Representative of mitophagy showing co - localization between mitophagy dye ( red ) lysosome dye ( green ) in DC control fibroblasts treated with vehicle or 3 mM NR . arrows : mitophagy . B mean value of / in each image was scored . At least 15 ( ~ 200 ) were counted per group . relative values in each group was normalized to Con1 .

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Representative images of mitophagy showing co-localization between the mitophagy dye (red) and the lysosome dye (green) in DC and control fibroblasts treated with vehicle or 3 mM NR. White arrows: mitophagy. B The mean value of fluorescence intensity/cells in each image was scored. At least 15 images (~ 200 cells) were counted per group. The relative values in each group was normalized to Con1.


C Representative immunoblots of expression of PINK1 PARKIN in DC treated with vehicle or 3 mM NR . of immunoblots from indicated is from three replicates .

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C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR. Quantification of immunoblots from the indicated proteins is from three replicates.


A PCR amplification efficiency at telomere in mock - FPG - treated DC - matched healthy control fibroblasts supplemented with vehicle or 3 mM NR . values are presented as mean ± SD of three replicates . relative telomere PCR amplification in each sample was normalized to 36B4 reference gene .

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A PCR amplification efficiency at the telomere in the mock- and FPG-treated DC and age-matched healthy control fibroblasts supplemented with vehicle or 3 mM NR. All values are presented as mean ± SD of three replicates. The relative telomere PCR amplification in each sample was normalized to the 36B4 reference gene.


B Telomere restriction fragment in DC control fibroblasts treated with vehicle or 3 mM NR . Genomic was isolated from indicated lines at 0 d or 14 d , with or without 3 mM NR treatment . At left , molecular mass markers , in kilobase pairs ( Kb ) .

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B Telomere restriction fragment analysis in DC and control fibroblasts treated with vehicle or 3 mM NR. Genomic DNA was isolated from the indicated cell lines at 0 d or 14 d, with or without 3 mM NR treatment. At left, DNA molecular mass markers, in kilobase pairs (Kb).


C , D Representative of γH2AX ( green ) , telomere immuno - FISH ( red ) in DC treated with vehicle or 3 mM NR . arrows : co - localization of γH2AX foci telomeric ( or TIF ) . Enlarged views of co - localizing foci are shown at right panel in Figure EV1 . of DC control with indicated TIFs per nuclei . ~ 100 per condition were scored .

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C, D Representative images of γH2AX (green), telomere immuno-FISH (red) in DC cells treated with vehicle or 3 mM NR. White arrows: co-localization of γH2AX foci and telomeric DNA (or TIF). Enlarged views of co-localizing foci are shown at the right panel and in Figure EV1. Percentage of DC and control cells with indicated TIFs per nuclei. ~ 100 cells per condition were scored.


A Cumulative population doubling of proliferation of representative scramble CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR . Each data point is represented as mean ± SD of three replicates .

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A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR. Each data point is represented as mean ± SD of three replicates.


B , C Representative of BrdU ( red ) DAPI ( blue ) of DC - matched healthy control fibroblasts treated with vehicle or NR . of BrdU - positive per condition . Each dot represents of with BrdU per image . ~ 400 were counted per condition . values are presented as mean ± SD .

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B, C Representative images of BrdU (red) and DAPI (blue) staining of DC and age-matched healthy control fibroblasts treated with vehicle or NR. Percentage of BrdU -positive cells per condition. Each dot represents the percentage of cells with BrdU staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.


D , E Representative of SPiDER - β - gal ( green ) DAPI ( blue ) of DC - matched healthy control treated with vehicle or 3 mM NR . of SA - β - gal - positive per condition . Each dot represents of with SA - β - gal per image . ~ 400 were counted per condition . values are presented as mean ± SD .

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D, E Representative images of SPiDER-β-gal (green) and DAPI (blue) staining of DC and age-matched healthy control cells treated with vehicle or 3 mM NR. Percentage of SA-β-gal-positive cells per condition. Each dot represents the percentage of cells with SA-β-gal staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.


F of IL - 6 , IL - 8 , MCP - 1 in both supernatant lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR . values are represented as mean ± SD of three replicates .

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F The levels of IL-6, IL-8, and MCP-1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR. All values are represented as mean ± SD of three replicates.


G , H Representative immunoblots of p21 p16 in DC fibroblasts treated with vehicle or 3 mM NR . of indicated are presented as mean ± SD from three immunoblots .

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G, H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR. Quantification of the indicated proteins are presented as mean ± SD from three immunoblots.


Immunofluorescence of LN229 , U87and SNB19 with antibodies against αv , β3 or β5 integrin subunits . Scale bars , 20μm for LN229 SNB19 , 50μm for U87 .

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Immunofluorescence staining of LN229, U87and SNB19 tumor cells with primary antibodies against αv, β3 or β5 integrin subunits. Scale bars, 20μm for LN229 and SNB19, and 50μm for U87.


Targeted gene delivery into LN229 , U87 SNB19 by RGD4C / AAVP - Luc carrying Luc reporter gene . were seeded in 48 well plates before transduction . Non - targeted / AAVP - Luc was used as negative control for targeted transduction . Results represent average Relative Luminescence Units ( RLU ) / 1μg . Data shown are representative of two experiments , n = 3 .

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Targeted gene delivery into LN229, U87 and SNB19 cells by RGD4C/AAVP-Luc carrying the Luc reporter gene. Cells were seeded in 48 well plates before transduction. Non-targeted/AAVP-Luc was used as negative control for targeted transduction. Results represent the average Relative Luminescence Units (RLU)/1μg protein. Data shown are representative of two experiments, n=3.


Western blot of endogenous Grp78 expression in LN229 , U87 SNB19 following treatment with increasing concentrations of TMZ . Grp78 level was normalized to GAPDH . Data shown in right panel are representative of two experiments , n = 3 .

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Western blot analysis of the endogenous Grp78 protein expression levels in LN229, U87 and SNB19 cells following treatment with increasing concentrations of TMZ. Grp78 level was normalized to GAPDH. Data shown in right panel are representative of two experiments, n=3.


Induction of Grp78 promoter activity by TMZ in RGD4C / AAVP - Grp78 - Luc . LN229 , U87 SNB19 stably transduced with RGD4C / AAVP - Grp78 - Luc or RGD4C / AAVP - CMV - Luc were grown in presence of TMZ for indicated times . Results represent average RLU / 1μg . Data shown are representative of two experiments , n = 3 . Two - way ANOVA with Bonferroni correction ( GraphPad Prism 6 ) was used for data .

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Induction of Grp78 promoter activity by TMZ in RGD4C/AAVP-Grp78-Luc cells. LN229, U87 and SNB19 cells stably transduced with RGD4C/AAVP-Grp78-Luc or RGD4C/AAVP-CMV-Luc were grown in the presence of TMZ for the indicated times. Results represent the average RLU/1μg protein. Data shown are representative of two experiments, n=3. Two-way ANOVA with Bonferroni correction (GraphPad Prism 6) was used for data analysis.


TMZ induction of phospho - eIF2α ATF6 - p90 expression . Human glioblastoma transduced with RGD4C / AAVP - Grp78 were analyzed by Western blot following treatment with TMZ ( 100 μM for LN229 SNB19 , 60 μM for U87 ) . GAPDH was used as a control .

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TMZ induction of phospho-eIF2α and ATF6-p90 expression. Human glioblastoma cells transduced with RGD4C/AAVP-Grp78 were analyzed by Western blot following treatment with TMZ (100 μM for LN229 and SNB19, 60 μM for U87). GAPDH was used as a control.


RT - PCR of constitutive expression splicing of XBP1 in glioblastoma transduced with RGD4C / AAVP - Grp78 , in presence of TMZ . Sizes of PCR products were 289 bp for unspliced XBP1 263 bp for spliced XBP1 , lower band is not specific . Time points were selected among those tested in Western blot in ( A ) , subsequently image containing 0 , 1 2 hours was juxtaposed to of 6 , 12 24 hours . A has been added between gel pieces that have been juxtaposed .

