Journal: Osteoarthritis and Cartilage Open

Article Title: Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage

doi: 10.1016/j.ocarto.2020.100128

Figure Lengend Snippet: Expression of ADAM15 is not altered in human OA cartilage, but is reduced with age. (A) Forest plot showing that donors used for (B–D) were age- and sex-matched. Mean age for all samples was calculated and subtracted from the mean age of OA females (n ​= ​5), OA males (n ​= ​3), normal females (n ​= ​6) and normal males (n ​= ​6). Data are depicted as differences in means (ΔMean) with 95% CI. ΔMeans were not significantly different (P ​= ​0.7886). (B) Chondrocytes were isolated from normal (n ​= ​12, 58–75 years of age) and OA (n ​= ​8, 58–84 years of age) cartilage and cells cultured in monolayer for 5 days. RNA was isolated and expression of ADAM15 quantified by RT-qPCR, relative to RPLP0 and to mean expression in normal chondrocytes (mean ​± ​SD). (C) RNA was extracted from normal (n ​= ​12, 58–75 years of age) and OA (n ​= ​8, 58–84 years of age) cartilage samples matching those in (B), and expression of ADAM15 quantified by RT-qPCR, relative to RPLP0 and to mean expression in normal cartilage (mean ​± ​SD). (D) Data shown in (C) were analysed by gender, irrespective of OA status, with expression normalised to the mean of expression in female donors (mean ​± ​SD). (E) Data shown in (C), along with data from an additional 4 normal donors (9–16 years of age), were analysed by age. Samples from normal cartilage are shown in closed circles (n ​= ​16) and those from OA cartilage shown in open circles (n ​= ​8). ∗P ​< ​0.05, ∗∗∗P ​< ​0.001.

Article Snippet: Primary antibodies were applied to detect ADAM15 (rabbit anti-ADAM15, Atlas Antibodies, HPA011633, 2 μg/ml) and/or perlecan (rat anti-perlecan, Millipore).

Techniques: Expressing, Isolation, Cell Culture, Quantitative RT-PCR

Journal: Osteoarthritis and Cartilage Open

Article Title: Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage

doi: 10.1016/j.ocarto.2020.100128

Figure Lengend Snippet: Secretome of Adam15 −/− chondrocytes is not significantly different from WT. (A) Volcano plot showing the −log10 of P-values versus the log2 of protein abundance in media of Adam15 −/− versus WT chondrocytes (n ​= ​3 animals per group). The hyperbolic curves represent a permutation-based FDR correction for multiple hypotheses (P ​= ​0.05, s0 ​= ​0.1). Detected proteins were below the curve after FDR, and thus were not significantly different between the groups. ECM proteins (based on Uniprot annotation) are shown in blue. (B) Abundance of collagens and aggrecan were not altered in the media of Adam15 −/− chondrocytes compared to WT. (C) Abundance of other selected extracellular matrix components and regulators was also not altered in the medium of Adam15 −/− chondrocytes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies were applied to detect ADAM15 (rabbit anti-ADAM15, Atlas Antibodies, HPA011633, 2 μg/ml) and/or perlecan (rat anti-perlecan, Millipore).

Techniques: Quantitative Proteomics

Journal: Osteoarthritis and Cartilage Open

Article Title: Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage

doi: 10.1016/j.ocarto.2020.100128

Figure Lengend Snippet: Cartilage expression of ADAM15 also decreases with age at the protein level. (A) Cartilage sections from donors aged 47, 54 and 90 were stained with antibodies against ADAM15 (red) and perlecan (green), and counterstained with DAPI. Exposure times for each channel were kept constant for all samples. (B) Expression of ADAM15 was semi-quantitatively evaluated in 3 normal (red) and 3 OA (orange) donors of various ages, with each dot representing integrated fluorescence intensity per single cell, calculated from at least six random fields of view on three sections per donor. Median fluorescence intensity with 95% CIs of the mean are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies were applied to detect ADAM15 (rabbit anti-ADAM15, Atlas Antibodies, HPA011633, 2 μg/ml) and/or perlecan (rat anti-perlecan, Millipore).

