Figure 3A Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmid constructs encoding EGFP or EGFP + Cre ( Cre ) . Upper boundary of layer V indicted with dotted line . EGFP signal is shown as green pseudocolor . DAPI staining ( blue ) marks cell nuclei . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . B Number of EGFP + Cre neurons migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * * P < 0 . 001 to EGFP . Data are presented as mean ± S . E . M . , n ( EGFP + Cre ) = 8 embryos ; n ( EGFP ) = 8 embryos . C Representative image of E18 . 5 coronal brain sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmid constructs encoding mRFP or mRFP + Cre ( Cre ) . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . mRFP signal is shown as green pseudocolor . DAPI staining ( blue ) marks cell nuclei . D Number of mRFP + Cre neurons migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * * P < 0 . 001 to mRFP . Data are presented as mean ± S . E . M . , n ( mRFP + Cre ) = 5 embryos ; n ( mRFP ) = 5 embryos . E Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos co - electroporated in utero at E14 . 5 with plasmids encoding a fluorescent marker ( green ) together and Cre + KCC2FL , Cre + KCC2CTD , or Cre + KCC2R952H . DAPI staining ( blue ) marks cell nuclei . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . F Number of transfected neurons migrating above ( II - IV , left ) and below ( V - VI / IZ - SVZ , right ) the upper border of layer V in embryos electroporated with constructs in ( E ) normalized to respective data from embryos electroporated with Cre . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 to Cre . Data are presented as mean ± S . E . M . , n ( Cre + KCC2FL ) = 13 embryos ; n ( Cre + KCC2CTD ) = 9 embryos ; n ( Cre + KCC2R952H ) = 9 embryos .

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

Figure 3A Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding EGFP or EGFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. EGFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. B Number of EGFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to EGFP. Data are presented as mean ± S.E.M., n (EGFP +Cre) = 8 embryos; n (EGFP) = 8 embryos.C Representative image of E18.5 coronal brain sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding mRFP or mRFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. mRFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. D Number of mRFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to mRFP. Data are presented as mean ± S.E.M., n (mRFP+Cre) = 5 embryos; n (mRFP) = 5 embryos. E Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos co-electroporated in utero at E14.5 with plasmids encoding a fluorescent marker (green) together and Cre+KCC2FL, Cre+KCC2CTD, or Cre+KCC2R952H. DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. F Number of transfected neurons migrating above (II-IV, left) and below (V-VI/IZ-SVZ, right) the upper border of layer V in embryos electroporated with constructs in (E) normalized to respective data from embryos electroporated with Cre. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 to Cre. Data are presented as mean ± S.E.M., n (Cre+KCC2FL) = 13 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos.


Figure 4A , B , C The layer thickness and neuronal number within each layer was assessed using layer - specific markers in Kcc2 - / - and Kcc2 + / + embryos at E18 . 5 . Cux1 was used to label layers II - IV ( A ) , Ctip2 to label layer V ( B ) , and Tbr1 to label layer VI ( C ) . Dashed white lines in the representative images in ( A - C ) indicate upper and lower layer boundaries . Sp , subplate . Statistical significance was determined using Kruskal - Wallis test with Dunn ' s post hoc ( neuronal numbers ) and one - way ANOVA with Holm - Sidak ' s post hoc ( layer thickness ) . Data are presented as mean ± S . E . M . , n = 11 embryos for both genotypes . Scale bar = 50 µm . D Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) in Kcc2 - / - and Kcc2 + / + embryos electroporated in utero at E14 . 5 with a plasmid encoding EGFP ( green ) . DAPI staining ( blue ) marks cell nuclei . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . E Neurons from the brain sections in ( D ) were quantified and the total number of EGFP + neurons in the Kcc2 - / - sections is presented as a percentage of pooled Kcc2 + / + and Kcc2 + / - values . Statistical significance was determined using one - tailed Student ' s t test , * P < 0 . 05 . Data are presented as mean ± S . E . M . , n ( Kcc2 + / + + Kcc2 + / - ) = 14 embryos ; n ( Kcc2 - / - ) = 9 embryos . F Number of EGFP + neurons in Kcc2 - / - embryos migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective pooled data from Kcc2 + / + and Kcc2 + / - embryos . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 to Kcc2 + / + + Kcc2 + / - . Data are presented as mean ± S . E . M . , n ( Kcc2 + / + + Kcc2 + / - ) = 14 embryos ; n ( Kcc2 - / - ) = 9 embryos .

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

Figure 4A, B, C The layer thickness and neuronal number within each layer was assessed using layer-specific markers in Kcc2-/-and Kcc2+/+ embryos at E18.5. Cux1 was used to label layers II-IV (A), Ctip2 to label layer V (B), and Tbr1 to label layer VI (C). Dashed white lines in the representative images in (A-C) indicate upper and lower layer boundaries. Sp, subplate. Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc (neuronal numbers) and one-way ANOVA with Holm-Sidak's post hoc (layer thickness). Data are presented as mean ± S.E.M., n = 11 embryos for both genotypes. Scale bar = 50 µm.D Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) in Kcc2-/- and Kcc2+/+ embryos electroporated in utero at E14.5 with a plasmid encoding EGFP (green). DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. E Neurons from the brain sections in (D) were quantified and the total number of EGFP+ neurons in the Kcc2-/- sections is presented as a percentage of pooled Kcc2+/+ and Kcc2+/- values. Statistical significance was determined using one-tailed Student's t test, *P < 0.05. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos. F Number of EGFP+ neurons in Kcc2-/- embryos migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective pooled data from Kcc2+/+ and Kcc2+/- embryos. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Kcc2+/+ + Kcc2+/-. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos.


Figure 1 ( B ) Representative images of GFAP + Sox2 + BrdU + RGLs , GFAP - Sox2 + BrdU + progenitors , Tbr2 + BrdU + IPCs in the DG . Scale bar = 20 μm . ( C ) Quantification of GFAP + Sox2 + BrdU + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 022 ) , GFAP - Sox2 + BrdU + progenitors ( WT vs . KO , Student ' s t - test , p = 0 . 0046 ) , Tbr2 + BrdU + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 4 mice ; KO , n = 6 mice . ( D ) Representative images of GFAP + Mcm2 + RGLs , Tbr2 + Mcm2 + IPCs , DCX + Mcm2 + NBs in the DG . Scale bar = 20 μm . ( E ) Quantification of GFAP + Mcm2 + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 016 ) , Tbr2 + Mcm2 + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) , DCX + Mcm2 + NBs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 3 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 1(B) Representative images of GFAP+Sox2+BrdU+ RGLs, GFAP-Sox2+BrdU+ progenitors, Tbr2+BrdU+ IPCs in the DG. Scale bar = 20 μm. (C) Quantification of GFAP+Sox2+BrdU+ RGLs (WT vs. KO, Student's t-test, p = 0.022), GFAP-Sox2+BrdU+ progenitors (WT vs. KO, Student's t-test, p = 0.0046), Tbr2+BrdU+ IPCs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 4 mice; KO, n = 6 mice. (D) Representative images of GFAP+Mcm2+ RGLs, Tbr2+Mcm2+ IPCs, DCX+Mcm2+ NBs in the DG. Scale bar = 20 μm. (E) Quantification of GFAP+Mcm2+ RGLs (WT vs. KO, Student's t-test, p = 0.016), Tbr2+Mcm2+ IPCs (WT vs. KO, Student's t-test, p = 0.003), DCX+Mcm2+ NBs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 3 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