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RT-PCR analysis of constitutive expression and splicing of XBP1 in glioblastoma cells transduced with RGD4C/AAVP-Grp78, in the presence of TMZ. Sizes of the PCR products were 289 bp for unspliced XBP1 and 263 bp for spliced XBP1, the lower size band is not specific. Time points were selected among those tested in the Western blot in (A), subsequently the image containing 0, 1 and 2 hours was juxtaposed to images of 6, 12 and 24 hours. A white line has been added between the gel pieces that have been juxtaposed.


killing in vitro by HSVtk / GCV approach . Glioblastoma stably transduced with RGD4C / AAVP - Grp78 - HSVtk or RGD4C / AAVP - CMV - HSVtk were treated with either GCV ( 10 μM ) or TMZ ( 100 μM for LN229 SNB19 , 60 μM for U87 ) or combination of both GCV TMZ . were stained with MitoSOX analyzed by FACS at day 4 post - treatment . Data shown are representative of three experiments , n = 3 . Two - way ANOVA with Tukey ' s multiple comparison ( GraphPad Prism 6 ) was used for data .

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Tumor cell killing in vitro by the HSVtk/GCV approach. Glioblastoma cells stably transduced with RGD4C/AAVP-Grp78-HSVtk or RGD4C/AAVP-CMV-HSVtk were treated with either GCV (10 μM) or TMZ (100 μM for LN229 and SNB19, 60 μM for U87) or combination of both GCV and TMZ. Cells were stained with MitoSOX and analyzed by FACS at day 4 post-treatment. Data shown are representative of three experiments, n=3. Two-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.


Immuofluorescent for αv integrin expression in brain sections including intracranial ( T ) surrounding healthy brain ( B ) . Scale bar , 100μm .

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Immuofluorescent staining for αv integrin expression in brain sections including intracranial tumor (T) and the surrounding healthy brain (B). Scale bar, 100μm.


Co - of CD31 ( red ) phage ( green ) in brain sections comprising healthy brain . Scale bar , 100μm . A high - magnification view from low - magnification insert of section is shown . Scale bar , 25 μm .

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Co-staining of CD31 (red) and phage (green) in brain sections comprising tumor and healthy brain. Scale bar, 100μm. A high-magnification view from the low-magnification insert of the tumor section is shown. Scale bar, 25 μm.


of relative homing ability of RGD4C / AAVP - Grp78 - HSVtk to brain tumors , brain tissue pancreas after intravenous administration . data were normalized both to non - targeted vector to tissue then expressed as relative TU . Data shown are representative of two experiments , n = 5 . One - way ANOVA with Tukey ' s multiple comparison ( GraphPad Prism 6 ) was used for data .

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Quantification of the relative homing ability of RGD4C/AAVP-Grp78-HSVtk to brain tumors, brain tissue and pancreas after intravenous administration. The data were normalized both to non-targeted vector and to tissue weight then expressed as relative TU. Data shown are representative of two experiments, n=5. One-way ANOVA with Tukey's multiple comparison test (GraphPad Prism 6) was used for data analysis.


In vivo BLI of Luc expression for evaluation of viability of representative - bearing mice from experimental groups before initiation of therapy ( day 9 post intracranial implantation ) at end of treatments ( day 27 post intracranial implantation ) .

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In vivo BLI of Luc expression for evaluation of tumor viability and tumor size of representative tumor-bearing mice from all experimental groups before initiation of therapy (day 9 post intracranial cell implantation) and at the end of treatments (day 27 post intracranial cell implantation).


Luc intensities before ( filled circles ) after therapy ( open circles ) , on days 9 27 post implantation , respectively . Data shown are representative of two experiments , n = 5 .

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Individual tumor Luc intensities before (filled circles) and after therapy (open circles), on days 9 and 27 post cell implantation, respectively. Data shown are representative of two experiments, n=5.


Average Luc activity showing glioblastoma progression in each experimental group . Pre - treatment day 9 was set at 100 % . Data shown are representative of two experiments , n = 5 .

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Average tumor Luc activity showing glioblastoma progression in each experimental group. Pre-treatment day 9 was set at 100%. Data shown are representative of two experiments, n=5.


Survival benefit for - bearing mice from treatment groups . Data shown are representative of two experiments , n = 5 . Statistical significance was determined by Kaplan - Meier method ( Kaplan - Meier survival fractions ) . Log - rank ( Mantel - Cox ) was used .

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Survival benefit for tumor-bearing mice from all treatment groups. Data shown are representative of two experiments, n=5. Statistical significance was determined by Kaplan-Meier method (Kaplan-Meier survival fractions). Log-rank (Mantel-Cox) test was used.


Hematoxylin eosin ( H & E ) immunostaining for proliferation marker Ki67 of representative sections from experimental groups , Scale bars , 200 μm for H & E 100 μm for Ki67 . T , ; B , brain . low - magnification inserts represent negative controls with secondary antibody alone on serial sections used for both Ki67 caspase - 3 immunostainings .

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Hematoxylin and eosin staining (H&E) and immunostaining for the proliferation marker protein Ki67 of representative tumor sections from all experimental groups, Scale bars, 200 μm for H&E and 100 μm for Ki67. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings.


Blood vessel with CD31 ( red ) . DAPI of sections is shown in blue . Scale bar , 200 μm . T , ; B , brain . low - magnification inserts represent negative controls with secondary antibody alone on serial sections used for both Ki67 caspase - 3 immunostainings .

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Blood vessel staining with CD31 (red). DAPI staining of the sections is shown in blue. Scale bar, 200 μm. T, tumor; B, brain. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings.


immunostaining for apoptosis using an anti - caspase - 3 antibody . Scale bar , 100 μm .

Tumor immunostaining for apoptosis using an anti- caspase-3 antibody. Scale bar, 100 μm.


- activated sorting ( FACS ) with antibodies against Sox - 2 , Nestin CD133 markers .

Fluorescence-activated cell sorting (FACS) analysis with antibodies against Sox-2, Nestin and CD133 stem cell markers.


Immunofluorescent for αv , β3 or β5 integrins in HSJD - GBM - 001 G26 GBM .

Immunofluorescent staining for αv, β3 or β5 integrins in primary HSJD-GBM-001 and G26 GBM cells.


Targeted transduction of HSJD - GBM - 001 G26 , over a time course , by RGD4C / AAVP - Luc . Non - targeted / AAVP - Luc was used as negative control . Results represent RLU / 1μg of triplicate wells . Data shown are representative of two experiments , n = 3 . Scale bar , 100 μm .

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Targeted transduction of HSJD-GBM-001 and G26 cells, over a time course, by RGD4C/AAVP-Luc. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein of triplicate wells. Data shown are representative of two experiments, n=3. Scale bar, 100 μm.


Immunofluorescent for αv , β3 or β5 integrins in human normal astrocytes , chondrocytes , skin lung fibroblasts .

Immunofluorescent staining for αv, β3 or β5 integrins in human normal astrocytes, chondrocytes, skin and lung fibroblasts.


Treatment of chondrocytes , astrocytes , lung skin fibroblasts with RGD4C / AAVP - Luc vector , alone or in presence of TMZ . Non - targeted / AAVP - Luc was used as negative control . Results represent RLU / 1μg . Data shown are representative of two experiments , n = 3 . Scale bars , 160 μm for chondrocytes 80 μm for other .

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Treatment of chondrocytes, astrocytes, lung and skin fibroblasts with RGD4C/AAVP-Luc vector, alone or in the presence of TMZ. Non-targeted/AAVP-Luc was used as negative control. Results represent RLU/1μg protein. Data shown are representative of two experiments, n=3. Scale bars, 160 μm for chondrocytes and 80 μm for all other cells.


BLI of luciferase of representative - bearing mice from experimental groups at day 19 post therapy initiation .

BLI of luciferase of representative tumor-bearing mice from all experimental groups at day 19 post therapy initiation.


Survival benefit for - bearing mice from treatment groups . Data shown are representative of two experiments , n = 6 . Statistical significance was determined by Kaplan - Meier method ( Kaplan - Meier survival fractions ) . Log - rank ( Mantel - Cox ) was used .

Survival benefit for tumor-bearing mice from all treatment groups. Data shown are representative of two experiments, n=6. Statistical significance was determined by Kaplan-Meier method (Kaplan-Meier survival fractions). Log-rank (Mantel-Cox) test was used.


H & E , Ki67 immunostaining , evaluation of apoptosis in tumors by using an anti - caspase - 3 antibody of representative sections from experimental groups . low - magnification inserts represent negative controls with secondary antibody alone on serial sections used for both Ki67 caspase - 3 immunostainings . Arrow points to apoptotic .

