( A ) Representative confocal of stage 17 embryonic heads highlighting expression of Myosin in ( blue ) FasII in axonal projections ( green ) in control ( elav > RFP ) in with reduced neuronal DIPkappa ( elav > DIPkappaRNAi ) as well as in , which mis - express DIPgamma or dpr1 in neuronal ( elav > DIPgamma , elav > dpr1 ) . Single channels focusing on projections ( FasII ) of MN2a / or MN3 to MHE , MHD or LR of an early stage 17 Drosophila are shown . Asterisks highlight location of MN2a MN3 , respectively . MN2a is identified by FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates Dfd expressing MHE muscle ( as shown in elav > RFP control ) , while MN3 is identified as MN underneath MN2a , which normally innervates MHD muscle ( as shown in elav > RFP control ) . panel on right represents a schematic drawing of confocal shown on left side , summarizing innervation of anterior ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control mis - expression conditions .

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(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced neuronal DIPkappa (elav>DIPkappaRNAi) as well as in animals, which mis-express DIPgamma or dpr1 in neuronal cells (elav>DIPgamma, elav>dpr1). Single channels focusing on the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right represents a schematic drawing of the confocal images shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.


( A ) Representative confocal of stage 17 embryonic heads highlighting expression of Dfd ( purple ) , Myosin in ( blue ) FasII in axonal projections ( green ) in control ( elav > RFP ) in with reduced Dfd expression in neurons ( elav > DfdRNAi ) or in ( Mef2 > DfdRNAi ) or both in both tissues ( elav ; Mef2 > DfdRNAi ) . single channels for projections ( FasII ) of MN2a / or MN3 to MHE , MHD or LR of an early stage 17 Drosophila are shown . Asterisks highlight location of MN2a MN3 , respectively . MN2a is identified by FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates Dfd expressing MHE muscle ( as shown in elav > RFP control ) , while MN3 is identified as MN underneath MN2a , which normally innervates MHD muscle ( as shown in elav > RFP control ) . panel on right side represents a schematic drawing of confocal image shown on left side , summarizing innervation of anterior ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control perturbation conditions .

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(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced Dfd expression in neurons (elav>DfdRNAi) or in muscles (Mef2>DfdRNAi) or both in both tissues (elav;Mef2>DfdRNAi). The single channels for the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and perturbation conditions.


( A ) Immuno - precipitations of extracts from asynchronous S2 using 1 μg of indicated antibodies at 4 ° C overnight . control antibody ( ab ) is a rabbit anti - yeast Mad2 .

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(A) Immuno-precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4°C overnight. The control antibody (ab) is a rabbit anti-yeast Mad2.


( B ) Single confocal section of a population of S2 . Examples of interphase are boxed in blue . Mitotic positive for anti - phosphoHistone3 ( pH3 ) are boxed in red .

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(B) Single confocal section of a population of S2 cells. Examples of interphase cells are boxed in blue. Mitotic cells positive for the anti-phosphoHistone3 (pH3) are boxed in red.


( C ) Single confocal section of a S2 . Anti - Incenp , anti - Cid mark centromeric inner KT , respectively .

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(C) Single confocal section of a S2 cell. Anti-Incenp, anti-Cid mark the centromeric chromatin and the inner KT, respectively.


( D - E ) Maximal projections of S2 imaged by STED microscopy . 7 z - sections for a total of 1 . 5 um ( D ) 4 z - sections for a total of 0 . 5 um ( E ) are shown . Anti - CenpC anti - Spc105R mark proximal distal region of different portions of outer KT , respectively .

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(D-E) Maximal projections of S2 cells imaged by STED microscopy. 7 z-sections for a total of 1.5 um (D) and 4 z-sections for a total of 0.5 um (E) are shown. Anti-CenpC and anti-Spc105R mark the proximal and the distal region of different portions of the outer KT, respectively.


( F - G ) Single confocal sections of S2 at indicated mitotic stages stained to detect Snap29 , pores ( WGA ) , CyclinB ( F ) , or Snap29 , Spc105R , lamina ( LaminA ) ( G ) . Note transient perinuclear enrichment of Snap29 in early prophase . WGA stains also speckles in hemocyte - derived S2 ( Rizki Rizki , 1983 ) . High magnification of insets in C - G shows localization of Snap29 relative to indicated markers .

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(F-G) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29, nuclear pores (WGA), and CyclinB (F), or Snap29, Spc105R, and the nuclear lamina (LaminA) (G). Note the transient perinuclear enrichment of Snap29 in early prophase. WGA stains also cytoplasmic speckles in hemocyte-derived S2 cells (Rizki Rizki, 1983). High magnification of the insets in C-G shows localization of Snap29 relative to the indicated markers.


( H - J ) Immuno - gold localization of Snap29 in cryo - sections of S2 in pro - metaphase . Pseudo - coloring in red identifies chromosomes ( Ch ) , in yellow organelles in green MTs . arrows in H - I indicate examples of signal in nucleoplasm that is not associated to membranes , but rather , to electron - dense material . MVB : multivesicular ; NE : envelope ; PM : plasma .

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(H-J) Immuno-gold localization of Snap29 in cryo-sections of S2 cells in pro-metaphase. Pseudo-coloring in red identifies chromosomes (Ch), in yellow membrane organelles and in green MTs. The white arrows in H-I indicate examples of signal in the nucleoplasm that is not associated to membranes, but rather, to electron-dense material. MVB: multivesicular body; NE: nuclear envelope; PM: plasma membrane.


( A ) Comparable maximum projections of confocal sections of dorsal wing pouch portion of third instar larval imaginal discs . A cartoon of full Snap29 is shown above control panel .

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(A) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs. A cartoon of full length Snap29 is shown above the control panel.


( B ) of Snap29 signal as in panel A , considering > 30 KTs per sample . Relative mRNA expression of downregulated genes versus control , measured by Q - PCR , is shown above graph . Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs .

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(B) Quantification of the Snap29 signal as in panel A, considering > 30 KTs per sample. Relative mRNA expression of the downregulated genes versus control, measured by Q-PCR, is shown above the graph. Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs.


( C ) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated indicated Snap29 form over - expressed . Cartoons of expressed Snap29 mutant forms are shown above panels . Anti - pH3 labels mitotic CenpC marks KTs . ( D ) of Snap29 as in panel C , considering > 28 KTs per sample . Note that expression of a form of Snap29 lacking SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi .

(C) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over-expressed. Cartoons of the expressed Snap29 mutant forms are shown above the panels. Anti-pH3 labels mitotic cells and CenpC marks KTs. (D) Quantification of Snap29 as in panel C, considering > 28 KTs per sample. Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells.


( A ) Selected frames of time - lapse imaging of control S2 or treated for 96 hrs with dsRNA against central portion of Snap29 . Note that depleted does not display a recognizable metaphase plate , or fail to segregate chromosomes correctly , or form tripolar spindles or micronuclei ( arrowheads ; arrowheads in control point to dividing normally ) .

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(A) Selected frames of time-lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29. Note that depleted cell does not display a recognizable metaphase plate, or fail to segregate all chromosomes correctly, or form tripolar spindles or micronuclei (arrowheads; arrowheads in control point to cells dividing normally).


( B ) Western blotting of extract from control depleted of Snap29 ( dsSnap29 ) relative to experiment in a .

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(B) Western blotting of extract from control cells and cells depleted of Snap29 (dsSnap29) relative to the experiment in a.


( C ) of mitotic phenotype of control dsSnap29 depleted , on time - lapse imaging of an average of > 28 per sample . P value is determined by two - t - considering defects together .

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(C) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells, based on time-lapse imaging of an average of > 28 individual cells per sample. P value is determined by two-tailed t-test considering all defects together.


