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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O 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 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

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 .

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 .

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 O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O 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 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

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 O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O 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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

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

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 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 O O

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


Representative western blot showing of total phosphorylated ZAP70 , PKCθ PLCγ1 in lysates of stimulated CTL , inhibited by PD - L1 in presence of 1μΜ of MB

O O O 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

Representative western blot showing the levels of total and phosphorylated ZAP70, PKCθ and PLCγ1 in the lysates of stimulated CTL, inhibited by PD-L1 in the presence of 1μΜ of MB


Representative western blot showing CD28 phosphorylation . CTL were stimulated with EG7 ( parental ) , EG7 - L1 EG7 - L1 in presence 100 nM of MB for 2 hours . immunoprecipitated ( IP ) with Anti - pY from lysates of indicated CTL - EG7 co - culture were subjected to SDS - PAGE separation blotted with aCD28 . β - actin served as loading control . EG7 - L1 : EG7 overexpressing PD - L1 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative western blot showing the levels CD28 phosphorylation. CTL were stimulated with EG7 cells (parental), EG7-L1 and EG7-L1 in the presence 100 nM of MB for 2 hours. Protein immunoprecipitated (IP) with Anti-pY from the lysates of the indicated CTL-EG7 co-culture were subjected to SDS-PAGE separation and blotted with aCD28. β-actin served as loading control. EG7-L1: EG7 overexpressing PD-L1 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


Phos - tag of CD28 phosphorylation in CTL . CTLs were stimulated with EG7 or EG7 - L1 in presence of 1μΜ MB . lysate were then separated by SDS - PAGE containing 50 μM of Phos - tag blotted with aCD2 antibody . Phosphorylated CD28 ( slow - moving ) species were visualized through

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

Phos-tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7-L1 in the presence of 1μΜ MB. Cell lysate were then separated by SDS-PAGE containing 50 μM of Phos-tag and blotted with aCD2 antibody. Phosphorylated CD28 (slow-moving) species were visualized through exposure


complementation of effect of MB on interaction between CD28 with P85 . Jurkat or Jurkat - PD - 1 were co - transfected with CD28 - C - Luc or CD28Y191F - C - Luc P85 - N - Luc . were seeded in a 96 - well plate coated with 10 μg / ml of aCD3 / aCD28 in presence of human 10 μg / ml of PD - L1 100 nM of MB for 6 hours . Luciferase activity was measured as readout for CD28 - P85 interaction 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O

Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat-PD-1 cells were co-transfected with CD28-C-Luc or CD28Y191F-C-Luc and P85-N-Luc. Cells were seeded in a 96-well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD-L1 protein and 100 nM of MB for 6 hours. Luciferase activity was measured as readout for CD28-P85 interaction 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


Schematic of xenograft mouse model for MB treatment . C57BL / 6J mice were inoculated with EG7 - L1 ( 2 × 106 , s . c . ) on right flank on day 1 , followed by injection ( 2 × 106 , i . v . ) of CD45 . 1 + CTL on day 3 day 6 respectively . mice were randomized into 3 groups ( n = 5 ) treated with vehicle , MB ( 20 mg / kg / day , i . g . ) , aPD1 ( 10 mg / kg , every other day , i . p . ) served as positive control . was measured through calipering every other day . EG7 - L1 : EG7 overexpressing PD - L1 . growth curve for ( A ) was shown as mean ± s . e . m . Representative image of on day 10 mentioned in ( A ) . on day 10 ( n = 5 ) mentioned in ( A ) . 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 . 001 ; * * * * P < 0 . 0001 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Schematic of the xenograft mouse model for MB treatment. C57BL/6J mice were inoculated with EG7-L1 cells (2 × 106 cells, s.c.) on the right flank on day 1, followed by injection (2 × 106 cells, i.v.) of CD45.1+ CTL on day 3 and day 6 respectively. The mice were randomized into 3 groups (n = 5) and treated with vehicle, MB (20 mg/kg/day, i.g.), aPD1 (10 mg/kg, every other day, i.p.) served as positive control. Tumor size was measured through calipering every other day. EG7-L1: EG7 overexpressing PD-L1.   Tumor growth curve for (A) was shown as mean ±s.e.m.   Representative image of the tumor on day 10 mentioned in (A).   Tumor weight on day 10 (n=5) mentioned in (A).   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 < 0.001; ****P < 0.0001.


