Total eFluor780 + events in the BAL of influenza - infected mice described in A , B 10 days p . i . ( n = 3 mice from each group ) .

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Total eFluor780+ events in the BAL of influenza-infected mice described in A, B 10 days p.i. (n = 3 mice from each group).


Representative flow cytometric analysis of Csf2ra ( CD116 ) expression on different fetal precursors from WT embryos . The solid gray histogram represents the fluorescence minus one ( FMO ) controls , the open black histogram represents stained samples . The gray number represents mean fluorescence intensity ( MFI ) of FMO controls , the black number represents MFI of stained samples . E10 . 5 YS cells were pooled from 8 - 10 embryos , E14 . 5 lung cells were pooled from 3 - 4 embryos , and E14 . 5 liver cells were from individual embryo .

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Representative flow cytometric analysis of Csf2ra (CD116) expression on different fetal precursors from WT embryos. The solid gray histogram represents the fluorescence minus one (FMO) controls, the open black histogram represents stained samples. The gray number represents mean fluorescence intensity (MFI) of FMO controls, the black number represents MFI of stained samples. E10.5 YS cells were pooled from 8-10 embryos, E14.5 lung cells were pooled from 3-4 embryos, and E14.5 liver cells were from individual embryo.


Representative flow cytometric analysis of Csf2ra expressions on pMΦ - and fetal Mo - derived AM in Csf2ra - / - recipients 4 weeks after transfer of pMΦ or fetal Mo from E14 . 5 fetal livers . AM from 4 - week old WT mice were included as controls .

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Representative flow cytometric analysis of Csf2ra expressions on pMΦ- and fetal Mo-derived AM in Csf2ra-/- recipients 4 weeks after transfer of pMΦ or fetal Mo from E14.5 fetal livers. AM from 4-week old WT mice were included as controls.


Heatmap of AM signature - up genes ( D ) , AM signature - down genes ( E ) , and AM - specific transcription factors ( F ) in the precursors and mature AM described above in A . the data are from one RNA - seq experiment ( 1 - 2 biological replicates per group ) .

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Heatmap of AM signature-up genes (D), AM signature-down genes (E), and AM-specific transcription factors (F) in the precursors and mature AM described above in A. the data are from one RNA-seq experiment (1 - 2 biological replicates per group).


qPCR ( n = 3 biological replicates ) analysis of Myb , Maf , Mafb , and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice .

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qPCR (n = 3 biological replicates) analysis of Myb, Maf, Mafb, and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice.


Oxygen consumption rate ( OCR ) of pMΦ and fetal Mo from E14 . 5 WT fetal liver at baseline and after treatment with oligomycin ( Oligo ) , FCCP or rotenone ( Rot ) and antimycin A ( AA ) .

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Oxygen consumption rate (OCR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with oligomycin (Oligo), FCCP or rotenone (Rot) and antimycin A (AA).


Extracellular acidification rate ( ECAR ) of pMΦ and fetal Mo from E14 . 5 WT fetal liver at baseline and after treatment with glucose , Oligo , or 2 - Deoxy - D - glucose ( 2 - DG ) .

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Extracellular acidification rate (ECAR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with glucose, Oligo, or 2-Deoxy-D-glucose (2-DG).


RNA - seq ( C , n = 2 biological replicates ) analysis of Eno1 , Slc2a6 , Txn1 , and Txnip gene expression in pMΦ and fetal Mo from E14 . 5 WT fetal liver .

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RNA-seq (C, n = 2 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


qPCR ( D , n = 3 biological replicates ) analysis of Eno1 , Slc2a6 , Txn1 , and Txnip gene expression in pMΦ and fetal Mo from E14 . 5 WT fetal liver .

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qPCR (D, n = 3 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


FACS analysis of relative TCRβ + T cell content in blood ( left panel ) and tumors ( right panel ) of mice at the end of treatments as indicated in A . αCD4 / αCD8 : n = 10 ; IgG : n = 6 .

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FACS analysis of relative TCRβ+ T cell content in blood (left panel) and tumors (right panel) of mice at the end of treatments as indicated in A. αCD4/αCD8: n=10; IgG: n=6.


Tumor burden ( left panel ) , tumor number ( middle panel ) and average tumor size ( right panel ) per mouse assessed during colon resection at the end of treatments . αCD4 / αCD8 : n = 10 ; IgG : n = 9 .

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Tumor burden (left panel), tumor number (middle panel) and average tumor size (right panel) per mouse assessed during colon resection at the end of treatments. αCD4/αCD8: n=10; IgG: n=9.


Immune cell contexture in healthy colon and colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Relative TCRβ + T cell content in colon ( n = 10 ) and tumor ( n = 22 ) . relative content of individual cell types was determined in tissue - derived single cell suspension using cell type specific antibodies and FACS analysis .

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Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative TCRβ+ T cell content in colon (n=10) and tumor (n=22). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Immune cell contexture in healthy colon and colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Anti - CD3 immunostaining on normal colon ( left panel ) and a representative colon tumor section derived from an Apcfl / fl - Cdx2CreERT2 mouse ( right panel : higher magnification of area indicated in middle panel ) .

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Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Anti-CD3 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).


Immune cell contexture in healthy colon and colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Relative CD11b + myeloid cell content in colon ( n = 16 ) and tumors ( n = 16 ) . relative content of individual cell types was determined in tissue - derived single cell suspension using cell type specific antibodies and FACS analysis .

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative CD11b+ myeloid cell content in colon (n=16) and tumors (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Immune cell contexture in healthy colon and colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Anti - Gr1 immunostaining on normal colon ( left panel ) and a representative colon tumor section derived from an Apcfl / fl - Cdx2CreERT2 mouse ( right panel : higher magnification of area indicated in middle panel ) .

Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Anti-Gr1 immunostaining on normal colon (left panel) and a representative colon tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse (right panel: higher magnification of area indicated in middle panel).


Immune cell contexture in healthy colon and colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Relative CD11b + MHCII - Gr1hi neutrophil ( E ) and CD11b + MHCII - Gr1lo monocyte ( F ) content in colon ( n = 13 ) and tumors ( n = 16 ) . relative content of individual cell types was determined in tissue - derived single cell suspension using cell type specific antibodies and FACS analysis .

