A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L - MICA - 129Met and an L - MICA - 129Val clone . L - con cells served as a negative and K562 cells as a positive control . The means of specific lysis of triplicates plus SD at different E : T ratios ( 200 : 1 to 3 : 1 ) were measured in an 51chromium - release assay . The MICA expression intensity and the binding of a recombinant NKG2D - Fc fusion protein to the target cells were determined in parallel by flow cytometry , and the MFIs are indicated .

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

A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L-MICA-129Met and an L-MICA-129Val clone. L-con cells served as a negative and K562 cells as a positive control. The means of specific lysis of triplicates plus SD at different E:T ratios (200:1 to 3:1) were measured in an 51chromium-release assay. The MICA expression intensity and the binding of a recombinant NKG2D-Fc fusion protein to the target cells were determined in parallel by flow cytometry, and the MFIs are indicated.


The IFNγ expression of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells exposed for 4 h to L - MICA - 129Met ( n = 23 ) or L - MICA - 129Val clones ( n = 23 ) was determined by flow cytometry . The IFNγ expression was analyzed after gating on CD56brightCD16 − NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16 − NK cells ( upper panels ) or the proportion of IFNγ + CD56brightCD16 − NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

The IFNγ expression of purified IL-2-stimulated (100 U/ml for 4 days) NK cells exposed for 4 h to L-MICA-129Met (n = 23) or L-MICA-129Val clones (n = 23) was determined by flow cytometry. The IFNγ expression was analyzed after gating on CD56brightCD16−NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16−NK cells (upper panels) or the proportion of IFNγ+CD56brightCD16−NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


The IFNγ release of purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days ) co - cultured with L - MICA - 129Met ( n = 34 ) or L - MICA - 129Val clones ( n = 32 ) for 24 h was measured in the supernatant by ELISA . In parallel , the MICA expression intensity on target cells was determined by flow cytometry . The linear regressions of IFNγ release ( pg / ml ) by NK cells and MICA expression on targets ( MFI ) are displayed for the L - MICA - 129Met clones ( left panel ) and the L - MICA - 129Val clones ( right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

The IFNγ release of purified IL-2-stimulated NK cells (100 U/ml for 4 days) co-cultured with L-MICA-129Met (n = 34) or L-MICA-129Val clones (n = 32) for 24 h was measured in the supernatant by ELISA. In parallel, the MICA expression intensity on target cells was determined by flow cytometry. The linear regressions of IFNγ release (pg/ml) by NK cells and MICA expression on targets (MFI) are displayed for the L-MICA-129Met clones (left panel) and the L-MICA-129Val clones (right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


NKG2D expression on purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 25 ) or L - MICA - 129Val clones ( n = 25 ) for 0 , 4 , and 24 h was analyzed by flow cytometry . NK cells ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating of CD3 − CD56 + cells . The means and SD of the percentage of NKG2D + NK cells ( left panel ) and of the MFI of NKG2D ( right panel ) are shown . Differences between the groups were analyzed by repeated measures ANOVA , and P - values for pairwise comparisons are indicated .

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

NKG2D expression on purified IL-2-stimulated NK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 25) or L-MICA-129Val clones (n = 25) for 0, 4, and 24 h was analyzed by flow cytometry. NK cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating of CD3−CD56+ cells. The means and SD of the percentage of NKG2D+ NK cells (left panel) and of the MFI of NKG2D (right panel) are shown. Differences between the groups were analyzed by repeated measures ANOVA, and P-values for pairwise comparisons are indicated.


MACS - separated CD8 + T cells were cultured in triplicate on an immobilized anti - CD3 mAb ( 0 . 005 μg / ml [ upper panel ] or 0 . 01 μg / ml [ lower panel ] ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , and OVA - Fc proteins at various concentrations ( 1 . 0 , 0 . 5 , 0 . 1 , 0 . 0 μg / ml ) . After 72 h , 25 % of the supernatant was harvested and IL - 2 concentrations were measured by ELISA . The harvested medium was replaced by the same volume containing 1 μCi 3H - labeled thymidine . After 12 h , the plates were completely harvested and the DNA - bound radioactivity was determined . The means and SD of the stimulation index ( SI ) are displayed ( n = 4 ) . Significant differences between MICA - 129Met / Val - Fc and OVA - Fc proteins were found when the antigen - specific signal ( anti - CD3 ) was limited ( * P < 0 . 05 , t - test ; upper left panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0372 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0366 ; upper right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0499 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0192 ; 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0164 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0357 ; lower left panel : 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0287 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0232 ; lower right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0171 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0484 ) .

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

MACS-separated CD8+ T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed (n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (*P < 0.05, t-test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484).


Purified CFSE - stained CD8 + T cells were stimulated by immobilized anti - CD3 ( 0 . 005 μg / ml ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , OVA - Fc proteins , or co - stimulatory mAb ( anti - CD28 , anti - NKG2D ) or an isotype control ( mIgG1 ) . The proliferation of CD3 + CD8 + T cells was assessed at 60 h by flow cytometry . Results of a representative out of 6 experiments are displayed . Untreated CFSE - stained CD8 + T cells are included for comparison . The percentage of proliferating cells and MFI for CFSE are indicated . The MFI of CFSE in unstimulated CD8 + T cells ( control ) was set to 100 % in individual experiments ( n = 6 ) , and the relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( * P = 0 . 0277 , Wilcoxon test ) between MICA - 129Met - Fc versus MICA - 129Val - Fc and OVA - Fc proteins were found at slightly higher anti - CD3 concentrations ( 0 . 1 and 0 . 05 μg / ml ) than at later time points ( see A ) . Anti - CD28 and anti - NKG2D mAb were used in parallel as a positive control , mean + SD are shown , and significant differences ( * P = 0 . 0277 , Wilcoxon test ) to the isotype control ( mIgG1 ) are indicated ( n = 6 ) .

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

Purified CFSE-stained CD8+T cells were stimulated by immobilized anti-CD3 (0.005 μg/ml) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, OVA-Fc proteins, or co-stimulatory mAb (anti-CD28, anti-NKG2D) or an isotype control (mIgG1). The proliferation of CD3+CD8+T cells was assessed at 60 h by flow cytometry. Results of a representative out of 6 experiments are displayed. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (*P = 0.0277, Wilcoxon test) between MICA-129Met-Fc versus MICA-129Val-Fc and OVA-Fc proteins were found at slightly higher anti-CD3 concentrations (0.1 and 0.05 μg/ml) than at later time points (see A).Anti-CD28 and anti-NKG2D mAb were used in parallel as a positive control, mean + SD are shown, and significant differences (*P = 0.0277, Wilcoxon test) to the isotype control (mIgG1) are indicated (n = 6).


NKG2D expression on purified CD8 + T cells exposed to L - MICA - 129Met ( n = 19 ) or L - MICA - 129Val clones ( n = 19 ) for 0 , 4 , and 24 h was analyzed by flow cytometry . CD8 + T cells ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3 + CD8 + T cells . The means and SD of the MFI of NKG2D ( left panel ) and of the percentage of NKG2D + CD8 + T cells ( right panel ) are displayed . Differences between the groups were analyzed by repeated measures ANOVA , and the P - values are indicated .

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

NKG2D expression on purified CD8+T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.


Purified CD8 + T cells were cultured on plate - bound anti - NKG2D ( 1 μg / ml ) or isotype control ( mIgG1 ) for 24 h before the NKG2D expression was measured by flow cytometry . Means and SD of MFI ( upper panel ) and percentage of NKG2D + cells ( lower panel ) are shown ( n = 6 ) . Differences between the groups were analyzed by t - tests , and the P - values are indicated .

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

Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.


