Purified CFSE - stained CD8 + T were stimulated by immobilized anti - CD3 ( 0 . 005 μg / ml ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , OVA - Fc , or co - stimulatory mAb ( anti - CD28 , anti - NKG2D ) or an isotype control ( mIgG1 ) . proliferation of CD3 + CD8 + T was assessed at 60 h by flow cytometry . Results of a representative out of 6 experiments are displayed . Untreated CFSE - stained CD8 + T are included for comparison . of proliferating MFI for CFSE are indicated . MFI of CFSE in unstimulated CD8 + T ( control ) was set to 100 % in experiments ( n = 6 ) , relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( * P = 0 . 0277 , Wilcoxon ) between MICA - 129Met - Fc versus MICA - 129Val - Fc OVA - Fc were found at slightly higher anti - CD3 concentrations ( 0 . 1 0 . 05 μg / ml ) than at later time points ( see A ) . Anti - CD28 anti - NKG2D mAb were used in parallel as a positive control , mean + SD are shown , significant differences ( * P = 0 . 0277 , Wilcoxon ) to 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 exposed to L - MICA - 129Met ( n = 19 ) or L - MICA - 129Val clones ( n = 19 ) for 0 , 4 , 24 h was analyzed by flow cytometry . CD8 + T ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target analyzed for NKG2D expression after gating on CD3 + CD8 + T . means SD of MFI of NKG2D ( left panel ) of of NKG2D + CD8 + T ( right panel ) are displayed . Differences between groups were analyzed by repeated measures ANOVA , P - values are indicated .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were subsequently CFSE - stained 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 are included for comparison . of proliferating MFI for CFSE are indicated . MFI of CFSE in unstimulated CD8 + T ( control ) was set to 100 % in experiments ( n = 6 ) , relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( Wilcoxon ) between CD8 + T pre - exposed to anti - NKG2D isotype control ( mIgG1 ) were found in these experiments at anti - CD3 concentrations of 0 . 01 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


B . Detection of sFn14 in conditioned media . HEK293E were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N - terminal HA - tag a C - terminal double FLAG - tag . Conditioned media lysates of transfected were collected analyzed by immunoblotting with indicated antibodies . Calnexin served as a loading control . Shown are representative blots from N = 3 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Detection of sFn14 in conditioned media. HEK293E cells were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N-terminal HA-tag and a C-terminal double FLAG-tag. Conditioned media and lysates of the transfected cells were collected and analyzed by immunoblotting with the indicated antibodies. Calnexin served as a loading control. Shown are representative blots from N=3 experiments.


C . Generation of sFn14 is sensitive to γ - secretase inhibitor DAPT . HEK293E were transfected with either Fn14 or C99 ( C - terminal fragment of APP ) , both containing a N - terminal HA - tag C - terminal double FLAG - tag . One day after transfection , were treated with γ - secretase inhibitor DAPT ( 1 μM ) , broad - spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or corresponding amount of vehicle DMSO as indicated . conditioned media lysates were blotted with anti - HA antibody . Shown are representative blots from N = 4 experiments .

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 B-CONTROLLED_VAR 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

C. Generation of sFn14 is sensitive to the γ-secretase inhibitor DAPT. HEK293E cells were transfected with either Fn14 or C99 (C-terminal fragment of APP), both containing a N-terminal HA-tag and C-terminal double FLAG-tag. One day after transfection, cells were treated with γ-secretase inhibitor DAPT (1 μM), broad-spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. The conditioned media and the lysates were blotted with anti-HA antibody. Shown are representative blots from N=4 experiments.


E . In vitro γ - secretase cleavage . HEK293E were transfected with epitope - tagged Fn14 or C99 . membranes were collected incubated under indicated conditions for γ - secretase activity . Reactions were terminated ultracentrifuged . Supernatant ( containing γ - secretase cleavage products ) was used for detecting ICD fragment while pellet was used to blot for full Fn14 or C99 . Blotting is done by anti - Flag antibody . Shown are representative blots from N = 3 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. In vitro γ-secretase cleavage assay. HEK293E cells were transfected with epitope-tagged Fn14 or C99. Cellular membranes were collected and incubated under indicated conditions for the γ-secretase activity assay. Reactions were terminated and ultracentrifuged. Supernatant (containing γ-secretase cleavage products) was used for detecting the ICD fragment while the pellet was used to blot for full length proteins Fn14 or C99. Blotting is done by anti-Flag antibody. Shown are representative blots from N=3 experiments.


F . sFn14 production requires proteolytic presenilin subunit of γ - secretase . HEK293 stably transfected with APP carrying Swedish double mutation ( K595N / M596L ) with a CRISPR / CAS9 - mediated knockout of presenilin 1 ( PS1 KO ) or of both PS1 PS2 ( PS1 / 2 dKO ) , were transiently transfected with epitope - tagged Fn14 . Conditioned media lysates of transfected were blotted with indicated antibodies . C83 is a small C - terminal fragment of APP ( containing C - terminal 83 ) , which is also subjected to γ - secretase processing . Upon longer exposure , C99 ( generated by BACE1 from APP ) is also visible in PS1 / 2 dKO condition . Shown are representative blots from N = 4 experiments . G . of results from panel F . sFn14 in conditioned media quantified normalized to WT sFn14 signal . Data Information : Panel ( G ) shows mean ± , along with p - values calculated by ordinary one - way ANOVA with Tukey ' s multiple comparison .

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

F. sFn14 production requires the proteolytic presenilin subunit of γ-secretase. HEK293 cells stably transfected with APP carrying the Swedish double mutation (K595N/M596L) and with a CRISPR/CAS9-mediated knockout of presenilin 1 (PS1 KO) or of both PS1 and PS2 (PS1/2 dKO), were transiently transfected with epitope-tagged Fn14. Conditioned media and lysates of the transfected cells were blotted with the indicated antibodies. C83 is a small C-terminal fragment of APP (containing the C-terminal 83 amino acids), which is also subjected to γ-secretase processing. Upon longer exposure, the C99 (generated by BACE1 from APP) is also visible in the PS1/2 dKO condition. Shown are representative blots from N=4 experiments. G. Quantification of results from panel F. sFn14 levels in the conditioned media quantified and normalized to WT sFn14 signal. Data Information: Panel (G) shows mean ± SEM, along with p-values calculated by ordinary one-way ANOVA test with Tukey's multiple comparison test.


A . Human breast cancer MDA - MB - 231 shows accumulation of Fn14 upon γ - secretase inhibition . were treated overnight with γ - secretase inhibitor DAPT ( 1 μM ) , broad spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or corresponding amount of vehicle DMSO as indicated . Lysates were blotted for Fn14 with an antibody that targets C - terminal end of , or against calnexin as loading control . asterisk labels an N - terminally truncated form of Fn14 . B . of blots from panel A . control condition , where were only treated with vehicle ( DMSO ) , was used as baseline , its average normalized to 1 . Data Information : data is shown as mean ± . panels have N = 3 biological replicates . For panels B tested conditions were compared against control ( DMSO ) condition by ordinary one - way ANOVA Dunnett ' s multiple comparison . p - values that are above 0 . 05 have not been included into panels .

O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Human breast cancer cell line MDA-MB-231 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. B. Quantification of blots from panel A. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels B the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


C . Human ovarian cancer SKOV - 3 shows accumulation of Fn14 upon γ - secretase inhibition . were treated overnight with γ - secretase inhibitor DAPT ( 1 μM ) , broad spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or corresponding amount of vehicle DMSO as indicated . Lysates were blotted for Fn14 with an antibody that targets C - terminal end of , or against calnexin as loading control . asterisk labels an N - terminally truncated form of Fn14 . D . of blot from panel C . control condition , where were only treated with vehicle ( DMSO ) , was used as baseline , its average normalized to 1 . Data Information : data is shown as mean ± . panels have N = 3 biological replicates . For panels D , tested conditions were compared against control ( DMSO ) condition by ordinary one - way ANOVA Dunnett ' s multiple comparison . p - values that are above 0 . 05 have not been included into panels .