Figure 2 ( B ) Representative images used for fate mapping of tdT + cells in the DG by co - stained with GFAP ( RGL ) , Tbr2 ( IPC ) , DCX ( IN ) , NeuN ( MN ) . Scale bar = 20 μm . ( C ) Quantification of composition of tdT + cells ( RGL : WT vs . KO , Student ' s t - test , p = 0 . 026 ; IPC : WT vs . KO , Student ' s t - test , p = 0 . 028 ; IN : WT vs . KO , Student ' s t - test , p = 0 . 049 ; N : WT vs . KO , Student ' s t - test , p = 0 . 048 ) in the DG of WT and cKO mice . WT , n = 3 mice ; cKO , n = 3 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 ( H ) Representative images of BrdU - retaining RGLs ( tdT + BrdU + ) in the DG . Scale bar = 20 μm . ( I ) Quantification of BrdU - retaining RGLs ( BrdU + tdT + GFAP + , WT vs . cKO , Student ' s t - test , p = 0 . 009 ) in the DG of WT and cKO mice . WT , n = 4 mice ; cKO , n = 3 mice .

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

Figure 2(B) Representative images used for fate mapping of tdT+ cells in the DG by co-stained with GFAP (RGL), Tbr2 (IPC), DCX (IN), NeuN (MN). Scale bar = 20 μm.(C) Quantification of composition of tdT+ cells (RGL: WT vs. KO, Student's t-test, p = 0.026; IPC: WT vs. KO, Student's t-test, p = 0.028; IN: WT vs. KO, Student's t-test, p = 0.049; N: WT vs. KO, Student's t-test, p = 0.048) in the DG of WT and cKO mice. WT, n = 3 mice; cKO, n = 3 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01(H) Representative images of BrdU-retaining RGLs (tdT+BrdU+) in the DG. Scale bar = 20 μm. (I) Quantification of BrdU-retaining RGLs (BrdU+tdT+GFAP+, WT vs. cKO, Student's t-test, p = 0.009) in the DG of WT and cKO mice. WT, n = 4 mice; cKO, n = 3 mice.


Figure 3 ( B ) Representative images of Nestin - GFP + GFAP + Mcm2 + RGLs in the DG . Scale bar = 50 μm . ( C ) Quantification of GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 001 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . ( D ) Percentage of GFAP + Nestin - GFP + Mcm2 + activated RGLs among total GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 0013 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 . ( I ) Representative images of NeuN + BrdU + adult - born neurons in the DG of 6 month - old mice . Scale bar = 50 μm . ( J ) Quantification of NeuN + BrdU + adult - born neurons ( WT vs . KO , Student ' s t - test , p = 0 . 0052 ) in the DG of WT and KO mice at 7 months of age . WT , n = 4 mice ; KO , n = 3 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 3(B) Representative images of Nestin-GFP+GFAP+Mcm2+ RGLs in the DG. Scale bar = 50 μm. (C) Quantification of GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.001) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. (D) Percentage of GFAP+Nestin-GFP+Mcm2+ activated RGLs among total GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.0013) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.(I) Representative images of NeuN+BrdU+ adult-born neurons in the DG of 6 month-old mice. Scale bar = 50 μm. (J) Quantification of NeuN+BrdU+ adult-born neurons (WT vs. KO, Student's t-test, p = 0.0052) in the DG of WT and KO mice at 7 months of age. WT, n = 4 mice; KO, n = 3 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.


Figure 4 ( A ) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling . Scale bar = 50 μm . ( B ) Percentage of BrdU + aNPCs among total aNPCs isolated from WT or KO mice ( WT vs . KO , Student ' s t - test , p = 0 . 0075 ) . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 4(A) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling. Scale bar = 50 μm. (B) Percentage of BrdU+ aNPCs among total aNPCs isolated from WT or KO mice (WT vs. KO, Student's t-test, p = 0.0075). WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; **, p < 0.01, ***, p < 0.001.


Figure 5 ( A ) DARTS analysis indicated HK1 as a molecular target of L - arginine in aNPCs . The aNPC lysates treated with L - arginine , digested with pronase and then analyzed by Western blot with anti - HK1 and GAPDH antibodies . ( B ) Quantification of HK1 expression level in aNPCs lysates treated with L - arginine and followed by digesting with pronase ( One - way ANOVA , F ( 3 , 8 ) = 3 . 61 , p = 0 . 043 ) . n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 . ( I ) Co - IP of HK1 with VDAC1 in vehicle or L - arginine - treated cells . ( J ) Quantification of the co - IP efficacy of HK1 and VDAC interaction in vehicle or L - arginine - treated cells ( IP : HK1 , vehicle vs . L - arginine , Student ' s t - test , p < 0 . 001 ; IP : VDAC1 , vehicle vs . L - arginine , Student ' s t - test , p = 0 . 0078 ) . Vehicle , n = 3 independent experiments ; L - arginine , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 5(A) DARTS analysis indicated HK1 as a molecular target of L-arginine in aNPCs. The aNPC lysates treated with L-arginine, digested with pronase and then analyzed by Western blot with anti-HK1 and GAPDH antibodies. (B) Quantification of HK1 expression level in aNPCs lysates treated with L-arginine and followed by digesting with pronase (One-way ANOVA, F(3, 8)= 3.61, p = 0.043). n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.(I) Co-IP of HK1 with VDAC1 in vehicle or L-arginine-treated cells. (J) Quantification of the co-IP efficacy of HK1 and VDAC interaction in vehicle or L-arginine-treated cells (IP: HK1, vehicle vs. L-arginine, Student's t-test, p < 0.001; IP: VDAC1, vehicle vs. L-arginine, Student's t-test, p = 0.0078). Vehicle, n = 3 independent experiments; L-arginine, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


Figure 6 ( C - E ) Quantification of glucose ( C , WT vs . KO , Student ' s t - test , p < 0 . 001 ) and glutamine ( E , WT vs . KO , Student ' s t - test , p = 0 . 94 ) consumption and lactate production ( D , WT vs . KO , Student ' s t - test , p = 0 . 019 ) in WT - and KO - aNPCs . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 * * * , p < 0 . 001 . ( J ) Representative profile of mitochondria stress test showing the OCR of WT - or KO - aNPCs . WT , n = 4 independent experiments , KO , n = 4 independent experiments .

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

Figure 6(C-E) Quantification of glucose (C, WT vs. KO, Student's t-test, p < 0.001) and glutamine (E, WT vs. KO, Student's t-test, p = 0.94) consumption and lactate production (D, WT vs. KO, Student's t-test, p = 0.019) in WT- and KO-aNPCs. WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05 ***, p < 0.001.(J) Representative profile of mitochondria stress test showing the OCR of WT- or KO-aNPCs. WT, n = 4 independent experiments, KO, n = 4 independent experiments.


