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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 HER-2-E0771 tumours at day 3 post adoptive CAR-T cell transfer and mCherry+CD45+CD3+CD8+ CAR-T cell numbers were determined by flow cytometry.


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

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

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


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

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

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

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


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

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 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

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


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

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

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

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


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

B-MEASURED_VAR I-MEASURED_VAR 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

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


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

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

O O 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 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

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


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

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

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


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

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

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


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

O O O O 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 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

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


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

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

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


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

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

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


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

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 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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(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 of cilia ( R ) of of ciliated ( S , n = 9 mice ) in mouse tracheal epithelium . To quantify of ciliated ( S ) , > 200 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 of ENKD1 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 ( B ) of of ciliated ( C , n = 3 independent experiments ) ciliary ( D , n = 70 cilia from three independent experiments ) for MEFs cultured in serum - free medium stained with antibodies against acetylated α - tubulin γ - tubulin DAPI . To quantify of ciliated ( C ) , > 100 were analyzed for each experiment . Scale bar , 5 µm .

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

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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.


of ciliary ( H , n = 100 cilia from three independent experiments ) for RPE1 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 ( I ) of of ciliated ( J , n = 3 independent experiments ) for RPE1 transfected with control or ENKD1 siRNAs , cultured in serum - free medium , stained with antibodies against Arl13b Centrin DAPI . To quantify of ciliated ( J ) , > 80 were analyzed for each experiment . Scale bar , 2 µm .

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

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(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 of RPE1 cultured in normal serum medium , stained with antibodies against ENKD1 Centrin 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 profiles of ENKD1 CEP164 in serum - starved RPE1 . arrow marks centriole subjected to 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 profiles of ENKD1 Ninein in serum - starved RPE1 . arrow marks centriole subjected to 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 ( D ) , of outer toroid diameters ( E , n = 20 from three independent experiments ) , a model illustrating relative positions of γ - tubulin , Ninein , ENKD1 , CEP164 , CEP912 , Centrin ( F ) within a centriole . RPE1 were serum - starved stained with antibodies against indicated . 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 of RPE1 cultured in normal serum or serum - free medium , stained with antibodies against ENKD1 γ - tubulin 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 of RPE1 cultured in serum - free medium , stained with antibodies against ENKD1 acetylated α - tubulin 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 of RPE1 transfected with GFP - ENKD1 or GFP vector , cultured in a serum - starved condition , stained with antibody against acetylated α - tubulin 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 of ENKD1 localization at centrosome in RPE1 cultured in serum - free medium ( 0 , 12 , 24 , or 48 h of serum starvation ) , stained with antibodies against ENKD1 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 ( B ) for RPE1 transfected with control or ENKD1 siRNAs , cultured in serum - free medium , stained with antibodies against CP110 acetylated α - tubulin 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.