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Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Relative CD11b+ MHCII- Gr1hi neutrophil (E) and CD11b+ MHCII- Gr1lo monocyte (F) content in colon (n=13) and tumors (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Immune cell contexture in healthy colon and colon tumors of Apcfl / fl - Cdx2CreERT2 mice . , Correlation of tumor T cell numbers to sample - matched tumor - derived neutrophil ( left panel ) and monocyte ( right panel ) numbers ( n = 16 ) . relative content of individual cell types was determined in tissue - derived single cell suspension using cell type specific antibodies and FACS analysis .

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Immune cell contexture in healthy colon and colon tumors of Apcfl/fl-Cdx2CreERT2 mice. , Correlation of tumor T cell numbers to sample-matched tumor-derived neutrophil (left panel) and monocyte (right panel) numbers (n=16). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Percentage of T cell subsets among total CD45 + hematopoietic cells in colon ( n = 10 ) and tumor ( n = 11 ) . H , CD4 + TCRβ + T cells . I , CD8 + TCRβ + T cells . J , Foxp3 + CD4 + TCRβ + regulatory T cells ( Tregs ) . relative content of individual cell types was determined in tissue - derived single cell suspension using cell type specific antibodies and FACS analysis .

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Percentage of T cell subsets among total CD45+ hematopoietic cells in colon (n=10) and tumor (n=11). H, CD4+ TCRβ+ T cells. I, CD8+ TCRβ+ T cells. J, Foxp3+ CD4+ TCRβ+ regulatory T cells (Tregs). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


Percentage of Tregs among total T cells ( CD45 + TCRβ + ) in normal colon ( n = 10 ) or colon tumors ( n = 11 ) . relative content of individual cell types was determined in tissue - derived single cell suspension using cell type specific antibodies and FACS analysis .

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Percentage of Tregs among total T cells (CD45+ TCRβ+) in normal colon (n=10) or colon tumors (n=11). relative content of individual cell types was determined in tissue-derived single cell suspension using cell type specific antibodies and FACS analysis.


In vitro co - culture of activated T cells with increasing ratios of neutrophils , monocytes or macrophages . T cell proliferation index is numbers of proliferated T cells after three days of indicated co - culture condition relative to the number of proliferated T cells when cultured alone . CD4 + and CD8 + T cells were derived from lymph nodes of wild - type mice . Neutrophils , monocytes and macrophages were derived from colon tumors of Apcfl / fl - Cdx2CreERT2 mice . Each dot represents an individual neutrophil ( n = 4 ) , monocyte ( n = 3 ) or macrophage ( n = 4 ) sample . Bars represent mean + / - SD .

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In vitro co-culture of activated T cells with increasing ratios of neutrophils, monocytes or macrophages. T cell proliferation index is numbers of proliferated T cells after three days of indicated co-culture condition relative to the number of proliferated T cells when cultured alone. CD4+ and CD8+ T cells were derived from lymph nodes of wild-type mice. Neutrophils, monocytes and macrophages were derived from colon tumors of Apcfl/fl-Cdx2CreERT2 mice. Each dot represents an individual neutrophil (n=4), monocyte (n=3) or macrophage (n=4) sample. Bars represent mean +/- SD.


Tumor neutrophil ( left panel ) and monocyte ( right panel ) content after αGr1 + CXCR2i ( n = 7 ) or IgG + DMSO ( n = 10 ) treatments .

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Tumor neutrophil (left panel) and monocyte (right panel) content after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=10) treatments.


Average tumor volume per mouse at the end of αGr1 + CXCR2i ( n = 9 ) or IgG + DMSO ( n = 12 ) treatments as indicated in B .

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Average tumor volume per mouse at the end of αGr1 + CXCR2i (n=9) or IgG + DMSO (n=12) treatments as indicated in B.


Anti - CD3 immunostaining on representative tumor sections derived from mice after indicated treatments .

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Anti-CD3 immunostaining on representative tumor sections derived from mice after indicated treatments.


FACS analysis of CD45 + TCRβ + cells , CD45 + TCRβ + CD8 + cells and CD45 + TCRβ + CD4 + Foxp3 + cells in extracted tumors after αGr1 + CXCR2i ( n = 8 ) or IgG + DMSO ( n = 7 ) treatments .

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FACS analysis of CD45+ TCRβ+ cells, CD45+ TCRβ+ CD8+ cells and CD45+ TCRβ+ CD4+ Foxp3+ cells in extracted tumors after αGr1 + CXCR2i (n=8) or IgG + DMSO (n=7) treatments.


FACS analysis of IFNγ ( left panel ) and IL17A ( right panel ) expression in tumor extracted T cells after αGr1 + CXCR2i ( n = 7 ) or IgG + DMSO ( n = 7 ) treatments .

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FACS analysis of IFNγ (left panel) and IL17A (right panel) expression in tumor extracted T cells after αGr1 + CXCR2i (n=7) or IgG + DMSO (n=7) treatments.


Measurement of cytokines present in cell culture supernatants of T cells co - cultured with either tumor - derived neutrophils ( T + Ne ; n = 3 ) or with tumor - derived monocytes ( T + Mo ; n = 3 ) . Cytokines with a log2 fold change > 0 . 6 or < - 0 . 4 were considered as differentially present in supernatants . Shown is a heatmap of scaled cytokines levels ( z - score ) differentially present in T + Ne compared to T + Mo . Adjusted p - values were calculated by moderated t - test ( limma ) and Benjamini post test and were < 0 . 05 only for MMP9 and RELMβ . Supernatants of T cells alone ( T only ; n = 2 ) served as internal control . T cells cultured with monocytes did not have differential presence of cytokines compared to T cells alone .