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H&E staining, Ki67 immunostaining, and evaluation of apoptosis in tumors by using an anti- caspase-3 antibody staining of representative tumor sections from all experimental groups. The low-magnification inserts represent negative controls with the secondary antibody alone on serial sections used for both Ki67 and caspase-3 immunostainings. Arrow points to apoptotic cells.


Immunostaining of tumors with an antibody against CD133 . Scale bars , 100 μm .

Immunostaining of tumors with an antibody against CD133. Scale bars, 100 μm.


Immunostaining for αv , β3 or β5 integrins in GL261 . Scale bar , 50 μm .

Immunostaining analysis for αv, β3 or β5 integrins in GL261 cells. Scale bar, 50 μm.


BLI of Luc of representative - bearing mice from experimental groups at day 34 post treatment .

BLI of Luc of representative tumor-bearing mice from all experimental groups at day 34 post treatment.


Total Luc intensities in each experimental group before treatment , on day 10 post implantation , after therapy , on day 34 post implantation . Data shown are representative of two experiments , n = 6 . Data was done by two - way ANOVA Bonferroni ' s multiple comparison .

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Total tumor Luc intensities in each experimental group before treatment, on day 10 post cell implantation, and after therapy, on day 34 post cell implantation. Data shown are representative of two experiments, n=6. Data analysis was done by two-way ANOVA and Bonferroni's multiple comparison test.


Weights of C57BL / 6J mice bearing intracranial GL261 tumors , from experimental groups , before after treatment . Data shown are representative of two experiments , n = 6 .

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Weights of C57BL/6J mice bearing intracranial GL261 tumors, from all experimental groups, before and after treatment. Data shown are representative of two experiments, n=6.


A . LRP6 level is reduced during serum starvation . HEK293 were incubated in serum - free medium for indicated hours . were lysed lysates were analyzed by immunoblotting .

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A. LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum-free medium for the indicated hours. Cells were lysed and the cell lysates were analyzed by immunoblotting.


B . Serum starvation reduced amount of LRP6 . HEK293 were incubated in serum - free medium for 3 h . Then were additionally incubated for 1 h after addition of 10 % serum . were lysed lysates were analyzed by immunoblotting ( left panel ) . of LRP6 / β - actin p - YAP / YAP of three independent immunoblots was quantified ( biological replicates , middle right panel , respectively ) . N . S . : non - significant , * * P < 0 . 01 * * * P < 0 . 001 . Error bars indicate standard deviation of biological triplicate measurements . Student ' s t - was used for statistical .

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B. Serum starvation reduced the amount of LRP6. HEK293 cells were incubated in serum-free medium for 3 h. Then cells were additionally incubated for 1 h after addition of 10% serum. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (biological replicates, middle and right panel, respectively). N.S.: non-significant, **P < 0.01 and ***P < 0.001. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.


C . Loss of LRP6 in serum starvation was seen in different lines . lines were incubated in serum - free medium for 2 h . were lysed , lysates were analyzed by immunoblotting . S . E . , short exposure ; L . E . , long exposure .

C. Loss of LRP6 in serum starvation was seen in different cell lines. Cell lines were incubated in serum-free medium for 2 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting. S.E., short exposure; L.E., long exposure.


D . Glucose starvation decreased LRP6 . HEK293 were incubated in glucose - free medium for 4 h . were lysed lysates were analyzed by immunoblotting .

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D. Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


E . decrease of LRP6 by serum starvation was mediated by lysosomal degradation pathway . HEK293 were incubated in serum - free medium for 4 h with DMSO or Bafilomycin A1 ( 100 nM ) . were lysed lysates were analyzed by immunoblotting .

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E. The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum-free medium for 4 h with DMSO or Bafilomycin A1 (100 nM). Cells were lysed and the cell lysates were analyzed by immunoblotting.


Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 were transfected with clathrin siRNA incubated in serum - free medium for 4 h . were lysed lysates were analyzed by immunoblotting .

Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 cells were transfected with clathrin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


G . Loss of LRP6 by serum starvation was mediated in a caveolin - 1 - dependent manner . HEK293 were transfected with caveolin siRNA incubated in serum - free medium for 4 h . were lysed lysates were analyzed by immunoblotting .

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G. Loss of LRP6 by serum starvation was mediated in a caveolin-1-dependent manner. HEK293 cells were transfected with caveolin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


H . Lipid components of serum were essential for maintaining LRP6 . HEK293 were incubated in serum - free medium with A2058 BSA ( Sigma ) or Fraction V BSA for 4 h . were lysed lysates were analyzed by immunoblotting ( left panel ) . of LRP6 / β - actin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . N . S . : non - significant * P < 0 . 05 . Student ' s t - was used for statistical .

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H. Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum-free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non-significant and *P < 0.05. Student's t-test was used for statistical analysis.


A . of LRP6 phosphorylated YAP showed inverse correlation under serum starvation . HEK293 were incubated in serum - free medium for indicated times . were lysed lysates were analyzed by immunoblotting .

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A. The levels of LRP6 and phosphorylated YAP showed inverse correlation under serum starvation. HEK293 cells were incubated in serum-free medium for the indicated times. Cells were lysed and the cell lysates were analyzed by immunoblotting.


B . Knockdown of LRP6 induced phosphorylation of YAP even under nutrient - rich condition . HEK293T were transfected with GFP siRNA as a control LRP6 siRNA under nutrient - rich conditions . of p - YAP / total - YAP of three independent immunoblot bands was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 . Student ' s t - was used for statistical .

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B. Knockdown of LRP6 induced the phosphorylation of YAP even under nutrient-rich condition. HEK293T cells were transfected with GFP siRNA as a control and LRP6 siRNA under nutrient-rich conditions. The ratio of p-YAP/total-YAP of three independent immunoblot bands was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.


C . Overexpression of LRP6 blocked increase of YAP phosphorylation under serum starvation conditions . HEK293T were transfected with empty vector ( - ) or LRP6 - EGFP as indicated in figure . One day after transfection , were incubated in serum - free medium for 4 h . were lysed lysates were analyzed by immunoblotting ( left panel ) . of LRP6 / β - actin p - YAP / YAP of three independent immunoblots was quantified ( middle right panel , respectively ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 * * * P < 0 . 001 . Student ' s t - was used for statistical .

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C. Overexpression of LRP6 blocked the increase of YAP phosphorylation under serum starvation conditions. HEK293T cells were transfected with empty vector (-) or LRP6-EGFP as indicated in the figure. One day after transfection, cells were incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (middle and right panel, respectively). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05 and ***P < 0.001. Student's t-test was used for statistical analysis.


D . Knockdown of LRP6 reduced YAP reporter activity . HEK293T were transfected with GFP siRNA as a control , LRP6 siRNA , pRL - TK 8XGTIIC luciferase reporter constructs . One day after transfection , luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * * P < 0 . 005 . Student ' s t - was used for statistical .

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D. Knockdown of LRP6 reduced YAP reporter activity. HEK293T cells were transfected with GFP siRNA as a control, LRP6 siRNA, pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.005. Student's t-test was used for statistical analysis.


E . Knockdown of LRP6 reduced expression of YAP target genes . Quantitative real - time PCR measuring expression of CTGF , ANKRD1 , INHBA , LRP6 mRNA in GFP LRP6 siRNA - transfected HEK293T was performed . of CTGF , ANKRD1 , INHBA , LRP6 mRNA was normalized against level of β - actin . Error bars indicate standard deviation of technical triplicate measurements . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Student ' s t - was used for statistical .

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E. Knockdown of LRP6 reduced the expression of YAP target genes. Quantitative real-time PCR assay measuring the expression of CTGF, ANKRD1, INHBA, and LRP6 mRNA in GFP and LRP6 siRNA-transfected HEK293T cells was performed. Quantification of CTGF, ANKRD1, INHBA, and LRP6 mRNA was normalized against the level of β-actin. Error bars indicate standard deviation of technical triplicate measurements. *P < 0.05, **P < 0.01 and ***P < 0.001. Student's t-test was used for statistical analysis.


F . Knockdown of LRP6 reduced interaction between YAP TEAD . HEK293T were transfected with EGFP - YAP , Myc - TEAD4 siRNA for LRP6 . were lysed lysates were immunoprecipitated with anti - Myc antibody analyzed by immunoblotting . WCL , whole - lysates .

