E . Protein samples ( 20μg ) from Jurkat cells treated as indicated were separated by SDS - PAGE and immunoblotted with the indicated antibodies . WB are representative of three independent experiments . F . Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown . Data represent mean ± SE of three independent experiments .

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E. Protein samples (20μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of three independent experiments.F. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown. Data represent mean ± SE of three independent experiments.


A Concentrations of amylase , lipase , CRP , and ANGPTL4 in the sera of patients ( n = 90 ) diagnosed with pancreatitis . The box plots show a typical display consisting of a median value depicted by the line in the center of the box ; an interquartile range ( IQR ; 25th to the 75th percentile ) depicted by the box ; and the maximum ( Q3 + 1 . 5 * IQR ) and minimum ( Q1 - 1 . 5 * IQR ) values depicted by the whisker . Statistical significance of Mann - Whitney U - tests is indicated ( * * P < 0 . 01 and * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA . The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient ( R ) . Each dot represents the levels of ANGPTL4 and CRP from an individual patient ( n = 90 ) .

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A Concentrations of amylase, lipase, CRP, and ANGPTL4 in the sera of patients (n = 90) diagnosed with pancreatitis. The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (** P < 0.01 and *** P < 0.001). Exact P values are shown in Appendix Table S2. ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA. The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient (R). Each dot represents the levels of ANGPTL4 and CRP from an individual patient (n = 90).


B Pancreatitis and adjacent normal regions ( n = 6 ) were stained by immunofluorescence for amylase and ANGPTL4 . Scale bar represents 30

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B Pancreatitis and adjacent normal regions (n = 6) were stained by immunofluorescence for amylase and ANGPTL4. Scale bar represents 30 µm


C H & E staining , ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild ( AP ) and severe acute pancreatitis ( SAP ) for 2 h ( n = 15 , each group ) . Scale bar represents 50 µm .

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C H&E staining, ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild (AP) and severe acute pancreatitis (SAP) for 2 h (n = 15, each group). Scale bar represents 50 µm.


D Amylase , lipase , and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas ( n = 15 , each group ) .

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D Amylase, lipase, and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas (n = 15, each group).


A AP was induced by 5 injections ( IP ) of cerulein at a dosage of 50 μg / kg . SAP was induced by AP and one injection of lipopolysaccharide ( LPS , IP ) at a dosage of 20 mg / kg . AP + ANGPTL4 ( 2 mg / kg ) was induced by AP and one injection of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . ANGPTL4 ( 4 mg / kg ) was induced by two injections of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . Mice were sacrificed 6 h after pancreatitis induction . ( H & E and immunofluorescent staining for IL - 1β ( red ) and amylase ( green ) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues ( n = 10 , each group ) . Scale bar represents 50

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A AP was induced by 5 injections (IP) of cerulein at a dosage of 50 μg/kg. SAP was induced by AP and one injection of lipopolysaccharide (LPS, IP) at a dosage of 20 mg/kg. AP+ANGPTL4 (2 mg/kg) was induced by AP and one injection of ANGPTL4 (IP) at a dosage of 2 mg/kg. ANGPTL4 (4 mg/kg) was induced by two injections of ANGPTL4 (IP) at a dosage of 2 mg/kg. Mice were sacrificed 6 h after pancreatitis induction. (H&E and immunofluorescent staining for IL-1β (red) and amylase (green) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues (n = 10, each group). Scale bar represents 50 µm


B Concentrations of the inflammatory cytokines TGF - β , IL - 1β , and IFN - γ as well as ANGPTL4 in the sera ( n = 10 , each group ) .

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B Concentrations of the inflammatory cytokines TGF-β, IL-1β, and IFN-γ as well as ANGPTL4 in the sera (n = 10, each group).


C TUNEL staining for apoptosis and vacuoles formation ( TEM ) . Red arrow , apoptotic cells ; white dotted line , vacuoles region .

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C TUNEL staining for apoptosis and vacuoles formation (TEM). Red arrow, apoptotic cells; white dotted line, vacuoles region.


D T . E . M . ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP , SAP and ANGPTL4 - treated models ( n = 5 , each group ) .

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D T.E.M. ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP, SAP and ANGPTL4-treated models (n = 5, each group).


A AP and SAP were induced in wild type ( WT ) and ANGPTL4 - / - mice ( n = 15 , each group ) , as described in the material and methods . H & E staining , TUNEL staining for apoptosis , and immunofluorescence staining for ANGPTL4 ( red ) and amylase ( green ) were performed in the pancreatic tissues . ANGPTL4 and IL - 6 protein expressions were also determined in pancreatic tissues from ANGPTL4 WT and - / - mice by Western blotting . Scale bar represents 100 µm .