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

Measurement of cytokines present in cell culture supernatants of T cells co-cultured with either tumor-derived neutrophils (T + Ne; n=3) or with tumor-derived monocytes (T + Mo; n=3). Cytokines with a log2 fold change >0.6 or <-0.4 were considered as differentially present in supernatants. Shown is a heatmap of scaled cytokines levels (z-score) differentially present in T + Ne compared to T + Mo. Adjusted p-values were calculated by moderated t-test (limma) and Benjamini post test and were <0.05 only for MMP9 and RELMβ. Supernatants of T cells alone (T only; n=2) served as internal control. T cells cultured with monocytes did not have differential presence of cytokines compared to T cells alone.


Co - immunostaining for MMP9 ( green ) and S100A9 ( red ) on a representative tumor section derived from an Apcfl / fl - Cdx2CreERT2 mouse . Right panel shows a higher magnification of indicated area of the left panel .

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Co-immunostaining for MMP9 (green) and S100A9 (red) on a representative tumor section derived from an Apcfl/fl-Cdx2CreERT2 mouse. Right panel shows a higher magnification of indicated area of the left panel.


mRNA expression levels of Mmp9 ( C ) or Tgfb1 , Tgfb2 and Tgfb3 ( D ) in indicated FACS sorted cell types isolated from mouse colon tumors ( n = 4 ) . Data are derived from RNA - sequencing and are displayed as reads per kilo base per million mapped reads ( rpkm ) .

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mRNA expression levels of Mmp9 (C) or Tgfb1, Tgfb2 and Tgfb3 (D) in indicated FACS sorted cell types isolated from mouse colon tumors (n=4). Data are derived from RNA-sequencing and are displayed as reads per kilo base per million mapped reads (rpkm).


Neutrophil - T cell co - cultures treated with vehicle ( T + Ne ) , 10µm Galunisertib ( T + Ne + TGFBRi ) , T cell proliferation after three days of indicated co - culture conditions . T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone .

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Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.


Neutrophil - T cell co - cultures treated with vehicle ( T + Ne ) , 10µm of the MMP inhibitor GM6001 ( T + Ne + MMPi ) Protein levels of free active TGFβ1 in cell culture supernatants of indicated conditions determined by ELISA .

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Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) Protein levels of free active TGFβ1 in cell culture supernatants of indicated conditions determined by ELISA.


Neutrophil - T cell co - cultures treated with vehicle ( T + Ne ) , 10µm of the MMP inhibitor GM6001 ( T + Ne + MMPi ) T cell proliferation after three days of indicated co - culture conditions . T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone .

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Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.


Neutrophil - T cell co - cultures treated with vehicle ( T + Ne ) , 10µm Galunisertib ( T + Ne + TGFBRi ) , 10µm of the MMP inhibitor GM6001 ( T + Ne + MMPi ) or with 10µm Galunisertib plus 10µm GM6001 ( T + Ne + TGFBRi + MMPi ) . T cell proliferation after three days of indicated co - culture conditions . T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone .

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

Neutrophil - T cell co-cultures treated with vehicle (T + Ne), 10µm Galunisertib (T + Ne + TGFBRi), 10µm of the MMP inhibitor GM6001 (T + Ne + MMPi) or with 10µm Galunisertib plus 10µm GM6001 (T + Ne + TGFBRi + MMPi). T cell proliferation after three days of indicated co-culture conditions. T cell proliferation index is numbers of proliferated T cells under indicated condition relative to the number of proliferated T cells when cultured alone.


Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg / kg of the MMP2 / 9 - inhibitor SB3 - CT ( MMPi ) . , Co - immunostaining for pSMAD3 ( green ) and IGFBP7 ( magenta ) on representative tumor - containing colon sections derived from Apcfl / fl - Cdx2CreERT2 mice treated with vehicle ( A ) MMPi ( B ) .

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Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). , Co-immunostaining for pSMAD3 (green) and IGFBP7 (magenta) on representative tumor-containing colon sections derived from Apcfl/fl-Cdx2CreERT2 mice treated with vehicle (A) MMPi (B).


Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg / kg of the MMP2 / 9 - inhibitor SB3 - CT ( MMPi ) . Quantification of pSMAD3 + cells ( left panel ) and pSMAD3 staining intensity per pSMAD3 + cell ( right panel ) on at least five arbitrarily selected tumor areas per section of MMPi ( n = 6 ) or vehicle ( n = 6 ) treated mice .

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Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). Quantification of pSMAD3+ cells (left panel) and pSMAD3 staining intensity per pSMAD3+ cell (right panel) on at least five arbitrarily selected tumor areas per section of MMPi (n=6) or vehicle (n=6) treated mice.


effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation . Apcfl / fl - Cdx2CreERT2 mice were treated with Tamoxifen and , one day post treatment , injected with either MMPi ( n = 10 ) or the TGFBRi SB431542 ( n = 10 ) at 40mg / kg for two weeks with five injections per week . Vehicle treated mice : n = 11 . Average tumor size per mouse was assessed during colon resection one week after the end of treatments ( E ) .

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effect of in vivo TGFBRi and MMPi treatment of mice for two weeks on colon tumor formation. Apcfl/fl-Cdx2CreERT2 mice were treated with Tamoxifen and, one day post treatment, injected with either MMPi (n=10) or the TGFBRi SB431542 (n=10) at 40mg/kg for two weeks with five injections per week. Vehicle treated mice: n=11. Average tumor size per mouse was assessed during colon resection one week after the end of treatments (E).


evaluation of S100A9 + neutrophil and CD8 + T cell infiltration in histologic sections of human CRC tumors . , Anti - S100A9 immunostaining on a representative human CRC section . Arrowheads indicate neutrophil infiltration at the tumor boarder . Co - immunostaining for CD8 ( brown ) and S100A9 ( gray - blue ) in the same CRC sample as shown in A .

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evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. , Anti-S100A9 immunostaining on a representative human CRC section. Arrowheads indicate neutrophil infiltration at the tumor boarder. Co-immunostaining for CD8 (brown) and S100A9 (gray-blue) in the same CRC sample as shown in A.


evaluation of S100A9 + neutrophil and CD8 + T cell infiltration in histologic sections of human CRC tumors . Quantification of S100A9 + cells ( upper panel ) and CD8 + T cells in different areas on CRC tumor sections containing tumor border with adjacent benign mucosa as determined by a specialized pathologist : " Tumor center " was tissue within tumor > 200μm from tumor border , " Tumor border " was tissue within tumor < 100μm from tumor border and " Adjacent benign mucosa " was mucosa tissue outside tumor < 100μm from tumor border . Per section , all identifiable tumor border and adjacent benign areas , as well as three arbitrary tumor center areas were scored . Sections were derived from 10 individual patients .

