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 .

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

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


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

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

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

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

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


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

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

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

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


HER - 2 - E0771 mammary tumour 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


HER - 2 - E0771 mammary tumour 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


HER - 2 - E0771 mammary tumour 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 .

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

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


HER - 2 - E0771 mammary tumour 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


HER - 2 - E0771 mammary tumour 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


HER - 2 - E0771 mammary tumour 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


HER - 2 - E0771 mammary tumours 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


HER - 2 - E0771 mammary tumours 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 .

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

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


HER - 2 - E0771 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 O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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

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 B-MEASURED_VAR O B-MEASURED_VAR 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

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 .

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

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


c - d ) HER - 2 - E0771 mammary tumour 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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


HER - 2 CAR - T 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 .

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

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


HER - 2 - E0771 mammary tumour 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 .

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


A , B ( A ) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II - S5P IF showing that SPT5 and Pol II - S5P occupied the FOS loci after serum starvation . Zoomed - in views of the merged regions are indicated by the white arrow . ( B ) Same as Fig 1A . but for FOS DNA FISH with concurrent SPT5 and NELFE IF .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (A) Confocal imaging of FOS DNA FISH with concurrent SPT5 and Pol II-S5P IF showing that SPT5 and Pol II-S5P occupied the FOS loci after serum starvation. Zoomed-in views of the merged regions are indicated by the white arrow. (B) Same as Fig 1A. but for FOS DNA FISH with concurrent SPT5 and NELFE IF.


E Confocal images showing that SPT5 , NELFA , Pol II - S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1 % 1 , 6 - hexanediol ( abbreviation as 1 , 6 - hex ) for 30 minutes . F Violin plot showing the number of cells containing nuclear puncta in Fig 1E . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . For SPT5 , p = 0 . 1301 . For NELFA , p = 0 . 2756 . For Pol II - S5P , p = 0 . 8179 . For AFF4 , * * * p < 0 . 0001 .

O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal images showing that SPT5, NELFA, Pol II-S5P and AFF4 formed nuclear puncta in HCT 116 cells before and after treated with 1% 1,6-hexanediol (abbreviation as 1,6-hex) for 30 minutes. F Violin plot showing the number of cells containing nuclear puncta in Fig 1E. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. For SPT5, p = 0.1301. For NELFA, p = 0.2756. For Pol II-S5P, p = 0.8179. For AFF4, ***p < 0.0001. G


G - I ChIP - qPCR showing that the occupancies of Pol II ( G ) , SPT5 ( H ) , and NELFE ( I ) at the promoters and gene bodies of HSP70 , GAPDH , and MYC in HeLa cells . The HEMO gene serves as a negative control for ChIP - qPCR . Error bars represent standard error of means . Results are plotted with four technical repeats from two biological replicates . Two - tailed , unpaired Student ' s t test was performed . For Pol II ChIP , HSP70 _ promoter , * * p = 0 . 0014 ; GAPDH _ promoter , p = 0 . 8385 ; GAPDH _ gene body , * * * p < 0 . 0001 ; MYC _ promoter , p = 0 . 1765 ; MYC _ gene body , * * * p = 0 . 0002 . For SPT5 ChIP , HSP70 _ promoter , * * p = 0 . 0033 ; GAPDH _ promoter , p = 0 . 9603 ; GAPDH _ gene body , * * p = 0 . 0012 ; MYC _ promoter , p = 0 . 9898 ; MYC _ gene body , * * * p < 0 . 0001 . For NELFE ChIP , HSP70 _ promoter , p = 0 . 3913 ; GAPDH _ promoter , p = 0 . 9347 ; MYC _ promoter , p = 0 . 8558 .

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

G-I ChIP-qPCR showing that the occupancies of Pol II (G), SPT5 (H), and NELFE (I) at the promoters and gene bodies of HSP70, GAPDH, and MYC in HeLa cells. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, **p = 0.0014; GAPDH_promoter, p = 0.8385; GAPDH_gene body, ***p < 0.0001; MYC_promoter, p = 0.1765; MYC_gene body, ***p = 0.0002. For SPT5 ChIP, HSP70_promoter, **p = 0.0033; GAPDH_promoter, p = 0.9603; GAPDH_gene body, **p = 0.0012; MYC_promoter, p = 0.9898; MYC_gene body, ***p < 0.0001. For NELFE ChIP, HSP70_promoter, p = 0.3913; GAPDH_promoter, p = 0.9347; MYC_promoter, p = 0.8558.


C Normalized FRAP curves for eGFP - SPT5 . The bleaching events occurred at 0 second . Confocal images were embedded within the plot showing FRAP of the in vitro eGFP - SPT5 clusters . Results shown are from three biological replicates . The arrowhead represents the bleached region .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Normalized FRAP curves for eGFP-SPT5. The bleaching events occurred at 0 second. Confocal images were embedded within the plot showing FRAP of the in vitro eGFP-SPT5 clusters. Results shown are from three biological replicates. The arrowhead represents the bleached region.


E Live cell imaging of HEK - 293T cells expressing eGFP - SPT5 together with mCherry - SPT5 , mCherry - SPT5ΔNTR , or mCherry - SPT5ΔCTR close to endogenous level . F Violin plot showing the clusters area ( top ) and circularity ( bottom ) of SPT5 with SPT5 , SPT5ΔNTR , or SPT5ΔCTR in Fig 2E . Each n > 20 . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Live cell imaging of HEK-293T cells expressing eGFP-SPT5 together with mCherry-SPT5, mCherry-SPT5ΔNTR, or mCherry-SPT5ΔCTR close to endogenous level. F Violin plot showing the clusters area (top) and circularity (bottom) of SPT5 with SPT5, SPT5ΔNTR, or SPT5ΔCTR in Fig 2E. Each n > 20. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001. G


H Confocal images showing FRAP analysis of the NELFA - IDR - eGFP and SPT5 - CTR - mCherry clusters in vitro . The arrowhead represents the bleached and recovered cluster . I Normalized FRAP curves for NELFA - IDR - eGFP and SPT5 - CTR - mCherry clusters . The bleaching events occurred at 0 second . Results shown are from two biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal images showing FRAP analysis of the NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters in vitro. The arrowhead represents the bleached and recovered cluster. I Normalized FRAP curves for NELFA-IDR-eGFP and SPT5-CTR-mCherry clusters. The bleaching events occurred at 0 second. Results shown are from two biological replicates.