Immunohistochemical of proliferation marker ki - 67 CD8 in nodules on day 10 ( n = 5 ) Statistical results of ( E ) . 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 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunohistochemical analysis of cell proliferation marker ki-67 and CD8 in tumor nodules on day 10 (n=5) Statistical results of (E). 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 < 0.001; ****P < 0.0001.


Expression of Perforin , GZMB IL - 2 by CD8 + CD45 . 1 + TILs were analyzed by flow cytometry . Statistical results of ( G ) . 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 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Perforin, GZMB and IL-2 by CD8+ CD45.1+ TILs were analyzed by flow cytometry.   Statistical results of (G).   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 < 0.001; ****P < 0.0001.


Schematic of EGFR L858R mice treated with MB . burdens recorded through Computed tomography ( CT ) scanning for EGFR L858R mice ( n = 5 , lung section = 3 ) . Mice were treated with MB ( 20 mg / kg / day , i . g . ) for 1 week , followed by imaged with CT again to record burden . Statistical results of ( J ) . Haematoxylin Eosin of lung section of EGFR L858R mice ( n = 5 , lung section = 3 ) in ( J ) . Statistical results of ( L ) . 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 . 001 ; * * * * P < 0 . 0001 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Schematic of EGFR L858R mice treated with MB.   Tumor burdens recorded through Computed tomography (CT) scanning for EGFR L858R mice (n=5, lung section = 3). Mice were treated with MB (20 mg/kg/day, i.g.) for 1 week, followed by imaged with CT again to record tumor burden.   Statistical results of (J).   Haematoxylin and Eosin staining of lung section of EGFR L858R mice (n = 5, lung section = 3) in (J).   Statistical results of (L).   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 < 0.001; ****P < 0.0001.


Effect of MB on proliferation of human CD8 + T . CFSE labeled human peripheral blood mononuclear ( PBMC ) were pre - incubated with DMSO , MB , 25 μg / ml of pembrolizumab or 20 μg / ml of Nivolumab for 1 h then seeded in 96 - well plate precoated with 10 μg / ml of aCD3 / aCD28 in presence of 10 μg / ml human PD - L1 . proliferation was analyzed through FACS of dilution of CFSE . Data information : Data are representative of three independent experiments , were analyzed by unpaired t - . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effect of MB on proliferation of human CD8+ T cells. CFSE labeled human peripheral blood mononuclear cells (PBMC) were pre-incubated with DMSO, MB, 25 μg/ml of pembrolizumab or 20 μg/ml of Nivolumab for 1 h and then seeded in the 96-well plate precoated with 10 μg/ml of aCD3/aCD28 in the presence of 10 μg/ml human PD-L1 Protein. Cell proliferation was analyzed through FACS analysis of dilution of CFSE. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


Cytokine cytolytic granule production in CD8 + T . Human peripheral blood mononuclear from healthy donators were stimulated with 5 μg / ml of phytohaemagglutinin for 2 days then rested for 1 day . were pretreated with DMSO , MB , 25 μg / ml of pembrolizumab ( Pem ) or 20 μg / ml of nivolumab ( Niv ) for 1 h then seeded in a 96 - well precoated with 10 μg / ml aCD3 / aCD28 , with media supplemented with 10 μg / ml of human PD - L1 . Cytokine cytolytic granule production were determined by intracellular flow cytometry by gating on CD8 + populations . Data information : Data are representative of three independent experiments , were analyzed by unpaired t - . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cytokine and cytolytic granule production in CD8+ T cells. Human peripheral blood mononuclear cells from healthy donators were stimulated with 5 μg/ml of phytohaemagglutinin for 2 days and then rested for 1 day. Cells were pretreated with DMSO, MB, 25 μg/ml of pembrolizumab (Pem) or 20 μg/ml of nivolumab (Niv) for 1 h and then seeded in a 96-well precoated with 10 μg/ml aCD3/aCD28, with media supplemented with 10 μg/ml of human PD-L1 Protein. Cytokine and cytolytic granule production were determined by intracellular flow cytometry by gating on CD8+ populations. Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