Figure 7 ( C ) Quantification of Nestin - GFP + GFAP + RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 99 ) . WT + Vehicle , n = 4 mice ; WT + IACS , n = 4 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 3 mice . ( D ) Quantification of Nestin - GFP + GFAP + BrdU + activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( E ) Quantification of Nestin - GFP + GFAP - BrdU + progenitors ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 028 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( F ) Quantification of Nestin - GFP + Tbr2 + BrdU + IPCs ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 02 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 4 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 . ( H ) Representative images of NeuN + BrdU + adult - born neurons in the DG . Scale bar = 20 μm . ( I ) Quantification of NeuN + BrdU + adult - born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 5 mice ; WT + IACS , n = 5 mice ; KO + Vehicle , n = 5 mice , KO + IACS , n = 5 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

Figure 7(C) Quantification of Nestin-GFP+GFAP+ RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.99). WT + Vehicle, n = 4 mice; WT + IACS, n = 4 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 3 mice. (D) Quantification of Nestin-GFP+GFAP+BrdU+ activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (E) Quantification of Nestin-GFP+GFAP-BrdU+ progenitors (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.028) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (F) Quantification of Nestin-GFP+Tbr2+BrdU+ IPCs (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.02) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 4 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.(H) Representative images of NeuN+BrdU+ adult-born neurons in the DG. Scale bar = 20 μm. (I) Quantification of NeuN+BrdU+ adult-born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 5 mice; WT + IACS, n = 5 mice; KO + Vehicle, n = 5 mice, KO + IACS, n = 5 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


Figure 1EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation ( A - C ) ( A , C ) The successive pellets ( 2K , 10K , 100K ) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side - by - side with the lysate of 0 . 4 x 106 producing cells : C . Images from two different representative experiments are shown . Amount of proteins recovered in 2K , 10K and 100K pellets analyzed in stain - free gel images was similar between pellets : AU ( protein amount in a given pellet / total protein amount in 2K + 10K + 100K ) for 2K , 10K and 100K pellets were 1 . 13 ± 0 . 28 , 0 , 96 ± 0 . 10 and 0 . 91 ± 0 . 33 , respectively in control cells ( A ) , and 1 . 22 ± 0 . 29 , 0 , 84 ± 0 . 35 and 0 . 94 ± 0 . 30 , respectively in infected cells ( C ) ( mean ± SD ) . EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD ) . ( B ) Whole - mount EM analysis showing representative images and size distribution of particles in 2K , 10K and 100K pellets ( Scale bar , 200 nm ) . The diameter ( nm ) of particles was determined with ImageJ for five different experiments . 387 , 363 and 385 particles were counted for 2K , 10K and 100K pellets , respectivelly . ( A - D ) EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient ( D ) . ( D ) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non - infected and infected Jurkat cells and analysed by Western blot for the indicated proteins .

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

Figure 1EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation (A-C) (A, C) The successive pellets (2K, 10K, 100K) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side-by-side with the lysate of 0.4 x 106 producing cells: C. Images from two different representative experiments are shown. Amount of proteins recovered in 2K, 10K and 100K pellets analyzed in stain-free gel images was similar between pellets: AU (protein amount in a given pellet/total protein amount in 2K+10K+100K) for 2K, 10K and 100K pellets were 1.13±0.28, 0,96±0.10 and 0.91±0.33, respectively in control cells (A), and 1.22±0.29, 0,84±0.35 and 0.94±0.30, respectively in infected cells (C) (mean±SD).EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD). (B) Whole-mount EM analysis showing representative images and size distribution of particles in 2K, 10K and 100K pellets (Scale bar, 200 nm). The diameter (nm) of particles was determined with ImageJ for five different experiments. 387, 363 and 385 particles were counted for 2K, 10K and 100K pellets, respectivelly.(A-D) EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient (D). (D) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non-infected and infected Jurkat cells and analysed by Western blot for the indicated proteins.


Figure 2 ( A ) Proteomic profiles of different proteins , showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells . Proteins with very similar profiles ( represented by the same colours ) are likely part of the same EV subtypes . Each profile consists of three independent data triplets ( F1 - F2 - F3A , F1 - F2 - F3B and F1 - F2 - F3C ) . ( C ) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1 - F2 - F3 subfractions as compared to expression in the total cell proteome . Solid horizontal lines indicate medians and dashed line indicate quartiles ( n = 3 ) . Mitochondria and ER markers are de - enriched , and plasma membrane markers are enriched in EV fractions , with progressively pronounced effects from F1 to F3 .

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

Figure 2(A) Proteomic profiles of different proteins, showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells. Proteins with very similar profiles (represented by the same colours) are likely part of the same EV subtypes. Each profile consists of three independent data triplets (F1- F2- F3A, F1- F2- F3B and F1-F2-F3C).(C) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1-F2-F3 subfractions as compared to expression in the total cell proteome. Solid horizontal lines indicate medians and dashed line indicate quartiles (n=3). Mitochondria and ER markers are de-enriched, and plasma membrane markers are enriched in EV fractions, with progressively pronounced effects from F1 to F3.


Figure 3EVs were purified by SEC from supernatants of Jurkat cells . ( A ) EVs were subjected to immuno - isolation with beads coupled to antibodies against CD81 or CD63 . Bead - associated ( Pull - down : PD ) vesicles and those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot analysis with antibodies specific for CD63 , Syntenin - 1 , CD81 , ADAM10 and CD3G . A representative blot ( top panel ) and quantification ( mean ± SD ) of the proportion of signal in FT as compared with total ( PD + FT ) from four independent experiments ( bottom panel ) are shown . ( B ) Representative images of double - immunogold labelling of EVs purified by SEC from Jurkat cells . Left panel , CD81 / CD63 ; right panel , CD81 / CD3E . Arrows show CD63 staining in CD81 + CD63 + EVs . EVs were purified by SEC from supernatants of Jurkat cells . ( C , D ) Multiplex bead - based flow cytometry assay for detection of EV surface markers . Antibody - coated capture beads were incubated with 2 x 109 particles . Captured EVs were detected with either APC - labeled anti - CD81 , anti - CD63 or anti - CD3E . ( C ) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads ( log10 scale ) . shown . ( D ) Heat - map representation of the median APC fluorescence values for the different bead populations detected with anti - CD63 or anti - CD3E antibodies relative to the values detected with anti - CD81 .

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

Figure 3EVs were purified by SEC from supernatants of Jurkat cells. (A) EVs were subjected to immuno-isolation with beads coupled to antibodies against CD81 or CD63. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for CD63, Syntenin-1, CD81, ADAM10 and CD3G. A representative blot (top panel) and quantification (mean ± SD) of the proportion of signal in FT as compared with total (PD+FT) from four independent experiments (bottom panel) are shown.(B) Representative images of double-immunogold labelling of EVs purified by SEC from Jurkat cells. Left panel, CD81/CD63; right panel, CD81/CD3E. Arrows show CD63 staining in CD81+CD63+ EVs.EVs were purified by SEC from supernatants of Jurkat cells. (C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81.