of of with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 transfected with control or ENKD1 siRNAs To quantify of with one CP110 dot ( C ) , > 120 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 ( D ) of of with one CEP97 dot ( E , n = 3 independent experiments ) for RPE1 transfected with control or ENKD1 siRNAs , cultured in serum - free medium , stained with antibodies against CEP97 acetylated α - tubulin DAPI . To quantify of with one CEP97 dot ( E ) , > 150 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 ( F ) , immunofluorescence ( G ) , of of with one CP110 dot ( H , n = 3 independent experiments ) for RPE1 transfected with control or ENKD1 siRNAs , cultured in serum - free medium for 120 h , stained with antibodies against CP110 acetylated α - tubulin DAPI . To quantify of with one CP110 dot ( C ) , > 80 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 ( A ) , immunofluorescence ( B ) of of with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 transfected with indicated siRNAs , cultured in serum - free medium for 48 h , stained with antibodies against CP110 acetylated α - tubulin DAPI . To quantify of with one CP110 dot ( C ) , > 180 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 of CP110 β - actin in RPE1 transfected with control or CP110 siRNAs 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 ( E ) of of ciliated ( F , n = 3 independent experiments ) for RPE1 transfected with indicated siRNAs , cultured in a serum - starved condition , stained with antibodies against γ - tubulin acetylated α - tubulin DAPI . To quantify of ciliated ( F ) , > 120 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 immunoblotting ( G ) ( H , n = 3 mice ) showing CP110 - CEP97 interaction in retinal tissues of wild - type Enkd1 knockout mice . of each CEP97 band was normalized to that of 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 immunoblotting ( I ) ( J , n = 3 independent experiments ) showing CP110 - CEP97 interaction in serum - starved RPE1 transfected with control or ENKD1 siRNAs . of each CEP97 band was normalized to that of 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 immunoblotting showing interaction between endogenous ENKD1 CP110 in mouse retinal tissues . Immunoprecipitation was performed with anti - ENKD1 ( upper panel ) or anti - CP110 ( lower panel ) antibody or 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 immunoblotting showing interaction of GFP - ENKD1 with Flag - CP110 ( B upper panel of D ) or HA - CP110 ( C bottom panel of D ) in HEK293T . Immunoprecipitation was performed with antibodies against GFP ( B 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 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 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 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 immunoblotting ( I ) ( J , n = 3 independent experiments ) showing interaction of HA - CP110 with Flag - CEP97 in HEK293T transfected with indicated plasmids . of each Flag - CEP97 band was normalized to that of 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 were treated with control , Plk4 or hSAS - 6 siRNA immunoblotting was performed with indicated antibodies . Asterisk indicates non - specific bands . positions of molecular markers ( kDa ) are shown on 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 were transfected with indicated siRNAs . peripheries are marked with dotted lines . Enlarged single ( marked with arrowheads in top row ) are shown at 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 . of dispersion of satellite components upon depletion of Plk4 but not hSAS - 6 knockdown . results of PCM1 ( C ) , hMsd1 / SSX2IP ( D ) or BBS4 ( E ) are shown . Data represent mean + SD ( > 200 , n = 3 ) . Statistical was performed using two - unpaired student ' s t - . * * 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 were arrested in G1 treated with control or Plk4 siRNA , followed by immunofluorescence microscopy by using indicated antibodies . Enlarged ( marked by arrowheads ) are shown on right . of PCM1 distribution is shown in G . > 200 were counted classified into two categories : normal ( around away from centrosome ) or dispersed . Data represent mean + SD ( n = 3 ) . Statistical was performed using two - unpaired student ' s t - . * * p < 0 . 01 . Scale bar , 1 μm ( F , right ) , 10 μm ( F ) .

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

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

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

H, 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 . Immunoprecipitation ( IP ) was performed with anti - GFP ( A ) or anti - myc antibodies ( B ) , followed by immunoblotting with indicated antibodies . DIC , dynein intermediate chain .

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

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

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

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 extracts were prepared from U2OS treated with PCM1 siRNA transfected with RNAi - resistant EGFP - PCM1 - WT * . 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 indicated antibodies .

O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 were transfected with two types of plasmids ( EGFP - mCherry - tagged ) that produce PCM1 - WT * PCM1 - S372A * . Immunoprecipitation subsequent immunoblotting were performed with indicated antibodies . data is shown on right . Statistical was performed using two - unpaired student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , n . s . not significant .

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 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

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 were treated with PCM1 siRNA 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 indicated antibodies .

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

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

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

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

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

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

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

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

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

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 B-MEASURED_VAR 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

(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 were deprived of total for 1hr followed by addition of for indicated time . Lysates were analysed by Western blot for phosphorylation of RagC known to belong to Akt mTOR pathways . Graph shows mean of quantitative analyses of Western blots after normalization for total or loading controls .

O O O O O 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 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

(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 were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , 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 B-CONTROLLED_VAR 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 , were selected by FACS . lines were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , 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 B-CONTROLLED_VAR 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.


active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E . kinase reaction was performed in presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Mass Spectrum for pRagC

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

The active fragment of mTOR 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 Rapamycin. Samples were analysed by Mass Spectrum for pRagC T394


active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E . kinase reaction was performed in presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Western blot for pRagC

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

The active fragment of mTOR 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 Rapamycin. Samples were analysed by Western blot for pRagC S21


( A ) HA - RagC HeLa Flp - In T - Rex stable lines were created ( See methods for details ) expression of RagC WT or mutants was induced by addition of tetracycline overnight . were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for of specified phosphorylation state of S6K1 . Graphs indicate mean ± S . E . M . of quantitative analyses of western blots after normalization for total ( * p < 0 . 05 , # no significant difference , two Student ' s t , n = 3 ) . ( B ) Expression of RagC WT , 3A or 3E was induced by addition of tetracycline overnight . were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for of specified phosphorylation state of S6K1 . Graphs indicate mean ± S . E . M . of quantitative analyses of western blots after normalization for total ( * p < 0 . 05 , # no significant difference , two Student ' s t , n = 3 )