( D ) Selected frames of time - lapse imaging of control U2OS U2OS treated for indicated time . Note that depleted at 48 - 66h display altered metaphase plate chromosome segregation ( arrowheads ) .

(D) Selected frames of time-lapse imaging of control U2OS cells and U2OS cells treated for the indicated time. Note that the depleted cell at 48-66h display altered metaphase plate and chromosome segregation (arrowheads).


( E ) Western blot of extracts relative to experiments in panel d .

(E) Western blot analysis of extracts relative to the experiments in panel d.


( F ) of time from prophase to entry in anaphase on of > 20 / sample by time - lapse microscopy . P values are determined by Kruskal - Wallis with Dunn ' s multiple comparison . OE : Over - expression .

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(F) Quantification of the time from prophase to entry in anaphase based on analysis of >20 individual cells/sample by time-lapse microscopy. P values are determined by Kruskal-Wallis test with Dunn's multiple comparison analysis. OE: Over-expression.


( A ) Single confocal sections of HeLa at metaphase treated for 8 min at 4 ° C immunostained to reveal presence of unattached kinetochores . CENPT labels KTs , while MAD1 identifies unattached ones . Enlargements of boxed areas displaying both chromosomes aligned not aligned at metaphase plate . ( B ) of number of KTs on attachment , position MAD1 positivity . Each point per category represents one experiment in which 10 random KTs / of 20 / sample were counted . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison . Note that in control most of KT are attached , MAD1 - negative aligned to metaphase plate , while in SNAP29KD half of KTs that are unattached , MAD1 - positive not aligned to metaphase plate .

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(A) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4°C and immunostained to reveal the presence of unattached kinetochores. CENPT labels all KTs, while MAD1 identifies unattached ones. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate. (B) Quantification of the number of KTs based on attachment, position and MAD1 positivity. Each point per category represents one experiment in which 10 random KTs/cell of 20 cells/sample were counted. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison test. Note that in control cells most of the KT are attached, MAD1-negative and aligned to the metaphase plate, while in SNAP29KD cells half of the KTs that are unattached, MAD1-positive and not aligned to the metaphase plate.


( C ) Single confocal sections of HeLa . CREST CENPT were used to label centromere region associated KTs , respectively . Enlargements of boxed areas displaying both chromosomes aligned not aligned at metaphase plate are shown . ( D ) Average of inter KT on in each of which 10 chromosomes / 10 / sample were counted . P values are obtained by Kruskal - Wallis with Dunn ' s multiple comparison . Note that both aligned not aligned KTs have a shorter inter KT compared to aligned control KTs .

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(C) Single confocal sections of HeLa cells. CREST and CENPT were used to label the centromere region and associated KTs, respectively. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown. (D) Average of the inter KT distance based on in each of which 10 chromosomes/cell and 10 cells/sample were counted. P values are obtained by Kruskal-Wallis test with Dunn's multiple comparison analysis. Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs.


( E ) Single confocal sections of HeLa at metaphase stained to visualize mitotic spindle . ( F ) Averaged spindle pole on > 20 with bipolar spindles / sample . P value is obtained by two - paired t - .

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(E) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA. (F) Averaged spindle pole length based on >20 cells with bipolar spindles/sample. The P value is obtained by two-tailed paired t-test.


( A - C , E ) Max projection of single control SNAP29 KD HeLa treated with 2 mM thymidine for 24 hrs , released in normal medium for 8 hrs arrested in prometaphase with Nocodazole for 2 hrs .

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(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.


( D ) Immunoblotting of HeLa extracts , treated as indicated .

(D) Immunoblotting of HeLa protein extracts, treated as indicated.


( F ) of 3 independent experiments evaluating localization of KT factors shown in A - C , e relative to CREST . 100 KTs from 10 control 200 KTs from 10 SNAP29 KD per sample were identified by CREST . P values are obtained by 2 - way ANOVA with Sidak ' s multiple comparison relative to localization of HEC1 , which is not lost on SNAP29KD .

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(F) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A-C, e relative to CREST. 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Sidak's multiple comparison analysis relative to localization of HEC1, which is not lost on SNAP29KD cells.


( A - C ) KNL1 localization in control , SNAP29 KD SNAP29 KD expressing full human SNAP29 , CFP tagged , full Drosophila Snap29 ( CFP - Snap29 ) , or mutant forms schematized above panels . Max projections of single in prophase are shown . ( D ) of 3 independent experiments evaluating localization of KNL1 relative to CREST . 200 KTs from 10 control 200 KTs from 10 SNAP29 KD per sample were identified by CREST . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison relative to localization of KNL1 in SNAP29KD .

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(A-C) KNL1 localization analysis in control, SNAP29 KD and SNAP29 KD expressing full length human SNAP29, CFP tagged, full length Drosophila Snap29 (CFP-Snap29), or the mutant forms schematized above the panels. Max projections of single cells in prophase are shown. (D) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST. 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells.


( E - F ) Single confocal sections of HeLa in interphase overexpressing indicated constructs . In expressing SNAP29 Q1 Q2 , SNAP29 KNL1 accumulate in large compartments that are often positive for p62 ( arrows ) .

(E-F) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs. In cells expressing SNAP29 Q1 Q2, SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 (arrows).


( A - B ) Confocal sections of wild - type Snap29 mutant eye - antennal imaginal discs ( eye disc portion to left ) of L3 larvae . Discs were labeled with phalloidin DAPI to visualize subcortical f - actin cytoskeleton nuclei , respectively . Arrowheads in B point to actin - rich unpolarized in apical , medial basal sections of same disc .

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(A-B) Confocal sections of wild-type and Snap29 mutant eye-antennal imaginal discs (eye disc portion to the left) of L3 larvae. Discs were labeled with phalloidin and DAPI to visualize the subcortical f-actin cytoskeleton and nuclei, respectively. Arrowheads in B point to actin-rich unpolarized cells in apical, medial and basal sections of the same disc.


( C ) High magnification of a single confocal cross - section of wild - type Snap29 mutant eye disc tissues . Two facing folds of tissue are separated by apical lumen . aPKC Dlg mark apical lateral plasma domains , respectively . In Snap29 mutant tissue apical - basal polarity is disrupted .

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(C) High magnification of a single confocal cross-section of wild-type and Snap29 mutant eye disc tissues. Two facing folds of the tissue are separated by the apical lumen. aPKC and Dlg mark the apical and lateral plasma membrane domains, respectively. In Snap29 mutant tissue the apical-basal polarity is disrupted.


( D ) Single confocal cross - sections of wild - type Snap29 eye - antennal discs , expressing Puckered - LacZ ( Puc - lacZ ) , a reporter of JNK signaling activation .

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(D) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs, expressing Puckered-LacZ (Puc-lacZ), a reporter of JNK signaling activation.


( E - H ) Single confocal cross - sections of wild - type Snap29 eye - antennal discs ( E - F ) , of otherwise wild - type Snap29 eye - antennal discs expressing apoptosis inhibitor p35 ( G - H ) . Anti - activated - Caspase 3 ( Casp3 ) marks apoptotic .

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(E-H) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs (E-F), and of otherwise wild-type and Snap29 eye-antennal discs expressing the bacterial apoptosis inhibitor p35 (G-H). Anti-activated-Caspase 3 (Casp3) marks apoptotic cells.


( a ) vBcl - 2 binding . After transfection , 293T were used for GST - pulldown purified were detected by silver .

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(a) vBcl-2 binding proteins. After transfection, 293T cells were used for GST-pulldown and the purified proteins were detected by silver staining.


( c ) UVRAG binds Beclin1 through its CCD . After transfection with Beclin1 - V5 , together with full - or fragments of Flag - UVRAG , 293T whole lysates ( WCLs ) were used for immunoprecipitation ( IP ) with anti - Flag followed by immunoblotting ( IB ) with anti - V5 .

