( a ) Accumulation of MRs in cells treated with the PI ( 3 ) K inhibitor 3 - MA . Left panels show HeLa cells treated with 3 - MA ( 4 mM ) for 3 days . Cells were fixed and stained . Images are colour - inverted for better visualization . The quantification of this experiment is shown on the upper right ( mean ± s . d . of three independent experiments ; n = total number of cells counted ) . The lower right panel shows an enlarged area of 3 - MA - treated cells in true colour . Scale bars are 20 μm .

O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

(a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm.


( b ) siRNA - mediated depletion of Atg6 ( Beclin - 1 ) and Atg7 from HeLa cells causes MR accumulations . The right panel shows the quantification of MRs per cell ( mean ± s . d . of three independent experiments ) ; the left panel the efficiency of Atg6 and Atg7 depletion .

O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O

(b) siRNA-mediated depletion of Atg6 (Beclin-1) and Atg7 from HeLa cells causes MR accumulations. The right panel shows the quantification of MRs per cell (mean ± s.d. of three independent experiments); the left panel the efficiency of Atg6 and Atg7 depletion.


( c ) Inhibition of lysosomal acidification leads to MR accumulation . Panels on the left show HeLa cells left untreated or treated with chloroquine ( 20 μM ) or ammonium chloride ( 10 mM ) for 3 days . Scale bar are 20 μm . Cells were fixed and stained as indicated . A quantification of this experiment is shown on the left ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

O O O O O O O O O B-SUBCELLULAR O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( a ) Cell - cycle lengths of different cell lines correlate well with the average amount of MRs . Indicated cell lines were grown on coverslips , fixed and MRs were counted after staining with anti - MKLP1 antibodies . Cell - cycle lengths were assessed as described in the Methods . Data are mean ± s . d . of three independent experiments .

O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Cell-cycle lengths of different cell lines correlate well with the average amount of MRs. Indicated cell lines were grown on coverslips, fixed and MRs were counted after staining with anti-MKLP1 antibodies. Cell-cycle lengths were assessed as described in the Methods. Data are mean ± s.d. of three independent experiments.


( b ) The degree of MR persistence depends on the time and not on cell - cycle duration . HeLa cells were either left untreated or were arrested in S - phase with hydroxyurea ( HU , 5 mM ) . Cells were fixed and stained as indicated . Scale bars are 20 μm . The right panel shows quantification of MR counting ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O 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) The degree of MR persistence depends on the time and not on cell-cycle duration. HeLa cells were either left untreated or were arrested in S-phase with hydroxyurea (HU, 5 mM). Cells were fixed and stained as indicated. Scale bars are 20 μm. The right panel shows quantification of MR counting (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( a ) Fibroblasts of individuals with mucolipidosis II ( I - cell disease ; upper right panel ) or Hurler syndrome ( lower right panel ) were compared with parental control cells ( left panels ) . The alleles are indicated . Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA . Arrowheads point to the accumulated MRs . Scale bars are 20 μm . ( b ) Quantification of a ( mean ± s . d . of three independent experiments ; n , total number of cells ; P , probability of a two - tailed paired t - test ) .

O O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels). The alleles are indicated. Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA. Arrowheads point to the accumulated MRs. Scale bars are 20 μm. (b) Quantification of a (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( G ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM . ( H ) Quantification of ∆ FosB + nuclei in cultured MSNs after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(G) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (H) Quantification of ∆FosB+ nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation , with different 1st and 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the right column . Scale bar , 25μM . ( J ) Quantification of ∆ FosB + nuclei in cultured MSNs after different 1st and 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(I) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation, with different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the right column. Scale bar, 25μM. (J) Quantification of ∆FosB+ nuclei in cultured MSNs after different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( K ) Representative immunofluorescence staining of ∆ FosB ( green ) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons ( Gad67Cre ; Alk4fl / fl ) and control mice ( Alk4fl / fl ) after exposure to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the second and fourth rows . Scale bar , 25μM . ( L ) Quantification of ∆ FosB + nuclei in cultured MSNs from mutant and control mice after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying ∆ FosB in the nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons (Gad67Cre;Alk4fl/fl) and control mice (Alk4fl/fl) after exposure to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (L) Quantification of ∆FosB+ nuclei in cultured MSNs from mutant and control mice after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( A ) Western blot analysis of Protein Kinase A phospho - substrates ( PKA Ⓟ - subs ) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation ( same treatment in both treatment days ) with forskolin ( Fsk ) in the absence or presence of the PKA inhibitor PKI14 - 22 ( PKI ) . Reprobing with anti - PCBP1 antibodies is shown in the middle blot . Lower blot shows the total amount of PCBP1 in the cell lysates . Quantification of three independent experiments is shown on the right . Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(A) Western blot analysis of Protein Kinase A phospho-substrates (PKA Ⓟ-subs) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with forskolin (Fsk) in the absence or presence of the PKA inhibitor PKI14-22 (PKI). Reprobing with anti-PCBP1 antibodies is shown in the middle blot. Lower blot shows the total amount of PCBP1 in the cell lysates. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( B ) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) , PKA inhibitor ( PKI ) and the ALK4 inhibitor SB431542 ( SB ) as indicated . Blots of nuclear and cytosolic fractions were reprobed with anti - PCNA and anti - αTubulin ( αTub ) antibodies , respectively . Quantification of three independent experiments is shown on the right . Results are expressed as average ± SEM of fold increase over vehicle ( first column ) normalized to PCBP1 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk), PKA inhibitor (PKI) and the ALK4 inhibitor SB431542 (SB) as indicated. Blots of nuclear and cytosolic fractions were reprobed with anti-PCNA and anti-αTubulin (αTub) antibodies, respectively. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle (first column) normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( C ) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) or their combination as indicated . The Act + Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control . Reprobing with anti - PCBP1 antibodies is shown in the middle blot . The lower blot shows unaltered Smad3 levels across all conditions . Quantification of four independent experiments ( biological replicates ) is shown on the right . Results are expressed as average ± SEM of fold increase of Smad3 / PCBP1 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to PCBP1 levels . ( D ) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel ( C ) . The lower blot shows unaltered PCBP1 levels across all conditions . Quantification of four independent experiments ( biological replicates ) is shown on the right . Results are expressed as average ± SEM of fold increase of PCBP1 / Smad3 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to Smad3 levels . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(C) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk) or their combination as indicated. The Act+Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control. Reprobing with anti-PCBP1 antibodies is shown in the middle blot. The lower blot shows unaltered Smad3 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of Smad3/PCBP1 co-immunoprecipitation (coIP) over vehicle (first column) normalized to PCBP1 levels. (D) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel (C). The lower blot shows unaltered PCBP1 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of PCBP1/Smad3 co-immunoprecipitation (coIP) over vehicle (first column) normalized to Smad3 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( E ) Representative immunofluorescence staining of Smad3 ( green ) and PCBP1 ( red ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O

