hexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This

Transcription

1 SUPPLEMENTAL FIGURE LEGEND Fig. S1. Generation and characterization of. (A) Coomassie staining of soluble hexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This protein was expressed in mammalian cell and was used as immunogen. M, protein ladder. (B) Purified GRP78-His protein and MCF7 whole cell lysate were resolved on SDS-PAGE and immunoblotted with (0.5 ug/ml) that was pre-incubated with 100 ug/ml bovine serum albumin (BSA) or GRP78-His. GRP78-His completely blocked the binding of to GRP78 on the immunoblot. (C) Breast carcinoma cell MCF7 was treated with 300 nm thapsigargin or DMSO for 16 hours and the whole cell lysates were immunoblotted using (signal in red) and antibody against HSP70 (signal in green). The same membrane was used for both antibodies. only recognized GRP78, which was significantly induced after thapsigargin treatment. (D) in immunoblot assays recognized the GRP78 protein band in mouse embryo fibroblast (MEF) lysates prepared from the Grp78 floxed/floxed mice. The GRP78 band was absent in the c78f/f MEFs where the Grp78 alleles were deleted by treatment of the MEFs with adenovirus expressing the cre-recombinase. The blot was re-probed with anti beta-actin antibody to confirm equivalent protein loading in both lanes. These results also show that only immunoreacted with GRP78 in mouse protein lysates, confirming its specificity. (E) Scatchard assay to determine the affinity of to GRP78. Fig. S2. induces GRP78 endocytosis through clathrin mediated pathway. (A) MCF7 cells were glucose starved for 3 days and then treated with 50 ug/ml for 1 hour at 37 C. Cells were fixed with paraformaldehyde and permeabilized with Triton X-100, followed by co-staining with and clathrin antibody. Co-localization of

2 GRP78/ and clathrin is shown. (B) Glucose starved MCF7 cells were incubated with 50 ug/ml at 4 C for an hour, followed by staining with EphB4 antibody on ice without fixation and permeabilization. Plasma membrane localization of GRP78 is shown. Cells were also treated with or the combination of and 2ug/mL or 5 ug/ml chlorpromazine for 1 hour at 37 C, followed by the same staining procedure described above. treatment significantly reduced the surface GRP78, whereas the endocytosis inhibitor chlorpromazine restored surface GRP78. Nuclei were counterstained with DAPI. Scale bar, 20 Fig. S3. induces apoptosis of tumor cells in vitro. (A) HT29 cells were glucose starved and treated with or control IgG for 5 days, and the cell viability was analyzed with clonogenic assay. Representative pictures and statistical analysis (normalized to PBS treated cells) are shown. treated cells had significantly less colony formed. Treated cells were also analyzed with M30-CytoDEATH (B) and Caspase activity assay (C). Experiments were performed at least in triplicate. Asterisk and double asterisks indicate P < 0.05 and P < 0.02 respectively, as determined by an unpaired 2-tail student T-test. Fig. S4. Surface GRP78 Forms Complex with PI3K. (A) Flag tagged GRP78 without KDEL was over-expressed in 293T cells, followed by surface protein biotinylation and immunoprecipitation (IP). The interaction of surface GRP78 with p85, but not EphB2, Erk1/2, or beta-actin was detected. In the bottom panel, biotinylated surface GRP78 could also be detected with fluorochrome-conjugated streptavidin. (B) The interaction of endogenous surface GRP78 with p85 was detected in 293T cells treated with thapsigargin. Same biotin-ip system was used as in (A).