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A AP and SAP were induced in wild type (WT) and ANGPTL4 -/- mice (n = 15, each group), as described in the material and methods. H&E staining, TUNEL staining for apoptosis, and immunofluorescence staining for ANGPTL4 (red) and amylase (green) were performed in the pancreatic tissues. ANGPTL4 and IL-6 protein expressions were also determined in pancreatic tissues from ANGPTL4 WT and -/- mice by Western blotting. Scale bar represents 100 µm.


B Levels of ANGPTL4 , amylase , lipase , and MPO in serum ( n = 15 , each group ) .

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B Levels of ANGPTL4, amylase, lipase, and MPO in serum (n = 15, each group).


C Immunofluorescence staining of TNF - α and IL - 6 , and the levels of the inflammatory cytokines TGF - β , IFN - γ , and IL - 1β in the sera from ANGPTL4 WT and - / - mice ( n = 10 , each group ) . Scale bar represents 150

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C Immunofluorescence staining of TNF-α and IL-6, and the levels of the inflammatory cytokines TGF-β, IFN-γ, and IL-1β in the sera from ANGPTL4 WT and -/- mice (n = 10, each group). Scale bar represents 150 µm


A For macrophage depletion models , mice ( six weeks ) were injected with GdCl3 ( 15 mg / kg ) . After the induction of macrophage depletion in the mice , pancreatitis was induced by cerulein , LPS , and ANGPTL4 for 24 h ( n = 10 , each group ) . H & E staining of pancreatic tissue , and levels of amylase and lipase in the serum . Scale bar represents 100

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A For macrophage depletion models, mice (six weeks) were injected with GdCl3 (15 mg/kg). After the induction of macrophage depletion in the mice, pancreatitis was induced by cerulein, LPS, and ANGPTL4 for 24 h (n = 10, each group). H&E staining of pancreatic tissue, and levels of amylase and lipase in the serum. Scale bar represents 100 µm


B Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues . Levels of ANGPTL4 in the serum were measured in the AP , SAP , and ANGPTL4 ( 4 mg / kg ) groups of macrophage WT or depletion mice h ( n = 10 , each group ) . Scale bar represents 100

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B Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues. Levels of ANGPTL4 in the serum were measured in the AP, SAP, and ANGPTL4 (4 mg/kg) groups of macrophage WT or depletion mice h (n = 10, each group). Scale bar represents 100 µm


C Immunofluorescent staining of the F4 / 80 macrophage marker and quantification of macrophage - positive cells in the AP and SAP groups from the ANGPTL4 - / - and WT mice ( n = 10 ) . Scale bar represents 150

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C Immunofluorescent staining of the F4/80 macrophage marker and quantification of macrophage-positive cells in the AP and SAP groups from the ANGPTL4 -/-and WT mice (n = 10). Scale bar represents 150 µm


A Cell morphology and the mRNA levels of inflammatory cytokines , including and TNF - α , IL - 6 , and IL - 1β in mouse primary macrophages treated with LPS and ANGPTL4 ( 100 ng / ml ) using qPCR and RT - PCR , and levels of TGF - β and NO in the LPS - and ANGPTL4 - treated primary macrophages ( n = 5 ) . Scale bar represents 100

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A Cell morphology and the mRNA levels of inflammatory cytokines, including and TNF-α, IL-6, and IL-1β in mouse primary macrophages treated with LPS and ANGPTL4 (100 ng/ml) using qPCR and RT-PCR, and levels of TGF-β and NO in the LPS- and ANGPTL4-treated primary macrophages (n = 5). Scale bar represents 100 µm


B Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2 - well silicone culture inserts that were placed in a cell culture μ - Dish and transwell at 24 h ( n = 5 ) . Scale bar represents 50

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B Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2-well silicone culture inserts that were placed in a cell culture μ-Dish and transwell at 24 h (n = 5). Scale bar represents 50 µm


C Mouse primary acinar cells and macrophages were labelled with fluorescence dyes ( macrophages , red and acinar cells , green ) . Co - cultured cells treated with 100 ng / ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h . Scale bars represent 80 µm ( red ) and 40 µm ( white ) .

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C Mouse primary acinar cells and macrophages were labelled with fluorescence dyes (macrophages, red and acinar cells, green). Co-cultured cells treated with 100 ng/ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h. Scale bars represent 80 µm (red) and 40 µm (white).


A Macrophage cells were treated with ANGPTL4 ( 100 ng / ml ) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay ( n = 3 ) . Mean pixel density was quantified using ImageJ software analysis .

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A Macrophage cells were treated with ANGPTL4 (100 ng/ml) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay (n=3). Mean pixel density was quantified using ImageJ software analysis.