A Western blot analysis of re - expression of mCherry - SPT5 , mCherry - SPT5ΔNTR or mCherry - SPT5ΔCTR after SPT5 depletion . α - Tubulin was used as a loading control . Results are representative of two biological replicates .

O O O O O O O O O O O O O O O O O O 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 Western blot analysis of re-expression of mCherry-SPT5, mCherry-SPT5ΔNTR or mCherry-SPT5ΔCTR after SPT5 depletion. α-Tubulin was used as a loading control. Results are representative of two biological replicates.


B Confocal imaging of nascent RNA labeled by EU in HEK - 293T cells expressing SPT5 , SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion .

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

B Confocal imaging of nascent RNA labeled by EU in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion.


G , H ChIP - qPCR showing the occupancies of Pol II at the promoters and gene bodies of GAPDH ( G ) , and MYC ( H ) in HEK - 293T cells expressing SPT5 , SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion . The HEMO gene serves as a negative control for ChIP - qPCR . Error bars represent standard error of means . Results are plotted with four technical repeats from two biological replicates . Two - tailed , unpaired Student ' s t test was performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . = not significant . For GAPDH _ promoter , p value ( from left to right , < 0 . 0001 , < 0 . 0001 , < 0 . 0001 , 0 . 0044 , 0 . 8873 , 0 . 9310 , 0 . 6855 , 0 . 9615 ) ; GAPDH _ gene body , p value ( from left to right , 0 . 0370 , 0 . 0214 , 0 . 0574 , 0 . 0256 , 0 . 9536 , 0 . 8038 , 0 . 7623 , 0 . 8773 ) ; For MYC _ promoter , p value ( from left to right , 0 . 0002 , 0 . 0014 , 0 . 0004 , 0 . 0048 , 0 . 0670 , 0 . 7501 , 0 . 3482 , 0 . 3284 ) ; MYC _ gene body , p value ( from left to right , 0 . 0004 , 0 . 0013 , 0 . 0081 , 0 . 0299 , 0 . 1332 , 0 . 5839 , 0 . 4404 , 0 . 6170 ) .

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

G, H ChIP-qPCR showing the occupancies of Pol II at the promoters and gene bodies of GAPDH (G), and MYC (H) in HEK-293T cells expressing SPT5, SPT5ΔNTR or SPT5ΔCTR after SPT5 depletion. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.=not significant. For GAPDH_promoter, p value (from left to right, < 0.0001, < 0.0001, < 0.0001, 0.0044, 0.8873, 0.9310, 0.6855, 0.9615); GAPDH_gene body, p value (from left to right, 0.0370, 0.0214, 0.0574, 0.0256, 0.9536, 0.8038, 0.7623, 0.8773); For MYC_promoter, p value (from left to right, 0.0002, 0.0014, 0.0004, 0.0048, 0.0670, 0.7501, 0.3482, 0.3284); MYC_gene body, p value (from left to right, 0.0004, 0.0013, 0.0081, 0.0299, 0.1332, 0.5839, 0.4404, 0.6170).


B Fluorescence microscopy images showing the phase - separated AFF4 - IDR - eGFP and SPT5 - CTR - mCherry droplets formed in 50 mM NaCl containing buffer in the presence of 10 % PEG - 8000 . The concentration ratios are indicated as CAFF4 - IDR / CSPT5 - CTR . C Dot plot showing the droplet area in Fig 4B . Fields per condition n = 5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence microscopy images showing the phase-separated AFF4-IDR-eGFP and SPT5-CTR-mCherry droplets formed in 50 mM NaCl containing buffer in the presence of 10% PEG-8000. The concentration ratios are indicated as CAFF4-IDR/CSPT5-CTR. C Dot plot showing the droplet area in Fig 4B. Fields per condition n = 5. D


D Live cell confocal images showing FRAP of the heterotypic eGFP - AFF4 + ENL and mCherry - SPT5 droplets . The arrowhead represents the bleached and recovered droplet . E Normalized FRAP curves for eGFP - AFF4 + ENL ( green ) and mCherry - SPT5 ( purple ) in the AFF4 / ENL / SPT5 heterotypic droplets . The bleaching events occurred at 0 second . Results shown are from three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Live cell confocal images showing FRAP of the heterotypic eGFP-AFF4 + ENL and mCherry-SPT5 droplets. The arrowhead represents the bleached and recovered droplet. E Normalized FRAP curves for eGFP-AFF4 + ENL (green) and mCherry-SPT5 (purple) in the AFF4/ENL/SPT5 heterotypic droplets. The bleaching events occurred at 0 second. Results shown are from three biological replicates.


C Confocal imaging of FOS RNA FISH with concurrent SPT5 IF in wild type ( WT ) and ENL KO HCT116 cells after serum treatment . Zoomed - in views of the merged regions are indicated by the white arrow .

O O O O O O O O O O O O O O O O O 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 Confocal imaging of FOS RNA FISH with concurrent SPT5 IF in wild type (WT) and ENL KO HCT116 cells after serum treatment. Zoomed-in views of the merged regions are indicated by the white arrow.


H Confocal images showing SPT5 in nuclear puncta in WT or ENL KO cells treatment with KL - 1 . I Violin plot showing the number of cells containing nuclear puncta in Fig 5H . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal images showing SPT5 in nuclear puncta in WT or ENL KO cells treatment with KL-1. I Violin plot showing the number of cells containing nuclear puncta in Fig 5H. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. ***p < 0.001.


J - M ( J ) Confocal images showing co - localization of Pol II - S5P with SPT5 in nuclear puncta in KL - 1 treated HCT 116 cells . Results shown are representative images from three independent experiments . ( K ) Boxplots showing the mean values of the Pearson correlation coefficient of co - localization ratios of SPT5 and Pol II - S5P in Fig 5J . Central band is median ; boxes represent 1st and 3rd quartile ( 25th and 75th percentile , respectively ) and whiskers 1 . 5 × interquartile range . Results are representative of three biological replicates , each n > 20 . Two - tailed , unpaired Student ' s t test was performed . KL - 1 vs . DMSO , * * p = 0 . 0050 . ( L ) Same as Fig 5J . but for SPT5 and NELFA . ( M ) Same as Fig 5K . but for SPT5 and NELFA . Two - tailed , unpaired Student ' s t test was performed . KL - 1 vs . DMSO , * p = 0 . 0100 .

