC; red) at day 5 (left panel) and 30 (right panel) after cardiotoxin (CTX) injection. Data information: Nuclei were counterstained with DAPI (blue).


C . Minimal Feret ' s diameter distribution of Control and CriptoMy - LOF myofibers at day 5 ( top panel ) and 30 ( bottom panel ) after CTX injection . Data are mean ± SEM ( n = 5 biological replicates ; P = ns , Student ' s t - test ) .

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C. Minimal Feret's diameter distribution of Control and CriptoMy-LOF myofibers at day 5 (top panel) and 30 (bottom panel) after CTX injection. Data are mean±SEM (n=5 biological replicates; P=ns, Student's t-test).


E . Representative pictures of double immunostaining with Laminin ( green ) and eMHC ( red ) at day 5 ( left panel ) and 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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E. Representative pictures of double immunostaining with Laminin (green) and eMHC (red) at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue).


F . Minimal Feret ' s diameter distribution of Control and

F. Minimal Feret's diameter distribution of Control and Cripto


B . Representative pictures of immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections with GFP ( green ) and CD206 ( white ; left panel ) , pSMAD3 ( red ; middle panel ) and GFP and pSMAD3 ( right panel ) . C - E . Quantification of GFP ( C ) , GFP / CD206 ( D ) and GFP / pSMAD3 ( E ) positive cells per area ( mm2 ) in mdx - Control and mdx - CriptoMy - LOF diaphragms . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n ≥ 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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B. Representative pictures of immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections with GFP (green) and CD206 (white; left panel), pSMAD3 (red; middle panel) and GFP and pSMAD3 (right panel). C-E. Quantification of GFP (C), GFP/CD206 (D) and GFP/pSMAD3 (E) positive cells per area (mm2) in mdx-Control and mdx-CriptoMy-LOF diaphragms. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


F - G . Representative pictures of Picrosirius red staining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections ( F ) and quantification of picrosirius red staining ( G ) . Data are expressed as percentage of stained area and are box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * P < 0 . 05 , Student ' s t - test ) .

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F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test).


H . Hydroxyproline ( HOP ) concentration in mdx - Control and mdx - CriptoMy - LOF diaphragm muscle sections . Data are expressed as HOP levels ( μg ) per muscle section volume ( mm3 ) and as box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * * P < 0 . 01 , Student ' s t - test ) .

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H. Hydroxyproline (HOP) concentration in mdx-Control and mdx-CriptoMy-LOF diaphragm muscle sections. Data are expressed as HOP levels (μg) per muscle section volume (mm3) and as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01, Student's t-test).


Representative images of Laminin ( red ) immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections of centrally nucleated myofibers . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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Representative images of Laminin (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections of centrally nucleated myofibers. Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


Representative mdx - Control and mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution of centrally nucleated myofibers . Data are expressed as mean ± SEM ( n = 5 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 ; Student ' s t - test )

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Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution of centrally nucleated myofibers. Data are expressed as mean±SEM (n=5 biological replicates; *P<0.05; ***P<0.001; Student's t-test)


Representative mdx - Control and mdx - CriptoMy - LOF diaphragm sections minimal Feret ' s diameter distribution Variance coefficient of centrally nucleated myofibers . box plots displaying minimum , first quartile , median , third quartile and

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Representative mdx-Control and mdx-CriptoMy-LOF diaphragm sections minimal Feret's diameter distribution Variance coefficient of centrally nucleated myofibers. box plots displaying minimum, first quartile, median, third quartile and maximum


L - M . Representative images of eMHC ( red ) immunostaining of mdx - Control and mdx - CriptoMy - LOF diaphragm sections ( L ) and quantification of eMHC + myofibers per area ( mm2 , M ) . Data are expressed as box plots displaying minimum , first quartile , median , third quartile and maximum ( n = 5 biological replicates ; * P < 0 . 05 ; Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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L-M. Representative images of eMHC (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (L) and quantification of eMHC+ myofibers per area (mm2, M). Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05; Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


A . Representative pictures of CD31 ( red ) immunostaining of Control and CriptoMy - LOF TA sections at day 5 ( left panel ) and 30 ( right panel ) after single injury ( CTX - I ) . B - D . Quantification of CD31 + capillary number per area ( mm2 ; B ) , average of capillary cross - sectional area ( µm2 ; C ) and percentage of small ( < 20 µm2 ) and large ( > 100 µm2 ) capillaries ( D ) in Control and CriptoMy - LOF injured muscles at indicated time points after single injury ( CTX - I ) . Data are mean ± SEM ( n = 5 biological replicates ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , Student ' s t - test ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I). B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


E . Representative pictures of CD31 ( red ) immunostaining of Control and CriptoMy - LOF TA sections at day 5 ( left panel ) and 30 ( right panel ) after re - injury ( CTX - II ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


F . Representative pictures of mdx - Control and mdx - CriptoMy - LOF diaphragm sections immunostained with VEcad ( red ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Scale bar : 100 μm . Magnification of the boxes is 3 . 5 x .

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F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.


G - H . Quantification of CD31 + capillary number per area ( mm2 ; G ) and average of capillary cross - sectional area ( µm2 ; H ) in Control and CriptoMy - LOF injured muscles at the indicated time points after re - injury ( CTX - II ) Data are mean ± SEM ( n = 6 biological replicates ; * P < 0 . 05 ; * * * P < 0 . 001 , Student ' s t - test ) .