B ANGPTL4 and C5a expression levels were measured in bone marrow - derived macrophages from pancreatitis - induced ANGPTL4 - / - and WT mice .

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B ANGPTL4 and C5a expression levels were measured in bone marrow-derived macrophages from pancreatitis-induced ANGPTL4-/- and WT mice.


C Phospho - kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1 - derived macrophage cells ( n = 3 ) . Cells were treated with ANGPTL4 ( 100 ng / ml ) for 6 h . After phospho - kinase array analysis , the expression of AMPKα , JNK , AKT , P70S6K , and EGFR was confirmed by western blotting .

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C Phospho-kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1-derived macrophage cells (n=3). Cells were treated with ANGPTL4 (100 ng/ml) for 6 h. After phospho-kinase array analysis, the expression of AMPKα, JNK, AKT, P70S6K, and EGFR was confirmed by western blotting.


D To explore whether ANGPTL4 affects JNK , AMPK , and PI3K / AKT signaling , each inhibitor ( SP600125 , compound C , and HS - 173 , 1 µM ) was treated to in THP1 - derived macrophages for 2 h after ANGPTL4 treatment ( 100 ng / ml ) for 6 h .

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D To explore whether ANGPTL4 affects JNK, AMPK, and PI3K/AKT signaling, each inhibitor (SP600125, compound C, and HS-173, 1 µM) was treated to in THP1-derived macrophages for 2 h after ANGPTL4 treatment (100 ng/ml) for 6 h.


E C5a , C5aR , TNF - α , IL - 6 , and IL - 1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS ( 100 ng / ml ) . In the transwell experiment , mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 ( 100 ng / ml ) . Expression of ANGPTL4 , C5aR , and C5a was determined in siANGPTL4 - treated THP1 - derived macrophage after LPS activation .

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E C5a, C5aR, TNF-α, IL-6, and IL-1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS (100 ng/ml). In the transwell experiment, mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 (100 ng/ml). Expression of ANGPTL4, C5aR, and C5a was determined in siANGPTL4-treated THP1-derived macrophage after LPS activation.


F In the transwell experiment , TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist ( W - 54011 , 10 nM ) pretreatment for 12 h prior to treatment with ANGPTL4 , LPS , and C5a ( 100 ng / ml ) for 24 h to macrophage in transwell . Scale bar represents 20

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F In the transwell experiment, TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist (W-54011, 10 nM) pretreatment for 12 h prior to treatment with ANGPTL4, LPS, and C5a (100 ng/ml) for 24 h to macrophage in transwell. Scale bar represents 20 µm


A Concentrations of C5a in the sera from pancreatitis patients , and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients ( n = 90 ) . The box plots show a typical display consisting of a median value depicted by the line in the center of the box ; an interquartile range ( IQR ; 25th to the 75th percentile ) depicted by the box ; and the maximum ( Q3 + 1 . 5 * IQR ) and minimum ( Q1 - 1 . 5 * IQR ) values depicted by the whisker . Statistical significance of Mann - Whitney U - tests is indicated ( * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient ( R ) . Each dot represents the levels of C5a and ANGPTL4 from an individual patient ( n = 90 ) . The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues . Scale bar represents 50

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A Concentrations of C5a in the sera from pancreatitis patients, and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients (n = 90). The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (*** P < 0.001). Exact P values are shown in Appendix Table S2. The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient (R). Each dot represents the levels of C5a and ANGPTL4 from an individual patient (n = 90). The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues. Scale bar represents 50 µm


B Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 - / - and WT animals and macrophage depletion / WT models with pancreatitis . Scale bar represents 150 µm . Levels of C5a were measured in serum ( n = 7 ) .

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B Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 -/- and WT animals and macrophage depletion/WT models with pancreatitis. Scale bar represents 150 µm. Levels of C5a were measured in serum (n = 7).


C After macrophages were activated with LPS ( 100 ng / ml ) , the cells were treated with either neutralizing ANGPTL4 or C5a antibody ( 100 ng / ml ) or an isotype control ( IgG ) . ANGPTL4 , C5a , TNF - α , and IL - 6 levels were measured in culture media ( n = 5 ) .

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

C After macrophages were activated with LPS (100 ng/ml), the cells were treated with either neutralizing ANGPTL4 or C5a antibody (100 ng/ml) or an isotype control (IgG). ANGPTL4, C5a, TNF-α, and IL-6 levels were measured in culture media (n = 5).


