A - C AID expression was analyzed in pancreas explants from C57BL / 6 mice . Samples were treated as indicated with 50 ng / ml TNF - α . ( C ) qRT - PCR of AID expression in pancreatic explants from wild - type mice ( n = 3 ) . * P = 0 . 0242 .

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A-C AID expression was analyzed in pancreas explants from C57BL/6 mice. Samples were treated as indicated with 50 ng/ml TNF-α. (C) qRT-PCR analysis of AID expression in pancreatic explants from wild-type mice (n = 3). *P = 0.0242.


D AID − / − or AID + / − mice were treated with 3 % DSS for 10 cycles , colonic sections were analyzed by histology inspection after H / E . Graphs represent mean frequency values of adenoma adenocarcinoma lesions of five independent experiments . n = 28 ( AID − / − ) ; 35 ( ) ; 23 ( AID + / − ) ; 25 ( ) . P - value : : 0 . 8 ; : 0 . 246 .

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D AID−/− or AID+/− mice were treated with 3% DSS for 10 cycles, and colonic sections were analyzed by histology inspection after H/E staining. Graphs represent mean frequency values of adenoma and adenocarcinoma lesions of five independent experiments. n = 28 (AID−/− males); 35 (females); 23 (AID+/− males); 25 (females). P-value: male: 0.8; female: 0.246.


GFP immunofluorescence in colonic pancreatic tissue from R26AID + / KIVillinCRE + / TG R26AID + / KIp48CRE + / KI mice . Scale bar : 50 μm .

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GFP immunofluorescence in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. Scale bar: 50 μm.


qRT - PCR of AID expression in colonic pancreatic tissue from R26AID + / KIVillinCRE + / TG R26AID + / KIp48CRE + / KI mice . n = 5 ( R26AID + / + VillinCRE + / TG ) ; 4 ( R26AID + / KIVillinCRE + / TG ) ; 2 ( R26AID + / + p48CRE + / KI ) ; 2 ( R26AID + / KIp48CRE + / KI ) . LPS + IL4 - stimulated B are shown as a positive control ( n = 2 ) . Bars show mean values ± normalized to LPS + IL4 - treated B .

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qRT-PCR analysis of AID expression in colonic and pancreatic tissue from R26AID+/KIVillinCRE+/TG and R26AID+/KIp48CRE+/KI mice. n = 5 (R26AID+/+ VillinCRE+/TG); 4 (R26AID+/KIVillinCRE+/TG); 2 (R26AID+/+p48CRE+/KI); 2 (R26AID+/KIp48CRE+/KI). LPS+IL4-stimulated B cells are shown as a positive control (n = 2). Bars show mean values ± SEM normalized to LPS+IL4-treated B cells.


Kaplan - Meier survival curves for R26AID + / KIVillinCRE + / TG ( left ) ( n = 47 ( R26AID + / + VillinCRE + / TG ) ; 38 ( R26AID + / KIVillinCRE + / TG ) ) R26AID + / KI p48CRE + / KI mice ( right ) ( n = 39 ( R26AID + / + p48CRE + / KI ) ; 23 ( R26AID + / KIp48CRE + / KI ) ) .

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Kaplan-Meier survival curves for R26AID+/KIVillinCRE+/TG (left) (n = 47 (R26AID+/+ VillinCRE+/TG); 38 (R26AID+/KIVillinCRE+/TG)) and R26AID+/KI p48CRE +/KI mice (right) (n = 39 (R26AID+/+p48CRE+/KI); 23 (R26AID+/KIp48CRE+/KI)).


A , B of AID mutagenic activity in Elastase1 ( A ) Elastase2a ( B ) by next - generation sequencing . Pancreatic was isolated from pools of R26AID + / KIp48CRE + / KI control R26AID + / + p48CRE + / KI 20 - week - old mice , then PCR - amplified with specific primers sequenced as previously described ( Perez - Duran et al , 2012 ) . Graphs show cytosine mutation frequency overall ( total ) or at AGCT hotspots . n = 2 . * P - value : Elastase1 : 0 . 0382 ; Elastase2a : 0 . 009 .

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A, B Analysis of AID mutagenic activity in Elastase1 (A) and Elastase2a (B) by next-generation sequencing. Pancreatic DNA was isolated from pools of R26AID+/KIp48CRE+/KI and control R26AID+/+p48CRE+/KI 20-week-old mice, and then PCR-amplified with specific primers and sequenced as previously described (Perez-Duran et al, 2012). Graphs show cytosine mutation frequency overall (total) or at AGCT hotspots. n = 2. *P-value: Elastase1: 0.0382; Elastase2a: 0.009.


C HTM - mediated of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days . Red lines show mean values . Results of two independent experiments are shown . * * * * P < 0 . 0001 . D Representative of γH2AX in pancreas explants from R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) ( 40 × magnification ) . arrow points a representative positive .

