EMBO/SourceData huggingface dataset:
Figure 6A 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, $$P = 0.004) (C) (n = 3).B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).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).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).
Figure 7A-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.
Figure 1G) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates (n=3). Β-actin serves as an internal loading control.
Figure 2B) 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.
Figure 3C-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.
Figure 6D) 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.
Figure 1Kaplan-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).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).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.Kaplan-Meier survival curves for patients carrying one or two MICA-129Met alleles (n = 214) stratified by treatment with ATG.
Figure 2The 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.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.
Figure 3The 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.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.
Figure 4The 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.
Figure 5NKG2D expression on purified IL-2-stimulated NK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 25) or L-MICA-129Val clones (n = 25) for 0, 4, and 24 h was analyzed by flow cytometry. NK cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating of CD3−CD56+ cells. The means and SD of the percentage of NKG2D+ NK cells (left panel) and of the MFI of NKG2D (right panel) are shown. Differences between the groups were analyzed by repeated measures ANOVA, and P-values for pairwise comparisons are indicated.
Figure 6MACS-separated CD8+ T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed (n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (*P < 0.05, t-test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484).Purified CFSE-stained CD8+T cells were stimulated by immobilized anti-CD3 (0.005 μg/ml) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, OVA-Fc proteins, or co-stimulatory mAb (anti-CD28, anti-NKG2D) or an isotype control (mIgG1). The proliferation of CD3+CD8+T cells was assessed at 60 h by flow cytometry. Results of a representative out of 6 experiments are displayed. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (*P = 0.0277, Wilcoxon test) between MICA-129Met-Fc versus MICA-129Val-Fc and OVA-Fc proteins were found at slightly higher anti-CD3 concentrations (0.1 and 0.05 μg/ml) than at later time points (see A).Anti-CD28 and anti-NKG2D mAb were used in parallel as a positive control, mean + SD are shown, and significant differences (*P = 0.0277, Wilcoxon test) to the isotype control (mIgG1) are indicated (n = 6).
Figure 7NKG2D expression on purified CD8+T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.These CD8+T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.
Figure 1B. Detection of sFn14 in conditioned media. HEK293E cells were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N-terminal HA-tag and a C-terminal double FLAG-tag. Conditioned media and lysates of the transfected cells were collected and analyzed by immunoblotting with the indicated antibodies. Calnexin served as a loading control. Shown are representative blots from N=3 experiments.C. Generation of sFn14 is sensitive to the γ-secretase inhibitor DAPT. HEK293E cells were transfected with either Fn14 or C99 (C-terminal fragment of APP), both containing a N-terminal HA-tag and C-terminal double FLAG-tag. One day after transfection, cells were treated with γ-secretase inhibitor DAPT (1 μM), broad-spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. The conditioned media and the lysates were blotted with anti-HA antibody. Shown are representative blots from N=4 experiments.E. In vitro γ-secretase cleavage assay. HEK293E cells were transfected with epitope-tagged Fn14 or C99. Cellular membranes were collected and incubated under indicated conditions for the γ-secretase activity assay. Reactions were terminated and ultracentrifuged. Supernatant (containing γ-secretase cleavage products) was used for detecting the ICD fragment while the pellet was used to blot for full length proteins Fn14 or C99. Blotting is done by anti-Flag antibody. Shown are representative blots from N=3 experiments.F. sFn14 production requires the proteolytic presenilin subunit of γ-secretase. HEK293 cells stably transfected with APP carrying the Swedish double mutation (K595N/M596L) and with a CRISPR/CAS9-mediated knockout of presenilin 1 (PS1 KO) or of both PS1 and PS2 (PS1/2 dKO), were transiently transfected with epitope-tagged Fn14. Conditioned media and lysates of the transfected cells were blotted with the indicated antibodies. C83 is a small C-terminal fragment of APP (containing the C-terminal 83 amino acids), which is also subjected to γ-secretase processing. Upon longer exposure, the C99 (generated by BACE1 from APP) is also visible in the PS1/2 dKO condition. Shown are representative blots from N=4 experiments. G. Quantification of results from panel F. sFn14 levels in the conditioned media quantified and normalized to WT sFn14 signal. Data Information: Panel (G) shows mean ± SEM, along with p-values calculated by ordinary one-way ANOVA test with Tukey's multiple comparison test.
Figure 3A. Human breast cancer cell line MDA-MB-231 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. B. Quantification of blots from panel A. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels B the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.C. Human ovarian cancer cell line SKOV-3 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. D. Quantification of blot from panel C. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels D, the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.
Figure 4A. MDA-MB-231 cells were transfected with an siRNA pool against human Fn14 or non-targeting control (Ctrl) siRNA. A day after transfection, the cells were treated with γ-secretase inhibitor DAPT (1 μM) or vehicle overnight. The lysate was collected and blotted against Fn14 (C-terminal antibody) or calnexin as loading control. Shown are representative blots from N=4 experiments. B. Quantification of blot in panel A. The control condition where the cells were only treated with vehicle DMSO and non-targeting siRNA (DMSO + siCtrl) was used as baseline, and its average normalized to 1. N=4 experiments. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against control (DMSO + siCtrl) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.
Figure 6A. Cellular Fn14 in ex vivo glioblastoma samples increased upon γ-secretase inhibition. Primary cells from four different glioblastomas were treated with DAPT (1 μM) or vehicle overnight. Lysates were blotted against Fn14 and calnexin as loading control. B. Quantification of the blot in panel A. For each glioblastoma, the relative (rel.) mean intensity of the normalized Fn14 vehicle condition was used for normalization. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against their corresponding control (DMSO) condition using two-tailed unpaired t-tests. The p-values that are above 0.05 have not been included into the panels. For all the panels, three biological replicates were performed.
Figure 7B. Plasma samples from 10 controls and GBM patients with an initial diagnosis of GBM (iGBM, 40 patients) or a recurrent GBM (rGBM, 20 patients) were collected and sFn14 levels were measured by ELISA. Data information: For panel B median of the measurements, along with interquartile range borders are displayed. In panel B, ordinary one-way ANOVA with Tukey's multiple comparison test was applied, but no value passed our p-value display threshold of 0.05.C. Plasma samples from 10 ovarian cancer patients with low/middle and 19 patients with high tumor burden were collected and sFn14 levels were measured by ELISA. The tumor burden was clinically determined by computer tomographic scans. Data information: For panel C, median of the measurements, along with interquartile range borders are displayed. For panel C, a two-tailed unpaired t-test was applied, but no value passed our p-value display threshold of 0.05.D. Serum samples from 10 patients with refractory multiple myeloma were collected pre- and post-treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days. sFn14 levels in these samples are displayed (N=10). Data information: For panel D, ratio-paired t-test was used to compare the sFn14 levels pre- and post-treatment.