Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42

Transcription

1 Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42 Gina L. Razidlo, Kevin M. Burton, and Mark A. McNiven SUPPORTING INFORMATION Figure S1. IL-6 promotes migration of pancreatic cancer cells. Figure S2. IL-6 stimulates activation of CDC42. Figure S3. JAK2 and STAT3 are required for IL-6 to activate CDC42 Movie S1. Live-cell fluorescence microscopy of a PANC-1 cell expressing GFP-Actin, before and after treatment with IL-6 (50 ng/ml). Example 1. Movie S2. Live-cell fluorescence microscopy of a PANC-1 cell expressing GFP-Actin, before and after treatment with IL-6 (50 ng/ml). Example 2.

2

3 Figure S1. IL-6 promotes migration of pancreatic cancer cells. A-C. IL-6 stimulates transwell migration of CFPAC (A), (B), and (C) pancreatic cancer cells. The indicated cell line was serum starved overnight and incubated in a chemotactic transwell migration assay in the presence or absence of IL-6 (50 ng/ml) for 20 hours. The percent of cells that migrated across the filter is shown. D. The effects of IL-6 on transwell migration are likely independent of proliferation. PANC-1 cells were incubated for the indicated times with IL-6 (50 ng/ml) or EGF (30 ng/ml) as a positive control, in the presence or absence of 10% FBS. IL-6 did not increase cell proliferation at the acute timepoints examined in the migration assays (t<24 hours). For A-E, graphed data represent the mean +/- SEM of 3-5 independent biological replicates. * indicates p<0.05, ** indicates p<0.01. E. An additional example of IL-6 inducing filopodial actin protrusions. PANC-1 cells were transfected to express GFP-Actin, and then imaged using live-cell fluorescence microscopy while stimulated with IL-6 (50 ng/ml). Image (a) represents a cell prior to IL-6 addition, (b) represents the same cell 30 minutes after IL-6 addition. Boxed regions are magnified at right (a and b ). (c) represents a kymograph from the region denoted with a white line. Scale bars = 10 µm. See also Figure 1 and Supplemental Movie 1-2.

4

5 Figure S2. IL-6 stimulates activation of CDC42. A. Dose curve of IL-6 demonstrating that IL-6 activates CDC42 from 1-50 ng/ml at a level comparable to that of the positive control, 10% FBS. PANC- 1 cells were treated with the indicated dose of IL-6, or 10% FBS, for 5 min, before GST-PBD pulldown and immunoblotting. Phosphorylated STAT3 was used as a positive control to show canonical IL-6 signaling, and phosphorylated ERK was used as a control to show canonical signaling by FBS. B, C. CFPAC pancreatic cancer cells were treated with IL-6 (50 ng/ml) as described in (A) and a GST-PBD pulldown was used to assess CDC42 activity (B) or RAC1 activity (C). Similar to PANC-1 cells, rapid activation of CDC42, but not RAC1, was observed in CFPAC pancreatic cancer cells. D-G. IL-6 stimulates CDC42 activation within 5 minutes in multiple pancreatic cancer cell lines. The indicated cell lines were treated with IL-6 (50 ng/ml) for 5 min, subjected to GST-PBD pulldown, and immunoblotted for CDC42. Graphed data represent quantitation of immunoblots of active CDC42 normalized to total CDC42, compared to unstimulated control cells in each experiment. Bars indicate the mean +/- SEM from 3-7 independent biological replicates. * indicates p<0.05. See also Figure 2.

6 Figure S3. JAK2 and STAT3 are required for IL-6 to activate CDC42. A. RNAi-mediated knockdown of JAK2 or STAT3 prevents activation of CDC42 by IL-6. PANC-1 cells were transfected with either a control non-targeting sirna or additional sirnas targeting JAK2 or STAT3, then stimulated with IL-6 (50 ng/ml) for 5 minutes. Active CDC42 was precipitated using a GST-PBD pulldown. Graphed data represent quantitation of immunoblots of active CDC42 normalized to total CDC42, compared to unstimulated control cells in each experiment. B. Quantification of the protein level of JAK2 following sirna-mediated knockdown using sirna 1 (Figure 4A) or sirna 2 (Figure S3A). C. Quantification of the protein level of STAT3 following sirna-mediated knockdown using sirna 1 (Figure 4C) or sirna 2 (Figure S3A). Graphed data represent quantitation of immunoblots normalized to Actin, compared to nontargeted control-transfected cells. For A-C, graphed data indicate the mean +/- SEM from 3-5 independent biological replicates. * indicates p<0.05, ** indicates p<0.01. See also Figure 4.