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 B-MEASURED_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 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 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 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 O O O O O O O O O O O O O O O O O O O O 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) The HA-RagC HeLa Flp-In T-Rex stable cell lines were created (See methods for details) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight. Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3). (B) Expression of RagC WT, 3A or 3E was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3)


( C ) Expression of RagC WT 3A was induced by addition of tetracycline overnight . were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for indicated time . Left : Lysates were analyzed by Western blot for of specified phosphorylation state of S6K1 . Right : Graph shows mean ± S . E . M . of quantitative analyses of Western blots after normalization for total , n = 3

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 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 O O O 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) Expression of RagC WT and 3A was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for indicated time. Left: Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Right: Graph shows mean ± S.E.M. of quantitative analyses of the Western blots after normalization for total protein, n=3


( D ) HEK - 293E stably expressing RagC WT , 3A or 3E were deprived of total for 1hr followed by addition of for 10 min . Lysates were analyzed by Western blot for of specified phosphorylation state of S6K1 . Graphs indicate mean ± S . E . M . of quantitative analyses of western blots after normalization for total ( * p < 0 . 05 , * * p < 0 . 01 , two Student ' s t , n = 3 )

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 O O O O O O O O O O O O O O O O O O O O O O O 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) HEK-293E cells stably expressing RagC WT, 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, ** p<0.01, two tailed Student's t test, n=3)


( A ) Expression of RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa followed by using a particle counter ( Horizontal lines , box limits whiskers represent median , 25th percentile ot 75th percentile minimum to maximum respectively . * p < 0 . 05 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 4 )

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

(A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4)


( B ) Phospho - RagC positively regulates wing compartment in Drosophila . Over - expression ( OE ) of dRagC WT , 3A , or 3E in wing posterior compartment using en - Gal4 driver posterior compartment sizes were measured in each genotype ( ( Horizontal lines , box limits whiskers represent median , 25th percentile ot 75th percentile minimum to maximum respectively . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Bonferroni multiple comparisons , n ≥ 63 flies per condition )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phospho-RagC positively regulates wing compartment size in Drosophila. Over-expression (OE) of dRagC WT, 3A, or 3E in wing posterior compartment using en-Gal4 driver and the posterior compartment sizes were measured in each genotype ((Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. ** p<0.01, *** p<0.001, **** p<0.0001, One-way ANOVA with Bonferroni multiple comparisons, n ≥ 63 flies per condition)


( C ) Empty vector ( EV ) , RagC WT , 3A or 3E were transiently expressed in stable RagC knockdown HEK - 293E followed by using a particle counter ( Horizontal lines , box limits whiskers represent median , 25th percentile ot 75th percentile minimum to maximum respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 2 for C2 n = 4 for other conditions )

O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Empty vector (EV), RagC WT, 3A or 3E were transiently expressed in stable RagC knockdown HEK-293E cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, ** p<0.01, **** p<0.0001, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=2 for C2 and n=4 for other conditions)


( D ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E followed 48 hr later by incubation with DMEM in presence or absence of serum for 4 hr . Lysates were analyzed by Western blot for phosphorylation of ULK1 total

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 B-MEASURED_VAR O

(D) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1


( E ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E followed 48 hr later by incubation with DMEM in presence or absence of serum for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . Lysates were analyzed by Western blot for of LC3 - II 14 - 3 - 3 . Graphs show mean ± S . E . M . of quantitative analyses of LC3 - II blotting after normalization for 14 - 3 - 3 . * p < 0 . 05 , two Student ' s t , n = 4

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 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 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

(E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4


( F ) GFP - LC3 was co - expressed with empty vector , RagC WT , 3A or 3E in HEK - 293E followed 48 hr later by serum deprivation for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . LC3 RagC were monitored by confocal microscopy . Scale bar , 10 µm

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(F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( G ) Quantitative of GFP ( F ) ( * * * * p < 0 . 0001 , two Student ' s t ; n ≥ 21 ) . Mean value is shown as a horizontal bar

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(G) Quantitative analysis of GFP puncta (F) (**** p<0.0001, two tailed Student's t test; n ≥ 21). Mean value is shown as a horizontal bar


( A ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were transiently expressed in HEK - 293E . RagC lysosome maker , Lamp1 , were monitored by confocal microscopy . Scale bar , 10 μm