3 Fig. S5. Surface GRP78 level in cancer cell lines. (A) Cancer cells were grown in complete medium or glucose depleted medium for 3 days and then subjected to cell surface biotinylation. The biotinylated surface GRP78 was enriched with streptavdin beads, resolved on SDS-PAGE, and detected with. (B) The band intensity on immunoblot was quantified with Odyssey and the ratio of surface GRP78 in total GRP78 is shown. Fig. S6. treatment of xenograft tumor leads to reduced vessel density and mtor signaling. treated A549 tumors were analyzed with CD31 antibody (A) and phosphorylated mtor (pmtor) antibody (B)s. Nuclei were counterstained with DAPI (blue). Asterisk and double asterisks indicate P < 0.02, and P < respectively, as determined by an unpaired 2-tail student T-test. Fig. S7. shows no toxicity to normal organs in tumor xenograft study. H&E staining pictures of the major mice organs in A549 xenograft study shows no apparent toxicity Fig. S8. Inhibition of primary tumor growth and metastasis by in 4T1 orthotopic model. (A) Pictures of primary 4T1 tumor in fat pad #4 (top) and metastasis in contralateral fat pad #9 (bottom) after 7 days of treatment. Tumor is demarcated by black line and #4 pad is indicated by black arrow. (B) Pictures of primary 4T1 tumor (demarcated with white dotted line) in fat pad #4 (indicated by white arrows) and metastasis (demarcated with red dotted line) in contralateral fat pad #9 (indicated by red arrows) after 14 days of treatment. Two representative pictures were shown for each group. inhibited primary tumor growth and secondary metastasis to #9 fat pads. (C) H&E staining and immunohistochemical staining of ps6 in 4T1 primary tumors harvested from mice treated with control IgG and

4 on day 14. Compared to the tumor from control IgG treated mouse, tumor from treated mouse is much smaller. ps6 staining (middle panel) is also much weaker in. Quantification data are presented as mean ± SEM (n=4). Signals were quantified with Image J. Double asterisks indicate P < Fig. S9. inhibits the growth of hormone refractory mouse prostate cancer cell CE1 in vivo. The anti-tumor activity of was tested in the xenograft tumor model of a PTEN deficient, hormone refractory mouse prostate cancer cell CE1. The study was performed as described in Figure 2A. Asterisk indicates P < 0.05, as determined by an unpaired 2-tail student T-test.

5 A M GRP78-His B Sample GRP78-His (10 ng) MCF7 lysate (10 ug) Blocking protein BSA GRP78-His BSA GRP78-His GRP78 Liu_FigS1 -actin D KDa f/f c78 f/f C MCF7 DMSO Tg GRP78 β-actin 35 GRP78 HSP70 E B/F kd=1.7 nm -actin Bound, pm

6 Liu_FigS2 A Clathrin DAPI /Clathrin/DAPI B EphB4 DAPI /EphB4/DAPI, 37C, 1 hour Chlorpromazine, 5 ug/ml, 37C, 1 hour Chlorpromazine, 2 ug/ml, 37C, 1 hour, 4C, 1 hour

7 Liu_FigS3 A PBS Ctrl IgG 1.2 Relative colony number * 0.0 Ctrl IgG B 10 5 C 3 Ctrl IgG M30-FITC Treatment Apoptotic cell percentage (%) 42.7 ± 5.3 Ctrl IgG 24.5 ± K 100K 150K 200K 250K FSC-A Caspase activity ** ** Caspase 8 Caspase 9

8 A B IP Liu_FigS p85 GRP78 EphB2 Erk1/2 -actin Sum p85 Actin GRP78 GRP78 p85 Biotin -actin Biotin 43 34

9 A Liu_FigS5 Whole cell lysate (5 ug) Surface protein fraction (42 ug input) Cell HT29 A549 MCF7 H249 HT29 A549 MCF7 H249 Glucose GRP actin B Surface GRP78 vs. total GRP78 (%) Glucose Cell HT29 A549 MCF7 H249

10 A CD31 DAPI Liu_FigS6 Ctrl IgG * 0 20 CD31 Coverage (%) B pmtor (Ser2448) DAPI Ctrl IgG ** pmtor positive area (%)

11 Ctrl IgG Liu_FigS7 Kidney Liver Heart Lung

12 Liu_FigS8 A 4T1, day 7, Ctrl IgG 4T1, day 7, Ctrl IgG Contralateral metastasis #9 fat pad Primary tumor #4 fat pad B 4T1 tumor in fat pads (day 14) Control Control #9 #4 #9 #4 C H&E ps6(s235/s236) ** ps6 positive area (%)

13 Liu_FigS9 300 CE1 Tumor size (mm 3 ) Ctrl IgG * Days of treatment