immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts . The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib ( 400nM ) and the SIRT1 inhibitor EX 527 ( 1 μM ) for 24 hours . Data are representative four replicates . All values are presented as mean ± SD of four replicates . One - way ANOVA was performed on DC cells in indicated conditions .

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immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.


Intracellular NAD levels in brain tissues from 6 - month - old G1 and G3 Tert - / - female mice .

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Intracellular NAD levels in brain tissues from 6-month- old G1 and G3 Tert-/- female mice.


immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6 - month - old G1 and G3 Tert - / - female mice . The CD38 antibody was validated using the CD38 - / - mouse brain tissues .

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immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues.


C Quantification of immunoblots from the indicated proteins in B . Data information : All values are presented as mean ± SD of G1 Tert - / - mouse brain tissues versus G3 Tert - / - mouse brain tissues . n = 4 in each group . P values on the basis of Student ' s t test .

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C Quantification of immunoblots from the indicated proteins in B. Data information: All values are presented as mean ± SD of G1 Tert-/- mouse brain tissues versus G3 Tert-/- mouse brain tissues. n=4 in each group. P values on the basis of Student's t test.


D NADase activity in G1 and G3 Tert - / - mouse brain lysate was determined at various time points during the reaction . NADase activity was not detectable in the extracts treated with the CD38 inhibitor , 78c , at a concentration of 50nM .

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D NADase activity in G1 and G3 Tert-/- mouse brain lysate was determined at various time points during the reaction. NADase activity was not detectable in the extracts treated with the CD38 inhibitor, 78c, at a concentration of 50nM.


A Representative immunoblots of the expression of PGC1 - 1α in DC cells treated with vehicle or 3 mM NR . Quantification of the indicated proteins is from three immunoblots .

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A Representative immunoblots of the expression of PGC1-1α in DC cells treated with vehicle or 3 mM NR. Quantification of the indicated proteins is from three immunoblots.


B - D Cellular and mitochondrial ROS in DC and age - matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates .

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B-D Cellular and mitochondrial ROS in DC and age-matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates.


Cellular and mitochondrial ROS in DC and age - matched healthy control cells in the scramble and CD38 knockdown DC cells ( D ) were measured by flow cytometry from three replicates .

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Cellular and mitochondrial ROS in DC and age-matched healthy control cells in the scramble and CD38 knockdown DC cells (D) were measured by flow cytometry from three replicates.


E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR . White arrows : mitochondria . Yellow arrow : autophagosome - like structures with engulfed mitochondria . Enlarged image within white frame is shown . F - H Quantifications of percentage of damaged mitochondria per cell ( F ) , mitochondrial length ( G ) , and mitochondrial diameter ( H ) . A minimum of 200 mitochondria counted per group .

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E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR. White arrows: mitochondria. Yellow arrow: autophagosome-like structures with engulfed mitochondria. Enlarged image within white frame is shown. F-H Quantifications of percentage of damaged mitochondria per cell (F), mitochondrial length (G), and mitochondrial diameter (H). A minimum of 200 mitochondria counted per group.


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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


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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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 staining for αv integrin expression in brain sections including intracranial tumor ( T ) and the 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 - 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 .

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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.


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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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 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 ) .

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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).


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 .

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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 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 .

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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 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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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 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 .

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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 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 .

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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.


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

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


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

Fluorescence-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 .

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

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Immunofluorescent 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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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 tumor - bearing mice from all experimental groups at day 19 post therapy initiation .

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BLI 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 .

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 .

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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 .

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


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

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Immunostaining 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 .

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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 .

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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 all experimental groups , before and 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 cells were incubated in serum - free medium for the indicated hours . Cells were lysed and the cell 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 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 .

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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 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 .

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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 .

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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 . 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 .

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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 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 .

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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 .

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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 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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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were transfected with siRNA for GFP and LRP6 . After transfection , cells were lysed and the cell 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 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 .

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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 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 .

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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 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 .

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

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 .

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

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 .

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

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 .

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

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

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 . 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 .

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

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.


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 .

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

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 .

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 .

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

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 .

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

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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 . 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 .

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

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 .

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

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

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 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 .

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

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 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 .

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

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 .

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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 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 .

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

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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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 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 .

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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 patient - matched breast tumor and 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.


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 .

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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.


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

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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 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

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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 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 .

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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 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 .

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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 tumor - 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 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

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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 ) 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 .

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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 stem cell signature ( Pece et al , 2010 ) in MDA231 - LM2 cancer cells 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 the same mammary stem cell signature in cancer cells 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 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

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

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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 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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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 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 .

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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 cells grown as oncospheres . NES , normalized enrichment score .

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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 .

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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 .

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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 .

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 .

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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 cells following treatment with JNKi or vehicle control . Values are mean from 10 wells per condition ± SD . Representative images 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 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 .

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

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SPP1 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 ) ) .

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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 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 .

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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.


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 .

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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 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 .

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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 analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients ( 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 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 .

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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.