( 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 .

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

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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 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 .

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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 ) the numbers of activated tumour infiltrating lymphocytes ( TILs ) were determined .

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


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

O 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 cells isolated from tumour bearing C57BL / 6 mice and the proportion of IFNγ + T cells 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 B-MEASURED_VAR 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 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

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

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

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

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

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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 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 .

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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 and donor - derived ( Ly5 . 2 + vα2 + ) tissue resident memory T cells ( TRMs ) determined by flow cytometry .

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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 and 23 .

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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 and Ly5 . 2 + ; OT - 1 ; Lck - Cre ; Ptpn2fl / fl donor T cell 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 the contralateral mammary fat pad were analysed for c ) CD11b + F4 / 80hiLy6ChiLy6Glo myeloid derived suppressor cells ( MDSC ) by flow cytometry .

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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 the contralateral mammary fat pad were analysed for CD4 + CD25 + FoxP3 + regulatory T cells ( Tregs ) by flow cytometry .

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

B-MEASURED_VAR O B-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

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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

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

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 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 .

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

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

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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 cells isolated from the 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 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 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 .

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

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

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 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 .

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

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

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

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

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 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 .

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

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

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 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 .

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

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 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 .

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

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

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

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

B-MEASURED_VAR O B-MEASURED_VAR 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

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 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 .

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

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

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 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 .

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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 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 .

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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 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 .

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

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 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 .

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

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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 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 .

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

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 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 .

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

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 ) , and knockout ( KO ) mice by PCR and agarose gel electrophoresis .

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


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

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


( 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 .

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(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 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 .

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(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 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 .

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O 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 ) 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 .

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

(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.


( R , S ) Scanning electron microscopy images of cilia ( R ) and quantification of the percentage of ciliated cells ( S , n = 9 mice ) in the mouse tracheal epithelium . To quantify the percentage of ciliated cells ( S ) , > 200 cells from six fields were analyzed for each mouse . Scale bar , 5 µm .

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(R, S) Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), >200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm.


( A ) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum - free medium for 48 h .

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(A) Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum-free medium for 48 h.


( B - D ) Immunofluorescence images ( B ) and quantification of the percentage of ciliated cells ( C , n = 3 independent experiments ) and ciliary length ( D , n = 70 cilia from three independent experiments ) for MEFs cultured in serum - free medium and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( C ) , > 100 cells were analyzed for each experiment . Scale bar , 5 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 B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O

(B-D) Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), >100 cells were analyzed for each experiment. Scale bar, 5 µm.


( E ) Immunoblot analysis of ENKD1 and β - actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum - free medium for 48 h .

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(E) Immunoblot analysis of ENKD1 and β-actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum-free medium for 48 h.


Immunofluorescence images ( F ) and quantification of the percentage of ciliated cells ( G , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against acetylated α - tubulin and γ - tubulin and DAPI . To quantify the percentage of ciliated cells ( G ) , > 150 cells were analyzed for each experiment . Scale bar , 5 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), >150 cells were analyzed for each experiment. Scale bar, 5 µm.


quantification of ciliary length ( H , n = 100 cilia from three independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free

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quantification of ciliary length (H, n = 100 cilia from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium


( I , J ) Immunofluorescence images ( I ) and quantification of the percentage of ciliated cells ( J , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against Arl13b and Centrin and DAPI . To quantify the percentage of ciliated cells ( J ) , > 80 cells were analyzed for each experiment . 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I, J) Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), >80 cells were analyzed for each experiment. Scale bar, 2 µm.


( K , L ) Immunofluorescence images ( K ) and quantification of the percentage of ciliated cells ( L , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP , GFP - ENKD1 , GFP - ENKD1 - N , GFP - ENKD1 - M , or GFP - ENKD1 - C , cultured in serum - free medium , and stained with the antibody against acetylated α - tubulin and DAPI . The siRNA - resistant forms of ENKD1 were used for these rescue experiments . To quantify the percentage of ciliated cells ( L ) , > 120 cells were analyzed for each experiment . 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K, L) Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP-ENKD1, GFP-ENKD1-N, GFP-ENKD1-M, or GFP-ENKD1-C, cultured in serum-free medium, and stained with the antibody against acetylated α-tubulin and DAPI. The siRNA-resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), >120 cells were analyzed for each experiment. Scale bar, 3 µm.


( A ) Immunofluorescence images of RPE1 cells cultured in normal serum medium , and stained with antibodies against ENKD1 and Centrin and DAPI . Scale bar , 1 µm .

