E , F Immunoblotting using anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAF siCTR - or siSHP2 - ( E ) or siPTPε - ( F ) transfected CAF treated or not with SOM230 for 30 min ( representative of n = 3 ) .

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E, F Immunoblotting using anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAF siCTR- or siSHP2- (E) or siPTPε- (F) transfected CAF treated or not with SOM230 for 30 min (representative of n = 3).


G , H Immunoblotting using an anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAFs treated with the indicated molecules ( PDGF receptor inhibitor , 5 - 10 μM ; Jak1 / 2 inhibitor ruxolitinib , 5 or 10 μM ; EGFR inhibitor , 150 or 300 nM and recombinant PDGF , 5 μg / ml ) ( representative of n = 3 ) .

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G, H Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs treated with the indicated molecules (PDGF receptor inhibitor, 5-10 μM; Jak1/2 inhibitor ruxolitinib, 5 or 10 μM; EGFR inhibitor, 150 or 300 nM and recombinant PDGF, 5 μg/ml) (representative of n = 3).


A - C MIA PaCa - 2 - GLuc cells were injected with or without CAFs into the pancreas of nude mice . Mice were treated with each indicated treatment ( SOM230 - LAR at day 7 and gemcitabine at days 7 , 10 , 14 and 17 ) , and the plasmatic luciferase activity was measured ( mean ± SD ) at days 7 , 14 and 21 ( n = 5 ) ( P = 0 . 003 for gemcitabine + SOM230LAR versus untreated ) ( A ) . Tumours were excised , photographed ( scale bars are 1 cm ) ( B ) , weighted ( P = 0 . 0008 ) ( C ) and paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( E ) .

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A-C MIA PaCa-2-GLuc cells were injected with or without CAFs into the pancreas of nude mice. Mice were treated with each indicated treatment (SOM230-LAR at day 7 and gemcitabine at days 7, 10, 14 and 17), and the plasmatic luciferase activity was measured (mean ± SD) at days 7, 14 and 21 (n = 5) (P = 0.003 for gemcitabine + SOM230LAR versus untreated) (A). Tumours were excised, photographed (scale bars are 1 cm) (B), weighted (P = 0.0008) (C) and paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (E).


D Human tumours were subcutaneously xenografted in nude mice and tumour volumes ( mean ± SD ) measured ( at day 37 , P = 0 . 0009 for gemcitabine + SOM230LAR versus untreated and P = 0 . 021 for gemcitabine + SOM230LAR versus gemcitabine ) . Mice were treated with the indicated treatments ( SOM230 - LAR at days 3 and 31 , and gemcitabine at day 3 and twice a week thereafter ) .

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D Human tumours were subcutaneously xenografted in nude mice and tumour volumes (mean ± SD) measured (at day 37, P = 0.0009 for gemcitabine + SOM230LAR versus untreated and P = 0.021 for gemcitabine + SOM230LAR versus gemcitabine). Mice were treated with the indicated treatments (SOM230-LAR at days 3 and 31, and gemcitabine at day 3 and twice a week thereafter).


E , F Tumours excised from mice as in ( A - C ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( E ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( F ) ( * P = 0 . 036 , * * P = 0 . 009 ) .

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E, F Tumours excised from mice as in (A-C) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (E). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (F) (*P = 0.036, **P = 0.009).


G , H Tumours excised from mice as in ( D ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( G ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( H ) ( * P = 0 . 027 ) .

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G, H Tumours excised from mice as in (D) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (G). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (H) (*P = 0.027).


A Membrane antibody array assay using CM from SOM230 - treated or not CAFs ( representative of n = 3 ) . Controls are circled in blue dashed line and IL - 6 in red square .

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A Membrane antibody array assay using CM from SOM230-treated or not CAFs (representative of n = 3). Controls are circled in blue dashed line and IL-6 in red square.


B , C Anti - IL - 6 ELISA assay ( mean ± SD ) using CM or protein extracts from SOM230 - treated or not CAF ( from left to right : § § § P = 0 . 0002 , § § § P = 0 . 0002 ) ( B ) , or CM from siCTR - or si4E - BP1 - transfected CAFs treated or not with SOM230 ( from left to right : * * * P = 0 . 0003 , $ $ P = 0 . 004 ) ( C ) ( n = 3 ) .