(E) Representative immunofluorescence staining of Smad3 (green) and PCBP1 (red) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM.


( F , G ) Quantification of PCBP1 + ( F ) or Smad3 + ( G ) nuclei in cultured MSNs after the indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± SEM of % of MAP2 + cells displaying PCBP1 ( F ) or Smad3 ( G ) in the nucleus . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) Quantification of PCBP1+ (F) or Smad3+ (G) nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying PCBP1 (F) or Smad3 (G) in the nucleus. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( H ) Quantification of PCBP1 / Smad3 co - localization in nuclei of cultured MSNs using conditions identical to panel ( F ) . Results are expressed as average ± SEM ( biological replicates ) of % of MAP2 + cells displaying co - localization of PCBP1 and Smad3 in the nucleus . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Quantification of PCBP1/Smad3 co-localization in nuclei of cultured MSNs using conditions identical to panel (F). Results are expressed as average ± SEM (biological replicates) of % of MAP2+ cells displaying co-localization of PCBP1 and Smad3 in the nucleus. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) RT - qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated ( same treatment in both treatment days ) . Results are expressed as average ± SEM of fold change over vehicle . No significant differences were found by one - way ANOVA followed with Tukey ' s multiple comparisons test ( p ≥ 0 . 05 ; N = 4 ) . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) RT-qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated (same treatment in both treatment days). Results are expressed as average ± SEM of fold change over vehicle. No significant differences were found by one-way ANOVA followed with Tukey's multiple comparisons test (p≥0.05; N=4). Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( J ) Representative images of PLA ( red ) between Smad3 and PCBP1 ( S3 / PCBP1 ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) , PKA inhibitor ( PKI ) or SB431542 ( SB ) as indicated . Counterstaining with MAP2 and DAPI is shown in the lower row . Scale bar , 25μM . ( K ) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments . Results are expressed as average ± SEM of number of PLA dots per cell . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Representative images of PLA (red) between Smad3 and PCBP1 (S3/PCBP1) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF), PKA inhibitor (PKI) or SB431542 (SB) as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (K) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of PLA dots per cell. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( D ) Representative images of RNA - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) and sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in HeLa cells exposed to consecutive stimulation ( same treatment in both treatment days ) as indicated . Counterstaining with phalloidin and DAPI is shown in the second and fourth columns . Scale bars , 20μM . ( E , F ) Quantification of RNA - PLA between PCBP1 ( E ) or Smad3 ( F ) and pE4 in HeLa cells after the indicated treatments . Results are expressed as average ± SEM of number of RNA - PLA dots per cell . Only nuclear dots were quantified in this analysis . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(D) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (E, F) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in HeLa cells after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( G ) Representative images of RNA - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) and sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in cultured MSNs exposed to consecutive stimulation as indicated . Counterstaining with phalloidin and DAPI is shown in the second and fourth columns . Scale bars , 20μM . ( H , I ) Quantification of RNA - PLA between PCBP1 ( E ) or Smad3 ( F ) and pE4 in cultured MSNs after the indicated treatments . Results are expressed as average ± SEM of number of RNA - PLA dots per cell . Only nuclear dots were quantified in this analysis . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(G) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in cultured MSNs exposed to consecutive stimulation as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (H, I) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( D ) Representative immunofluorescence staining of ∆ FosB ( red ) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with green fluorescent protein ( GFP , expressed by the lentiviruses ) and DAPI is shown in the second and fourth rows . Scale bar , 25μM . ( E ) Quantification of ∆ FosB + nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments . Results are expressed as average ± SEM of % of GFP + cells displaying ∆ FosB in the nucleus . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; two - way ANOVA followed with Tukey ' s multiple comparisons test in E ) . N denotes biological replicates .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O B-CELL_TYPE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Representative immunofluorescence staining of ∆FosB (red) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with green fluorescent protein (GFP, expressed by the lentiviruses) and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (E) Quantification of ∆FosB+ nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments. Results are expressed as average ± SEM of % of GFP+ cells displaying ∆FosB in the nucleus. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; two-way ANOVA followed with Tukey's multiple comparisons test in E). N denotes biological replicates.