O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Human ovarian cancer cell line SKOV-3 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. D. Quantification of blot from panel C. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels D, the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


A . MDA - MB - 231 were transfected with an siRNA pool against human Fn14 or non - targeting control ( Ctrl ) siRNA . A day after transfection , were treated with γ - secretase inhibitor DAPT ( 1 μM ) or vehicle overnight . lysate was collected blotted against Fn14 ( C - terminal antibody ) or calnexin as loading control . Shown are representative blots from N = 4 experiments . B . of blot in panel A . control condition where were only treated with vehicle DMSO non - targeting siRNA ( DMSO + siCtrl ) was used as baseline , its average normalized to 1 . N = 4 experiments . Data information : data is shown as mean ± . tested conditions were compared against control ( DMSO + siCtrl ) condition by ordinary one - way ANOVA Dunnett ' s multiple comparison . p - values that are above 0 . 05 have not been included into panels .

O O O O O O O O O O O O O O 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 O O O O O O O O O O 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. MDA-MB-231 cells were transfected with an siRNA pool against human Fn14 or non-targeting control (Ctrl) siRNA. A day after transfection, the cells were treated with γ-secretase inhibitor DAPT (1 μM) or vehicle overnight. The lysate was collected and blotted against Fn14 (C-terminal antibody) or calnexin as loading control. Shown are representative blots from N=4 experiments. B. Quantification of blot in panel A. The control condition where the cells were only treated with vehicle DMSO and non-targeting siRNA (DMSO + siCtrl) was used as baseline, and its average normalized to 1. N=4 experiments. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against control (DMSO + siCtrl) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


A . Fn14 in ex vivo glioblastoma samples increased upon γ - secretase inhibition . from four different glioblastomas were treated with DAPT ( 1 μM ) or vehicle overnight . Lysates were blotted against Fn14 calnexin as loading control . B . of blot in panel A . For each glioblastoma , relative ( rel . ) mean of normalized Fn14 vehicle condition was used for normalization . Data information : data is shown as mean ± . tested conditions were compared against their corresponding control ( DMSO ) condition using two - unpaired t - . p - values that are above 0 . 05 have not been included into panels . For panels , three biological replicates were performed .

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 O O O O O O

A. Cellular Fn14 in ex vivo glioblastoma samples increased upon γ-secretase inhibition. Primary cells from four different glioblastomas were treated with DAPT (1 μM) or vehicle overnight. Lysates were blotted against Fn14 and calnexin as loading control. B. Quantification of the blot in panel A. For each glioblastoma, the relative (rel.) mean intensity of the normalized Fn14 vehicle condition was used for normalization. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against their corresponding control (DMSO) condition using two-tailed unpaired t-tests. The p-values that are above 0.05 have not been included into the panels. For all the panels, three biological replicates were performed.


B . Plasma samples from 10 controls GBM with an initial diagnosis of GBM ( iGBM , 40 ) or a recurrent GBM ( rGBM , 20 ) were collected sFn14 were measured by ELISA . Data information : For panel B median of measurements , along with interquartile range borders are displayed . In panel B , ordinary one - way ANOVA with Tukey ' s multiple comparison was applied , but no value passed our p - value display threshold of 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Plasma samples from 10 controls and GBM patients with an initial diagnosis of GBM (iGBM, 40 patients) or a recurrent GBM (rGBM, 20 patients) were collected and sFn14 levels were measured by ELISA. Data information: For panel B median of the measurements, along with interquartile range borders are displayed. In panel B, ordinary one-way ANOVA with Tukey's multiple comparison test was applied, but no value passed our p-value display threshold of 0.05.


C . Plasma samples from 10 ovarian cancer with low / middle 19 with high burden were collected sFn14 were measured by ELISA . burden was clinically determined by computer tomographic scans . Data information : For panel C , median of measurements , along with interquartile range borders are displayed . For panel C , a two - unpaired t - was applied , but no value passed our p - value display threshold of 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Plasma samples from 10 ovarian cancer patients with low/middle and 19 patients with high tumor burden were collected and sFn14 levels were measured by ELISA. The tumor burden was clinically determined by computer tomographic scans. Data information: For panel C, median of the measurements, along with interquartile range borders are displayed. For panel C, a two-tailed unpaired t-test was applied, but no value passed our p-value display threshold of 0.05.


D . Serum samples from 10 with refractory multiple myeloma were collected pre - post - treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days . sFn14 in these samples are displayed ( N = 10 ) . Data information : For panel D , - paired t - was used to compare sFn14 pre - post - treatment .

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

D. Serum samples from 10 patients with refractory multiple myeloma were collected pre- and post-treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days. sFn14 levels in these samples are displayed (N=10). Data information: For panel D, ratio-paired t-test was used to compare the sFn14 levels pre- and post-treatment.


B of expression of miR - 19a miR - 19b in bones of mice treated with PTH ( n = 15 ) or Scl - Ab ( n = 14 ) compared to veh ( n = 13 ) control . C of expression of miR - 19a miR - 19b in calvarial osteoblasts upon in vitro stimulation with PTH or Wnt3a compared to veh control ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of the expression of miR-19a and miR-19b in bones of mice treated with PTH (n=15) or Scl-Ab (n=14) compared to veh (n=13) control. C Quantification of the expression of miR-19a and miR-19b in calvarial osteoblasts upon in vitro stimulation with PTH or Wnt3a compared to veh control (n=3).


D Alkaline phosphatase of differentiating calvarial osteoblasts transfected with a scrambled ( scr ) control miRNA , anti - sense microRNA ( anti - miR ) inhibiting miR - 19a or miR - 19b or a combination thereof ( anti - miR - 19a / b ) ( representative image of 4 independent experiments ) . E of alkaline phosphatase of calvarial osteoblasts transfected with scr , anti - miR - 19a , anti - miR - 19b or anti - miR - 19a / b ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Alkaline phosphatase staining of differentiating calvarial osteoblasts transfected with a scrambled (scr) control miRNA, anti-sense microRNA (anti-miR) inhibiting miR-19a or miR-19b or a combination thereof (anti-miR-19a/b) (representative image of 4 independent experiments). E Quantification of alkaline phosphatase staining of calvarial osteoblasts transfected with scr, anti-miR-19a, anti-miR-19b or anti-miR-19a/b (n=4).


F , G Expression of osteoblast marker genes ( F ) Runx2 ( G ) Type I Collagen ( Col1a1 ) in calvarial osteoblasts transfected with scr , anti - miR - 19a , anti - miR - 19b or anti - miR - 19a / b ( n = 4 ) .

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

F, G Expression analysis of osteoblast marker genes (F) Runx2 and (G) Type I Collagen (Col1a1) in calvarial osteoblasts transfected with scr, anti-miR-19a, anti-miR-19b or anti-miR-19a/b (n=4).


H of von Kossa - stained histological sections of proximal tibiae of 12 - week - old C57Bl / 6J mice double labeling to visualize bone formation ( insets ) after 4 - of treatment with anti - miR - 19a / b ( n = 12 ) , scr control oligonucleotides ( n = 11 ) or veh ( n = 8 ) . Scale bars indicate 1 mm ( black ) 50 μm ( ) . I Bone histomorphometric of proximal tibiae of same as in ( H ) Abbreviations : BV / TV , bone volume / tissue volume ; MS / BS , mineralizing surface / bone surface ; BFR / BS , bone formation rate / bone surface ; MAR , mineral apposition rate ; OS / BS , osteoid surface / bone surface ; Ob . S / BS , osteoblast surface / bone surface ; N . Ob / BS , number of osteoblasts / bone surface ; ES / BS , eroded surface / bone surface ; Oc . S / BS , osteoclast surface / bone surface ; N . Oc / BS , number of osteoclasts / bone surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male C57Bl/6J mice and fluorescence double labeling to visualize bone formation (insets) after 4-weeks of treatment with anti-miR-19a/b (n=12), scr control oligonucleotides (n=11) or veh (n=8). Scale bars indicate 1 mm (black) and 50 μm (white). I Bone histomorphometric analysis of the proximal tibiae of the same animals as in (H) Abbreviations: BV/TV, bone volume/tissue volume; MS/BS, mineralizing surface/bone surface; BFR/BS, bone formation rate/bone surface; MAR, mineral apposition rate; OS/BS, osteoid surface/bone surface; Ob.S/BS, osteoblast surface/bone surface; N.Ob/BS, number of osteoblasts/bone surface; ES/BS, eroded surface/bone surface; Oc.S/BS, osteoclast surface/bone surface; N.Oc/BS, number of osteoclasts/bone surface.