D The neutralizing ANGPTL4 antibody ( 10 mg / kg ) was injected twice ( IP ) into the AP and SAP models . H & E staining of pancreatic tissues and the concentrations of ANGPTL4 , C5a , TNF - α , and amylase in the serum ( n = 10 , each group ) . Scale bar represents 50

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

D The neutralizing ANGPTL4 antibody (10 mg/kg) was injected twice (IP) into the AP and SAP models. H&E staining of pancreatic tissues and the concentrations of ANGPTL4, C5a, TNF-α, and amylase in the serum (n = 10, each group). Scale bar represents 50 µm


( A ) . Human neutrophils stained for CD66b after incubation with RBCs infected with GFP + P . falciparum parasites ( white arrows ) . Nuclei were stained with DAPI ( blue ) , neutrophils were stained with CD66b ( red ) , GFP expressing parasites ( green ) . Scale bar , 5 µm . The upper and lower panel show two different cells .

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

(A). Human neutrophils stained for CD66b after incubation with RBCs infected with GFP+ P. falciparum parasites (white arrows). Nuclei were stained with DAPI (blue), neutrophils were stained with CD66b (red), GFP expressing parasites (green). Scale bar, 5 µm. The upper and lower panel show two different cells.


( B ) . Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P . falciparum parasites ( black arrows ) .

O O O O B-SMALL_MOLECULE O O O O B-ORGANISM B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O

(B). Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P. falciparum parasites (black arrows).


( C ) . Flow cytometry analysis of human neutrophils incubated with opsonized or non - opsonized GFP + - late - stage iRBCs . Free neutrophils were used as control .

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

(C). Flow cytometry analysis of human neutrophils incubated with opsonized or non-opsonized GFP+-late-stage iRBCs. Free neutrophils were used as control.


( D ) . Quantification of GFP + iRBCs phagocytosed by neutrophils . The phagocytosis was analysed by flow cytometry ( FACS ) . Data information :

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

(D). Quantification of GFP+ iRBCs phagocytosed by neutrophils. The phagocytosis was analysed by flow cytometry (FACS). Data information:


( A ) . Interaction between neutrophils and opsonized ( red ) or non - opsonized ( blue ) GFP + iRBCs by time in a " pulse - chase " experiment . The percentage of GFP + - neutrophils following 5 min of co - culture was set as 100 % .

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

(A). Interaction between neutrophils and opsonized (red) or non-opsonized (blue) GFP+ iRBCs by time in a "pulse-chase" experiment. The percentage of GFP+-neutrophils following 5 min of co-culture was set as 100%.


( C ) . Parasitemia ( percentage % ) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils .

O O O O B-EXP_ASSAY O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O

(C). Parasitemia (percentage %) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils.


( D ) . Parasitemia ( percentage % ) of cultures of RBC infected by different P . falciparum lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) . The parasites were kept in culture for six days in the presence ( Neut . ) or absence ( Cont . ) of neutrophils .

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

(D). Parasitemia (percentage %) of cultures of RBC infected by different P. falciparum lines (NF54, Dd2, SL, PfEMP1-KO). The parasites were kept in culture for six days in the presence (Neut.) or absence (Cont.) of neutrophils.


( E ) . Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) .

O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O B-CELL_TYPE O O O O O O B-ORGANISM O B-ORGANISM O O O B-ORGANISM O O O O

(E). Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines (NF54, Dd2, SL, PfEMP1-KO).


( F ) . Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression . Neutrophils were stained with an anti - CD11b antibody , while iRBCs were labeled with SYBR green . Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population , excluding the neutrophils that were SYBR green negative , but CD11b positive .

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

(F). Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression. Neutrophils were stained with an anti-CD11b antibody, while iRBCs were labeled with SYBR green. Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population, excluding the neutrophils that were SYBR green negative, but CD11b positive.


( G ) . Short term neutrophil killing of late - stage NF54 - luciferase positive RBCs in the presence ( red ) or absence ( blue ) of catalase .

O O O O O O B-CELL_TYPE O O O O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O

(G). Short term neutrophil killing of late-stage NF54-luciferase positive RBCs in the presence (red) or absence (blue) of catalase.


( H ) . Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase .

O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O

(H). Parasitemia of cultures of iRBCs challenged with neutrophils in the presence or absence of catalase.


( I ) . Short - term luciferase - based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D .

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

(I). Short-term luciferase-based killing assay using neutrophils from three different donors in the presence or absence of 5 uM cytochalasin D.


( A ) . Representative flow cytometric analysis of human neutrophils cultured alone ( Control ) , incubated with late - stage NF54 parasites ( wild type ) or PfEMP1 Knock out ( PfEMP1 KO ) GFP + iRBC .

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

(A). Representative flow cytometric analysis of human neutrophils cultured alone (Control), incubated with late-stage NF54 parasites (wild type) or PfEMP1 Knock out (PfEMP1 KO) GFP+ iRBC.