( A - F ) RT - qPCR analysis of FosB ( A , D ) , c - Fos ( B , E ) and ∆ FosB ( C , F ) mRNAs in NAc of Gad67Cre ; Alk4fl / fl ( A - C ) mutant mice 1 hour ( A - C ) or 24 hour ( D - F ) after the second saline ( Sal ) or cocaine ( Coc ) injection of TIPS . Results are expressed as average ± SEM of fold increase over the saline group ( Sal / Sal ) of Gad67Cre mice . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(A-F) RT-qPCR analysis of FosB (A, D), c-Fos (B, E) and ∆FosB (C, F) mRNAs in NAc of Gad67Cre;Alk4fl/fl (A-C) mutant mice 1 hour (A-C) or 24 hour (D-F) after the second saline (Sal) or cocaine (Coc) injection of TIPS. Results are expressed as average ± SEM of fold increase over the saline group (Sal/Sal) of Gad67Cre mice. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( G ) Representative immunofluorescence staining of ∆ FosB in Nac of control ( Gad67Cre ) and mutant ( Gad67Cre ; Alk4fl / fl ) mice , 24 hours after the second injection of the TIPS protocol . Scale bars 20 μm . ( H ) Quantification of ∆ FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre ; Alk4fl / fl mice . Results are expressed as average ± SEM of the number of ∆ FosB + cells per mm2 . At least 10 images from both left and right hemispheres were analyzed per mouse and condition . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(G) Representative immunofluorescence staining of ∆FosB in Nac of control (Gad67Cre) and mutant (Gad67Cre;Alk4fl/fl) mice, 24 hours after the second injection of the TIPS protocol. Scale bars 20 μm. (H) Quantification of ∆FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre;Alk4fl/fl mice. Results are expressed as average ± SEM of the number of ∆FosB+ cells per mm2. At least 10 images from both left and right hemispheres were analyzed per mouse and condition. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) Western blot analysis of FosB and ∆ FosB in in NAc of Gad67Cre and Gad67Cre ; Alk4fl / fl mice 24 hour after the second injection of TIPS . Blots were probed with an antibody that recognizes both FosB ( upper band , 48kDa ) and ∆ FosB ( lower band , 38 kDa ) . Lower blot shows reprobing with anti - β - actin . ( J , K ) Quantification of FosB ( J ) and ∆ FosB ( K ) levels in NAc of Gad67Cre and Gad67Cre ; Alk4fl / fl mice 24 hour after the second injection of TIPS . Results are expressed as average ± SEM of fold increase of FosB and ∆ FosB over the saline group ( Sal / Sal ) of Gad67Cre mice normalized to β - actin levels . N = 4s . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons test . N denotes biological replicates .

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

(I) Western blot analysis of FosB and ∆FosB in in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Blots were probed with an antibody that recognizes both FosB (upper band, 48kDa) and ∆FosB (lower band, 38 kDa). Lower blot shows reprobing with anti-β-actin. (J, K) Quantification of FosB (J) and ∆FosB (K) levels in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Results are expressed as average ± SEM of fold increase of FosB and ∆FosB over the saline group (Sal/Sal) of Gad67Cre mice normalized to β-actin levels. N=4s. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


A A neutral red - stained mouse pancreas is excised and digested with collagenase . Islets are then separated by hand picking and further digested into single islet cells . Scale bars : 10 mm ( left image ) ; 200 µm ( middle image ) ; 60 µm ( right image ) .

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

A A neutral red-stained mouse pancreas is excised and digested with collagenase. Islets are then separated by hand picking and further digested into single islet cells. Scale bars: 10 mm (left image); 200 µm (middle image); 60 µm (right image).


B Epidydimal fat pads are isolated and processed mechanically and enzymatically . The obtained mixture , containing MVF and ECM , is filtrated for the purification of MVF . Scale bars : 20 mm ( left image ) ; 25 µm ( right image ) .

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B Epidydimal fat pads are isolated and processed mechanically and enzymatically. The obtained mixture, containing MVF and ECM, is filtrated for the purification of MVF. Scale bars: 20 mm (left image); 25 µm (right image).


C Western blots of CD31 , α - SMA and GAPDH in whole cell extracts of eEND , HDMEC , HUVEC and MVF .

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C Western blots of CD31, α-SMA and GAPDH in whole cell extracts of eEND, HDMEC, HUVEC and MVF.


D Immunofluorescence stainings of CD31 / α - SMA and CD31 / PDGFR - β in MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 20 µm .

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D Immunofluorescence stainings of CD31 / α-SMA and CD31 / PDGFR-β in MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 20 µm.


F PI and PI + MVF on days - 5 , - 3 and 0 . Scale bar : 200 µm .

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F PI and PI+MVF on days -5, -3 and 0. Scale bar: 200 µm.


G Surface morphology of FI , CI , PI and PI + MVF on day 0 as visualized by scanning electron microscopy . Scale bar : 50 µm .

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G Surface morphology of FI, CI, PI and PI+MVF on day 0 as visualized by scanning electron microscopy. Scale bar: 50 µm.


A Neutral red and trypan blue stainings of FI , CI , PI and PI + MVF . CI incubated for 24 h with 0 . 2 % H2O2 served as control . Scale bar : 100 µm .

O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

A Neutral red and trypan blue stainings of FI, CI, PI and PI+MVF. CI incubated for 24 h with 0.2% H2O2 served as control. Scale bar: 100 µm.


B Representative flow cytometric scatterplots of propidium iodide / annexin V - stained cells from PI + MVF and CI + H2O2 ( positive control ) . C Quantitative analysis of propidium iodide / annexin V - stained cells from FI , CI , PI , PI + MVF and CI + H2O2 subdivided in necrotic , necroptotic , apoptotic and vital cells in % of total cell number ( n = 3 each ) .

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

B Representative flow cytometric scatterplots of propidium iodide / annexin V-stained cells from PI+MVF and CI+H2O2 (positive control). C Quantitative analysis of propidium iodide / annexin V-stained cells from FI, CI, PI, PI+MVF and CI+H2O2 subdivided in necrotic, necroptotic, apoptotic and vital cells in % of total cell number (n = 3 each).