J of von Kossa - stained histological sections of fourth lumbar vertebral of 12 - week - old mice after 4 - of treatment with anti - miR - 19a / b , scr or veh . Scale bar indicates 1 mm . K of bone mass ( BV / TV ) of fourth lumbar vertebral of 12 - week - old mice after 4 - of treatment with anti - miR - 19a / b ( n = 12 ) , scr ( n = 12 ) or veh ( n = 8 ) .

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

J Images of von Kossa-stained histological sections of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b, scr or veh. Scale bar indicates 1 mm. K Quantification of bone mass (BV/TV) of the fourth lumbar vertebral body of 12-week-old mice after 4-weeks of treatment with anti-miR-19a/b (n=12), scr (n=12) or veh (n=8).


A of von Kossa - stained histological sections of bone bone marrow ( upper panel ) of hematoxylin - eosin - stained liver sections ( lower panel ) of 12 - week - old mice after treatment with anti - miR - 19a / b ( n = 12 ) , scrambled ( scr ) control oligonucleotides ( n = 11 ) or vehicle ( veh ) ( n = 8 ) . Scale bars indicate 100 μm ( upper panel ) 50 μm ( lower panel ) . B , C of femur ( B ) width ( C ) after treatment with anti - miR - 19a / b ( n = 8 ) , scr ( n = 7 ) or veh ( n = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of von Kossa-stained histological sections of the bone and bone marrow (upper panel) and of hematoxylin-eosin-stained liver sections (lower panel) of 12-week-old mice after treatment with anti-miR-19a/b (n=12), scrambled (scr) control oligonucleotides (n=11) or vehicle (veh) (n=8). Scale bars indicate 100 μm (upper panel) and 50 μm (lower panel). B, C Quantification of the femur length (B) and width (C) after treatment with anti-miR-19a/b (n=8), scr (n=7) or veh (n=8).


D , E of expression of miR - 19a ( D ) miR - 19b ( E ) in bone , bone marrow liver after treatment with anti - miR - 19a / b ( n = 4 ) , scr ( n = 4 ) or veh ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E Quantification of the expression of miR-19a (D) and miR-19b (E) in the bone, bone marrow and liver after treatment with anti-miR-19a/b (n=4), scr (n=4) or veh (n=3).


F - H of expression of housekeeping genes glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) , beta - 2 microglobulin ( b2m ) , beta - actin 1 ( β - actin 1 ) beta - actin 2 ( β - actin 2 ) in bone ( F ) , bone marrow ( G ) liver ( H ) after treatment with anti - miR - 19a / b ( n = 4 ) , scr ( n = 4 ) or veh ( n = 3 ) .

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

F-H Quantification of the expression of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (Gapdh), beta-2 microglobulin (b2m), beta-actin 1 (β-actin 1) and beta-actin 2 (β-actin 2) in the bone (F), bone marrow (G) and liver (H) after treatment with anti-miR-19a/b (n=4), scr (n=4) or veh (n=3).


I , J CTG - of luminescence signal as surrogate parameter of metabolically active osteoblasts of MC3T3 - E1 ( I ) of hepatocytes of HepG2 ( J ) treated with anti - miR - 19a / b , scr or veh after stimulation with staurosporine to induce as positive control . 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 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

I, J CTG assay-based quantification of luminescence signal intensity as surrogate parameter of metabolically active osteoblasts of the MC3T3-E1 cell line (I) and of hepatocytes of the HepG2 cell line (J) treated with anti-miR-19a/b, scr or veh and after stimulation with staurosporine to induce cell death as positive control. n= 6.


A Immunoblot of Tgif1 expression in calvarial osteoblasts transfected with scrambled ( scr ) , or with anti - miR - 19a , anti - miR - 19b anti - miR - 19a / b ( left ) , or with miR - 19a , mir - 19b mir - 19a / b mimics ( right ) as indicated . Immunoblot for Actin was used as loading control . Normalized fold expression molecular in kilo Dalton ( kD ) are indicated ( representative image of 3 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot of Tgif1 protein expression in calvarial osteoblasts transfected with scrambled (scr), or with anti-miR-19a, anti-miR-19b and anti-miR-19a/b (left), or with miR-19a, mir-19b and mir-19a/b mimics (right) as indicated. Immunoblot for Actin was used as loading control. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).


C of von Kossa - stained histological sections of proximal tibiae of 12 - week - old mice of genotypes Dmp1 - Cre - ; Tgif1fl / fl treated weekly with scr ( n = 8 ) or anti - miR - 19a / b ( n = 10 ) Dmp1 - CreTg ; Tgif1fl / fl treated weekly with scr ( n = 7 ) or anti - miR - 19a / b ( n = 14 ) for 4 . Scale bar indicates 1 mm . D Bone histomorphometric of proximal tibiae of same as in ( C ) . For abbreviations see legend to figure 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male mice of the genotypes Dmp1-Cre-;Tgif1fl/fl treated weekly with scr (n=8) or anti-miR-19a/b (n=10) and Dmp1-CreTg;Tgif1fl/fl treated weekly with scr (n=7) or anti-miR-19a/b (n=14) for 4 weeks. Scale bar indicates 1 mm. D Bone histomorphometric analysis of the proximal tibiae of the same animals as in (C). For abbreviations see legend to figure 1.


E of von Kossa - stained histological sections of fourth lumbar vertebral bodies of same as in ( C ) ( F ) of BV / TV . Scale bar represents 1 mm .

O O O O O O O O O O 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 Images of von Kossa-stained histological sections of the fourth lumbar vertebral bodies of the same animals as in (C) and (F) quantification of the BV/TV. Scale bar represents 1 mm.


A Immunoblot of Tgif1 expression in calvarial osteoblasts after stimulation with PTH ( + ) or vehicle ( - ) after transfection with scrambled ( scr ) , anti - miR - 19a , anti - miR - 19b or anti - miR - 19a / b as indicated . Immunoblot for Actin was used as loading control . Normalized fold expression molecular in kilo Dalton ( kD ) are indicated ( representative image of 3 independent experiments ) .

A Immunoblot of Tgif1 protein expression in calvarial osteoblasts after stimulation with PTH (+) or vehicle (-) and after transfection with scrambled (scr), anti-miR-19a, anti-miR-19b or anti-miR-19a/b as indicated. Immunoblot for Actin was used as loading control. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).


B of von Kossa - stained histological sections of proximal tibiae of 12 - week - old wild type mice after treatment with scr oligonucleotides vehicle ( veh ) ( n = 8 ) , anti - miR - 19a / b veh ( n = 10 ) , scr intermittent PTH ( n = 12 ) or a co - treatment with anti - miR - 19a / b PTH ( n = 10 ) for 4 . Scale bar indicates 1 mm . C Histomorphometric of proximal tibiae of same as in ( B ) after treatment with intermittent PTH or veh / or weekly injections of anti - miR - 19a / b or scr control for 4 . For abbreviations see legend to figure 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of von Kossa-stained histological sections of the proximal tibiae of 12-week-old male wild type mice after treatment with scr oligonucleotides and vehicle (veh) (n=8), anti-miR-19a/b and veh (n=10), scr and intermittent PTH (n=12) or a co-treatment with anti-miR-19a/b and PTH (n=10) for 4 weeks. Scale bar indicates 1 mm. C Histomorphometric analysis of the proximal tibiae of the same animals as in (B) after treatment with intermittent PTH or veh and/or weekly injections of anti-miR-19a/b or scr control for 4 weeks. For abbreviations see legend to figure 1.