Comparison of efficacy of MB with PD1 inhibitors currently used in clinic to activate T . JP - luc were stimulated with Raji - L1 or parental Raji preloaded with 1 μg / ml of superantigen ( SEE ) in presence of MB at indicated or 25μg / ml of pembrolizumab ( Pem ) or 20μg / ml of nivolumab ( Niv ) for 6 hours . Luciferase activity was measured by luminometer . Data are representative of three independent experiments , were analyzed by unpaired t - . 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 , were analyzed by unpaired t - . Error bars denote s . e . m . * * P < 0 . 01 ; * * * * P < 0 . 0001 .

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

Comparison of the efficacy of MB with PD1 inhibitors currently used in clinic to activate T cell. JP-luc cells were stimulated with Raji-L1 or parental Raji cells preloaded with 1 μg/ml of superantigen (SEE) in the presence of MB at indicated concentration or 25μg/ml of pembrolizumab (Pem) or 20μg/ml of nivolumab (Niv) for 6 hours. Luciferase activity was measured by luminometer. Data are representative of three independent experiments, and were analyzed by unpaired t-test. 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, and were analyzed by unpaired t-test. Error bars denote s.e.m. **P < 0.01; ****P < 0.0001.


Curated heatmaps of top 76 up 72 down regulated genes . Highlighted genes were chosen on biological interest . coloured bar represents expression level from low ( green ) to high ( red ) .

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Curated heatmaps of top 76 up and 72 down regulated genes. Highlighted genes were chosen based on biological interest. The coloured bar represents the expression level from low (green) to high (red).


Real - time qPCR of organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1 , B Blimp - 1 , C FcRn D Lct increase of adult markers E Sis , F Treh G Arg2 ( n = 3 wells from single organoid culture ( see material methods ) ; experiment was repeated in four to eight independent organoid cultures with similar results ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Real-time qPCR analysis of fetal organoids cultured for one month showing decrease in relative expression of indicated neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct and increase of the adult markers E Sis, F Treh and G Arg2 (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four to eight independent organoid cultures with similar results).


Enzyme activity of organoids for H Lactase , I sucrase , J maltase , K trehalase L arginase . Activity is given in µM glucose / µg · min - 1 ( experiment was generated from single organoid culture ( see material methods ) repeated in 3 independent organoid cultures with similar results ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Enzyme activity assay of fetal organoids for H Lactase, I sucrase, J maltase, K trehalase and L arginase. Activity is given in µM glucose/µg protein·min-1 (experiment was generated from single organoid culture (see material and methods) and repeated in 3 independent organoid cultures with similar results).


Relative expression detected by Real - time qPCR in organoids ( ) adult organoids ( ) . Neonatal markers A Ass1 , B Blimp - 1 , C FcRn D Lct in organoids cultured for one month compare to adult organoids . Mature markers E Sis , F Treh G Arg2 increase in organoids to adult ( n = 3 wells from single organoid culture ( see material methods ) ; experiment was repeated in four independent organoid cultures with similar results ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative expression detected by Real-time qPCR in fetal organoids ( ) and adult organoids ( ). Neonatal markers A Ass1, B Blimp-1, C FcRn and D Lct in fetal organoids cultured for one month compare to levels adult organoids. Mature markers E Sis, F Treh and G Arg2 increase in fetal organoids to adult levels (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in four independent organoid cultures with similar results).


Enzyme activity of ( ) adult ( ) organoids . H Lactase activity is higher in organoids decreasing to adult at day 28 , while I sucrase , J maltase , K trehalase L arginase increase to adult . Activity is given in µM glucose / µg · min - 1 ( n = 3 independent organoid cultures ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Enzyme activity assay of fetal ( ) and adult ( ) organoids. H Lactase activity is higher in fetal organoids decreasing to adult levels at day 28, while I sucrase, J maltase, K trehalase and L arginase increase to adult levels. Activity is given in µM glucose/µg protein·min-1 (n=3 independent organoid cultures).