D Schematic illustration of the mitochondrial ROS - sensor mito - roGFP2 - Orp1 . The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels , as shown by representative fluorescence images of FI . Scale bar : 70 µm .

O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O

D Schematic illustration of the mitochondrial ROS-sensor mito-roGFP2-Orp1. The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels, as shown by representative fluorescence images of FI. Scale bar: 70 µm.


E Quantitative analysis of the ROS sensor ratio ( 405 nm / 488 nm ) of FI , CI , PI and PI + MVF ( n = 25 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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E Quantitative analysis of the ROS sensor ratio (405 nm / 488 nm) of FI, CI, PI and PI+MVF (n = 25 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


F Representative Western blot analysis of β - actin and HO - 1 in whole cell extracts from FI , CI , PI and PI + MVF . G Quantitative analysis of HO - 1 expression in FI , CI , PI and PI + MVF ( n = 3 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O

F Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from FI, CI, PI and PI+MVF. G Quantitative analysis of HO-1 expression in FI, CI, PI and PI+MVF (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


H Representative immunofluorescence stainings of HO - 1 / insulin and HO - 1 / CD31 in PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 50 µm .

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

H Representative immunofluorescence stainings of HO-1 / insulin and HO-1 / CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 µm.


I Representative Western blot analysis of β - actin and HO - 1 in whole cell extracts from PI and MVF .

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I Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from PI and MVF.


A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI , CI , PI and PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 60 µm . B - D Quantitative analysis of insulin - ( B ) , glucagon - ( C ) and somatostatin - positive cells ( D ) in FI , CI , PI and PI + MVF in % of all islet or organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O

A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI, CI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 60 µm. B-D Quantitative analysis of insulin- (B), glucagon- (C) and somatostatin-positive cells (D) in FI, CI, PI and PI+MVF in % of all islet or organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


E Western blot analysis of β - actin and insulin in whole cell extracts from FI , CI , PI and PI + MVF .

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E Western blot analysis of β-actin and insulin in whole cell extracts from FI, CI, PI and PI+MVF.


Insulin secretion ( µU / mL ) ( normalized to DNA content ) of FI , CI , PI and PI + MVF exposed to 1 . 1 mM and 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test as well as an unpaired Student ' s t - test were used for statistical analysis . * P < 0 . 05 vs . insulin secretion at 1 . 1 mM glucose within each individual group ; + P < 0 . 05 vs . insulin secretion of PI at 16 . 5 mM glucose .

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Insulin secretion (µU/mL) (normalized to DNA content) of FI, CI, PI and PI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test as well as an unpaired Student's t-test were used for statistical analysis. *P < 0.05 vs. insulin secretion at 1.1 mM glucose within each individual group; +P < 0.05 vs. insulin secretion of PI at 16.5 mM glucose.


A Representative immunofluorescence stainings of insulin and CD31 in CI , FI , PI and PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm . B Quantitative analysis of CD31 - positive cells in FI , CI , PI and PI + MVF in % of all islet or islet organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

A Representative immunofluorescence stainings of insulin and CD31 in CI, FI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm. B Quantitative analysis of CD31-positive cells in FI, CI, PI and PI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


C Representative Western blot analysis of CD31 and β - actin in whole cell extracts from CI , FI , PI and PI + MVF .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O

C Representative Western blot analysis of CD31 and β-actin in whole cell extracts from CI, FI, PI and PI+MVF.


D Quantitative analysis of CD31 expression in FI , CI , PI and PI + MVF in % of FI ( n = 3 ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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D Quantitative analysis of CD31 expression in FI, CI, PI and PI+MVF in % of FI (n = 3). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


E Confocal microscopic images of CD31 and cell nuclei ( blue ) in PI + MVF , demonstrating endothelial cells ( arrowheads ) . Scale bar : 100 µm .

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E Confocal microscopic images of CD31 and cell nuclei (blue) in PI+MVF, demonstrating endothelial cells (arrowheads). Scale bar: 100 µm.


F Representative immunofluorescence staining of Ki67 and CD31 in PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark endothelial cells expressing Ki67 . Scale bar : 100 µm .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-TISSUE O B-TISSUE O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O

F Representative immunofluorescence staining of Ki67 and CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark endothelial cells expressing Ki67. Scale bar: 100 µm.


G Quantitative analysis of CD31 / Ki67 - positive cells in FI , CI , PI and PI + MVF in % of all endothelial cells ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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G Quantitative analysis of CD31 / Ki67-positive cells in FI, CI, PI and PI+MVF in % of all endothelial cells (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


H Flow cytometric analysis of BrdU - positive cells from PI + MVF treated with DMSO ( vehicle ) or linsitinib during the entire fusion process ( n = 5 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle .

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H Flow cytometric analysis of BrdU-positive cells from PI+MVF treated with DMSO (vehicle) or linsitinib during the entire fusion process (n = 5 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.


I Representative Western blot analysis of pAkt , Akt , pERK1 / 2 and ERK1 / 2 from MVF cell extracts . MVF were treated with vehicle or insulin for 3 h . Quantitative analysis of pERK1 / 2 / ERK1 / 2 ( J ) ratio in PI + MVF treated with vehicle or insulin for 3 h . Data are expressed as relative intensities ( n = 3 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle .

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

I Representative Western blot analysis of pAkt, Akt, pERK1/2 and ERK1/2 from MVF cell extracts. MVF were treated with vehicle or insulin for 3 h. Quantitative analysis of pERK1/2 / ERK1/2 (J) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.