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C HTM-mediated quantification of γH2AX intensities per nuclei in pancreas explants cultured in vitro for 6 days. Red lines show mean values. Results of two independent experiments are shown. ****P < 0.0001.D Representative images of γH2AX staining in pancreas explants from R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom) (40× magnification). White arrow points a representative positive cell.


Representative of Ki67 in pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI ( top ) or R26AID + / KIp48CRE + / KI mice ( bottom ) . Detail is shown on right . Scale bar : 100 μm . Graph shows of Ki67 - positive epithelial per mm2 of tissue ( n = 8 ) . * P = 0 . 0266 .

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Representative images of Ki67 staining in pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom). Detail is shown on the right. Scale bar: 100 μm. Graph shows quantification of Ki67-positive epithelial cells per mm2 of tissue (n = 8). *P = 0.0266.


Representative immunofluorescence of 20 - week - old R26AID + / KIp48CRE + / KI mice : blue , DAPI ; red , Ki67 ; green , CK8 . Scale bar : 50 μm . Detail is shown on bottom .

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Representative immunofluorescence staining of 20-week-old R26AID+/KIp48CRE+/KI mice: blue, DAPI; red, Ki67; green, CK8. Scale bar: 50 μm. Detail is shown on the bottom.


Quantitative FACS of RAE expression in pancreatic explants from R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Left : Graph shows mean . Each dot represents an mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 8 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 077 . Right : Representative FACS for RAE in explants from R26AID + / + p48CRE + / KI ( red ) R26AID + / KIp48CRE + / KI mice ( black ) .

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Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Graph shows mean fluorescence intensity. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 8 (R26AID+/KIp48CRE+/KI). P = 0.077. Right: Representative FACS staining for RAE in explants from R26AID+/+p48CRE+/KI (red) and R26AID+/KIp48CRE+/KI mice (black).


of RAE expression by ddPCR in aged ( 75 - week - old ) mice . Data are presented as of positive drops normalized to mean control value . Each point represents an mouse shows mean amplification from two independent experiments n = 7 ( R26AID + / + p48CRE + / KI ) ; 9 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 012 .

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Analysis of RAE expression by ddPCR in aged (75-week-old) mice. Data are presented as the percentage of positive drops normalized to the mean control value. Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 (R26AID+/+p48CRE+/KI); 9 (R26AID+/KIp48CRE+/KI). *P = 0.012.


of killing activity . explants of pancreatic from R26AID + / + p48CRE + / KI or R26AID + / KIp48CRE + / KI mice were cultured with NK , killing activity was assessed as described in Materials Methods ( n = 4 ) . P = 0 . 19 .

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Analysis of killing activity. Primary explants of pancreatic cells from R26AID+/+p48CRE+/KI or R26AID+/KIp48CRE+/KI mice were cultured with primary NK cells, and killing activity was assessed as described in Materials and Methods (n = 4). P = 0.19.


Hematoxylin - eosin ( HE ) of pancreas from aged ( 75 - week - old ) R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Left : Representative HE showing an immune infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 200 μm . Right : of number of foci per mm2 of tissue . n = 13 ( R26AID + / + p48CRE + / KI ) ; 21 ( R26AID + / KIp48CRE + / KI ) . * * P = 0 . 0098 .

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Hematoxylin-eosin (HE) staining of pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative HE staining showing an immune infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 200 μm. Right: Quantification of number of foci per mm2 of tissue. n = 13 (R26AID+/+p48CRE+/KI); 21 (R26AID+/KIp48CRE+/KI). **P = 0.0098.


CD3 immunohistochemistry of pancreas from 75 - week - old R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Left : Representative image of a CD3 infiltrate in a R26AID + / KIp48CRE + / KI mouse . Scale bar : 100 μm . Right : of number of CD3 - positive per mm2 of tissue . n = 12 ( R26AID + / + p48CRE + / KI ) ; 13 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0149 .

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CD3 immunohistochemistry of pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Representative image of a CD3 infiltrate in a R26AID+/KIp48CRE+/KI mouse. Scale bar: 100 μm. Right: Quantification of the number of CD3-positive cells per mm2 of tissue. n = 12 (R26AID+/+p48CRE+/KI); 13 (R26AID+/KIp48CRE+/KI). *P = 0.0149.


Representative immunofluorescence of CD3 CD8 in pancreas from 20 - week - old R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice . Scale bar : 20 μm .

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Representative immunofluorescence staining of CD3 and CD8 in pancreas from 20-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Scale bar: 20 μm.