D of von Kossa - stained histological sections of fourth lumbar vertebral bodies of same as in ( B ) . Scale bars indicate 1 mm . E Histomorphometric of fourth lumbar vertebral . For abbreviations see legend to figure 1 , scr , veh n = 8 ; anti - miR - 19a / b , veh n = 10 ; scr , PTH n = 12 ; anti - miR - 19a / b , veh n = 10 . F of serum carboxy - terminal collagen crosslinks ( CTX ) in same as in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of von Kossa-stained histological sections of the fourth lumbar vertebral bodies of the same animals as in (B). Scale bars indicate 1 mm. E Histomorphometric analysis of the fourth lumbar vertebral body. For abbreviations see legend to figure 1, scr, veh n=8; anti-miR-19a/b, veh n= 10; scr, PTH n=12; anti-miR-19a/b, veh n=10. F Analysis of serum carboxy-terminal collagen crosslinks (CTX) in the same animals as in (B).


B - D Micro - Computed Tomography ( μCT ) of distal femora ( B , C ) midshaft femoral cross sections ( D ) of same as in A , after termination of experiment . E - G of trabecular BV / TV by μCT in femora ( E ) , tibiae ( F ) , fourth lumbar vertebral bodies ( G ) of same as in ( A - D ) 70 days after OVX .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Micro-Computed Tomography (μCT) of the distal femora (B, C) and midshaft femoral cross sections (D) of the same animals as in A, after termination of the experiment. E-G Quantification of trabecular BV/TV by μCT in femora (E), tibiae (F), and the fourth lumbar vertebral bodies (G) of the same animals as in (A-D) 70 days after OVX.


J Expression of osteoclast marker genes Trap Cathepsin K ( CatK ) in bone marrow macrophages isolated from sham - operated ( n = 2 ) or ovariectomized mice treated with scr ( n = 3 ) or anti - miR - 19a / b ( n = 3 ) after 4 days of differentiation .

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

J Expression analysis of the osteoclast marker genes Trap and Cathepsin K (CatK) in bone marrow macrophages isolated from sham-operated (n=2) or ovariectomized mice treated with scr (n=3) or anti-miR-19a/b (n=3) after 4 days of differentiation.


C Representative of TRAP - stained osteoclast cultures of bone marrow transfected with scrambled ( scr ) control oligonucleotide or anti - miR - 19a / b ( representative of 3 independent experiments ) . Scale bar indicates 200 μm . D of multinucleated TRAP - positive multi - nucleated ( MNCs ) after 4 days of osteoclast differentiation of bone marrow transfected with scr ( n = 4 ) or anti - miR - 19a / b ( n = 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of TRAP-stained osteoclast cultures of bone marrow cells transfected with scrambled (scr) control oligonucleotide or anti-miR-19a/b (representative images of 3 independent experiments). Scale bar indicates 200 μm. D Quantification of multinucleated TRAP-positive multi-nucleated cells (MNCs) after 4 days of osteoclast differentiation of bone marrow cells transfected with scr (n=4) or anti-miR-19a/b (n=8).


E , F Representative ( E ) of MNCs ( F ) after transfection with scr or anti - miR - 19a / b stimulation with conditioned medium ( CM ) collected from Ocy454 that were stimulated with vehicle ( veh ) or PTH ( n = 6 ) . Scale bar indicates 200 μm .

E, F Representative images (E) and quantification of MNCs (F) after transfection with scr or anti-miR-19a/b and stimulation with conditioned medium (CM) collected from Ocy454 cells that were stimulated with vehicle (veh) or PTH (n=6). Scale bar indicates 200 μm.


L Immunoblot of Crtc2 expression in cytoplasm nucleus of Ocy454 transfected with control GapmeR or GapmeR against PP2A - Cβ stimulated with veh or PTH . Immunoblot for Lamin A / C was used as loading control purity control of fraction . Normalized fold expression molecular in kilo Dalton ( kD ) are indicated ( representative image of 3 independent experiments ) .

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

L Immunoblot of Crtc2 protein expression in the cytoplasm and nucleus of Ocy454 cells transfected with control GapmeR or GapmeR against PP2A-Cβ and stimulated with veh or PTH. Immunoblot for Lamin A/C was used as loading control and purity control of the nuclear fraction. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).


M Immunoblot of Crtc2 expression in cytoplasm nucleus of Ocy454 transfected with scr or anti - miR - 19a / b stimulated with veh or PTH . Immunoblot for Lamin A / C was used as loading control purity control of fraction . Normalized fold expression molecular in kilo Dalton ( kD ) are indicated ( representative image of 3 independent experiments ) .

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

M Immunoblot of Crtc2 protein expression in the cytoplasm and nucleus of Ocy454 cells transfected with scr or anti-miR-19a/b and stimulated with veh or PTH. Immunoblot for Lamin A/C was used as loading control and purity control of the nuclear fraction. Normalized fold expression and molecular weight in kilo Dalton (kD) are indicated (representative image of 3 independent experiments).


A , B Relative miR - 19a ( A ) miR - 19b ( B ) expression in long bones of sham - operated ( n = 4 ) ovariectomized ( OVX ) ( n = 4 ) mice . C expression of Tgif1 mRNA in long bones of sham - operated ( n = 11 ) OVX ( n = 6 ) mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative miR-19a (A) and miR-19b (B) expression in long bones of sham-operated (n=4) and ovariectomized (OVX) (n=4) mice. C expression of Tgif1 mRNA in long bones of sham-operated (n=11) and OVX (n=6) mice.


D , E Relative expression of miR - 19a ( D ) miR - 19b ( E ) in Ocy454 upon estrogen deficiency ( ED ) control ( C ) treatment ( n = 6 ) .

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

D, E Relative expression of miR-19a (D) and miR-19b (E) in Ocy454 cells upon estrogen deficiency (ED) and control (C) treatment (n=6).


H Expression of Rankl mRNA in Ocy454 transfected with scrambled ( scr ) control oligonucleotide or anti - miR - 19a / b in context of ED ( n = 9 ) .

O O O O O O O O O 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 Expression of Rankl mRNA in Ocy454 cells transfected with scrambled (scr) control oligonucleotide or anti-miR-19a/b in the context of ED (n=9).


I Representative of von Kossa - stained human bone samples obtained from non - fractured femoral heads of postmenopausal women after assignment to groups of relatively higher bone mass ( HBM , n = 4 ) relatively lower bone mass ( LBM , n = 4 ) . J Histomorphometric of bone mass ( BV / TV , bone volume / tissue volume ) , Trabecular number ( Tb . N / mm ) , trabecular thickness ( Tb . Th ) trabecular separation ( Tb . Sp ) of same human bone samples as in ( I ) .

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

I Representative images of von Kossa-stained human bone samples obtained from non-fractured femoral heads of postmenopausal women after assignment to groups of relatively higher bone mass (HBM, n=4) and relatively lower bone mass (LBM, n=4). J Histomorphometric analysis of bone mass (BV/TV, bone volume/tissue volume), Trabecular number (Tb.N/mm), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of the same human bone samples as in (I).


K , L Representative µCT of 3D reconstructed human bone samples ( K ) of bone mineral density ( BMD ) ( L ) . HBM , n = 4 , LBM , n = 4 .

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

K, L Representative µCT images of 3D reconstructed human bone samples (K) and quantification of bone mineral density (BMD) (L). HBM, n=4, LBM, n=4.


( A ) Waterfall plot showing of volume change in olaparib - treated tumors compared to volume on day 1 . + 20 % - 30 % are marked by doted lines to indicate range of PR , SD PD . box underneath summarizes different characteristics of each model clinical context at moment of PDX implantation . TNBC , Triple Negative Breast Cancer ; ER + BC , Estrogen Receptor positive Breast Cancer ; P , ; M , metastasis . Error bars indicate from independent tumors ( n ≥ 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Waterfall plot showing the percentage of tumor volume change in olaparib-treated tumors compared to the tumor volume on day 1. +20% and -30% are marked by doted lines to indicate the range of PR, SD and PD. The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation. TNBC, Triple Negative Breast Cancer; ER+BC, Estrogen Receptor positive Breast Cancer; P, primary; M, metastasis. Error bars indicate SEM from independent tumors (n ≥ 3).