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(A) Empty vector (EV), HA-RagC WT, 3A or 3E were transiently expressed in HEK-293E cells. RagC and lysosome maker, Lamp1, were monitored by confocal fluorescence microscopy. Scale bar, 10 μm


( B ) Empty vector ( EV ) , HA - RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK - 293E . mTOR HA - RagC were monitored by confocal microscopy . Scale bar , 10 µm

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(B) Empty vector (EV), HA-RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK-293E cells. mTOR and HA-RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( C ) Empty vector ( EV ) , Flag - RagC WT , 3A or 3E were transiently expressed with HA - RagA in HEK - 293E . Total lysates amounts of mTOR , Raptor HA - RagA in Flag - RagC immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * p < 0 . 05 , two Student ' s t , n ≥ 3 )

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(C) Empty vector (EV), Flag-RagC WT, 3A or 3E were transiently expressed with HA-RagA in HEK-293E cells. Total cell lysates and amounts of mTOR, Raptor and HA-RagA in the Flag-RagC immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (* p<0.05, two tailed Student's t test, n ≥ 3)


( D ) Expression of HA - RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa . Total lysates ( TCL ) amount of mTOR in Raptor immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * * * p < 0 . 001 , Student ' s t , n = 3 )

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(D) Expression of HA-RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells. Total cell lysates (TCL) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (*** p<0.001, Student's t test, n=3)


Figure 1 . Identification of non - canonical p53 binding sites in human subtelomeres . ( A B ) ChIP - seq tracks for p53 in camptothecin ( CPT ) treated or untreated HCT116 were aligned with RNAPII , CTCF , RAD21 , SMC1 for human chromosome18q ( A ) or 13q ( B ) subtelomeres . Position relative to centromeric end of terminal TTAGGG repeat tract are indicated in bp at top . Subtelomeric paralogy segment tracks , internal telomere - like tracks ( red CCCTAA ) , annotated RefSeq gene tracks are indicated above ChIP - seq tracks . ( C ) Alignment of human subtelomeric elements containing non - canonical p53 recognition sequences . position of RepeatMasker - defined LTR10c element is shown ; also note ( TG ) n microsatellite repeat adjacent to p53 binding site . ( D ) Schematic organization of non - canonical p53 binding sites found in human subtelomeres .

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Figure 1. Identification of non-canonical p53 binding sites in human subtelomeres. (A and B) ChIP-seq tracks for p53 in camptothecin (CPT) treated or untreated HCT116 cells were aligned with RNAPII, CTCF, RAD21, and SMC1 for human chromosome18q (A) or 13q (B) subtelomeres. Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top. Subtelomeric DNA paralogy segment tracks, internal telomere-like sequence tracks (red CCCTAA), and annotated RefSeq gene tracks are indicated above the ChIP-seq tracks. (C) Alignment of human subtelomeric elements containing non-canonical p53 recognition sequences. The position of the RepeatMasker-defined LTR10c element is shown; also note the (TG)n microsatellite repeat adjacent to the p53 binding site. (D) Schematic organization of non-canonical p53 binding sites found in human subtelomeres.


Stress - induced p53 binding at human subtelomeres . ( A ) Western blot of HCT116 p53 - / - p53 + / + treated with DMSO ( D ) or 50 µM etoposide ( E ) for 24 hrs . lysates were assayed for total p53 , phospho - Ser15 p53 ( p - p53 ) , γH2AX , or GAPDH .

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Stress-induced p53 binding at human subtelomeres. (A) Western blot of HCT116 p53-/- and p53+/+ cells treated with DMSO (D) or 50 µM etoposide (E) for 24 hrs. Cell lysates were assayed for total p53, phospho-Ser15 p53 (p-p53), γH2AX, or GAPDH.


( B ) ChIP - qPCR was assayed in HCT116 p53 - / - or p53 + / + treated with DMSO or etoposide as in panel A , assayed by ChIP with antibodies to p53 ( Ab6 ) or control IgG , followed by qPCR for p53 binding site in subtelomere 18q ( top panel ) or 13q ( lower panel ) .

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(B) ChIP-qPCR was assayed in HCT116 p53-/- or p53 +/+ cells treated with DMSO or etoposide as in panel A, and assayed by ChIP with antibodies to p53 (Ab6) or control IgG, followed by qPCR for p53 binding site in subtelomere 18q (top panel) or 13q (lower panel).


