Fang et al. NMuMG. PyVmT unstained Anti-CCR2-PE MDA-MB MCF MCF10A

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1 A NMuMG PyVmT Fang et al. unstained Anti-CCR2-PE 4T1 Control MCF1A MCF MDA-M unstained Secondary antibody only Anti-CCR2 SUPPLEMENTAL FIGURE 1. Representative flow cytometry histograms of CCR2 expression in A. mouse cell lines and. human cell lines. (A) NMuMG, PyVmT, 4T1 and Control Raw cell lines were immunostained with antibodies to CCR2 conjugated to phycoerytherin (PE) as indicated at the x-axis. () MCF1A, MCF-7 and MDA-M-231 cell lines were immunostained with antibodies to CCR2 and detected with secondary antibodies conjugated to Alexa Fluor-488 as indicated at the x-axis. Flow cytometry data was analyzed by Flow Jo software. Values are expressed as mean percent +sem. 3

2 A + 1hr 4hr 8hr 24hr TGF + 1hr 4hr 8hr 24hr Fang et al. P- 4T1 MCF-7 P42/44MAPK + 1hr 4hr 8hr 24hr + 1hr 4hr 8hr 24hr P- PyVmT MDA-M-231 P42/44MAPK DAPI /DAPI Anti-rabbit-568 DAPI Secondary antibody only SUPPLEMENTAL FIGURE 2. stimulates phosphorylation of and p42/44mapk proteins in mouse and human breast cancer cell lines. A. Mouse (4T1 and PyVmT) and human (MCF-7 and MDA-M-231) breast cancer cell lines were incubated in serum free medium () in the presence or absence of 2 ng/ml over a period of 24 hours and analyzed by western blot for phosphorylation of and p42/44mapk proteins. The cell lines were treated with 5 ng/ml as a positive control for 24 hours.. 4T1 mammary carcinoma cells were stimulated with for 4 hours and analyzed for expression in the nucleus by immunofluorescence. Cells were imaged using a Motic AE31 microscope with Q capture software at 2 x magnification. 31

3 (hr) p-akt Akt p-fak FAK p-src Src Actin SUPPLEMENTAL FIGURE 3. has low to no significant effect on Src, AKT and FAK, other signaling pathways related to survival and motility. 4T1 mammary carcinoma cells were incubated in serum free medium () in the presence or absence of 2 ng/ml over a period of 24 hours and analyzed by western blot for phosphorylation of src, AKT, and FAK proteins. 32

4 Fang et al. A Par Con p- MAPK Actin Par Con p- MAPK Actin SUPPLEMENTAL FIGURE 4. Effect of knockdown on induced signaling in MDA-M-231 and PyVmT mammary carcinoma cells. Parental mammary carcinoma cells or cells transiently transfected with sirnas or control scramble sirnas were treated with 2 ng/ml for A. 1 hour for MDA-M-231 cells and. 4 hours for PyVmT cells, and analyzed for changes in phosphorylation of and MAPK proteins by immunoblot analysis. 34

5 A Sm3-1 Par Uninf Ad-Con Ad-Sm Sm3-6 Par Uninf Ad-Con Ad-Sm p- Fang et al. MAPK Actin % cleaved caspase Uninf Ad-Con ** ** Ad-Sm3 Uninf Ad-Con Ad-Sm3 Par Gentamycin Gentamycin/ C 7 Sm3-1 ** Sm3-6 ** % wound closure Uninf Ad-Con Ad-Sm3 Uninf Ad-Con Ad-Sm3 Par Sm3-1 Sm3-6 SUPPLEMENTAL FIGURE 5. re-expression in knockdown cells restores induced survival and migration. deficient 4T1 carcinoma cells (Sm3-1, Sm3-6) were infected with adenovirus overexpressing wildtype (Ad-Sm3) or adenovirus vehicle control (Ad-Con), in the presence or absence of (2 ng/ml), and analyzed for changes in A. expression of indicated proteins. gentamycin induced apoptosis and C. wound closure, in comparison with Uninfected control cells (Uninf) and Parental 4T1 cells (Par). Statistical analysis was determined by Two Tailed T-test,**p<.1. Values are expressed as mean+sem. 35

6 Fang et al. A. 6 **** % cleaved caspase Gentamcyin Gentamycin/. 36 **** % wound closure ng/ml 4 ng/ml 8 ng/ml SUPPLEMENTAL FIGURE 6. Effect of on MCF1A cells. A. MCF1A cells were treated with 2 ng/ml in the presence or absence of gentamycin and examined for cleaved caspase 3 expression.. MCF1A cells were treated with increasing concentrations of and examined for changes in wound closure. Statistical analysis was determined by Two Tailed T-test,****p>.5. Values are expressed as mean+sem. 37

7 Fang et al. A - S S DAPI CCR2/ RI DAPI Rabbit-alexa 488 DAPI CCR2/ RI Secondary Antibody Control C S nm Pertussis (nm) P- P- p42/44mapk p42/44mapk SUPPLEMENTAL FIGURE 7. receptor I inhibitors have no affect on CCR2 signaling. A. 4T1 cells were incubated in serum free () media in the presence or absence of 1 mm S431542, (positive control) or for 5 minutes. Cells were then fixed, and stained with antibodies to CCR2 (anti-ccr2-pe) and RI conjugated to anti-rabbit-alexa 488. Cells were counterstained with DAPI, and analyzed for CCR2 and RI expression by confocal microscopy. Cells were imaged by confocal microscopy using a Leica TCS SPE confocal upright microscope and LSM software at 63x magnification.. 4T1 cells were treated with S in the presence or absence of and analyzed for changes in CCR2/ receptor I receptor clustering by confocal microscopy.. 4T1 mammary carcinoma cells were treated with 2 ng/ml in the presence or absence of pertussis toxin or TGF-b type I receptor inhibitor S for 8 hours and analyzed for expression of phosphorylated or p42/44mapk by western blot. 38

8 A 4T1 alone Tgfbr2 Flox/flox -CM Tgfbr2 FspKO -CM No inhibitor I gg anti- No inhibitor I gg anti- No inhibitor I gg anti- p-p42/44mapk p42/44mapk p- * * (ng/ml) Tgfbr2 flox/flox Tgfbr2 FspKO 4T1 SUPPLEMENTAL FIGURE 8. derived from Tgfbr2 FspKO fibroblasts regulate CCR2 paracrine signaling interactions with breast cancer cells. A. 4T1 mammary carcinoma cells were treated with serum free medium (4T1 alone) or conditioned medium (CM) from Tgfbr2 flox/flox or Tgfbr2 FspKO fibroblasts, in the presence or absence of 1 g/ml goat IgG control or anti- for 8 hours and analyzed for changes in phosphorylated and MAPK proteins by immunoblot analysis.. Levels of secretion in media from the indicated cell lines were analyzed by ELISA. Statistical analysis was determined by Two Tailed T-test,*p<.1. Values are expressed as mean+sem. 36

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