Maturation is accelerated in dexamethasone treated organoids ( ) compared to control ( ) , as revealed by real - time qPCR . Decrease in relative expression of neonatal marker A Blimp - 1 increase in expression of adult marker B Sis occur earlier in dexamethasone treated

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 B-CONTROLLED_VAR O

Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. Decrease in relative expression of neonatal marker A Blimp-1 and increase in expression of adult marker B Sis occur earlier in dexamethasone treated organoids


Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of culture . Activity is given in µM glucose / µg · min - 1 ( n = 3 independent organoid cultures ) .

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

Dexamethasone treatment accelerates increase in activity of sucrase maltase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).


Maturation is accelerated in dexamethasone treated organoids ( ) compared to control ( ) , as revealed by real - time qPCR . expression of adult marker E Treh F Arg2 remains unchanged ( n = 3 wells from single organoid

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Maturation is accelerated in dexamethasone treated fetal organoids ( ) compared to control ( ), as revealed by real-time qPCR. expression of adult marker E Treh and F Arg2 remains unchanged (n=3 individual wells from single organoid culture


Dexamethasone treatment accelerates increase in activity of G trehalase H arginase reaching adult organoid level ( ) at day 13 of culture . Activity is given in µM glucose / µg · min - 1 ( n = 3 independent organoid cultures ) .

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Dexamethasone treatment accelerates increase in activity of G trehalase and H arginase reaching adult organoid level ( ) at day 13 of the culture. Activity is given in µM glucose/µg protein·min-1 (n = 3 independent organoid cultures).


Microscopic of organoids at day 3 , 13 , 20 30 of culture . Scale bars : 500 µm .

Microscopic images of fetal organoids at day 3, 13, 20 and 30 of culture. Scale bars: 500 µm.


of spheroids decreases during culture ( n = 3 wells from single organoid culture ( see material methods ) ; experiment was repeated in ten independent organoid cultures with similar results ) .

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Percentage of fetal spheroids decreases during culture (n=3 individual wells from single organoid culture (see material and methods); experiment was repeated in ten independent organoid cultures with similar results).


Immmunohistochemistry of organoids for C Ass1 D Sis E Arg2 . of 3 slides per , 7 to 10 organoids / spheroids per slide . Scale bars : 50µm .

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Immmunohistochemistry of fetal organoids for C Ass1 D Sis and E Arg2. Quantification of 3 slides per staining, 7 to 10 organoids / spheroids per slide. Scale bars: 50µm.


( C ) eight subunits of CCT chaperonin were enriched in U2OS - A3A samples . detected by MS are displayed by fold enrichment ( log2 ) from U2OS - A3A compared to parental ( x - axis ) , by abundance within U2OS - A3A samples ( y - axis ) . Significantly enriched ( p < 0 . 05 by one - t - , n = 3 ) are denoted in blue . Components of CCT are highlighted labeled .

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(C) All eight subunits of the CCT chaperonin complex were enriched in U2OS-A3A samples. Proteins detected by MS are displayed by fold enrichment (log2) from U2OS-A3A cells compared to parental cells (x-axis), and by protein abundance within the U2OS-A3A samples (y-axis). Significantly enriched proteins (p<0.05 by one-tailed t-test, n=3) are denoted in dark blue. Components of the CCT complex are highlighted and labeled.


( A ) A3A co - precipitates with CCT subunits . U2OS HepaRG with dox - inducible A3A - HA transgenes were treated with dox prior to immunoprecipitation of HA . HA immunoprecipitates were analyzed by immunoblot with antibodies for HA subunits of CCT .

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(A) A3A co-precipitates with CCT complex subunits. U2OS and HepaRG cells with dox-inducible A3A-HA transgenes were treated with dox prior to immunoprecipitation of HA. HA immunoprecipitates were analyzed by immunoblot with antibodies for HA and subunits of the CCT complex.


( C ) A3A interacts with CCT in leukemia . HA immunoprecipitation in hematopoietic lines with inducible A3A - HA was analyzed by immunoblot . HA , CCT1 , CCT5 antibodies were used to evaluate co - precipitation of CCT subunits with A3A .