( C ) HCT116 p53 - / - p53 + / + were cultured in DMEM with 10 % FBS or Hank ' s buffered saline solution ( HBSS ) lacking FBS then assayed by ChIP with either IgG control ( grey ) or two different p53 - specific antibodies Ab6 ( red ) or FL ( black ) . ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q , 13q , or p21 promoter .

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(C) HCT116 p53-/- and p53+/+ cells were cultured in DMEM with 10% FBS or Hank's buffered saline solution (HBSS) lacking FBS and then assayed by ChIP with either IgG control (grey) or two different p53-specific antibodies Ab6 (red) or FL(black). ChIP was assayed by qPCR with primers proximal to p53 binding sites at 18q, 13q, or p21 promoter.


. ( D ) Doxycyclin ( Dox ) - inducible p53 H1299 were treated without ( - ) or with ( + ) Dox in media containing 10 % serum or in serum free HBSS , then subject to ChIP with control IgG or p53 - specific Ab6 antibody . ChIP was assayed by qPCR using primers specific for p53 binding site at p21 ( top panel ) , p53 - negative control IGX1A ( middle panel ) , or p21 positive control ( lower panel ) . Error bars represent SD , * indicates p value < . 05 . p - values were determined by Student t - .

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. (D) Doxycyclin (Dox)-inducible p53 H1299 cells were treated without (-) or with (+) Dox in media containing 10% serum or in serum free HBSS, and then subject to ChIP assay with control IgG or p53-specific Ab6 antibody. ChIP DNA was assayed by qPCR using primers specific for p53 binding site at p21 (top panel), p53-negative control IGX1A (middle panel), or p21 positive control (lower panel). Error bars represent SD, and * indicates p value < .05. p-values were determined by Student t-test.


. Transcriptional activity of subtelomeric p53 - response elements . ( A ) Luciferase constructs containing either empty vector ( top ) , p53 - response elements from 18q subtelomere ( middle ) or MDM2 promoter ( lower ) were assayed in HCT p53 - / - transfected with two independent preparations of empty vector or p53 WT expression vector .

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. Transcriptional activity of subtelomeric p53-response elements. (A) Luciferase test constructs containing either empty test vector (top), p53-response elements from 18q subtelomere (middle) or MDM2 promoter (lower) were assayed in HCT p53-/- cells transfected with two independent preparations of empty vector or p53 WT expression vector.


( B ) RT - qPCR measure of p21 ( top panel ) or PARD6G ( lower panel ) in HCT116 p53 - / - or p53 + / + after treatment with DMSO or 50 µM etoposide .

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(B) RT-qPCR measure of p21 (top panel) or PARD6G (lower panel) in HCT116 p53-/- or p53+/+ cells after treatment with DMSO or 50 µM etoposide.


( C ) RT - qPCR of TERRA transcripts from subtelomere 13q ( upper ) or 2q ( lower ) .

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(C) RT-qPCR analysis of TERRA transcripts from subtelomere 13q (upper) or 2q (lower).


( D ) RT - qPCR of TERRA transcripts from 18q using two independent primer sets pr1 ( top panel ) or pr2 ( lower panel ) from HCT116 p53 + / + cultured with serum ( grey bars ) or in HBSS lacking serum ( black bars ) . Error bars represent SD * indicates p value < . 05 using student t - comparing treated untreated samples .

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(D) RT-qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 (top panel) or pr2 (lower panel) from HCT116 p53+/+ cells cultured with serum (grey bars) or in HBSS lacking serum (black bars). Error bars represent SD and * indicates p value <.05 using student t-test comparing treated and untreated samples.


. p53 - dependent viability telomere protection in response to damage . ( A ) Western blot of total p53 , γH2AX , GAPDH in HCT116 p53 - / - or p53 + / + treated with 1 µM or 50 µM etoposide for 24 hrs .

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. p53-dependent cell viability and telomere protection in response to DNA damage. (A) Western blot of total p53, γH2AX, and GAPDH in HCT116 p53-/- or p53+/+ cells treated with 1 µM or 50 µM etoposide for 24 hrs.


s . ( B ) cycle of HCT116 p53 - / - or p53 + / + untreated or treated with 50 µM etoposide for 24 hrs . cycle was determined by FACS of propidium iodide stained .

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s. (B) Cell cycle analysis of HCT116 p53-/- or p53+/+ cells untreated or treated with 50 µM etoposide for 24 hrs. Cell cycle was determined by FACS analysis of propidium iodide stained cells.