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(A) Immunofluorescence images of RPE1 cells cultured in normal serum medium, and stained with antibodies against ENKD1 and Centrin and DAPI. Scale bar, 1 µm.


( B ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 1 µm .

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(B) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and CEP164 in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 1 µm.


( C ) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum - starved RPE1 cells . The arrow marks the centriole subjected to the line scan . Scale bar , 2 µm .

(C) Immunofluorescence images and fluorescence intensity profiles of ENKD1 and Ninein in serum-starved RPE1 cells. The arrow marks the centriole subjected to the line scan. Scale bar, 2 µm.


( D - F ) 3D - SIM super - resolution images ( D ) , quantification of the outer toroid diameters ( E , n = 20 cells from three independent experiments ) , and a model illustrating the relative positions of γ - tubulin , Ninein , ENKD1 , CEP164 , CEP912 , and Centrin ( F ) within a centriole . RPE1 cells were serum - starved and stained with antibodies against the indicated proteins . Scale bar , 1 µm .

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(D-F) 3D-SIM super-resolution images (D), quantification of the outer toroid diameters (E, n = 20 cells from three independent experiments), and a model illustrating the relative positions of γ-tubulin, Ninein, ENKD1, CEP164, CEP912, and Centrin (F) within a centriole. RPE1 cells were serum-starved and stained with antibodies against the indicated proteins. Scale bar, 1 µm.


( G ) Immunofluorescence images of RPE1 cells cultured in normal serum or serum - free medium , and stained with antibodies against ENKD1 and γ - tubulin and DAPI . Scale bar , 1 µm .

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(G) Immunofluorescence images of RPE1 cells cultured in normal serum or serum-free medium, and stained with antibodies against ENKD1 and γ-tubulin and DAPI. Scale bar, 1 µm.


( H ) Immunofluorescence images of RPE1 cells cultured in serum - free medium , and stained with antibodies against ENKD1 and acetylated α - tubulin and DAPI . Scale bar , 1 µm .

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(H) Immunofluorescence images of RPE1 cells cultured in serum-free medium, and stained with antibodies against ENKD1 and acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( I ) Immunofluorescence images of RPE1 cells transfected with GFP - ENKD1 or GFP vector , cultured in a serum - starved condition , and stained with the antibody against acetylated α - tubulin and DAPI . Scale bar , 1 µm .

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(I) Immunofluorescence images of RPE1 cells transfected with GFP-ENKD1 or GFP vector, cultured in a serum-starved condition, and stained with the antibody against acetylated α-tubulin and DAPI. Scale bar, 1 µm.


( K ) 3D - SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum - free medium ( 0 , 12 , 24 , or 48 h of serum starvation ) , and stained with antibodies against ENKD1 and acetylated α - tubulin . Scale bar , 1 µm .

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(K) 3D-SIM images of ENKD1 localization at the centrosome in RPE1 cells cultured in serum-free medium (0, 12, 24, or 48 h of serum starvation), and stained with antibodies against ENKD1 and acetylated α-tubulin. Scale bar, 1 µm.


Immunofluorescence images ( B ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI .

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Immunofluorescence images (B) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI.


quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot ( C ) , > 120 cells were analyzed for each experiment .

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quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot (C), >120 cells were analyzed for each experiment.


( D , E ) Immunofluorescence images ( D ) and quantification of the percentage of cells with one CEP97 dot ( E , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CEP97 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CEP97 dot ( E ) , > 150 cells were analyzed for each experiment . Scale bar , 1 µm .

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(D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm.


( F - H ) Immunoblot analysis ( F ) , immunofluorescence images ( G ) , and quantification of the percentage of cells with one CP110 dot ( H , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium for 120 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 80 cells were analyzed for each experiment . Scale bar , 1 µm .

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(F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.


( A - C ) Immunoblot analysis ( A ) , immunofluorescence images ( B ) and quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in serum - free medium for 48 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 180 cells were analyzed for each experiment . Scale bar , 1 µm .

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(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm.


( D ) Immunoblot analysis of CP110 and β - actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum - free medium for 48 h .

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(D) Immunoblot analysis of CP110 and β-actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum-free medium for 48 h.


( E , F ) Immunofluorescence images ( E ) and quantification of the percentage of ciliated cells ( F , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in a serum - starved condition , and stained with antibodies against γ - tubulin and acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( F ) , > 120 cells were analyzed for each experiment . Scale bar , 10 µm .

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(E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.


( G , H ) Immunoprecipitation and immunoblotting ( G ) and quantification ( H , n = 3 mice ) showing the CP110 - CEP97 interaction in the retinal tissues of wild - type and Enkd1 knockout mice . The intensity of each CEP97 band was normalized to that of the β - actin band .