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(C) A3A interacts with CCT in leukemia cells. HA immunoprecipitation in hematopoietic cell lines with inducible A3A-HA was analyzed by immunoblot. HA, CCT1, and CCT5 antibodies were used to evaluate co-precipitation of CCT subunits with A3A.


( A ) CCT co - precipitates with A3A but not A3B or A3G . 293T were transfected with empty vector ( V ) , A3A - HA , A3B - HA , or A3G - HA . CCT IP was performed from transfected evaluated by immunoblot using antibodies for CCT5 HA .

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(A) CCT co-precipitates with A3A but not A3B or A3G. 293T cells were transfected with empty vector (V), A3A-HA, A3B-HA, or A3G-HA. CCT IP was performed from transfected cells and evaluated by immunoblot analysis using antibodies for CCT5 and HA.


( B ) A3A , but not other APOBEC3 enzymes , interacts with CCT subunits . After transfection of 293T with HA - tagged APOBEC3 expression plasmids , IP was performed with anti - HA antibody analyzed by immunoblot using antibodies to HA CCT subunits . Arrows mark location of each APOBEC3 on HA immunoblot .

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(B) A3A, but not other APOBEC3 enzymes, interacts with CCT complex subunits. After transfection of 293T cells with HA-tagged APOBEC3 expression plasmids, IP was performed with anti-HA antibody and analyzed by immunoblot using antibodies to HA and CCT subunits. Arrows mark location of each APOBEC3 protein on HA immunoblot.


( F ) CCT co - precipitates with endogenous A3A . Immunoprecipitation of CCT in BICR6 was analyzed by immunoblot using antibodies to endogenous A3A CCT1 . Chemiluminescent exposure times described as short long are indicated beside blot . IgG antibody was used as a control throughout .

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(F) CCT co-precipitates with endogenous A3A. Immunoprecipitation of the CCT complex in BICR6 cells was analyzed by immunoblot using antibodies to endogenous A3A and CCT1. Chemiluminescent exposure times described as short and long are indicated beside the blot. IgG antibody was used as a control throughout.


( A ) Knockdown of one CCT member results in depletion of other CCT subunits . Following siCCT4 or siCCT7 transfection , lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1 , CCT4 , CCT5 , CCT7 . GAPDH was used as a loading control .

(A) Knockdown of one CCT complex member results in depletion of other CCT subunits. Following siCCT4 or siCCT7 transfection, cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1, CCT4, CCT5, and CCT7. GAPDH was used as a loading control.


( B ) Decreased viability upon CCT depletion A3A expression . CCT4 ( left ) CCT7 ( right ) subunits were depleted by siRNA transfection in U2OS with dox - inducible A3A transgenes . Immunoblot of lysates showed decreased expression of targeted CCT subunit , but no alteration in A3A expression . Scr indicates non - targeted siRNA , used as control . Ku - 86 is a loading control . Following siRNA transfection to deplete CCT subunits , U2OS - A3A were treated with dox . Viability was determined by colorimetric change after addition of a water - soluble tetrazolium salt . viability was normalized to untreated controls . Statistical was performed using a paired two - t - , n = 3 biological replicates ; error bars , .

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(B) Decreased cell viability upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS cells with dox-inducible A3A transgenes. Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A cells were treated with dox. Viability was determined by colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM.


( D ) Viability of with catalytically inactive A3A . U2OS - A3A - C106S were induced with dox to express catalytically inactive A3A mutant ( C106S ) , depleted of CCT4 by siRNA , or combination . were subsequently evaluated by live - dead assessed by FACS . Bar chart shows quantitation of FACS results averaged over three biological replicates . Statistical was performed using a paired two - t - , n = 3 ; error bars , . Immunoblot of lysates showed decreased expression of CCT4 after siRNA targeting . Ku - 86 is a loading control .

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(D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4 by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control.