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evaluation of S100A9+ neutrophil and CD8+ T cell infiltration in histologic sections of human CRC tumors. Quantification of S100A9+ cells (upper panel) and CD8+ T cells in different areas on CRC tumor sections containing tumor border with adjacent benign mucosa as determined by a specialized pathologist: "Tumor center" was tissue within tumor >200μm from tumor border, "Tumor border" was tissue within tumor <100μm from tumor border and "Adjacent benign mucosa" was mucosa tissue outside tumor <100μm from tumor border. Per section, all identifiable tumor border and adjacent benign areas, as well as three arbitrary tumor center areas were scored. Sections were derived from 10 individual patients.


Gene expression scores were generated as described in material and methods . Boxes are lower and upper quartiles with median as solid lines ; horizontal lines define minimum and maximum ; dots define outliers . , Neutrophil scores in tumor samples categorized according to CMS subtypes .

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Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. , Neutrophil scores in tumor samples categorized according to CMS subtypes.


Gene expression scores were generated as described in material and methods . Boxes are lower and upper quartiles with median as solid lines ; horizontal lines define minimum and maximum ; dots define outliers . Comparison of T cell gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets ( median , lower and upper quartiles ; horizontal lines define minimum and maximum ; dots define outliers ) . Samples are categorized according to the medians ( high = > median ; low = neutrophil and TGFβ gene expression scores . Upper panel : TCGA dataset . Lower panel GSE39582 dataset .

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Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. Comparison of T cell gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets (median, lower and upper quartiles; horizontal lines define minimum and maximum; dots define outliers). Samples are categorized according to the medians (high = >median; low = neutrophil and TGFβ gene expression scores. Upper panel: TCGA dataset. Lower panel GSE39582 dataset.


Proliferation of activated blood T cells in vitro in co - culture with increasing ratios of CRC patient - derived autologous blood neutrophils . CD3 + T cells and CD66b + neutrophils were isolated and cultured individually for each patient . Bar graphs display relative numbers of proliferated T cells after three days of co - culture . Each dot per condition represents a neutrophil sample from an individual patient ( n = 13 ) . Bars represent mean + SD .

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Proliferation of activated blood T cells in vitro in co-culture with increasing ratios of CRC patient-derived autologous blood neutrophils. CD3+ T cells and CD66b+ neutrophils were isolated and cultured individually for each patient. Bar graphs display relative numbers of proliferated T cells after three days of co-culture. Each dot per condition represents a neutrophil sample from an individual patient (n=13). Bars represent mean + SD.


Proliferation of activated blood T cells in vitro in co - culture with autologous blood neutrophils . Cells were derived from both CRC patients ( n = 2 ) and healthy volunteers ( n = 5 ) . T cells and neutrophils were cultured at a 1 : 1 ratio and treated with vehicle ( T + Ne ) or 10µm Galunisertib ( T + Ne + TGFBRi ) . Each line represents a neutrophil sample from an individual healthy volunteer .

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Proliferation of activated blood T cells in vitro in co-culture with autologous blood neutrophils. Cells were derived from both CRC patients (n=2) and healthy volunteers (n=5). T cells and neutrophils were cultured at a 1:1 ratio and treated with vehicle (T + Ne) or 10µm Galunisertib (T + Ne + TGFBRi). Each line represents a neutrophil sample from an individual healthy volunteer.


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 .

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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 .

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 .

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 .

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.


Ferrozine - based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points . * P < 0 . 05 ( unpaired Student ' s t - test versus each time points with FeSO4 100 µM ) .

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Ferrozine-based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points. *P < 0.05 (unpaired Student's t-test versus each time points with FeSO4 100 µM).


Representative confocal microscope images of L6 cells stained with IP - 1 ( Iron Probe 1 ) or transfected with IRE - CFP ( Iron Regulatory Element ) reporter after iron treatment ( FeSO4 , 250 µM ) for 24 h .

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Representative confocal microscope images of L6 cells stained with IP-1 (Iron Probe 1) or transfected with IRE-CFP (Iron Regulatory Element) reporter after iron treatment (FeSO4, 250 µM) for 24 h.


Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment ( 250 µM , 24 h ) with iron chelator DPD ( 500 µM ) .

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Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment (250 µM, 24 h) with iron chelator DPD (500 µM).


Relative gene expressions - ferritin heavy chain ( FTH ) , ferritin light chain ( FTL ) , ferroportin ( SLC40A1 , transferrin receptor 1 ( tfr1 , TFRC ) - normalized to 18sr RNA expression after iron treatment ( FeSO4 , 250 µM ) for 24 h . * P < 0 . 05 ( unpaired Student ' s t - test versus Basal ) .

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Relative gene expressions - ferritin heavy chain (FTH), ferritin light chain (FTL), ferroportin (SLC40A1, transferrin receptor 1 (tfr1, TFRC) - normalized to 18sr RNA expression after iron treatment (FeSO4, 250 µM) for 24 h. *P < 0.05 (unpaired Student's t-test versus Basal).


Representative western blot images and quantification of ferritin ( heavy chain ) and tfr1 ( indicated with arrowhead ) over GAPDH after iron treatment ( 250 µM , 24 h ) .

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Representative western blot images and quantification of ferritin (heavy chain) and tfr1 (indicated with arrowhead) over GAPDH after iron treatment (250 µM, 24 h).


Glucose uptake of L6 cells with insulin stimulation ( 10 nM or 100 nM , 20 min ) after iron treatment ( 250 µM , 1 h or 24 h ) .

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Glucose uptake of L6 cells with insulin stimulation (10 nM or 100 nM, 20 min) after iron treatment (250 µM, 1 h or 24 h).


Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) over GAPDH with insulin stimulation ( 10 nM or 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) .