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(c) UVRAG binds Beclin1 through its CCD. After transfection with Beclin1-V5, together with full-length or fragments of Flag-UVRAG, 293T whole cell lysates (WCLs) were used for immunoprecipitation (IP) with anti-Flag followed by immunoblotting (IB) with anti-V5.


( d ) Interactions between endogenous UVRAG Beclin1 between endogenous UVRAG PI ( 3 ) KC3 . 293T lysates were used for immunoprecipitation with preimmune sera or anti - Beclin1 , followed by immunoblotting with anti - UVRAG or anti - Beclin1 , or for immunoprecipitation with preimmune sera or anti - UVRAG , followed by immunoblotting with anti - PI ( 3 ) KC3 .

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(d) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI(3)KC3. 293T cell lysates were used for immunoprecipitation with preimmune sera or anti-Beclin1, followed by immunoblotting with anti-UVRAG or anti-Beclin1, or for immunoprecipitation with preimmune sera or anti-UVRAG, followed by immunoblotting with anti-PI(3)KC3.


( e ) UVRAGCCD is required for Beclin1 interaction . After transfection with Beclin1 - V5 ( lanes 2 4 ) together with Flag - UVRAGΔCCD ( lanes 1 2 ) or Flag - UVRAGΔC2 ( lanes 3 4 ) , WCLs were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 .

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(e) UVRAGCCD is required for Beclin1 interaction. After transfection with Beclin1-V5 (lanes 2 and 4) together with Flag-UVRAGΔCCD (lanes 1 and 2) or Flag-UVRAGΔC2 (lanes 3 and 4), WCLs were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5.


( f ) Beclin1 binds UVRAG through its CCD . After transfection with Flag - UVRAG together with full - or fragments of Beclin1 - V5 , WCLs were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag .

(f) Beclin1 binds UVRAG through its CCD. After transfection with Flag-UVRAG together with full-length or fragments of Beclin1-V5, WCLs were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag.


( g ) An increase in UVRAG stability induced by Beclin1 . After transfection with Flag - UVRAG empty vector or Flag - UVRAG Beclin1 - V5 , WCLs were treated with cycloheximide ( CHX , 20 μg ml − 1 ) for indicated time , followed by immunoblotting with anti - Flag .

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(g) An increase in UVRAG stability induced by Beclin1. After transfection with Flag-UVRAG and empty vector or Flag-UVRAG and Beclin1-V5, WCLs were treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time, followed by immunoblotting with anti-Flag.


( h ) UVRAG directly interacts with Beclin1 . MBP - UVRAGCCD ( lane 1 ) , His6 - Beclin1CCD ( lane 2 ) or mixtures of both in a dependent manner ( lanes 3 - 8 ) , were subjected to native gel electrophoresis . numbers indicate ( nmol ) of . Arrows indicate purified MBP - UVRAGCCD , His6 - Beclin1CCD between these two .

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(h) UVRAG directly interacts with Beclin1. MBP-UVRAGCCD (lane 1), His6-Beclin1CCD (lane 2) or mixtures of both proteins in a concentration dependent manner (lanes 3-8), were subjected to native gel electrophoresis. The numbers indicate the concentration (nmol) of protein. Arrows indicate the purified MBP-UVRAGCCD, His6-Beclin1CCD and complex between these two.


( i ) Beclin1CCD UVRAGCCD associate into a α - helical heterodimer . After removing N - terminal His6 MBP tags , Beclin1CCD preformed Beclin1CCD - UVRAGCCD were subjected to circular dichroism spectroscopic . raw data for this experiment are presented in Supplementary Information , Fig . S5 .

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(i) Beclin1CCD and UVRAGCCD associate into a α-helical heterodimer. After removing the N-terminal His6 and MBP tags, the Beclin1CCD and the preformed Beclin1CCD-UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.


( a ) Bcl - 2 binding domain of Beclin1 is sufficient to bind vBcl - 2 . At 48 h posttransfection with wild - type mutant Beclin1 - V5 , together with HA - vBcl - 2 , WCLs of 293T were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - HA .

(a) The Bcl-2 binding domain of Beclin1 is sufficient to bind vBcl-2. At 48 h posttransfection with wild-type and mutant Beclin1-V5, together with HA-vBcl-2, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-HA.


( b ) loss of Beclin1 interaction of vBcl - 2AAA mutant . At 48 h posttransfection with wild - type HA - vBcl - 2 or vBcl - 2AAA mutant , together with Beclin1 - V5 , WCLs of 293T were used for immunoprecipitation with anti - V5 , followed by immunoblotting with anti - HA .

(b) The loss of Beclin1 interaction of vBcl-2AAA mutant. At 48 h posttransfection with wild-type HA-vBcl-2 or vBcl-2AAA mutant, together with Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5, followed by immunoblotting with anti-HA.


( c ) vBcl - 2 interaction with wild - type UVRAG in presence of Beclin1 . At 48 h posttransfection with Flag - UVRAG together with Beclin1 - V5 HA - vBcl - 2 or vBcl - 2AAA mutant , WCLs of 293T were used for immunoprecipitation with anti - HA followed by immunoblotting with anti - Flag .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) vBcl-2 interaction with wild-type UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAG together with Beclin1-V5 and HA-vBcl-2 or vBcl-2AAA mutant, WCLs of 293T cells were used for immunoprecipitation with anti-HA followed by immunoblotting with anti-Flag.


( d ) vBcl - 2 interaction with PI ( 3 ) KC3 in presence of Beclin1 . At 48 h posttransfection with HA - vBcl - 2 together with Flag - PI ( 3 ) KC3 Beclin1 - V5 , WCLs of 293T were used for immunprecipitation with anti - HA followed by immunoblotting with anti - Flag .

(d) vBcl-2 interaction with PI(3)KC3 in the presence of Beclin1. At 48 h posttransfection with HA-vBcl-2 together with Flag-PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunprecipitation with anti-HA followed by immunoblotting with anti-Flag.


( e ) PI ( 3 ) KC3 N - terminal C2 domain binds Beclin1 . At 48 h posttransfection with Beclin1 - V5 together with full - or fragments of Flag - PI ( 3 ) KC3 , WCLs of 293T were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - V5 . CAT , catalytic domain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PI(3)KC3 N-terminal C2 domain binds Beclin1. At 48 h posttransfection with Beclin1-V5 together with full-length or fragments of Flag-PI(3)KC3, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5. CAT, catalytic domain.


( f ) Beclin1CCD Beclin1ECD are sufficient for binding PI ( 3 ) KC3 . At 48 h posttransfection with Flag - PI ( 3 ) KC3 together with full - or fragments of Beclin1 - V5 , WCLs of 293T were used for immunoprecipitation with anti - V5 followed by immunoblotting with anti - Flag . CT , C terminus ( aa 337 - 448 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beclin1CCD and Beclin1ECD are sufficient for binding PI(3)KC3. At 48 h posttransfection with Flag-PI(3)KC3 together with full-length or fragments of Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag. CT, C terminus (aa 337-448).


( g ) PI ( 3 ) KC3 interaction with UVRAG in presence of Beclin1 . At 48 h posttransfection with Flag - UVRAGΔCCD or UVRAGCCD together with HA - PI ( 3 ) KC3 Beclin1 - V5 , WCLs of 293T were used for immunoprecipitation with anti - Flag followed by immunoblotting with anti - HA .

(g) PI(3)KC3 interaction with UVRAG in the presence of Beclin1. At 48 h posttransfection with Flag-UVRAGΔCCD or UVRAGCCD together with HA- PI(3)KC3 and Beclin1-V5, WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA.