Figure 4 ( B ) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions . Each profile consists of two independent data triplets ( F1 - F2 - F3A and F1 - F2 - F3B ) . Abundance profiles of the same groups of proteins showed the same clustering behaviour , although in this second dataset , the proportion of proteins recovered in the F2 was more variable . These differences , however , did not affect the results of the NNP or movement analysis . ( C ) Unbiased identification of significant translocation events triggered by HIV - 1 infection . Each protein is scored for magnitude of translocation ( M score , x - axis ) and reproducibility of translocation direction ( R score , y - axis ) across the two replicates . MR plot analysis reveals significant translocations in the top right quadrant . Proteins with M > 1 and R > 0 . 9 are candidate hits for changing localization ( estimated FDR = 8 % ) .

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

Figure 4(B) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions. Each profile consists of two independent data triplets (F1- F2- F3A and F1- F2- F3B). Abundance profiles of the same groups of proteins showed the same clustering behaviour, although in this second dataset, the proportion of proteins recovered in the F2 was more variable. These differences, however, did not affect the results of the NNP or movement analysis.(C) Unbiased identification of significant translocation events triggered by HIV-1 infection. Each protein is scored for magnitude of translocation (M score, x-axis) and reproducibility of translocation direction (R score, y-axis) across the two replicates. MR plot analysis reveals significant translocations in the top right quadrant. Proteins with M>1 and R>0.9 are candidate hits for changing localization (estimated FDR = 8%).


Figure 5 ( A , B ) ( A ) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected ( red boxes ) Jurkat cells . The successive pellets ( 2K , 10K , 100K ) were analyzed by Western blot , using antibodies against SPN , MOV10 , SERINC3 and p24 ( HIV - 1 protein ) . The 2K , 10K , and 100K pellets obtained from 20 x 106 cells were loaded on the gel . Representative images ( A ) and quantifications in three to five independent experiments ( B ) are shown . For each pellet , arbitrary units ( AU ) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet . In cell lysates from the same experiments , AU ( given protein / total amount of protein ) for SPN and MOV10 were 1 . 29 ± 0 . 15 and 0 . 84 ± 0 . 33 times the levels in control cells , respectively ( Mean ± SD ) . ( C ) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV - 1 infected ( red boxes ) cells . Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN . SERINC3 was recovered in the middle fractions . ( T = top ; B = bottom ) . ( D , E ) Quantification of MOV10 , SPN , SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV - 1 infected samples . AU = band intensity in a given pellet / sum ( band intensity in all six pellets ) . ( F ) Quantification of SERINC3 signals in the different fractions from control and infected samples . AU = band intensity in a given pellet / sum ( band intensity in all 12 pellets ) . Lines represent the medians of three independent experiments . ( G , H ) EVs from supernatant of HIV - infected Jurkat cells were subjected to immuno - isolation with beads coupled to antibody against SPN . Bead - associated ( Pull - down : PD ) vesicles and those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot analysis with antibodies specific for SPN , MOV10 , CD63 , SERINC3 and viral p24 . A representative image ( G ) and quantification ( H ) Mean ± SD of the proportion of signal in PD as compared with total ( PD + FT ) in three independent experiments are shown . ( I ) EVs remaining in the FT , or EVs secreted by the same number of cells but not subjected to immuno - isolation ( input ) were used to infect a reporter cell line . Mean ± SD of six independent experiments is shown .

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

Figure 5(A, B) (A) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected (red boxes) Jurkat cells. The successive pellets (2K, 10K, 100K) were analyzed by Western blot, using antibodies against SPN, MOV10, SERINC3 and p24 (HIV-1 protein). The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (A) and quantifications in three to five independent experiments (B) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet. In cell lysates from the same experiments, AU (given protein/total amount of protein) for SPN and MOV10 were 1.29±0.15 and 0.84±0.33 times the levels in control cells, respectively (Mean±SD).(C) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV-1 infected (red boxes) cells. Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN. SERINC3 was recovered in the middle fractions. (T = top; B = bottom). (D, E) Quantification of MOV10, SPN, SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV-1 infected samples. AU = band intensity in a given pellet / sum (band intensity in all six pellets). (F) Quantification of SERINC3 signals in the different fractions from control and infected samples. AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Lines represent the medians of three independent experiments. (G, H) EVs from supernatant of HIV-infected Jurkat cells were subjected to immuno-isolation with beads coupled to antibody against SPN. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for SPN, MOV10, CD63, SERINC3 and viral p24. A representative image (G) and quantification (H) Mean ± SD of the proportion of signal in PD as compared with total (PD+FT) in three independent experiments are shown.(I) EVs remaining in the FT, or EVs secreted by the same number of cells but not subjected to immuno-isolation (input) were used to infect a reporter cell line. Mean± SD of six independent experiments is shown.


Figure 6Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 ( A ) GFP + Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells . Results are expressed as a ratio to the number of GFP + cells after treatment with supernatant from infected control cells . . Mean ± SD of three independent experiments is shown . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( B ) Number of particles secreted were quantified by NTA . Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( C ) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation . Six fractions were recovered and analyzed by Western blot for the presence of SPN , SERINC3 and p24 . ( T = top ; B = bottom ) . ( D ) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations ( exp 1 : open symbols , exp 2 : closed symbols ) . AU = band intensity in a given pellet / sum ( band intensity in all 12 pellets ) . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed by Western blot with antibodies against SPN , and p24 . The 2K , 10K , and 100K pellets obtained from 20 x 106 cells were loaded on the gel . Representative images ( E ) Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed and quantification of SPN and p24 signals ( F ) are shown . For each pellet , arbitrary units ( AU ) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K + 10K + 100K from control cells . Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( G ) Multiplex bead - based flow cytometry assay for detection of EV surface markers . Antibody - coated capture beads were incubated with EV samples . Captured EVs were detected with a mixture of APC - labeled anti - CD9 , anti - CD63 , and anti - CD81 . Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction . Results were relativized to the values obtained with control sample for each experiment . Dotted line represents values obtained for EVs isolated from control cell CM .

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

Figure 6Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 (A) GFP+ Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells. Results are expressed as a ratio to the number of GFP+ cells after treatment with supernatant from infected control cells. . Mean ± SD of three independent experiments is shown.Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (B) Number of particles secreted were quantified by NTA. Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells.Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (C) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation. Six fractions were recovered and analyzed by Western blot for the presence of SPN, SERINC3 and p24. (T = top; B = bottom). (D) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations (exp 1: open symbols, exp 2: closed symbols). AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed by Western blot with antibodies against SPN, and p24. The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (E)Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed and quantification of SPN and p24 signals (F) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K+10K+100K from control cells.Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (G) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with EV samples. Captured EVs were detected with a mixture of APC-labeled anti-CD9, anti-CD63, and anti-CD81. Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction. Results were relativized to the values obtained with control sample for each experiment. Dotted line represents values obtained for EVs isolated from control cell CM.