Quantitative analysis of pAkt / Akt ( K ) ratio in PI + MVF treated with vehicle or insulin for 3 h . Data are expressed as relative intensities ( n = 3 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . vehicle .

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Quantitative analysis of pAkt / Akt (K) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.


L Representative bright field image of angiogenic sprouting from PI + MVF on 3 day of the sprouting assay . Scale bar : 150 µm . Insert : Immunofluorescence staining of insulin ( red ) and CD31 ( green ) of sprouted PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 30 µm .

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

L Representative bright field image of angiogenic sprouting from PI+MVF on 3 day of the sprouting assay. Scale bar: 150 µm. Insert: Immunofluorescence staining of insulin (red) and CD31 (green) of sprouted PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 30 µm.


M Quantitative analysis of sprouting area of FI , CI , PI and PI + MVF in % of the initial size ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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M Quantitative analysis of sprouting area of FI, CI, PI and PI+MVF in % of the initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


B Bright field images of hFI , hCI , hPI and hPI + MVF . Scale bar : 100 µm .

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B Bright field images of hFI, hCI, hPI and hPI+MVF. Scale bar: 100 µm.


C Representative immunohistochemical stainings of insulin and CD31 in hFI , hCI , hPI and hPI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm .

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C Representative immunohistochemical stainings of insulin and CD31 in hFI, hCI, hPI and hPI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm.


D - G Quantitative analysis of insulin - ( D ) , glucagon - ( E ) , somatostatin - ( F ) and CD31 - positive cells ( G ) in hFI , hCI , hPI and hPI + MVF in % of all islet or islet organoid cells ( n = 20 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . hFI ; # P < 0 . 05 vs . hCI ; + P < 0 . 05 vs . hPI .

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D-G Quantitative analysis of insulin-(D), glucagon-(E), somatostatin-(F) and CD31-positive cells (G) in hFI, hCI, hPI and hPI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.


H Representative bright field images of angiogenic sprouting of hFI , hCI , hPI and hPI + MVF on day 3 of the sprouting assay . Scale bar : 150 µm .

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H Representative bright field images of angiogenic sprouting of hFI, hCI, hPI and hPI+MVF on day 3 of the sprouting assay. Scale bar: 150 µm.


I Quantitative analysis of sprouting area of hFI , hCI , hPI and hPI + MVF in % of initial size ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . hFI ; # P < 0 . 05 vs . hCI ; + P < 0 . 05 vs . hPI .

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I Quantitative analysis of sprouting area of hFI, hCI, hPI and hPI+MVF in % of initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.


J Quantitative analysis of C - peptide secretion ( pg / mL ) ( normalized to DNA concentration ) of hFI and hPI + MVF exposed to 1 . 1 mM and 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD .

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J Quantitative analysis of C-peptide secretion (pg/mL) (normalized to DNA concentration) of hFI and hPI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD.


B Observation window of a dorsal skinfold chamber with 8 neutral red - stained transplanted FI on day 0 . Scale bar : 5 mm .

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B Observation window of a dorsal skinfold chamber with 8 neutral red-stained transplanted FI on day 0. Scale bar: 5 mm.


C Take rate ( % of transplanted islets ) on day 14 after islet transplantation into the dorsal skinfold chamber ( n = 8 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . + P < 0 . 05 vs . PI .

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C Take rate (% of transplanted islets) on day 14 after islet transplantation into the dorsal skinfold chamber (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. +P < 0.05 vs. PI.


D Representative intravital fluorescent microscopic images of FI , CI , PI and PI + MVF within the dorsal skinfold chamber on day 0 and 14 . FITC - labeled dextran is used for the visualization of blood - perfused microvessels . Grafts are marked by broken lines . Scale bar : 150 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O

D Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 0 and 14. FITC-labeled dextran is used for the visualization of blood-perfused microvessels. Grafts are marked by broken lines. Scale bar: 150 μm.


E , F Quantitative analysis of the functional capillary density ( cm / cm - 2 ) ( E ) and revascularized area ( mm2 ) ( F ) of transplanted FI , CI , PI and PI + MVF on day 0 , 3 , 6 , 10 and 14 ( n = 8 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O

E, F Quantitative analysis of the functional capillary density (cm/cm-2) (E) and revascularized area (mm2) (F) of transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14 (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


G Representative intravital fluorescent microscopic images of FI , CI , PI and PI + MVF within the dorsal skinfold chamber on day 14 . Rhodamine 6G is used to visualize blood perfused vital endocrine cells ( bright signals ) . Scale bar : 150 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-TISSUE O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

G Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 14. Rhodamine 6G is used to visualize blood perfused vital endocrine cells (bright signals). Scale bar: 150 µm.


H Quantitative analysis of the rhodamine 6G - positive area ( % of islet size ) within transplanted FI , CI , PI and PI + MVF on day 0 , 3 , 6 , 10 and 14 . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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H Quantitative analysis of the rhodamine 6G-positive area (% of islet size) within transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14. Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


A Representative immunofluorescent stainings of insulin / glucagon , insulin / somatostatin and insulin / CD31 in FI , CI , PI and PI + MVF on day 14 after transplantation . Cell nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark intra - islet endothelial cells . Scale bar : 70 µm . B - E Quantitative analysis of insulin - ( B ) , glucagon - ( C ) , somatostatin - ( D ) and intra - islet CD31 - positive cells ( E ) within FI , CI , PI and PI + MVF on day 14 after transplantation ( n = 10 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE B-GENEPROD O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O

A Representative immunofluorescent stainings of insulin / glucagon, insulin / somatostatin and insulin / CD31 in FI, CI, PI and PI+MVF on day 14 after transplantation. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark intra-islet endothelial cells. Scale bar: 70 µm. B-E Quantitative analysis of insulin- (B), glucagon- (C), somatostatin- (D) and intra-islet CD31-positive cells (E) within FI, CI, PI and PI+MVF on day 14 after transplantation (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


B Quantitative analysis of the body weight ( g ) of nondiabetic and diabetic mice transplanted with FI or PI + MVF from day - 8 to day 28 ( n = 7 each ) . Mean ± SD .