These CD8 + T cells were subsequently CFSE - stained and cultured on plates coated with anti - CD3 ( 0 . 005 μg / ml ) in combination with anti - CD28 ( 0 . 5 μg / ml ) as a positive control or anti - NKG2D ( 0 . 5 μg / ml ) . Proliferation was measured after 60 h by flow cytometry . Untreated CFSE - stained CD8 + T cells are included for comparison . The percentage of proliferating cells and the MFI for CFSE are indicated . The MFI of CFSE in unstimulated CD8 + T cells ( control ) was set to 100 % in individual experiments ( n = 6 ) , and the relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( Wilcoxon test ) between CD8 + T cells pre - exposed to anti - NKG2D and isotype control ( mIgG1 ) were found in these experiments at anti - CD3 concentrations of 0 . 01 and 0 . 005 μg / ml .

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

These CD8+T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.


A - D Appearance of autophagic structures ( marked with GFP - Atg8a ) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1 , Atg5 , Atg6 , Atg7 or Atg12 . All constructs are expressed in the epidermis under the control of the A58 - Gal4 driver . ( A ) Time points from movies of wounded epidermis . The wounds have closed by 2 h in all cases . ( B ) Higher magnification of the areas marked by magenta boxes at t = 120 min . ( C ) Quantification of the appearance of GFP - Atg8a puncta in the imaged area ( 10000 µm2 ) 3 control larvae , shown for each individual larva . ( D ) Quantification of number of GFP - Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure ; n = 4 - 10 larvae each genotype , for the detail see Data analysis .

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

A-D Appearance of autophagic structures (marked with GFP-Atg8a) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1, Atg5, Atg6, Atg7 or Atg12. All constructs are expressed in the epidermis under the control of the A58-Gal4 driver. (A) Time points from movies of wounded epidermis. The wounds have closed by 2 h in all cases. (B) Higher magnification of the areas marked by magenta boxes at t=120 min. (C) Quantification of the appearance of GFP-Atg8a puncta in the imaged area (10000 µm2) 3 control larvae, shown for each individual larva. (D) Quantification of number of GFP-Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure; n=4-10 larvae each genotype, for the detail see Data analysis.


Control of epidermal autophagy by TOR signalling . ( A ) Epidermis of third instar larvae expressing the autophagosome marker GFP - Atg8a together with constructs for up - or down - regulating the autophagy pathway in the epidermis . Healthy epidermis contains few autophagosomes , but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes . ( B ) Higher magnification of the areas marked by magenta boxes in ( A ) .

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

Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A).


Control of epidermal autophagy by TOR signalling . ( C ) Quantification of Atg8a puncta in an area of 10000 µm2 , n = 6 - 8 larvae each genotype . We assumed unequal sample size and unequal variances and calculations were performed . Values are presented as box plots . Box plot elements are : center line , median ; box limits , upper and lower quartiles ; whiskers , 1 . 5x interquartile range ; points , outliers .

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

Control of epidermal autophagy by TOR signalling. (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.


A - D Fluorescence loss in photobleaching ( FLIP ) to test free cytoplasmic GFP motility within the epidermis . A small area ( magenta circle ; 179 or 1098 µm2 ) was laser - illuminated for the indicated times ( 3 or 20 min ) in control or Atg1 - expressing epidermis also expressing free GFP . ( A ) Snapshots before bleaching and at 3 points of recovery . ( B ) Kymographs along the broken line in ( A ) during recovery . ( C , D ) Quantification of fluorescence recovery after bleaching , shown separately for 3 and 20 min bleaching protocols .

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

A-D Fluorescence loss in photobleaching (FLIP) to test free cytoplasmic GFP motility within the epidermis. A small area (magenta circle; 179 or 1098 µm2) was laser-illuminated for the indicated times (3 or 20 min) in control or Atg1-expressing epidermis also expressing free GFP. (A) Snapshots before bleaching and at 3 points of recovery. (B) Kymographs along the broken line in (A) during recovery. (C, D) Quantification of fluorescence recovery after bleaching, shown separately for 3 and 20 min bleaching protocols.


B - D Atg1 - induced syncytium formation is abolished when Atg5 function is downregulated ( compare to rows 2 and 3 in Fig . 5A , B ) ; same representation as shown in Fig . 5A - D .

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

B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.


C , D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy . ( C ) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493 / 503 to mark lipid accumulations . ( D ) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions . A - D , n = 7 - 20 larvae each genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.


( a , b ) Bacteria in the spleens of Slamf1 − / − Rag1 − / − and Rag1 − / − BALB / c mice ( a ) or Slamf1 − / − and Slamf1 + / + BALB / c mice ( b ) 48 h after intraperitoneal injection of virulent S . typhimurium 14028s or attenuated S . typhimurium SseB − . CFU , colony - forming units ; ND , not detectable . Data are representative of four independent experiments ( mean and s . d . ) .

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(a,b) Bacteria in the spleens of Slamf1−/−Rag1−/− and Rag1−/− BALB/c mice (a) or Slamf1−/− and Slamf1+/+ BALB/c mice (b) 48 h after intraperitoneal injection of virulent S. typhimurium 14028s or attenuated S. typhimurium SseB−. CFU, colony-forming units; ND, not detectable. Data are representative of four independent experiments (mean and s.d.).


( c - e ) Killing of bacteria by peritoneal macrophages from Slamf1 + / + and Slamf1 − / − BALB / c mice exposed to S . typhimurium SseB − ( c ) , E . coli F18 ( d ) or S . aureus ( e ) , assessed by gentamycin assay . Data are representative of five independent experiments ( mean and s . d . ) .

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(c-e) Killing of bacteria by peritoneal macrophages from Slamf1+/+ and Slamf1−/− BALB/c mice exposed to S. typhimurium SseB− (c), E. coli F18 (d) or S. aureus (e), assessed by gentamycin assay. Data are representative of five independent experiments (mean and s.d.).


( f ) Uptake of bacteria by Slamf1 + / + ( red solid lines ) or Slamf1 − / − ( black solid lines ) BALB / c peritoneal macrophages incubated at 37 ° C with E . coli - eGFP or S . typhimurium - eGFP or by Slamf1 + / + macrophages incubated for 60 min at 4 ° C with the bacteria ( dotted lines ) . Right , mean fluorescence intensity ( MFI ) . Data are representative of three independent experiments ( mean and s . d . ) .

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(f) Uptake of bacteria by Slamf1+/+ (red solid lines) or Slamf1−/− (black solid lines) BALB/c peritoneal macrophages incubated at 37 °C with E. coli-eGFP or S. typhimurium-eGFP or by Slamf1+/+ macrophages incubated for 60 min at 4 °C with the bacteria (dotted lines). Right, mean fluorescence intensity (MFI). Data are representative of three independent experiments (mean and s.d.).


( a ) NOX2 activity in Slamf1 + / + and Slamf1 - / - BALB / c peritoneal macrophages stimulated for 0 - 100 min with E . coli F18 , S . aureus or PMA , assessed with lucigenin . Data are representative of five independent experiments ( mean ± s . d . ) .

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(a) NOX2 activity in Slamf1+/+ and Slamf1-/- BALB/c peritoneal macrophages stimulated for 0-100 min with E. coli F18, S. aureus or PMA, assessed with lucigenin. Data are representative of five independent experiments (mean ± s.d.).


( b ) Phagosomal pH of Slamf1 + / + , Slamf1 - / - and gp91phox - / - B6 primary macrophages loaded for 0 - 200 min with pHrodo - coated E . coli or S . aureus , analyzed by flow cytometry . Data are representative of three independent experiments ( mean ± s . d . ) .

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(b) Phagosomal pH of Slamf1+/+, Slamf1-/- and gp91phox-/- B6 primary macrophages loaded for 0-200 min with pHrodo-coated E. coli or S. aureus, analyzed by flow cytometry. Data are representative of three independent experiments (mean ± s.d.).


( c ) NOX2 activity in primary macrophages in response to LPS , purified OmpC , peptidoglycan ( PGN ) or PMA , assessed with lucigenin . TLR4 - KO , TLR4 - deficient ( strain del / Jtht ; C3H ) . Data are representative of five independent experiments ( mean ± s . d . ) .