( C ) clonal survival for HCT116 p53 - / - or p53 + / + treated with DMSO or 1 , 1 . 5 or 2 µM etoposide for 24 hr prior to replating .

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(C) Cell clonal survival assay for HCT116 p53-/- or p53+/+ cells treated with DMSO or 1, 1.5 or 2 µM etoposide for 24 hr prior to replating.


. ( D ) of clonal survival relative total numbers ( top panel ) or normalized to DMSO controls ( lower panel ) .

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. (D) Quantification of clonal survival assay relative total cell numbers (top panel) or normalized to DMSO controls (lower panel).


E ) TIF for HCT116 p53 + / + or p53 - / - treated with 1 µM etoposide for 3 hrs . Telomere FISH with TelC probe ( red ) , γH2AX ( green ) , Dapi ( blue ) .

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E) TIF assay for HCT116 p53+/+ or p53-/- cells treated with 1 µM etoposide for 3 hrs. Telomere DNA FISH with TelC probe (red), γH2AX (green), Dapi (blue).


( F ) of TIF for % with > 4 TIFs per for p53 + / + ( black ) or p53 - / - ( red ) .

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(F) Quantification of TIF assay for % cells with > 4 TIFs per cell for p53+/+ (black) or p53-/- (red) cell.


G ) Telomere showing p53 - / - or p53 + / + treated with either DMS0 , 1 or 50 µM etoposide . Ethidium bromide ( EtBr ) stained agarose gels ( left panel ) , Southern blot hybridized with Tel C probe ( middle panel ) or with α Satellite promote ( right panel ) . of telomereDNA signal relative to invariant bands in α Sat . Error bars represent SD * indicates p values < . 05 determined by student t - ( TLA ) , or chi - square ( TIF ) .

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G) Telomere length assay showing p53-/- or p53+/+ cells treated with either DMS0, 1 or 50 µM etoposide. Ethidium bromide (EtBr) stained agarose gels (left panel), Southern blot hybridized with Tel C probe (middle panel) or with α Satellite DNA promote (right panel). Quantification of telomereDNA signal relative to invariant bands in α Sat. Error bars represent SD and * indicates p values <.05 determined by student t-test (TLA), or chi-square (TIF assay).


( H ) Two - dimensional agarose gel of telomeric from HCT p53 + / + or or p53 - / - treated with 50 µM etoposide for 24 hrs . Ethidium gel ( top ) Southern blot probed with TelG probe ( lower panel ) . Single stranded telomere ( red arrow ) recombination / replication - associated structures ( blue arrow ) are indicated .

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(H) Two-dimensional agarose gel analysis of telomeric DNA from HCT p53+/+ or or p53-/- treated with 50 µM etoposide for 24 hrs. Ethidium gel (top) and Southern blot probed with TelG probe (lower panel). Single stranded telomere DNA (red arrow) and recombination/ replication-associated structures (blue arrow) are indicated.


Figure 5 . p53 - dependent histone modification changes in response to damage stress . HCT116p53 - / - ( left panels ) or p53 + / + ( right panels ) were treated with DMSO ( black ) or 50 µM etoposide ( red ) for 24 hrs assayed by ChIP - qPCR at various locations relative to TTAGGG repeats across chromosome18qsubtelomere ( panel A ) or 13qsubtelomere ( panel B ) or for control regions ( p21 promoter , GKN1 / 2 , PARKIN ) as indicated below . ChIP - for p53 , H3K9Ac , H3K27Ac , γH2AX , or IgG control were quantified as % input . Error bars indicates SD * indicates p value < . 05 using student t - comparing ChIP values in p53 + / + relative to p53 - / - for specific primer pairs .

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Figure 5. p53-dependent histone modification changes in response to DNA damage stress. HCT116p53-/- (left panels) or p53 +/+ (right panels) were treated with DMSO (black) or 50 µM etoposide (red) for 24 hrs and assayed by ChIP-qPCR at various locations relative to the TTAGGG repeats across the chromosome18qsubtelomere (panel A) or 13qsubtelomere (panel B) or for control regions (p21 promoter, GKN1/2, PARKIN) as indicated below. ChIP- for p53, H3K9Ac, H3K27Ac, γH2AX, or IgG control were quantified as % input. Error bars indicates SD and * indicates p value<.05 using student t-test comparing ChIP values in p53+/+ relative to p53-/- cells for specific primer pairs.