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F. Knockdown of LRP6 reduced the interaction between YAP and TEAD. HEK293T cells were transfected with EGFP-YAP, Myc-TEAD4 and siRNA for LRP6. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


G . Overexpression of LRP6 rescued reduced YAP reporter activity under serum starvation conditions . HEK293T were transfected with an empty vector ( - ) or VSVG - LRP6 , with pRL - TK 8XGTIIC luciferase reporter constructs . One day after transfection , were incubated in serum - free medium for 4 h luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * * P < 0 . 001 . Student ' s t - was used for statistical .

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G. Overexpression of LRP6 rescued the reduced YAP reporter activity under serum starvation conditions. HEK293T cells were transfected with an empty vector (-) or VSVG-LRP6, and with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were incubated in serum-free medium for 4 h and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.001. Student's t-test was used for statistical analysis.


H . Knockdown of LRP6 induced localization of YAP . HEK293A were transfected with siRNA for GFP or LRP6 immunofluorescence was performed . Figure shows representative image of multiple areas . DAPI was used for nucleus . Scale bars : 10 μm . of YAP is shown in right panel . was performed by counting that have - localized YAP per image ( n = 8 ) . Error bars indicate standard deviation . * * * P < 0 . 001 . Student ' s t - was used for statistical .

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H. Knockdown of LRP6 induced cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP or LRP6 and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear-localized YAP per image (n=8). Error bars indicate standard deviation. ***P < 0.001. Student's t-test was used for statistical analysis.


I . Overexpression of LRP6 induced localization of YAP under starvation conditions . HEK293A were transfected with LRP6 - EGFP 1 d after transfected were incubated with or without serum - containing medium for 4 h , immunofluorescence was performed . Figure shows representative image of multiple areas . DAPI was used for nucleus . Scale bars : 10μm . of YAP is shown in right panel . was performed by counting that have localized YAP per image ( n = 6 ) . Error bars indicate standard deviation . * P < 0 . 05 . Student ' s t - was used for statistical .

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I. Overexpression of LRP6 induced nuclear localization of YAP under starvation conditions. HEK293A cells were transfected with LRP6-EGFP and 1 d after transfected cells were incubated with or without serum-containing medium for 4 h, and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear localized YAP per image (n=6). Error bars indicate standard deviation. *P < 0.05. Student's t-test was used for statistical analysis.


A . Knockdown of LRP6 activated LATS1 . HEK293T were transfected with siRNA for GFP LRP6 . After transfection , were lysed lysates were analyzed by immunoblotting .

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A. Knockdown of LRP6 activated LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6. After transfection, cells were lysed and the cell lysates were analyzed by immunoblotting.


B . Overexpression of LRP6 reduced amount of active form of LATS1 that was induced by serum starvation . HEK293T were transfected with empty vector ( - ) or VSVG - LRP6 . 1 d after transfection , were incubated in serum - free medium for 4 h . After incubation , were lysed , lysates were analyzed by immunoblotting .

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B. Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation. HEK293T cells were transfected with empty vector (-) or VSVG-LRP6. 1 d after transfection, cells were incubated in serum-free medium for 4 h. After incubation, cells were lysed, and the cell lysates were analyzed by immunoblotting.


C . Merlin LATS1 / 2 were necessary for LRP6 - knockdown - mediated YAP phosphorylation . HEK293T were transfected with siRNA for GFP , LRP6 , Merlin , LATS1 / 2 . were lysed lysates were analyzed by immunoblotting .

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C. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated YAP phosphorylation. HEK293T cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.


D . Merlin LATS1 / 2 were necessary for LRP6 - knockdown - mediated decrease of YAP reporter activity . HEK293T were seeded transfected with siRNA for GFP , LRP6 , Merlin LATS1 / 2 . After 1 d , these were transfected again with pRL - TK 8XGTIIC luciferase reporter constructs . One day after transfection , were lysed luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * P < 0 . 01 N . S : Non - significant . Student ' s t - was used for statistical .

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D. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated decrease of YAP reporter activity. HEK293T cells were seeded and transfected with siRNA for GFP, LRP6, Merlin and LATS1/2. After 1 d, these cells were transfected again with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were lysed and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. **P< 0.01 and N.S: Non-significant. Student's t-test was used for statistical analysis.


E . Merlin LATS1 / 2 were necessary for LRP6 knockdown - mediated - localization of YAP . HEK293A were transfected with siRNA for GFP , LRP6 , Merlin , LATS1 / 2 , immunofluorescence was performed . figure shows representative of multiple areas . DAPI was used for nucleus . Scale bars : 10 μm . of that have YAP is shown in Fig EV3B .

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E. Merlin and LATS1/2 were necessary for the LRP6 knockdown-mediated-cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B.


F . YAP phosphorylation by loss of LRP6 was dependent on interaction between Merlin LATS1 / 2 . HEK293T were transfected with siRNA for GFP , LRP6 , Merlin , after 1 d were transfected again with HA - Merlin HA - Merlin ∆ FERM . were lysed lysates were analyzed by immunoblotting . arrow indicates endogenous Merlin HA - Merlin , arrowhead indicates HA - Merlin ∆ FERM .

F. YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and Merlin, and after 1 d were transfected again with HA-Merlin and HA-Merlin∆FERM. Cells were lysed and the cell lysates were analyzed by immunoblotting. The arrow indicates endogenous Merlin and HA-Merlin, and the arrowhead indicates HA-Merlin∆FERM.


G . MST1 / 2 were necessary for LRP6 - knockdown - mediated activation of LATS1 / 2 . HEK293T were transfected with siRNA for GFP , LRP6 MST1 / 2 . were lysed lysates were analyzed by immunoblotting .

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G. MST1/2 were necessary for the LRP6-knockdown-mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6 and MST1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.


H . MAP4K4 / 6 / 7 were necessary for LRP6 knockdown mediated activation of LATS1 / 2 . HEK293T were transfected with siRNA for GFP , LRP6 , MAP4K4 / 6 / 7 . were lysed lysates were analyzed by immunoblotting .

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H. MAP4K4/6/7 were necessary for the LRP6 knockdown mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MAP4K4/6/7. Cells were lysed and the cell lysates were analyzed by immunoblotting.


A . interaction between overexpressed Merlin LRP6 was reduced under serum starvation condition . HEK293T were transfected with Flag - Merlin LRP6 - EGFP . After transfection , were incubated in serum - free medium for overnight with Bafilomycin A1 ( 20 nM ) . lysates were immunoprecipitated with anti - EGFP antibody analyzed by immunoblotting with indicated antibodies . WCL , whole - lysates .

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A. The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag-Merlin and LRP6-EGFP. After transfection, cells were incubated in serum-free medium for overnight with Bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.


interaction between endogenous Merlin LRP6 was reduced under serum starvation . HEK293T were incubated in serum - free medium for 4 h with Bafilomycin A1 ( 100 nM ) . lysates were immunoprecipitated with anti - LRP6 antibody analyzed by immunoblotting with indicated antibodies . WCL , whole - lysates .

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The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-LRP6 antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.


interaction between endogenous Merlin LRP6 was reduced under serum starvation . HEK293T were incubated in serum - free medium for 4 h with Bafilomycin A1 ( 100 nM ) . lysates were immunoprecipitated with anti - Merlin antibody analyzed by immunoblotting with indicated antibodies . WCL , whole - lysates .

The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.


D . interaction between overexpressed Merlin LATS1 was increased under serum starvation . HEK293T were transfected with Myc - LATS1 EGFP - Merlin . After transfection , were incubated in serum - free medium for overnight then were lysed . lysates were immunoprecipitated with anti - EGFP antibody analyzed by immunoblotting . WCL , whole - lysates .

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D. The interaction between overexpressed Merlin and LATS1 was increased under serum starvation. HEK293T cells were transfected with Myc-LATS1 and EGFP-Merlin. After transfection, cells were incubated in serum-free medium for overnight and then were lysed. The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


E . Knockdown of LRP6 enhanced interaction between overexpressed Merlin LATS1 . HEK293T were transfected with Myc - LATS1 , EGFP - Merlin LRP6 siRNA . were lysed lysates were immunoprecipitated with anti - EGFP antibody analyzed by immunoblotting . WCL , whole - lysates .

E. Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1. HEK293T cells were transfected with Myc-LATS1, EGFP-Merlin and LRP6 siRNA. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


F . Knockdown of LRP6 enhances interaction between endogenous Merlin LATS1 . HEK293T were transfected with siRNA for GFP or LRP6 . After transfection , were lysed lysates were immunoprecipitated with anti - Merlin antibody analyzed by immunoblotting . WCL , whole - lysates .