( a ) UVRAG expression LC3 process in HCT116 . same amounts of 293T , NIH3T3 , HCT116 MCF7 were used for immunoblotting with anti - UVRAG anti - tubulin . WCLs of 293T transfected with Flag - tagged UVRAG were included as controls . WCLs of HCT116 . vector HCT116 . UVRAG were incubated with or without starvation conditions used for immunoblotting with anti - LC3 anti - tubulin . raw data for this experiment is presented in Supplementary information , Fig . S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UVRAG expression and LC3 process in HCT116 cells. The same protein amounts of 293T, NIH3T3, HCT116 and MCF7 cells were used for immunoblotting with anti-UVRAG and anti-tubulin. WCLs of 293T cells transfected with Flag-tagged UVRAG were included as controls. WCLs of HCT116.vector and HCT116.UVRAG were incubated with or without starvation conditions and used for immunoblotting with anti-LC3 and anti-tubulin. The raw data for this experiment is presented in the Supplementary information, Fig. S5.


( b ) Electron microscopy of autophagy in HCT116 . HCT116 . vector HCT116 . UVRAG were incubated under normal or starvation conditions for 4 h subjected to scanning electron microscopy . Scale bars ( from left to right panels ) represent 2 nm , 0 . 5 nm , 0 . 2 nm 0 . 2 nm . arrows indicate autophagosome or autolysosome .

(b) Electron microscopy of autophagy in HCT116 cells. HCT116.vector and HCT116.UVRAG cells were incubated under normal or starvation conditions for 4 h and subjected to scanning electron microscopy. Scale bars (from left to right panels) represent 2 nm, 0.5 nm, 0.2 nm and 0.2 nm. The arrows indicate autophagosome or autolysosome.


( c ) UVRAG induces autophagosome formation in HCT116 . After transfection with GFP - LC3 vector , HCT116 . vector HCT116 . UVRAG were maintained under normal conditions or starved for 3 h . GFP - LC3 was detected using an inverted microscope . Arrows indicate autophagosomes . scale bars represent 10 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 O O O O O O O O

(c) UVRAG induces autophagosome formation in HCT116 cells. After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions or starved for 3 h. GFP-LC3 was detected using an inverted fluorescence microscope. Arrows indicate autophagosomes. The scale bars represent 10 μm.


( d ) After transfection with GFP - LC3 vector , HCT116 . vector HCT116 . UVRAG were maintained under normal conditions , starved for 3 h in absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Autophagome was quantified as mean ± s . d . of combined results from three independent experiments .

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

(d) After transfection with GFP-LC3 vector, HCT116.vector and HCT116.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Autophagome was quantified as the mean ± s.d. of combined results from three independent experiments.


( a ) HCT116 . vector HCT116 . UVRAG were labelled with LysoTracker subjected to flow cytometry . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAG ΔCCD were treated with or without wortmannin ( 1 μM ) for 2 h their lysates were used to measure lysosomal acid phosphatase enzymatic activity . Data represents mean ± s . d . of three experiments .

O O O O O O O O O O O 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

(a) HCT116.vector and HCT116.UVRAG cells were labelled with LysoTracker and subjected to flow cytometry analysis. HCT116.vector, HCT116.UVRAG, and HCT116.UVRAG ΔCCD cells were treated with or without wortmannin (1 μM) for 2 h and their lysates were used to measure the lysosomal acid phosphatase enzymatic activity. Data represents mean ± s.d. of three experiments.


( b ) UVRAG induces autophagosome formation in NIH3T3 . After transfection with GFP - LC3 vector , NIH3T3 . vector NIH3T3 . UVRAG were maintained under normal conditions , starved for 3 h in absence or presence of 10 mM 3 - methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3 - methyladenine . Data represents mean ± s . d . of three experiments .

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

(b) UVRAG induces autophagosome formation in NIH3T3 cells. After transfection with GFP-LC3 vector, NIH3T3.vector and NIH3T3.UVRAG cells were maintained under normal conditions, starved for 3 h in the absence or presence of 10 mM 3-methyladenine or treated with or without 2 μM rapamycin or rapamycin + 10 mM 3-methyladenine. Data represents mean ± s.d. of three experiments.


( a ) Autophagic activity of UVRAG mutants . HCT116 expressing wild - type or mutant UVRAG were transfected with GFP - LC3 expression vector incubated under normal or starvation conditions for 3 h . Autophagy was quantified as described in Fig . 3 .

O O O O O O O O O O O O O O O O O O O O O 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) Autophagic activity of UVRAG mutants. HCT116 cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 expression vector and incubated under normal or starvation conditions for 3 h. Autophagy was quantified as described in Fig. 3.


( b ) Effect of wild - type mutant UVRAG on Beclin1 - mediated autophagy . MCF7 . vector , MCF7 . Beclin1 MCF7 . Beclin1ΔECD were cotransfected with wild - type UVRAG or its mutant expression vector together with GFP - LC3 expression vector . At 16 - 18 h posttransfection , were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as described in Fig . 3 . scale bars represent 20 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 O O O O O O O O O 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) Effect of wild-type and mutant UVRAG on Beclin1-mediated autophagy. MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells were cotransfected with wild-type UVRAG or its mutant expression vector together with GFP-LC3 expression vector. At 16-18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as described in Fig. 3. The scale bars represent 20 μm.


( c , d ) Interdependence between UVRAG Beclin1 for autophagosome induction . HCT116 . vector HCT116 . UVRAG were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . scale bars represent 20 μm . In figures , data represents mean ± s . d . of three experiments . raw data for c d are presented in Supplementary Information , Fig S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, d) Interdependence between UVRAG and Beclin1 for autophagosome induction. HCT116.vector and HCT116.UVRAG cells were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.


( c , d ) Interdependence between UVRAG Beclin1 for autophagosome induction . MCF7 . vector , MCF7 . Beclin1 MCF7 . Beclin1ΔECD ( d ) were transfected with control siRNA , Beclin1 siRNA or UVRAG siRNA together with GFP - LC3 expression vector as indicated . Autophagy was quantified as described in Fig . 3 . WCLs of these were used for immunoblotting with anti - UVRAG , anti - Beclin1 or anti - tubulin . scale bars represent 20 μm . In figures , data represents mean ± s . d . of three experiments . raw data for c d are presented in Supplementary Information , Fig S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, d) Interdependence between UVRAG and Beclin1 for autophagosome induction.MCF7.vector, MCF7.Beclin1 and MCF7.Beclin1ΔECD cells (d) were transfected with control siRNA, Beclin1 siRNA or UVRAG siRNA together with GFP-LC3 expression vector as indicated. Autophagy was quantified as described in Fig. 3. WCLs of these cells were used for immunoblotting with anti-UVRAG, anti-Beclin1 or anti-tubulin. The scale bars represent 20 μm. In all figures, data represents mean ± s.d. of three experiments. The raw data for c and d are presented in the Supplementary Information, Fig S5.


( a ) Increased Beclin1 - PI ( 3 ) KC3 interaction by UVRAG continued interaction of UVRAG with Beclin1 on stress conditions . HCT116 . vector , HCT116 . UVRAG HCT116 . UVRAGΔCCD were used for immunoprecipitation with anti - PI ( 3 ) KC3 followed by immunoblotting with anti - Beclin1 . WCLs were used for immunoblotting with anti - Beclin1 anti - PI ( 3 ) KC3 . HCT116 . UVRAG expressing Flag - tagged UVRAG were incubated under nutrient - rich conditions ( lanes 1 2 ) , starvation for 4 h ( lane 3 ) , or 2 μM rapamycin treatment for 2 h ( lane 4 ) . WCLs were then used for immunoprecipitation with anti - Beclin1 ( lanes 2 , 3 4 ) or preimmune antibody ( lane 1 ) followed by immunoblotting with anti - Flag . raw data for this experiment are presented in Supplementary Information , Fig . S5 .

