Effect of TIMP3 on apoptosis.

<p><b>A:</b> Death receptor dependent apoptosis was analyzed in Cal78 cells and Cal78 cells transduced with LacZ or TIMP3 cultured for 24 h and stimulated with 100 ng/ml FasL, TNF-a or TRAIL for 16 hours by measurement of caspase 3 and 7 activities and <b>B,C:</b> cleavage of caspase 3 and PARP in western blot analyses. GAPDH serves as an internal control. <b>D:</b> TIMP3-induced apoptosis was evaluated in Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 cultured for 24 to 72 h. Apoptosis was assessed by measurement of caspase 3 and 7 activities and <b>E:</b> by histone fragmentation assay.</p

TIMP3-induced apoptosis is influenced by serum factors.

<p><b>A:</b> Influence of TIMP3 on activation of cRaf, ERK1/2, RSK1 and Akt was determined in Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 by spot array measurements. <b>B:</b> Phosphorylation of cRaf, ERK1/2, RSK1 and Akt was confirmed by western blotting. <b>C:</b> Influence of serum withdrawal on apoptosis rates after 24 hours. Apoptosis in lentiviral transduced cells was measured relative to Cal78 cells cultured with 10% FCS. Values less than p<0.05 (*or °) were considered statistically significant. <b>D:</b> Influence of specific growth factors on apoptosis rates under serum-free conditions. Prior to the assessment of apoptosis, Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 were stimulated with 100 ng/ml EGF, TGF-ß or FGF-2 and cultured for 24 hours. Apoptosis was defined by caspase 3 and 7 activities. *Indicates statistical significance (p<0.05). <b>E:</b> Influence of serum withdrawal on autophagocytosis in Cal78 cells after 24 hours. Autophagocytosis in TIMP3 overexpressing CAL78 cells was determined relative to Cal78 cells transduced with a control construct (LacZ) cultured with 10% FCS, 1% FCS or ITS by Western blot analysis of the typical marker proteins LC3 and Beclin.</p

Dose-dependent effect of TIMP3 on apoptosis.

<p><b>A–C:</b> Determination of the apoptosis response in different cell clones (clone 1 to 4) by caspase-3/7 activity and corresponding TIMP3 expression in these cell clones. <b>D,E:</b> Activation of initiator caspases-8 and -9 in transduced Cal78 cells with LacZ or TIMP3 cultured for 72 h. <b>F:</b> The effect of exogeneous TIMP3 on apoptosis after stimulation of Cal78 cells with recombinant human (rh) TIMP3 up to 200 nM for 96 h. Values less than p<0.05 (*) were considered statistically significant.</p

Proteoglycan-bound TIMP3 induces apoptosis.

<p><b>A:</b> Cal78 or Cal78 transduced with TIMP3 or LacZ were cultured and subsequently treated with heparinase I and III for 2 hours. Supernatants from non-treated and treated cells were transferred onto Cal78 cells for 72 hours prior assessment of apoptosis <b>B:</b> Cal78 or Cal78 transduced with TIMP3 or LacZ were incubated with or without heparinase I and III for 72 hours until evaluation of apoptotic cell death. Inactive heparinases (without CaCl₂) were used as a treatment control. Apoptosis was assessed by measurement of caspase 3 and 7 activities. Values less than p<0.05 (*) were considered statistically significant. <b>C:</b> Evaluation of TIMP3 release from cell surface of transduced cells by heparinase. Western Blot analysis of TIMP3-V5 from cell extracts and corresponding supernatants 4 hours after treatment with heparinase. <b>D:</b> Quantification of the Western Blot bands of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070709#pone-0070709-g003" target="_blank">figure 3C</a>. The band of soluble TIMP3-V5 from supernatants was shown versus bounded TIMP3-V5 from the cell lysates. <b>E:</b> Western blot analysis of the supernatants shown in figure C with a specific TIMP3 antibody. 75 ng/ml rh TIMP3 serves as an indication of the amount of TIMP3 in supernatants of Cal78 cells.</p