Supplementary Figure 1. Intracellular distribution of the EPE peptide. HeLa cells were serum-starved (16 h, 0.1%), and treated with EPE peptide,

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1 Supplementary Figure 1. Intracellular distribution of the EPE peptide. HeLa cells were serum-starved (16 h, 0.1%), and treated with EPE peptide, conjugated with either TAT or Myristic acid and biotin for indicated times. Cells were processed as described under Material and Methods. Intracellular distribution of peptide was visualized using fluorescent microscopy. The scale bars in the top left boxes in each panel are of 20 µm. 1

2 Supplementary Figure 2. Optimization of the nuclear-translocation-inhibiting peptide. HeLa cells were grown in 0.1% FCS for 24 hours and then pretreated with Scramble (Scr), SPS (WT), APA or EPE peptides (all 10 µm for 2 hours) or left untreated. Then the cells were either left untreated (NT) or stimulated with TPA (250 nm, 15 min., all other treatments). Then the cells were fixed, stained with anti gerk1/2 Ab and visualized using fluorescent microscopy as described under Materials and Methods. The scale bar in the top left box is of 20µm. 2

3 Supplementary Figure 3. Using cellular fractionation to confirm that the EPE peptide inhibits the nuclear translocation of ERK1/2. Serum-starved (0.1% FCS, 16 hr) A2352, HeLa, MDA-MB-231, or LOXMVI (Lox) cells were pretreated with EPE or scrambled peptides (10 M, 2 hr). Cells were then stimulated with EGF (50 ng/ml) or left untreated, and then harvested. Subcellular fractionation of cytoplasm and nucleus was performed as below, and fractions were subjected to Western blot analysis with the indicated Abs. Molecular weight markers appear in the right side of the panels of each antibody. Subcellular Fractionation was performed as follows: Harvested cells were resuspended in 200 l of buffer H containing 0.1% Nonidet P-40. The lysates were mixed vigorously and centrifuged immediately to yield supernatants containing the cytosolic fraction. Nuclear proteins were extracted by resuspending the nuclear pellets in 200 l of extraction buffer, waiting on ice for 5 min, brief sonication (2 x 5 sec, 40 W, 4 o C), vigorous mixing, and centrifugation. Both cytosolic and nuclear fractions were subjected to Western blotting. 3

Supplementary Figure 4. The EPE peptide has a similar effect on the nuclear translocation of both ERK1 and ERK2. A2185 (A) and A2352 (B) cells were serum-starved (16 hours, 0.

4 Supplementary Figure 4. The EPE peptide has a similar effect on the nuclear translocation of both ERK1 and ERK2. A2185 (A) and A2352 (B) cells were serum-starved (16 hours, 0.1% FBS), and then either pretreated with the EPE peptides (10 M, 2 hours) or left untreated. Then, the cells were stimulated with TPA (100 µm, 20 min), or DMSO contorl (NS). The cells were stained with either anti ERK1 (Santa Cruz, C16) or anti ERK2 (Santa Cruz, C14) Abs and DAPI. Scale bar in top left boxes in A and B are of 20 µm (C) Bar graphs showing average and standard errors of percentage of cells in which ERK1 or ERK2 were mostly (>80%) nuclear. Quantification was done by counting 5 fields each containing >50 cells per field. 4

Supplementary Figure 5. The EPE peptide does not affect the stimulated translocation of JNK and p38 into the nucleus. (A) The EPE peptide does not affect the nuclear translocation of JNK2 and p38.

5 Supplementary Figure 5. The EPE peptide does not affect the stimulated translocation of JNK and p38 into the nucleus. (A) The EPE peptide does not affect the nuclear translocation of JNK2 and p38. HeLa cells were serum-starved (16 h, 0.1%), pretreated with EPE peptide, or scrambled peptide (10 M, 2 h), and then either stimulated with either TPA (250nM), Anisomycin (Anis, 10 g/ml) for the indicated times, or left untreated as control (NT, upper panels). The cells were stained with p38 or gjnk Abs and DAPI. Scale bar in the top left box 20 µm. (B) Extracts from cells treated as in A were analyzed by Western blotting with the indicated Abs. Molecular weight markers appear in the right panels. 5