Techniques: Expressing, Staining, Fluorescence

Journal: Osteoarthritis and Cartilage Open

Article Title: Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage

doi: 10.1016/j.ocarto.2020.100128

Figure Lengend Snippet: Expression of Adam15 is reduced with age in murine joints. (A) RNA was extracted from joints of male mice 6 ​h after DMM or sham surgery and expression of Adam15 quantified by RT-qPCR, relative to Rplp0 and average expression in sham-operated joints (mean ​± ​SD, n ​= ​8 animals per group). (B) RNA was extracted from joints of male mice 8 and 12 weeks after DMM or sham surgery or from age-matched naïve controls, and expression of Adam15 quantified by RT-qPCR, relative to Rplp0 and average expression in naïve joints (mean ​± ​SD, n ​= ​5–13 animals per group). (C–I) RNA was extracted from joints of male mice aged 0.2–18 months as indicated, and expression of Adam15 (C), Adam17 (D), Sirt1 (E), Runx2 (F), Cdkn1a (G), Cdkn2a (H) and Il6 (I) were quantified by RT-qPCR, relative to Rplp0 (mean ​± ​SD, n ​= ​6–13 animals per group). Data are expressed relative to average expression in 18-month old mice (C) or 0.2 month old mice (D–I). ∗P ​≤ ​0.05, ∗∗P ​≤ ​0.01, ∗∗∗∗, P ​≤ ​0.0001.

Article Snippet: Primary antibodies were applied to detect ADAM15 (rabbit anti-ADAM15, Atlas Antibodies, HPA011633, 2 μg/ml) and/or perlecan (rat anti-perlecan, Millipore).

Techniques: Expressing, Quantitative RT-PCR

Journal: Osteoarthritis and Cartilage Open

Article Title: Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage

doi: 10.1016/j.ocarto.2020.100128

Figure Lengend Snippet: IL-13 stimulates expression of ADAM15. (A) After 24 ​h of serum starvation, HTB94 ​cells were treated for a further 24 ​h with Dexamethasone (1 ​μM, DMSO control), phorbol 12-myristate 13-acetate (DMSO control), dihydrotestosterone (DHT, 0.1 ​μM, methanol control), estradiol (0.1 ​μM, ethanol control), mirin (50 ​μM, DMSO control), resveratrol (10, 50, 100 ​μM, ethanol control), EX-527 (1, 10 ​μM, DMSO control) or 3-MA (5 ​μM, DMSO control). RNA was extracted and expression of ADAM15 quantified by RT-qPCR, relative to RPLP0 and mean expression the respective controls (mean ​± ​SD, n ​= ​3). (B) After 24 ​h of serum starvation, HTB94 ​cells were treated for a further 24 ​h with M-CSF (100 ​ng/ml), GM-CSF (50 ​ng/ml), TNF (100 ​ng/ml), IFNγ (100 ​ng/ml), IL-4 (20 ​ng/ml), IL-6 (20 ​ng/ml), IL-10 (10 ​ng/ml), IL-13 (20 ​ng/ml), LPS (100 ​ng/ml), activin A (50 ​ng/ml), BMP-7 (100 ​ng/ml), CTGF (100 ​ng/ml), FGF2 (100 ​ng/ml), FGF18 (100 ​ng/ml) or TGFβ (10, 100 and 1000 ​pg/ml), for 24 ​h. RNA was extracted and expression of ADAM15 quantified by RT-qPCR, relative to RPLP0 and the average expression in control (left panel) or DMSO-treated (right panel) cells (mean ​± ​SD, n ​= ​3). (C) After 24 ​h of serum starvation, HTB94 ​cells were treated with IL-13 (100 ​ng/ml) for 48 ​h, cells lysed in SDS sample buffer, and expression of ADAM15 and actin analysed by immunoblotting. (D) Band intensities from (B) were quantified and expression of ADAM15 normalised to actin plotted (mean ​± ​SD, n ​= ​6). Arrow indicates predicted position of active ADAM15. (E) RNA was extracted from joints of male mice aged 0.2–18 months as indicated, and expression of Il13 quantified by RT-qPCR, relative to Rplp0 and to average expression in 18-month old mice (mean ​± ​SD, n ​= ​6–13). ∗P ​< ​0.05, ∗∗P ​< ​0.01.