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Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) over GAPDH with insulin stimulation (10 nM or 100 nM, 5 min) after iron treatment (250 µM, 24 h).


Representative confocal microscope image of L6 cells stably expressing tandem fluorescent - eGFP - mCherry LC3B treated with iron ( FeSO4 , 250 µM ) for the indicated time points ( 4 h , 8 h , 24 h ) . Quantification of mean LC3B puncta per cell from ( A ) . Experiments were repeated three times , and one representative experiment is presented here .

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Representative confocal microscope image of L6 cells stably expressing tandem fluorescent-eGFP-mCherry LC3B treated with iron (FeSO4, 250 µM) for the indicated time points (4 h, 8 h, 24 h). Quantification of mean LC3B puncta per cell from (A). Experiments were repeated three times, and one representative experiment is presented here.


Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment ( FeSO4 , 250 µM ) at multiple time points .

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Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment (FeSO4, 250 µM) at multiple time points.


Representative western blot images and quantification of LC3B - II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine ( CQ , 30 µM ) . * Representative western blot images and quantification of LC3B - II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM .

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Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine (CQ, 30 µM). * Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM.


Representative confocal microscope z - stack image of mCherry - LC3B L6 cells after iron treatment ( 250 µM , 24 h ) .

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Representative confocal microscope z-stack image of mCherry-LC3B L6 cells after iron treatment (250 µM, 24 h).


Quantification of LC3B puncta size from ( F ) . Experiments were performed three times , and one representative experiment is presented here .

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Quantification of LC3B puncta size from (F). Experiments were performed three times, and one representative experiment is presented here.


Quantification of autophagosomes motility from live cell microscopy ( Supplementary Movie 1 ) from mCherry - LC3B L6 cells under basal , iron ( 250 µM , 24 h ) and starvation ( amino acid free ) condition . Experiments were performed three times , and one representative experiment is presented here . * P < 0 . 05 ( unpaired Student ' s t - test compared to Iron 24h ) .

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Quantification of autophagosomes motility from live cell microscopy (Supplementary Movie 1) from mCherry-LC3B L6 cells under basal, iron (250 µM, 24 h) and starvation (amino acid free) condition. Experiments were performed three times, and one representative experiment is presented here. *P < 0.05 (unpaired Student's t-test compared to Iron 24h).


Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment ( 250 µM , 24 h ) .

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Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment (250 µM, 24 h).


Representative confocal microscope images of eGFP - mCherry - LC3B L6 cells with Lysotracker DeepRed after iron treatment ( 250 µM , 24 h ) .

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Representative confocal microscope images of eGFP-mCherry-LC3B L6 cells with Lysotracker DeepRed after iron treatment (250 µM, 24 h).


Representative western blot images of phospho - ULK1 ( S757 ) , phospho - S6Kp70 ( T389 ) , total S6K , GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points . Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points .

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Representative western blot images of phospho-ULK1 (S757), phospho-S6Kp70 (T389), total S6K, GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points.


Representative epi - immunofluorescent images of L6 cells transfected with myc - RHEB Q46L and immuno - stained against LC3B and myc after iron treatment ( FeSO4 , 250 µM ) for 24 h .

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Representative epi-immunofluorescent images of L6 cells transfected with myc-RHEB Q46L and immuno-stained against LC3B and myc after iron treatment (FeSO4, 250 µM) for 24 h.


Ferrozine - based colorimetric measurement of intracellular iron in wild - type ( wt ) L6 and RHEB - Q64L L6 cells after iron treatment ( 50 µM or 250 µM ) for 24 h .

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Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h.


Representative confocal images of wt L6 and RHEB - Q64L L6 cells pulsed with Lysotracker Deep Red and immuno - stained against LC3B after iron treatment ( 250 µM , 24 h ) .

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Representative confocal images of wt L6 and RHEB-Q64L L6 cells pulsed with Lysotracker Deep Red and immuno-stained against LC3B after iron treatment (250 µM, 24 h).


Quantification of LC3B - free lysosome numbers in ( E ) . Experiments were repeated three times , and one representative experiment is presented here . * P < 0 . 05 ( multiple unpaired Student ' s t - test versus Basal in wt and RHEB Q64L cells )

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Quantification of LC3B-free lysosome numbers in (E). Experiments were repeated three times, and one representative experiment is presented here. *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)


Representative TEM images of wt L6 and RHEB - Q64L cells after iron treatment ( 250 µM , 24 h ) .

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Representative TEM images of wt L6 and RHEB-Q64L cells after iron treatment (250 µM, 24 h).


Representative western blot images and quantifications of phospho - IRS1 ( Y612 , indicated by arrowhead ) and AKT ( T308 , indicated by arrowhead ) to GAPDH in wt and RHEB - Q64L L6 cells stimulated with insulin ( 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) . * P < 0 . 05 ( multiple unpaired Student ' s t - test versus Basal in wt and RHEB Q64L cells )

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Representative western blot images and quantifications of phospho-IRS1 (Y612, indicated by arrowhead) and AKT (T308, indicated by arrowhead) to GAPDH in wt and RHEB-Q64L L6 cells stimulated with insulin (100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)


Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment ( 250 µM , 24 h ) followed by 24 h iron withdrawal . Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal .

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Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment (250 µM, 24 h) followed by 24 h iron withdrawal. Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment (250 µM, 24 h) followed by 24 h withdrawal.


Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) to GAPDH in L6 cells iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal .

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Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in L6 cells iron treatment (250 µM, 24 h) followed by 24 h withdrawal.


Representative epi - immunofluorescent microscope images of L6 cells immuno - stained against LC3B and LAMP1 after iron treatment ( 250 µM , 24 h ) followed by 3 h withdrawal .

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Representative epi-immunofluorescent microscope images of L6 cells immuno-stained against LC3B and LAMP1 after iron treatment (250 µM, 24 h) followed by 3 h withdrawal.


Quantification of autophagosome - free lysosomes in ( E ) . Experiments were performed three times , and one representative experiment is presented here .

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Quantification of autophagosome-free lysosomes in (E). Experiments were performed three times, and one representative experiment is presented here.


Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h or 24 h .

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Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h or 24 h.


Representative western blot images and quantification of phospho - mTOR S2448 , total mTOR and actin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h .

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Representative western blot images and quantification of phospho-mTOR S2448, total mTOR and actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h.


Representative western blot images of phospho - mTOR S2448 , total mTOR phsophpo - TSC2 T1462 , total TSC2 , phospho - AKT S473 , total AKT , actin in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without MK2206 ( AKT inhibitor . )

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Representative western blot images of phospho-mTOR S2448, total mTOR phsophpo-TSC2 T1462, total TSC2, phospho-AKT S473, total AKT, actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without MK2206 (AKT inhibitor.)


Representative western blot images of phospho - UVRAG S550 and total UVRAG in L6 cells transfected with FLAG - UVRAG and FLAG pulldown after iron treatment ( 250 µM , 4 h and 24 h ) followed by withdrawal for 1 h and 3 h .

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Representative western blot images of phospho-UVRAG S550 and total UVRAG in L6 cells transfected with FLAG-UVRAG and FLAG pulldown after iron treatment (250 µM, 4 h and 24 h) followed by withdrawal for 1 h and 3 h.


Representative western blot images of endogenous phospho - UVRAG S550 , total UVRAG expression in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without torin1 ( 200 nM ) .

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Representative western blot images of endogenous phospho-UVRAG S550, total UVRAG expression in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without torin1 (200 nM).


Live cell imaging analysis of L6 cells transfected with LAMP1 - RFP from Movie EV2 : lysosomal number in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) .

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Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: lysosomal number in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).


Live cell imaging analysis of L6 cells transfected with LAMP1 - RFP from Movie EV2 : speed ( F ) in L6 cells after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) .

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Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: speed (F) in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).


Representative confocal images of time - lapse captures of lysosomes at 6 sec intervals for 30 sec . White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds , and the traces of puncta were shown as lines in final panel .

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Representative confocal images of time-lapse captures of lysosomes at 6 sec intervals for 30 sec. White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds, and the traces of puncta were shown as lines in final panel.


Prussian Perl blue staining in liver and skeletal muscle after iron injections .

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Prussian Perl blue staining in liver and skeletal muscle after iron injections.


Representative western blot images and quantification of ferritin and tfr1 ( indicated by arrowhead ) to tubulin in skeletal muscles 24 h after iron injections .

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Representative western blot images and quantification of ferritin and tfr1 (indicated by arrowhead) to tubulin in skeletal muscles 24 h after iron injections.


Insulin tolerance test 24 h after iron injections .

Insulin tolerance test 24 h after iron injections.


Glucose tolerance test ( GTT ) 24 h after iron injections .

Glucose tolerance test (GTT) 24 h after iron injections.


Insulin tolerance test 24 h after iron injections . Quantification of area under curve in ITT ( E ) .

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Insulin tolerance test 24 h after iron injections. Quantification of area under curve in ITT (E).


Glucose tolerance test ( GTT ) 24 h after iron injections . Quantification of area under curve for panel ( F ) .

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Glucose tolerance test (GTT) 24 h after iron injections. Quantification of area under curve for panel (F).


Representative western blot images and quantification of phospho - IRS1 ( Y612 ) and phospho - AKT ( T308 ) to GAPDH in skeletal muscles 24 h after iron injection followed by i . p . insulin injection .

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Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in skeletal muscles 24 h after iron injection followed by i.p. insulin injection.


Representative confocal microscope images of skeletal muscle tissue sections immuno - stained against LC3B ( Alexa 647 ) and LAMP1 ( Alexa 555 ) Quantification of lysosomes ( LAMP1 puncta ) in skeletal muscles 24 h after iron injections .

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Representative confocal microscope images of skeletal muscle tissue sections immuno-stained against LC3B (Alexa 647) and LAMP1 (Alexa 555) Quantification of lysosomes (LAMP1 puncta) in skeletal muscles 24 h after iron injections.


Representative TEM images of skeletal muscle 24 h after iron injections .

Representative TEM images of skeletal muscle 24 h after iron injections.


Representative western blot images of phospho - UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections .

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Representative western blot images of phospho-UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections.


Representative western blot images and quantification of LC3 - B and p62 to tubulin in skeletal muscles 24 h after iron injections .

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Representative western blot images and quantification of LC3-B and p62 to tubulin in skeletal muscles 24 h after iron injections.


( B - C ) Western blot analysis ( B ) and densitometric quantification ( C ) of OTUD1 in aortas of control and Ang II - induced mice . β - actin was used as the loading control ( n = 5 biological replicates ) . ( D ) mRNA levels of OTUD1 in aortas of control and Ang II - induced mice were determined by RT - qPCR . The values were normalized to Rn18s ( n = 5 biological replicates ) .

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(B-C) Western blot analysis (B) and densitometric quantification (C) of OTUD1 in aortas of control and Ang II-induced mice. β-actin was used as the loading control (n=5 biological replicates). (D) mRNA levels of OTUD1 in aortas of control and Ang II-induced mice were determined by RT-qPCR. The values were normalized to Rn18s (n=5 biological replicates).


( E ) Representative immunofluorescence staining of CD31 ( green ) and OTUD1 ( red ) of aortic sections ( scale bar = 25 μm ) . ( F ) Representative immunofluorescence staining of α - SMA ( green ) and OTUD1 ( red ) of aortic sections ( scale bar = 25 μm ) .

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(E) Representative immunofluorescence staining of CD31 (green) and OTUD1 (red) of aortic sections (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and OTUD1 (red) of aortic sections (scale bar=25 μm).


( G - H ) Time - course of OTUD1 induction in response to Ang II in HUVECs . HUVECs were exposed to 1 μM Ang II for indicated time . Western blot analysis ( G ) and densitometric quantification ( H ) of OTUD1 were shown ( n = 3 biological replicates ) . GAPDH was used as the loading control .

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(G-H) Time-course of OTUD1 induction in response to Ang II in HUVECs. HUVECs were exposed to 1 μM Ang II for indicated time. Western blot analysis (G) and densitometric quantification (H) of OTUD1 were shown (n = 3 biological replicates). GAPDH was used as the loading control.