( E ) Increased damage signaling upon CCT depletion A3A expression . Depletion of CCT7 in K562 - A3A was achieved by siRNA targeting . Expression of A3A was induced by dox treatment . Intracellular with anti - γH2AX antibody was analyzed by flow cytometry . Bar chart shows of FACS results obtained over three biological replicates . Statistical was performed using a two - t - , n = 3 ; error bars , . Immunoblot of lysates showed decreased expression of CCT7 after siRNA targeting A3A expression upon dox treatment . Ku - 86 is a loading control . * * p - value < 0 . 01 , * * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 , n . s . non - significant .

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(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.


( C ) of APOBEC3 mutational signatures among three types that have previously been associated with high APOBEC3 activity . Breast , bladder , cervical cancer samples from TCGA database were divided into CCT - mutated non - mutated genomes , as above . number of mutations contributing to SBS 2 SBS 13 in each type was quantified displayed as an average . Comparison by Wilcoxon rank sum yielded p - values denoted .

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(C) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity. Breast, bladder, and cervical cancer samples from the TCGA database were divided into CCT-mutated and non-mutated genomes, as above. The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average. Comparison by Wilcoxon rank sum test yielded p-values denoted.


Blue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . Immunodetection of indicated after

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Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGE


Blue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . ( C ) in - gel activity for CI ( D ) CIV ( shown is a representative gel from two technical two biological replicates ) .

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Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates).


BNGE of whole - zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) its respective scaf1 + / + counterpart . Immunodetection of indicated

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BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. Immunodetection of the indicated proteins


BNGE of whole - zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) its respective scaf1 + / + counterpart . ( G ) in - gel activity of CI ( H ) CIV ( representative gel from two technical three biological replicates ) .

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BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates).


I , 2D BNGE / SDS electrophoresis : 1st dimension with digitonin ( Dig ) 2nd dimension with SDS , followed by immunoblotting with indicated antibodies to identify detected by commercial anti - SCAF1 antibody . Asterisks indicate missing bands in scaf1Δ1 / Δ1 .

I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.


A , Quantitative data - independent scanning ( DiS ) mass spectrometry profiles for CI , CIII CIV . Vertical numbers indicate BNGE gel slices . Left right profiles correspond to scaf1 + / + scaf1Δ1 / Δ1 , respectively . Red corresponds to E - score from two proteotypic Scaf1 - derived tryptic peptides spanning sequences ascribed to CIII interacting site ( in green ) to CIV interacting site ( in yellow ) . Thick blue , marked with an asterisk , indicates putative proteolytic site in Scaf1 .

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A, Quantitative data-independent scanning (DiS) mass spectrometry protein profiles for CI, CIII and CIV. Vertical numbers indicate the BNGE gel slices. Left and right profiles correspond to scaf1+/+ and scaf1Δ1/Δ1 animals, respectively. Red heatmap corresponds to the E-score from two proteotypic Scaf1-derived tryptic peptides spanning sequences ascribed to the CIII interacting site (in green) and to the CIV interacting site (in yellow). Thick blue line, marked with an asterisk, indicates the putative proteolytic site in Scaf1.


A , B , Representative from scaf1 + / + scaf1 - / - ( A ) ( B ) adult zebrafish .

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A, B, Representative images from scaf1+/+ and scaf1-/- (A) female and (B) male adult zebrafish.


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c-f, Size of scaf1Δ1/ Δ1 and scaf1Δ2/ Δ2 (scaf1-/-) fish in comparison with their respective scaf1+/+ lines, (C) length and (E) weight of females (Δ1 +/+ n=10, Δ1 -/- n=12, Δ2 +/+ n=24, Δ2 -/- n=18); (D) length and (F) weight of males (Δ1 +/+ n=16, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=23).


G - K , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( G , I ) Adipose tissue per total section ( average of 3 sections / biological replicate ) ( H , J ) adipocyte ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) of ( G , H ) ( Δ1 n = 8 , Δ2 n = 8 , same number of for homozygous mutants controls ) ( I , J ) ( Δ1 n = 8 , Δ2 n = 7 , same number of for homozygous mutants controls ) . K , Representative of ventral fat deposits in ( dotted lines ) .

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G-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines).