( B ) Graph showing of volume change during olaparib treatment in PDXs from cohort - 1 . Olaparib - sensitive models are represented with discontinuous lines . Acquisition of PARPi - resistance in PDX302 , STG201 PDX093 after prolonged exposure to olaparib is shown .

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 B-CONTROLLED_VAR O O O

(B) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort-1. Olaparib-sensitive models are represented with discontinuous lines. Acquisition of PARPi-resistance in PDX302, STG201 and PDX093 after prolonged exposure to olaparib is shown.


( A ) of BRCA1 promoter hypermethylation , BRCA1 mRNA presence of BRCA1 foci by immunofluorescence are shown T127 T162 were used as positive controls for hypermethylated BRCA1 promoter . Error bars indicate from independent tumors ( n ≥ 2 ) . Dashed indicates mean of BRCA1 mRNA in normal breast . PARPi - response is shown in summary underneath : box : PD ; black box : PR / CR . Alterations in HRR - related genes in PDX are also indicated .

(A) Levels of BRCA1 promoter hypermethylation, levels BRCA1 mRNA and the presence of BRCA1 nuclear foci by immunofluorescence are shown T127 and T162 were used as positive controls for hypermethylated BRCA1 promoter. Error bars indicate SEM from independent tumors (n ≥ 2). Dashed line indicates mean of BRCA1 mRNA levels in normal breast. PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Alterations in HRR-related genes in PDX are also indicated.


( B ) Western blot of PALB2 detected in U2OS PDXs . Three biological replicates of PDX093 are shown ; PDX302 is used as PALB2 wild type PDX control .

O O O O O O 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) Western blot of PALB2 detected in U2OS cells and PDXs. Three biological replicates of PDX093 are shown; PDX302 is used as PALB2 wild type PDX control.


( C ) YFP - PALB2 recruitment to - induced DSBs is impaired in HeLa expressing PALB2 p . M296Nfs ( n = 4 , unpaired t - at 16min ) .

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

(C) YFP-PALB2 recruitment to laser-induced DSBs is impaired in HeLa cells expressing PALB2 p.M296Nfs (n=4, unpaired t-test at 16min).


Gene - targeting efficiency using Cas9 / mClover - LMNA1 homologous recombination of ( D ) siRNA PALB2 ( n = 4 , one - way ANOVA ) or ( E ) with no PALB2 depletion complemented with wild type p . M296Nfs siRNA - resistant constructs ( n = 7 , one - way ANOVA ) . Western blots of PALB2 wild type PALB2 p . M296Nfs for each condition are shown . Error bars indicate from independent experiments .

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

Gene-targeting efficiency using Cas9/mClover-LMNA1 homologous recombination assay of (D) siRNA PALB2 cells (n=4, one-way ANOVA) or (E) cells with no PALB2 depletion complemented with wild type and p.M296Nfs siRNA-resistant constructs (n=7, one-way ANOVA). Western blots of PALB2 wild type and PALB2 p.M296Nfs for each condition are shown. Error bars indicate SEM from independent experiments.


( A ) of geminin - positive , RAD51 foci - containing detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate from independent tumors ( n ≥ 2 ) . PARPi - response is shown in summary underneath : box : PD ; black box : PR / CR . Immunofluorescence of RAD51 foci in PARPi - vehicle - treated PDX tumors is shown . Alterations in HRR - related genes are also summarized : hBRCA1 : BRCA1 promoter hypermethylation lack of BRCA1 expression BRCA1 foci formation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of geminin-positive, RAD51 nuclear foci-containing cells detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Immunofluorescence staining of RAD51 foci in PARPi- and vehicle-treated PDX tumors is shown. Alterations in HRR-related genes are also summarized: hBRCA1: BRCA1 promoter hypermethylation and lack of BRCA1 expression and BRCA1 nuclear foci formation.


( B ) Restoration of RAD51 foci formation in PARPi acquired - resistant PDXs . Immunofluorescence of BRCA1 RAD51 foci in PARPi - treated tumors from STG201 , PDX302 corresponding PARPi acquired - resistant models ( STG201OR PDX302OR ) . Scale bars : 10µm .

O O O O O O O O O O O O O O O O O O O O O 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) Restoration of RAD51 foci formation in PARPi acquired-resistant PDXs. Immunofluorescence staining of BRCA1 and RAD51 foci in PARPi-treated tumors from STG201, PDX302 and the corresponding PARPi acquired-resistant models (STG201OR and PDX302OR). Scale bars: 10µm.


( C ) of geminin - positive that exhibit γH2AX foci following treatment with vehicle olaparib . Paired t in PARPi - sensitive ( CR / PR ) vs . PARPi - resistant ( PD ) PDXs .

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(C) Quantification of geminin-positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib. Paired t test in PARPi-sensitive (CR/PR) vs. PARPi-resistant (PD) PDXs.


( A ) of geminin - positive , RAD51 foci - containing in FFPE samples from untreated PDX tumors . Color bars indicate presence of pathogenic variants in indicated genes . Error bars indicate from independent tumors ( n ≥ 2 ) . PARPi - response is shown in summary underneath : black box : PR / CR ; box : PD . Box colors indicate PARP inhibitor treatment . Boxes with two colors indicate same response to both treatments . Olaparib 100mg / kg & 50mg / kg indicates that both doses were tested resulted in same response categorization .

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(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells in FFPE samples from untreated PDX tumors. Color bars indicate the presence of pathogenic variants in the indicated genes. Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: black box: PR/CR; white box: PD. Box colors indicate the PARP inhibitor treatment. Boxes with two colors indicate the same response to both treatments. Olaparib 100mg/kg & 50mg/kg indicates that both doses were tested and resulted in the same response categorization.


( B ) ROC curves of RAD51 score HRD score , for PARPi response prediction capacity in PDX cohort - 2 . Bootstrap statistical .

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(B) ROC curves of the RAD51 score and HRD score, for PARPi response prediction capacity in the PDX cohort-2. Bootstrap statistical test.


( C ) Immunofluorescence of 53BP1 BRCA1 foci [ with an antibody towards N - terminus ( B1 - NT ) or C - terminus ( B1 - CT ) of BRCA1 ] in three BRCA1 - mutant , PARPi - resistant models from PDX cohort - 2 . location of mutation within gene is indicated . Scale bars : 10µm .

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(C) Immunofluorescence staining of 53BP1 and BRCA1 nuclear foci [with an antibody towards the N-terminus (B1-NT) or C-terminus (B1-CT) of BRCA1] in three BRCA1-mutant, PARPi-resistant models from PDX cohort-2. The location of the mutation within the gene is indicated. Scale bars: 10µm.


( B ) of geminin - positive , RAD51 or γH2AX foci - containing in FFPE samples from with HBOC syndrome . box underneath summarizes ' s young onset ( < 35y : < 35 years ) , her family history ( FH , purple box for PALB2 - related tumors ) presence of BRCA1 foci in analyzed samples .

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(B) Percentage of geminin-positive, RAD51 or γH2AX nuclear foci-containing cells in FFPE tumor samples from patients with HBOC syndrome. The box underneath summarizes the patient's young onset (<35y: <35 years), her family history (FH, purple box for PALB2-related tumors) and the presence of BRCA1 nuclear foci in the analyzed tumor samples.


( C ) Immunofluorescence of γH2AX , BRCA1 RAD51 foci in three representative FFPE tumors from : one RAD51 - BRCA1 - positive ( Pt03 ) , one RAD51 - BRCA1 - negative ( Pt11 ) one PALB2 - related , with BRCA1 but not RAD51 foci ( Pt20 . 1 ) . Scale bars : 10µm .

