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

O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - disease ; upper right panel ) or Hurler syndrome ( lower right panel ) were compared with parental control ( left panels ) . alleles are indicated . were fixed stained with indicated antibodies DAPI to visualize . Arrowheads point to accumulated MRs . Scale bars are 20 μm . ( b ) of a ( mean ± s . d . of three independent experiments ; n , total number of ; P , probability of a two - paired t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 DAPI is shown in lower row . Scale bar , 25μM . ( H ) of ∆ FosB + nuclei in cultured MSNs after indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± of % of MAP2 + displaying ∆ FosB in nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of ∆ FosB ( green ) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons ( Gad67Cre ; Alk4fl / fl ) 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 DAPI is shown in second fourth rows . Scale bar , 25μM . ( L ) of ∆ FosB + nuclei in cultured MSNs from mutant control mice after indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± of % of MAP2 + displaying ∆ FosB in nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

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

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

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of Smad3 ( green ) 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 DAPI is shown in lower row . Scale bar , 25μM .

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

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

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated ( same treatment in both treatment days ) . Results are expressed as average ± of fold change over vehicle . No significant differences were found by one - way ANOVA followed with Tukey ' s multiple comparisons ( 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 . N denotes biological replicates .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of PLA ( red ) between Smad3 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 DAPI is shown in lower row . Scale bar , 25μM . ( K ) of PLA between Smad3 PCBP1 in cultured MSNs after indicated treatments . Results are expressed as average ± of number of PLA dots per . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

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

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

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(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 of FosB ( A , D ) , c - Fos ( B , E ) ∆ FosB ( C , F ) mRNAs in NAc of Gad67Cre ; Alk4fl / fl ( A - C ) mutant mice 1 hour ( A - C ) or 24 hour ( D - F ) after second saline ( Sal ) or cocaine ( Coc ) injection of TIPS . Results are expressed as average ± of fold increase over 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 . N denotes biological replicates .

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

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

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(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 digested with collagenase . Islets are then separated by picking further digested into single islet . Scale bars : 10 mm ( left image ) ; 200 µm ( middle image ) ; 60 µm ( right image ) .

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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 processed mechanically enzymatically . obtained mixture , containing MVF ECM , is filtrated for 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 GAPDH in whole extracts of eEND , HDMEC , HUVEC 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 CD31 / PDGFR - β in MVF . 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 PI + MVF on days - 5 , - 3 0 . Scale bar : 200 µm .

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


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

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

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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 mitochondrial ROS - sensor mito - roGFP2 - Orp1 . maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 , as shown by representative of FI . Scale bar : 70 µm .

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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 of ROS sensor ( 405 nm / 488 nm ) of FI , CI , PI PI + MVF ( n = 25 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * 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 of β - actin HO - 1 in whole extracts from FI , CI , PI PI + MVF . G Quantitative of HO - 1 expression in FI , CI , PI PI + MVF ( n = 3 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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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 HO - 1 / CD31 in PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 50 µm .

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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 of β - actin HO - 1 in whole extracts from PI 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 insulin / somatostatin in FI , CI , PI PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 60 µm . B - D Quantitative of insulin - ( B ) , glucagon - ( C ) somatostatin - positive ( D ) in FI , CI , PI PI + MVF in % of islet or organoid ( n = 20 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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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 of β - actin insulin in whole extracts from FI , CI , PI 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 content ) of FI , CI , PI PI + MVF exposed to 1 . 1 mM 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc as well as an unpaired Student ' s t - were used for statistical . * P < 0 . 05 vs . insulin secretion at 1 . 1 mM glucose within each 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 CD31 in CI , FI , PI PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm . B Quantitative of CD31 - positive in FI , CI , PI PI + MVF in % of islet or islet organoid ( n = 20 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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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 of CD31 β - actin in whole extracts from CI , FI , PI PI + MVF .

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


D Quantitative of CD31 expression in FI , CI , PI PI + MVF in % of FI ( n = 3 ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * 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 of CD31 nuclei ( blue ) in PI + MVF , demonstrating endothelial ( 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 of Ki67 CD31 in PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark endothelial expressing Ki67 . Scale bar : 100 µm .

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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 of CD31 / Ki67 - positive in FI , CI , PI PI + MVF in % of endothelial ( n = 10 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * 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 of BrdU - positive from PI + MVF treated with DMSO ( vehicle ) or linsitinib during entire fusion process ( n = 5 each ) . Mean ± SD . Unpaired Student ' s t - was used for statistical . * 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 of pAkt , Akt , pERK1 / 2 ERK1 / 2 from MVF extracts . MVF were treated with vehicle or insulin for 3 h . Quantitative of pERK1 / 2 / ERK1 / 2 ( J ) 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 - was used for statistical . * P < 0 . 05 vs . vehicle .