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

(a) Increased Beclin1-PI(3)KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells were used for immunoprecipitation with anti-PI(3)KC3 followed by immunoblotting with anti-Beclin1. WCLs were used for immunoblotting with anti-Beclin1 and anti-PI(3)KC3. HCT116.UVRAG cells expressing Flag-tagged UVRAG were incubated under nutrient-rich conditions (lanes 1 and 2), starvation for 4 h (lane 3), or 2 μM rapamycin treatment for 2 h (lane 4). WCLs were then used for immunoprecipitation with anti-Beclin1 (lanes 2, 3 and 4) or preimmune antibody (lane 1) followed by immunoblotting with anti-Flag. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.


( b ) Increased PI ( 3 ) KC3 activity by UVRAG . At 48 h posttransfection with Flag - tagged PI ( 3 ) KC3 with Beclin1 ( lane 1 ) , with Beclin1 + UVRAG ( lane 2 ) or with Beclin1 + UVRAGΔCCD vector ( lane 3 ) , WCLs of 293T were used for immunoprecipitation with anti - Flag followed by an for PI ( 3 ) KC3 lipid kinase activity , as described in Methods . At 16 h posttransfection with p40 ( phox ) PX - EGFP fusion , HCT116 . vector HCT116 . UVRAG were detected using an inverted microscope . p40 ( phox ) PX - EGFP - positive vesicles were quantified as mean ± s . d . of combined results from three independent experiments . scale bars represent 10 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Increased PI(3)KC3 activity by UVRAG. At 48 h posttransfection with Flag-tagged PI(3)KC3 with Beclin1 (lane 1), with Beclin1 + UVRAG (lane 2) or with Beclin1 + UVRAGΔCCD vector (lane 3), WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by an assay for PI(3)KC3 lipid kinase activity, as described in the Methods. At 16 h posttransfection with p40(phox) PX-EGFP fusion, HCT116.vector and HCT116.UVRAG cells were detected using an inverted fluorescence microscope. p40(phox) PX-EGFP-positive vesicles were quantified as the mean ± s.d. of combined results from three independent experiments. The scale bars represent 10 μm.


( c ) Suppression of autophagy by γHV68 vBcl - 2 . At 18 h posttransfection with GFP - LC3 vector , NIH3T3 . vector , NIH3T3 . vBcl - 2 NIH3T3 . vBcl - 2AAA , were incubated under normal or starvation conditions for 4 h . Subsequently , GFP - LC3 was detected using an inverted microscope autophagy was quantified as mean ± s . d . of combined results from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Suppression of autophagy by γHV68 vBcl-2. At 18 h posttransfection with GFP-LC3 vector, NIH3T3.vector, NIH3T3.vBcl-2 and NIH3T3.vBcl-2AAA, cells were incubated under normal or starvation conditions for 4 h. Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s.d. of combined results from three independent experiments.


( a ) Proliferation rates . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD HCT116 . UVRAGCCD ( 1 × 105 per well ) were seeded into 6 - well microtitre plates . numbers were counted on days indicated . results represent means of three independent assays .

(a) Proliferation rates. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 105 per well) were seeded into 6-well microtitre plates. Cell numbers were counted on the days indicated. The results represent means of three independent assays.


( b ) Anchorage independence . HCT116 . vector , HCT116 . UVRAG , HCT116 . UVRAGΔC2 , HCT116 . UVRAGΔCCD HCT116 . UVRAGCCD ( 1 × 104 ) were incubated in soft agar as described in Methods . After ten days , colonies were photographed counted . scale bars represent 50 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 O O O O O O O

(b) Anchorage independence. HCT116.vector, HCT116.UVRAG, HCT116.UVRAGΔC2, HCT116.UVRAGΔCCD and HCT116.UVRAGCCD cells (1 × 104) were incubated in soft agar as described in the Methods. After ten days, colonies were photographed and counted. The scale bars represent 50 μm.


( c ) Tumorigenicity . HCT116 . vector , HCT116 . UVRAG HCT116 . UVRAGΔCCD ( 1 × 107 ) were subcutaneously injected into 5 nude mice , separately . At 16 days postinoculation , mice tumours were photographed tumour weights were measured . Arrows indicate location of tumours .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tumorigenicity. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells (1 × 107) were subcutaneously injected into 5 nude mice, separately. At 16 days postinoculation, mice and tumours were photographed and tumour weights were measured. Arrows indicate location of tumours.


Lymphocyte subsets from 12 month old Ptpn2fl / fl ; p53 + / - Lck - Cre ; Ptpn2fl / f ; p53 + / - mice were analyzed by flow cytometry . Significance in ( a ) was determined using two - sided Fisher ' s exact .

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

Lymphocyte subsets from 12 month old Ptpn2fl/fl;p53+/- and Lck-Cre;Ptpn2fl/f;p53+/- mice were analyzed by flow cytometry. Significance in (a) was determined using two-sided Fisher's exact test.


AT - 3 - OVA breast cancer were injected into fourth inguinal mammary fat pads of Ptpn2fl / fl Lck - Cre ; Ptpn2fl / fl mice ( c ) tumour growth monitored over 26 days .

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

AT-3-OVA breast cancer cells were injected into the fourth inguinal mammary fat pads of female Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl mice and (c) tumour growth monitored over 26 days.


At day 26 ( d26 ) numbers of activated tumour infiltrating lymphocytes ( TILs ) were determined .

At day 26 (d26) the numbers of activated tumour infiltrating lymphocytes (TILs) were determined.


proportion of IFNγ + versus IFNγ + TNF + d26 TILs was determined by flow cytometry .

The proportion of IFNγ+ versus IFNγ+TNF+ d26 TILs was determined by flow cytometry.


d26 TILS were incubated with AT - 3 - OVA tumour isolated from tumour bearing C57BL / 6 mice proportion of IFNγ + T was determined .

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

d26 TILS were incubated with AT-3-OVA tumour cells isolated from tumour bearing C57BL/6 mice and the proportion of IFNγ+ T cells was determined.


AT - 3 - OVA mammary tumour ( 1x106 ) were injected into fourth inguinal mammary fat pads of Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . Tumour - bearing Ly5 . 1 + mice were monitored for a ) tumour growth over 21

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

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. Tumour-bearing Ly5.1+ mice were monitored for a) tumour growth over 21 days


AT - 3 - OVA mammary tumour ( 1x106 ) were injected into fourth inguinal mammary fat pads of Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . After 21 days TILs were processed for flow cytometry donor T numbers ( Ly5 . 1 - Ly5 . 2 + ) determined .

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

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. After 21 days TILs were processed for flow cytometry and donor T cell numbers (Ly5.1-Ly5.2+) determined.


AT - 3 - OVA mammary tumour ( 1x106 ) were injected into fourth inguinal mammary fat pads of Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . After 9 days proportion of Ly5 . 2 + IFNγ + TNF + versus Ly5 . 2 + GrzB + TILs was determined .

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. After 9 days the proportion of Ly5.2+IFNγ+TNF+ versus Ly5.2+GrzB+ TILs was determined.


AT - 3 - OVA mammary tumour ( 1x106 ) were injected into fourth inguinal mammary fat pads of Ly5 . 1 + mice . Seven days after tumour injection FACS - purified 2x106 naïve CD8 + CD44loCD62Lhi lymph node T from Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred into tumour - bearing Ly5 . 1 + mice . d ) for survival over 86 days .

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

AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. d) for survival over 86 days.


Gene expression in tumours from mice treated with Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl T 21 days post - adoptive transfer versus those re - emerging in mice treated with Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl T .

Gene expression in tumours from mice treated with Ly5.2+;OT-1;Ptpn2fl/fl T cells 21 days post-adoptive transfer versus those re-emerging in mice treated with Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl T cells.