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B Quantitative analysis of the body weight (g) of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day -8 to day 28 (n = 7 each). Mean ± SD.


C AUC of the body weight of nondiabetic and diabetic mice transplanted with FI or PI + MVF from day 0 to day 28 ( n = 7 each ) . Mean ± SD .

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C AUC of the body weight of nondiabetic and diabetic mice transplanted with FI or PI+MVF from day 0 to day 28 (n = 7 each). Mean ± SD.


D Quantitative analysis of blood glucose level ( mg / dL ) of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls from day - 8 to day 28 ( n = 7 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF .

O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O B-DISEASE B-ORGANISM O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O B-TISSUE O

D Quantitative analysis of blood glucose level (mg/dL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day -8 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.


E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls from day 0 to day 28 ( n = 7 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF .

O O O O B-TISSUE B-SMALL_MOLECULE O O B-DISEASE B-ORGANISM O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O B-TISSUE O

E AUC of the blood glucose levels of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls from day 0 to day 28 (n = 7 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.


F Quantitative analysis of blood glucose level ( mg / dL ) according to the IPGTT of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls to the indicated time points ( n = 5 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI .

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

F Quantitative analysis of blood glucose level (mg/dL) according to the IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls to the indicated time points (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI.


G AUC of IPGTT of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls ( n = 5 each ) . Mean ± SD . One - way ANOVA and Tukey ' s multiple comparisons post hoc test were used for statistical analysis . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF .

O O O B-EXP_ASSAY O B-DISEASE B-ORGANISM O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O B-TISSUE O B-TISSUE O

G AUC of IPGTT of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls (n = 5 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. PI+MVF.


H Plasma insulin level ( µU / mL ) of diabetic mice transplanted with FI or PI + MVF and nondiabetic controls at 180 min after IPGTT ( n = 4 each ) . Mean ± SD .

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H Plasma insulin level (µU/mL) of diabetic mice transplanted with FI or PI+MVF and nondiabetic controls at 180 min after IPGTT (n = 4 each). Mean ± SD.


I Representative immunofluorescent stainings of insulin / CD31 in FI and PI + MVF on day 28 after islet transplantation . Scale bar : 50 µm .

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I Representative immunofluorescent stainings of insulin / CD31 in FI and PI+MVF on day 28 after islet transplantation. Scale bar: 50 µm.


J Quantitative analysis of insulin - ( β - cells ) , glucagon - ( α - cells ) , somatostatin - ( δ - cells ) and CD31 - positive ( endothelial ) cells within transplanted FI and PI + MVF on day 28 after islet transplantation . Data are expressed in % of total islet cells ( n = 25 ) . Mean ± SD .

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J Quantitative analysis of insulin- (β-cells), glucagon- (α-cells), somatostatin- (δ-cells) and CD31-positive (endothelial) cells within transplanted FI and PI+MVF on day 28 after islet transplantation. Data are expressed in % of total islet cells (n = 25). Mean ± SD.


K Quantitative analysis of islet cells in transplanted FI and PI + MVF per HPF ( n = 70 each ) . Mean ± SD . Unpaired Student ' s t - test was used for statistical analysis . * P < 0 . 05 vs . FI .

O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O 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-TISSUE O

K Quantitative analysis of islet cells in transplanted FI and PI+MVF per HPF (n = 70 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. FI.


( A ) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 7 weeks reveals formation and elimination of dendritic spines ( white and empty arrowheads , respectively ) in WT and APP - KO mice . All mice were housed in standard conditions . Scale bar - 10 μm . ( B - E ) Graphical representations of the relative spine density , spine turnover rate ( TOR ) , elimination and formation over the imaging period . Non - linear regression ( F test ) has been used for fitting the data points and comparing the difference between groups . N = 4 mice per group * * p < 0 . 01 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O B-TISSUE I-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O

(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 7 weeks reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. All mice were housed in standard conditions. Scale bar - 10 μm. (B-E) Graphical representations of the relative spine density, spine turnover rate (TOR), elimination and formation over the imaging period. Non-linear regression (F test) has been used for fitting the data points and comparing the difference between groups. N=4 mice per group ** p<0.01.


( A ) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 46 days reveals formation and elimination of dendritic spines ( white and empty arrowheads , respectively ) in WT and APP - KO mice . Prior to the exposure to environmental enrichment ( EE ) , all mice were housed in standard conditions . Scale bar - 10 μm . ( B - C ) Graphical representations of the relative spine density and spine elimination over the period of the exposure of mice to EE . Non - linear regression ( F test ) has been used for fitting the data points . Repeated one - way ANOVA was performed followed by Dunnett test . WT n = 5 mice and APP - KO n = 6 mice ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O B-TISSUE I-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O 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-ORGANISM O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 46 days reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. Prior to the exposure to environmental enrichment (EE), all mice were housed in standard conditions. Scale bar - 10 μm. (B-C) Graphical representations of the relative spine density and spine elimination over the period of the exposure of mice to EE. Non-linear regression (F test) has been used for fitting the data points. Repeated one-way ANOVA was performed followed by Dunnett test. WT n=5 mice and APP-KO n=6 mice; * p<0.05, ** p<0.01, NS - no significant difference.