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B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).


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D Panc - 1 cell viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment as the percentage of the respective untreated cells ( = 100 % ) ( n = 3 ; from left to right : * * P = 0 . 004 , # # P = 0 . 001 , § P = 0 . 023 , § P = 0 . 019 ) .

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D Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment as the percentage of the respective untreated cells (= 100%) (n = 3; from left to right: **P = 0.004, ##P = 0.001, §P = 0.023, §P = 0.019).


E , F Immunoblotting using an anti - cleaved caspase - 3 , anti - PARP or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells incubated with CAF - CM pre - incubated or not with an anti - IL - 6 neutralizing antibody ( E ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( F ) .

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E, F Immunoblotting using an anti-cleaved caspase-3, anti-PARP or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells incubated with CAF-CM pre-incubated or not with an anti-IL-6 neutralizing antibody (E), or with human recombinant IL-6 (representative of n = 3) (F).


G , H Immunoblotting using an anti - survivin or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells stimulated or not with CAF - CM pre - incubated or not with anti - IL - 6 neutralizing antibody ( G ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( H ) .

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G, H Immunoblotting using an anti-survivin or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells stimulated or not with CAF-CM pre-incubated or not with anti-IL-6 neutralizing antibody (G), or with human recombinant IL-6 (representative of n = 3) (H).


I Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of protein extracts from SOM230 - treated or SOM230 - untreated CAF incubated or not with IL - 6 neutralizing antibody ( representative of n = 3 ) .

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I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).


A - C Immunohistofluorescence using an anti - α - SMA or anti - IL - 6 antibody in paraffin - embedded sections from MIA PaCa - 2 - Gluc and CAF cells orthotopic co - xenografted tumours from Fig5A ( A ) or from human tumour subcutaneously xenografted from Fig5D ( B ) in nude mice treated or not with gemcitabine + SOM230 ( representative of five different tumours ) . Representative co - localization of α - SMA with IL - 6 in human PDAC samples ( C ) ( n = 15 ) . Scale bar = 100 μm .

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A-C Immunohistofluorescence using an anti-α-SMA or anti-IL-6 antibody in paraffin-embedded sections from MIA PaCa-2-Gluc and CAF cells orthotopic co-xenografted tumours from Fig5A (A) or from human tumour subcutaneously xenografted from Fig5D (B) in nude mice treated or not with gemcitabine + SOM230 (representative of five different tumours). Representative co-localization of α-SMA with IL-6 in human PDAC samples (C) (n = 15). Scale bar = 100 μm.


D , E Plasma was collected from MIA PaCa - 2GLuc and CAFs ( * P = 0 . 034 , * * P = 0 . 007 ) ( D ) , or human pancreatic tumour ( * P = 0 . 028 , * * P = 0 . 004 ) ( E ) , xenografts in nude mice and human IL - 6 plasmatic concentrations ( mean ± SD ) were quantified by ELISA ( n = 5 mice / group ) . * treated versus untreated mice .

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D, E Plasma was collected from MIA PaCa-2GLuc and CAFs (*P = 0.034, **P = 0.007) (D), or human pancreatic tumour (*P = 0.028, **P = 0.004) (E), xenografts in nude mice and human IL-6 plasmatic concentrations (mean ± SD) were quantified by ELISA (n = 5 mice / group). * treated versus untreated mice.


G ) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates ( n = 3 ) . Β - actin serves as an internal loading control .

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G) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates (n=3). Β-actin serves as an internal loading control.


B ) Images represent localization of KTR for ERK , JNK , or p38 under basal growth conditions . Scales bars = 20µm .

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B) Images represent localization of KTR for ERK, JNK, or p38 under basal growth conditions. Scales bars=20µm.


C - E ) Kinase activity for ERK ( C ) , JNK ( D ) , or p38 ( E ) in three representative single cells ( upper panels ) in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) as measured by C / N ratio . Lower panel shows mean C / N ratio in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) . Thick line represents mean with shaded area = SD . N for each condition and kinase is shown in population average plots .