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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 of pAkt / Akt ( K ) 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 - was used for statistical . * 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 sprouting . Scale bar : 150 µm . Insert : Immunofluorescence of insulin ( red ) CD31 ( green ) of sprouted PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 30 µm .

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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 of sprouting of FI , CI , PI PI + MVF in % of initial ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * 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 of hFI , hCI , hPI hPI + MVF . Scale bar : 100 µm .

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


C Representative immunohistochemical stainings of insulin CD31 in hFI , hCI , hPI hPI + MVF . 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 of insulin - ( D ) , glucagon - ( E ) , somatostatin - ( F ) CD31 - positive ( G ) in hFI , hCI , hPI hPI + MVF in % of islet or islet organoid ( n = 20 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * 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 of angiogenic sprouting of hFI , hCI , hPI hPI + MVF on day 3 of sprouting . 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 of sprouting of hFI , hCI , hPI hPI + MVF in % of initial ( day 0 ) ( n = 10 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . hFI ; # P < 0 . 05 vs . hCI ; + P < 0 . 05 vs . hPI .

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 of C - peptide secretion ( pg / mL ) ( normalized to ) of hFI hPI + MVF exposed to 1 . 1 mM 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 dorsal skinfold chamber ( n = 8 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . + 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 microscopic of FI , CI , PI PI + MVF within dorsal skinfold chamber on day 0 14 . FITC - labeled dextran is used for visualization of blood - perfused microvessels . Grafts are marked by broken lines . Scale bar : 150 μm .

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

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

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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 of rhodamine 6G - positive ( % of islet ) within transplanted FI , CI , PI PI + MVF on day 0 , 3 , 6 , 10 14 . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * 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 insulin / CD31 in FI , CI , PI PI + MVF on day 14 after transplantation . nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark intra - islet endothelial . Scale bar : 70 µm . B - E Quantitative of insulin - ( B ) , glucagon - ( C ) , somatostatin - ( D ) intra - islet CD31 - positive ( E ) within FI , CI , PI PI + MVF on day 14 after transplantation ( n = 10 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

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

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

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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 nondiabetic controls ( n = 5 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . PI + MVF .

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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 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 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 of insulin - ( β - ) , glucagon - ( α - ) , somatostatin - ( δ - ) CD31 - positive ( endothelial ) within transplanted FI PI + MVF on day 28 after islet transplantation . Data are expressed in % of total islet ( 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 of islet in transplanted FI PI + MVF per HPF ( n = 70 each ) . Mean ± SD . Unpaired Student ' s t - was used for statistical . * P < 0 . 05 vs . FI .

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

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

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

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(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 - microelectrode biosensors .

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(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 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 followed by Bonferroni ' s multiple comparisons n > 6 mice per group ; * p < 0 . 05 , * * p < 0 . 01 as compared to WT .

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

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(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 ) of total that mice traveled for 10 min .

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


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

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(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 , 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 4 groups of mice shown in panel B . Statistics : Gehan - Breslow - Wilcoxon .

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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 for calreticulin in masses of control ( PBS ) BoxA - treated mice . Scale bar 50 μm . distribution of calreticulin positive pixels describes location of calreticulin ( intracellular vs ecto - calreticulin ) . Statistics : Mann - Whitney , n = 4 .

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(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 in medium of AB1 treated with BoxA or 1 μM MTX for 24 hours . Statistics : One - way Anova with Dunnett ' s post - ; n = 3 . Data information : columns represent average 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 ) of phosphorylated eIF2α ( p - eIF2α ) over eIF2α in MM 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 - ; n = 2 . Data information : columns represent average 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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(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 caspase 3 ( red ) in MM treated for 24 hours with MTX ( 1 μM ) or increasing concentrations of BoxA . Nuclei were stained with Hoechst ( blue ) cytosol with phalloidin ( green ) . Scale bar 50 μm .

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(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 pretreated with PBS or BoxA shown in panel H ( Log rank Gehan Breslow Wilcoxon , p = 0 . 59 ) .

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

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

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(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 .

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


( A ) Confocal of Proximity Ligation Assays performed on CD47 CXCR4 , CD47 RAGE , CD47 TLR4 . Representative from one experiment out of 3 performed are shown . MM 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 . of red signal in PLA was quantified as described in Materials Methods in ( n as indicated ) . Insets are enlargements of y axis . Mean SD are indicated . Statistics : Kolmogorov - Smirnov .