TNF - α expression . Total was isolated from pancreas of aged ( 75 - week - old ) R26AID + / KIp48CRE + / KI control mice , TNF - α expression was quantified by qRT - PCR . Each dot represents an mouse . n = 8 ( R26AID + / + p48CRE + / KI ) ; 15 ( R26AID + / KIp48CRE + / KI ) . * P = 0 . 0392 .

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TNF-α expression. Total RNA was isolated from pancreas of aged (75-week-old) R26AID+/KIp48CRE+/KI and control mice, and TNF-α expression was quantified by qRT-PCR. Each dot represents an individual mouse. n = 8 (R26AID+/+p48CRE+/KI); 15 (R26AID+/KIp48CRE+/KI). *P = 0.0392.


detection . Pancreas from 75 - week - old R26AID + / + p48CRE + / KI R26AID + / KIp48CRE + / KI mice were stained with anti - caspase - 3 . Left : Representative from an R26AID + / + p48CRE + / KI mouse ( top ) an R26AID + / KIp48CRE + / KI mouse ( bottom ) . Scale bar : 50 μm . Right : of number of per mm2 of tissue . Each dot represents an mouse . n = 9 ( R26AID + / + p48CRE + / KI ) ; 11 ( R26AID + / KIp48CRE + / KI ) . P = 0 . 0545 .

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Cell death detection. Pancreas from 75-week-old R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice were stained with anti-caspase-3. Left: Representative staining from an R26AID+/+p48CRE+/KI mouse (top) and an R26AID+/KIp48CRE+/KI mouse (bottom). Scale bar: 50 μm. Right: Quantification of number of cells per mm2 of tissue. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 11 (R26AID+/KIp48CRE+/KI). P = 0.0545.


( E ) of different manifesting after interference in comparison to control : MN2a axons projecting to MHE muscle , representing wild - type ( wt ) innervation pattern ( magenta ) ; MN2a axons projecting to other than MHE termed abnormal innervation ( grey ) ; abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects ( grey ) . Note , each genetic experiment was performed in parallel to an adequate control experiment using same driver crossed to a that controls expression of either UAS - RFP or UAS - GFPRNAi . Each experiment was performed in triplicates , innervation rates were calculated from n = 56 for elav > RFP , n = 21 for elav > DfdRNAi , n = 10 for elav > hthRNAi n = 7 for elav > mirrRNAi . In case of Dfd knock - down , two different driver lines were used , pan - neural elav - GAL4 motoneuron - specific OK6 - GAL4 drivers , respectively . Both result in similar , highlighting that elav - GAL4 driven effects are specific to MNs . p - values between two genetic conditions were calculated by a two - sided Fisher . * * * : p < 0 . 005 .

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(E) Quantification of different phenotypes manifesting after RNA interference in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern (magenta); MN2a axons projecting to muscles other than the MHE termed abnormal innervation (light grey); abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects (dark grey). Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. Each experiment was performed in triplicates, innervation rates were calculated from n=56 for elav>RFP, n=21 for elav>DfdRNAi, n=10 for elav>hthRNAi and n=7 for elav>mirrRNAi. In the case of Dfd knock-down, two different driver lines were used, the pan-neural elav-GAL4 and the motoneuron-specific OK6-GAL4 drivers, respectively. Both result in similar phenotypes, highlighting that the elav-GAL4 driven effects are specific to MNs. p-values between two genetic conditions were calculated by a two-sided Fisher test. ***: p < 0.005.


( B ) Representative confocal of stage 17 embryonic heads highlighting expression of Dfd ( purple ) , Myosin in ( blue ) FasII in axonal projections ( green ) in control ( elav > RFP ) mis - expressing Hox TFs Lab , Dfd Scr by means of elav - GAL4 driver . A zoom on projections ( FasII ) of MN2a MN3 to MHE MHD 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 mis - expression conditions .

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(B) 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 animals mis-expressing the Hox TFs Lab, Dfd and Scr by means of the elav-GAL4 driver. A zoom on the projections (FasII staining) of MN2a and MN3 to the MHE and MHD 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 mis-expression conditions.


( 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 .

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(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 ) .

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(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 .

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(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 .

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

(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 .

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

(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 .

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

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

(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 .

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

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

(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 B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 .

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

(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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(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 .

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(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 .

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(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 B-MEASURED_VAR 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

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

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

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

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

(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 .

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(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 .

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(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 B-CONTROLLED_VAR 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

(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 B-CONTROLLED_VAR 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

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

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

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

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

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

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 .

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 B-CONTROLLED_VAR O O O O

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

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

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 .

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 .

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

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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 .

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

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 .

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

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

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 .

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

B-MEASURED_VAR I-MEASURED_VAR 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

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 B-CONTROLLED_VAR 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

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 B-CONTROLLED_VAR O O O O 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 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 ) .

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

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 .

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

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 .

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

B-CONTROLLED_VAR 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

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 .

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

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

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.