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

J-M (J) Confocal images showing co-localization of Pol II-S5P with SPT5 in nuclear puncta in KL-1 treated HCT 116 cells. Results shown are representative images from three independent experiments. (K) Boxplots showing the mean values of the Pearson correlation coefficient of co-localization ratios of SPT5 and Pol II-S5P in Fig 5J. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of three biological replicates, each n > 20. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, **p = 0.0050. (L) Same as Fig 5J. but for SPT5 and NELFA. (M) Same as Fig 5K. but for SPT5 and NELFA. Two-tailed, unpaired Student's t test was performed. KL-1 vs. DMSO, *p = 0.0100.


B Fluorescence microscopy images showing that 2 μM SPT5 - CTR - eGFP formed phase - separated droplets in the NaCl containing buffer . C Droplet number ( top ) and droplet area ( bottom ) analyses are shown . Central band is median ; boxes represent 1st and 3rd quartile ( 25th and 75th percentile , respectively ) and whiskers 1 . 5 × interquartile range . Results are representative of two biological replicates , each n > 6 . Two - tailed , unpaired Student ' s t test was performed . For droplet number , SPT5 - CTR + P - TEFb ( - ATP ) vs . SPT5 - CTR , p = * * 0 . 0015 . SPT5 - CTR + P - TEFb ( + vs . - ATP ) , * * p = 0 . 0032 . For droplet area , SPT5 - CTR + P - TEFb ( + vs . - ATP )

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

B Fluorescence microscopy images showing that 2 μM SPT5-CTR-eGFP formed phase-separated droplets in the NaCl containing buffer. C Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. For droplet number, SPT5-CTR + P-TEFb (- ATP) vs. SPT5-CTR, p = **0.0015. SPT5-CTR + P-TEFb (+ vs.- ATP), **p = 0.0032. For droplet area, SPT5-CTR + P-TEFb (+ vs. - ATP)


E Fluorescence microscopy images showing the phase - separated droplets formed in 50 mM NaCl containing buffer with 10 μM AFF4 - IDR - eGFP and 10 μM SPT5 - CTR - mCherry or SPT5 - CTR - TE - mCherry in the presence of 10 % PEG - 8000 . F Droplet number ( top ) and droplet area ( bottom ) analyses are shown . Central band is median ; boxes represent 1st and 3rd quartile ( 25th and 75th percentile , respectively ) and whiskers 1 . 5 × interquartile range . Results are representative of two biological replicates , each n > 6 . Two - tailed , unpaired Student ' s t test was performed . AFF4 - IDR + SPT5 - CTR - TE vs . AFF4 - IDR + SPT5 - CTR , * * p = 0 . 0029 .

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E Fluorescence microscopy images showing the phase-separated droplets formed in 50 mM NaCl containing buffer with 10 μM AFF4-IDR-eGFP and 10 μM SPT5-CTR-mCherry or SPT5-CTR-TE-mCherry in the presence of 10% PEG-8000. F Droplet number (top) and droplet area (bottom) analyses are shown. Central band is median; boxes represent 1st and 3rd quartile (25th and 75th percentile, respectively) and whiskers 1.5× interquartile range. Results are representative of two biological replicates, each n > 6. Two-tailed, unpaired Student's t test was performed. AFF4-IDR + SPT5-CTR-TE vs. AFF4-IDR + SPT5-CTR, **p = 0.0029.


A Confocal images showing co - localization of AFF4 with SPT5 in nuclear puncta in AFF4 wild type ( WT ) or R258W cells . B Dotplot ( left ) showing the AFF4 or SPT5 puncta area in AFF4 WT or R258W cells ( Fig 7A ) . Two - tailed , unpaired Student ' s t test was performed . AFF4 R258W vs . WT , * p = 0 . 0254 . Violin plot ( right ) showing the Pearson correlation coefficient of co - localization ratio ( Fig 7A ) . Each n > 20 . Results are representative of three biological replicates . Two - tailed , unpaired Student ' s t test was performed . AFF4 R258W vs . WT , * p = 0 . 0166 .

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A Confocal images showing co-localization of AFF4 with SPT5 in nuclear puncta in AFF4 wild type (WT) or R258W cells. B Dotplot (left) showing the AFF4 or SPT5 puncta area in AFF4 WT or R258W cells (Fig 7A). Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0254. Violin plot (right) showing the Pearson correlation coefficient of co-localization ratio (Fig 7A). Each n > 20. Results are representative of three biological replicates. Two-tailed, unpaired Student's t test was performed. AFF4 R258W vs. WT, *p = 0.0166. C


C Droplet experiments showing that CHOPS syndrome patient missense mutations reinforced AFF4 - ALF domain droplet formation . D Dotplot showing the droplet areas in Fig 7C . Fields per condition n = 5 . Two - tailed , unpaired Student ' s t test was performed . T254S , T254A or R258W vs . WT , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E AFF4 - ALF - mCherry condensed fraction as a function of AFF4 - ALF - mCherry concentration for experiments in Fig 7C . Error bars represent standard deviations . Fields per condition n = 5 . Two - tailed , unpaired Student ' s t test was performed . T254S , T254A or R258W vs . WT , * p < 0 . 05 , * * p < 0 . 01 .

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C Droplet experiments showing that CHOPS syndrome patient missense mutations reinforced AFF4-ALF domain droplet formation. D Dotplot showing the droplet areas in Fig 7C. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01, ***p < 0.001. E AFF4-ALF-mCherry condensed fraction as a function of AFF4-ALF-mCherry concentration for experiments in Fig 7C. Error bars represent standard deviations. Fields per condition n = 5. Two-tailed, unpaired Student's t test was performed. T254S, T254A or R258W vs. WT, *p < 0.05, **p < 0.01. F


K - N ChIP - qPCR showing the occupancies of Pol II ( K ) , AFF4 ( L ) , SPT5 ( M ) , and PAF1 ( N ) at the promoter and gene body of HSP70 in AFF4 WT and R258W HEK - 293T cells after heat shock for 2 hours . The HEMO gene serves as a negative control for ChIP - qPCR . Error bars represent standard error of means . Results are plotted with four technical repeats from two biological replicates . Two - tailed , unpaired Student ' s t test was performed . For Pol II ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene body , * * * p = 0 . 0002 . For AFF4 ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene body , * * * p < 0 . 0001 . For SPT5 ChIP , HSP70 _ promoter , * * * p < 0 . 0001 ; HSP70 _ gene body , * * p = 0 . 0086 . For PAF1 ChIP , HSP70 _ promoter , * * * p = 0 . 0007 ; HSP70 _ gene body , * * p = 0 . 0021 .

