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

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


Figure 6MACS - separated CD8 + T cells were cultured in triplicate on an immobilized anti - CD3 mAb ( 0 . 005 μg / ml [ upper panel ] or 0 . 01 μg / ml [ lower panel ] ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , and OVA - Fc proteins at various concentrations ( 1 . 0 , 0 . 5 , 0 . 1 , 0 . 0 μg / ml ) . After 72 h , 25 % of the supernatant was harvested and IL - 2 concentrations were measured by ELISA . The harvested medium was replaced by the same volume containing 1 μCi 3H - labeled thymidine . After 12 h , the plates were completely harvested and the DNA - bound radioactivity was determined . The means and SD of the stimulation index ( SI ) are displayed ( n = 4 ) . Significant differences between MICA - 129Met / Val - Fc and OVA - Fc proteins were found when the antigen - specific signal ( anti - CD3 ) was limited ( * P < 0 . 05 , t - test ; upper left panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0372 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0366 ; upper right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0499 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0192 ; 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0164 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0357 ; lower left panel : 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0287 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0232 ; lower right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0171 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0484 ) . Purified CFSE - stained CD8 + T cells were stimulated by immobilized anti - CD3 ( 0 . 005 μg / ml ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , OVA - Fc proteins , or co - stimulatory mAb ( anti - CD28 , anti - NKG2D ) or an isotype control ( mIgG1 ) . The proliferation of CD3 + CD8 + T cells was assessed at 60 h by flow cytometry . Results of a representative out of 6 experiments are displayed . Untreated CFSE - stained CD8 + T cells are included for comparison . The percentage of proliferating cells and MFI for CFSE are indicated . The MFI of CFSE in unstimulated CD8 + T cells ( control ) was set to 100 % in individual experiments ( n = 6 ) , and the relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( * P = 0 . 0277 , Wilcoxon test ) between MICA - 129Met - Fc versus MICA - 129Val - Fc and OVA - Fc proteins were found at slightly higher anti - CD3 concentrations ( 0 . 1 and 0 . 05 μg / ml ) than at later time points ( see A ) . Anti - CD28 and anti - NKG2D mAb were used in parallel as a positive control , mean + SD are shown , and significant differences ( * P = 0 . 0277 , Wilcoxon test ) to the isotype control ( mIgG1 ) are indicated ( n = 6 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6MACS-separated CD8+ T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed (n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (*P < 0.05, t-test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484).Purified CFSE-stained CD8+T cells were stimulated by immobilized anti-CD3 (0.005 μg/ml) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, OVA-Fc proteins, or co-stimulatory mAb (anti-CD28, anti-NKG2D) or an isotype control (mIgG1). The proliferation of CD3+CD8+T cells was assessed at 60 h by flow cytometry. Results of a representative out of 6 experiments are displayed. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (*P = 0.0277, Wilcoxon test) between MICA-129Met-Fc versus MICA-129Val-Fc and OVA-Fc proteins were found at slightly higher anti-CD3 concentrations (0.1 and 0.05 μg/ml) than at later time points (see A).Anti-CD28 and anti-NKG2D mAb were used in parallel as a positive control, mean + SD are shown, and significant differences (*P = 0.0277, Wilcoxon test) to the isotype control (mIgG1) are indicated (n = 6).


Figure 7NKG2D expression on purified CD8 + T cells exposed to L - MICA - 129Met ( n = 19 ) or L - MICA - 129Val clones ( n = 19 ) for 0 , 4 , and 24 h was analyzed by flow cytometry . CD8 + T cells ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3 + CD8 + T cells . The means and SD of the MFI of NKG2D ( left panel ) and of the percentage of NKG2D + CD8 + T cells ( right panel ) are displayed . Differences between the groups were analyzed by repeated measures ANOVA , and the P - values are indicated . Purified CD8 + T cells were cultured on plate - bound anti - NKG2D ( 1 μg / ml ) or isotype control ( mIgG1 ) for 24 h before the NKG2D expression was measured by flow cytometry . Means and SD of MFI ( upper panel ) and percentage of NKG2D + cells ( lower panel ) are shown ( n = 6 ) . Differences between the groups were analyzed by t - tests , and the P - values are indicated . 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 .

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Figure 7NKG2D expression on purified CD8+T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.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.


Figure 1B . 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B. 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 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 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 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.


Figure 3A . 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 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 .

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Figure 3A. 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 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.


Figure 4A . 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 .

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Figure 4A. 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.


Figure 6A . 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 .

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Figure 6A. 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.


Figure 7B . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7B. 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 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 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.


Figure 1B 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 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B 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 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 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 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 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).


Figure 2A 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A 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 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 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 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.


Figure 3A 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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 3A 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 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 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.


Figure 4A 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A 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 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 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).


Figure 5B - 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B-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 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.


Figure 6C 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6C 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 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 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 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).


Figure 7A , 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 ) and miR - 19b ( E ) in Ocy454 cells upon estrogen deficiency ( ED ) and control ( C ) treatment ( n = 6 ) . 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 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 images of 3D reconstructed human bone samples ( K ) and quantification of bone mineral density ( BMD ) ( L ) . HBM , n = 4 , LBM , n = 4 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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 7A, 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) and miR-19b (E) in Ocy454 cells upon estrogen deficiency (ED) and control (C) treatment (n=6).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 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 images of 3D reconstructed human bone samples (K) and quantification of bone mineral density (BMD) (L). HBM, n=4, LBM, n=4.


Figure 1 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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 1(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 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.


Figure 2 ( 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 cells and PDXs . Three biological replicates of PDX093 are shown ; PDX302 is used as PALB2 wild type PDX control . ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(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 cells and PDXs. Three biological replicates of PDX093 are shown; PDX302 is used as PALB2 wild type PDX control.(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 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.


Figure 3 ( 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 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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 3(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 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) 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.


Figure 4 ( 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 the RAD51 score and HRD score , for PARPi response prediction capacity in the PDX cohort - 2 . Bootstrap statistical test . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(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 the RAD51 score and HRD score, for PARPi response prediction capacity in the PDX cohort-2. Bootstrap statistical test.(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.


Figure 5 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5(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 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.


figf1 ( 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 .

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figf1(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.


figf2Transverse 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 .

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figf2Transverse 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.


figf3Accumulation 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 .

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figf3Accumulation 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.


figf4In 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 .

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figf4In 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.


Figure 1C , 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 ( 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1C,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 (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 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.


Figure 2A , 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 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A,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 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) 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.


Figure 3A , 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 ) 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A,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) 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 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.


Figure 1 ( 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 .

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Figure 1(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.


Figure 2 ( 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 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 .

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Figure 2(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 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.


Figure 3 . ( 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 the YDR261C locus with a novel DNC transcript . Annotations as in ( B )

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Figure 3. (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 the YDR261C locus with a novel DNC transcript. Annotations as in (B)


Figure 4 ( 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 ) 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(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) 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).


Figure 5 ( 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 .

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Figure 5(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.


Figure 1A - 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A-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 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 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 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 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.


Figure 2A - 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 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 .

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Figure 2A-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 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.


Figure 3A - 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 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 + 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A-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 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+ 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.