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G-H. Quantification of CD31+ capillary number per area (mm2; G) and average of capillary cross-sectional area (µm2; H) in Control and CriptoMy-LOF injured muscles at the indicated time points after re-injury (CTX-II) Data are mean±SEM (n=6 biological replicates; *P<0.05; ***P<0.001, Student's t-test).


I - J . Quantification of capillary number per area ( mm2 ; I ) and average of capillary cross - sectional area ( µm2 ; J ) in mdx - Control and mdx - CriptoMy - LOF diaphragm muscles . Data are mean ± SEM ( n = 5 biological replicates ; * * * P < 0 . 001 , Student ' s t - test ) .

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I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).


A . Representative pictures of double immunostaining with VEcad ( red ; left and middle panel ) and KLF4 ( green , left panel ) or TCF4 ( green , middle panel ) , and with CD31 ( red ; right panels , confocal pictures ) and TCF4 ( white , right panel ) on Control and CriptoMy - LOF TA sections at day 5 after injury . B - D . Quantification of VEcad / KLF4 ( B ) , VEcad / TCF4 ( C ) and CD31 / TCF4 ( D ) double positive cells per area ( mm2 ) in Control and CriptoMy - LOF TA sections at day 5 after injury . Data information : Nuclei were counterstained with DAPI ( blue ) .

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A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury. B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury. Data information: Nuclei were counterstained with DAPI (blue).


E . Representative Confocal pictures of CD31 + ( red ) and PDGFRα + ( green ) immunostaining on Control and CriptoMy - LOF TA sections at day 5 after injury . F - G . Quantification of CD31 + / PDGFRα + ( F ) and CD31 - / PDGFRα + ( G ) cells per area ( mm2 ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury. F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). Data information: Nuclei were counterstained with DAPI (blue).


H - I . Representative pictures of triple immunostaining with VEcad ( red ) , KLF4 ( green ) and pSMAD3 ( white ) on Control and CriptoMy - LOF TA sections at day 5 after injury ( H ) and quantification of VEcad / KLF4 / pSMAD3 triple positive cells per area ( mm2 ; I ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I). Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) and KLF4 ( green ) on Control and CriptoMy - LOF TA sections at day 5 after re - injury ( CTX - II Data information : Nuclei were counterstained with DAPI ( blue ) .

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Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) and KLF4 ( green ) on diaphragm sections of mdx - Control and mdx - CriptoMy - LOF Data information : Nuclei were counterstained with DAPI ( blue ) .

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Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF Data information: Nuclei were counterstained with DAPI (blue).


Quantification of VEcad / KLF4 double positive cells per area ( mm2 ) in Control and CriptoMy - LOF TA sections at day 5 after re - injury ( CTX -

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Quantification of VEcad/KLF4 double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II


Quantification of VEcad / KLF4 double positive cells per area ( mm2 ) in diaphragm sections of mdx - Control and mdx - CriptoMy -

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Quantification of VEcad/KLF4 double positive cells per area (mm2) in diaphragm sections of mdx-Control and mdx-CriptoMy-LOF


A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes ( CellROX green , Molecular Probes , USA ) in HC ( n = 4 ) and DM ( n = 4 ) platelets . ( * * p = 0 . 032 vs . HC ) .

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A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).


B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC ( # 1 - 3 ) and DM ( # 1 - 8 ) patient platelets . Quantification analysis on HC ( n = 5 ) and DM ( n = 18 ) individuals . ( * * p = 0 . 0025 vs . HC ) . GAPDH served as the loading control .

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B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.


C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets . Graph indicates HC and DM platelets positive for phosphorylated p53 signal . The y - axis indicates percentage of phosphorylated p53 positive cell from total cells ( * * p = 0 . 0064 vs . HC ) . Representative figure from n = 5 .

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C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5.


D Mitochondrial membrane potential ( ΔΨm ) was measured by MitoTracker staining ( 50nM for 15 min ) in HC and DM patient platelets . Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets . The y - axis indicates fold of signal intensity in HC and DM platelet ( * * p = 1 . 1608E - 05 vs HC ) . Signal intensity of each group was converted to fold change compared with HC values . Representative figure from HC ( n = 6 ) and DM ( n = 4 ) .

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D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4).


E ΔΨm and platelet apoptosis were measured by flow cytometry analysis . ΔΨm was detected using TMRM and apoptosis was assessed with Annexin - V ( PS externalization ) . Representative figure from n = 3 .

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E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.


F Western blot analysis of apoptosis - related pp53 , p53 , cytochrome c , and active caspase 3 in HC and DM platelets . Tubulin was used as the loading control . Representative figure from n = 3 .

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F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3.


G Western blot analysis showing release of cytochrome c ( Cyto c ) into the cytosol in DM . Representative figure from n = 3 .

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G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3.


H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets ( DM ) compared to a typical healthy mitochondria ( HC ) . Arrows indicate disrupted outer membranes . I Quantification of mitochondria in HC ( n = 33 ) and DM ( n = 17 ) patient platelets . The y - axis indicates average number of mitochondria in HC and DM single platelet EM view ( * * p = 5 . 50811E - 05 vs . HC ) . J Quantification of healthy and damaged mitochondria in HC and DM platelets .

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H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.