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(c) NOX2 activity in primary macrophages in response to LPS, purified OmpC, peptidoglycan (PGN) or PMA, assessed with lucigenin. TLR4-KO, TLR4-deficient (strain del/Jtht; C3H). Data are representative of five independent experiments (mean ± s.d.).


( a ) Fluorescence microscopy of E . coli - containing phagosomes in primary macrophages transfected with RFP - conjugated LAMP - 1 , showing colocalization with E . coli - eGFP or S . aureus - eGFP . Right , quantification of LAMP - 1 + phagosomes in the microscopy at left .

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(a) Fluorescence microscopy of E. coli-containing phagosomes in primary macrophages transfected with RFP-conjugated LAMP-1, showing colocalization with E. coli-eGFP or S. aureus-eGFP. Right, quantification of LAMP-1+ phagosomes in the microscopy at left.


( b ) Immunoblot analysis of LAMP - 1 in phagosomes isolated by sucrose - gradient flotation from RAW264 . 7 macrophages after phagocytosis by Slamf1 - or mock - transfected RAW264 . 7 macrophages of beads coated with E . coli outer membrane extract . WCL , whole - cell lysate . Right , quantification of LAMP - 1 in the immunoblot at left .

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(b) Immunoblot analysis of LAMP-1 in phagosomes isolated by sucrose-gradient flotation from RAW264.7 macrophages after phagocytosis by Slamf1- or mock-transfected RAW264.7 macrophages of beads coated with E. coli outer membrane extract. WCL, whole-cell lysate. Right, quantification of LAMP-1 in the immunoblot at left.


( c ) Localization of MHC class II - eGFP ( MHCII GFP ) in phagosomes of primary macrophages from Slamf1 + / + or Slamf1 − / − MHC class II - eGFP ( B6 ) mice and 3 - μm beads coated with E . coli outer membrane extract . ( d ) Entry of 3 - μm beads coated with E . coli outer membrane extract into lysosomes loaded with Texas red - dextran . Right ( c , d ) , quantification of fluorescence in the microscopy at left . Numbers in bottom right corners ( a , c , d ) indicate time ( in min ) . Original magnification ( a - c ) , × 60 . DIC , differential interference contrast . Data are representative of three combined experiments ( a ) or at least three independent experiments ( b - d ) with at least 100 beads or 80 bacteria in each ( error bars , s . d . ) .

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(c) Localization of MHC class II-eGFP (MHCII GFP) in phagosomes of primary macrophages from Slamf1+/+ or Slamf1−/− MHC class II-eGFP (B6) mice and 3-μm beads coated with E. coli outer membrane extract. (d) Entry of 3-μm beads coated with E. coli outer membrane extract into lysosomes loaded with Texas red-dextran. Right (c,d), quantification of fluorescence in the microscopy at left. Numbers in bottom right corners (a,c,d) indicate time (in min). Original magnification (a-c), ×60. DIC, differential interference contrast. Data are representative of three combined experiments (a) or at least three independent experiments (b-d) with at least 100 beads or 80 bacteria in each (error bars, s.d.).


( a , b ) Association of EEA - 1 ( a ) or Rab5 ( b ) with phagosomes generated in primary macrophages by 3 - μm beads coated with E . coli outer membrane extract ; numbers in bottom corners indicate time ( in min ) . α - , anti - . Original magnification , × 60 . Right ( a , b ) , quantification of fluorescence . Data are representative of at least three independent experiments ( error bars , s . d . ) .

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(a,b) Association of EEA-1 (a) or Rab5 (b) with phagosomes generated in primary macrophages by 3-μm beads coated with E. coli outer membrane extract; numbers in bottom corners indicate time (in min). α-, anti-. Original magnification, ×60. Right (a,b), quantification of fluorescence. Data are representative of at least three independent experiments (error bars, s.d.).


( a ) Colocalization of SLAM and bacteria in RAW264 . 7 macrophages transfected with SLAM - mCherry and allowed to phagocytose E . coli - eGFP or S . aureus - eGFP for 0 - 120 min ( time ( in min ) , bottom right corners at left ) . Original magnification , × 60 . Right , quantification of fluorescence at left . Data are representative of two independent experiments with a minimum of 40 bacteria per time point ( error bars , s . d . ) .

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(a) Colocalization of SLAM and bacteria in RAW264.7 macrophages transfected with SLAM-mCherry and allowed to phagocytose E. coli-eGFP or S. aureus-eGFP for 0-120 min (time (in min), bottom right corners at left). Original magnification, ×60. Right, quantification of fluorescence at left. Data are representative of two independent experiments with a minimum of 40 bacteria per time point (error bars, s.d.).


( b ) Immunoblot analysis of phagosome isolates from RAW264 . 7 macrophages transiently transfected with cDNA encoding Myc - tagged SLAM ( Slamf1 - Myc ) or mock transfected and allowed to phagocytose beads coated with E . coli outer membrane extract for 60 or 120 min . kDa , kilodaltons . Data are representative of at least three independent experiments .

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(b) Immunoblot analysis of phagosome isolates from RAW264.7 macrophages transiently transfected with cDNA encoding Myc-tagged SLAM (Slamf1-Myc) or mock transfected and allowed to phagocytose beads coated with E. coli outer membrane extract for 60 or 120 min. kDa, kilodaltons. Data are representative of at least three independent experiments.


( a ) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter ( as described in Results ) , plus a renilla luciferase reporter , then exposed to heat - killed bacteria ( top ) .

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(a) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter (as described in Results), plus a renilla luciferase reporter, then exposed to heat-killed bacteria (top).


( b ) Luciferase activity in Jurkat cells transfected with the fusion in a ( Slamf1 - CD3ζ ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ ( ΔIgV - Slamf1 - CD3ζ ) , and luciferase reporters as in a , then exposed to heat - killed E . coli F18 .

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(b) Luciferase activity in Jurkat cells transfected with the fusion in a (Slamf1-CD3ζ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ (ΔIgV-Slamf1-CD3ζ), and luciferase reporters as in a, then exposed to heat-killed E. coli F18.


( c ) Luciferase activity in Jurkat cells transfected as in a , then left uninoculated ( − ) or inoculated with 10 × 108 E . coli F18 ( + ) , followed by the addition of monoclonal antibody ( mAb ; amount , under graph ) 9D1 to SLAM ( α - SLAM ) or rat immunoglobulin G2b isotype - matched control antibody ( Rat IgG2b ) .

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(c) Luciferase activity in Jurkat cells transfected as in a, then left uninoculated (−) or inoculated with 10 × 108 E. coli F18 (+), followed by the addition of monoclonal antibody (mAb; amount, under graph) 9D1 to SLAM (α-SLAM) or rat immunoglobulin G2b isotype-matched control antibody (Rat IgG2b).


( d ) Luciferase activity in Jurkat cells transfected as in a , then exposed to medium alone ( Med ) , wild - type E . coli ( JM101 ) , or JM101 E . coli mutants lacking both Omp C and Omp F ( HN705 ) or lacking either OmpC ( ΔOmpC ) or OmpF ( ΔOmpF ) . Data are representative of at least three independent experiments ( error bars ( a ) , s . d . ) .

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(d) Luciferase activity in Jurkat cells transfected as in a, then exposed to medium alone (Med), wild-type E. coli (JM101), or JM101 E. coli mutants lacking both Omp C and Omp F (HN705) or lacking either OmpC (ΔOmpC) or OmpF (ΔOmpF). Data are representative of at least three independent experiments (error bars (a), s.d.).


( a , b ) Production of phagosomal PtdIns ( 3 ) P in E . coli - containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP - tagged PX domain of p40phox ( p40 PX - eGFP ) and treated with DsRed - expressing E . coli ( E . coli - DsRed ; a ) or beads coated with E . coli outer membrane extract ( b ) . Numbers in bottom right corners indicate time ( in min ) . Original magnification , × 60 . Right , quantification of fluorescence in microscopy at left .