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(G, H) Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110-CEP97 interaction in the retinal tissues of wild-type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β-actin band.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the CP110 - CEP97 interaction in serum - starved RPE1 cells transfected with control or ENKD1 siRNAs . The intensity of each CEP97 band was normalized to that of the β - actin band .

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(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110-CEP97 interaction in serum-starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β-actin band.


( A ) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues . Immunoprecipitation was performed with the anti - ENKD1 ( upper panel ) or anti - CP110 ( lower panel ) antibody or the corresponding control IgG .

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(A) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti-ENKD1 (upper panel) or anti-CP110 (lower panel) antibody or the corresponding control IgG.


( B - D ) Immunoprecipitation and immunoblotting showing the interaction of GFP - ENKD1 with Flag - CP110 ( B and upper panel of D ) or HA - CP110 ( C and bottom panel of D ) in HEK293T cells . Immunoprecipitation was performed with antibodies against GFP ( B and C ) , Flag ( upper panel of D ) , or HA ( bottom panel of D ) .

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(B-D) Immunoprecipitation and immunoblotting showing the interaction of GFP-ENKD1 with Flag-CP110 (B and upper panel of D) or HA-CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D).


( E ) Identification of the domains of ENKD1 mediating its interaction with CP110 .

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(E) Identification of the domains of ENKD1 mediating its interaction with CP110.


( F ) Identification of the domains of CP110 mediating its interaction with ENKD1 .

(F) Identification of the domains of CP110 mediating its interaction with ENKD1.


( H ) GST pull - down showing the interaction of purified GST - CP110 - N with purified His - ENKD1 - M .

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(H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M.


( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the interaction of HA - CP110 with Flag - CEP97 in HEK293T cells transfected with the indicated plasmids . The intensity of each Flag - CEP97 band was normalized to that of the β - actin band .

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(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA-CP110 with Flag-CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag-CEP97 band was normalized to that of the β-actin band.


A . Evaluation of siRNA - mediated depletion . U2OS cells were treated with control , Plk4 or hSAS - 6 siRNA and immunoblotting was performed with the indicated antibodies . Asterisk indicates non - specific bands . The positions of molecular weight markers ( kDa ) are shown on the right .

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A. Evaluation of siRNA-mediated depletion. U2OS cells were treated with control, Plk4 or hSAS-6 siRNA and immunoblotting was performed with the indicated antibodies. Asterisk indicates non-specific bands. The positions of molecular weight markers (kDa) are shown on the right.


B . U2OS cells were transfected with the indicated siRNAs . Cell peripheries are marked with dotted lines . Enlarged single cell images ( marked with arrowheads in the top row ) are shown at the bottom . Scale bars , 5 μm ( bottom ) , 10 μm ( top ) .

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B. U2OS cells were transfected with the indicated siRNAs. Cell peripheries are marked with dotted lines. Enlarged single cell images (marked with arrowheads in the top row) are shown at the bottom. Scale bars, 5 μm (bottom),10 μm (top).


C , D , E . Quantification of the dispersion of satellite components upon depletion of Plk4 but not hSAS - 6 knockdown . The results of PCM1 ( C ) , hMsd1 / SSX2IP ( D ) or BBS4 ( E ) are shown . Data represent the mean + SD ( > 200 cells , n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * p < 0 . 01 , n . s . ( not significant ) .

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C, D, E. Quantification of the dispersion of satellite components upon depletion of Plk4 but not hSAS-6 knockdown. The results of PCM1 (C), hMsd1/SSX2IP (D) or BBS4 (E) are shown. Data represent the mean + SD (>200 cells, n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. **p<0.01, n.s. (not significant).


F , G . U2OS cells were arrested in G1 and treated with control or Plk4 siRNA , followed by immunofluorescence microscopy by using indicated antibodies . Enlarged images ( marked by arrowheads ) are shown on the right . Quantification of PCM1 distribution is shown in G . > 200 cells were counted and classified into two categories : normal ( around and away from the centrosome ) or dispersed . Data represent the mean + SD ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * p < 0 . 01 . Scale bar , 1 μm ( F , right ) , 10 μm ( F ) .

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F, G. U2OS cells were arrested in G1 and treated with control or Plk4 siRNA, followed by immunofluorescence microscopy by using indicated antibodies. Enlarged images (marked by arrowheads) are shown on the right. Quantification of PCM1 distribution is shown in G. >200 cells were counted and classified into two categories: normal (around and away from the centrosome) or dispersed. Data represent the mean + SD (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. **p<0.01. Scale bar, 1 μm (F, right), 10 μm (F).