( C ) Dot blot of telomere repeat or Alu repeat from p53 + / + or p53 - / - HCT116 treated with DMSO or etoposide ( ETP ) ( as described in panel A ) , that were subject to ChIP with antibodies to IgG , p53 , γH2AX , TRF2 , or TRF1 . Quantitation of three independent ChIP - assays for TRF1 TRF2 is shown in panel to right . (

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(C) Dot blot analysis of telomere repeat or Alu repeat DNA from p53+/+ or p53-/- HCT116 cells treated with DMSO or etoposide (ETP) (as described in panel A), that were subject to ChIP with antibodies to IgG, p53, γH2AX, TRF2, or TRF1. Quantitation of three independent ChIP-assays for TRF1 and TRF2 is shown in the panel to the right. (


( E ) ChIP - qPCR for TRF2 is shown for treated as in panel A using primers across subtelomeres of 18q ( left ) or 13q ( right ) .

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(E) ChIP-qPCR for TRF2 is shown for cells treated as in panel A using primers across the subtelomeres of 18q (left) or 13q (right).


( A ) HCT116 p53 + / + or p53 - / - treated with 50 µM etoposide were assayed by Western blot with antibodies for p53 , γH2AX , H3K9Ac , panH3 , GAPDH at times 0 , 8 , 16 , or 24 hr post - treatment .

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(A) HCT116 p53+/+ or p53-/- cells treated with 50 µM etoposide were assayed by Western blot with antibodies for p53, γH2AX, H3K9Ac, panH3, and GAPDH at times 0, 8, 16, or 24 hr post-treatment.


( B ) ChIP - qPCR for γH2AX ( upper panels ) or IgG control ( lower panels ) for treated as indicated in panel A . ( C - D ) RT - qPCR for treated as shown in panel A .

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(B) ChIP-qPCR for γH2AX (upper panels) or IgG control (lower panels) for cells treated as indicated in panel A. (C-D) RT-qPCR analysis for cells treated as shown in panel A.


C - D ) RT - qPCR for treated as shown in panel A . ( C ) RT - PCR for p21 ( top ) , or PARD6G mRNA ( lower ) . ( D ) RT - PCR for primer pairs spanning subtelomere18q as indicated below . Lower panel shows No - RT control for 18qsubtelomere primers . RT - qPCR was quantified as relative to Actin with error bars indicating SD * indicating p value < . 05 using student t - comparing etoposide ( 24 hr ) relative to untreated for specific primer pairs .

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C-D) RT-qPCR analysis for cells treated as shown in panel A. (C) RT-PCR for the p21 (top), or PARD6G mRNA (lower). (D) RT-PCR for primer pairs spanning subtelomere18q as indicated below. Lower panel shows the No-RT control for 18qsubtelomere primers. RT-qPCR was quantified as relative to Actin with error bars indicating SD and * indicating p value <.05 using student t-test comparing etoposide (24 hr) relative to untreated for specific primer pairs.


( E ) Meta - data of published GRO - Seq ChIP Seq data sets . Gro - Seq Minus ( red / maroon ) or Plus ( purple / pink ) strand from Control or Nutlin - 3a treated MCF7 was mapped to human 18q subtelomere . Histone H3K27ac , H3K4me1 , H3K4me3ChIP - Seq data sets from untreated MCF7 are also shown for these subtelomeric regions . Right panel shows a zoom in of approximately 1000bp ( blue on left panel ) surrounding p53 response element within subtelomere .

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(E) Meta-data analysis of published GRO-Seq and ChIP Seq data sets. Gro-Seq Minus (red/maroon) or Plus (purple/pink) RNA strand from Control or Nutlin -3a treated MCF7 cells was mapped to human 18q subtelomere. Histone H3K27ac, H3K4me1, and H3K4me3ChIP-Seq data sets from untreated MCF7 are also shown for these subtelomeric regions. Right panel shows a zoom in of approximately 1000bp (light blue on left panel) surrounding the p53 response element within the subtelomere.


( F ) Summary of GRO - Seq ChIP - Seq metadata analyses from human subtelomeres with p53 binding sites . Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at p53 response element itself .

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

(F) Summary of GRO-Seq and ChIP-Seq metadata analyses from human subtelomeres with p53 binding sites. Each p53 response element is located upstream of a region of bidirectional transcription which is modulated in a p53 dependent manner along with local bidirectional transcription at the p53 response element itself.