( A ) Representative images of H & E staining of aortic sections of the mice from four groups ( scale bar = 25 μm ) . E , endothelium ; M , media ; A , adventitia . ( B ) The quantification of arterial wall thickness in panel A ; n = 5 biological replicates . ( C ) Representative images of Masson ' s Trichrome staining of aortic sections of the mice ( scale bar = 50 μm ) . ( D ) The quantification of collagen deposition in panel C ; n = 5 biological replicates .

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(A) Representative images of H&E staining of aortic sections of the mice from four groups (scale bar=25 μm). E, endothelium; M, media; A, adventitia. (B) The quantification of arterial wall thickness in panel A; n=5 biological replicates. (C) Representative images of Masson's Trichrome staining of aortic sections of the mice (scale bar=50 μm). (D) The quantification of collagen deposition in panel C; n=5 biological replicates.


( E - F ) Western blot analysis ( E ) of VE - cadherin , Vimentin , Twist , Snail , and OTUD1 and densitometric quantification ( F ) of VE - cadherin , Vimentin , Twist and Snail in aortic tissues of the mice . β - actin was used as the loading control ( n = 5 biological replicates ) .

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(E-F) Western blot analysis (E) of VE-cadherin, Vimentin, Twist, Snail, and OTUD1 and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues of the mice. β-actin was used as the loading control (n=5 biological replicates).


( G - H ) mRNA levels of Cdh5 , Vim , Twist1 , Snai1 ( G ) , Tgfb1 and Col1a1 ( H ) in aortic tissues of the mice were examined by real - time qPCR assay . The values were normalized to Rn18s ( n = 5 biological replicates ) .

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(G-H) mRNA levels of Cdh5, Vim, Twist1, Snai1 (G), Tgfb1 and Col1a1 (H) in aortic tissues of the mice were examined by real-time qPCR assay. The values were normalized to Rn18s (n=5 biological replicates).


( B ) HUVECs transfected with si - OTUD1 or NC were challenged with 1 μM Ang II for 24 h . Western blot analysis of VE - cadherin , Vimentin , α - SMA , Twist and Snail were performed . GAPDH was used as the loading control .

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(B) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( E ) HUVECs transfected with Flag - OTUD1 or EV were challenged with 1 μM Ang II for 24 h . Western blot analysis of VE - cadherin , Vimentin , α - SMA , Twist and Snail were performed . GAPDH was used as the loading control .

(E) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( C ) Co - immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag - OTUD1 . Exogenous Flag - OTUD1 was immunoprecipitated by anti - Flag antibody . IgG , immunoglobulin G .

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(C) Co-immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag-OTUD1. Exogenous Flag-OTUD1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G.


( G ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub and Flag - OTUD1 and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( H ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub , and Flag - OTUD1 or Flag - OTUD1 ( C320S ) , and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( I ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , Flag - OTUD1 , and HA - Ub , HA - Ub - K48 ( K48 only ) , or HA - Ub - K63 ( K63 only ) , respectively , and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody .

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(G) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (H) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and Flag-OTUD1 or Flag-OTUD1(C320S), and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (I) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, Flag-OTUD1, and HA-Ub, HA-Ub-K48 (K48 only), or HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody.


( A - B ) 293T cells were transfected with various amount of Flag - OTUD1 . Western blot analysis ( A ) and densitometric quantification ( B ) of SMAD3 . GAPDH was used as the loading control ( n = 3 biological replicates ) .

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(A-B) 293T cells were transfected with various amount of Flag-OTUD1. Western blot analysis (A) and densitometric quantification (B) of SMAD3. GAPDH was used as the loading control (n=3 biological replicates).


( E - F ) Western blot analysis ( E ) and densitometric quantification ( F ) of SMAD3 in aortic tissues of the mice from four groups . β - actin was used as the loading control ( n = 5 biological replicates ) .

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(E-F) Western blot analysis (E) and densitometric quantification (F) of SMAD3 in aortic tissues of the mice from four groups. β-actin was used as the loading control (n=5 biological replicates).


( G ) Co - immunoprecipitation of SMAD3 and SMAD4 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub , and HA - Ub - K63 ( K63 only ) , respectively , and then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody .

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(G) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody.


( H ) Co - immunoprecipitation of SMAD3 and SMAD4 in 293T cells co - transfected with GFP - SMAD3 and Flag - OTUD1 and then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody . IgG , immunoglobulin G .

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(H) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3 and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. IgG, immunoglobulin G.


( A - B ) Representative images ( A ) of H & E staining of aortic sections ( scale bar = 25 μm ) and quantification of arterial wall thickness ( B ) ; n = 5 biological replicates . E , endothelium ; M , media ; A , adventitia . ( C - D ) Representative images ( C ) of Masson ' s Trichrome staining of aortic sections ( scale bar = 25 μm ) and quantification of collagen deposition ( D ) ; n = 5 biological replicates .

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(A-B) Representative images (A) of H&E staining of aortic sections (scale bar=25 μm) and quantification of arterial wall thickness (B); n=5 biological replicates. E, endothelium; M, media; A, adventitia. (C-D) Representative images (C) of Masson's Trichrome staining of aortic sections (scale bar=25 μm) and quantification of collagen deposition (D); n=5 biological replicates.


( E - F ) Western blot analysis ( E ) and densitometric quantification ( F ) of VE - cadherin , Vimentin , Twist and Snail in aortic tissues . β - actin was used as the loading control ( n = 5 biological replicates ) .

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(E-F) Western blot analysis (E) and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues. β-actin was used as the loading control (n=5 biological replicates).


( C ) Immunocytochemistry showing post - synaptic marker PSD - 95 ( red ) , pre - synaptic marker Synapsin - I ( green ) , dendritic marker Map2 ( blue ) and co - localization of PSD - 95 and Synapsin - I from dissociated Nedd4 - 2 WT or Nedd4 - 2 cKO hippocampal neurons at DIV 16 . Representative secondary dendrites ( left ) and quantification of colocalized synaptic puncta number and area ( right ) ( n = 36 for Nedd4 - 2 WT and Nedd4 - 2 cKO neurons ) are shown . Scale bar : 5 µm .