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(a,b) Production of phagosomal PtdIns(3)P in E. coli-containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP-tagged PX domain of p40phox (p40 PX-eGFP) and treated with DsRed-expressing E. coli (E. coli-DsRed; a) or beads coated with E. coli outer membrane extract (b). Numbers in bottom right corners indicate time (in min). Original magnification, ×60. Right, quantification of fluorescence in microscopy at left.


( c ) HPLC analysis of the production of PtdIns ( 3 ) P in primary macrophages labeled with 3H - tagged myoinositol and treated with beads coated with E . coli outer membrane extract .

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(c) HPLC analysis of the production of PtdIns(3)P in primary macrophages labeled with 3H-tagged myoinositol and treated with beads coated with E. coli outer membrane extract.


( d ) PtdIns ( 3 ) P production in RAW264 . 7 cells stably expressing SLAM or a mock construct , transfected with eGFP - tagged p40 PX and treated with beads coated with E . coli outer membrane extract . Data are from three combined experiments ( a ) or are representative of at least three independent experiments ( b , d ) or two independent experiments ( c ) with at least 100 beads or bacteria per experiment ( error bars , s . d . ) .

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(d) PtdIns(3)P production in RAW264.7 cells stably expressing SLAM or a mock construct, transfected with eGFP-tagged p40 PX and treated with beads coated with E. coli outer membrane extract. Data are from three combined experiments (a) or are representative of at least three independent experiments (b,d) or two independent experiments (c) with at least 100 beads or bacteria per experiment (error bars, s.d.).


( a , b ) Immunoassay of 293 cells transfected with various combinations of full - length SLAM ( a ) or hemagglutinin - tagged ( - HA ) tail - less SLAM ( b ) , EAT - 2A , V5 - tagged ( - V5 ) Vps34 - Vps15 , and beclin - 1 ; proteins immunoprecipitated ( IP ) from lysates with monoclonal antibody to SLAM , as well as whole - cell lysates ( WCL ) , were analyzed by immunoblot ( IB ) with anti - beclin - 1 , anti - Vps34 , anti - V5 , anti - hemagglutinin , anti - SLAM or anti - EAT2 .

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(a,b) Immunoassay of 293 cells transfected with various combinations of full-length SLAM (a) or hemagglutinin-tagged (-HA) tail-less SLAM (b), EAT-2A, V5-tagged (-V5) Vps34-Vps15, and beclin-1; proteins immunoprecipitated (IP) from lysates with monoclonal antibody to SLAM, as well as whole-cell lysates (WCL), were analyzed by immunoblot (IB) with anti-beclin-1, anti-Vps34, anti-V5, anti-hemagglutinin, anti-SLAM or anti-EAT2.


( c ) Microscopy of RAW264 . 7 cells transiently transfected with cDNA encoding GFP - tagged beclin - 1 and SLAM - mCherry , then treated with beads coated with E . coli outer membrane extract ( at a ratio of 10 : 1 , beads / cells ) and fixed after 60 min . Data are representative of six ( a ) or two ( b , c ) independent experiments .

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(c) Microscopy of RAW264.7 cells transiently transfected with cDNA encoding GFP-tagged beclin-1 and SLAM-mCherry, then treated with beads coated with E. coli outer membrane extract (at a ratio of 10:1, beads/cells) and fixed after 60 min. Data are representative of six (a) or two (b,c) independent experiments.


B . Two - hybrid interaction between GBD - AC40 and WT or mutant GAD - IN1578 - 635 . Alanine substitutions in IN1578 - 635 are indicated . Cells were plated in two - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were detected ( Figs EV1A - B ) . + , interaction ; - , no interaction .

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B. Two-hybrid interaction between GBD-AC40 and WT or mutant GAD-IN1578-635. Alanine substitutions in IN1578-635 are indicated. Cells were plated in two-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1A-B). +, interaction; -, no interaction.


C . Two - hybrid interaction between GBD - AC40 and different IN1 regions fused to GAD , as indicated . Cells were plated in ten - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were detected ( Figs EV1C - D ) . + , interaction ; - , no interaction .

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C. Two-hybrid interaction between GBD-AC40 and different IN1 regions fused to GAD, as indicated. Cells were plated in ten-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1C-D). +, interaction; -, no interaction.


D . In vitro interaction between AC40 and IN1 proteins co - expressed in E . coli . Co - precipitation of WT or mutated ( K617A , S621A , or L622A ) IN1 - C - tag using AC40 - Twin - Strep - tag as bait from bacterial protein extracts co - expressing ( + ) or not ( - ) these proteins . Expected sizes are 41 kDa for AC40 - Twin - Strep - tag and 100 kDa for IN1 - C - tag ( WT and mutants ) .

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D. In vitro interaction between AC40 and IN1 proteins co-expressed in E. coli. Co-precipitation of WT or mutated (K617A, S621A, or L622A) IN1-C-tag using AC40-Twin-Strep-tag as bait from bacterial protein extracts co-expressing (+) or not (-) these proteins. Expected sizes are 41 kDa for AC40-Twin-Strep-tag and 100 kDa for IN1-C-tag (WT and mutants).


A . Localization of GFP2 - IN1 bNLS variants in yeast cells by direct fluorescence microscopy . Nup49 - mCherry signal was used to visualize the location of the nuclear envelope . Corresponding DIC images are shown . Scale bar is 5 μm .

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A. Localization of GFP2-IN1 bNLS variants in yeast cells by direct fluorescence microscopy. Nup49-mCherry signal was used to visualize the location of the nuclear envelope. Corresponding DIC images are shown. Scale bar is 5 μm.


B . Retrotransposition frequency ( log scale ) of pGAL1 - Ty1his3AI bearing the indicated substitutions of conserved residues in a spt3 - 101 rad52 ∆ strain . IN1 catalytic core domain mutant D154A is defective for integration and NLS mutant ( KKR628 - 630GGT ) for nuclear import . Values are mean ± SD , n = 4 experiments , each performed with four independent colonies . * * * p ≤ 0 . 001 , otherwise not significant ( Student t - test ) .

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B. Retrotransposition frequency (log scale) of pGAL1-Ty1his3AI bearing the indicated substitutions of conserved residues in a spt3-101 rad52∆ strain. IN1 catalytic core domain mutant D154A is defective for integration and NLS mutant (KKR628-630GGT) for nuclear import. Values are mean±SD, n=4 experiments, each performed with four independent colonies. ***p ≤ 0.001, otherwise not significant (Student t-test).


A . Co - immunoprecipitation of ectopic IN1 using C160 - HA as bait , from yeast protein extracts expressing WT or mutated IN1 - Strep ( K617A , S621A , or L622A ) from the GAL1 promoter in the presence of galactose . Expected sizes are 160 kDa for C160 - HA and 100 kDa for IN1 - Strep ( WT and mutants ) .

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A. Co-immunoprecipitation of ectopic IN1 using C160-HA as bait, from yeast protein extracts expressing WT or mutated IN1-Strep (K617A, S621A, or L622A) from the GAL1 promoter in the presence of galactose. Expected sizes are 160 kDa for C160-HA and 100 kDa for IN1-Strep (WT and mutants).


B . Quantitative ChIP analysis of HA - IN1 enrichment at Pol III - transcribed genes . Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input . Pol III transcribed - genes : tDNA - Ile and tDNA - Leu families ( 16 and 22 genes , respectively ) and the unique SCR1 gene . GAL1 ORF serves as a control . Data represent means ± SD ( n ≥ 3 ) .

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B. Quantitative ChIP analysis of HA-IN1 enrichment at Pol III-transcribed genes. Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input. Pol III transcribed-genes: tDNA-Ile and tDNA-Leu families (16 and 22 genes, respectively) and the unique SCR1 gene. GAL1 ORF serves as a control. Data represent means ± SD (n ≥ 3).


C . Fold enrichment of WT HA - IN1 over input at all the genes where HA - IN1 has been detected by ChIP - seq analysis . Each dot represents a gene . Grey , tDNAs ; Red , other Pol III - transcribed genes ; Blue , Pol II - transcribed genes . The dashed line indicates the separation between Pol II and Pol III - transcribed genes .