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(C) Immunofluorescence staining of γH2AX, BRCA1 and RAD51 foci in three representative FFPE tumors from patients: one RAD51- and BRCA1-positive tumor (Pt03), one RAD51- and BRCA1-negative tumor (Pt11) and one PALB2-related tumor, with BRCA1 but not RAD51 nuclear foci (Pt20.1). Scale bars: 10µm.


( a - f ) Muscle biopsy from subject 4 . 1 subjected to electron microscopy ( transverse sections ) . In many , there is material between layers of basal lamina ( a , arrows ; scale bar , 500 nm ) or overt exocytic vacuoles ( b , arrow ; scale bar , 2 μm ) . Some show a single centralized nucleus ( c ; scale bar , 2 μm ) . Mitochondria are of variable distribution . In some , they form a loop around nucleus in periphery of fiber , resembling a necklace as in c ; in others , they form clusters ( d ; scale bar , 2 μm ) . appearance of cristae is often abnormal ( e ; scale bar , 2 μm ; f ; scale bar , 500 nm ) . N , nucleus ; M , mitochondrion .

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(a-f) Muscle biopsy from subject 4.1 subjected to electron microscopy (transverse sections). In many fibers, there is material between the layers of basal lamina (a, arrows; scale bar, 500 nm) or overt exocytic vacuoles (b, arrow; scale bar, 2 μm). Some fibers show a single centralized nucleus (c; scale bar, 2 μm). Mitochondria are of variable size and distribution. In some fibers, they form a loop around the nucleus in the periphery of the fiber, resembling a necklace as in c; in others, they form clusters (d; scale bar, 2 μm). The appearance of cristae is often abnormal (e; scale bar, 2 μm; f; scale bar, 500 nm). N, nucleus; M, mitochondrion.


Transverse sections from muscle biopsies from subject 3 . 1 ( top ) a normal control ( bottom ) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 counterstained with antibody to M - band titin , titin M8 . Accumulation of NBR1 in ( autophagosomes , arrowheads ) of MURF2 , as well as fiber inhomogeneity with marked fiber atrophy , characterizes Vici syndrome muscle . Numerous of very small cross - sectional ( arrows ) with high content of MURF2 NBR1 are frequently seen . Scale bar , 10 μm .

Transverse sections from muscle biopsies from subject 3.1 (top) and a normal control (bottom) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 and counterstained with antibody to the M-band titin, titin M8. Accumulation of NBR1 in puncta (autophagosomes, arrowheads) and of MURF2, as well as fiber inhomogeneity with marked fiber atrophy, characterizes Vici syndrome muscle. Numerous fibers of very small cross-sectional area (arrows) with high content of MURF2 and NBR1 puncta are frequently seen. Scale bar, 10 μm.


Accumulation of autophagy adaptors NBR1 SQSTM1 phagophore component LC3 is induced in fibroblasts from two controls a subject with Vici syndrome ( subject 4 . 1 ) after 12 h of treatment with rapamycin or dual treatment with rapamycin bafilomycin .

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Accumulation of autophagy adaptors NBR1 and SQSTM1 and the phagophore membrane component LC3 is induced in fibroblasts from two controls and a subject with Vici syndrome (subject 4.1) after 12 h of treatment with rapamycin or dual treatment with rapamycin and bafilomycin.


In control fibroblasts subjected to 6 h of bafilomycin treatment , lysosomal structures were detected by with monoclonal antibody to LAMP1 . Numerous LC3 - positive autophagosomes are found engulfed by LAMP1 - positive vesicular structures ( arrowheads ) . In contrast , fibroblasts from individuals with Vici syndrome consistently show smaller LC3 - positive that only sporadically colocalize with LAMP1 , with many isolated LC3 - positive ( arrows ) . Note that LC3 signal in Vici syndrome occurs mostly at rim of LAMP1 - positive structures , not centrally . Scale bar , 5 μm .

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In control fibroblasts subjected to 6 h of bafilomycin treatment, lysosomal structures were detected by staining with monoclonal antibody to LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, fibroblasts from individuals with Vici syndrome consistently show smaller LC3-positive puncta that only sporadically colocalize with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici syndrome cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar, 5 μm.


C , D . Immunolabeling of HH25 chick sections 50 hours post - engraftment of A375P : : GFP , using an anti - GFP antibody ( in green in C , in brown in D , A375P : GFP ) , an anti - HNK1 antibody ( in , migrating early post - migrating NCCs ) , an anti - Ki67 antibody ( in red in C , cycling ) , an anti - SOX10 antibody ( in red in D , also stains chick endogenous NCCs ) an anti - MITF antibody ( in yellow in D ) . Nuclei were stained with Hoechst ( in blue ) . In C , upper left photo shows a grafted prior to cryosection . In D , right panels are enlargements of lower left panel . Scale bar : 200 µm .

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C,D. Immunolabeling of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody (in green in C, in brown in D, A375P:GFP cells), an anti-HNK1 antibody (in white, migrating and early post-migrating NCCs), an anti-Ki67 antibody (in red in C, cycling cells), an anti-SOX10 antibody (in red in D, also stains chick endogenous NCCs) and an anti-MITF antibody (in yellow in D). Nuclei were stained with Hoechst (in blue). In C, the upper left photo shows a grafted embryo prior to cryosection. In D, right panels are enlargements of the lower left panel. Scale bar: 200 µm.


F . 3D views ( - sheet imaging ) of HH25 chick engrafted with different melanoma samples , showing different patterns of localization 50 hours post - engraftment .

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F. 3D views (light-sheet imaging) of HH25 chick embryos engrafted with different melanoma patient samples, showing different patterns of tumor cell localization 50 hours post-engraftment.


G . Immunofluorescent labeling of HH25 chick sections 50 hours post - engraftment of OF - MEL - 020 sample , labeled with CFSE ( in green ) prior to graft . An anti - SOX10 antibody ( in red ) an anti - MITF antibody ( in yellow ) were used . Nuclei were stained with Hoechst ( in blue ) . Right panels are enlargements of dotted in left panel .

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G. Immunofluorescent labeling of HH25 chick embryo sections 50 hours post-engraftment of OF-MEL-020 patient sample, labeled with CFSE (in green) prior to the graft. An anti-SOX10 antibody (in red) and an anti-MITF antibody (in yellow) were used. Nuclei were stained with Hoechst (in blue). Right panels are enlargements of the dotted area in the left panel.


A , B . Immunofluorescent labeling ( A ) of HH25 chick sections 50 hours post - engraftment of A375P : : GFP , using an anti - GFP antibody , an anti - MITF antibody ( pink ) an anti - SOX10 ( ) antibody . Nuclei were stained with Hoechst . Right panels are enlargements of dotted in left panel . In B , mean fraction of MITF - negative ( MITFneg ) , - low ( MITFlow ) - positive ( MITFpos ) A375P : : GFP in mass in migrating was quantified ( n = 16 slices from 3 ) . Error bars show . Mann - Whitney comparing tumors vs migrating for each class of MITF expression was performed , p - values are indicated on graph .

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A,B. Immunofluorescent labeling (A) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody, an anti-MITF antibody (pink) and an anti-SOX10 (white) antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in left panel. In B, the mean fraction of MITF-negative (MITFneg), -low (MITFlow) and -positive (MITFpos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 16 slices from 3 embryos). Error bars show SEM. Mann-Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed, p-values are indicated on the graph.


C , D . Immunofluorescent labeling ( C ) of HH25 chick sections 50 hours post - engraftment of A375P : : GFP , using an anti - GFP antibody an anti - Ki67 antibody . Nuclei were stained with Hoechst . Right panels are enlargements of dotted in left panel . In D , mean fraction of Ki67 - positive ( Ki67pos ) A375P : : GFP in mass in migrating was quantified ( n = 25 slices from 3 ) . Arrows point at having a mesenchymal . Error bars show . Student t - , * * p = 0 . 0070 .