Article Snippet: Primary antibodies were applied to detect ADAM15 (rabbit anti-ADAM15, Atlas Antibodies, HPA011633, 2 μg/ml) and/or perlecan (rat anti-perlecan, Millipore).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 1. ADAM15-dependent mechano-induced downregulation of lncRNA H19 is affected by inhibition of p21-activated kinases (PAKs). (A, B) Synovial fibroblasts either silenced with an ADAM15 siRNA or a non-silencing negative siRNA were mechanically strained (1 Hz and 15% elongation) for 1–9 h and H19 levels determined by RT-qPCR. (A) Fold changes of H19 in ADAM15-expressing versus non-expressing cells were calculated using the 2−ΔΔCt. Shown is the mean ± SD of 6 different RASFs. (B) time course of GAPDH- normalized Ct values for H19 upon mechanical strain, showing increase of Ct values (i.e. lower H19 amounts) in ADAM15 expressing cells (black dots) compared to ADAM15 non-expressing cells (open circle) in one representative RASF cell line. (C) RT-qPCR for H19 of RASFs stimulated for 1 and 3 h in the presence of either DMEM medium, or inhibitors for JNK (SP600125), Src family kinases (dasatinib), CAMKII (KN- 93), calmodulin (TFP) and PAKs (IPA-3). **p < 0.005, ***p < 0.0005, Student’s t-test, comparing ADAM15- expressing versus non-expressing cells or inhibitor versus DMEM.

Article Snippet: For detection of ADAM15 and NCAD, cells (1 × 104) were grown on 8-chamberslides for 48 h, fixed with 4% PFA, permeabilized with PBS/0.1% Triton X-100 for 5 min, blocked with 1% horse serum and incubated with mouse ADAM15 (R&D Systems, #MAB935, 1:50) and sheep-anti N-cadherin antibodies.

Techniques: Inhibition, Quantitative RT-PCR, Expressing

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 2. ADAM15- and N-cadherin (NCAD) dependent activation of PAK2 upon mechanical strain. (A–D) Immunoblots for pPAK2. (A) of RASFs seeded at different cell densities and mechanically strained for 30 and 60 min. (B) from strained RASFs with PAK2 silencing (si) and treated with a negative control siRNA (neg). (C, D) immunoblots for pPAK2, ADAM15 and NCAD from RASFs treated with ADAM15 and NCAD siRNA; right panels, showing the mean ± SD of densitometric analysis of 6 different RASF cell lines. ***p < 0.0005, Student’s t-test. GAPDH served as a loading control.

Article Snippet: For detection of ADAM15 and NCAD, cells (1 × 104) were grown on 8-chamberslides for 48 h, fixed with 4% PFA, permeabilized with PBS/0.1% Triton X-100 for 5 min, blocked with 1% horse serum and incubated with mouse ADAM15 (R&D Systems, #MAB935, 1:50) and sheep-anti N-cadherin antibodies.

Techniques: Activation Assay, Western Blot, Negative Control, Control

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 3. Interaction of ADAM15 with N-cadherin (NCAD) in synovial fibroblasts. (A) Co- immunoprecipitations (IP) of RASFs with prior silencing of ADAM15 using siRNA (I) and negative control siRNA (N) using NCAD antibodies (upper panels) or ADAM15 antibodies (lower panels) and detection of ADAM15 or NCAD with the respective antibodies. (B) Analogous to (A) Co-IPs using NCAD and ADAM15 antibodies in chondrocyte cell lines transfected with full length ADAM15 plasmid (+) or with ADAM15 lacking the cytoplasmic tail (Δcyto) and an empty vector (-). Mouse IgG served as a control. Input: 10% total lysate. (C) confocal microscopy of RASFs double immunofluorescently stained for NCAD (green) and ADAM15 (red). White inset indicates area of the magnified merged image. (D) In situ proximity ligation assays for ADAM15 and NCAD, showing direct interaction of both molecules in densely seeded cells only (arrows). Cell nuclei were counterstained with DAPI. Objective 40x, scale bar = 10 μm.

Article Snippet: For detection of ADAM15 and NCAD, cells (1 × 104) were grown on 8-chamberslides for 48 h, fixed with 4% PFA, permeabilized with PBS/0.1% Triton X-100 for 5 min, blocked with 1% horse serum and incubated with mouse ADAM15 (R&D Systems, #MAB935, 1:50) and sheep-anti N-cadherin antibodies.

Techniques: Negative Control, Transfection, Plasmid Preparation, Control, Confocal Microscopy, Staining, In Situ, Ligation

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 5. Binding of PAK2 to ADAM15/NCAD complex at cell membrane is crucially dependent on ADAM15. (A) IPs of strained RASFs using ADAM15 or NCAD antibodies and detection of PAK2 and Nck. TL = total lysate. IgG, mouse IgG served as background control. (B) cell surface biotinylation and enrichment of membrane fractions on streptavidin beads of strained cells with ADAM15-silenced or nonsilenced (N). Contr: non biotinylated cell lysates enriched on streptavidin beads served as background control. (C, D) IPs using ADAM15 antibodies in (C) RASFs with silenced and nonsilenced ADAM15 expression (N) and (D) in chondrocyte cell lines transfected with full length ADAM15 (full-A15) or ADAM15 lacking the cytoplasmic domain (Δcyto). (E, F) analogous to (C, D) IPs using N-cadherin antibodies, showing precipitates of PAK2 and Nck in ADAM15-expressing cells only. (E) right, densitometric evaluation of PAK2 from experiments obtained from 4 donors.