H , I . siRNA - treated U2OS cells were transfected with empty vectors ( EV ) or plasmids containing Plk4 or siRNA - resistant Plk4 - myc WT * or KD * and immunostained with the indicated antibodies . Cell peripheries are marked with solid lines , in which cells with yellow lines are successfully transfected ( positive in myc signals ) , while those with white lines represent non - transfected cells . Quantification of PCM1 distribution is shown in I . Data represent the mean + SD ( > 200 cells , n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * * * p < 0 . 0001 , * * p < 0 . 01 , n . s . ( not significant ) . Scale bar , 5 μm ( H ) .

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H, I. siRNA-treated U2OS cells were transfected with empty vectors (EV) or plasmids containing Plk4 or siRNA-resistant Plk4-myc WT* or KD* and immunostained with the indicated antibodies. Cell peripheries are marked with solid lines, in which cells with yellow lines are successfully transfected (positive in myc signals), while those with white lines represent non-transfected cells. Quantification of PCM1 distribution is shown in I. Data represent the mean + SD (>200 cells, n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. ****p<0.0001, **p<0.01, n.s. (not significant). Scale bar, 5 μm (H).


A , B . Either empty vector plasmids ( EV ) or those producing EGFP - PCM1 ( A ) or myc - Plk4 ( B ) were transfected into U2OS cells . Immunoprecipitation ( IP ) was performed with anti - GFP ( A ) or anti - myc antibodies ( B ) , followed by immunoblotting with the indicated antibodies . DIC , the dynein intermediate chain .

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A, B. Either empty vector plasmids (EV) or those producing EGFP-PCM1 (A) or myc-Plk4 (B) were transfected into U2OS cells. Immunoprecipitation (IP) was performed with anti-GFP (A) or anti-myc antibodies (B), followed by immunoblotting with the indicated antibodies. DIC, the dynein intermediate chain.


C . Bacterially expressed and purified His - PCM1 ( a . a . 1 - 1128 ) was incubated with MBP alone or MBP - Plk4 ( WT or KD ) in vitro , and pulled down by magnetic beads coupled with an anti - MBP antibody , followed by staining with Coomassie Blue ( left ) or immunoblotting with an anti - His antibody ( right ) . Asterisks show non - specific bands . The mobility difference between Plk4 - WT and Plk4 - KD was reported previously [ 43 , 48 ] . We reproducibly observed the upward mobility shift of His - PCM1 upon binding to MBP - Plk4 - WT , but not Plk4 - KD .

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C. Bacterially expressed and purified His-PCM1 (a.a. 1-1128) was incubated with MBP alone or MBP-Plk4 (WT or KD) in vitro, and pulled down by magnetic beads coupled with an anti-MBP antibody, followed by staining with Coomassie Blue (left) or immunoblotting with an anti-His antibody (right). Asterisks show non-specific bands. The mobility difference between Plk4-WT and Plk4-KD was reported previously [43, 48]. We reproducibly observed the upward mobility shift of His-PCM1 upon binding to MBP-Plk4-WT, but not Plk4-KD.


E . Total cell extracts were prepared from U2OS cells treated with PCM1 siRNA and transfected with RNAi - resistant EGFP - PCM1 - WT * . Cells were cultured asynchronously ( As ) or arrested in G1 , G2 or M phase , followed by immunoprecipitation with an anti - GFP antibodies . Pulled down samples were immunoblotted with the indicated antibodies .

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E. Total cell extracts were prepared from U2OS cells treated with PCM1 siRNA and transfected with RNAi-resistant EGFP-PCM1-WT*. Cells were cultured asynchronously (As) or arrested in G1, G2 or M phase, followed by immunoprecipitation with an anti-GFP antibodies. Pulled down samples were immunoblotted with the indicated antibodies.


A , B , C , D . siRNA - treated U2OS cells ( PCM1 for A and B ; Plk4 for C and D ) were transfected with plasmids producing various EGFP - PCM1 constructs ( WT * , S372A * , S372D * or S372E * ) , fixed and stained with the indicated antibodies ( arrowheads point to the centrosome ) ( A and C ) . Quantification data are shown in B and D . > 200 cells were counted and classified into three categories : normal ( around and away from the centrosome ) , aggregated or dispersed . Data represent the mean + SD ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , n . s . ( not significant ) . Note that the percentage of cells displaying " dispersed " is analysed . Scale bars , 5 μm ( A and C ) .