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F. Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1. HEK293T cells were transfected with siRNA for GFP or LRP6. After transfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


A . Endogenous LRP6 OGT interact with each other . HEK293T were lysed lysates were immunoprecipitated with anti - LRP6 antibody blotted with OGT . WCL , whole - lysates .

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A. Endogenous LRP6 and OGT interact with each other. HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti-LRP6 antibody and blotted with OGT. WCL, whole-cell lysates.


B . LRP6 is O - GlcNAcylated . LRP6 - EGFP - transfected HEK293T were treated in Thiamet G ( 30 μM ) or OSMI - 1 ( 50 μM ) for 6 h with Bafiolmycin A1 ( 100nM ) . After treatment , were lysed lysates were immunoprecipitated with anti - EGFP antibody analyzed by immunoblotting . WCL , whole - lysates .

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B. LRP6 is O-GlcNAcylated. LRP6-EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI-1 (50 μM) for 6 h with Bafiolmycin A1 (100nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


C . LRP6 is O - GlcNAcylated . lysates from HEK293T were incubated with sWGA - beads in absence or presence of free GlcNAc ( 25 mM ) for 3 h . After incubation , sWGA - beads were precipitated analyzed by immunoblotting . WCL , whole - lysates .

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C. LRP6 is O-GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.


D . Blocking of O - GlcNAcylation reduced amount of LRP6 . HEK293 were treated with OSMI - 1 ( 50 μM ) Bafilomycin A1 ( 100 nM ) for 6 h . were lysed lysates were analyzed by immunoblotting .

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D. Blocking of O-GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI-1 (50 μM) and Bafilomycin A1 (100 nM) for 6 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


E . O - GlcNAcylation on LRP6 was reduced under serum starvation condition . HEK293T were transfected with LRP6 - EGFP . were incubated in serum - free medium with Bafilomycin A1 ( 100 nM ) for 4 h . After incubation , were lysed lysates were immunoprecipitated with anti - EGFP antibody analyzed by immunoblotting . WCL , whole - lysates . of O - GlcNAc / EGFP from three independent immunoblots was quantified ( biological replicates , bottom panel ) . * P < 0 . 05 . Error bars indicate standard deviation of biological triplicate measurements . Student ' s t - was used for statistical .

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E. The O-GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6-EGFP. Cells were incubated in serum-free medium with Bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. The ratio of O-GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.


F . Increase of O - GlcNAcylation enhanced interaction between ectopically expressed LRP6 Merlin . LRP6 - EGFP Flag - Merlin transfected HEK293T were incubated in serum - free medium with Bafilomycin A1 ( 20 nM ) Thiamet G ( 30μM ) overnight . were lysed lysates were immunoprecipitated with anti - EGFP antibody analyzed by immunoblotting . WCL , whole - lysates .

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F. Increase of O-GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6-EGFP and Flag-Merlin transfected HEK293T cells were incubated in serum-free medium with Bafilomycin A1 (20 nM) and Thiamet G (30μM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


G . Enhancement of global O - GlcNAcylation blocked serum starvation - mediated increase of interaction between overexpressed LATS1 Merlin . HEK293T were transfected with Myc - LATS1 Flag - Merlin . After transfection , were incubated in serum - free medium with Thiamet G ( 30μM ) Bafilomycin A1 ( 20nM ) overnight . were lysed lysates were immunoprecipitated with anti - Myc antibody analyzed by immunoblotting . WCL , whole - lysates .

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G. Enhancement of global O-GlcNAcylation blocked the serum starvation-mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc-LATS1 and Flag-Merlin. After transfection, cells were incubated in serum-free medium with Thiamet G (30μM) and Bafilomycin A1 (20nM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


A . Nutrient starvation reduced level of LRP6 in mouse liver . Mice were starved for 36 h liver tissues were analyzed by immunoblotting ( left panel ) . of LRP6 / Vinculin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * * P < 0 . 01 . Student ' s t - was used for statistical .

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A. Nutrient starvation reduced the level of LRP6 in mouse liver. Mice were starved for 36 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01. Student's t-test was used for statistical analysis.


B . Refeeding of nutrients rescued LRP6 in mouse liver . Mice were starved then refed for 6 h liver tissues were analyzed by immunoblotting ( left panel ) . of LRP6 / Vinculin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 . Student ' s t - was used for statistical .

B. Refeeding of nutrients rescued LRP6 in mouse liver. Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.


C . level of LRP6 was not reduced during nutrient starvation in mouse brain . Mice were starved brain tissues were analyzed by immunoblotting .

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C. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved and brain tissues were analyzed by immunoblotting.


D . LRP6 was O - GlcNAcylated in mouse liver . Liver tissues were incubated with sWGA - beads in absence or presence of free GlcNAc ( 25 mM ) for 3 h . After incubation , sWGA - beads were precipitated analyzed by immunoblotting . WCL , whole - lysates .

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D. LRP6 was O-GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.


E . O - GlcNAcylation of LRP6 was reduced during starvation in mouse liver . LRP6 in liver tissue lysates from both fed starved mice was equilibrated by loading twice as much lysate from livers of starved mice on gel . Liver tissues were incubated in sWGA - beads for 3 h . After incubation , sWGA - beads were precipitated analyzed by immunoblotting . WCL , whole - lysates .

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E. O-GlcNAcylation of LRP6 was reduced during starvation in mouse liver. LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel. Liver tissues were incubated in sWGA-beads for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.


Kaplan - Meier of overall survival in breast cancer with recurrent lethal disease , classified according to p - JNK expression . p - JNK was determined by immunostaining of breast cancer samples in a tissue microarray ; p - JNK low , n = 31 ; p - JNK high , n = 10 . P value hazard ( HR ) were calculated by log - rank .

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Kaplan-Meier analysis of overall survival in breast cancer patients with recurrent lethal disease, classified according to p-JNK expression. p-JNK was determined by immunostaining of breast cancer samples in a tissue microarray; p-JNK low, n = 31; p-JNK high, n = 10. P value and hazard ratio (HR) were calculated by log-rank test.


Immunostaining of p - c - Jun ( arrows ) in - matched breast lung metastasis . Scale bar , 50 μm .

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Immunostaining of p-c-Jun (arrows) in patient-matched breast tumor and lung metastasis. Scale bar, 50 μm.


of p - c - Jun positive ( p - c - Jun + ) per in matched tumors lung metastases . Boxes depict median from 12 with upper lower quartiles .

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Quantification of p-c-Jun positive (p-c-Jun+) cells per tumor area in matched primary tumors and lung metastases. Boxes depict median from 12 patients with upper and lower quartiles.


of p - c - Jun positive ( p - c - Jun + ) cancer within mammary tumors matched metastases in mice injected with MDA231 - LM2 .

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Quantification of p-c-Jun positive (p-c-Jun+) cancer cells within mammary tumors and matched metastases in mice injected with MDA231-LM2 cells.


Immunofluorescence of p - c - Jun in micrometastatic ( day 7 ) macrometastatic ( day 21 ) nodules in a xenograft mouse model injected intravenously with MDA231 - LM2 . DAPI was used to stain nuclei , cancer express GFP as a marker . Scale bar , 50 μm . of p - c - Jun + cancer

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Immunofluorescence analysis of p-c-Jun in micrometastatic (day 7) and macrometastatic (day 21) nodules in a xenograft mouse model injected intravenously with MDA231-LM2 cells. DAPI was used to stain cell nuclei, and cancer cells express GFP as a marker. Scale bar, 50 μm. Quantification of p-c-Jun+ cancer cells


Mammary growth curves from mice described in panel G . Each value represents mean ± . For MDA231 - LM2 , n = 12 tumors in 6 mice for each experimental group . For SUM159 - LM1 , n = 16 tumors in 8 mice for each experimental group .

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Mammary tumor growth curves from mice described in panel G. Each value represents the mean ± SEM. For MDA231-LM2, n = 12 tumors in 6 mice for each experimental group. For SUM159-LM1, n = 16 tumors in 8 mice for each experimental group.


Lung metastatic burden in MDA231 - LM2 or SUM159 - LM1 - bearing mice treated with JNKi . of lung metastases . FOV , field of view ; LS , lung section . Each value represents mean ± . For metastatic foci per FOV , 8 - 10 fields were quantified per mouse . MDA231 - LM2 - bearing mice : n = 6 mice per group . SUM159 - LM1 - bearing mice , vehicle n = 8 mice ; JNKi n = 7 mice .