( A ) Typical confocal images of apical dendrites with spines ( z - projections ) from layer V pyramidal neurons in the somatosensory cortex of WT and APP - KO mice ( top and bottom ) housed in standard and enriched environments ( left and right ) . For classification of spine types , 3D reconstructions by Imaris have been applied . Thin , mushroom and stubby spines are encoded in blue , green and red , respectively . Scale bar represents 2 μm . ( B - C ) Summary plots of thin and mushroom spine fractions in WT and APP - KO mice exposed to standard ( SE ) and enriched environments ( EE ) . . Two - tailed Student t - test was used and n = 6 mice in all experimental groups ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-TISSUE I-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O 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-GENEPROD O O B-ORGANISM O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Typical confocal images of apical dendrites with spines (z-projections) from layer V pyramidal neurons in the somatosensory cortex of WT and APP-KO mice (top and bottom) housed in standard and enriched environments (left and right). For classification of spine types, 3D reconstructions by Imaris have been applied. Thin, mushroom and stubby spines are encoded in blue, green and red, respectively. Scale bar represents 2 μm. (B-C) Summary plots of thin and mushroom spine fractions in WT and APP-KO mice exposed to standard (SE) and enriched environments (EE). . Two-tailed Student t-test was used and n=6 mice in all experimental groups; * p<0.05, ** p<0.01, NS - no significant difference.


( A ) Extracellular D - serine concentrations in cortex were detected by enzyme - based microelectrode biosensors .

(A) Extracellular D-serine concentrations in cortex were detected by enzyme-based microelectrode biosensors.


( B , C ) High performance liquid chromatography was performed to quantify total D - serine and L - serine contents in mouse brain of two genotypes . APP - KO mice were fed with D - serine over one month . One - way ANOVA was used for analysis followed by Bonferroni ' s multiple comparisons test and n > 6 mice per group ; * p < 0 . 05 , * * p < 0 . 01 as compared to WT .

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

(B, C) High performance liquid chromatography was performed to quantify total D-serine and L-serine contents in mouse brain of two genotypes. APP-KO mice were fed with D-serine over one month. One-way ANOVA was used for analysis followed by Bonferroni's multiple comparisons test and n>6 mice per group; * p<0.05, ** p<0.01 as compared to WT.


( A ) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex of APP - KO mice housed under standard or enriched environment . Note that both groups of mice received D - serine after the second imaging time point ( 8 d ) ; white and empty arrowheads point to newly formed and eliminated spines , respectively . Scale bar - 10 μm . ( B ) Spine TOR prior and during continuous D - serine treatment . ( C , D ) Summary plots of the fraction of spine elimination and formation in APP - KO mice before and after D - serine treatment ( 8 d and 46 d , respectively ) . ( E ) Relative spine densities in D - serine treated APP - KO mice housed under standard and enriched environments . ( F , G ) Summary plots of the fraction of thin and mushroom spines in control and D - serine treated APP - KO mice . For illustration purpose , the control data from Figure 2 B and C are presented also here . Non - linear regression ( F test ) has been used for fitting the data points . Two - tailed Student t - test was used in ( C - D , F - G ) and repeated one - way ANOVA was performed followed by Dunnett test in ( B and E ) . N = 5 mice in each group ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

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

(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex of APP-KO mice housed under standard or enriched environment. Note that both groups of mice received D-serine after the second imaging time point (8 d); white and empty arrowheads point to newly formed and eliminated spines, respectively. Scale bar - 10 μm. (B) Spine TOR prior and during continuous D-serine treatment. (C, D) Summary plots of the fraction of spine elimination and formation in APP-KO mice before and after D-serine treatment (8 d and 46 d, respectively). (E) Relative spine densities in D-serine treated APP-KO mice housed under standard and enriched environments. (F, G) Summary plots of the fraction of thin and mushroom spines in control and D-serine treated APP-KO mice. For illustration purpose, the control data from Figure 2 B and C are presented also here. Non-linear regression (F test) has been used for fitting the data points. Two-tailed Student t-test was used in (C-D, F-G) and repeated one-way ANOVA was performed followed by Dunnett test in (B and E). N=5 mice in each group; * p<0.05, ** p<0.01, NS - no significant difference.


( A ) Quantification of total distance that mice traveled for 10 min .

(A) Quantification of total distance that mice traveled for 10 min.


( B ) The ratios of mice explored between novel and familiar objects . Lines in boxes indicate mean levels and whiskers represent the data from minimal to maximal range . The box extends from 25 % to 75 % of values . Wilcoxon signed - rank test was performed to compare data to the hypothetical value 1 . N = 9 - 12 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM 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) The ratios of mice explored between novel and familiar objects. Lines in boxes indicate mean levels and whiskers represent the data from minimal to maximal range. The box extends from 25% to 75% of values. Wilcoxon signed-rank test was performed to compare data to the hypothetical value 1. N=9-12 mice per group; * p<0.05, ** p<0.01, NS - no significant difference.


( D ) Kaplan - Meier survival curves . Statistics : log rank Gehan Breslow Wilcoxon test , p < 0 . 0001 , n = 20 per group .

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(D) Kaplan-Meier survival curves. Statistics: log rank Gehan Breslow Wilcoxon test, p < 0.0001, n=20 per group.


( C ) Kaplan - Meier survival curves of the 4 groups of mice shown in panel B . Statistics : Gehan - Breslow - Wilcoxon test .

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(C) Kaplan-Meier survival curves of the 4 groups of mice shown in panel B. Statistics: Gehan-Breslow-Wilcoxon test.


( B ) Representative immunohistochemical staining for calreticulin in tumor masses of control ( PBS ) and BoxA - treated mice . Scale bar 50 μm . The distribution of calreticulin positive pixels describes the location of calreticulin ( intracellular vs ecto - calreticulin ) . Statistics : Mann - Whitney test , n = 4 .

