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

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

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


B . Plasma samples from 10 controls 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 .

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

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


C . Plasma samples from 10 ovarian cancer 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 .

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

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


D . Serum samples from 10 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 O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


B 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 ) .

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

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


D Alkaline phosphatase 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 ) .

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

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


F , G Expression 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 ) .

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

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


H 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 .

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

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


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

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


A 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 ) .

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

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


D , E 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 ) .

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

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 ) .

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

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

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


A Immunoblot of Tgif1 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 ) .

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

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


C 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 .

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

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

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


A Immunoblot of Tgif1 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 ) .

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

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


B 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 .

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

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

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


B - D Micro - Computed Tomography ( μCT ) of 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 .

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

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


J Expression 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 .

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


C Representative 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 ) .

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


E , F Representative 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 .

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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 ) .

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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 ) .

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


A , B Relative miR - 19a ( A ) 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 .

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


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

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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 ) .

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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 ) .

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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 .

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


( A ) Waterfall plot showing 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 ) .

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(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 .

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


( A ) 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 .

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(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 .

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(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 ) .

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(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 .

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


( A ) 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 .

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(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 .

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(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 .

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


( A ) 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 .

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


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

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


( C ) Immunofluorescence 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 .

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


( B ) 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 .

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


( C ) Immunofluorescence 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 .

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


( a - f ) Muscle biopsy from subject 4 . 1 subjected to electron microscopy ( transverse sections ) . In many 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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(a-f) Muscle biopsy from subject 4.1 subjected to electron microscopy (transverse sections). In many fibers, there is material between the layers of basal lamina (a, arrows; scale bar, 500 nm) or overt exocytic vacuoles (b, arrow; scale bar, 2 μm). Some fibers show a single centralized nucleus (c; scale bar, 2 μm). Mitochondria are of variable size and distribution. In some fibers, they form a loop around the nucleus in the periphery of the fiber, resembling a necklace as in c; in others, they form clusters (d; scale bar, 2 μm). The appearance of cristae is often abnormal (e; scale bar, 2 μm; f; scale bar, 500 nm). N, nucleus; M, mitochondrion.


Transverse sections from muscle biopsies from subject 3 . 1 ( top ) 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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Transverse sections from muscle biopsies from subject 3.1 (top) and a normal control (bottom) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 and counterstained with antibody to the M-band titin, titin M8. Accumulation of NBR1 in puncta (autophagosomes, arrowheads) and of MURF2, as well as fiber inhomogeneity with marked fiber atrophy, characterizes Vici syndrome muscle. Numerous fibers of very small cross-sectional area (arrows) with high content of MURF2 and NBR1 puncta are frequently seen. Scale bar, 10 μm.


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


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


C , D . Immunolabeling of HH25 chick 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 .

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


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

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


G . Immunofluorescent labeling of HH25 chick 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 .

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


A , B . Immunofluorescent labeling ( A ) of HH25 chick 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 .

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

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


C , D . Immunofluorescent labeling ( C ) of HH25 chick 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 .

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

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 .

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


A , B . 3D views ( A ) 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 .

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


C , D . Survival rate ( left axis ) 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 .

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


( F - H ) of HH25 chick 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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


( C ) Flow cytometry 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 .

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


. ( B ) NET - seq data at 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

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


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

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


( C ) A representative image of transcription initiation observed as 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 .

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


( D , E ) 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 ) .

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


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


A - D J - Lat 8 . 4 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 )

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


A - D J - Lat 8 . 4 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 )

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


A - D J - Lat 8 . 4 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 )

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


A - D J - Lat 8 . 4 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 ) .

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A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment; 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 .

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


A - D J - Lat 8 . 4 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 )

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


A - D J - Lat 8 . 4 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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A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, metabolic activity was assessed by a WST-1 assay (C, D).Data information: Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value 100% (C, D). The selected doses were indicated by "an arrow". Plasmatic concentrations after usual dosage (Cmax) of each drug in human therapy are indicated below the LRA names and by a box in the graph.


A - D J - Lat 8 . 4 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 )

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


A - D J - Lat 8 . 4 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 ) .

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A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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


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


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

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

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


( A ) RFP - ATG8g - labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ ( oD600 = 0 . 2 ) at 2 dpi in the presence or absence of ConA ( bars = 20 μm ) . Puncta were calculated from z - stacks ( 15 ) of n = 6 individuals using ImageJ . Central band of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Different letters indicate statistically different groups ( P < 0 . 05 ) as determined by one - way ANOVA . The experiment was repeated twice with similar results .

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

(A) RFP-ATG8g-labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ (oD600=0.2) at 2 dpi in the presence or absence of ConA (bars = 20 μm). Puncta were calculated from z-stacks (15) of n=6 individuals using ImageJ. Central band of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically different groups (P < 0.05) as determined by one-way ANOVA. The experiment was repeated twice with similar results.


( B ) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N . benthamiana plants at 1 and 2dpi . Agrobacterium - mediated transient expression of AIMp - RFP serves as a control for autophagy suppression . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results .

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(B) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N. benthamiana plants at 1 and 2dpi. Agrobacterium-mediated transient expression of AIMp-RFP serves as a control for autophagy suppression. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( C ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ was co - infiltrated with Agrobacteria containing the luciferase reporter constructs . Coexpression of RFP - AIMp serves as a control for autophagy inhibition . Expression of the latter was confirmed with western blot ( inset ) . Renilla ( RLUC ) and Firefly ( FLUC ) luciferase activities were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . The experiment was repeated more than 3 times with similar results .

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

(C) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ was co-infiltrated with Agrobacteria containing the luciferase reporter constructs. Coexpression of RFP-AIMp serves as a control for autophagy inhibition. Expression of the latter was confirmed with western blot (inset). Renilla (RLUC) and Firefly (FLUC) luciferase activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P<0.001) was revealed by Student's t-test. The experiment was repeated more than 3 times with similar results.


( D ) RT - qPCR analysis of NbATG8 - 1 . 1 / 1 . 2 , NbATG8 - 2 . 1 / 2 . 2 and NbJoka2 transcript levels upon challenge of N . benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants . Values represent expression relative to mock control of respective time point and were normalized to actin . Values are biological replicates ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) was revealed by Student ' s t - test .

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

(D) RT-qPCR analysis of NbATG8-1.1/1.2, NbATG8-2.1/2.2 and NbJoka2 transcript levels upon challenge of N. benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants. Values represent expression relative to mock control of respective time point and were normalized to actin. Values are biological replicates (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (*P < 0.05, **P < 0.01, *** P < 0.001) was revealed by Student's t-test.


( E ) Bacterial density in leaves of N . benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp - RFP . Leaves were syringe - infiltrated with OD600 = 0 . 0004 , and colony - forming units were counted at 6 dpi . Compared to empty vector control ( EV ) , AIMp expressing plants ( n = 6 ) harbour significantly more bacteria . Bacterial growth was repeated with the same result in 12 plants over two independent experiments . Red and yellow data points indicate independent repeats of the experiment . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . Expression of RFP - AIMp was verified at 6 dpi with an anti - RFP blot ( inset ) .

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

(E) Bacterial density in leaves of N. benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp-RFP. Leaves were syringe-infiltrated with OD600 = 0.0004, and colony-forming units were counted at 6 dpi. Compared to empty vector control (EV), AIMp expressing plants (n=6) harbour significantly more bacteria. Bacterial growth was repeated with the same result in 12 plants over two independent experiments. Red and yellow data points indicate independent repeats of the experiment. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P < 0.001) was revealed by Student's t-test. Expression of RFP-AIMp was verified at 6 dpi with an anti-RFP blot (inset).