( B ) . Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP + iRBC .

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

(B). Flow cytometric quantification of the effect of trypsin treatment on neutrophil interaction with wild type and PfEMP1 KO GFP+ iRBC.


( C ) . Luciferase - based killing assay and neutrophil elimination of late - stage wild type ( wt ) and PfEMP1ko iRBC .

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

(C). Luciferase-based killing assay and neutrophil elimination of late-stage wild type (wt) and PfEMP1ko iRBC.


( B ) . Short - term ( 6h ) neutrophil killing of unselected NF54 and antibody - selected PFD1235w expressing parasite lines .

O O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O

(B). Short-term (6h) neutrophil killing of unselected NF54 and antibody-selected PFD1235w expressing parasite lines.


( C ) . Flow cytometric quantification of neutrophil interaction with MitoTracker ( APC + ) stained NF54 iRBCs and antibody - selected PFD1235w expressing iRBC .

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

(C). Flow cytometric quantification of neutrophil interaction with MitoTracker (APC+) stained NF54 iRBCs and antibody-selected PFD1235w expressing iRBC.


( D ) . Percent reduction in parasitemia of NF54 and antibody - selected PFD1235w expressing parasites after 5 days of co - culture with neutrophils , compared to unchallenged

O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE O O O O

(D). Percent reduction in parasitemia of NF54 and antibody-selected PFD1235w expressing parasites after 5 days of co-culture with neutrophils, compared to unchallenged parasites


( E ) . var gene transcription profiles measured by qRT - PCR of antibody - selected PFD1235w expressing parasite line cultured in the absence ( upper panel ) or presence ( lower panel ) of neutrophils . Steady state mRNA levels of each individual var gene was calculated as in A . I .

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

(E). var gene transcription profiles measured by qRT-PCR of antibody-selected PFD1235w expressing parasite line cultured in the absence (upper panel) or presence (lower panel) of neutrophils. Steady state mRNA levels of each individual var gene was calculated as in A. I.


( A ) QRT - PCR assay of PAX6 , SOX1 , NKX2 - 1 , NKX2 - 2 , TBR1 and TBR2 during dorsal ( up ) and ventral ( down ) neural differentiation . ( B ) QRT - PCR assay of SOX1 - OT V1 during dorsal ( up ) and ventral ( down ) neural differentiation Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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

(A) QRT-PCR assay of PAX6, SOX1, NKX2-1, NKX2-2, TBR1 and TBR2 during dorsal (up) and ventral (down) neural differentiation. (B) QRT-PCR assay of SOX1-OT V1 during dorsal (up) and ventral (down) neural differentiation Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( F and G ) Immunostaining assay of PAX6 ( green ) and SOX1 ( red ) in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( F ) , and quantiﬁcation of SOX1 + cells ( G ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of dorsal neural differentiation (F), and quantiﬁcation of SOX1+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( H and I ) Immunostaining assay of MAP2 ( green ) in control and V1 - PAKI cells on day 35 of dorsal neural differentiation ( H ) , and quantiﬁcation of MAP2 + cells ( I ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and I) Immunostaining assay of MAP2 (green) in control and V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( J and K ) Immunostaining assay of PAX6 ( green ) and SOX1 ( red ) in control and V1 - PAKI cells on day 16 of ventral neural differentiation ( J ) , and quantiﬁcation of SOX1 + cells ( K ) , p = 0 . 0003 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

(J and K) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of ventral neural differentiation (J), and quantiﬁcation of SOX1+ cells (K), p=0.0003. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( L and M ) Immunostaining assay of GABA ( green ) in control and V1 - PAKI cells on day 35 of ventral neural differentiation ( L ) , and quantiﬁcation of GABA + cells ( M ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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

(L and M) Immunostaining assay of GABA (green) in control and V1-PAKI cells on day 35 of ventral neural differentiation (L), and quantiﬁcation of GABA+ cells (M), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( C and D ) Immunostaining assay of MAP2 ( green ) in control and SOX1KO cells on day 35 of dorsal neural differentiation ( C ) , and quantiﬁcation of MAP2 + cells ( D ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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

(C and D) Immunostaining assay of MAP2 (green) in control and SOX1KO cells on day 35 of dorsal neural differentiation (C), and quantiﬁcation of MAP2+ cells (D), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( F and G ) Immunostaining assay of GABA ( green ) in control and SOX1KO cells on day 35 of ventral neural differentiation ( F ) , and quantiﬁcation of GABA + cells ( G ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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

(F and G) Immunostaining assay of GABA (green) in control and SOX1KO cells on day 35 of ventral neural differentiation (F), and quantiﬁcation of GABA+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( H and I ) Immunostaining assay of MAP2 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 35 of dorsal neural differentiation ( H ) , and quantiﬁcation of MAP2 + cells ( I ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( V1 - PAKI + ptSOX1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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