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

(B) Representative immunohistochemical staining for calreticulin in tumor masses of control (PBS) and BoxA-treated mice. Scale bar 50 μm. The distribution of calreticulin positive pixels describes the location of calreticulin (intracellular vs ecto-calreticulin). Statistics: Mann-Whitney test, n=4.


( C ) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours . Statistics : One - way Anova with Dunnett ' s post - test ; n = 3 . Data information : columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(C) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours. Statistics: One-way Anova with Dunnett's post-test; n=3. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( D ) Ratio of phosphorylated eIF2α ( p - eIF2α ) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours ; β - actin is shown as loading control . Statistics : One - way Anova with Dunnett ' s post - test ; n = 2 . Data information : columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(D) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours; β-actin is shown as loading control. Statistics: One-way Anova with Dunnett's post-test; n=2. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( G ) Confocal immunofluorescence microscopy of cleaved caspase 3 ( red ) in MM cells treated for 24 hours with MTX ( 1 μM ) or increasing concentrations of BoxA . Nuclei were stained with Hoechst ( blue ) and cytosol with phalloidin ( green ) . Scale bar 50 μm .

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

(G) Confocal immunofluorescence microscopy of cleaved caspase 3 (red) in MM cells treated for 24 hours with MTX (1 μM) or increasing concentrations of BoxA. Nuclei were stained with Hoechst (blue) and cytosol with phalloidin (green). Scale bar 50 μm.


( I ) Kaplan - Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H ( Log rank Gehan Breslow Wilcoxon test , p = 0 . 59 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(I) Kaplan-Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H (Log rank Gehan Breslow Wilcoxon test, p=0.59).


( E ) Representative immunofluorescence staining of CD47 ( red ) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours . Nuclei are counterstained with DAPI . Scale bar 50 μm .

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

(E) Representative immunofluorescence staining of CD47 (red) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours. Nuclei are counterstained with DAPI. Scale bar 50 μm.


( A ) Ecto - calreticulin exposure ( expressed as MFI ) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours . Error bars indicate standard deviation . Data were pooled from two different experiments . Data information : bars and error bars represent mean ± SD ; statistics : One - way Anova with Dunnett ' s post - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Ecto-calreticulin exposure (expressed as MFI) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours. Error bars indicate standard deviation. Data were pooled from two different experiments. Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( I ) Kaplan - Meier survival curves of mice shown in panel H . Statistics : Gehan - Breslow - Wilcoxon test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O

(I) Kaplan-Meier survival curves of mice shown in panel H. Statistics: Gehan-Breslow-Wilcoxon test.


( A ) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4 , CD47 and RAGE , CD47 and TLR4 . Representative images from one experiment out of 3 performed are shown . MM cells were incubated overnight with 400 nM BoxA or PBS . Nuclei are in blue ( DAPI ) , phalloidin is in green . Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4 . Scale bar , 20 μm . The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells ( n as indicated ) . Insets are enlargements of the y axis . Mean and SD are indicated . Statistics : Kolmogorov - Smirnov test .

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

(A) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4, CD47 and RAGE, CD47 and TLR4. Representative images from one experiment out of 3 performed are shown. MM cells were incubated overnight with 400 nM BoxA or PBS. Nuclei are in blue (DAPI), phalloidin is in green. Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4. Scale bar, 20 μm. The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells (n as indicated). Insets are enlargements of the y axis. Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.


( C ) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12 . The experiment shown is representative of 2 performed , in biological triplicate . Data information : bars and error bars represent mean ± SD ; statistics : one - way Anova plus Dunnett ' s post - test . In all panels , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12. The experiment shown is representative of 2 performed, in biological triplicate. Data information: bars and error bars represent mean ± SD; statistics: one-way Anova plus Dunnett's post-test. In all panels, *p<0.05, **p<0.01, ****p<0.0001.


Impact of MB on cytotoxicity of OT - 1 CTLs against EG7 or EG7 - L1 . Splenocytes from OT - 1 mice in culture were stimulated with 10 nM of OVA257 - 264 ( SIINFEKL ) for 3 days to generate mature CTLs . CTLs were incubated with CFSE labeled EG7 - L1 cells in the presence of MB at indicated concentrations . Cytotoxicity was determined by flow cytometry analysis . Data are representative of three independent experiments . ( effector to target ratio = 5 : 1 , unpaired t - test ) . EG7 - L1 : EG7 overexpressing PD - L1 . Statistical results of ( B ) . ELISA measured of LDH release to assess the cytotoxicity of OT - I CTLs on EG7 - L1 in the presence of MB . Anti - PD1 antibody ( aPD - 1 ) served as positive control . Data information : Data are representative of three independent experiments . Unpaired t - test ; Error bars denote s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O B-CELL_LINE O B-CELL_LINE O B-GENEPROD O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-CELL_LINE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O B-CELL_LINE O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Impact of MB on cytotoxicity of OT-1 CTLs against EG7 or EG7-L1. Splenocytes from OT-1 mice in culture were stimulated with 10 nM of OVA257-264 (SIINFEKL) for 3 days to generate mature CTLs. CTLs were incubated with CFSE labeled EG7-L1 cells in the presence of MB at indicated concentrations. Cytotoxicity was determined by flow cytometry analysis. Data are representative of three independent experiments. (effector to target ratio = 5:1, unpaired t-test). EG7-L1: EG7 overexpressing PD-L1.   Statistical results of (B).   ELISA measured of LDH release to assess the cytotoxicity of OT-I CTLs on EG7-L1 in the presence of MB. Anti-PD1 antibody (aPD-1) served as positive control.   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001.