Article Snippet: For detection of ADAM15 and NCAD, cells (1 × 104) were grown on 8-chamberslides for 48 h, fixed with 4% PFA, permeabilized with PBS/0.1% Triton X-100 for 5 min, blocked with 1% horse serum and incubated with mouse ADAM15 (R&D Systems, #MAB935, 1:50) and sheep-anti N-cadherin antibodies.

Techniques: Binding Assay, Membrane, Control, Expressing, Transfection

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 6. Cadherin-11, but not Cadherin-2 (NCAD) is a target of mechano-downregulated H19. (A, B) RASFs with ADAM15-nonsilenced (N) or silenced (I) were mechanically strained and (A) RT-qPCR, fold changes of CDH11 or NCAD from 5 different RASFs and (B) immunoblots for CDH11 and NCAD, right, densitometry for CDH11 and NCAD from 4 different RASFs. (C, D) unstimulated RASFs with H19 silenced versus nonsilenced, (C) RT-qPCR, fold changes of CDH11 and NCAD from 5 different RASFs, (D) immunoblots for CDH11 and NCAD, right, densitometry from 4 different RASFs. (E) RT-qPCR for CDH11 and NCAD from mechanically strained (1 h) RASFs incubated with PAK inhibitor IPA-3 or DMEM medium. (F) densitometric evaluation of immunoblots for CDH11 and NCAD from 4 different RASFs (mean ± SD) treated with DMEM or IPA-3 for 48 h. GAPDH served as loading control. **p < 0.005, ***p < 0.0005, Student’s t-test, comparing DMEM versus inhibitor.

Article Snippet: For detection of ADAM15 and NCAD, cells (1 × 104) were grown on 8-chamberslides for 48 h, fixed with 4% PFA, permeabilized with PBS/0.1% Triton X-100 for 5 min, blocked with 1% horse serum and incubated with mouse ADAM15 (R&D Systems, #MAB935, 1:50) and sheep-anti N-cadherin antibodies.

Techniques: Quantitative RT-PCR, Western Blot, Incubation, Control

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 7. ADAM15/H19-dependent downregulation of miR-130a-3p, which binds to 3’ UTR of cadherin-11 and regulates its expression. (A) RNA pull-downs using transfected miR-130a-3p or scrambled mimics, purification on streptavidin beads and detection of binding using qPCR of H19, CDH11 and NCAD, showing Ct values of 5 different RASFs (mean ± SD). (B) RT-qPCR of miR-130a-3p in ADAM15-expressing or silenced RASFs, showing the mean ± SD of fold changes from 5 different RASFs. (C) RT-qPCR for miR-130a-3p in RASFs silenced with two different H19 siRNAs (I and II) vs. negative control siRNA (N). (D, E) RASFs prior transfected with 130a-3p mimics or scramble were strained and (D) RT-qPCR for CDH11 and NCAD and (E) immunoblots for CDH11 and NCAD; untreated = strained without any treatment; right panel, densitometry for CDH11 from 4 different RASFs. GAPDH served as loading control. **p < 0.005, ***p < 0.0005, Student’s t-test.

Article Snippet: For detection of ADAM15 and NCAD, cells (1 × 104) were grown on 8-chamberslides for 48 h, fixed with 4% PFA, permeabilized with PBS/0.1% Triton X-100 for 5 min, blocked with 1% horse serum and incubated with mouse ADAM15 (R&D Systems, #MAB935, 1:50) and sheep-anti N-cadherin antibodies.