(H and I) Immunostaining assay of MAP2 (green) after SOX1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001 (V1-PAKI), p=0.0001 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( J and K ) Immunostaining assay of GABA ( green ) after SOX1 overexpression in V1 - PAKI cells on day 35 of ventral neural differentiation ( J ) , and quantiﬁcation of GABA + cells ( K ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0002 ( V1 - PAKI + ptSOX1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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

(J and K) Immunostaining assay of GABA (green) after SOX1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (J), and quantiﬁcation of GABA+ cells (K), p=0.0001 (V1-PAKI), p=0.0002 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Enrichment of H3K14Ac ( left ) and H3K27Ac ( right ) on SOX1 promoter region in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( A ) , p = 0 . 0046 ( H3K14Ac ) , p = 0 . 0223 ( H3K27Ac ) and ventral neural differentiation ( B ) , p = 0 . 0499 ( H3K14Ac ) , p = 0 . 0022 ( H3K27Ac ) . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) Enrichment of H3K14Ac (left) and H3K27Ac (right) on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (A), p=0.0046 (H3K14Ac), p=0.0223 (H3K27Ac) and ventral neural differentiation (B), p=0.0499 (H3K14Ac), p=0.0022 (H3K27Ac). Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01 versus Ctrl group (unpaired two-tailed Student's t-test).


( C Immunostaining assay of SOX1 ( red ) after 4 HDAC inhibitors ( 4i ) , including RG2833 ( 2833 , 2μM ) , CAY10683 ( CAY , 2μM ) , PCI - 34051 ( 34051 , 5μM ) and Bufexamac ( Buf , 12 . 5μM ) , treatment or remove one inhibitor from these 4 HDAC inhibitors ( 4i ) in V1 - PAKI cells on day 16 of dorsal neural differentiation Scale bar , 100 μm .

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

(C Immunostaining assay of SOX1 (red) after 4 HDAC inhibitors (4i), including RG2833 (2833, 2μM), CAY10683 (CAY, 2μM), PCI-34051 (34051, 5μM) and Bufexamac (Buf, 12.5μM), treatment or remove one inhibitor from these 4 HDAC inhibitors (4i) in V1-PAKI cells on day 16 of dorsal neural differentiation Scale bar, 100 μm.


( G and H ) Immunostaining assay of SOX1 ( red ) after Bufexamac treatment in V1 - PAKI cells on day 16 of dorsal neural differentiation ( G ) , and quantiﬁcation of SOX1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( + Buf ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ; ( one - way ANOVA ) .

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

(G and H) Immunostaining assay of SOX1 (red) after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).


( J and K ) Immunostaining assay of MAP2 ( green ) after Bufexamac treatment in V1 - PAKI cells on day 35 of dorsal neural differentiation ( J ) , and quantiﬁcation of MAP2 + cells ( K ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( + Buf ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ( one - way ANOVA ) .

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

(J and K) Immunostaining assay of MAP2 (green) after Bufexamac treatment in V1-PAKI cells on day 35 of dorsal neural differentiation (J), and quantiﬁcation of MAP2+ cells (K), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group (one-way ANOVA).


( L ) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1 - PAKI cells on day 16 of dorsal ( left , p = 0 . 0007 ( V1 - PAKI ) , p = 0 . 0002 ( + Buf ) ) and ventral ( right , p = 0 . 0083 ( V1 - PAKI ) , p = 0 . 0276 ( + Buf ) ) neural differentiation . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ; ( one - way ANOVA ) .

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

(L) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal (left, p=0.0007 (V1-PAKI), p=0.0002 (+Buf)) and ventral (right, p=0.0083 (V1-PAKI), p=0.0276 (+Buf)) neural differentiation. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).


( B and C ) RIP analysis for endogenous interaction between HDAC10 and SOX1 - OT V1 on day 16 of dorsal neural differentiation ( B ) , p = 0 . 0085 and ventral neural differentiation ( C ) , p = 0 . 0152 . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus IgG group ( unpaired two - tailed Student ' s t - test ) .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B and C) RIP analysis for endogenous interaction between HDAC10 and SOX1-OT V1 on day 16 of dorsal neural differentiation (B), p=0.0085 and ventral neural differentiation (C), p=0.0152. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus IgG group (unpaired two-tailed Student's t-test).