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K-N ChIP-qPCR showing the occupancies of Pol II (K), AFF4 (L), SPT5 (M), and PAF1 (N) at the promoter and gene body of HSP70 in AFF4 WT and R258W HEK-293T cells after heat shock for 2 hours. The HEMO gene serves as a negative control for ChIP-qPCR. Error bars represent standard error of means. Results are plotted with four technical repeats from two biological replicates. Two-tailed, unpaired Student's t test was performed. For Pol II ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p = 0.0002. For AFF4 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, ***p < 0.0001. For SPT5 ChIP, HSP70_promoter, ***p < 0.0001; HSP70_gene body, **p = 0.0086. For PAF1 ChIP, HSP70_promoter, ***p = 0.0007; HSP70_gene body, **p = 0.0021.


B ) Western blot detection of DOT1L and TUBULIN ( TUB ) in whole testicular protein extracts from CTL and Dot1l - KO ( KO ) adult mice .

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B) Western blot detection of DOT1L and TUBULIN (TUB) in whole testicular protein extracts from CTL and Dot1l-KO (KO) adult mice.


D ) Western blot detection of DOT1L and H3K79me2 in Dot1l - KO ( KO ) and CTL germ cells . Normalization was performed by detecting the membrane with anti - TUBULIN ( TUB ) . SC = primary spermatocytes , RS = round spermatids .

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D) Western blot detection of DOT1L and H3K79me2 in Dot1l-KO (KO) and CTL germ cells. Normalization was performed by detecting the membrane with anti-TUBULIN (TUB). SC = primary spermatocytes, RS = round spermatids.


B ) Histology of Dot1l - KO and CTL testes ( periodic acid - Schiff staining ) . Scale bars indicate 200µm .

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B) Histology of Dot1l-KO and CTL testes (periodic acid-Schiff staining). Scale bars indicate 200µm.


D ) Results from the tests of fertility ( natural mating ) of Dot1l - KO and CTL males ( 7 males in each group mated for ~ 3 months with wild - type females ) . Schematic diagrams representing the number of litters and the litter size from progeny of Dot1l - KO or CTL males ( mean ± SEM ) . The p - value obtained with a Student t - test is indicated on each graph .

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D) Results from the tests of fertility (natural mating) of Dot1l-KO and CTL males (7 males in each group mated for ~3 months with wild-type females). Schematic diagrams representing the number of litters and the litter size from progeny of Dot1l-KO or CTL males (mean ± SEM).The p-value obtained with a Student t-test is indicated on each graph.


C ) Scatter plots ( mean values following angular transformation ± SEM ) of the percentage of motile and progressively motile spermatozoa in Dot1l - KO and CTL adult mice ( N = 12 biological replicates for CTL and 10 biological replicates for Dot1l - KO ) obtained following CASA ( computer - assisted sperm analysis ) . * * * p - value < 0 . 0005 ( Mann Whitney test performed after angular transformation of the percentages ) .

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C) Scatter plots (mean values following angular transformation ± SEM) of the percentage of motile and progressively motile spermatozoa in Dot1l-KO and CTL adult mice (N= 12 biological replicates for CTL and 10 biological replicates for Dot1l-KO) obtained following CASA (computer-assisted sperm analysis). ***p-value <0.0005 (Mann Whitney test performed after angular transformation of the percentages).


F ) Western blot detection of transition protein 2 ( TNP2 ) in spermatozoa from Dot1l - KO and CTL males . The same quantity of material was loaded in each well ( i . e . extracts from 2 million spermatozoa ) .

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F) Western blot detection of transition protein 2 (TNP2) in spermatozoa from Dot1l-KO and CTL males. The same quantity of material was loaded in each well (i.e. extracts from 2 million spermatozoa).


G ) Coomassie - stained protamine extracts from CTL and Dot1l - KO spermatozoa following acid urea gel electrophoresis ( same gel , two different intensities ) . The same quantity of material has been loaded in each well ( i . e . extracts from 1 . 4 million spermatozoa ) . Protamine 1 and 2 bands ( PRM1 and PRM2 , respectively ) are detected at the bottom of the gel . The rectangle highlights the bands that are likely immature forms of Protamine 2 ( i . e . non - cleaved precursor forms ) . The right panel shows a western blot detection using anti - PRM2 antibody which confirms that one of the high molecular weight band only observed in Dot1l - KO spermatozoa is an immature form of Protamine 2 ( Pre - PRM2 ) . The corresponding Coomassie - stained gel is also shown .

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G) Coomassie-stained protamine extracts from CTL and Dot1l-KO spermatozoa following acid urea gel electrophoresis (same gel, two different intensities). The same quantity of material has been loaded in each well (i.e. extracts from 1.4 million spermatozoa). Protamine 1 and 2 bands (PRM1 and PRM2, respectively) are detected at the bottom of the gel. The rectangle highlights the bands that are likely immature forms of Protamine 2 (i.e. non-cleaved precursor forms). The right panel shows a western blot detection using anti-PRM2 antibody which confirms that one of the high molecular weight band only observed in Dot1l-KO spermatozoa is an immature form of Protamine 2 (Pre-PRM2). The corresponding Coomassie-stained gel is also shown.


H ) Quantification of histones TH2B and H3 ( N = 11 and 10 biological replicates for CTL and KO , respectively ) , of TNP2 ( N = 8 biological replicates for CTL and for KO ) and of PRM1 / PRM2 ratio ( N = 3 biological replicates for CTL and for KO ) in Dot1l - KO and CTL spermatozoa , related to Fig 3E - G . The data shown are normalized to control mean values ( ± SEM ) . * p - value < 0 . 05 , * * * p - value < 0 . 0005 ( unpaired t - tests ) . A . U . = arbitrary units .

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H) Quantification of histones TH2B and H3 (N= 11 and 10 biological replicates for CTL and KO, respectively), of TNP2 (N= 8 biological replicates for CTL and for KO) and of PRM1/PRM2 ratio (N= 3 biological replicates for CTL and for KO) in Dot1l-KO and CTL spermatozoa, related to Fig 3E-G. The data shown are normalized to control mean values (± SEM). *p-value <0.05, ***p-value <0.0005 (unpaired t-tests). A.U. = arbitrary units.


