Fig 1: Strain-induced ATP release is dependent on activated pannexin-1 (PANX1). (A) Immunoblots from SF, strained for 0–9 h, with prior downregulation of ADAM15 by siRNA (I) or non-silencing siRNA (N), showing increased phosphorylations of PANX1 and Src in ADAM15-expressing SF. (B) Immunoblots of strained SF in the presence of dasatinib. (C) ATP release and (D) total ATP of strained SF in the presence of dasatinib (1 µM) and the PANX1 channel inhibitor carbenoxolone (carbenoxo, 100 µM). Data show the mean ±SD from one representative experiment out of at least 3 independent experiments. *** p < 0.0005, by Student’s t-test, for comparison of DMEM with inhibitor-treated SF. (E) ATP release and (F) total ATP from SF with downregulated ADAM15 (siA15), double knockdown of ADAM15/HOTAIR (siA15+Hot), single knockdown of HOTAIR (siHot), and negative siRNA (Neg). ** p < 0.005; *** p < 0.0005, using Student’s t-test.
Fig 2: ATP upregulates the expression of ADAM15. Western blot analysis of ADAM15 expression in RASFs stimulated with ATP-?-S (200 µM) for 48 h. Tubulin served as a loading control. Representative results out of three independent experiments are shown. Densitometric evaluation of protein bands, normalized to tubulin, are shown as numbers above the gel lanes.
Fig 3: Secretome analysis of ADAM15 knockdown HTB84 cells. (A) HTB94 cells were transfected with 20 nM siRNA targeting ADAM15 (siADAM15) or same concentration of non-targeting control RNA (NT). ADAM15 expression was measured by qPCR at 0, 24, 48, and 72 h after treatment with ADAM15 or NT siRNAs, and displayed as a percentage of ADAM15 expression in cells transfected with NT siRNAs at each time point. Protein levels of ADAM15 in HTB94 cells treated with NT siRNAs (control) or ADAM15 siRNAs were analysed by immunoblotting at 72 h (insert). (B) Number of proteins annotated as a membrane or secreted proteins identified by secretome analysis or hiSPECS analysis in the conditioned media of ADAM15 knockdown HTB94 cells (left). Topology of single-span membrane proteins detected by secretome or SPECS analysis in the conditioned media of ADAM15 knockdown cells (right). (C) Volcano plot showing the -log10 of p-values versus the log2 of protein ratio between ADAM15 KD and HTB94 control cells (ct) of proteins detected in the conditioned media by serum-free secretome analysis (n = 6). AXL, which is the only protein above the FDR curves (displayed as black hyperbolic curves) is displayed as a blue solid dot, while proteins below the FDR are displayed by grey open dots. (D) Protein levels of full-length AXL in the cell lysates (flAXL) or shed AXL conditioned media (sAXL) of HTB94 cells treated with non-targeting or ADAM15 siRNAs (siADAM15) cells analysed by immunoblotting. (E) Bands corresponding to sAXL in the conditioned media from 3 independent experiments were quantified and normalized to the mean of sAXL values in conditioned media of cells treated with non-targeting siRNAs. A two-sided Student’s t-test was used to statistically evaluate changes in sAXL (** p < 0.01). (F) AXL mRNA expression levels in HTB94 cells treated with non-targeting or ADAM15 siRNAs plotted as relative 2-??CT (*** p < 0.005, Student’s t-test) (G). Volcano plot showing the -log10 of p-values versus the log2 of protein ratio between ADAM15 KD and HTB94 control cells (ct) of proteins detected in the conditioned media by hiSPECS analysis (n = 6).
Fig 4: Interaction of ADAM15 with transient receptor potential vanilloid 4 (TRPV4) channel and colocalization in the cell membrane. (A,B) Immunoprecipitations (IP) of (A) SF with downregulated ADAM15 by siRNA (I) or non-silencing siRNA (N) and (B) chondrocyte cell line transfected with full-length ADAM15 (+) or ADAM15 lacking the cytoplasmic domain (?C), using TRPV4 or ADAM15 antibodies, or IgG as control. GAPDH served as a loading control. wcl—whole-cell lysate. (C) Confocal microscopy of double immunofluorescence stainings of SF, using ADAM15 and TRPV4 antibodies. Objective 40x, size bar = 20 µm. The white box marks the magnified area. (D) Immunoblots of the cell surface biotinylated and purified membrane fractions of SF with ADAM15-silenced by siRNA (I) or non-silencing siRNA (N), showing TRPV4 in ADAM15-expressing cell membranes only. Co = non-biotinylated cell lysates, purified on streptavidin magnetic beads, served as the background control (N). Representative results out of three independent experiments are shown. (E) Diagram of summarized results: cyclic strain results in ADAM15-mediated activation of JNK, the downregulation of HOTAIR and subsequent upregulation of SIRT1, leading to decreased ROS, increased NAD+ levels and ATP release. In parallel, ADAM15-mediated Src activation results in the phosphorylation of PANX1, thereby activating PANX1-mediated ATP release. The interaction with ADAM15 inhibits the constitutive cycling of the mechanosensitive calcium channel TRPV4 to the endosome (dashed double arrow). The knockdown of ADAM15 not only blocks mechanical force-induced JNK- and HOTAIR-dependent upregulation of SIRT1 completely but also eliminates all respective downstream effects on NAD+, ROS and ATP, including its release as a purinergic mediator of inflammation. ECM—extracellular matrix.
Fig 5: Strain-induced ATP release is dependent on ADAM15 and calcium signaling. (A) ATP release and (B) total ATP of SF strained for 9 h with prior downregulation of ADAM15 by siRNA and negative siRNA as control. Each dot represents the mean value of one individual donor, the horizontal bar (-) the median of 7 different donors. * p < 0.05 by Wilcoxon signed-rank test, comparing ADAM15-expressing versus non-expressing SF. (C) ATP release and (D) total ATP from SF stimulated with DMEM and inhibitors of TRPV4, CaM, JNK or SIRT1. *** p < 0.0005, by Student’s t-test, when comparing DMEM with the inhibitor. Representative results out of at least three independent experiments are shown.
Supplier Page from Abcam for Anti-ADAM15 antibody [EPR5619]