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(C) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from dissociated Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons at DIV 16. Representative secondary dendrites (left) and quantification of colocalized synaptic puncta number and area (right) (n = 36 for Nedd4-2 WT and Nedd4-2 cKO neurons) are shown. Scale bar: 5 µm.


( F ) A train of high frequency stimulation ( HFS ; 100 Hz for 1s ) induced early - phase LTP ( E - LTP ) at Schaffer collateral synapses in Nedd4 - 2 WT ( n = 7 slices from 6 mice ) and Nedd4 - 2 cKO ( n = 6 slices from 5 mice ) mice . Representative fEPSP traces were recorded before and 60 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing the fEPSP slopes measured 50 - 60 min after a HFS at Schaffer collateral synapses was on the right . ( G ) Late - phase LTP ( L - LTP ) induced by four trains of HFS in Nedd4 - 2 WT slices ( n = 7 slices from 7 mice ) and Nedd4 - 2 cKO slices ( n = 9 slices from 8 mice ) . Representative fEPSP traces were recorded before and 180 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing the fEPSP slopes measured 160 - 180 min after L - LTP induction at Schaffer collateral synapses was on the right .

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(F) A train of high frequency stimulation (HFS; 100 Hz for 1s) induced early-phase LTP (E-LTP) at Schaffer collateral synapses in Nedd4-2 WT (n = 7 slices from 6 mice) and Nedd4-2 cKO (n = 6 slices from 5 mice) mice. Representative fEPSP traces were recorded before and 60 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 50-60 min after a HFS at Schaffer collateral synapses was on the right. (G) Late-phase LTP (L-LTP) induced by four trains of HFS in Nedd4-2 WT slices (n = 7 slices from 7 mice) and Nedd4-2 cKO slices (n = 9 slices from 8 mice). Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses was on the right.


( B ) Barnes maze test from Nedd4 - 2 WT ( n = 8 ) or Nedd4 - 2 cKO ( n = 8 ) mice ( 6 - 8 weeks old ) . Schematic representation of Barnes maze ( top left ) . Mice were trained for 4 days with three training trials on each day to locate the escape box ( black circle ) . Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping ( top right ) . Schematic representation of Barnes maze on probe trials ( bottom left ) . Quadrant occupancy on probe trial at day5 and at day12 ( bottom right ) were assessed by the time spent in the target area ( green quadrant ) .

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(B) Barnes maze test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of Barnes maze (top left). Mice were trained for 4 days with three training trials on each day to locate the escape box (black circle). Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping (top right). Schematic representation of Barnes maze on probe trials (bottom left). Quadrant occupancy on probe trial at day5 and at day12 (bottom right) were assessed by the time spent in the target area (green quadrant).


( B ) Western blots of F - actin to G - actin ratio with or without cLTP induction from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured neurons . Schematic representation of the experimental design for cLTP inductions ( top left ) . Representative western blots ( bottom left ) and quantification of F - actin to G - actin from Nedd4 - 2 WT or Nedd4 - 2 cKO ( right ) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis ( n = 11 and 8 for Nedd4 - 2 WT and Nedd4 - 2 cKO cultures , respectively ) . For quantification , data from cLTP induction were normalized to data from control ( CTL ) condition .

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(B) Western blots of F-actin to G-actin ratio with or without cLTP induction from Nedd4-2 WT or Nedd4-2 cKO cultured neurons. Schematic representation of the experimental design for cLTP inductions (top left). Representative western blots (bottom left) and quantification of F-actin to G-actin from Nedd4-2 WT or Nedd4-2 cKO (right) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis (n = 11 and 8 for Nedd4-2 WT and Nedd4-2 cKO cultures, respectively). For quantification, data from cLTP induction were normalized to data from control (CTL) condition.


( A ) Western blots of Nedd4 - 2 , total cofilin , phospho ( p ) - cofilin and GAPDH from Nedd4 - 2 WT or Nedd4 - 2 cKO forebrains at postnatal day 14 . Representative western blots and quantification of total cofilin relative to GAPDH and p - cofilin relative to total cofilin ( n = 6 for Nedd4 - 2 WT and Nedd4 - 2 cKO brains ) were shown .

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(A) Western blots of Nedd4-2, total cofilin, phospho (p)-cofilin and GAPDH from Nedd4-2 WT or Nedd4-2 cKO forebrains at postnatal day 14. Representative western blots and quantification of total cofilin relative to GAPDH and p-cofilin relative to total cofilin (n = 6 for Nedd4-2 WT and Nedd4-2 cKO brains) were shown.


( B ) Immunocytochemistry of cofilin , p - cofilin and dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons at DIV 16 . Representative dendrites of Nedd4 - 2 WT and Nedd4 - 2 cKO neurons and quantification of total cofilin relative to MAP2 and p - cofilin relative to total cofilin are shown ( n = 25 and 26 for Nedd4 - 2 WT and Nedd4 - 2 cKO neurons , respectively ) . Scale bar : 5 µm .

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(B) Immunocytochemistry of cofilin, p-cofilin and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16. Representative dendrites of Nedd4-2 WT and Nedd4-2 cKO neurons and quantification of total cofilin relative to MAP2 and p-cofilin relative to total cofilin are shown (n = 25 and 26 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively). Scale bar: 5 µm.


( C ) Immunocytochemistry showing cofilin , p - cofilin , and dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16 . Representative dendrites and quantification from Nedd4 - 2 WT or Nedd4 - 2 cKO cultures are shown ( n = 36 - 39 neurons ) . Scale bar : 5 µm . For quantification , data from cLTP induction were normalized to data from control ( CTL ) condition .

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(C) Immunocytochemistry showing cofilin, p-cofilin, and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16. Representative dendrites and quantification from Nedd4-2 WT or Nedd4-2 cKO cultures are shown (n = 36-39 neurons). Scale bar: 5 µm. For quantification, data from cLTP induction were normalized to data from control (CTL) condition.