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Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Quantification of lung metastases. FOV, field of view; LS, lung section. Each value represents the mean ± SEM. For metastatic foci per FOV, 8-10 fields were quantified per mouse. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice, vehicle n = 8 mice; JNKi n = 7 mice.


Lung metastatic burden in MDA231 - LM2 or SUM159 - LM1 - bearing mice treated with JNKi . Representative histology examples of metastasis in lungs , detected by expression of human vimentin . Scale bar , 100 μm .

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Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Representative histology examples of metastasis in lungs, detected by expression of human vimentin. Scale bar, 100 μm.


Metastatic burden determined by photon flux in lungs of MDA231 - LM2 - injected NSG mice treated with vehicle JNKi . Boxes show median with upper lower quartiles . Whiskers represent minimum maximum values . Vehicle control , n = 8 ; JNKi - treated , n = 8 . Examples of bioluminescence in

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Metastatic burden determined by photon flux in lungs of MDA231-LM2-injected NSG mice treated with vehicle and JNKi. Boxes show the median with upper and lower quartiles. Whiskers represent minimum and maximum values. Vehicle control, n = 8; JNKi-treated, n = 8. Examples of bioluminescence in mice


Scatter plot elucidating fold change ( FC ) in gene expression between samples treated with JNKi compared to vehicle ( Y - axis ) expression of constitutively active JNK ( MKK7 - JNK ) versus vector control ( X - axis ) from biological triplicates . Representative examples of genes that are specifically induced by JNK in metastatic breast cancer are highlighted in red .

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Scatter plot elucidating fold change (FC) in gene expression between samples treated with JNKi compared to vehicle (Y-axis) and expression of constitutively active JNK (MKK7-JNK) versus vector control (X-axis) from biological triplicates. Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red.


GSEA graphs showing ( C ) enrichment of a mammary signature ( Pece et al , 2010 ) in MDA231 - LM2 cancer expressing MKK7 - JNK NES , normalized enrichment score .

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GSEA graphs showing (C) enrichment of a mammary stem cell signature (Pece et al, 2010) in MDA231-LM2 cancer cells expressing MKK7-JNK NES, normalized enrichment score.


GSEA graphs showing under - representation of same mammary signature in cancer treated with JNKi . NES , normalized enrichment score .

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GSEA graphs showing under-representation of the same mammary stem cell signature in cancer cells treated with JNKi. NES, normalized enrichment score.


GSEA showing significant enrichment of four distinct signatures of mammary within data sets from MDA231 - LM2 either expressing MKK7 - JNK or treated with JNKi . Mammary signatures are indicated as follows : a , up in mammary b , up in mammary ; c , up in adult mammary d , up in mammary repopulating

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GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231-LM2 cells either expressing MKK7-JNK or treated with JNKi. Mammary stem cell signatures are indicated as follows: a, up in mammary stem cells b, up in fetal mammary stem cells ; c, up in adult mammary stem cells and d, up in mammary repopulating units


Heat map of JNK signature composed of 68 genes that are induced by expression of MKK7 - JNK repressed by JNKi ,

Heat map of the JNK signature composed of 68 genes that are induced by expression of MKK7-JNK and repressed by JNKi,


Proportion of JNK signature - positive tumors in breast cancer from METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK - S , JNK signature as in panel F . Subtype numbers : Luminal , n = 1213 ; HER2 , n = 240 ; Basal - like , n = 331 .

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Proportion of JNK signature-positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK-S, JNK signature as in panel F. Subtype patient numbers: Luminal, n = 1213; HER2, n = 240; Basal-like, n = 331.


Gene expression profile of MDA231 - LM2 grown as oncospheres or as a monolayer . Examples of genes that are both upregulated in spheres induced by JNK signaling are highlighted in red .

Gene expression profile of MDA231-LM2 cells grown as oncospheres or as a monolayer. Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red.


GSEA graph showing enrichment of JNK signature in MDA231 - LM2 cancer grown as oncospheres . NES , normalized enrichment score .

GSEA graph showing enrichment of JNK signature in MDA231-LM2 cancer cells grown as oncospheres. NES, normalized enrichment score.


Immunoblot of phosphorylated JNK ( p - JNK ) , JNK , phosphorylated c - Jun ( p - c - Jun ) c - Jun in oncospheres from SUM159 - LM1 . Tubulin was included as a loading control .

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Immunoblot of phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in oncospheres from SUM159-LM1 cells. Tubulin was included as a loading control.


GSEA of a basal - like breast cancer signature induced in MDA231 - LM2 oncospheres .

GSEA of a basal-like breast cancer signature induced in MDA231-LM2 oncospheres.


Normalized enrichment score ( GSEA ) of mammary signatures in oncospheres from MDA231 - LM2 .

Normalized enrichment score (GSEA) of mammary stem cell signatures in oncospheres from MDA231-LM2 cells.


Generation of oncospheres in SUM159 expressing constitutively active JNK or inactive mutated JNK . Values are mean from 10 wells per group ± SD .

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Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK. Values are mean from 10 wells per group ± SD.


Oncosphere formation in SUM159 - LM1 following treatment with JNKi or vehicle control . Values are mean from 10 wells per condition ± SD . Representative of

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Oncosphere formation in SUM159-LM1 cells following treatment with JNKi or vehicle control. Values are mean from 10 wells per condition ± SD. Representative images of spheres


Oncosphere formation in JNKi - treated cancer from breast cancer ( pleural effusions or ascites ) . ( I ) of oncospheres per well in 4 different . Values are mean from 10 wells ± SD . ( J ) Representative oncospheres from samples . Scale bar , 50 μm .

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Oncosphere formation in JNKi-treated cancer cells from breast cancer patients (pleural effusions or ascites). (I) Quantification of oncospheres per well in 4 different patients. Values are mean from 10 wells ± SD. (J) Representative oncospheres from patient samples. Scale bar, 50 μm.


Matrigel invasion of MDA231 - LM2 treated with JNKi . Left , examples of invading cancer stained with crystal violet ( arrows ) . Scale bar , 50 μm . Right , of invading per field of view ( FOV ) showing mean from 8 fields ± .

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Matrigel invasion assay of MDA231-LM2 cells treated with JNKi. Left, examples of invading cancer cells stained with crystal violet (arrows). Scale bar, 50 μm. Right, quantification of invading cells per field of view (FOV) showing mean from 8 fields ± SEM.


SPP1 TNC expression in indicated breast cancer treated with increasing concentrations of JNKi .

SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi.


SPP1 TNC expression in MDA231 - LM2 transduced with pLVX - puro lentivirus without a transgene ( control ) or expressing constitutively - active JNK ( MKK7 - JNK ) or mutated inactive JNK ( MKK7 - JNK ( mut ) ) .

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SPP1 and TNC expression in MDA231-LM2 cells transduced with pLVX-puro lentivirus without a transgene (control) or expressing constitutively-active JNK (MKK7-JNK) or mutated inactive JNK (MKK7-JNK(mut)).


SPP1 TNC expression in pleural effusion ( 1 2 ) ascites ( 3 4 ) samples from four different treated with JNKi ( 4 μM , 48h ) in culture . Values are mean ± SD from triplicates . results from four samples were included to calculate significance of JNKi on gene expression . SPP1 , P = 0 . 0079 ; TNC , P = 0 . 0002 .

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SPP1 and TNC expression in pleural effusion (1 and 2) and ascites (3 and 4) samples from four different patients treated with JNKi (4 μM, 48h) in culture. Values are mean ± SD from triplicates. The results from all four patient samples were included to calculate significance of JNKi on gene expression. SPP1, P = 0.0079; TNC, P = 0.0002.


of SPP1 TNC mRNA in SUM159 - LM1 grown as monolayer or spheres treated with vehicle or JNKi . For panels A - D , values represent mean of qPCR triplicates ± SD .

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Analysis of SPP1 and TNC mRNA levels in SUM159-LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi. For panels A-D, values represent the mean of qPCR triplicates ± SD.


ChIP of c - Jun binding to sequences in SPP1 or TNC promoters . of SPP1 TNC promoter fragments pulled down by c - Jun IgG antibodies . Values represent mean of qPCR triplicates ± SD .