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C. Fold enrichment of WT HA-IN1 over input at all the genes where HA-IN1 has been detected by ChIP-seq analysis. Each dot represents a gene. Grey, tDNAs ; Red, other Pol III-transcribed genes ; Blue, Pol II-transcribed genes. The dashed line indicates the separation between Pol II and Pol III-transcribed genes.


D . WT and mutant IN1 association with all Pol III - transcribed genes . Values obtained from ChIP seq analysis have been normalized in log2 RPKM ( reads per kilobase per million mapped reads ) . Control is anti - HA immunoprecipitation of chromatin extracts in cells expressing IN1 - Strep . Immunopurified DNA from 3 - 4 independent experiments was combined in one replicate . Data are presented as boxplots showing the median , the first quartile , and the third quartile . Error bars show minimum and maximum values .

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D. WT and mutant IN1 association with all Pol III-transcribed genes. Values obtained from ChIP seq analysis have been normalized in log2 RPKM (reads per kilobase per million mapped reads). Control is anti-HA immunoprecipitation of chromatin extracts in cells expressing IN1-Strep. Immunopurified DNA from 3-4 independent experiments was combined in one replicate. Data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values.


E . Genome - wide occupancy profiles ( top ) and heatmaps ( bottom ) of WT and HA - IN1 mutants over a 1kb window upstream and downstream of all tDNAs . Pearson correlation to WT values corresponding to Fig EV2B are indicated .

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E. Genome-wide occupancy profiles (top) and heatmaps (bottom) of WT and HA-IN1 mutants over a 1kb window upstream and downstream of all tDNAs. Pearson correlation to WT values corresponding to Fig EV2B are indicated.


F . WT and mutant IN1 association to all Pol - II transcribed genes respectively . Control , replicates and data presentation , as described for panel D . Regions used for calculation are gene bodies .

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F. WT and mutant IN1 association to all Pol-II transcribed genes respectively. Control, replicates and data presentation, as described for panel D. Regions used for calculation are gene bodies.


G . Recruitment of IN1 along the SCR1 gene locus . Top . ChIP ‐ qPCR analysis as described for panel Fig 3B . Data represent means ± SD ( n = 3 ) . Bottom . Schematic representation of the SCR1 locus with DNA amplicon positions . TATA , A and B boxes and terminator of transcription ( T ) are indicated .

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G. Recruitment of IN1 along the SCR1 gene locus. Top. ChIP‐qPCR analysis as described for panel Fig 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the SCR1 locus with DNA amplicon positions. TATA, A and B boxes and terminator of transcription (T) are indicated.


A . Co - immunoprecipitation of ectopic IN1 using TAP - tagged - A190 as bait from yeast protein extracts expressing WT or the indicated IN1 - HBH mutants from a pTet - Off promoter in the absence of doxycycline . Expected sizes are 204 kDa for A190 - HA and 82 kDa for IN1 - Strep ( WT and mutants ) .

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A. Co-immunoprecipitation of ectopic IN1 using TAP-tagged-A190 as bait from yeast protein extracts expressing WT or the indicated IN1-HBH mutants from a pTet-Off promoter in the absence of doxycycline. Expected sizes are 204 kDa for A190-HA and 82 kDa for IN1-Strep (WT and mutants).


B . Genome browser visualization of HA - IN1 occupancy at the RDN1 locus . Occupancy of WT HA - IN1 and K617A , S621A and L622A HA - IN1 mutants is represented in each panel . Control , as described for panel 3D .

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B. Genome browser visualization of HA-IN1 occupancy at the RDN1 locus. Occupancy of WT HA-IN1 and K617A, S621A and L622A HA-IN1 mutants is represented in each panel. Control, as described for panel 3D.


C . Top . IN1 is recruited at Pol I - transcribed genes . ChIP ‐ qPCR analysis as described for panel 3B . Data represent means ± SD ( n = 3 ) . Bottom . Schematic representation of the RDN1 locus with DNA amplicon positions .

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C. Top. IN1 is recruited at Pol I-transcribed genes. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the RDN1 locus with DNA amplicon positions.


D . Recruitment of WT and mutant HA - IN1 ( K617A , S621A , or L622A ) at the 18S rDNA locus transcribed by Pol I . ChIP ‐ qPCR analysis as described for panel 3B . Data represent means ± SD ( n = 3 ) .

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D. Recruitment of WT and mutant HA-IN1 (K617A, S621A, or L622A) at the 18S rDNA locus transcribed by Pol I. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3).


A . Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 ( red triangle ) and a primer in the locus of interest ( blue triangle ) . Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant ( IN1 K617A , S621A and L622A ) Ty1his3AI from the GAL1 promoter . Total genomic DNA was extracted from His + cells obtained from independent cultures .

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A. Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 (red triangle) and a primer in the locus of interest (blue triangle). Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant (IN1 K617A, S621A and L622A) Ty1his3AI from the GAL1 promoter. Total genomic DNA was extracted from His+ cells obtained from independent cultures.


B . Genome - Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap . Score is computed in column Z - score . ORFs , all Pol II - transcribed genes except gene at subtelomeres ; Pol I , one RDN37 copy ; up 1kb Pol III , 1kb upstream of all Pol III transcribed genes ; Subtelomeres , genomic coordinates corresponding to chromatin covered by Sir2 and Sir3 , when they are co - overexpressed ( Hocher et al , 2018 ) ; Random , 100 000 random Ty1 computed insertions in the genome .

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B. Genome-Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap. Score is computed in column Z-score. ORFs, all Pol II-transcribed genes except gene at subtelomeres; Pol I, one RDN37 copy; up 1kb Pol III, 1kb upstream of all Pol III transcribed genes; Subtelomeres, genomic coordinates corresponding to chromatin covered by Sir2 and Sir3, when they are co-overexpressed (Hocher et al, 2018); Random, 100 000 random Ty1 computed insertions in the genome.


C . Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A . Four total genomic DNA preparation from panel A were randomly tested by PCR .

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C. Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A. Four total genomic DNA preparation from panel A were randomly tested by PCR.


D . Ty1 insertion profile upstream of tDNAs . Total genomic DNA extracted in ( B ) was prepared for Ty1 de novo integration event sequencing . Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs ( position 0 in the graph ) . Each position is divided by the number of insertions at this position ( weight ) . The Smoothing curves indicate the general trend . Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from ( Brogaard et al , 2012 ) .

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D. Ty1 insertion profile upstream of tDNAs. Total genomic DNA extracted in (B) was prepared for Ty1 de novo integration event sequencing. Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs (position 0 in the graph). Each position is divided by the number of insertions at this position (weight). The Smoothing curves indicate the general trend. Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from (Brogaard et al, 2012).


E . Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap . Score is computed in row Z - score . Random , as described for panel B . High level of WT Ty1 - HIS3 integration in II - L is due to the presence of the tRNA gene tF ( GAA ) B .

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E. Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap. Score is computed in row Z-score. Random, as described for panel B. High level of WT Ty1-HIS3 integration in II-L is due to the presence of the tRNA gene tF(GAA)B.


A . Two - hybrid interaction between GBD - AC40 ( left ) or GBD - Sir4 ( right ) and different GAD - IN5 or GAD - IN1 constructions . Cells were plated in five - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were

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A. Two-hybrid interaction between GBD-AC40 (left) or GBD-Sir4 (right) and different GAD-IN5 or GAD-IN1 constructions. Cells were plated in five-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected


B . Genome - wide occupancy profiles ( top ) and heatmaps ( bottom ) of IN5 , WT , and indicated mutants , + / - 1kb upstream and downstream of all tDNAs .

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B. Genome-wide occupancy profiles (top) and heatmaps (bottom) of IN5, WT, and indicated mutants, +/- 1kb upstream and downstream of all tDNAs.