Figure 1A CAFs isolation from human pancreatic tumour resections ( left panel ) and in vitro primo - culture ( right panel ) . B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti - vimentin ( left panel ) or anti - α - SMA ( right panel ) antibody ( one representative field of n = 3 ) . D , E Panc - 1 cell viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment ( CM from PaSC , or from CAF , or from CAF ± SOM230 , SOM230 ) as a percentage of the respective gemcitabine - untreated cells ( = 100 % ) ( n = 4 ; from left to right : * * P = 0 . 007 , # # P = 0 . 006 in D ; * P = 0 . 032 , # P = 0 . 041 , # P = 0 . 047 in E ) . F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti - cleaved caspase - 3 or anti - PARP antibody ( representative of n = 3 ) . G Panc - 1 was analysed by flow cytometry . Percentages ( mean ± SD ) of annexin V - positive cells are indicated ( n = 3 ; from left to right : * * P = 0 . 005 , # # P = 0 . 004 , * * P = 0 . 006 ) .

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Figure 1A CAFs isolation from human pancreatic tumour resections (left panel) and in vitro primo-culture (right panel).B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti-vimentin (left panel) or anti-α-SMA (right panel) antibody (one representative field of n = 3).D, E Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment (CM from PaSC, or from CAF, or from CAF ± SOM230, SOM230) as a percentage of the respective gemcitabine-untreated cells (= 100%) (n = 4; from left to right: **P = 0.007, ##P = 0.006 in D; *P = 0.032, #P = 0.041, #P = 0.047 in E).F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti-cleaved caspase-3 or anti-PARP antibody (representative of n = 3).G Panc-1 was analysed by flow cytometry. Percentages (mean ± SD) of annexin V-positive cells are indicated (n = 3; from left to right: **P = 0.005, ##P = 0.004, **P = 0.006).


Figure 2Immunoblotting of protein extracts from PaSCs or CAFs treated ( + ) or not with SOM230 ( 10 − 7 M ) for 48 h , using the anti - puromycin antibody ( representative of n = 3 ) . Polysomes profiles of CAFs treated or not with SOM230 for 48 h ( representative of n = 3 ) . Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of protein extracts from PaSCs or CAFs treated ( + ) with SOM230 for 30 min ( representative of n = 3 ) . Immunoblotting of protein extracts from CAFs treated ( + ) or not with SOM230 ( 10 − 7 M ) for the indicated times , using anti - P - Akt , anti - P - S6 or anti - 4E - BP1 antibody ( representative of n = 3 ) . Protein concentration in untreated or SOM230 - treated CAF - CM or extracts normalized per 1 × 106 cells . Results ( mean ± SD ) are presented as a percentage of the untreated CAFs ( = 100 % ) ( n = 3 ; from left to right : # # P = 0 . 008 , # # P = 0 . 007 ) . Immunoblotting using an anti - 4E - BP1 or anti - GAPDH ( loading control ) antibody of protein extracts from siCTR - or si4E - BP1 - transfected CAFs ( representative of n = 3 ) . Immunoblotting of equal amounts of protein from siCTR - or si4E - BP1 - transfected CAFs treated ( + ) or not with SOM230 for 48 h , using the anti - puromycin antibody ( representative of n = 3 ) . Panc - 1 cell viability was assessed by MTT . Panc - 1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230 - treated CAFs transfected with the siCTR or si4E - BP - 1 . Results ( mean ± SD ) are presented as a percentage of the untreated CAFs ( = 100 % ) ( n = 3 ; * * P = 0 . 003 , § § P = 0 . 002 ) . Caspase - 3 and PARP cleavage induced by gemcitabine in Panc - 1 was evaluated by Western blot using the respective antibodies ( representative of n = 3 ) . Arrow indicates cleaved forms of PARP .

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

Figure 2Immunoblotting of protein extracts from PaSCs or CAFs treated (+) or not with SOM230 (10−7 M) for 48 h, using the anti-puromycin antibody (representative of n = 3).Polysomes profiles of CAFs treated or not with SOM230 for 48 h (representative of n = 3).Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from PaSCs or CAFs treated (+) with SOM230 for 30 min (representative of n = 3).Immunoblotting of protein extracts from CAFs treated (+) or not with SOM230 (10−7 M) for the indicated times, using anti-P-Akt, anti-P-S6 or anti-4E-BP1 antibody (representative of n = 3).Protein concentration in untreated or SOM230-treated CAF-CM or extracts normalized per 1 × 106 cells. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; from left to right: ##P = 0.008, ##P = 0.007).Immunoblotting using an anti-4E-BP1 or anti-GAPDH (loading control) antibody of protein extracts from siCTR- or si4E-BP1-transfected CAFs (representative of n = 3).Immunoblotting of equal amounts of protein from siCTR- or si4E-BP1-transfected CAFs treated (+) or not with SOM230 for 48 h, using the anti-puromycin antibody (representative of n = 3).Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with the siCTR or si4E-BP-1. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; **P = 0.003, §§P = 0.002).Caspase-3 and PARP cleavage induced by gemcitabine in Panc-1 was evaluated by Western blot using the respective antibodies (representative of n = 3). Arrow indicates cleaved forms of PARP.


Figure 3A Expression of the somatostatin receptor subtype 1 ( sst1 ) analysed by RT - qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients ( mean ± SD ) . B Immunoblotting of protein extracts from PaSCs and CAFs , using anti - sst1 , anti - α - SMA , anti - P - S6 or anti - GAPDH ( loading control ) antibody ( representative of n = 3 ) . C Immunoblotting of protein extracts from BON , CAFs and indicated pancreatic cancer cell lines , using anti - sst1 or anti - GAPDH ( loading control ) antibody ( representative of n = 3 ) . D Immunofluorescence confocal microscopy analyses of α - SMA and sst1 in CAFs . Co - localization of α - SMA and sst1 is shown ( merge ) ( one representative field of n = 3 ) ( scale bar = 100 μm ) . E - G Representative immunofluorescence confocal microscopy analyses of sst1 co - localization with cytokeratin - 19 ( n = 42 PDAC samples ) ( E ) , α - SMA ( n = 42 PDAC samples ) ( F ) or with P - 4E - BP1 ( n = 25 PDAC samples ) ( G ) in serial slides ( scale bar = 100 μm ) . Dashed white lines represent the limits between stroma and tumour epithelial glands . H , I Immunoblotting of protein extracts from siCTR - or sisst1 - transfected CAFs treated ( + ) or not with SOM230 for 48 h ( sst1 , puromycin and GAPDH as loading control , H ) or for 30 min ( P - Akt , P - S6 , 4E - BP1 and GAPDH as loading control , I ) ( representative of n = 3 ) . J Panc - 1 cell viability was assessed by MTT . Panc - 1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230 - treated CAFs transfected with siCTR or si - sst1 . Results ( mean ± SD ) are presented as the percentage of the untreated cells ( = 100 % ) ( n = 3 ; * * P = 0 . 003 , § § P = 0 . 002 ) .