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C,D. Immunofluorescent labeling (C) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody and an anti-Ki67 antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in the left panel. In D, the mean fraction of Ki67-positive (Ki67pos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 25 slices from 3 embryos). Arrows point at cells having a mesenchymal morphology. Error bars show SEM. Student t-test, **p = 0.0070.


H - K . 3D views ( H , J ) of volumes ( I , K ) of HH25 chick engrafted with GLO ( H , I ) or GLO - R ( J , K ) treated with a combination of Vemurafenib Cobimetinib , or Vorinostat alone , or a combination of three molecules . number of analyzed for each experimental condition are indicated on graphs . Bars error bars indicate mean + . Student t - , * * : p < 0 . 01 , ns : not significant . Exact p - values are indicated on graphs .

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H-K. 3D views (H, J) and quantification of tumor volumes (I, K) of HH25 chick embryos engrafted with GLO (H, I) or GLO-R (J, K) cells and treated with a combination of Vemurafenib and Cobimetinib, or Vorinostat alone, or a combination of the three molecules. The number of embryos analyzed for each experimental condition are indicated on the graphs. Bars and error bars indicate mean + SEM. Student t-tests, **: p < 0.01, ns: not significant. Exact p-values are indicated on the graphs.


A , B . 3D views ( A ) of variations in mean volume ( B ) of HH25 chick engrafted with BRAFwt ( OF - MEL - 028 ) or BRAFV600E ( OF - MEL - 020 , OF - MEL - 027 ) samples treated with excipient or a combination of Vemurafenib Cobimetinib . number of analyzed for each sample is indicated on graphs . Student t - for OF - MEL - 020 ; Mann - Whintey for OF - MEL - 028 , * : p < 0 . 05 , ns : not significant . Exact p - values are indicated on graphs .

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A,B. 3D views (A) and quantification of variations in the mean tumor volume (B) of HH25 chick embryos engrafted with BRAFwt (OF-MEL-028) or BRAFV600E (OF-MEL-020, OF-MEL-027) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib. The number of embryos analyzed for each patient sample is indicated on the graphs. Student t-test for OF-MEL-020; Mann-Whintey test for OF-MEL-028, *: p < 0.05, ns: not significant. Exact p-values are indicated on the graphs.


C , D . Survival rate ( left axis ) mean surface ( BSA , right axis ) of chick injected with increasing doses of Dabrafenib ( C ) Trametinib ( D ) . maximum tolerated dose ( MTD , in red on X - axis ) was defined as higher dose of drug associated with a survival rate higher than 75 % a mean BSA similar ( i . e . , non - statistically different ) from treated with excipient ( 17 . 1 0 . 0228 mg / kg respectively ) . N = 10 replicates per experimental group . Error bars indicate . ns , non - significant using Student ' s t - compared with excipient .

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C,D. Survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of Dabrafenib (C) and Trametinib (D). The maximum tolerated dose (MTD, in red on the X-axis) was defined as the higher dose of drug associated with a survival rate higher than 75% and a mean BSA similar (i.e., non-statistically different) from embryos treated with excipient (17.1 and 0.0228 mg/kg respectively). N=10 replicates per experimental group. Error bars indicate SEM. ns, non-significant using Student's t-test compared with excipient.


( F - H ) of HH25 chick engrafted with 3 different samples ( OF - MEL - 033 ( F ) , OF - MEL - 034 ( G ) , OF - MEL - 035 ( H ) ) treated with excipient or a combination of Dabrafenib Trametinib . clinical response of each after a 3 months treatment with Dabrafenib / Trametinib is indicated above graphs . Error bars indicate . Mann - Whitney , exact p - values are indicated on graphs .

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(F-H) of HH25 chick embryos engrafted with 3 different patient samples (OF-MEL-033 (F), OF-MEL-034 (G), OF-MEL-035 (H)) and treated with excipient or a combination of Dabrafenib and Trametinib. The clinical response of each patient after a 3 months treatment with Dabrafenib/Trametinib is indicated above the graphs. Error bars indicate SEM. Mann-Whitney tests, exact p-values are indicated on the graphs.


( D , E ) Scatter plot visualization of YFP vs mCherry signals in GCG1 ORC2 promoter FPR screens . Each data point represents median signal of a deletion mutant normalized to plate median . Highlighted data points represent mutants of Hda1C ( purple ) , SAGA acetylation module ( blue ) , SAGA deubiquitination module ( orange ) , cac2 ∆ ( yellow ) . regression is marked in black . mutants favoring DNC transcription are found above regression . mutants favoring coding transcription are below regression . Technical repeat = 1 , biological observations = 50 , 000 before filtering .

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(D, E) Scatter plot visualization of YFP vs mCherry fluorescence signals in the GCG1 and ORC2 promoter FPR screens. Each data point represents the median signal of a deletion mutant normalized to individual plate median. Highlighted data points represent the mutants of Hda1C (purple), SAGA acetylation module (blue), SAGA deubiquitination module (orange), and cac2∆ (yellow). The regression line is marked in black. The mutants favoring DNC transcription are found above the regression line. The mutants favoring coding transcription are below the regression line. Technical repeat = 1, biological observations = 50,000 individual cells before filtering.


( C ) Flow cytometry of mCherry / YFP in mutants with GCG1pr FPR background . data are normalized to signal values of wild type ( FPR ) .

(C) Flow cytometry quantification of mCherry/YFP fluorescence in the mutants with GCG1pr FPR background. The data are normalized to the signal values of the wild type (FPR) strain.


( D ) Endogenous transcript by RT - qPCR in mutants . bars represent fold gene expression of coding divergent transcript normalized to expression of reference gene , UBC6 .

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(D) Endogenous transcript analysis by RT-qPCR in the mutants. The bars represent fold gene expression ratio of coding and divergent transcript normalized to the expression of the reference gene, UBC6.


. ( B ) NET - seq data at YPL172C locus . signal represents NET - seq reads showing nascent transcription at genomic position of divergent non - coding strand ( black ) coding strand ( grey ) . bar graph depicts NET - seq reads as FPKM values for coding DNC transcript in strains

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. (B) NET-seq data at the YPL172C locus. The signal represents NET-seq reads showing nascent transcription at the genomic position of the divergent non-coding strand (black) and the coding strand (grey). The bar graph depicts NET-seq reads as FPKM values for the coding and DNC transcript in the strains


. ( C ) NET - seq data at YDR261C locus with a novel DNC transcript . Annotations as in ( B )

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. (C) NET-seq data at the YDR261C locus with a novel DNC transcript. Annotations as in (B)


( C ) A representative image of transcription initiation observed as spots in recorded live - movie of wild type . movie output using Python were analyzed snapshot was obtained using Image J software . red green channel denotes GCG1 SUT098 transcript initiation , respectively . represent maximum projections , right bottom sidebars indicate sideviews in yz xz directions , respectively . Scale : 5 µm .

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(C) A representative image of transcription initiation observed as fluorescence spots in the recorded live-cell movie of wild type strain. The movie output using Python were analyzed and the snapshot was obtained using Image J software. The red and green channel denotes GCG1 and SUT098 transcript initiation, respectively. Images represent maximum intensity projections, and the right and bottom sidebars indicate sideviews in the yz and xz directions, respectively. Scale: 5 µm.


( D , E ) of transcription dynamics by duration frequency ( time between initiation ) of transcription . Bar graphs show transcription parameters of SUT098 in candidate mutants compared to wild type . error bars represent calculated by live - image of 179 , 135 , 60 129 single - biological repeats for WT , hda1 ∆ , hda3 ∆ cac2 ∆ strains , respectively . Statistical significance was calculated by random sampling with replacement method using a bootstrapping algorithm in Python ( asterisk * denotes p - value < 0 . 05 ) .

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(D, E) Quantification of transcription dynamics by the duration and frequency (time between initiation) of transcription. Bar graphs show the transcription parameters of SUT098 in candidate mutants compared to the wild type strain. The error bars represent SEM calculated by live-cell image analysis of 179, 135, 60 and 129 single-cell biological repeats for WT, hda1∆, hda3∆ and cac2∆ strains, respectively. Statistical significance was calculated by random sampling with replacement method using a bootstrapping algorithm in Python (the asterisk * denotes p-value <0.05).