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ChIP analysis of c-Jun binding to sequences in SPP1 or TNC promoters. Analysis of SPP1 and TNC promoter fragments pulled down by c-Jun and IgG antibodies. Values represent the mean of qPCR triplicates ± SD.


Correlation of SPP1 TNC with JUN expression in dissected metastatic nodules from breast cancer ( GSE14020 ) . N = 65 metastasis samples .

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Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients (GSE14020). N = 65 metastasis samples.


Lung metastasis in Spp1 + / - mice implanted with MDA231 - LM2 expressing control shRNA ( control ) , Spp1 - / - mice implanted with MDA231 - LM2 independently expressing one of two different SPP1 shRNAs ( SPP1 - deficient ) . Ex vivo lung luminescence was analyzed , panel H . Boxes show median value with upper lower quartiles . Whiskers represent minimum maximum values . Control n = 16 mice , SPP1 deficient ( 1 ) n = 14 mice SPP1 deficient ( 2 ) n = 15 mice . P values were determined by a two - Mann - Whitney . ( I ) Representative histological of metastatic foci ( top ) ex vivo lung bioluminescence ( bottom ) . In histological samples , immunohistochemistry was used to determine metastatic nodules on expression of human vimentin ( arrows ) . Scale bar , 100 µm .

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Lung metastasis in Spp1+/- mice implanted with MDA231-LM2 cells expressing control shRNA (control), and Spp1-/- mice implanted with MDA231-LM2 cells independently expressing one of two different SPP1 shRNAs (SPP1-deficient). Ex vivo lung luminescence was analyzed, panel H. Boxes show the median value with upper and lower quartiles. Whiskers represent minimum and maximum values. Control n = 16 mice, SPP1 deficient (1) n = 14 mice and SPP1 deficient (2) n = 15 mice. P values were determined by a two-tailed Mann-Whitney test. (I) Representative histological images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). In histological samples, immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin (arrows). Scale bar, 100 µm.


SUM159 - LM1 expressing either control shRNA ( control ) , or one of two different TNC shRNAs ( shTNC 1 2 ) , were injected intravenously into NSG mice . ( J ) Metastatic colonization was quantified by lung bioluminescence . Control shTNC ( 1 ) n = 8 mice per group , shTNC ( 2 ) n = 7 mice . P value was determined by a two - Mann - Whitney . ( K ) Representative of metastatic foci ( top ) ex vivo lung bioluminescence ( bottom ) . Arrows denote vimentin - expressing cancer in metastases .

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SUM159-LM1 cells expressing either control shRNA (control), or one of two different TNC shRNAs (shTNC 1 and 2), were injected intravenously into NSG mice. (J) Metastatic colonization was quantified by lung bioluminescence. Control and shTNC (1) n = 8 mice per group, shTNC (2) n = 7 mice. P value was determined by a two-tailed Mann-Whitney test. (K) Representative images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). Arrows denote vimentin-expressing cancer cells in metastases.


Kaplan - Meier of breast cancer ( combined GSE2603 GSE5327 datasets ) showing a link between high SPP1 TNC expression poor lung metastasis - free survival . samples , SPP1 TNC low n = 70 ; SPP1 TNC high n = 70 . HR , Hazard .

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Kaplan-Meier analysis of breast cancer patients (combined GSE2603 and GSE5327 datasets) showing a link between high SPP1 and TNC expression and poor lung metastasis-free survival. Patient samples, SPP1 and TNC low n = 70; SPP1 and TNC high n = 70. HR, Hazard ratio.


Gene expression profile of MDA231 - LM2 treated with paclitaxel ( PAX ) for 48 hours . Highlighted are examples of JNK signature genes that are induced by PAX treatment .

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Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment.


GSEA normalized enrichment scores of four independent mammary signatures that arise in response to paclitaxel treatment of MDA231 - LM2 .

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GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells.


Immunofluorescence of p - c - Jun + cancer in lung metastases from mice injected intravenously with MDA231 - LM2 treated with PAX at week 3 post injection . Lungs were harvested 72h after PAX treatment . Cancer express GFP as a marker . DAPI was used for . Scale bar , 50 µm .

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Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm.


of cancer expressing p - c - Jun in panel D . Values are mean from three mice ± . Nine metastatic foci were analyzed per mouse .

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Quantification of cancer cells expressing p-c-Jun in panel D. Values are mean from three mice ± SEM. Nine metastatic foci were analyzed per mouse.


Western blot analyzing phosphorylated JNK ( p - JNK ) , JNK , phosphorylated c - Jun ( p - c - Jun ) c - Jun in MDA231 - LM2 treated with PAX for 48 hours . Tubulin was used as a loading control .

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Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.


SPP1 TNC expression in SUM159 - LM1 treated with increasing concentrations of PAX qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD.


SPP1 TNC expression in SUM159 - LM1 treated with increasing concentrations of doxorubicin ( DOXO ) . qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD.


SPP1 TNC expression in SUM159 - LM1 cancer treated with 4 μM 5 - Fluorouracil or 4 μM methotrexate . were treated for 48 hours . qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD.


SPP1 TNC expression in SUM159 - LM1 treated with paclitaxel JNKi . qPCR triplicates ± SD . SPP1 TNC repression by JNKi , P < 0 . 05 for both vehicle PAX - treated .

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SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells.


Growth curves of mammary tumors in vehicle - or PAX - treated Spp1 + / - mice implanted with indicated expressing control shRNA , Spp1 - / - mice implanted with indicated expressing SPP1 shRNA . MDA231 - LM2 : control , PAX SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 14 tumors . SUM159 - LM1 : control , PAX SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 15 tumors .

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Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors.


of lung metastasis from described in panels A B . number of metastatic foci per field of view ( FOV ) or per lung section ( LS ) was determined . MDA231 - LM2 - bearing mice : control , PAX SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice . SUM159 - LM1 - bearing mice : control , PAX SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice .

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

Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice.


in mice at day 40 after implantation of MDA231 - LM2 breast cancer in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin ( Adriamycin ) cyclophosphamide ( AC regimen ) . Control , AC SPP1 def . n = 16 tumors per group , SPP1 def . + AC n = 14 tumors .

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Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors.


Lung metastasis was quantified in mice that were implanted with MDA231 - LM2 tumors subjected to AC chemotherapy . Each value represents mean ± ; n = 5 mice per group . Ten random fields per lung section were analyzed for of metastatic . Representative histological of metastatic nodules in lungs of mice from experiment described in panel E . Metastases were determined by expression of human vimentin ( arrows ) . Scale bar , 100 µm .

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Lung metastasis was quantified in mice that were implanted with MDA231-LM2 tumors and subjected to AC chemotherapy. Each value represents the mean ± SEM; n = 5 mice per group. Ten random fields per lung section were analyzed for percentage of metastatic area. Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E. Metastases were determined by expression of human vimentin (arrows). Scale bar, 100 µm.


Mammary growth curves in PAX - treated NSG mice bearing control or TNC knockdown MDA231 - LM2 tumors . Values are means ± , n = 16 tumors per group . * * * * P < 0 . 0001 . Lung metastases quantified in bearing

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

Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P < 0.0001. Lung metastases quantified in tumor bearing mice


Representative histology of human vimentin - positive lung metastases ( arrows )

Representative histology of human vimentin-positive lung metastases (arrows)


Mammary growth curves in NSG mice implanted with MDA231 - LM2 or SUM159 - LM1 treated with paclitaxel as single agent or in combination with JNKi . Each value represents mean ± . MDA231 - LM2 : n = 12 tumors per group . SUM159 - LM1 : vehicle , JNKi PAX n = 12 tumors per group ; PAX + JNKi n = 10 tumors . * * P < 0 . 01 , * * * P < 0 . 001 .

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Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001.


Metastatic burden in lungs of MDA231 - LM2 SUM159 - LM1 - bearing mice treated with paclitaxel as single therapy or in combination with JNKi . Each value represents mean ± . MDA231 - LM2 - bearing mice : n = 6 mice per group . SUM159 - LM1 - bearing mice : vehicle , JNKi PAX n = 6 mice per group ; PAX + JNKi n = 5 . Ten fields were quantified per lung section . FOV , field of view , LS , lung section . PAX , paclitaxel . Representative histological of metastatic foci ( arrows ) in lungs of MDA231 - LM2 - implanted mice described in panel C , stained for human vimentin expression in differentially treated groups . Scale bar , 100 μm .

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Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel. Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm.