B16 . F10 - OVA melanoma ( 1x105 ) were engrafted onto abraded skin in flanks of Ly5 . 1 + mice . 24 h after tumour engraftment naïve CD8 + CD62LhiCD44lo lymph node T from Ly5 . 2 + OT - 1 ; Ptpn2fl / fl versus Ly5 . 2 + OT - 1 ; Lck - Cre ; Ptpn2fl / fl mice were adoptively transferred tumour incidence monitored .

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

B16.F10-OVA melanoma cells (1x105) were engrafted onto the abraded skin in the flanks of Ly5.1+ mice. 24 h after tumour cell engraftment naïve CD8+CD62LhiCD44lo lymph node T cells from Ly5.2+ OT-1;Ptpn2fl/fl versus Ly5.2+ OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred and tumour incidence monitored.


Epidermal lymphocytes from tumour - free mice were stained for CD69hiCD103hi donor - derived ( Ly5 . 2 + vα2 + ) tissue resident memory T ( TRMs ) determined by flow cytometry .

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

Epidermal lymphocytes from tumour-free mice were stained for CD69hiCD103hi and donor-derived (Ly5.2+vα2+) tissue resident memory T cells (TRMs) determined by flow cytometry.


Tumour sizes in B16 . F10 - OVA melanoma bearing mice were determined between day 21 23 .

Tumour sizes in B16.F10-OVA melanoma bearing mice were determined between day 21 and 23.


TILs were assessed for Ly5 . 2 + ; OT - 1 ; Ptpn2fl / fl Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl donor T numbers by flow cytometry .

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

TILs were assessed for Ly5.2+;OT-1;Ptpn2fl/fl and Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl donor T cell numbers by flow cytometry.


CD8 + HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Lck + / - splenocytes were stained for intracellular p ( Y418 ) - SFK p ( Y418 ) - SFK MFIs were determined by flow cytometry .

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

CD8+ HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes were stained for intracellular p(Y418)-SFK and p(Y418)-SFK MFIs were determined by flow cytometry.


HER - 2 - specific Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Lck + / - CAR - T were incubated with HER - 2 expressing 24JK sarcoma ( 24JK - HER - 2 ) or HER - 2 negative 24JK sarcoma CD44 , CD25 , PD - 1 LAG - 3 MFIs on CD8 + CAR - T were determined by flow cytometry .

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

HER-2-specific Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with HER-2 expressing 24JK sarcoma cells (24JK-HER-2) or HER-2 negative 24JK sarcoma cells and CD44, CD25, PD-1 and LAG-3 MFIs on CD8+ CAR-T cells were determined by flow cytometry.


Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl , or Lck - Cre ; Ptpn2fl / fl ; Lck + / - HER - 2 CAR - T were incubated with 24JK - HER - 2 or 24JK sarcoma proportion of CD8 + IFNγ + versus CD8 + IFNγ + TNF + CAR - T determined by flow cytometry .

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

Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 24JK-HER-2 or 24JK sarcoma cells and the proportion of CD8+IFNγ+ versus CD8+IFNγ+TNF+ CAR-T cells determined by flow cytometry.


Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl or Lck - Cre ; Ptpn2fl / fl ; Lck + / - HER - 2 CAR - T were incubated with 5 μM CTV - labelled ( CTVbright ) 24JK - HER - 2 0 . 5 μM CTV - labelled ( CTVdim ) 24JK sarcoma . Antigen - specific target lysis was monitored for depletion of CTVbright 24JK - HER - 2 by flow cytometry .

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

Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl or Lck-Cre;Ptpn2fl/fl;Lck+/- HER-2 CAR-T cells were incubated with 5 μM CTV-labelled (CTVbright) 24JK-HER-2 and 0.5 μM CTV-labelled (CTVdim) 24JK sarcoma cells. Antigen-specific target cell lysis was monitored for the depletion of CTVbright 24JK-HER-2 cells by flow cytometry.


HER - 2 - specific Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl Lck - Cre ; Ptpn2fl / fl ; Lck + / - CAR - T were incubated with plate - bound α - CD3 CD25 MFIs on CD8 + CD44hiCD62Llo versus CD8 + CD44hiCD62Lhi CAR - T determined by flow cytometry .

HER-2-specific Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl;Lck+/- CAR-T cells were incubated with plate-bound α-CD3 and CD25 MFIs on CD8+CD44hiCD62Llo versus CD8+CD44hiCD62Lhi CAR-T cells determined by flow cytometry.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD44hiCD62Lhi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl or Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Lck + / - splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer monitored for tumour growth .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl or Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth.


TILs isolated from HER - 2 - E0771 mammary tumour versus lymphocytes isolated from mammary fat pad were analysed for c ) CD11b + F4 / 80hiLy6ChiLy6Glo myeloid derived suppressor ( MDSC ) by flow cytometry .

TILs isolated from HER-2-E0771 mammary tumour versus lymphocytes isolated from the contralateral mammary fat pad were analysed for c) CD11b+ F4/80hiLy6ChiLy6Glo myeloid derived suppressor cells (MDSC) by flow cytometry.


TILs isolated from HER - 2 - E0771 mammary tumour versus lymphocytes isolated from mammary fat pad were analysed for CD4 + CD25 + FoxP3 + regulatory T ( Tregs ) by flow cytometry .

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

TILs isolated from HER-2-E0771 mammary tumour versus lymphocytes isolated from the contralateral mammary fat pad were analysed for CD4+CD25+FoxP3+ regulatory T cells (Tregs) by flow cytometry.


Tgfb Il10 mRNA in HER - 2 - E0771 tumours mammary fat pads were assessed by quantitative real time PCR .

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

Tgfb and Il10 mRNA levels in HER-2-E0771 tumours and mammary fat pads were assessed by quantitative real time PCR.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . f ) 13 or g ) six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD44hiCD62Lhi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with f ) IL - 2 ( 50 , 000 IU / day ) or g ) saline on days 0 - 4 after adoptive CAR - T transfer monitored for tumour growth .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. f) 13 or g) six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with f) IL-2 (50,000 IU/day) or g) saline on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified Lck - Cre ; Ptpn2fl / fl CD8 + CD44hiCD62Lhi central memory HER - 2 CAR - T . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer monitored for survival .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified Lck-Cre;Ptpn2fl/fl CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for survival.


HER - 2 - E0771 tumours at day 10 post adoptive CAR - T transfer or from HER - 2 - E0771 tumours that had re - emerged after being cleared by Lck - Cre ; Ptpn2fl / fl CD8 + HER - 2 CAR - T were analyzed for HER - 2 expression by immunohistochemistry . Scale bars : 200 μm ( full ) 70 μm ( zoom ) .

HER-2-E0771 tumours at day 10 post adoptive CAR-T cell transfer or from HER-2-E0771 tumours that had re-emerged after being cleared by Lck-Cre;Ptpn2fl/fl CD8+ HER-2 CAR-T cells were analyzed for HER-2 expression by immunohistochemistry. Scale bars: 200 μm (full size) and 70 μm (zoom).


Normal mammary tissue or HER - 2 - E0771 tumours at day 10 post - adoptive CAR - T transfer , or from those had re - emerged after being cleared by Lck - Cre ; Ptpn2fl / fl CD8 + HER - 2 CAR - T were analysed for HER - 2 expression by quantitative PCR .

Normal mammary tissue or HER-2-E0771 tumours at day 10 post-adoptive CAR-T cell transfer, or from those had re-emerged after being cleared by Lck-Cre;Ptpn2fl/fl CD8+ HER-2 CAR-T cells were analysed for HER-2 expression by quantitative PCR.


HER - 2 - E0771 versus HER - 2 negative E0771 breast cancer ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice 50 days after adoptive CAR - T transfer mice were monitored for tumour growth .

