1. Supplementary information
Foerster et al.
Targeting the actin cytoskeleton: selective anti-tumor action via trapping PKCε
Figure legends
(S1) Participation of the MPT in ChA induced apoptosis. Mitochondria were isolated from either untreated (controls) or ChA treated MDA-MB-231 cells. Parallel measurements of ΔΨ by Rhodamine123+ (125nM) quenching (a) and mitochondrial swelling (MPT) by absorbance at 540 nm (b). Mitochondria of control and ChA 300 nM (24 h) treated cells were incubated with Ca2+ (400µM) in the presence of Cyclosporin A (5µM). FCCP addition (500 nM) after 50 min served as internal control for ΔΨ disruption. The experiment was independently repeated two times.
(S2) Other actin overpolymerizing compounds also lead to the inclusion of PKCε in actin bundles. MCF-7 cells transfected with mGFP-β-actin were treated with Doliculide 500 nM (Doli 500 nM), Jasplakinolide 300 nM (Jasp 300 nM) or solvent control for 6 hours. Cells were fixed with 4% PFA and stained with anti-PKCε antibody (SantaCruz) over night followed by incubation with anti-rabbit-Alexa-633 secondary antibody and Hoechst Actin cytoskeleton: green; PKCε: red; Nuclei: blue. The experiment was independently repeated three times.
(S3) PKCα does not co-localize with ChA induced actin bundles. MCF7 cells transfected with mGFP-β-actin were treated with 300 nM ChA for 6 h and 24h. Cells were fixed with 4% PFA and stained with anti-PKCα antibody (SantaCruz) over night followed by incubation with anti-rabbit-Alexa-633 secondary antibody and Hoechst Actin cytoskeleton: green; PKCα: red; Nuclei: blue. The experiment was independently repeated three times.
(S4) PKCε colocalizes with actin in the cytoskeletal fraction of ChA treated MCF7 cells. Cytosolic and cytoskeletal fractions of MCF7 cells were separated via centrifugation. Protein lysates were resolved in SDS-PAGE and immunoblotted using antibodies against PKCα, PKCε, Actin and GAPDH.
(S5) Downregulation of PKCε induces apoptosis. MDA-MB-231 cells were transfected with PKCε siRNA for 72 hours. The downregulation was confirmed via Western blot (inlet and quantification). Apoptosis was quantified using an Annexin V/FITC staining (72 hours after transfection). The experiment was independently repeated two times.
(S6) ChA induced disruption of the actin cytoskeleton in MCF10-A. MCF10-A cells were transfected with mGFP-actin and treated with 300 nM ChA for 24h.

2. S1 A
500
400
RFU
Ctrl
300
Ch300
200
FCCP
100 10 20 30 40 50 60 70
time [min] B 10 20 30 40 50
Ctrl
Ch300
OD 540
0.30
0.35
0.40
0.45
0.50
0.55
time [min]

3. S2
Ctrl
Doli 500 nM
Jasp 500 nM

4. S3 S4 Ctrl mGFP-β-actin PKCα Nuclei Merge 6h 24h Ch300 GAPDH PKCε
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300
Cyto
CSK 6h
24h

5. S5 S6
Ctrl
Ch300