Supplemental Figures S1 S5

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Eugenia Bennett
5 years ago
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1 Beyond reduction of atherosclerosis: PON2 provides apoptosis resistance and stabilizes tumor cells Ines Witte (1), Sebastian Altenhöfer (1), Petra Wilgenbus (1), Julianna Amort (1), Albrecht M. Clement (2), Andrea Pautz (1), Huige Li (1), Ulrich Förstermann (1), Sven Horke * (1) (1) Institute of Pharmacology, Obere Zahlbacher Str. 67, (2) Institute of Pathobiochemistry, Duesbergweg 6, University Medical Center, Mainz, Germany ( ) Both authors contributed equally. *Correspondence should be addressed to Sven Horke, Institute of Pharmacology, University Medicine Mainz, Obere Zahlbacher Str. 67, Mainz, Germany; Tel.: ; Fax: ; horke@uni-mainz.de Supplemental Figures S1 S5

A) N C P N C P N C P N C P B) C) CHOP mrna (%) 2 15 1 5 EA.hy EA.

2 A) N C P N C P N C P N C P B) C) CHOP mrna (%) EA.hy EA.hy-PON2-GFP Control Actino Stauro Figure S1: Efficiency of PON2 and JNK sirna and the effect of staurosporine and actinomycin D on CHOP mrna. (A) Indicated cells were untreated (naïve; N), treated with scrambled (control; C) or PON2-specific sirnas (P) (35 nm). Three days later PON2 and - tubulin protein levels were determined. Similar results were obtained for Huh7, HepG2 cells and Jurkat T-cells; the latter expressed very low PON2 amounts. (B) Indicated cells were treated with scrambled or JNK sirnas (35 nm). Next day, JNK mrna levels were quantified by qrt-pcr. (C) Naïve or PON2-GFP overexpressing EA.hy 926 cells were left untreated (control) or were treated with actinomycin D (1 µg/ml) or staurosporine (.3 µm) for 16 h, followed by CHOP mrna quantification by qrt-pcr.

3 Figure S2: PON2 localizes to the ER and mitochondria. Cells grown on glass plates were stained with reduced MitoTracker Orange CM-H 2 TMRos (1µM; 3min; 37 C; Molecular Probes), methanol-acetone fixed, re-hydrated in PBS +.1% Triton X-1 (PBS-T (also used in all subsequent steps); 5 min; RT), blocked with 5% mouse IgGs (Santa Cruz) and mouse- IgG1κ (MOPC-21; Sigma; 1:5), incubated with rabbit-anti-pon2 (1:1) or mouse-anticalnexin (Dianova; 1:2; 1h; RT), washed 3x, stained with goat-anti-rabbit AlexaFluor-488 (1:4) and goat-anti-mouse AlexaFluor-647 (1:1; Molecular Probes), washed 3x and mounted with ProLong Gold antifade reagent (Molecular Probes). Confocal laser scanning microscopy was performed with a Zeiss LSM-71, equipped with ZEN28 software (Zeiss), using a Plan/Apochromat 63x/1.4 oil DIC objective. Appropriate wavelengths were sequentially scanned; absence of cross-emission was verified (not shown). The lower panel shows a magnification of the upper panel, scale bar = 1 µm. A representative result is shown.

4 A) B) 5 6 Caspase-8 activation Caspase-9 activation 4 2 Control STS Z-VAD STS + Z-VAD Control STS Z-VAD STS + Z-VAD C) D) 5 Caspase-3/7 activation Control STS Z-VAD STS + Z-VAD Figure S3: Caspase-inhibition does not prevent staurosporine-induced cytochrome C release from mitochondria. (A-C) Naïve EA.hy 926 cells were left untreated (control) or were treated with staurosporine (STS; 1 µm; 16h) in the absence or presence of pan-caspase inhibitor Z-VAD-fmk (1 µm). Subsequently, activity of caspase-8 (A), caspase-9 (B) or caspase-3/7 (C) was monitored. The graphs show mean±sem of one representative experiment out of 3, each performed in duplicates. (C) Cells treated as before were analyzed for mitochondrial cytochrome C release in a FACS-based protocol. A left-shift of the peak indicates cytochrome C liberation.

a plate reader using excitation / emission wavelengths of 45 / 58 nm. Graph (mean±sem) shows one representative out of 3 experiments with similar results.

5 A) B) Figure S4: PON2 overexpressing cells show decreased mitochondrial superoxide production. (A) Naïve or PON2-HA overexpressing cells were washed, loaded with Mito-HE (1 µm), washed and stimulated with antimycin A (15 µm), followed by immediate analysis in a plate reader using excitation / emission wavelengths of 45 / 58 nm. Graph (mean±sem) shows one representative out of 3 experiments with similar results. (B) Intact mitochondria isolated from naïve or PON2 overexpressing cells were enriched (3 µg/ml), loaded with 1µM MitoSOX-Red were stimulated with antimycin A (15 µm) and analyzed in a platereader using excitation / emission wavelengths 45/58 nm.

6 Figure S5: Simplified scheme illustrating how PON2 alters pro-apoptotic signaling. Stress in the ER activates PERK, ATF6 and IRE1 and is accompanied by O - 2 generation, which induces expression of pro-apoptotic CHOP via JNK. While leaving stress transducers unaltered, PON2 diminishes oxidative stress and induction of CHOP, which otherwise causes cardiolipin peroxidation and caspase activation. Pro-apoptotic stimuli are signaled to the - mitochondria, where they cause uncoupling of the electron transport chain (ETC) and O 2 generation. This key event is reduced by PON2. Otherwise, excess O - 2 gives rise to H 2 O 2, which results in cardiolipin peroxidation, cytochrome C liberation and apoptosis. STS = staurosporine; Act D = actinomycin D; Doxo = doxorubicin; CytoC = cytochrome C; TNF-R = TNF receptors; DRs = death receptors; patf6 = processed ATF6; dashed = pathways unknown or unaddressed in this study.

Supplementary Figure 1. AnnexinV FITC and Sytox orange staining in wild type, Nlrp3 /, ASC / and casp1/11 / TEC treated with TNF /CHX.

Supplementary Figure 1. AnnexinV FITC and Sytox orange staining in wild type, Nlrp3 /, ASC / and casp1/11 / TEC treated with TNF /CHX. Phase contrast and widefield fluorescence microscopy (20x magnification).