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: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

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: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

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. 4. Mechano-induced recruitment of PAK2 together with Nck to the cell membrane. (A, B) RASFs grown on Bioflex plates and either unstimulated or strained for 30 min were fluorescently stained for (A) NCAD and Nck and (B) NCAD and PAK2. Adjacent to images: quantification of the pixel density of NCAD /Nck or NCAD /PAK2 along the white arrow. (C) double staining of NCAD and PAK2 in RASFs with prior silencing of Nck, right, pixel density along arrow. Cell nuclei were counterstained with DAPI. Objective 40x, scale bar = 10 μm. (D) immunoblots for PAK2, pPAK2, Nck and NCAD. RASFs pre-treated with Nck siRNA (I) and a nonsilencing siRNA (N) were mechanically strained for 15 and 30 min, the cell surface biotinylated with non-membrane-permeable biotin reagent and purified on streptavidin-conjugated magnetic beads. Contr: non biotinylated cell lysates enriched on streptavidin beads served as background control. TL = total lysates from 30 min time point. GAPDH was used to control the purity of the plasma membrane fractions.

Article Snippet: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

Techniques: Membrane, Staining, Double Staining, Western Blot, Purification, Magnetic Beads, Control, Clinical Proteomics

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: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

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. 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: Slices were glued with mounting medium to a glass slide and cells fixed with ice cold methanol for 5 min and aceton for 45 s. After blocking with 1% horse serum in PBS for 1 h, cells were incubated with sheep-anti N-cadherin (R&D Systems, #AF6426, 1:100) and rabbit anti-Nck (Cell Signaling Technology, #2319, 1:100), phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cell Signaling Technology, #2606, 1:100), anti-phosphoPAK2 PAK2 (R&D Systems, #MAB6849; 1:50), antibodies overnight and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-rabbit conjugated antibodies (1:500; Invitrogen).

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