C . Genome browser visualization of different HA - IN occupancy for chromosome V ( chrV : 431129 . . 443275 ) . Occupancy of the indicated integrase is represented in each panel . Control , as panel 3D . The region contains tH ( GUG ) E2 , tK ( CUU ) E2 , tV ( ACC ) E1 , tI ( AAU ) E1 , SNR52 and SCR1 , all transcribed by Pol III .

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C. Genome browser visualization of different HA-IN occupancy for chromosome V (chrV:431129..443275). Occupancy of the indicated integrase is represented in each panel. Control, as panel 3D. The region contains tH(GUG)E2, tK(CUU)E2, tV(ACC)E1, tI(AAU)E1, SNR52 and SCR1, all transcribed by Pol III.


D . Detection of Ty5 , Ty5ΔTD5 + bNLS or Ty1 de novo integration events at HMR and HML loci , SCR1 and upstream of all glycine tDNAs , by PCR using a primer in HIS3 and a primer in the locus of interest . Retrotransposition was induced in cells transformed with WT or mutated pGAL1 - Ty5his3AI ( Ty5 or Ty5ΔTD5 + bNLS ) and pGAL1 - Ty1his3AI ( Ty1 ) . Total genomic DNA was extracted from His + cells obtained from independent cultures . Nucleosome position ( green circles ) is indicated on the right of the panels ( Brogaard et al , 2012 ) .

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D. Detection of Ty5, Ty5ΔTD5+bNLS or Ty1 de novo integration events at HMR and HML loci, SCR1 and upstream of all glycine tDNAs, by PCR using a primer in HIS3 and a primer in the locus of interest. Retrotransposition was induced in cells transformed with WT or mutated pGAL1-Ty5his3AI (Ty5 or Ty5ΔTD5+bNLS) and pGAL1-Ty1his3AI (Ty1). Total genomic DNA was extracted from His+ cells obtained from independent cultures. Nucleosome position (green circles) is indicated on the right of the panels (Brogaard et al, 2012).


( B ) Violin plots displaying RNA - seq readings by FPKM ( normalized Fragments Per Kilobase Million ) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls ( n = 49 , 42 ; unpaired Kolmogorov - Smirnov test ; continuous lines : median ; dashed lines : quartiles ) .

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(B) Violin plots displaying RNA-seq readings by FPKM (normalized Fragments Per Kilobase Million) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls (n = 49,42; unpaired Kolmogorov-Smirnov test; continuous lines: median; dashed lines: quartiles).


( D ) Mean and SEM of mRNA levels of Xbp1s , Bip / Hspa5 and Chop / Ddit3 were determined in dissected hippocampus of i . p . treated mice by quantitative RT - PCR . ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups ) .

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(D) Mean and SEM of mRNA levels of Xbp1s, Bip/Hspa5 and Chop/Ddit3 were determined in dissected hippocampus of i.p. treated mice by quantitative RT-PCR. (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups).


( E ) Animals received bilateral intra - hippocampal injections of 2 μl tunicamcyin ( 5 mg / mL ) or vehicle for 24 h . Graphs indicate mean and s . e . m . of Xbp1s mRNA levels in the hippocampus ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups .

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(E) Animals received bilateral intra-hippocampal injections of 2 μl tunicamcyin (5 mg/mL) or vehicle for 24 h. Graphs indicate mean and s.e.m. of Xbp1s mRNA levels in the hippocampus (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups.


( F - G ) Mean and SEM of relative levels of S - nitrosylated IRE1 ( f ) and total IRE1 ( g ) in brains from young and aged mice . Histograms show relative levels of SNO - IRE1 / input IRE1α measured by biotin - switch assay and total IRE1 / GAPDH quantified by densitometry on standard immunoblots for young ( 3 - month - old ) versus aged ( 24 month - old ) mouse brains ( n = 3 , unpaired Student ' s t test ) . Data information :

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(F-G) Mean and SEM of relative levels of S-nitrosylated IRE1 (f) and total IRE1 (g) in brains from young and aged mice. Histograms show relative levels of SNO-IRE1/input IRE1α measured by biotin-switch assay and total IRE1/GAPDH quantified by densitometry on standard immunoblots for young (3-month-old) versus aged (24 month-old) mouse brains (n = 3, unpaired Student's t test). Data information:


Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( C ) Representative photomicrographs of β - galactosidase staining of hippocampal slices derived from young ( 3 months ) , middle - aged ( 12 months ) and aged ( 22 - 24 months ) of IRE1WT or IRE1cKO animals . Magnification : 20x ; scale bar : 200 μm . Arrows indicate β - galactosidase staining .

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Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (C) Representative photomicrographs of β-galactosidase staining of hippocampal slices derived from young (3 months), middle-aged (12 months) and aged (22-24 months) of IRE1WT or IRE1cKO animals. Magnification: 20x; scale bar: 200 μm. Arrows indicate β-galactosidase staining.


Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( D ) Mean and SEM of percentage of β - galactosidase staining in CA3 region ( n = 3 - 5 animals / group ) .

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Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (D) Mean and SEM of percentage of β-galactosidase staining in CA3 region (n = 3-5 animals/group).


( F ) Representative confocal images of dendritic spines ( arrows ) in the CA1 region of middle - aged IRE1WT animals injected with AAV - Mock or AAV - Cre . 60x magnification , ( G ) Mean and SEM of spine density per μm ( n = 3 , 3 ; 4 , 4 animals ) . P computed by unpaired Student ' s t test within each age - matched group .

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(F) Representative confocal images of dendritic spines (arrows) in the CA1 region of middle-aged IRE1WT animals injected with AAV-Mock or AAV-Cre. 60x magnification, (G) Mean and SEM of spine density per μm (n = 3, 3; 4, 4 animals). P computed by unpaired Student's t test within each age-matched group.


( E ) Representative images of β - galactosidase staining of hippocampal slices derived from aged ( 22 - 24 months ) TgXBP1s and WT animals . ( F ) Mean and SEM of percentage of area stained by β - galactosidase in the hippocampus of aged ( 20 - 24 months ) TgXBP1s compared to littermate controls .

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(E) Representative images of β-galactosidase staining of hippocampal slices derived from aged (22-24 months) TgXBP1s and WT animals. (F) Mean and SEM of percentage of area stained by β-galactosidase in the hippocampus of aged (20-24 months) TgXBP1s compared to littermate controls.


( D ) Left panel : Representative images showing dendritic spines of aged ( 22 - 24 months ) animals injected with AAV2 - eGFP ( mock ) or with AAV2 - XBP1s ( arrows indicate dendritic spines ) . Young animal dendrite is shown for reference . 60x magnification . Scale bar : 5μm . Right panel : Mean and s . e . m . of spine density per μm for indicated experimental groups ( n = 24 , 53 and 35 dendrites from 3 , 5 and 6 animals ,

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(D) Left panel: Representative images showing dendritic spines of aged (22-24 months) animals injected with AAV2-eGFP (mock) or with AAV2-XBP1s (arrows indicate dendritic spines). Young animal dendrite is shown for reference. 60x magnification. Scale bar: 5μm. Right panel: Mean and s.e.m. of spine density per μm for indicated experimental groups (n = 24, 53 and 35 dendrites from 3, 5 and 6 animals, respectively


( E ) Left panel : Representative images of β - galactosidase staining of hippocampal slices derived from aged mice injected with AAV2 - Mock or AAV2 - XBP1s ( arrow indicate β - galactosidase staining ) . Magnification 20x ; scale bar , 200 μm . Right panel : Histograms show mean and s . e . m . of percentage of β - galactosidase - stained area . n = 6 , 6 ; unpaired Student ' s t test .

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(E) Left panel: Representative images of β-galactosidase staining of hippocampal slices derived from aged mice injected with AAV2-Mock or AAV2-XBP1s (arrow indicate β-galactosidase staining). Magnification 20x; scale bar, 200 μm. Right panel: Histograms show mean and s.e.m. of percentage of β-galactosidase-stained area. n = 6, 6; unpaired Student's t test.