( D and E ) Enrichment of HDAC10 on SOX1 promoter region in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( D ) , p = 0 . 0052 and ventral neural differentiation ( E ) , p = 0 . 0001 . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D and E) Enrichment of HDAC10 on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (D), p=0.0052 and ventral neural differentiation (E), p=0.0001. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( G and H ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression in V1 - PAKI cells on day 16 of dorsal neural differentiation ( G ) , and quantiﬁcation of SOX1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression in V1 - PAKI cells on day 16 of ventral neural differentiation ( I ) , and quantiﬁcation of SOX1 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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

(G and H) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of ventral neural differentiation (I), and quantiﬁcation of SOX1+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of dorsal neural differentiation ( A ) , and quantiﬁcation of ASCL1 + cells ( B ) , p = 0 . 0021 ( V1 - PAKI ) , p = 0 . 0024 ( SOX1KO ) . Scale bar , 100 μm . ( C and D ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of ventral neural differentiation ( C ) , and quantiﬁcation of ASCL1 + cells ( D ) , p = 0 . 0003 ( V1 - PAKI ) , p = 0 . 0004 ( SOX1KO ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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

(A and B) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of dorsal neural differentiation (A), and quantiﬁcation of ASCL1+ cells (B), p=0.0021 (V1-PAKI), p=0.0024 (SOX1KO). Scale bar, 100 μm. (C and D) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of ventral neural differentiation (C), and quantiﬁcation of ASCL1+ cells (D), p=0.0003 (V1-PAKI), p=0.0004 (SOX1KO). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( E and F ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of dorsal neural differentiation ( E ) , and quantiﬁcation of ASCL1 + cells ( F ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( G and H ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of ventral neural differentiation ( G ) , and quantiﬁcation of ASCL1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of MAP2 ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of dorsal neural differentiation ( I ) , and quantiﬁcation of MAP2 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + dox ) . Scale bar , 100 μm . ( K and L ) Immunostaining assay of GABA ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of ventral neural differentiation ( K ) , and quantiﬁcation of GABA + cells ( L ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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

(E and F) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of dorsal neural differentiation (E), and quantiﬁcation of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (G and H) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantiﬁcation of ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of MAP2 (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (I), and quantiﬁcation of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. (K and L) Immunostaining assay of GABA (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (K), and quantiﬁcation of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Immunostaining assay of SOX1 ( red ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal ( A ) and ventral ( B ) organoids differentiation . Scale bar , 100 μm . ( C ) Immunostaining assay of MAP2 ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( D ) Immunostaining assay of GABA ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm .

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

(A and B) Immunostaining assay of SOX1 (red) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal (A) and ventral (B) organoids differentiation. Scale bar, 100 μm. (C) Immunostaining assay of MAP2 (green) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (D) Immunostaining assay of GABA (green) in control, V1-PAKI and SOX1KO cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.


( E and F ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal ( E ) and ventral ( F ) organoids differentiation . Scale bar , 100 μm . ( G ) Immunostaining assay of MAP2 ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( H ) Immunostaining assay of GABA ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm .

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

(E and F) Immunostaining assay of SOX1 (red) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal (E) and ventral (F) organoids differentiation. Scale bar, 100 μm. (G) Immunostaining assay of MAP2 (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (H) Immunostaining assay of GABA (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.


B ) Total T cells were enriched from spleens of Ptpn6fl / fl / Ptpn11fl / flWbm , CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm , CD4cre Ptpn6fl / fl mice . Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates , Gapdh used as loading control .

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

B) Total T cells were enriched from spleens of Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl mice. Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates, Gapdh used as loading control.


CD4cre Ptpn6fl / fl and control mice were subcutaneously injected with MC38 cells . Tumour growth in individual mice is shown for the indicated genotypes and treatments ; number of mice eradicating the tumour is shown within the graphs ( C ) .

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

CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Tumour growth in individual mice is shown for the indicated genotypes and treatments; number of mice eradicating the tumour is shown within the graphs (C).


CD4cre Ptpn6fl / fl and control mice were subcutaneously injected with MC38 cells . Survival curves are shown ( D ) .

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

CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Survival curves are shown (D).


Tumour growth in individual mice challenged with MC38 and subsequently treated with anti - PD - 1 antibody or isotype control is shown for each indicated genotype ; number of mice eradicating the tumour is shown within the graphs ( G ) .

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

Tumour growth in individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype; number of mice eradicating the tumour is shown within the graphs (G).


individual mice challenged with MC38 and subsequently treated with anti - PD - 1 antibody or isotype control is shown for each indicated genotype Survival curves of CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm and control mice is shown ( H ) .

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

individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype Survival curves of CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm and control mice is shown (H).