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

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(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 in presence of MB at indicated concentrations . Cytotoxicity was determined by flow cytometry . Data are representative of three independent experiments . ( effector to target = 5 : 1 , unpaired t - ) . EG7 - L1 : EG7 overexpressing PD - L1 . Statistical results of ( B ) . ELISA measured of LDH release to assess cytotoxicity of OT - I CTLs on EG7 - L1 in presence of MB . Anti - PD1 antibody ( aPD - 1 ) served as positive control . Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


Impact of MB on cytotoxicity of PD - 1 - / - ( PD - 1KO ) OT - 1 CTLs against EG7 or PD - L1 expressing EG7 stable . Data are representative of three independent experiments . ( unpaired t - ) . Statistical results of ( E ) . Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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Impact of MB on cytotoxicity of PD-1-/- (PD-1KO) OT-1 CTLs against EG7 or PD-L1 expressing EG7 stable cell line. Data are representative of three independent experiments. (unpaired t-test). Statistical results of (E). 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.


Cytotoxicity of OT - I CTLs against B16 - F10 - OVA ( designated B16 - OVA ) in presence of 1 μΜ of MB or DMSO . Cytotoxicity was determined by relative unoccupied by examined under microscope ( scale bar = 50 μm ) . Data are representative of three independent experiments , were analyzed by unpaired t - . Statistical results of ( G ) . Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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Cytotoxicity of OT-I CTLs against B16-F10-OVA cells (designated B16-OVA) in the presence of 1 μΜ of MB or DMSO. Cytotoxicity was determined by the relative area unoccupied by cells examined under microscope (scale bar = 50 μm). Data are representative of three independent experiments, and were analyzed by unpaired t-test.   Statistical results of (G).   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.


Effect of MB on proliferation of WT CTLs , PD - 1KO CTLs . Splenic were stained with 5 μM of CFSE seeded into aCD3 / aCD28 coated 96 - well plates . 10 μg / ml of PD - L1 were administered in media . proliferation were checked by monitoring dilution of CFSE 48 hours after CD3 / CD28 stimulation by gating on CD8 positive population through FACS . WT : splenic from wildtype C57BL / J mice . PD - 1KO : splenic from PD - 1 knockout mice . Statistical results of ( A ) Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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Effect of MB on the proliferation of WT CTLs, PD-1KO CTLs. Splenic cells were stained with 5 μM of CFSE and seeded into aCD3/aCD28 coated 96-well plates. 10 μg/ml of PD-L1 were administered in media. Cell proliferation were checked by monitoring dilution of CFSE 48 hours after CD3/CD28 stimulation by gating on CD8 positive population through FACS analysis. WT: splenic cell from wildtype C57BL/J mice. PD-1KO: splenic cell from PD-1 knockout mice.   Statistical results of (A)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


FACS of effect of MB on activation of OT - I CTLs . OT - I CTLs were stimulated with precoated aCD3 / aCD28 10 μg / ml of mouse PD - L1 in presence of 100 nM MB or 10 μg / ml aPD1 ( served as positive control ) . After 24 hours , surface expression of CD25 CD69 on OT - 1 CTLs were determined through FACS . Statistical results of ( C ) Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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FACS analysis of the effect of MB on the activation of OT-I CTLs. OT-I CTLs were stimulated with precoated aCD3/aCD28 and 10 μg/ml of mouse PD-L1 protein in the presence of 100 nM MB or 10 μg/ml aPD1 (served as positive control). After 24 hours, surface expression of CD25 and CD69 on OT-1 CTLs were determined through FACS analysis.   Statistical results of (C)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


Effect of MB on activation status of Jurkat T . Luciferase activity is suppressed in JP - luc by co - culture with Raji - L1 preloaded with 1 μg / ml superantigen ( SEE ) . Treatment with MB enhanced luciferase expression ( SEE - Loaded Raji ( PD - L1 negative ) served as positive control ) . IC50 is calculated to be 117 nM . JP - luc : Jurkat harboring NFAT - luciferase transgene overexpressing PD - 1 ; Raji - L1 : Raji overexpressing PD - L1 Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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Effect of MB on the activation status of Jurkat T cells. Luciferase activity is suppressed in JP-luc by co-culture with Raji-L1 preloaded with 1 μg/ml superantigen (SEE). Treatment with MB enhanced luciferase expression (SEE-Loaded Raji (PD-L1 negative) served as positive control). IC50 is calculated to be 117 nM. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1; Raji-L1: Raji overexpressing PD-L1 Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