6 Supplementary Figure 6. Full scans of panels in Figure 2C. For details see the legend of Figure 2. 6

7 Supplementary Figure 7. The EPE peptide attenuates ERK-dependent activation of Elk1. HeLa cells were transfected with plasmids FR-Luc, pfa2-elk1, pfc2-dbd and prl-tk (see below). Cells were pretreated with EPE peptide or scrambled peptide (10 M, 2 hours) or left untreated, and then stimulated with EGF (50 ng/ml, 24 hr). The Bar-graph represents the ratio of luminescence of pfr-luc / Renilla as indication of Elk1 fold activation. The data shown represents average ± standard error of three different experiments (* p<0.005, t-test 2 tails). Luciferase reporter assay was performed as follows: The following plasmids (obtained from the Forchheimer Plasmid Collection of the Weizmann Institute of Science, Rehovot, Israel) were used for the experiment: FR-Luc (reporter plasmid), pfa2-elk1 (fusion trans-activator plasmid), pfc2-dbd (negative control) and prl-tk (Renilla). The plasmid were transfected into HeLa cells with PEI as previously described. After 24 hours, cells were serum starved, pretreated with the EPE or scramble peptides (10 M, 2 hours), or left untreated. Cells were then stimulated with EGF (50 ng/ml 24 hours) or left untreated. The assay was performed using Dual-Luciferase reporter assay (Promega, Madison, WI) according to the manufacturer s instruction. 7

8 Supplementary Figure 8. The EPE peptide reduces the expression and phosphorylation of c-fos in A2185 cells. (A) Effect of the EPE peptide on c-fos. A2185 cells were grown in full medium and then serum starved (0.1 %, 16 hours), pretreated either with EPE peptide or scrambled peptide (10 M, 1.5 hours), and then stimulated with TPA (50 or 100 nm, 60 min.) or left untreated (NS). Cell extracts were subjected to Western blot analysis with the indicated Abs. The anti g-cfos and p-c-fos Abs were purchased from Santa Cruz (CA). Molecular weight markers appear in the right side of each panel. (B) Quantification of the results of the 100 nm TPA (lanes 1,3,4 and 6) in A. Mean and standard errors are from 2 independent experiments. 8

9 Supplementary Figure 9. Dose response of the peptides on melanoma cells viability. LOXIMVI and A2352 melanoma cells were treated with the indicated doses of EPE or scrambled peptides. The data is presented as the ratio: amount of viable cell at 72 hours/initial cell number, where initial cell number was considered as

10 Supplementary Figure 10. Comparison of the EPE peptide with the ERK inhibitor GDC-0994 (A) Effect on cell viability. A2352, LOX-IMVI, and HeLa cells were grown in 1% FCS and treated with EPE peptide, scrambled peptide, GDC-0994 (all at 10 M), DMSO control (0.1%), or no treatment administrating fresh medium every 24 hours. Viable cells were assayed by Methylene Blue at 72 hours after cell seeding. Experiments were done twice in triplicate. Data presented as fold change of viable cells compared to DMSO control (* p < 0.01, Student s t-test 2 tails). Initial cell number was considered as 1. The inhibitor was purchased from Selleckchem (B) Effects on ERK and ERK targets. The same cells were grown serum starved for 16 hours and treated with EPE peptide 10 M, GDC M, or DMSO control for 2 hours, stimulated with 100 nm TPA, and assayed with the indicated antibodies (left). Molecular weight markers are in the right panels. Experiments where done twice. Quantification of band density of pelk in EPE and ERKi stimulated (mean and standard errors, 15 min, lanes 4 and 6), from 2 independent experiments, relative to DMSO control stimulated for 15 minutes (lanes 2). Data presented as fold change. 10

11 Supplementary Figure 11. The EPE peptide prevents nuclear accumulation/translocation of ERK1/2 in PLX4032-resistant melanoma cells. (A) A2352 PLX4032-resistant (PR) that were prepared as described under Material and Methods) and (B) A4132 PLX4032 resistant melanoma cell from patient were serumstarved (16 hours, 0.1%), pretreated either with the EPE or scrambled peptide (10 M, 2 hours) and then either stimulated with TPA (100 nm, 15 min) or left untreated (NT) as control. The cells were stained with gerk1/2 Ab (C-14) and DAPI. Scale bars in the bottom left boxes in A and B are of 20 µm. 11

12 Supplementary Figure 12. EPE peptide results in cytoplasmic retention of ERK1/2 in tumor xenographt. MDA-MB-231 and LOXIMVI xenographts were immunostained with gerk1/2 Ab, and pictures of tumor tissues were taken as described (Material and Methods). Scale bars for lower magnification (top and bottom left boxes - 50 µm. ERK1/2 localization was quantified by visual examination.the bar-graph represents number of cells with cytoplasmic/nuclear ERK1/2 staining in 10 random microscopic fields, at least 40 cells per field. 12

Supplemental Fig. 1: PEA-15 knockdown efficiency assessed by immunohistochemistry and qpcr

Supplemental figure legends Supplemental Fig. 1: PEA-15 knockdown efficiency assessed by immunohistochemistry and qpcr A, LβT2 cells were transfected with either scrambled or PEA-15 sirna. Cells were then