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

HER-2-E0771 versus HER-2 negative E0771 breast cancer cells (2x105) were injected into the contralateral fourth inguinal mammary fat pads of female HER-2 TG mice 50 days after adoptive CAR-T cell transfer and mice were monitored for tumour growth.


At day 28 number of CD45 + CD3 + CD8 + mCherry + CAR - T in HER - 2 - E0771 versus E0771 tumours dLNs were determined by flow cytometry .

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

At day 28 the number of CD45+CD3+CD8+ mCherry+ CAR-T cells in HER-2-E0771 versus E0771 tumours and dLNs were determined by flow cytometry.


Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl HER - 2 CAR - T isolated from spleens of HER - 2 TG mice at f ) 70

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

Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells isolated from the spleens of HER-2 TG mice at f) 70 days


Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl HER - 2 CAR - T isolated from spleens of HER - 2 TG mice at g ) 10 days post adoptive transfer were stained for CD8 , CD44 CD62L analysed by flow cytometry .

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

Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells isolated from the spleens of HER-2 TG mice at g) 10 days post adoptive transfer were stained for CD8, CD44 and CD62L and analysed by flow cytometry.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer . Lymphocytes were isolated from tumours dLN at day 10 post adoptive transfer mCherry + CD45 + CD3 + CD8 + CAR - T numbers were determined by flow cytometry .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. Lymphocytes were isolated from the tumours and dLN at day 10 post adoptive transfer and mCherry+CD45+CD3+CD8+ CAR-T cell numbers were determined by flow cytometry.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer . CTV - labelled CD8 + HER - 2 CAR - T were incubated with 24JK - HER - 2 or 24JK sarcoma CTV dilution assessed by flow cytometry to monitor proliferation .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. CTV-labelled CD8+ HER-2 CAR-T cells were incubated with 24JK-HER-2 or 24JK sarcoma cells and CTV dilution assessed by flow cytometry to monitor proliferation.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer . CXCR3 MFIs on CD8 + CD44hiCD62Lhi versus CD8 + CD44hiCD62Llo CAR - T ( c ) CXCR5 , CCR7 , CCR5 MFIs on CD8 + CD44hiCD62Lhi CAR - T ( d ) were determined by flow cytometry .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. CXCR3 MFIs on CD8+CD44hiCD62Lhi versus CD8+CD44hiCD62Llo CAR-T cells (c) and CXCR5, CCR7, CCR5 MFIs on CD8+CD44hiCD62Lhi CAR-T cells (d) were determined by flow cytometry.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer . Cxcl9 Cxcl10 mRNA in HER - 2 - E0771 tumours were assessed by quantitative real time PCR .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. Cxcl9 and Cxcl10 mRNA levels in HER-2-E0771 tumours were assessed by quantitative real time PCR.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer . Lymphocytes were isolated from HER - 2 - E0771 tumours at day 3 post adoptive CAR - T transfer mCherry + CD45 + CD3 + CD8 + CAR - T numbers were determined by flow cytometry .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. Lymphocytes were isolated from HER-2-E0771 tumours at day 3 post adoptive CAR-T cell transfer and mCherry+CD45+CD3+CD8+ CAR-T cell numbers were determined by flow cytometry.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer . Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Lck + / - or Lck - Cre ; Ptpn2fl / fl ; Stat5fl / + HER - 2 CAR - T were incubated with plate - bound α - CD3 CXCR3 MFIs on CD8 + CD44hiCD62Llo CAR - T determined by flow cytometry .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- or Lck-Cre;Ptpn2fl/fl;Stat5fl/+ HER-2 CAR-T cells were incubated with plate-bound α-CD3 and CXCR3 MFIs on CD8+CD44hiCD62Llo CAR-T cells determined by flow cytometry.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer . Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl ; Stat5fl / + HER - 2 CAR - T were incubated with plate - bound α - CD3 stimulated with murine recombinant IL - 2 IL - 15 for indicated time points . Intracellular p ( Y694 ) - STAT - 5 MFIs in CD8 + CD44hiCD62Llo were determined by flow cytometry .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer. Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Stat5fl/+ HER-2 CAR-T cells were incubated with plate-bound α-CD3 and stimulated with murine recombinant IL-2 and IL-15 for the indicated time points. Intracellular p(Y694)-STAT-5 MFIs in CD8+CD44hiCD62Llo were determined by flow cytometry.


HER - 2 - E0771 mammary tumours ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl ; Stat5fl / + or Lck - Cre ; Ptpn2fl / fl ; Lck + / - splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer monitored for tumour growth . Lymphocytes were isolated from tumours on day 3 post adoptive transfer mCherry + CD45 + CD8 + CAR - T numbers were determined by flow cytometry .

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

HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. Lymphocytes were isolated from the tumours on day 3 post adoptive transfer and mCherry+CD45+ CD8+ CAR-T cell numbers were determined by flow cytometry.


HER - 2 - E0771 mammary tumours ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl ; Stat5fl / + or Lck - Cre ; Ptpn2fl / fl ; Lck + / - splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer monitored for tumour growth .

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

HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth.


HER - 2 - E0771 mammary tumours ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 6x106 FACS - purified CD8 + CD62LhiCD44hi central memory HER - 2 CAR - T generated from Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl , Lck - Cre ; Ptpn2fl / fl ; Stat5fl / + or Lck - Cre ; Ptpn2fl / fl ; Lck + / - splenocytes . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer monitored for tumour growth . Lymphocytes were isolated from tumours on day 16 post adoptive transfer mCherry + CD45 + CD8 + CAR - T numbers were determined by flow cytometry . In ( c ) TILs were stained for intracellular IFNγ TNF after PMA / Ionomycin treatment .

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

HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. Lymphocytes were isolated from the tumours on day 16 post adoptive transfer and mCherry+CD45+ CD8+ CAR-T cell numbers were determined by flow cytometry. In (c) TILs were stained for intracellular IFNγ and TNF after PMA/Ionomycin treatment.


HER - 2 - E0771 generated to inducibly overexpress PTPN2 in response to doxycycline ( E0771 - HER - 2 - PTPN2hi ) were pre - incubated ( 24 h ) with vehicle or doxycycline ( DOX ) subsequently stimulated with IFNγ for indicated times . STAT - 1 Y701 phosphorylation ( p - STAT - 1 ) PTPN2 were assessed by immunoblotting .

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 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

HER-2-E0771 cells generated to inducibly overexpress PTPN2 in response to doxycycline (E0771-HER-2-PTPN2hi) were pre-incubated (24 h) with vehicle or doxycycline (DOX) subsequently stimulated with IFNγ for the indicated times. STAT-1 Y701 phosphorylation (p-STAT-1) and PTPN2 levels were assessed by immunoblotting.


Cxcl9 Cxcl10 mRNA in vehicle versus DOX - treated IFNγ - stimulated HER - 2 - E0771 were assessed by quantitative real time PCR .

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

Cxcl9 and Cxcl10 mRNA levels in vehicle versus DOX-treated and IFNγ-stimulated HER-2-E0771 cells were assessed by quantitative real time PCR.


E0771 - HER - 2 - PTPN2hi mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation ( 4 Gy ) on day 6 adoptive transfer of 6x106 FACS - purified central memory Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl HER - 2 CAR - T . Mice were then injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 post adoptive CAR - T transfer In ( g ) CD45 + CD8 + mCherry + TILs were quantified by flow cytometry at day 4 post adoptive transfer .

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

E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. Mice were then injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer In (g) CD45+CD8+mCherry+ TILs were quantified by flow cytometry at day 4 post adoptive transfer.