Impact of MB on luciferase activity of various engineered Jurkat T . J - luc : Jurkat harboring NFAT - luciferase transgene ; J - luc - sgPD - 1 : J - luc treated with lentivirus expressing sgPD - 1 / CAS9 simultaneously Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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Impact of MB on luciferase activity of various engineered Jurkat T cells. J-luc: Jurkat cell harboring NFAT-luciferase transgene; J-luc-sgPD-1: J-luc cells treated with lentivirus expressing sgPD-1/CAS9 simultaneously Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


qRT - PCR of IL - 2 mRNA level in JP - luc stimulated with precoated aCD3 / aCD28 ( 10 μg / ml ) in presence of 10 μg / ml of PD - L1 MB at indicated concentrations Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

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qRT-PCR analysis of IL-2 mRNA level in JP-luc cells stimulated with precoated aCD3/aCD28 (10 μg/ml) in the presence of 10 μg/ml of PD-L1 and MB at indicated concentrations Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


MB enhancing IL - 2 expression by JP - luc stimulated with Raji - L1 for 24 - hour quantified through ELISA ( aPD1 served as positive control ) . MB enhancing production of cytokine cytolytic granule by OT - I CTLs . CTLs were incubated with EG7 - L1 in presence of transport inhibitor ( PTI ) MB at indicated concentrations . Expression of cytokine cytolytic granule were determined by flow cytometry . aPD1 served as positive control . EG7 - L1 : EG7 overexpressing PD - L1 . Statistical results of ( I ) Data information : Data are representative of three independent experiments . Unpaired t - ; Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001

B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MB enhancing IL-2 expression by JP-luc stimulated with Raji-L1 for 24-hour quantified through ELISA analysis (aPD1 served as positive control).   MB enhancing production of cytokine and cytolytic granule by OT-I CTLs. CTLs were incubated with EG7-L1 cells in the presence of protein transport inhibitor (PTI) and MB at indicated concentrations. Expression of cytokine and cytolytic granule were determined by flow cytometry. aPD1 served as positive control. EG7-L1: EG7 overexpressing PD-L1.   Statistical results of (I)   Data information: Data are representative of three independent experiments. Unpaired t-test; Error bars denote s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001


Confocal of Jurkat co - expressing PD - 1 - EGFP SHP2 - mCherry . Jurkat co - expressing PD - 1 - EGFP SHP2 - mCherry were incubated with 10 μg / ml of human PD - L1 in presence of 100 nM 1 μM of MB for 2 minutes , fixed with 4 % paraformaldehyde stained with DAPI . Colocalization signal were examined with confocal microscope ( scale bar = 2 µm )

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal cell images of Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry. Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry were incubated with 10 μg/ml of human PD-L1 protein in the presence of 100 nM and 1 μM of MB for 2 minutes, fixed with 4% paraformaldehyde and stained with DAPI. Colocalization signal were examined with confocal microscope (scale bar = 2 µm)


complementation of effect of MB on interaction between PD - 1 with SHP2 . Jurkat co - expressing PD1 - C - Luc or PD1Y248F - C - Luc SHP2 - N - luc were seeded in a 96 - well precoated with 10 μg / ml of aCD3 / aCD28 with media supplemented with 10 μg / ml human PD - L1 in presenc of MB at indicated concentrations for 6 hours followed by of luciferase activity Data information : Data are representative of three independent experiments , were analyzed by unpaired t - . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001

O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence complementation analysis of the effect of MB on the interaction between PD-1 with SHP2. Jurkat cells co-expressing PD1-C-Luc or PD1Y248F-C-Luc and SHP2-N-luc were seeded in a 96-well precoated with 10 μg/ml of aCD3/aCD28 with media supplemented with 10 μg/ml human PD-L1 protein in the presenc of MB at indicated concentrations for 6 hours followed by analysis of luciferase activity Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001


Co - IP of impact of MB on interaction between PD - 1 SHP2 . 293T co - expressing PD - 1 SHP2 - FLAG were treated with MB at indicated for 6 hours

O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O

Co-IP analysis of impact of MB on interaction between PD-1 and SHP2. 293T cells co-expressing PD-1 and SHP2-FLAG were treated with MB at indicated concentration for 6 hours


Ability of MB to block interaction between PD - 1 with SHP2 revealed through ELISA . Y248 phosphorylated human PD - 1 ITSM peptide was coated at 4 μg / ml in 96 plate overnight . Recombinant human SHP2 incubated with coated peptide in presence of MB for 2 hours . Bound SHP2 were measured by monitoring activity of horse - peroxidase conjugated on secondary antibody targeting SHP2 Data information : Data are representative of three independent experiments , were analyzed by unpaired t - . Error bars denote s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001

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Ability of MB to block interaction between PD-1 with SHP2 revealed through ELISA. Y248 phosphorylated human PD-1 ITSM peptide was coated at 4 μg/ml in 96 plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 hours. Bound SHP2 were measured by monitoring the activity of horse-peroxidase conjugated on secondary antibody targeting SHP2 Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001