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

Figure 3A Expression of the somatostatin receptor subtype 1 (sst1) analysed by RT-qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients (mean ± SD).B Immunoblotting of protein extracts from PaSCs and CAFs, using anti-sst1, anti-α-SMA, anti-P-S6 or anti-GAPDH (loading control) antibody (representative of n = 3).C Immunoblotting of protein extracts from BON, CAFs and indicated pancreatic cancer cell lines, using anti-sst1 or anti-GAPDH (loading control) antibody (representative of n = 3).D Immunofluorescence confocal microscopy analyses of α-SMA and sst1 in CAFs. Co-localization of α-SMA and sst1 is shown (merge) (one representative field of n = 3) (scale bar = 100 μm).E-G Representative immunofluorescence confocal microscopy analyses of sst1 co-localization with cytokeratin-19 (n = 42 PDAC samples) (E), α-SMA (n = 42 PDAC samples) (F) or with P-4E-BP1 (n = 25 PDAC samples) (G) in serial slides (scale bar = 100 μm). Dashed white lines represent the limits between stroma and tumour epithelial glands.H, I Immunoblotting of protein extracts from siCTR- or sisst1-transfected CAFs treated (+) or not with SOM230 for 48 h (sst1, puromycin and GAPDH as loading control, H) or for 30 min (P-Akt, P-S6, 4E-BP1 and GAPDH as loading control, I) (representative of n = 3).J Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with siCTR or si-sst1. Results (mean ± SD) are presented as the percentage of the untreated cells (= 100%) (n = 3; **P = 0.003, §§P = 0.002).


Figure 4A - D Immunoblotting using an anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAFs pre - treated ( + ) or not overnight with PTX ( 100 ng / ml ) ( A ) , gallein ( 10 μM ) ( B ) , NSC87877 ( 10 μM ) ( C ) , Src inhibitor ( 5 μM ) ( D ) , treated ( + ) or not with SOM230 ( 10 − 7 M ) for 30 min ( representative of n = 3 ) . E , F Immunoblotting using anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAF siCTR - or siSHP2 - ( E ) or siPTPε - ( F ) transfected CAF treated or not with SOM230 for 30 min ( representative of n = 3 ) . G , H Immunoblotting using an anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAFs treated with the indicated molecules ( PDGF receptor inhibitor , 5 - 10 μM ; Jak1 / 2 inhibitor ruxolitinib , 5 or 10 μM ; EGFR inhibitor , 150 or 300 nM and recombinant PDGF , 5 μg / ml ) ( representative of n = 3 ) .

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Figure 4A-D Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs pre-treated (+) or not overnight with PTX (100 ng/ml) (A), gallein (10 μM) (B), NSC87877 (10 μM) (C), Src inhibitor (5 μM) (D), treated (+) or not with SOM230 (10−7 M) for 30 min (representative of n = 3).E, F Immunoblotting using anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAF siCTR- or siSHP2- (E) or siPTPε- (F) transfected CAF treated or not with SOM230 for 30 min (representative of n = 3).G, H Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs treated with the indicated molecules (PDGF receptor inhibitor, 5-10 μM; Jak1/2 inhibitor ruxolitinib, 5 or 10 μM; EGFR inhibitor, 150 or 300 nM and recombinant PDGF, 5 μg/ml) (representative of n = 3).


Figure 5A - C MIA PaCa - 2 - GLuc cells were injected with or without CAFs into the pancreas of nude mice . Mice were treated with each indicated treatment ( SOM230 - LAR at day 7 and gemcitabine at days 7 , 10 , 14 and 17 ) , and the plasmatic luciferase activity was measured ( mean ± SD ) at days 7 , 14 and 21 ( n = 5 ) ( P = 0 . 003 for gemcitabine + SOM230LAR versus untreated ) ( A ) . Tumours were excised , photographed ( scale bars are 1 cm ) ( B ) , weighted ( P = 0 . 0008 ) ( C ) and paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( E ) . D Human tumours were subcutaneously xenografted in nude mice and tumour volumes ( mean ± SD ) measured ( at day 37 , P = 0 . 0009 for gemcitabine + SOM230LAR versus untreated and P = 0 . 021 for gemcitabine + SOM230LAR versus gemcitabine ) . Mice were treated with the indicated treatments ( SOM230 - LAR at days 3 and 31 , and gemcitabine at day 3 and twice a week thereafter ) . E , F Tumours excised from mice as in ( A - C ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( E ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( F ) ( * P = 0 . 036 , * * P = 0 . 009 ) . G , H Tumours excised from mice as in ( D ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( G ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( H ) ( * P = 0 . 027 ) .

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

Figure 5A-C MIA PaCa-2-GLuc cells were injected with or without CAFs into the pancreas of nude mice. Mice were treated with each indicated treatment (SOM230-LAR at day 7 and gemcitabine at days 7, 10, 14 and 17), and the plasmatic luciferase activity was measured (mean ± SD) at days 7, 14 and 21 (n = 5) (P = 0.003 for gemcitabine + SOM230LAR versus untreated) (A). Tumours were excised, photographed (scale bars are 1 cm) (B), weighted (P = 0.0008) (C) and paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (E).D Human tumours were subcutaneously xenografted in nude mice and tumour volumes (mean ± SD) measured (at day 37, P = 0.0009 for gemcitabine + SOM230LAR versus untreated and P = 0.021 for gemcitabine + SOM230LAR versus gemcitabine). Mice were treated with the indicated treatments (SOM230-LAR at days 3 and 31, and gemcitabine at day 3 and twice a week thereafter).E, F Tumours excised from mice as in (A-C) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (E). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (F) (*P = 0.036, **P = 0.009).G, H Tumours excised from mice as in (D) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (G). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (H) (*P = 0.027).


Figure 6A Membrane antibody array assay using CM from SOM230 - treated or not CAFs ( representative of n = 3 ) . Controls are circled in blue dashed line and IL - 6 in red square . B , C Anti - IL - 6 ELISA assay ( mean ± SD ) using CM or protein extracts from SOM230 - treated or not CAF ( from left to right : § § § P = 0 . 0002 , § § § P = 0 . 0002 ) ( B ) , or CM from siCTR - or si4E - BP1 - transfected CAFs treated or not with SOM230 ( from left to right : * * * P = 0 . 0003 , $ $ P = 0 . 004 ) ( C ) ( n = 3 ) . B , C Anti - IL - 6 ELISA assay ( mean ± SD ) using CM or protein extracts from SOM230 - treated or not CAF ( from left to right : § § § P = 0 . 0002 , § § § P = 0 . 0002 ) ( B ) , or CM from siCTR - or si4E - BP1 - transfected CAFs treated or not with SOM230 ( from left to right : * * * P = 0 . 0003 , $ $ P = 0 . 004 ) ( C ) ( n = 3 ) . D Panc - 1 cell viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment as the percentage of the respective untreated cells ( = 100 % ) ( n = 3 ; from left to right : * * P = 0 . 004 , # # P = 0 . 001 , § P = 0 . 023 , § P = 0 . 019 ) . E , F Immunoblotting using an anti - cleaved caspase - 3 , anti - PARP or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells incubated with CAF - CM pre - incubated or not with an anti - IL - 6 neutralizing antibody ( E ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( F ) . G , H Immunoblotting using an anti - survivin or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells stimulated or not with CAF - CM pre - incubated or not with anti - IL - 6 neutralizing antibody ( G ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( H ) . I Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of protein extracts from SOM230 - treated or SOM230 - untreated CAF incubated or not with IL - 6 neutralizing antibody ( representative of n = 3 ) .