( A , B ) Bar plots showing the quantification of post - translational modifications ( PTMs ) in histones H3 and H4 in ES ( elongating / condensing spermatids ) by LC - MS / MS . After normalization to be at constant amount of histone H3 or H4 in each analyzed sample mass spectrometry signals were divided by the average signal in both conditions ( CTL and KO ) , so as to be able to represent all peptides in the same figure , whatever their MS intensity . Quantitative data obtained on biological replicates ( N = 3 for CTL and 3 for KO ) were plotted as dots , and the height of the bars indicates the average value ( mean ± standard deviation , SD ) . T - tests were performed to estimate the possible significant difference of modified peptide abundances between the two genotypes . * p - value < 0 . 05 , * * p - value < 0 . 005 and * * * p - value < 0 . 0005 . When interpreting our mass spectrometry data against the database MS _ histoneDB , we were able to identify various H3 variants , namely canonical H3 and variants H3 . 3 , testis - specific H3t and H3mm13 . " H3 " indicates sequences shared among several variants ; " H3 . 3 " , " H3t " or " H3mm13 " , each variant . A . U . = arbitrary units .

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(A, B) Bar plots showing the quantification of post-translational modifications (PTMs) in histones H3 and H4 in ES (elongating/condensing spermatids) by LC-MS/MS. After normalization to be at constant amount of histone H3 or H4 in each analyzed sample mass spectrometry signals were divided by the average signal in both conditions (CTL and KO), so as to be able to represent all peptides in the same figure, whatever their MS intensity. Quantitative data obtained on biological replicates (N= 3 for CTL and 3 for KO) were plotted as dots, and the height of the bars indicates the average value (mean ± standard deviation, SD). T-tests were performed to estimate the possible significant difference of modified peptide abundances between the two genotypes. *p-value <0.05, ** p-value<0.005 and ***p-value <0.0005. When interpreting our mass spectrometry data against the database MS_histoneDB, we were able to identify various H3 variants, namely canonical H3 and variants H3.3, testis-specific H3t and H3mm13. "H3" indicates sequences shared among several variants; "H3.3", "H3t" or "H3mm13", each variant. A.U. = arbitrary units.


D ) H3K79me2 enrichment profile at selected deregulated genes , in two biological replicates of ChIP - Seq performed in wild type RS ( Rep # 1 and 2 ) . The input value is shown in grey .

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D) H3K79me2 enrichment profile at selected deregulated genes, in two biological replicates of ChIP-Seq performed in wild type RS (Rep#1 and 2). The input value is shown in grey.


Representative confocal images of CAD cells exposed for 4 h to the indicated treatments . Cells were fixed and then stained with WGA ( green ) and DAPI ( blue ) . Percentage of TNT - connected CAD cells with each of the indicated treatments .

Representative confocal images of CAD cells exposed for 4 h to the indicated treatments. Cells were fixed and then stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments.


Top : Schematic of the co - culture system used to assess vesicle transfer from donor ( labeled with DiD ) to acceptor ( labeled with CTG ) cells . After labeling , cells were detached , replated in 1 : 1 ratio and cultured in control conditions or treated with 200 µM H2O2 or 200 ng / mL Wnt7a for 4 h . Middle : Representative confocal images of co - cultured donor and acceptor CAD cells . Each image corresponds to the projection of the entire Z - stack . Bottom : The insets correspond to the projection of selected Z - stacks of the area marked with a yellow square in each condition to better illustrate the transferred vesicles in acceptor cells . Yellow arrows point to DiD - labeled vesicles inside acceptor cells . Percentage of acceptor cells containing DiD - labeled vesicles in the co - culture systems with the indicated treatments .

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Top: Schematic of the co-culture system used to assess vesicle transfer from donor (labeled with DiD) to acceptor (labeled with CTG) cells. After labeling, cells were detached, replated in 1:1 ratio and cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Middle: Representative confocal images of co-cultured donor and acceptor CAD cells. Each image corresponds to the projection of the entire Z-stack. Bottom: The insets correspond to the projection of selected Z-stacks of the area marked with a yellow square in each condition to better illustrate the transferred vesicles in acceptor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.


Top : Schematic of the culture system used to assess vesicle transfer by secretion . Donor cells ( labeled with DiD ) were cultured in control conditions or treated with 200 µM H2O2 or 200 ng / mL Wnt7a for 4 h . Then acceptor cells ( labeled with CTG ) were treated for 4 h with the conditioned media from donor cells . Bottom : Representative confocal images of acceptor CAD cells treated with the conditioned media from donor cells . Yellow arrows point to DiD - labeled vesicles inside acceptor cells . Percentage of acceptor cells containing DiD - labeled vesicles after being cultured for 4 h with the conditioned media of treated donor cells .

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Top: Schematic of the culture system used to assess vesicle transfer by secretion. Donor cells (labeled with DiD) were cultured in control conditions or treated with 200 µM H2O2 or 200 ng/mL Wnt7a for 4 h. Then acceptor cells (labeled with CTG) were treated for 4 h with the conditioned media from donor cells. Bottom: Representative confocal images of acceptor CAD cells treated with the conditioned media from donor cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor cells.


Representative confocal images of CAD cells cultured in control conditions or treated with 0 . 2 % DMSO , 200 µM H2O2 , 200 ng / mL Wnt7a , 10 μM KN - 93 , 200 ng / mL Wnt7a plus 10 μM KN - 93 , 1 μM TAT or 200 ng / mL Wnt7a plus 1 μM TAT for 4 h . Cells were fixed and stained with WGA ( green ) and DAPI ( blue ) . Percentage of TNT - connected CAD cells with each of the indicated treatments .

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Representative confocal images of CAD cells cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Cells were fixed and stained with WGA (green) and DAPI (blue). Percentage of TNT-connected CAD cells with each of the indicated treatments.


Representative confocal images of co - cultured donor and acceptor CAD cells . Cells were co - cultured in control conditions or treated with 0 . 2 % DMSO , 200 µM H2O2 , 200 ng / mL Wnt7a , 10 μM KN - 93 , 200 ng / mL Wnt7a plus 10 μM KN - 93 , 1 μM TAT or 200 ng / mL Wnt7a plus 1 μM TAT for 4 h . Yellow arrows point to DiD - labeled vesicles inside acceptor cells . Percentage of acceptor cells containing DiD - labeled vesicles in the co - culture systems with the indicated treatments .