I , J ) 10 - 12 days following MC38 tumour inoculation and treatment with anti - PD - 1 antibody or isotype control , CD4cre Ptpn6 / 11wt / wt and CD4cre Ptpn6fl / fl / Ptpn11fl / flWbm mice were sacrificed , and TILs analysed . Graphs depict frequencies of CD8 + T cells ( gated as CD45 + TCRβ + CD8 + ) expressing IFN - γ and TNFα upon re - stimulation ( G ) and percentages of CD8 + T cells ( H ) .

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

I, J) 10-12 days following MC38 tumour inoculation and treatment with anti-PD-1 antibody or isotype control, CD4cre Ptpn6/11wt/wt and CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm mice were sacrificed, and TILs analysed. Graphs depict frequencies of CD8+ T cells (gated as CD45+ TCRβ+ CD8+) expressing IFN-γ and TNFα upon re-stimulation (G) and percentages of CD8+ T cells (H).


B ) Ptpn6 and Ptpn11 copy number , as estimated through high resolution mass spectrometry in GzmBcre Ptpn6 / 11fl / fl and control CTLs .

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

B) Ptpn6 and Ptpn11 copy number, as estimated through high resolution mass spectrometry in GzmBcre Ptpn6/11fl/fl and control CTLs.


C and D ) GzmBcre R26R - YFP reporter mice were injected subcutaneously with MC38 cells and analysed after 11 days . Graphs show the percentage of YFP + cells among CD45 + CD8 + TILs ( C ) and the percentage of PD - 1 + cells among CD45 + CD8 + YFP + TILs ( D ) . Results depict n = 4 mice ( C and D ) .

O O O O B-GENEPROD O O B-GENEPROD O B-ORGANISM O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O

C and D) GzmBcre R26R-YFP reporter mice were injected subcutaneously with MC38 cells and analysed after 11 days. Graphs show the percentage of YFP+ cells among CD45+ CD8+ TILs (C) and the percentage of PD-1+ cells among CD45+ CD8+ YFP+ TILs (D). Results depict n = 4 mice (C and D).


GzmBcre Ptpn6 / 11fl / fl and control Ptpn6 / 11fl / fl mice were subcutaneously injected with MC38 cells . Survival curves and statistical comparisons between different groups are shown ( E ) .

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

GzmBcre Ptpn6/11fl/fl and control Ptpn6/11fl/fl mice were subcutaneously injected with MC38 cells. Survival curves and statistical comparisons between different groups are shown (E).


The tumour start depicts the time ( days ) following tumour engraftment after which the tumour is palpable , while tumour endpoint depicts the time ( days ) from tumour start until the reaching the maximal allowed tumour size ( F ) .

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

The tumour start depicts the time (days) following tumour engraftment after which the tumour is palpable, while tumour endpoint depicts the time (days) from tumour start until the reaching the maximal allowed tumour size (F).


C ) Numbers of CTLs ( gated on CD8 + T cells ) from Ptpn6 / 11fl / fl or GzmBcre Ptpn6 / 11fl / fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells .

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

C) Numbers of CTLs (gated on CD8+ T cells) from Ptpn6/11fl/fl or GzmBcre Ptpn6/11fl/fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells.


D ) Quantitative high - resolution mass spectrometry was used to resolve the proteome of Ptpn6 / 11fl / fl and GzmBcre Ptpn6 / 11fl / fl CTLs ( day 7 ) . The plot depicts protein copy number in control and GzmBcre Ptpn6 / 11fl / fl cells . Significantly altered proteins are indicated in red ; selected examples are annotated . Results are based on n = 3 biological replicates and two - tailed , unequal - variance t - test on log10 transformed copy number per cell values was used to compare differences between experimental groups .

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

D) Quantitative high-resolution mass spectrometry was used to resolve the proteome of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (day 7). The plot depicts protein copy number in control and GzmBcre Ptpn6/11fl/fl cells. Significantly altered proteins are indicated in red; selected examples are annotated. Results are based on n = 3 biological replicates and two-tailed, unequal-variance t-test on log10 transformed copy number per cell values was used to compare differences between experimental groups.


G ) Graph illustrates the percentage of DAPI + dead cells of Ptpn6 / 11fl / fl and GzmBcre Ptpn6 / 11fl / fl CTLs ( gated on CD8 + T cells ) at the indicated days as measured by flow cytometry .

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

G) Graph illustrates the percentage of DAPI+ dead cells of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (gated on CD8+ T cells) at the indicated days as measured by flow cytometry.


H ) Protein copy number of Tnfrsf1b and Mcl - 1 in GzmB cre Ptpn6 / 11fl / fl and control CTLs , as estimated through high resolution mass spectrometry .

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

H) Protein copy number of Tnfrsf1b and Mcl-1 in GzmB cre Ptpn6/11fl/fl and control CTLs, as estimated through high resolution mass spectrometry.