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

Figure 6A Membrane antibody array assay using CM from SOM230-treated or not CAFs (representative of n = 3). Controls are circled in blue dashed line and IL-6 in red square.B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).D Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment as the percentage of the respective untreated cells (= 100%) (n = 3; from left to right: **P = 0.004, ##P = 0.001, §P = 0.023, §P = 0.019).E, F Immunoblotting using an anti-cleaved caspase-3, anti-PARP or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells incubated with CAF-CM pre-incubated or not with an anti-IL-6 neutralizing antibody (E), or with human recombinant IL-6 (representative of n = 3) (F).G, H Immunoblotting using an anti-survivin or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells stimulated or not with CAF-CM pre-incubated or not with anti-IL-6 neutralizing antibody (G), or with human recombinant IL-6 (representative of n = 3) (H).I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).


Figure 7A - C Immunohistofluorescence using an anti - α - SMA or anti - IL - 6 antibody in paraffin - embedded sections from MIA PaCa - 2 - Gluc and CAF cells orthotopic co - xenografted tumours from Fig5A ( A ) or from human tumour subcutaneously xenografted from Fig5D ( B ) in nude mice treated or not with gemcitabine + SOM230 ( representative of five different tumours ) . Representative co - localization of α - SMA with IL - 6 in human PDAC samples ( C ) ( n = 15 ) . Scale bar = 100 μm . D , E Plasma was collected from MIA PaCa - 2GLuc and CAFs ( * P = 0 . 034 , * * P = 0 . 007 ) ( D ) , or human pancreatic tumour ( * P = 0 . 028 , * * P = 0 . 004 ) ( E ) , xenografts in nude mice and human IL - 6 plasmatic concentrations ( mean ± SD ) were quantified by ELISA ( n = 5 mice / group ) . * treated versus untreated mice .

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Figure 7A-C Immunohistofluorescence using an anti-α-SMA or anti-IL-6 antibody in paraffin-embedded sections from MIA PaCa-2-Gluc and CAF cells orthotopic co-xenografted tumours from Fig5A (A) or from human tumour subcutaneously xenografted from Fig5D (B) in nude mice treated or not with gemcitabine + SOM230 (representative of five different tumours). Representative co-localization of α-SMA with IL-6 in human PDAC samples (C) (n = 15). Scale bar = 100 μm.D, E Plasma was collected from MIA PaCa-2GLuc and CAFs (*P = 0.034, **P = 0.007) (D), or human pancreatic tumour (*P = 0.028, **P = 0.004) (E), xenografts in nude mice and human IL-6 plasmatic concentrations (mean ± SD) were quantified by ELISA (n = 5 mice / group). * treated versus untreated mice.


Figure 1G ) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates ( n = 3 ) . Β - actin serves as an internal loading control .

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Figure 1G) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates (n=3). Β-actin serves as an internal loading control.


Figure 2B ) Images represent localization of KTR for ERK , JNK , or p38 under basal growth conditions . Scales bars = 20µm . C - E ) Kinase activity for ERK ( C ) , JNK ( D ) , or p38 ( E ) in three representative single cells ( upper panels ) in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) as measured by C / N ratio . Lower panel shows mean C / N ratio in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) . Thick line represents mean with shaded area = SD . N for each condition and kinase is shown in population average plots .

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Figure 2B) Images represent localization of KTR for ERK, JNK, or p38 under basal growth conditions. Scales bars=20µm.C-E) Kinase activity for ERK (C), JNK (D), or p38 (E) in three representative single cells (upper panels) in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red) as measured by C/N ratio. Lower panel shows mean C/N ratio in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red). Thick line represents mean with shaded area=SD. N for each condition and kinase is shown in population average plots.


Figure 3C - E ) Mean kinase activation for ERK € , JNK ( D ) , or p38 ( E ) in both live ( grey ) and dead ( red ) cells measured over 24 - hours in response to H2O2 . Shaded areas = SD . N for each condition is shown .

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Figure 3C-E) Mean kinase activation for ERK €, JNK (D), or p38 (E) in both live (grey) and dead (red) cells measured over 24-hours in response to H2O2. Shaded areas=SD. N for each condition is shown.


Figure 6D ) Mean JNK activity as measured by C / N ratio ( bold line ) ± SD ( shaded area ) in response to 300 µM H2O2 with or without p38 inhibition ( grey ) . Bar graph shows integrated JNK activity ( C / N ratio ) over 24 - hours . Data represents mean ± SD . The number of analyzed cells , n , is shown for each condition . Statistics performed using 2 - tailed t - test . Data information : * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant .

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Figure 6D) Mean JNK activity as measured by C/N ratio (bold line)±SD (shaded area) in response to 300 µM H2O2 with or without p38 inhibition (grey). Bar graph shows integrated JNK activity (C/N ratio) over 24-hours. Data represents mean±SD. The number of analyzed cells, n, is shown for each condition. Statistics performed using 2-tailed t-test. Data information: ** p<0.01, *** p<0.001, ns=not significant.


Figure 1Kaplan - Meier survival curves stratified by the patient MICA - 129 genotype for all patients ( n = 446 ) . The survival curve is displayed for the first 66 months ; 7 . 6 % of the patients were followed longer . Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2 . The HR indicates the risk per MICA - 129Met allele carried by the patients ( additive risk model ) . The numbers of patients carrying the three genotypes and the number of events ( in brackets ) are indicated . Kaplan - Meier survival curves for patients receiving a graft matched for the MICA - 129 genotype ( n = 404 ) . Kaplan - Meier survival curves for patients who experienced aGVDH ( any grade , n = 244 ) . Kaplan - Meier survival curves for patients not experiencing aGVHD ( n = 189 ) . The HR indicates the risk of patients carrying two MICA - 129Met alleles ( recessive risk model ) . Kaplan - Meier survival curves for patients who did not receive a T - cell - depleting treatment with ATG ( n = 193 ) . Kaplan - Meier survival curves for patients treated with ATG ( n = 250 ) . Kaplan - Meier survival curves for patients with the MICA - 129Val / Val genotype ( n = 229 ) stratified by treatment with ATG . Kaplan - Meier survival curves for patients carrying one or two MICA - 129Met alleles ( n = 214 ) stratified by treatment with ATG .