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

Representative confocal images of co-cultured donor and acceptor CAD cells. Cells were co-cultured in control conditions or treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. Yellow arrows point to DiD-labeled vesicles inside acceptor cells. Percentage of acceptor cells containing DiD-labeled vesicles in the co-culture systems with the indicated treatments.


Representative confocal images of CAD cells stained for vinculin ( red ) and HCS CellMask ™ Blue after being treated with 0 . 2 % DMSO , 200 µM H2O2 , 200 ng / mL Wnt7a , 10 μM KN - 93 , 200 ng / mL Wnt7a plus 10 μM KN - 93 , 1 μM TAT or 200 ng / mL Wnt7a plus 1 μM TAT for 4 h . The ring circumscribed between the green lines mark the area where the vinculin puncta were counted for each cell . This area is defined by the board of the HCS CellMask ™ Blue staining and the vinculin staining around it . Average number of vinculin puncta per cell with the indicated treatments .

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

Representative confocal images of CAD cells stained for vinculin (red) and HCS CellMask™ Blue after being treated with 0.2% DMSO, 200 µM H2O2, 200 ng/mL Wnt7a, 10 μM KN-93, 200 ng/mL Wnt7a plus 10 μM KN-93, 1 μM TAT or 200 ng/mL Wnt7a plus 1 μM TAT for 4 h. The ring circumscribed between the green lines mark the area where the vinculin puncta were counted for each cell. This area is defined by the board of the HCS CellMask™ Blue staining and the vinculin staining around it. Average number of vinculin puncta per cell with the indicated treatments.


Representative confocal images of CAD cells expressing the indicated GFP - fusion proteins . Top : Cells showing WGA ( white ) and DAPI ( blue ) staining . Bottom : Cells showing phalloidin ( red ) and DAPI ( blue ) staining . Note that the expression pattern of βCaMKII WT and the mutants K43R and A303R , is rather punctate than diffused .

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Representative confocal images of CAD cells expressing the indicated GFP-fusion proteins. Top: Cells showing WGA (white) and DAPI (blue) staining. Bottom: Cells showing phalloidin (red) and DAPI (blue) staining. Note that the expression pattern of βCaMKII WT and the mutants K43R and A303R, is rather punctate than diffused.


Percentage of TNT - connected CAD cells 24 h after transfection with indicated GFP - fusion proteins .

Percentage of TNT-connected CAD cells 24 h after transfection with indicated GFP-fusion proteins.


Percentage of transfection rate of indicated GFP - fusion proteins in CAD cells .

Percentage of transfection rate of indicated GFP-fusion proteins in CAD cells.


Percentage of acceptor cells containing DiD - labeled vesicles when acceptor cells were co - cultured with donor cells expressing the indicated GFP - fusion proteins .

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

Percentage of acceptor cells containing DiD-labeled vesicles when acceptor cells were co-cultured with donor cells expressing the indicated GFP-fusion proteins.


Top : Schematic of the co - culture system used to assess vesicle transfer in transfected cells . Donor cell were transfected with the indicated GFP - fusion proteins and then labeled with DiD . Acceptor cells were transfected with a H2B - mCherry plasmid . After 24 h , the cells were detached , replated in 1 : 1 ratio and cultured for 4 h . Cells were then fixed and stained with HCS CellMask ™ Blue . Bottom : Representative confocal images of co - cultured donor and acceptor CAD cells . Yellow arrows point to DiD - labeled vesicles inside acceptor cells .

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Top: Schematic of the co-culture system used to assess vesicle transfer in transfected cells. Donor cell were transfected with the indicated GFP-fusion proteins and then labeled with DiD. Acceptor cells were transfected with a H2B-mCherry plasmid. After 24 h, the cells were detached, replated in 1:1 ratio and cultured for 4 h. Cells were then fixed and stained with HCS CellMask™ Blue. Bottom: Representative confocal images of co-cultured donor and acceptor CAD cells. Yellow arrows point to DiD-labeled vesicles inside acceptor cells.


Time lapses showing a TNT connecting two transfected cells . Top : GFP - vector transfected cells . Middle : GFP - βCaMKII WT transfected cells . Bottom : GFP - βCaMKII T287D transfected cells . Cells were stained with WGA ( white ) . Yellow arrowheads point out a TNT in each condition . Scale bars represent 10 μm .

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Time lapses showing a TNT connecting two transfected cells. Top: GFP-vector transfected cells. Middle: GFP-βCaMKII WT transfected cells. Bottom: GFP-βCaMKII T287D transfected cells. Cells were stained with WGA (white). Yellow arrowheads point out a TNT in each condition. Scale bars represent 10 μm.


TNT ' s average duration between GFP - vector , GFP - βCaMKII WT and GFP - βCaMKII T287D transfected cells . Graph represents the average lifetime of 15 - 20 TNTs per condition ± SEM . Statistical significance was calculated with respect to control ( Ctrl ) ; * * p ≤ 0 . 01 , NS = not significant ( one - way ANOVA ) .

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TNT's average duration between GFP-vector, GFP-βCaMKII WT and GFP-βCaMKII T287D transfected cells. Graph represents the average lifetime of 15-20 TNTs per condition ± SEM. Statistical significance was calculated with respect to control (Ctrl); ** p ≤ 0.01, NS = not significant (one-way ANOVA).


CODIM super - resolution images ( 3 right side panels ) of TNTs shown in the confocal expanded field images ( left panels ) of transfected cells showing expression of indicated GFP - tagged proteins ( green ) . Cells were stained for F - actin with phalloidin ( red ) . Scale bars in the expanded fields represent 5 µm and 1 µm in the insets .

CODIM super-resolution images (3 right side panels) of TNTs shown in the confocal expanded field images (left panels) of transfected cells showing expression of indicated GFP-tagged proteins (green). Cells were stained for F-actin with phalloidin (red). Scale bars in the expanded fields represent 5 µm and 1 µm in the insets.


Confocal images and 3D reconstruction showing the intracellular localization of endogenous βCaMKII ( green ) in CAD cells . Cells were stained with DAPI ( blue ) and phalloidin ( red ) . Yellow arrows point to βCaMKII - positive puncta inside a TNT . Scale bars in the expanded fields represent 10 µm in the micrographs and 1 µm in the 3D projection .

Confocal images and 3D reconstruction showing the intracellular localization of endogenous βCaMKII (green) in CAD cells. Cells were stained with DAPI (blue) and phalloidin (red). Yellow arrows point to βCaMKII-positive puncta inside a TNT. Scale bars in the expanded fields represent 10 µm in the micrographs and 1 µm in the 3D projection.