( F ) Left panel : Immunofluorescence of γH2aX ( red ) counterstained with nuclei ( blue ) comparing aged mice injected with AAV - Mock or AAV - XBP1s . Magnification : 40x ; Scale bar : 50μm . Right panel : Histograms show mean and s . e . m . of percentage of γH2A - positive cells ; unpaired Student ' s t test comparing the aged groups ( n = 6 , 6 animals / group ; unpaired Student ' s t test ) .

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(F) Left panel: Immunofluorescence of γH2aX (red) counterstained with nuclei (blue) comparing aged mice injected with AAV-Mock or AAV-XBP1s. Magnification: 40x; Scale bar: 50μm. Right panel: Histograms show mean and s.e.m. of percentage of γH2A- positive cells; unpaired Student's t test comparing the aged groups ( n = 6, 6 animals/group; unpaired Student's t test).


A . Whole chromosomes imaged by conventional TEM . Data information : Scale bars :

A. Whole chromosomes imaged by conventional TEM. Data information: Scale bars: 1µm


B . Slice from a large tomographic volume ; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk ; the inset illustrates that the slice of a plate corresponds to a line in the x - y plane ( perpendicular to the direction of the electron beam ) . Data information : Scale bars : 200

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B. Slice from a large tomographic volume; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk; the inset illustrates that the slice of a plate corresponds to a line in the x-y plane (perpendicular to the direction of the electron beam). Data information: Scale bars: 200 nm


C - G Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes . In addition to a typical slice through the x - y plane ( yellow ) , two slices through the x - z ( red ) and y - z ( green ) planes ( orthogonal to the x - y plane ) are shown in ( C ) . Data information : Scale bars : 200 nm D ) , 100 nm ( E - G ) , 50 nm ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes. In addition to a typical slice through the x-y plane (yellow), two slices through the x-z (red) and y-z (green) planes (orthogonal to the x-y plane) are shown in (C). Data information: Scale bars: 200 nm D), 100 nm (E-G), 50 nm (C).


A . Nucleosomes ( N ) decorating a relaxed plate ( P1 ) ; insets in the upper right show additional examples . Nucleosomes are not visible as individual units in typical compact plates ( P2 ) . Short compact interdigitated solenoids are shown in the main image ( S ) and in the bottom - left insets . Data information : Scale bars : 50

A. Nucleosomes (N) decorating a relaxed plate (P1); insets in the upper right show additional examples. Nucleosomes are not visible as individual units in typical compact plates (P2). Short compact interdigitated solenoids are shown in the main image (S) and in the bottom-left insets. Data information: Scale bars: 50 nm


B - E Slice ( B ) and segmentations in three different orientations ( C ) of a large relaxed plate decorated with many nucleosomes on its right side . Slice ( D ) and segmentation ( E ) of a relaxed plate forming a tube decorated with nucleosomes . Data information : Scale bars : 50 nm insets , B - E )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Slice (B) and segmentations in three different orientations (C) of a large relaxed plate decorated with many nucleosomes on its right side. Slice (D) and segmentation (E) of a relaxed plate forming a tube decorated with nucleosomes. Data information: Scale bars: 50 nm insets, B-E)


A , B Plates with several layers that are not closely appressed . C - E Large multilayer plates having the size of human metaphase chromatids [ ~ 0 . 6 µm diameter ] ( C ) ; the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin - plate model In other slices ( D , E ) , the multilayer structures shown in ( C ) are more compact and the individual layers are not visible as separate elements . Data information : Scale bars : 50 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plates with several layers that are not closely appressed. C-E Large multilayer plates having the size of human metaphase chromatids [~0.6 µm diameter ] (C); the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin-plate model In other slices (D,E), the multilayer structures shown in (C) are more compact and the individual layers are not visible as separate elements. Data information: Scale bars: 50 nm.


A - C Slices showing lateral association between two plates and the corresponding 3D segmentations . D . Edge - to - edge interactions form cylindrical structures . Data information : Scale bars : 50 nm .

O O O O O O O O O O O O O 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-C Slices showing lateral association between two plates and the corresponding 3D segmentations. D. Edge-to-edge interactions form cylindrical structures. Data information: Scale bars: 50 nm.


A - C The cation concentrations used are indicated for each experiment ; AU , arbitrary units .

A-C The cation concentrations used are indicated for each experiment; AU, arbitrary units.


Dox - induced prolongation of Nanog expression in Nanogtg embryos up to E9 . 5 results in lack of blood ( left ) and downregulation of erythropoietic gene expression . The center and right panels show whole - mount in situ hybridization for Hbb - bh1 ( in embryos with intact yolk sacs ) and for the long non - coding RNA Redrum . Asterisks mark the aorta - gonad - mesonephros region ( AGM ) and arrows the tailbud . Embryos of the same genotype but not treated with dox were used as controls ( - dox ) . Scale bars , 500 µm .

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

Dox-induced prolongation of Nanog expression in Nanogtg embryos up to E9.5 results in lack of blood (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-mount in situ hybridization for Hbb-bh1 (in embryos with intact yolk sacs) and for the long non-coding RNA Redrum. Asterisks mark the aorta-gonad-mesonephros region (AGM) and arrows the tailbud. Embryos of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm.


Endomucin staining of vessels in control ( - dox ) or treated ( + dox ) E9 . 5 Nanogtg embryos . On the right , higher magnifications of the boxed areas . Scale bar , 500 µm .

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

Endomucin staining of vessels in control (-dox) or treated (+dox) E9.5 Nanogtg embryos. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm.


Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox - treated E9 . 5 Nanogtg embryos . Quantification of the CD71 + Ter119 + population in controls ( - dox , black dots ; n = 8 ) and Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 yolk sacs . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg embryos . * * * P . < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars standard deviation ( SD ) .

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

Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox-treated E9.5 Nanogtg embryos. Quantification of the CD71+ Ter119+ population in controls (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. ***P. < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD).


Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls ( - dox ) and Nanog expressing ( + dox ) E9 . 5 Nanogtg embryos . Quantification of different progenitor populations in yolk sacs from control ( - dox , black dots ; n = 8 ) and Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 embryos . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg embryos . Horizontal line represents mean values and error bars SD .

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

Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls (-dox) and Nanog expressing (+dox) E9.5 Nanogtg embryos. Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 embryos. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. Horizontal line represents mean values and error bars SD.


Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71 + Ter119 + population of control ( - dox ; n = 7 ) and Nanog - expressing ( + dox ; n = 4 ) E9 . 5 embryos . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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 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

Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71+ Ter119+ population of control (-dox; n=7) and Nanog-expressing (+dox; n=4) E9.5 embryos. **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Freshly dissected dox - treated Nanogtg E10 . 5 embryos without ( control ) and with ( chimera ) contribution of wt - ESGFP cells ( left , brightfield ; right , GFP ) . Arrows mark the presence of blood in chimeric embryos that is absent from controls . Scale bar , 500 µm .

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

Freshly dissected dox-treated Nanogtg E10.5 embryos without (control) and with (chimera) contribution of wt-ESGFP cells (left, brightfield; right, GFP). Arrows mark the presence of blood in chimeric embryos that is absent from controls. Scale bar, 500 µm.


Representative FACS plots showing of red blood cell maturation as determined by CD71 / Ter119 staining in single dox - treated E10 . 5 control ( left ) and chimeric ( right ) embryos .

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

Representative FACS plots showing of red blood cell maturation as determined by CD71/Ter119 staining in single dox-treated E10.5 control (left) and chimeric (right) embryos.


