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SLO-permeabilization

By Todd Cruz,2014-08-30 08:09
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SLO-permeabilization

Supplemental Figure 1

    SLO-permeabilization does not compromise endosomal integrity. 10 kDa FITC-Dextran

    6 (Invitrogen) at 5 mg/ml was added to 6 x 10KG-1 cells for the indicated times. Cells were

    then permeabilized with SLO as described in Material and Methods or lysed in 100 l of 1%

    CE in TBS, pH 7.4. After centrifugation, 20 l of supernatants from SLO-permeabilized 129

    cells, lysed pellet fractions from SLO-permeabilization or cell lysates were analyzed in triplicates for FITC-Dextran content in MicroFluor 1 plates with an ELISA plate reader (Excitation/Emission = 485/535 nm). The graph shows the mean value (? SD) of two independent experiments.

Supplemental Figure 2

    6 Cytochalasin D inhibits the uptake of Firefly luciferase. 6 x 10KG-1 cells were pretreated

    with cytochalasin D at 10 g/ml (Cyto D, ;) or with DMSO as control (Ctr, ;) for 30 min.

    Firefly luciferase was then added at 49 g/ml to each sample for 30 min. After extensive

    washes with PBS, cells were lysed in 100 l of 1% CE in TBS, pH 7.4. After centrifugation, 129

    20 l of supernatants were analyzed for luciferase activity with a luminometer. The graph shows the mean value (? SD) of triplicate samples. Results are representative of at least three independent experiments.

Supplemental Figure 3

    ExoA does not affect Firefly luciferase uptake or in vitro refolding. A. PBS control (;) or

    ExoA (;) at 330 g/ml was internalized together with Firefly luciferase for 15 min at 37ºC. After extensive washing with PBS, cells were lysed in 100 l of 1% CE in TBS, pH 7.4. After 129

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    centrifugation, 20 l of supernatants were analyzed for luciferase activity with a luminometer. The graph shows the percent of luciferase activity compared to control cells (;) (? SD) of

    triplicate samples. Results are representative of at least three independent experiments. B. Cells

    left untreated (;) or that had internalized ExoA (;) or BSA (;) at 330 g/ml for 15 min at 37ºC

    were permeabilized with SLO as described in Materials and Methods. After centrifugation at 14,000 g for 5 min at 4ºC, supernatants were used for in vitro refolding of chemically unfolded

    Firefly luciferase as described in Materials and Methods. 20 l of refolding reactions were

    analyzed for luciferase activity with a luminometer; refolding in DPBS buffer alone was used as a control. The graph shows the mean value (? SD) of triplicate samples. Results are representative of at least three independent experiments.

Supplemental Figure 4

    Denaturation by guanidinium chloride followed by dialysis results in soluble inactive Renilla reniformis luciferase. The enzyme was chemically unfolded in 6M Guanidine-HCl in

    ICT buffer for 2 hrs and then dialyzed overnight against PBS. Luciferase activities of 0.1 ng of

    folded (;) or unfolded (;) enzyme in PBS were measured with a luminometer. Lack of

    significant detectable activity of the unfolded luciferase sample demonstrates efficient unfolding.

    The graph shows the mean value (? SD) of triplicate samples and it is representative of at least three independent experiments.

Supplemental Figure 5

    Prolonged treatment of KG-1 cells demonstrates efficient inhibition of cytosolic refolding at low radicicol concentrations. KG-1 cells were pretreated with DMSO or radicicol for 15 hrs

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    6 KG-1 cells for each sample were resuspended in 1% FBS at the indicated concentrations. 6 x 10

    IMDM and incubated for 15 min with unfolded Renilla reniformis luciferase at the final concentration of 20 g/ml. After extensive washes in serum-free medium, cells were

    permeabilized with SLO as described in Materials and Methods. After centrifugation at 14,000 g for 5 min at 4ºC, 20 l of supernatants were analyzed for luciferase activity. (Ctr : DMSO

    overnight treated cells not permeabilized with SLO; ; indicates permeabilized samples). The

    graph shows the mean value (? SD) of triplicate samples and it is representative of at least three independent experiments.

Supplemental Figure 6

    Radicicol treatment does not affect macropinocytosis. KG-1 cells were pretreated for 1 hr

    with DMSO as control or with radicicol at the indicated concentrations. After extensive washes

     in serum-free medium, cells were resuspended in 1% FBS in IMDMand Lucifer Yellow (final

    concentration 250 g/ml) was added for the indicated time points (solid lines). After extensive washing, Lucifer Yellow fluid phase uptake was evaluated by flow cytometric analysis. Cells incubated with Lucifer Yellow at 4ºC (dashed lines) served as a control.

Supplemental Figure 7

    6 Hsp90 ; knock-down does not affect macropinocytosis. 1.5 x 10control or Hsp90 ; shRNA

    KG-1 cells were resuspended in 1.5 ml 1% FBS IMDM. Lucifer Yellow at the final concentration of 250 g/ml was added for the indicated time points (solid lines). After extensive washes, Lucifer Yellow fluid phase uptake was evaluated by flow cytometric analysis. Incubation performed at 4ºC (dashed lines) served as a control.

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