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Figure 1Kaplan-Meier survival curves stratified by the patient MICA-129 genotype for all patients (n = 446). The survival curve is displayed for the first 66 months; 7.6% of the patients were followed longer. Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2. The HR indicates the risk per MICA-129Met allele carried by the patients (additive risk model). The numbers of patients carrying the three genotypes and the number of events (in brackets) are indicated.Kaplan-Meier survival curves for patients receiving a graft matched for the MICA-129 genotype (n = 404).Kaplan-Meier survival curves for patients who experienced aGVDH (any grade, n = 244).Kaplan-Meier survival curves for patients not experiencing aGVHD (n = 189). The HR indicates the risk of patients carrying two MICA-129Met alleles (recessive risk model).Kaplan-Meier survival curves for patients who did not receive a T-cell-depleting treatment with ATG (n = 193).Kaplan-Meier survival curves for patients treated with ATG (n = 250).Kaplan-Meier survival curves for patients with the MICA-129Val/Val genotype (n = 229) stratified by treatment with ATG.Kaplan-Meier survival curves for patients carrying one or two MICA-129Met alleles (n = 214) stratified by treatment with ATG.


Figure 2The linear regression of MICA expression intensity and binding of a recombinant NKG2D - Fc fusion protein both determined as MFI by flow cytometry is displayed for L - MICA - 129Met ( n = 79 , left panel ) and L - MICA - 129Val clones ( n = 81 , right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated . Purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells ( 106 ) were stimulated with immobilized MICA - 129Met - mIgG2a - Fc or MICA - 129Val - mIgG2a - Fc or OVA - mIgG2a - Fc fusion proteins ( 10 μg / ml ) for 3 , 10 , or 30 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb , an anti - phospho - SRC family ( Tyr419 ) kinases Ab , and an anti - β - actin mAb as a loading control . The arrow points toward phosphorylated SRC family kinases . Blots obtained from three independent experiments were analyzed by densitometry , and the means plus SD of the ratio between phospho - SRC family kinase and β - actin signals is displayed . The difference between NK cells stimulated for 10 min by MICA - 129Met - Fc or MICA - 129Val - Fc proteins was assessed by t - test . Purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days , 106 ) were incubated with the SRC kinase inhibitor PP2 ( 25 μM ) , the vehicle DMSO , or medium only ( Ø ) for 30 min before being added to immobilized MICA - 129Met - Fc , MICA - 129Val - Fc , or OVA - Fc fusion proteins ( 10 μg / ml ) for 10 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb and an anti - β - actin mAb as a loading control . The blot is representative for two independent experiments . In parallel , degranulation of the NK cells was measured by anti - CD107a staining in flow cytometry . The difference between DMSO - and PP2 - treated cells with respect to CD107a + cells and the MFI of CD107a is indicated in the histograms . The results are representative for two independent experiments .

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

Figure 2The linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met (n = 79, left panel) and L-MICA-129Val clones (n = 81, right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (106) were stimulated with immobilized MICA-129Met-mIgG2a-Fc or MICA-129Val-mIgG2a-Fc or OVA-mIgG2a-Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases.Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t-test.Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.


Figure 3The degranulation ( CD107a expression ) of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells in response to immobilized MICA - 129Met - Fc and MICA - 129Val - Fc , and as negative control , OVA - Fc fusion proteins was determined by flow cytometry after 1 h ( n = 3 ) . Displayed are means and SD of the MFI ( left panel ) and the percentage of CD107a + cells ( right panel ) . Differences between MICA - 129Met - Fc - and MICA - 129Val - Fc - induced NK - cell degranulation were analyzed by two - way ANCOVA adjusted for MICA protein concentration , and the respective P - values are indicated . The degranulation of IL - 2 - stimulated LAK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 27 ) or L - MICA - 129Val clones ( n = 27 ) for 1 h was determined by flow cytometry . CD107a cell surface expression was analyzed after gating on CD56 + NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of CD107a on NK cells ( upper panels ) or the proportion of CD107a + NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated . A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L - MICA - 129Met and an L - MICA - 129Val clone . L - con cells served as a negative and K562 cells as a positive control . The means of specific lysis of triplicates plus SD at different E : T ratios ( 200 : 1 to 3 : 1 ) were measured in an 51chromium - release assay . The MICA expression intensity and the binding of a recombinant NKG2D - Fc fusion protein to the target cells were determined in parallel by flow cytometry , and the MFIs are indicated .

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

Figure 3The degranulation (CD107a expression) of purified IL-2-stimulated (100 U/ml for 4 days) NK cells in response to immobilized MICA-129Met-Fc and MICA-129Val-Fc, and as negative control, OVA-Fc fusion proteins was determined by flow cytometry after 1 h (n = 3). Displayed are means and SD of the MFI (left panel) and the percentage of CD107a+ cells (right panel). Differences between MICA-129Met-Fc- and MICA-129Val-Fc-induced NK-cell degranulation were analyzed by two-way ANCOVA adjusted for MICA protein concentration, and the respective P-values are indicated.The degranulation of IL-2-stimulated LAK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 27) or L-MICA-129Val clones (n = 27) for 1 h was determined by flow cytometry. CD107a cell surface expression was analyzed after gating on CD56+ NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of CD107a on NK cells (upper panels) or the proportion of CD107a+ NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L-MICA-129Met and an L-MICA-129Val clone. L-con cells served as a negative and K562 cells as a positive control. The means of specific lysis of triplicates plus SD at different E:T ratios (200:1 to 3:1) were measured in an 51chromium-release assay. The MICA expression intensity and the binding of a recombinant NKG2D-Fc fusion protein to the target cells were determined in parallel by flow cytometry, and the MFIs are indicated.


Figure 4The IFNγ expression of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells exposed for 4 h to L - MICA - 129Met ( n = 23 ) or L - MICA - 129Val clones ( n = 23 ) was determined by flow cytometry . The IFNγ expression was analyzed after gating on CD56brightCD16 − NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16 − NK cells ( upper panels ) or the proportion of IFNγ + CD56brightCD16 − NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated . The IFNγ release of purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days ) co - cultured with L - MICA - 129Met ( n = 34 ) or L - MICA - 129Val clones ( n = 32 ) for 24 h was measured in the supernatant by ELISA . In parallel , the MICA expression intensity on target cells was determined by flow cytometry . The linear regressions of IFNγ release ( pg / ml ) by NK cells and MICA expression on targets ( MFI ) are displayed for the L - MICA - 129Met clones ( left panel ) and the L - MICA - 129Val clones ( right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

Figure 4The IFNγ expression of purified IL-2-stimulated (100 U/ml for 4 days) NK cells exposed for 4 h to L-MICA-129Met (n = 23) or L-MICA-129Val clones (n = 23) was determined by flow cytometry. The IFNγ expression was analyzed after gating on CD56brightCD16−NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16−NK cells (upper panels) or the proportion of IFNγ+CD56brightCD16−NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.The IFNγ release of purified IL-2-stimulated NK cells (100 U/ml for 4 days) co-cultured with L-MICA-129Met (n = 34) or L-MICA-129Val clones (n = 32) for 24 h was measured in the supernatant by ELISA. In parallel, the MICA expression intensity on target cells was determined by flow cytometry. The linear regressions of IFNγ release (pg/ml) by NK cells and MICA expression on targets (MFI) are displayed for the L-MICA-129Met clones (left panel) and the L-MICA-129Val clones (right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.