Quantification of the CD71 + Ter119 + population in single dox - treated E10 . 5 embryos ( D ; control , n = 18 ; chimera , n = 10 ) , untreated embryos ( E ; control , n = 7 ; chimera , n = 12 ) , and in GFP - cells ( not derived from wild type ES cells ) from dox - treated embryos ( F ; control , n = 18 ; chimera , n = 10 ) . * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Quantification of the CD71+ Ter119+ population in single dox-treated E10.5 embryos (D; control, n=18; chimera, n=10), untreated embryos (E; control, n=7; chimera, n=12), and in GFP- cells (not derived from wild type ES cells) from dox-treated embryos (F; control, n=18; chimera, n=10). ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Quantification of colony - forming units generated by wild type ( wt ) and knockout ( Nanog - / - ) ES cells after culture of EBs for 5 ( D5 ) , 6 ( D6 ) , or 7 ( D7 ) days and plating disaggregated cells in different hemogenic - promoting conditions ) . Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media . CFU - GEMM , progenitors giving rise to granulocytes , erythrocytes , monocytes , and megakaryocytes ; BFU - E , burst forming units - erythroid ; Ery - P , colony forming primitive erythroid ; CFU - GM , granulocyte - monocyte precursors ; CFU - M , monocyte precursors ; CFU - G , granulocyte precursors . No CFU - GEMM are detected at D5 and no BFU - E at D7 . For both wt and knockout cells , n = 3 each with 3 technical replicates . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 00005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Quantification of colony-forming units generated by wild type (wt) and knockout (Nanog-/-) ES cells after culture of EBs for 5 (D5), 6 (D6), or 7 (D7) days and plating disaggregated cells in different hemogenic-promoting conditions). Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media. CFU-GEMM, progenitors giving rise to granulocytes, erythrocytes, monocytes, and megakaryocytes; BFU-E, burst forming units-erythroid; Ery-P, colony forming primitive erythroid; CFU-GM, granulocyte-monocyte precursors; CFU-M, monocyte precursors; CFU-G, granulocyte precursors. No CFU-GEMM are detected at D5 and no BFU-E at D7. For both wt and knockout cells, n=3 each with 3 technical replicates. *P < 0.05, **P < 0.005, ***P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control ( wt , right ) and knockout ( Nanog - / - , left ) ES cells ( n = 3 ) during 10 days of EB differentiation in hematopoietic - cytokine - enriched medium . Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic - gene expression .

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

RT-qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control (wt, right) and knockout (Nanog-/-, left) ES cells (n=3) during 10 days of EB differentiation in hematopoietic-cytokine-enriched medium. Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic-gene expression.


Representative FACS plots showing the distribution of different populations distinguished by CD71 / Ter119 staining in whole bone marrow from untreated ( - dox ) or treated ( + dox ) adult mice . S0 ( double negative cell ) , S1 ( proerythroblast ) , S2 ( basophylic erythroblast ) , S3 ( polychromatic erythroblast ) , and S4 ( orthochromatic erythroblast ) are different stages of blood maturation . Quantification of the S1 - S4 erythroid populations ( - dox , n = 4 ; + dox , n = 5 ) . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FACS plots showing the distribution of different populations distinguished by CD71/Ter119 staining in whole bone marrow from untreated (-dox) or treated (+dox) adult mice. S0 (double negative cell), S1 (proerythroblast), S2 (basophylic erythroblast), S3 (polychromatic erythroblast), and S4 (orthochromatic erythroblast) are different stages of blood maturation. Quantification of the S1-S4 erythroid populations (-dox, n=4; +dox, n=5). *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Representative FACS plots showing the distribution of CD16 / 32 and CD34 hematopoietic precursors sorted from the cKit + Sca1 - LIN - bone marrow of untreated ( - dox ) or treated ( + dox ) adult Nanogtg mice .

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

Representative FACS plots showing the distribution of CD16/32 and CD34 hematopoietic precursors sorted from the cKit+Sca1-LIN- bone marrow of untreated (-dox) or treated (+dox) adult Nanogtg mice.


Quantification of precursor populations based on CD16 / 32 and CD34 sorting , as total number of cells per individual femur ( - dox , n = 5 ; + dox , n = 6 ) . * P < 0 . 05 , * * P < 0 . 005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Quantification of precursor populations based on CD16/32 and CD34 sorting, as total number of cells per individual femur (-dox, n=5; +dox, n=6). *P < 0.05, **P < 0.005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte - erythroid progenitors ( MEP ) ( - dox , n = 8 ; + dox , n = 5 ) . * P < 0 . 05 , * * * * P < 0 . 00005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

RT-qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte-erythroid progenitors (MEP) (-dox, n=8; +dox, n=5). *P < 0.05, ****P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before ( left ) and after ( right ) dox treatment . Percentage of host derived cells ( CD45 . 1 + ) are shown in black , and of donor derived cells ( CD45 . 1 / CD45 . 2 double + ) in red . Individual mice are indicated on the x - axis ( n = 7 ) .

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

Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before (left) and after (right) dox treatment. Percentage of host derived cells (CD45.1+) are shown in black, and of donor derived cells (CD45.1/CD45.2 double +) in red. Individual mice are indicated on the x-axis (n=7).


Contribution of Nanogtg transplanted cells to LSK , CMP , GMP and MEP populations purified from bone marrow . Percentage of host derived cells ( CD45 . 1 + ) are show in black , and of donor derived cells ( CD45 . 1 / CD45 . 2 double + ) in blue . Individual mice are indicated on the x - axis ( n = 7 ) .

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

Contribution of Nanogtg transplanted cells to LSK, CMP, GMP and MEP populations purified from bone marrow. Percentage of host derived cells (CD45.1+) are show in black, and of donor derived cells (CD45.1/CD45.2 double +) in blue. Individual mice are indicated on the x-axis (n=7).


Expected and observed number of mesodermal ( Flk1 + ) cells of the E7 . 0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression , based on single cell RNA - seq data ( Scialdone et al . , 2016 ) . Statistical significance was calculated with a hypergeometric test .

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

Expected and observed number of mesodermal (Flk1+) cells of the E7.0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression, based on single cell RNA-seq data (Scialdone et al., 2016). Statistical significance was calculated with a hypergeometric test.


PCA showing the distribution of Flk1 + E7 . 0 mesoderm cells expressing Nanog ( green ) or Tal1 ( red ) . The single cell expressing both genes is shown in yellow and indicated by an arrow .

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

PCA showing the distribution of Flk1+ E7.0 mesoderm cells expressing Nanog (green) or Tal1 (red). The single cell expressing both genes is shown in yellow and indicated by an arrow.


E6 . 5 Nanogtg embryos after 8 hours ex - utero culture in the presence ( + dox ) or absence ( - dox ) of doxycycline . Scale bar , 100 µm .

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

E6.5 Nanogtg embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm.


RT - qPCR quantification of the relative expression of Nanog , Tal1 , and Klf1 in individual untreated embryos ( - dox ) or treated embryos ( + dox ) ( n = 5 ) . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

RT-qPCR quantification of the relative expression of Nanog, Tal1, and Klf1 in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Whole mount in situ hybridization of Tal1 in E7 . 5 untreated ( - dox ) or in utero treated ( + dox ) Nanogtg embryos . Scale bar , 100 µm .

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

Whole mount in situ hybridization of Tal1 in E7.5 untreated (-dox) or in utero treated (+dox) Nanogtg embryos. Scale bar, 100 µm.


RT - qPCR determination of relative expression in wild type and CRISPR - deleted embryos ( n = 5 ) of Tal1 ( wt , n = 19 ; deleted , n = 13 ) , Klf1 ( wt , n = 3 ; deleted , n = 6 ) , Gfi1b ( wt , n = 10 ; deleted , n = 8 ) , Runx1 ( wt , n = 13 ; deleted , n = 5 ) and Stil ( wt , n = 19 ; deleted , n = 13 ) . * * P < 0 . 005 , Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

RT-qPCR determination of relative expression in wild type and CRISPR-deleted embryos (n=5) of Tal1 (wt, n=19; deleted, n=13), Klf1 (wt, n=3; deleted, n=6), Gfi1b (wt, n=10; deleted, n=8), Runx1 (wt, n=13; deleted, n=5) and Stil (wt, n=19; deleted, n=13). **P < 0.005, Student's t-test. Horizontal line represents mean values and error bars SD.