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A, B, C, D. siRNA-treated U2OS cells (PCM1 for A and B; Plk4 for C and D) were transfected with plasmids producing various EGFP-PCM1 constructs (WT*, S372A*, S372D* or S372E*), fixed and stained with the indicated antibodies (arrowheads point to the centrosome) (A and C). Quantification data are shown in B and D. >200 cells were counted and classified into three categories: normal (around and away from the centrosome), aggregated or dispersed. Data represent the mean + SD (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. ***p<0.001, **p<0.01, *p<0.05, n.s. (not significant). Note that the percentage of cells displaying "dispersed" is analysed. Scale bars, 5 μm (A and C).


E . PCM1 siRNA - treated U2OS cells were transfected with two types of plasmids ( EGFP - and mCherry - tagged ) that produce PCM1 - WT * and PCM1 - S372A * . Immunoprecipitation and subsequent immunoblotting were performed with the indicated antibodies . Quantification data is shown on the right . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , n . s . not significant .

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E. PCM1 siRNA-treated U2OS cells were transfected with two types of plasmids (EGFP- and mCherry-tagged) that produce PCM1-WT* and PCM1-S372A*. Immunoprecipitation and subsequent immunoblotting were performed with the indicated antibodies. Quantification data is shown on the right. Statistical analysis was performed using two-tailed unpaired student's t-tests. *p<0.05, **p<0.01, n.s. not significant.


F . U2OS cells were treated with PCM1 siRNA and transfected with empty vectors ( EV ) or EGFP plasmids that produce PCM1 - WT * or PCM1 - S372A * . Immunoprecipitation was performed with an anti - GFP antibody , followed by immunoblotting with the indicated antibodies .

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F. U2OS cells were treated with PCM1 siRNA and transfected with empty vectors (EV) or EGFP plasmids that produce PCM1-WT* or PCM1-S372A*. Immunoprecipitation was performed with an anti-GFP antibody, followed by immunoblotting with the indicated antibodies.


A . Immunofluorescence microscopy was performed using the indicated antibodies in hTERT - RPE - 1 cells treated with control , PCM1 or Plk4 siRNA . Regions around the basal body ( insets ) are enlarged on the right hand side . Scale bars , 5 μm ( left ) and 1 μm ( right ) . B . Quantification . At least 150 cells were counted ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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A. Immunofluorescence microscopy was performed using the indicated antibodies in hTERT-RPE-1 cells treated with control, PCM1 or Plk4 siRNA. Regions around the basal body (insets) are enlarged on the right hand side. Scale bars, 5 μm (left) and 1 μm (right).B. Quantification. At least 150 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. ***p<0.001, ****p<0.0001.


C . hTERT - RPE - 1 cells treated with PCM1 siRNA were transfected by an empty vector ( EV ) or plasmids producing PCM1 - WT * , - S327A * or - S372E * , and immunofluorescence microscopy performed . Enlarged images of the regions around the basal body are shown in insets . Scale bars , 5 μm and 1 μm ( inset ) .

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C. hTERT-RPE-1 cells treated with PCM1 siRNA were transfected by an empty vector (EV) or plasmids producing PCM1-WT*, -S327A* or -S372E*, and immunofluorescence microscopy performed. Enlarged images of the regions around the basal body are shown in insets. Scale bars, 5 μm and 1 μm (inset).


D . Quantification . At least 200 cells were counted ( n = 3 ) . Statistical analysis was performed using two - tailed unpaired student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n . s . not significant .

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D. Quantification. At least 200 cells were counted (n=3). Statistical analysis was performed using two-tailed unpaired student's t-tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant.


( B ) The active fragment of mTOR or active Akt2 was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC - 0068 .

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(B) The active fragment of mTOR or active Akt2 was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC-0068.


( C ) HEK - 293E Cells were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls .

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(C) HEK-293E Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.


( D ) HEK - 293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls .

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

(D) HEK-293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.


( E ) Control or iRapKO MEFs were treated with or without 2 μM 4 - Hydroxytamoxifen ( 4 - OHT ) for 4 days to induce Raptor knockout . Treated cells were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot .

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

(E) Control or iRapKO MEFs were treated with or without 2 μM 4-Hydroxytamoxifen (4-OHT) for 4 days to induce Raptor knockout. Treated cells were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.


( F ) SIN1 WT was expressed in SIN1 KO MEFs , and cells were selected by FACS . Cell lines were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot .

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

(F) SIN1 WT was expressed in SIN1 KO MEFs, and cells were selected by FACS. Cell lines were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.