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Fig. S1. Maintenance and enlargement of the jasplakinolide-induced F-actin aggregate. (A,B) Vero cells were treated with a single pulse of Jpk at low concentration (50 nM) for 24 hours and 48 hours. FAG was not observed after 48 hours. (C-F) When the culture medium of these Jpk-treated cells was replaced every 12 hours (+Jpk 12/24/36 hours) by new medium containing Jpk, FAG lasted longer and was larger (compare C with A and D with B). Cells were stained with TRITC-phalloidin (A-F) and DAPI (blue) (E,F). Bars, 10 µm.
Fig. S2. The formation of the F-actin aggregate in neural cells. Mouse c17.2 neural stem cells cells (NSC; A,B) and primary cultures of rat astrocytes (C,D) or hippocampal neurons (E,F) were treated with Jpk (+Jpk, 500 nM/45 minutes for NSC and astrocytes, or 5 mM/45 minutes for neurons) and Jpk was then washed out (−Jpk/48 hours). Cells were stained with TRITC-phalloidin. Bar, 10 µm.
Fig. S3. (A) Cell viability is not compromised by the presence of the F-actin aggregate. Cell viability was examined by determining the mitochondrial function (MTT reduction ability) of Jpk (50 nM)-treated (+Jpk) and Jpk (500 nM/45 minutes)-washout (−Jpk) Vero cells for the indicated times. No significant differences were observed. Data represent means ± s.d. for three independent experiments. (B) The F-actin aggregate triggers apoptosis in hippocampal neurons but not in Vero cells. FAG was generated by treating Vero and hippocampal neurons first with Jpk at higher concentrations (+Jpk, 5 mM/45 minutes) and then the actin toxin was washed out for 24 hours (-Jpk). Cells were double stained with TRITC-phalloidin (red) and DAPI (blue). Bar, 10 µm.
Fig. S4. Distribution of cytoskeletal proteins from cell homogenates containing the F-actin aggregates in a discontinuous sucrose density gradient. (A) Homogenates were separated by centrifugation in a discontinuous sucrose density gradient (see Materials and Methods). Fractions were collected from the top (#1) to the bottom (#10). Each fraction was immunoblotted for β-actin, Arp3, ADF/cofilin and myosin IIA. (B) TRITC-phalloidin staining of gathered sucrose fractions (indicated at the top of each panel). Bar, 10 µm.
Fig. S5. (A) Actin in the F-actin aggregate is non-dynamic. Vero cells were transfected with YFP-actin plasmid. After 24 hours, cells were treated with Jpk (+Jpk, 50 nM/6 hours) to induce the formation of FAG. Subsequently, FAG (B, white circle) or an equivalent portion of the cytoplasm in untreated cells (A, white circle) was subjected to FRAP and movies were recorded to visualize the recovery of fluorescence over time (see Movie 1). Figure shows representative frames obtained from videos (time indicated in seconds). (B) The Jpk-induced F-actin aggregate is resistant to latrunculin B and does not interfere with the reassembly of actin stress fibres that occurs after removal of the toxin. Untreated Vero cells (Control) or cells treated with Jpk (+Jpk, 50 nM/6 hours) containing FAG were incubated with LtB (+LtB, 500 nM/30 and 60 minutes). In Vero cells without or with FAG the actin toxin was washed out (-LtB, 5, 30, and 60 minutes). Bars, 10 µm.
Fig. S6. The presence of the F-actin aggregate does not alter the formation of lamellipodia and stress fibres. Untreated Vero cells (Control) or cells treated with Jpk (+Jpk, 50 nM/6 hours) containing FAG were cultured for 4 hours in the absence of FBS to clarify the cytoplasm of stress fibres (-FBS). The subsequent addition of phorbol myristate acetate (PMA) (1 mM/60 seconds) or lysophosphatidic acid (LPA) (1 mM/60 seconds) quickly induced the formation of lamellipodia and stress fibres, respectively. Cells were stained with TRITC-phalloidin. Bar, 10 µm.
Fig. S7. Disruption of microtubules prolongs the life-span of the F-actin aggregate. Vero cells were treated with Jpk (+Jpk, 50 nM/48 hours) alone (A) or pre-treated with Jpk (+Jpk, 50 nM/6 hours) and then co-incubated with nocodazole (30 mM) (+Jpk +NZ) (B) or taxol (30 mM) (+Jpk +TX) until 48 hours (C). Cells were stained with TRITC-phalloidin (A-C). Insets show the MT organization stained with anti-β-tubulin antibodies in untreated (A), NZ (B)- or TX (C)-treated cells after 48 hours. Bar, 20 µm.
Fig. S8. The activation of heat shock proteins accelerates the dissolution of the F-actin aggregate. Vero cells were treated with Jpk (+Jpk, 500 nM/45 minutes) and then the toxin was removed for 16 or 24 hours (A,B). 17-DMAG (10 µM) (C,D) or 17-DMAG plus lactac stin (10 µM) (E,F) was added when Jpk was washed-out and maintained for the same times. Note that 17-DMAG accelerated the dissolution of FAG, which in contrast was normalized by the presence of lactacystin. Bar, 10 µm
Movie 1. FRAP confocal recording of YFP-actin in untreated Vero cells (Control) and in cells containing FAG (Jpk).
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