L - N , Effect of Scaf1 loss of function on fertility . ( L ) Number of eggs per clutch ( Δ1 + / + n = 12 , Δ1 - / - n = 13 , Δ2 + / + n = 13 , Δ2 - / - n = 10 ) . ( M ) of mature ovary follicles per ovary section ( average of three sections / biological replicate ) ( Δ1 n = 5 , Δ2 n = 8 ; same number of for homozygous scaf1 + / + scaf1 - / - ) . ( N ) Representative of H & E - stained ovaries . Dotted lines delineate adipose tissue .

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L-N, Effect of Scaf1 loss of function on female fertility. (L) Number of eggs per clutch (Δ1 +/+ n=12, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=10). (M) Quantification of mature ovary follicles per ovary section (average of three sections/biological replicate) (Δ1 n=5, Δ2 n=8; same number of animals for homozygous scaf1+/+ and scaf1-/-). (N) Representative images of H&E-stained ovaries. Dotted lines delineate adipose tissue.


Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) scaf1 + / + fish ( n = 3 ) . ( A ) Representative showing mitochondria .

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Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (A) Representative images showing mitochondria.


Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) scaf1 + / + fish ( n = 3 ) . ( B ) Mitochondria ( 100 - 150 mitochondria per biological sample ) . ( C ) Cristae lumen width ( average of three cristae per mitochondria , 20 mitochondria per biological sample ) . Different biological replicates are represented with different color tones .

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Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (B) Mitochondria size (100-150 mitochondria per biological sample). (C) Cristae lumen width (average of three cristae per mitochondria, 20 mitochondria per biological sample). Different biological replicates are represented with different color tones.


D , E , Mitochondrial copy number per copy number in muscle in ( D ) ( E ) ( Δ1 n = 6 , Δ2 n = 6 same number for their respective controls ) .

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D, E, Mitochondrial DNA copy number per nuclear copy number in muscle in females (D) and males (E) (Δ1 n=6, Δ2 n=6 and same number for their respective controls).


F , Survival curve of 4 days post - fertilization treated with different concentrations of indicated OXPHOS inhibitors ( three experimental replicates per biological replicate three biological replicates ) .

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F, Survival curve of 4 days post-fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors (three experimental replicates per biological replicate and three biological replicates).


G , H , Oxygen consumption of 48 hours post - fertilization using XFe24 Seahorse analyzer , ( G ) Representative oxygen consumption rate ( OCR ) profile along time ( H ) maximum OCR ( Δ1 n = 11 , Δ2 n = 11 , n = 10 , n = 11 respectively for their controls ) .

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G, H, Oxygen consumption of 48 hours post-fertilization embryos using the XFe24 Seahorse analyzer, (G) Representative oxygen consumption rate (OCR) profile along time and (H) maximum OCR (Δ1 n=11, Δ2 n=11, n=10, n=11 respectively for their controls).


I , Maximum uncoupled ( FCCP ) OCR in isolated mitochondria from adult fish ( Δ1 n = 4 Δ2 n = 4 , same number for their respective controls ) with indicated site I [ pyruvate ( Pyr ) , glutamate ( Glu ) , malate ( Mal ) ] or site II [ succinate ( Succ ) ] substrates .

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I, Maximum uncoupled (FCCP) OCR in isolated mitochondria from adult fish (male and females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with the indicated site I [pyruvate (Pyr), glutamate (Glu), malate (Mal)] or site II [succinate (Succ)] substrates.


J , K , Respiratory control ( RCR ) ( State 3 / State4 ) ( J ) P / O ( K ) in isolated mitochondria from adult fish ( Δ1 n = 4 , same number for their respective controls ) with indicated substrates .

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J, K, Respiratory control ratio (RCR)(State 3/State4) (J) and P/O ratio (K) in isolated mitochondria from adult fish (male and females Δ1 n=4, and same number for their respective controls) with the indicated substrates.


A , Representative of scaf1 - / - scaf1 + / + fish fed with indicated diets .

A, Representative images of scaf1-/- and scaf1+/+ fish fed with the indicated diets.


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B, C, Changes in (B) length and (C) weight over time (Δ1 +/+ n=10, Δ1 -/- n=10, Δ2 +/+ n=10, Δ2 -/- n=12-13.