( A ) Flow cytometry data . Bars represent mCherry / YFP of respective yeast mutants normalized to signal values of H3 wild type FPR . error bars show calculated from biological triplicates with three technical repeats . Unpaired two - student ' s t - indicates statistical significance of mutant compared to respective wild type . asterisks * , * * * * * indicate p - values < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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(A) Flow cytometry data analysis. Bars represent the mCherry/YFP fluorescence ratio of the respective yeast mutants normalized to the signal values of the H3 wild type FPR strain. The error bars show SEM calculated from biological triplicates with three technical repeats. Unpaired two-tailed student's t-test analysis indicates the statistical significance of the mutant compared to the respective wild type. The asterisks *, ** and *** indicate p-values <0.05, <0.01 and <0.001, respectively.


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment , viral production was measured by quantifying p24 antigen production in culture supernatants ( A )

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A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment , metabolic activity was assessed by a WST - 1 ( B )

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A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, metabolic activity was assessed by a WST-1 assay (B)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment viral expression was analyzed by FACS ( C )

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A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment viral protein expression was analyzed by FACS (C)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment ; initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR ( D ) .

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

A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment; and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR (D).


E J - Lat 8 . 4 were mock - treated or treated with 5 - AzadC ( 400nM ) or TNFα ( 10ng / ml ) as positive control . At 24 h , 48 h or 72 h post - treatment , initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR .

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E J-Lat 8.4 cells were mock-treated or treated with 5-AzadC (400nM) or TNFα (10ng/ml) as positive control. At 24 h, 48 h or 72 h post-treatment, initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR.


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of HDACIs . At 24 h post - treatment , viral production was measured by quantifying p24 antigen production in culture supernatants ( A , B )

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A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of HDACIs . At 24 h post - treatment , metabolic activity was assessed by a WST - 1 ( C , D ) . Data information : Means standard errors of means from three independent biological duplicates ( n = 6 ) are indicated . result obtained with mock - treated was arbitrarily set at a value 100 % ( C , D ) . selected doses were indicated by " an arrow " . Plasmatic concentrations after usual dosage ( Cmax ) of each drug in human therapy are indicated below LRA names by a box in graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, metabolic activity was assessed by a WST-1 assay (C, D).Data information: Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value 100% (C, D). The selected doses were indicated by "an arrow". Plasmatic concentrations after usual dosage (Cmax) of each drug in human therapy are indicated below the LRA names and by a box in the graph.


A - D J - Lat 8 . 4 was mock - treated or treated with 5 - AzadC / or SAHA for different periods of time as indicated . Samples were harvested at indicated times . Viral production was measured by quantifying p24 antigen production in culture supernatants ( A , B )

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A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. Viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)


A - D J - Lat 8 . 4 was mock - treated or treated with 5 - AzadC / or SAHA for different periods of time as indicated . Samples were harvested at indicated times . metabolic activity was assessed by a WST - 1 ( C , D ) . Means standard errors of means from three independent biological duplicates ( n = 6 ) are indicated . result obtained with mock - treated was arbitrarily set at a value of 100 % ( C , D ) .

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 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

A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. metabolic activity was assessed by a WST-1 assay (C, D). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 100% (C, D).


E From data of ex vivo cultures of CD8 + - depleted PBMCs isolated from 24 HIV + presented in Appendix Table S2 , extracellular HIV - 1 genomic for each LRA treatment are represented . One night after purification , were mock - treated or simultaneously treated with 5 - AzadC ( 1µM ) / or SAHA ( 1µM ) . Six days after treatment , of viral in culture supernatants was determined ( in copies / ml ) . results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented .

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

E From data of ex vivo cultures of CD8+-depleted PBMCs isolated from 24 HIV+ patients presented in Appendix Table S2, the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented. One night after cell purification, cells were mock-treated or simultaneously treated with 5-AzadC (1µM) and/or SAHA (1µM). Six days after treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


A - F J - Lat 8 . 4 ( A , C , E ) 15 . 4 ( B , D , F ) lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested analysed as follows : viral p24 production in supernatant was measured ( A B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: viral p24 production in cell supernatant was measured (A and B)


A - F J - Lat 8 . 4 ( A , C , E ) 15 . 4 ( B , D , F ) lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested analysed as follows : FACS analyses were performed percentages of GFP + are presented as histograms ( C D )

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A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows:FACS analyses were performed and the percentages of GFP+ cells are presented as histograms (C and D)


A - F J - Lat 8 . 4 ( A , C , E ) 15 . 4 ( B , D , F ) lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested analysed as follows : initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR results were normalized using β - actin gene primers are presented as histograms representing fold - inductions compared to mock - treated condition ( E F ) . Means standard errors of means from three independent biological duplicates ( n = 6 ) are indicated . result obtained with mock - treated was arbitrarily set at a value of 1 ( E , F ) .

O O O O O O O O O O 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 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

A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR and results were normalized using the β-actin gene primers and are presented as histograms representing fold-inductions compared to the mock-treated condition (E and F). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (E, F).


One night after purification , CD8 + - depleted PBMCs from 12 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added to cultures . Six days after 5 - AzadC treatment , WST - 1 was performed . Mock value was arbitrary fixed at 100 % for each .

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 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

One night after cell purification, CD8+-depleted PBMCs from 12 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added to the cultures. Six days after 5-AzadC treatment, WST-1 assay was performed. Mock value was arbitrary fixed at 100% for each individual.


Figure 6 : Representation of reactivation status of ex vivo cultures of CD8 + - depleted PBMCs isolated from HIV + . A - C From data of ex vivo cultures of CD8 + - depleted PBMCs isolated from HIV + ( Table 2 ) , extracellular HIV - 1 genomic for each LRA treatment are represented from cultures ( A ) , from cultures presenting no viral reactivation in mock condition ( C ) , from cultures exhibiting reactivation in mock condition ( B ) . One night after purification , were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . Six days after 5 - AzadC treatment , of viral in culture supernatants was determined ( in copies / ml ) . results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented .

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

Figure 6: Representation of reactivation status of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients.A-C From data of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients (Table 2), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


A Reactivation status of cultures presenting no viral reactivation in mock condition isolated from aviremic cART - treated HIV + ( shown in Appendix Table S2 ) . B Reactivation status of cultures presenting no viral reactivation in mock condition isolated from aviremic cART - treated HIV + ( shown in Table 2 ) . Data information : results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented by a .

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A Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Appendix Table S2).B Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Table 2).Data information: The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented by a line.


Figure 8 : Representation of reactivation status of ex vivo cultures of resting CD4 + T . A - C From data of ex vivo cultures of resting CD4 + T isolated from HIV + ( Table 3 ) , extracellular HIV - 1 genomic for each LRA treatment are represented from cultures ( A ) , from cultures presenting no viral reactivation in mock condition ( C ) , from cultures exhibiting reactivation in mock condition ( B ) . One night after purification , were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . Six days after 5 - AzadC treatment , of viral in culture supernatants was determined ( in copies / ml ) . results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented .

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

Figure 8: Representation of reactivation status of ex vivo cultures of resting CD4+ T cells.A-C From data of ex vivo cultures of resting CD4+ T cells isolated from HIV+ patients (Table 3), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


A - H One night after purification , CD8 + - depleted PBMCs or HLA DR - CD69 - CD25 - CD4 + T from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . activation status of CD4 + T subset was assessed 6 days after 5 - AzadC treatment by flow cytometry of activation markers relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented .

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

A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.


One night after purification , HLA DR - CD69 - CD25 - CD4 + T from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . median of CD4 receptor of viable CD4 + T subset was assessed 6 days after 5 - AzadC treatment by flow cytometry relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented .

O O O 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 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 O O O O O O O

One night after cell purification, HLA DR-CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The median fluorescence intensity of CD4 receptor of viable CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.