E0771 - HER - 2 - PTPN2hi mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation ( 4 Gy ) on day 6 adoptive transfer of 6x106 FACS - purified central memory Ptpn2fl / fl versus Lck - Cre ; Ptpn2fl / fl HER - 2 CAR - T . Mice were then injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 post adoptive CAR - T transfer ( h ) tumour growth was monitored .

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

E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. Mice were then injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and (h) tumour growth was monitored.


CD8 + LY CAR - T generated from human PBMCs were treated with PTPN2 - inhibitor ( + ) or vehicle ( - ) followed by incubation with plate - bound α - LY , LY - negative MDA - MB - 435 LY - expressing OVCAR - 3 . proportion of IFNγ + versus IFNγ + TNF + CD8 + CAR - T was determined by flow cytometry .

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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

CD8+ LY CAR-T cells generated from human PBMCs were treated with PTPN2-inhibitor (+) or vehicle (-) followed by incubation with plate-bound α-LY, LY-negative MDA-MB-435 cells and LY-expressing OVCAR-3 cells. The proportion of IFNγ+ versus IFNγ+TNF+ CD8+CAR-T cells was determined by flow cytometry.


HER - 2 CAR - T generated from C57BL / 6 Lck - Cre ; Ptpn2fl / fl splenocytes were transfected with GFP versus Ptpn2 siSTABLE ™ FITC - conjugated siRNAs intracellular PTPN2 were determined by flow cytometry .

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

HER-2 CAR-T cells generated from C57BL/6 and Lck-Cre;Ptpn2fl/fl splenocytes were transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs and intracellular PTPN2 levels were determined by flow cytometry.


c - d ) HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 10x106 HER - 2 CAR - T generated from C57BL / 6 splenocytes transfected with GFP versus Ptpn2 siSTABLE ™ FITC - conjugated siRNAs two days before adoptive CAR - T transfer . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer tumour growth was monitored .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-d) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 HER-2 CAR-T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs two days before adoptive CAR-T cell transfer. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and tumour growth was monitored.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 10x106 HER - 2 CAR - T generated from C57BL / 6 splenocytes transfected with GFP versus Ptpn2 siSTABLE ™ FITC - conjugated siRNAs two days before adoptive CAR - T transfer . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 after adoptive CAR - T transfer tumour growth was monitored . CD45 + CD3 + CD8 + mCherry + CAR - T numbers were determined in HER - 2 - E0771 positive tumours spleens by flow cytometry 21 days post adoptive transfer .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 HER-2 CAR-T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs two days before adoptive CAR-T cell transfer. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and tumour growth was monitored. CD45+CD3+CD8+ mCherry+ CAR-T cells numbers were determined in HER-2-E0771 positive tumours and spleens by flow cytometry 21 days post adoptive transfer.


HER - 2 CAR - T generated from C57BL / 6 Lck - Cre ; Ptpn2fl / fl splenocytes were transfected with Cas9 control or Ptpn2 sgRNAs using Lonza 4D - Nucleofector after two days intracellular PTPN2 determined by flow cytometry .

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

HER-2 CAR-T cells generated from C57BL/6 and Lck-Cre;Ptpn2fl/fl splenocytes were transfected with Cas9 and control or Ptpn2 sgRNAs using the Lonza 4D-Nucleofector and after two days intracellular PTPN2 levels determined by flow cytometry.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 10x106 control HER - 2 CAR - T or those in which PTPN2 had been deleted by CRISPR RNP . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 post adoptive CAR - T transfer tumour growth monitored .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 control HER-2 CAR-T cells or those in which PTPN2 had been deleted by CRISPR RNP. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and tumour growth monitored.


HER - 2 - E0771 mammary tumour ( 2x105 ) were injected into fourth inguinal mammary fat pads of HER - 2 TG mice . Six days after tumour injection HER - 2 TG mice received total irradiation ( 4 Gy ) followed by adoptive transfer of 10x106 control HER - 2 CAR - T or those in which PTPN2 had been deleted by CRISPR RNP . Mice were injected with IL - 2 ( 50 , 000 IU / day ) on days 0 - 4 post adoptive CAR - T transfer tumour growth monitored . g ) CD45 + CD8 + mCherry + CAR - T numbers were determined in HER - 2 - E0771 tumours by flow cytometry 19 days post adoptive transfer .

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

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 control HER-2 CAR-T cells or those in which PTPN2 had been deleted by CRISPR RNP. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and tumour growth monitored. g) CD45+ CD8+ mCherry+ CAR-T cells numbers were determined in HER-2-E0771 tumours by flow cytometry 19 days post adoptive transfer.


( B ) Identification of wild - type ( WT ) , heterozygous ( HZ ) , knockout ( KO ) mice by PCR agarose gel electrophoresis .

O O O O 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) Identification of wild-type (WT), heterozygous (HZ), and knockout (KO) mice by PCR and agarose gel electrophoresis.


( C ) Immunoblotting ( IB ) of ENKD1 α - tubulin in wild - type Enkd1 knockout mice .

(C) Immunoblotting (IB) of ENKD1 and α-tubulin in wild-type and Enkd1 knockout mice.


( D - F ) Immunofluorescence ( D ) of density of cilia ( E , n = 20 fields from three mice ) ciliary ( F , n = 100 cilia from three mice ) in mouse retinas stained with antibodies against acetylated α - tubulin ( Ace - α - tubulin ) γ - tubulin DAPI . 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 O O O O O O O O O O O O O O O O O O

(D-F) Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α-tubulin (Ace-α-tubulin) and γ-tubulin and DAPI. Scale bar, 2 µm.


( G - H ) ERG recording ( G ) a - b wave amplitudes ( H , n = 5 mice ) show difference in ERG a - b wave amplitude between wild - type Enkd1 knockout mice .

O O O O O O O O O O O O O O O O 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-H) ERG recording images (G) and a-b wave amplitudes (H, n = 5 mice) show the difference in the ERG a-b wave amplitude between wild-type and Enkd1 knockout mice.


( I - J ) VEP recording ( I ) N2 - P2 wave amplitudes ( J , n = 5 mice ) show difference in VEP N2 - P2 wave amplitude between wild - type Enkd1 knockout mice .

(I-J) VEP recording images (I) and N2-P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2-P2 wave amplitude between wild-type and Enkd1 knockout mice.


( K - M ) Immunofluorescence ( K ) of of ciliated ( L , n = 3 mice ) ciliary ( M , n = 100 cilia from three mice ) in mouse kidneys stained with antibody against acetylated α - tubulin DAPI . To quantify of ciliated ( L ) , > 200 from 12 were analyzed for each mouse . Scale bar , 3 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 O O O O O O O O O O O O O O O O O O O O O O

(K-M) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (L), >200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm.


( N - Q ) ( N ) of of flagellated sperm ( O , n = 9 mice ) , flagellar ( P , n = 100 sperm from nine mice ) , of sperm with normal motility among flagellated sperm ( Q , n = 9 mice ) for wild - type Enkd1 knockout mice . To quantify of flagellated sperm ( O ) , > 200 sperm were analyzed for each mouse . To quantify of sperm with normal motility among flagellated sperm ( Q ) , > 200 flagellated sperm were analyzed for each mouse . In panel N , arrows indicate two spermatozoa without tails . Scale bar , 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(N-Q) Images (N) and quantification of the percentage of flagellated sperm (O, n = 9 mice), flagellar length (P, n = 100 sperm from nine mice), and the percentage of sperm with normal motility among flagellated sperm (Q, n = 9 mice) for wild-type and Enkd1 knockout mice. To quantify the percentage of flagellated sperm (O), >200 sperm were analyzed for each mouse. To quantify the percentage of sperm with normal motility among flagellated sperm (Q), >200 flagellated sperm were analyzed for each mouse. In panel N, the arrows indicate two spermatozoa without tails. Scale bar, 50 µm.