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C-E) Kinase activity for ERK (C), JNK (D), or p38 (E) in three representative single cells (upper panels) in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red) as measured by C/N ratio. Lower panel shows mean C/N ratio in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red). Thick line represents mean with shaded area=SD. N for each condition and kinase is shown in population average plots.


C - E ) Mean kinase activation for ERK € , JNK ( D ) , or p38 ( E ) in both live ( grey ) and dead ( red ) cells measured over 24 - hours in response to H2O2 . Shaded areas = SD . N for each condition is shown .

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C-E) Mean kinase activation for ERK €, JNK (D), or p38 (E) in both live (grey) and dead (red) cells measured over 24-hours in response to H2O2. Shaded areas=SD. N for each condition is shown.


D ) Mean JNK activity as measured by C / N ratio ( bold line ) ± SD ( shaded area ) in response to 300 µM H2O2 with or without p38 inhibition ( grey ) . Bar graph shows integrated JNK activity ( C / N ratio ) over 24 - hours . Data represents mean ± SD . The number of analyzed cells , n , is shown for each condition . Statistics performed using 2 - tailed t - test . Data information : * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant .

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D) Mean JNK activity as measured by C/N ratio (bold line)±SD (shaded area) in response to 300 µM H2O2 with or without p38 inhibition (grey). Bar graph shows integrated JNK activity (C/N ratio) over 24-hours. Data represents mean±SD. The number of analyzed cells, n, is shown for each condition. Statistics performed using 2-tailed t-test. Data information: ** p<0.01, *** p<0.001, ns=not significant.


Kaplan - Meier survival curves stratified by the patient MICA - 129 genotype for all patients ( n = 446 ) . The survival curve is displayed for the first 66 months ; 7 . 6 % of the patients were followed longer . Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2 . The HR indicates the risk per MICA - 129Met allele carried by the patients ( additive risk model ) . The numbers of patients carrying the three genotypes and the number of events ( in brackets ) are indicated .

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Kaplan-Meier survival curves stratified by the patient MICA-129 genotype for all patients (n = 446). The survival curve is displayed for the first 66 months; 7.6% of the patients were followed longer. Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2. The HR indicates the risk per MICA-129Met allele carried by the patients (additive risk model). The numbers of patients carrying the three genotypes and the number of events (in brackets) are indicated.


Kaplan - Meier survival curves for patients receiving a graft matched for the MICA - 129 genotype ( n = 404 ) .

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Kaplan-Meier survival curves for patients receiving a graft matched for the MICA-129 genotype (n = 404).


Kaplan - Meier survival curves for patients who experienced aGVDH ( any grade , n = 244 ) .

Kaplan-Meier survival curves for patients who experienced aGVDH (any grade, n = 244).


Kaplan - Meier survival curves for patients not experiencing aGVHD ( n = 189 ) . The HR indicates the risk of patients carrying two MICA - 129Met alleles ( recessive risk model ) .

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Kaplan-Meier survival curves for patients not experiencing aGVHD (n = 189). The HR indicates the risk of patients carrying two MICA-129Met alleles (recessive risk model).


Kaplan - Meier survival curves for patients who did not receive a T - cell - depleting treatment with ATG ( n = 193 ) . Kaplan - Meier survival curves for patients treated with ATG ( n = 250 ) .

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Kaplan-Meier survival curves for patients who did not receive a T-cell-depleting treatment with ATG (n = 193).Kaplan-Meier survival curves for patients treated with ATG (n = 250).


Kaplan - Meier survival curves for patients with the MICA - 129Val / Val genotype ( n = 229 ) stratified by treatment with ATG .

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Kaplan-Meier survival curves for patients with the MICA-129Val/Val genotype (n = 229) stratified by treatment with ATG.


Kaplan - Meier survival curves for patients carrying one or two MICA - 129Met alleles ( n = 214 ) stratified by treatment with ATG .

Kaplan-Meier survival curves for patients carrying one or two MICA-129Met alleles (n = 214) stratified by treatment with ATG.