Confocal images of untransfected and GFP - βCaMKII WT transfected CAD cells ( green ) , treated or not with Wnt7a . Top : Cells were stained with phalloidin ( red ) and DAPI ( blue ) . Middle : Phalloidin staining ( white ) of the cells is shown . Bottom : Pseudocolor images of cells stained with phalloidin highlight the differences in F - actin levels among the treatments . Scale bars represent 5 μm . Anti - actin immunoblot and bar graph showing changes in the ratio between G - actin ( in supernatant fraction , s ) and F - actin ( in the pellet fraction , p ) of CAD cells untransfected or transfected with GFP - βCaMKII WT plasmid and treated or not with Wnt7a .

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Confocal images of untransfected and GFP-βCaMKII WT transfected CAD cells (green), treated or not with Wnt7a. Top: Cells were stained with phalloidin (red) and DAPI (blue). Middle: Phalloidin staining (white) of the cells is shown. Bottom: Pseudocolor images of cells stained with phalloidin highlight the differences in F-actin levels among the treatments. Scale bars represent 5 μm. Anti-actin immunoblot and bar graph showing changes in the ratio between G-actin (in supernatant fraction, s) and F-actin (in the pellet fraction, p) of CAD cells untransfected or transfected with GFP-βCaMKII WT plasmid and treated or not with Wnt7a.


Confocal images of CAD cells transfected with GFP - βCaMKII WT plasmid ( green ) and treated or not with Wnt7a . Cells were fixed and then stained with phalloidin ( red ) and DAPI ( blue ) . The insets ( right panels ) show a magnified image of the area depicted in the expanded field images ( left ) . 3D reconstruction shows co - localization of GFP / phalloidin signals . Scale bars represent 10 µm .

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Confocal images of CAD cells transfected with GFP-βCaMKII WT plasmid (green) and treated or not with Wnt7a. Cells were fixed and then stained with phalloidin (red) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field images (left). 3D reconstruction shows co-localization of GFP/phalloidin signals. Scale bars represent 10 µm.


Top : Schematic of the culture systems used to assess cell - to - cell contact - dependent or secretion - mediated transfer of vesicles in primary neurons . After dissection , neurons were labeled in suspension with DiI ( donor , D ) or CTG ( acceptor , A ) . In the co - culture system , neurons were plated in 1 : 1 ratio and cultured for 20 h and then treated with 200 ng / mL of Wnt5a or Wnt7a for 4 h . In the secretion - based system , donor neurons cultured alone for 20 h were treated with 200 ng / mL of Wnt5a or Wnt7a for 4 h , and the medium was then collected and replaced for the medium of acceptor neurons cultured separately . Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h . Bottom : Representative confocal images of co - cultured donor and acceptor neurons . The insets ( right panels ) show a magnification of the area depicted in the expanded field images ( left ) . Yellow arrows point to DiI - labeled vesicles inside acceptor neurons .

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Top: Schematic of the culture systems used to assess cell-to-cell contact-dependent or secretion-mediated transfer of vesicles in primary neurons. After dissection, neurons were labeled in suspension with DiI (donor, D) or CTG (acceptor, A). In the co-culture system, neurons were plated in 1:1 ratio and cultured for 20 h and then treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h. In the secretion-based system, donor neurons cultured alone for 20 h were treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h, and the medium was then collected and replaced for the medium of acceptor neurons cultured separately. Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h. Bottom: Representative confocal images of co-cultured donor and acceptor neurons. The insets (right panels) show a magnification of the area depicted in the expanded field images (left). Yellow arrows point to DiI-labeled vesicles inside acceptor neurons.


Percentage of acceptor neurons containing DiI - labeled vesicles after being co - cultured with donor neurons and incubated for 4 h with the indicated treatments . Percentage of acceptor neurons containing DiI - labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons .

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

Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments. Percentage of acceptor neurons containing DiI-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons.


Confocal image showing a donor ( D ) and an acceptor ( A ) neuron connected by TNT - like structures ( yellow arrowhead in the merged panel ) . Neurons were stained with WGA ( white ) and DAPI ( blue ) . The insets ( right panels ) show a magnified image of the area depicted in the expanded field ( left ) .

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Confocal image showing a donor (D) and an acceptor (A) neuron connected by TNT-like structures (yellow arrowhead in the merged panel). Neurons were stained with WGA (white) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field (left).


Representative confocal images of co - cultured donor ( D , labeled with DiI ) and acceptor ( A , labeled with CTG ) neurons treated or not with 200 ng / mL of Wnt5a , 200 ng / mL sFRP - 2 or 200 ng / mL Wnt5a plus 200 ng / mL sFRP - 2 for 4 h . Yellow arrows point to DiI - labeled vesicles inside acceptor neurons . Percentage of acceptor neurons containing DiI - labeled vesicles after being co - cultured with donor neurons and incubated for 4 h with the indicated treatments .

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

Representative confocal images of co-cultured donor (D, labeled with DiI) and acceptor (A, labeled with CTG) neurons treated or not with 200 ng/mL of Wnt5a, 200 ng/mL sFRP-2 or 200 ng/mL Wnt5a plus 200 ng/mL sFRP-2 for 4 h. Yellow arrows point to DiI-labeled vesicles inside acceptor neurons. Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments.


Primary cortical neurons at 1 DIV were fixed and labeled with MAP - 2 ( green ) , β - III - tubulin ( red ) and WGA ( white ) . The inset shows a magnified image of the area depicted in the merged panel . Yellow arrowheads point out TNT - like structures .

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Primary cortical neurons at 1 DIV were fixed and labeled with MAP-2 (green), β-III-tubulin (red) and WGA (white). The inset shows a magnified image of the area depicted in the merged panel. Yellow arrowheads point out TNT-like structures.


Transfer of α - syn fibrils is shown in neurons at 1 DIV . Cells were loaded in suspension with either Alexa - 568 α - syn fibrils ( red ) or Alexa - 488 α - syn fibrils ( green ) and cultured together . The insets ( right panels ) show a magnification of the area depicted in the expanded field image ( left ) . Yellow arrows point to red and green α - syn puncta contained in the soma of a neuron . Yellow arrowhead points to a TNT - like connection .

