Figure 2TMZ 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 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 . 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 .

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Figure 2TMZ 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 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.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.


Figure 3Immuofluorescent staining for αv integrin expression in brain sections including intracranial tumor ( T ) and the surrounding healthy brain ( B ) . Scale bar , 100μm . 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 . 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 .

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Figure 3Immuofluorescent staining for αv integrin expression in brain sections including intracranial tumor (T) and the surrounding healthy brain (B). Scale bar, 100μm.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.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.


Figure 4In 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 ) . 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 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 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 .

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Figure 4In 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).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 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 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.


Figure 5Hematoxylin 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 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 . Tumor immunostaining for apoptosis using an anti - caspase - 3 antibody . Scale bar , 100 μm .

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Figure 5Hematoxylin 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 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.Tumor immunostaining for apoptosis using an anti- caspase-3 antibody. Scale bar, 100 μm.


Figure 6Fluorescence - activated cell sorting ( FACS ) analysis with antibodies against Sox - 2 , Nestin and CD133 stem cell markers . Immunofluorescent staining for αv , β3 or β5 integrins in primary HSJD - GBM - 001 and G26 GBM cells . 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 .

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Figure 6Fluorescence-activated cell sorting (FACS) analysis with antibodies against Sox-2, Nestin and CD133 stem cell markers.Immunofluorescent staining for αv, β3 or β5 integrins in primary HSJD-GBM-001 and G26 GBM cells.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.


Figure 7Immunofluorescent staining for αv , β3 or β5 integrins in human normal astrocytes , chondrocytes , skin and lung fibroblasts . 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 .

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Figure 7Immunofluorescent staining for αv, β3 or β5 integrins in human normal astrocytes, chondrocytes, skin and lung fibroblasts.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.


Figure 8BLI of luciferase of representative tumor - bearing mice from all experimental groups at day 19 post therapy initiation . 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 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 .

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Figure 8BLI of luciferase of representative tumor-bearing mice from all experimental groups at day 19 post therapy initiation.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 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.


Figure 9Immunostaining analysis for αv , β3 or β5 integrins in GL261 cells . Scale bar , 50 μm . BLI of Luc of representative tumor - bearing mice from all experimental groups at day 34 post treatment . 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 . 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 . 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 .

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Figure 9Immunostaining analysis for αv, β3 or β5 integrins in GL261 cells. Scale bar, 50 μm.BLI of Luc of representative tumor-bearing mice from all experimental groups at day 34 post treatment.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.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.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.


Figure 1A . 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 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 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 cells were incubated in glucose - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting . 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 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 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 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 .

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Figure 1A. 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 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 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 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.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 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 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 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.


Figure 2A . 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 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 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 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 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 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 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 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 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 .

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

Figure 2A. 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 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 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 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 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 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 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 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 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.


Figure 3A . 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 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 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 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 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 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 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 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 .

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

Figure 3A. 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 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 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 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 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 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 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 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.


Figure 4A . 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 . 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 . 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 . 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 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 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 .

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

Figure 4A. 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.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.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. 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 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 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.


Figure 5A . 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 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 . 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 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 . 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 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 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 .

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

Figure 5A. 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 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. 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 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. 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 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 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.


Figure 6A . 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 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 . 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 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 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 .

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Figure 6A. 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 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. 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 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 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.


Figure 1Kaplan - 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 patient - matched breast tumor and lung metastasis . Scale bar , 50 μm . 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 . 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 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 cellsMammary 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 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 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 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

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

Figure 1Kaplan-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 patient-matched breast tumor and lung metastasis. Scale bar, 50 μm.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.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 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 cellsMammary 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 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 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 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


Figure 2Scatter 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 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 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 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 unitsHeat 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 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 .

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Figure 2Scatter 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 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 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 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 unitsHeat 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 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.


Figure 3Gene 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 cells grown as oncospheres . NES , normalized enrichment score . 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 . Normalized enrichment score ( GSEA ) of mammary stem cell signatures in oncospheres from MDA231 - LM2 cells . 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 cells following treatment with JNKi or vehicle control . Values are mean from 10 wells per condition ± SD . Representative images of spheresOncosphere 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 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 .

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Figure 3Gene 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 cells grown as oncospheres. NES, normalized enrichment score.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.Normalized enrichment score (GSEA) of mammary stem cell signatures in oncospheres from MDA231-LM2 cells.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 cells following treatment with JNKi or vehicle control. Values are mean from 10 wells per condition ± SD. Representative images of spheresOncosphere 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 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.


Figure 4SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi . 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 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 . 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 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 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 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 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 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 .

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Figure 4SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi.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 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.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 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 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 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 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 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.


Figure 5Gene 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 stem cell signatures that arise in response to paclitaxel treatment of MDA231 - LM2 cells . 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 . 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 ) and c - Jun in MDA231 - LM2 cells treated with PAX for 48 hours . Tubulin was used as a loading control . SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD . SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of doxorubicin ( DOXO ) . qPCR triplicates ± SD . 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 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 .

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Figure 5Gene 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 stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells.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.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) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD.SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD.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 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.


Figure 6Growth 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 . 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 . 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 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 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 miceRepresentative histology of human vimentin - positive lung metastases ( arrows )

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Figure 6Growth 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.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.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 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 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 miceRepresentative histology of human vimentin-positive lung metastases (arrows)


Figure 7Mammary 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 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 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 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 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 .

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Figure 7Mammary 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 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 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 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 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.


Figure 1 ( 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 ) 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 ) 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 ) 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 .

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Figure 1(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) 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) 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) 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.


Figure 2NOD2 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 .

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Figure 2NOD2 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.


Figure 3LL37 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 and IRGM protein levels were measured in cytosolic fractions by western blot analysis . ( 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 .

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Figure 3LL37 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 and IRGM protein levels were measured in cytosolic fractions by western blot analysis.(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.


Figure 4 ( 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 . 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 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 and IRGM protein was assessed by western blot . One representative experiment out of three experiments is presented . ( 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 .

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Figure 4(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.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 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 and IRGM protein was assessed by western blot. One representative experiment out of three experiments is presented.(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.


Figure 5 ( 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 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 .

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Figure 5(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 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.