Techniques: Expressing, Transfection, Purification, Binding Assay, Quantitative RT-PCR, Negative Control, Western Blot, Control

Journal: Scientific reports

Article Title: Mechanical forces trigger invasive behavior in synovial fibroblasts through N-cadherin/ADAM15 -dependent modulation of LncRNA H19.

doi: 10.1038/s41598-025-94012-2

Figure Lengend Snippet: Fig. 8. CDH11-mediated increased cell invasion can be blocked by CDH11 silencing and transfection with miR-130a-3p mimics. (A, B) RASFs were treated with H19 siRNA or negative nonsilencing siRNA (N) and (A) MTT-based cell viability assays and (B) cells transmigrated through matrigel were stained with DAPI, the whole transwell photographed and all cells counted. (C) upper panel, invaded cells prior transfected with 130a- 3p mimics, CDH11 siRNA or scramble control, shown is the mean ± SD from 5 different RASFs. (C) lower panel, immunoblots for CDH11 from cells transfected and strained as in upper panel. *p < 0.05, **p < 0.005, ***p < 0.0005, Student’s test. (D) diagram of summarized results: mechanical strain results in NCAD/ADAM15- mediated phosphorylation of PAK2, resulting in the downregulation of lncRNA H19 and miR-130a-3p and subsequent upregulation of CDH11, which in turn promotes an aggressive phenotype: increased cell invasion. Inhibition of PAK signaling by PAK inhibitor IPA-3 blocks mechano-induced H19 downregulation and subsequently CDH11 upregulation. In addition, mechano-induced PAK2 phosphorylation is accompanied by recruitment of SH2/SH3 adapter Nck and PAK2 to the NCAD/ADAM15 complex, but binding of Nck/PAK2 is restricted to the cytoplasmic domain of ADAM15. The mechano-induced redistribution of PAK2 to the cell membrane does not occur when Nck is silenced.

Article Snippet: For detection of ADAM15 and NCAD, cells (1 × 104) were grown on 8-chamberslides for 48 h, fixed with 4% PFA, permeabilized with PBS/0.1% Triton X-100 for 5 min, blocked with 1% horse serum and incubated with mouse ADAM15 (R&D Systems, #MAB935, 1:50) and sheep-anti N-cadherin antibodies.

Techniques: Transfection, Staining, Control, Western Blot, Phospho-proteomics, Inhibition, Binding Assay, Membrane

Journal: Nature Communications

Article Title: Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding

doi: 10.1038/s41467-024-45558-8

Figure Lengend Snippet: a HeLa cells were transfected with mCh-MT1-MMP. 3 h post transfection, cells were treated with metalloproteinase inhibitors as indicated. Protein lysates were made 24 h after the transfection and analysed by WB. Graph shows the quantification of the 34 kDa cleavage product of MT1-MMP. Cells treated with the lowest concentration of DMSO were set to 1. Data is from n = 3 independent experiments, mean +/− SD. One sample two-sided t -test. b MDA-MB-231 cells were transiently transfected with mCh-MT1-MMP, TKS4- or TKS5-GFP and ADAM12-myc. 24 h after transfection, cells were fixed and stained with anti-mCh, anti-GFP and anti-Myc antibodies and analysed by confocal microscopy. mCh-MT1-MMP, TKS4/5-GFP and ADAM12-myc colocalise on endosomes. Displayed is a maximum intensity projection of 2 slices à 0.9 µm section thickness. Representative of 13 (TKS4) and 6 (TKS5) confocal image acquisitions. See also Supplementary Fig. for ADAM15. c Co-immunoprecipitation experiment using HeLa cells transfected with mCh-MT1-MMP, ADAM15-myc-SNAP and TKS5-GFP wt or µµ or GFP (ctrl). ADAM15-myc-SNAP preferentially co-immunoprecipitates with mCh-MT1-MMP in the presence of wt, but not mutated TKS5-GFP (“µµ”) or GFP alone. Graphs show the relative mean intensity +/−SD of the ADAM15-myc-SNAP signal quantified from cells transfected with TKS5-GFP ( n = 4), TKS4-GFP ( n = 3) or a combination of both ( n = 4). Representative WBs for TKS4-GFP and TKS4 + 5-GFP Co-IP experiments are shown in Supplementary Fig. . One-way ANOVA with Tukey’s multiple comparisons test. d MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells were treated with 25 or 50 mM NH 4 Cl for 20 h. Total cell lysates were analysed by WB. Graph shows the quantification of the 34 kDa cleavage product of MT1-MMP. Data is from three independent experiments, mean +/− SD, one sample two-sided t -test.

Article Snippet: ADAM15 (NM_003815) was purchased from Origene as human tagged ORF clone (RC200642) and subcloned as a SNAP-tagged fusion.

Techniques: Transfection, Concentration Assay, Staining, Confocal Microscopy, Immunoprecipitation, Co-Immunoprecipitation Assay