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Transfer of α-syn fibrils is shown in neurons at 1 DIV. Cells were loaded in suspension with either Alexa-568 α-syn fibrils (red) or Alexa-488 α-syn fibrils (green) and cultured together. The insets (right panels) show a magnification of the area depicted in the expanded field image (left). Yellow arrows point to red and green α-syn puncta contained in the soma of a neuron. Yellow arrowhead points to a TNT-like connection.


A TNT - like connection containing Alexa - 488 α - syn positive puncta is found in neurons at 1 DIV stained with phalloidin ( white ) and DAPI ( blue ) . The insets ( right panels ) show the 3D reconstruction of the area depicted in the expanded field image ( left ) .

A TNT-like connection containing Alexa-488 α-syn positive puncta is found in neurons at 1 DIV stained with phalloidin (white) and DAPI (blue). The insets (right panels) show the 3D reconstruction of the area depicted in the expanded field image (left).


Representative confocal images of 1 DIV co - cultured donor ( D , loaded with Alexa - 568 α - syn fibrils ) and acceptor ( A , labeled with CTG ) neurons . After 20 h post - plating , neurons were treated with 200 ng / mL Wnt5a for 4 h . The insets ( right panels ) show a magnification of the area depicted in the expanded field ( left ) . Yellow arrows point to α - syn puncta inside acceptor neurons .

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Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. After 20 h post-plating, neurons were treated with 200 ng/mL Wnt5a for 4 h. The insets (right panels) show a magnification of the area depicted in the expanded field (left). Yellow arrows point to α-syn puncta inside acceptor neurons.


Percentage of acceptor neurons containing α - syn puncta for each of the indicated treatments .

Percentage of acceptor neurons containing α-syn puncta for each of the indicated treatments.


Time lapses of neurons loaded with Alexa - 568 α - syn fibrils and cultured in control conditions or treated with 200 ng / mL Wnt5a . Neuron - to - neuron transfer of α - syn puncta ( yellow arrows ) through a TNT - like structure ( yellow arrowheads ) is shown upon Wnt5a treatment .

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Time lapses of neurons loaded with Alexa-568 α-syn fibrils and cultured in control conditions or treated with 200 ng/mL Wnt5a. Neuron-to-neuron transfer of α-syn puncta (yellow arrows) through a TNT-like structure (yellow arrowheads) is shown upon Wnt5a treatment.


Representative confocal images of 1 DIV co - cultured donor ( D , loaded with Alexa - 568 α - syn fibrils ) and acceptor ( A , labeled with CTG ) neurons . Neurons were obtained from WT or βCaMKII K . O . mice . Yellow arrows point to α - syn puncta inside acceptor neurons in both conditions .

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Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. Neurons were obtained from WT or βCaMKII K.O. mice. Yellow arrows point to α-syn puncta inside acceptor neurons in both conditions.


A Super - resolution fluorescence microscopy images showing relative distribution between MATR3 and DAPI . Scale bars , 5μm ( Upper ) or 0 . 5μm ( Lower ) . Arrows point to the representative MATR3 / DAPI co - staining regions . B Coefficient of correlation between MATR3 and histone modification H3K9me3 ( n = 105 ) , H3K9me2 ( n = 98 ) , H3K27me3 ( n = 107 ) , H3K27ac ( n = 97 ) and H3K4me3 ( n = 99 ) in AML12 cells . Quantifications were performed on randomly selected ROIs in cell nuclei . Each point represents one cell .

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A Super-resolution fluorescence microscopy images showing relative distribution between MATR3 and DAPI. Scale bars, 5μm (Upper) or 0.5μm (Lower). Arrows point to the representative MATR3/DAPI co-staining regions. B Coefficient of correlation between MATR3 and histone modification H3K9me3 (n=105), H3K9me2 (n=98), H3K27me3 (n=107), H3K27ac (n=97) and H3K4me3 (n=99) in AML12 cells. Quantifications were performed on randomly selected ROIs in cell nuclei. Each point represents one cell.


C ( Upper ) Schematic diagram of dox - inducible shRNA system for MATR3 knockdown and MATR3 rescue in AML12 cells . ( Lower ) Western blotting detected the expression level of MATR3 after 3 days of Dox treatment ( + Dox ) and followed by 3 days of Dox removal ( ± Dox ) in AML12 cells . Rep , replicate .

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C (Upper) Schematic diagram of dox-inducible shRNA system for MATR3 knockdown and MATR3 rescue in AML12 cells. (Lower) Western blotting detected the expression level of MATR3 after 3 days of Dox treatment (+Dox) and followed by 3 days of Dox removal (±Dox) in AML12 cells. Rep, replicate.


D ( Upper ) Representative cross - section images showing distribution of histone modifications upon Ctrl and MATR3 knock down ( + Dox ) . ( Lower ) Quantify the distribution pattern of histone modifications by Standard Deviation of Pixel Intensity in cell nuclei . For H3K9me3 , n = 102 ( Ctrl ) or 84 ( + Dox ) ; for H3K9me2 , n = 100 ( Ctrl ) or 101 ( + Dox ) ; for H3K27me3 , n = 98 ( Ctrl ) or 98 ( + Dox ) ; for H3K27ac , n = 117 ( Ctrl ) or 124 ( + Dox ) ; for H3K27me3 , n = 98 ( Ctrl ) or 98 ( + Dox ) ; for H3K4me3 , n = 107 ( Ctrl ) or 97 ( + Dox ) . Each point represents one cell . The P values were calculated using unpaired two - tailed Student ' s t test ; ns , not significant , * p < 0 . 05 , * * * * p < 0 . 0001 . Error bars indicate mean ± s . e . m .

D (Upper) Representative cross-section images showing distribution of histone modifications upon Ctrl and MATR3 knock down (+Dox). (Lower) Quantify the distribution pattern of histone modifications by Standard Deviation of Pixel Intensity in cell nuclei. For H3K9me3, n=102 (Ctrl) or 84 (+Dox); for H3K9me2, n=100 (Ctrl) or 101 (+Dox); for H3K27me3, n=98 (Ctrl) or 98 (+Dox); for H3K27ac, n=117 (Ctrl) or 124 (+Dox); for H3K27me3, n=98 (Ctrl) or 98 (+Dox); for H3K4me3, n=107 (Ctrl) or 97 (+Dox). Each point represents one cell. The P values were calculated using unpaired two-tailed Student's t test; ns, not significant, *p<0.05, ****p<0.0001. Error bars indicate mean ± s.e.m.