The linear regression of MICA expression intensity and binding of a recombinant NKG2D - Fc fusion protein both determined as MFI by flow cytometry is displayed for L - MICA - 129Met ( n = 79 , left panel ) and L - MICA - 129Val clones ( n = 81 , right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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The linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met (n = 79, left panel) and L-MICA-129Val clones (n = 81, right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


Purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells ( 106 ) were stimulated with immobilized MICA - 129Met - mIgG2a - Fc or MICA - 129Val - mIgG2a - Fc or OVA - mIgG2a - Fc fusion proteins ( 10 μg / ml ) for 3 , 10 , or 30 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb , an anti - phospho - SRC family ( Tyr419 ) kinases Ab , and an anti - β - actin mAb as a loading control . The arrow points toward phosphorylated SRC family kinases . Blots obtained from three independent experiments were analyzed by densitometry , and the means plus SD of the ratio between phospho - SRC family kinase and β - actin signals is displayed . The difference between NK cells stimulated for 10 min by MICA - 129Met - Fc or MICA - 129Val - Fc proteins was assessed by t - test .

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Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (106) were stimulated with immobilized MICA-129Met-mIgG2a-Fc or MICA-129Val-mIgG2a-Fc or OVA-mIgG2a-Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases.Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t-test.


Purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days , 106 ) were incubated with the SRC kinase inhibitor PP2 ( 25 μM ) , the vehicle DMSO , or medium only ( Ø ) for 30 min before being added to immobilized MICA - 129Met - Fc , MICA - 129Val - Fc , or OVA - Fc fusion proteins ( 10 μg / ml ) for 10 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb and an anti - β - actin mAb as a loading control . The blot is representative for two independent experiments .

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Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.


In parallel , degranulation of the NK cells was measured by anti - CD107a staining in flow cytometry . The difference between DMSO - and PP2 - treated cells with respect to CD107a + cells and the MFI of CD107a is indicated in the histograms . The results are representative for two independent experiments .

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In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.


The degranulation ( CD107a expression ) of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells in response to immobilized MICA - 129Met - Fc and MICA - 129Val - Fc , and as negative control , OVA - Fc fusion proteins was determined by flow cytometry after 1 h ( n = 3 ) . Displayed are means and SD of the MFI ( left panel ) and the percentage of CD107a + cells ( right panel ) . Differences between MICA - 129Met - Fc - and MICA - 129Val - Fc - induced NK - cell degranulation were analyzed by two - way ANCOVA adjusted for MICA protein concentration , and the respective P - values are indicated .

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

The degranulation (CD107a expression) of purified IL-2-stimulated (100 U/ml for 4 days) NK cells in response to immobilized MICA-129Met-Fc and MICA-129Val-Fc, and as negative control, OVA-Fc fusion proteins was determined by flow cytometry after 1 h (n = 3). Displayed are means and SD of the MFI (left panel) and the percentage of CD107a+ cells (right panel). Differences between MICA-129Met-Fc- and MICA-129Val-Fc-induced NK-cell degranulation were analyzed by two-way ANCOVA adjusted for MICA protein concentration, and the respective P-values are indicated.


The degranulation of IL - 2 - stimulated LAK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 27 ) or L - MICA - 129Val clones ( n = 27 ) for 1 h was determined by flow cytometry . CD107a cell surface expression was analyzed after gating on CD56 + NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of CD107a on NK cells ( upper panels ) or the proportion of CD107a + NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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

The degranulation of IL-2-stimulated LAK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 27) or L-MICA-129Val clones (n = 27) for 1 h was determined by flow cytometry. CD107a cell surface expression was analyzed after gating on CD56+ NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of CD107a on NK cells (upper panels) or the proportion of CD107a+ NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


C . Generation of sFn14 is sensitive to 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 .

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 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

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 .

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 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

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

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


A . Human breast cancer 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 .

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 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 O O O O O O O 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

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


C . Human ovarian cancer 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 .

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

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


A . MDA - MB - 231 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 .

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 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 O O O O O

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


A . 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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 .

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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 .

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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 .

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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 ) .

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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 .

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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 ) .

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 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

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.