Apoptosis was determined by of TUNEL - positive per field of view ( FOV ) in MDA231 - LM2 tumors ( from described in ( C ) ) treated with JNKi / or PAX . Values are means from 6 mice per condition ± .

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Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM.


GSEA showing enrichment of JNK signature in samples from breast cancer from TOP trial ( GSE16446 ) , stratified according to pathological response to neoadjuvant chemotherapy . NES , normalized enrichment score . pCR , pathological complete response .

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GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial (GSE16446), stratified according to pathological response to neoadjuvant chemotherapy. NES, normalized enrichment score. pCR, pathological complete response.


Kaplan - Meier analyses of chemotherapy - treated breast cancer associating JNK signature ( JNK - S ) with metastasis - free survival ( TOP trial data set , JNK - S low n = 54 ; JNK - S high n = 53 ( G ) ) or overall survival ( METABRIC data set , JNK - S low n = 135 ; JNK - S high n = 135 ( H ) ) . HR , hazard . P values were determined by log - rank .

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Kaplan-Meier analyses of chemotherapy-treated breast cancer patients associating JNK signature (JNK-S) with metastasis-free survival (TOP trial data set, JNK-S low n = 54 patients; JNK-S high n = 53 patients (G)) or overall survival (METABRIC data set, JNK-S low n = 135 patients; JNK-S high n = 135 patients (H)). HR, hazard ratio. P values were determined by log-rank test.


( A ) Intracellular detection of NOD2 ( bold in histogram plot ) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar ( R1 , dot plot ) . One representative experiment out of three experiments is presented .

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(A) Intracellular detection of NOD2 (bold line in the histogram plot) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar cells (R1, dot plot). One representative experiment out of three experiments is presented.


( B ) were stimulated with 10 μg / mL of MDP for 24 h , NOD2 IκBα were measured in fractions by western blot . fold increase relative to unstimulated normalized to tubulin of one representative experiment out of three is reported .

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(B) Cells were stimulated with 10 μg/mL of MDP for 24 h, and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis. The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported.


( C ) upregulation of NOD2 gene expression was assessed by quantitative PCR . results are depicted as mean fold change in NOD2 gene expression relative to unstimulated ( n = 11 ) .

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(C) The upregulation of NOD2 gene expression was assessed by quantitative PCR. The results are depicted as the mean fold change in NOD2 gene expression relative to unstimulated cells (n = 11).


( D ) Subcellular localization of NOD2 was ascertained in unstimulated ( left , × 80 , 000 ) MDP ‐ stimulated ( right , × 63 , 000 ) using an anti ‐ NOD2 antibody detected with a secondary antibody coupled to 5 ‐ nm gold particles , indicated with arrowheads . Bar represents 150 nm . were obtained from one experiment .

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(D) Subcellular localization of NOD2 was ascertained in unstimulated (left, ×80,000) and MDP‐stimulated cells (right, ×63,000) using an anti‐NOD2 antibody and detected with a secondary antibody coupled to 5‐nm gold particles, indicated with arrowheads. Bar represents 150 nm. Micrographs were obtained from one experiment.


NOD2 ligation in alveolar macrophages induces pro ‐ inflammatory cytokine release . were incubated for 24 h in presence of 10 μg / mL of MDP or 100 ng / mL of LPS . Production of IL ‐ 1β , IL ‐ 6 , TNF ‐ α , IL ‐ 10 was measured in culture supernatants using Milliplex technology . Depicted are box plots with median values quartiles for each cytokine . data are representative of two independent experiments ( n = 11 ) ; * p 0 . 05 * * p 0 . 01 using two ‐ Wilcoxon signed ‐ rank .

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NOD2 ligation in alveolar macrophages induces pro‐inflammatory cytokine release. Cells were incubated for 24 h in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS. Production of IL‐1β, IL‐6, TNF‐α, and IL‐10 was measured in culture supernatants using Milliplex technology. Depicted are box plots with median values and quartiles for each cytokine. The data are representative of two independent experiments (n = 11); *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.


LL37 IRGM gene expression is upregulated following NOD2 activation ( A ‐ D ) . Alveolar macrophages were incubated in presence of 10 μg / mL of MDP or 100 ng / mL of LPS for 24 h . Upregulation of ( A ) LL37 ( D ) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using Taqman system ΔΔCT method for relative . fold change in gene expression relative to unstimulated is depicted . Data are representative of two independent experiments ( n = 7 - 12 ) . Bold lines indicate median values .

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LL37 and IRGM gene expression is upregulated following NOD2 activation (A‐D). Alveolar macrophages were incubated in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS for 24 h. Upregulation of (A) LL37 and (D) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. The fold change in gene expression relative to unstimulated cells is depicted. Data are representative of two independent experiments (n = 7-12). Bold lines indicate median values.


( B , E ) LL37 IRGM were measured in fractions by western blot .

(B, E) LL37 and IRGM protein levels were measured in cytosolic fractions by western blot analysis.


( C , F ) Data are expressed as fold increases relative to unstimulated normalized to tubulin calculated by densitometry are shown as means ± . One representative experiment out of three experiments is presented .

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(C, F) Data are expressed as the protein fold increases relative to unstimulated cells and normalized to tubulin calculated by densitometry and are shown as means ± SEM. One representative experiment out of three experiments is presented.


( A ) Intracellular burden was measured by quantifying CFU , intracellular growth index was calculated after 1 4 days post infection relative to phagocytosed at day 0 . Data are shown as medians quartiles are representative of n = 7 experiments .

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(A) Intracellular bacterial burden was measured by quantifying CFU, and the intracellular growth index was calculated after 1 and 4 days post infection relative to phagocytosed bacteria at day 0. Data are shown as medians and quartiles and are representative of n = 7 experiments.


production of ( B ) TNF ‐ α , ( C ) IL ‐ 6 ( D ) IL ‐ 1β was measured in culture supernatants using Milliplex technology . Box plots depicting median values quartiles are representative of n = 9 experiments .

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The production of (B) TNF‐α, (C) IL‐6 and (D) IL‐1β was measured in culture supernatants using Milliplex technology. Box plots depicting median values and quartiles are representative of n = 9 experiments.


( E ) LL37 ( F ) IRGM gene expression was assessed by quantitative PCR using Taqman system ΔΔCT method for relative . Fold changes in gene expression relative to unstimulated are reported . Bold lines indicate median values of n = 9 . * p 0 . 05 * * p 0 . 01 using two ‐ Wilcoxon signed ‐ rank .

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(E) LL37 and (F) IRGM gene expression was assessed by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. Fold changes in gene expression relative to unstimulated cells are reported. Bold lines indicate median values of n = 9. *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.


( G ) LL37 IRGM was assessed by western blot . One representative experiment out of three experiments is presented .

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(G) LL37 and IRGM protein was assessed by western blot. One representative experiment out of three experiments is presented.


( H ) amount of normalized to tubulin relative to infected ‐ only was calculated by densitometry . Data are depicted as means ± are representative of n = 3 experiments .

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(H) The amount of protein normalized to tubulin relative to infected‐only cells was calculated by densitometry. Data are depicted as the means ± SEM and are representative of n = 3 experiments.


( A , B , D , E , G , H ) subcellular localization of autophagy was ascertained in untreated MDP ‐ treated using ( A , B ) anti ‐ IRGM , ( D , E ) anti ‐ LC3 , ( G , H ) anti ‐ ATG16L1 antibodies detected with a secondary antibody coupled to 5 ‐ nm gold particles indicated with arrowheads ; bar represents 150 nm .

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(A, B, D, E, G, H) The subcellular localization of autophagy proteins was ascertained in untreated and MDP‐treated cells using (A, B) anti‐IRGM, (D, E) anti‐LC3, and (G, H) anti‐ATG16L1 antibodies and detected with a secondary antibody coupled to 5‐nm gold particles indicated with arrowheads; bar represents 150 nm.


( C , F , I ) Gold particles co ‐ localizing with were manually counted in ten macrophages of each condition . Means ± are depicted differences between treatments are indicated , * p 0 . 05 using a two ‐ paired t ‐ . ( B ) Transmission electron microscopy ( ) magnification × 50 , 000 ; of are magnification × 80 , 000 . Data were generated from one experiment .

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(C, F, I) Gold particles co‐localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p 0.05 using a two‐tailed paired t‐test. (B) Transmission electron microscopy (TEM) magnification ×50,000; the rest of the micrographs are TEM magnification ×80,000. Data were generated from one experiment.