Supplementary information

Supplementary Materials and Methods

Preparation of low endotoxin recombinant HSPB1 proteins

HSPB1 was cloned into the pProExHTa vector (Invitrogen). The resulting plasmid was transformed into E. coli BL21 (DE3). His-HSPB1 proteins were purified by DEAE-Sepharose anion-exchange chromatography and size exclusion chromatography as described previously (Lee et al, 2005). Endotoxins were removed using the ProteospinTM Endotoxin Removal Kit (NORGEN).

Hitidine pull-down assays

Histidine-tagged DNA proteins, bound to Ni2+NTA agarose beads, were incubated with VEGF in NETN buffer (100mM NaCl, 1mM EDTA (pH 8.0), 20 mM Tris-HCl (pH 8.0), 0.2% Nonidet P-40, 10 mM imidazole) for 2h at 4°C. After washing the resin with buffer containing 10mM imidazole, proteins were recovered by suspension in non-reducing sample buffer and then subjected to Immunoblot analysis using anti-VEGF antibodies (R&D).

Enzyme-linked immunosorbent assays

Medical records and patient sera were collected from The Catholic University of Korea after the Institutional Review Board (IRB) had approved the study protocol [190 lung cancer patients (stage I: n = 33, II: n = 9, III: n = 37, IV: n = 117; Male: n = 127, Female: n = 63; average age: 65.8)]. The concentrations of HSPB1, HSP70, and VEGF were quantified by enzyme-linked immunosorbent assay (ELISA) using commercial mouse HSPB1, human HSPB1, and HSP70 kits (Stressgen), and mouse VEGF and human VEGF kits (R&D Systems) in accordance with the manufacturer’s instructions.

Tumor xenografts

Six-week-old male Balb/c athymic nude mice (SLC Inc.) received a subcutaneous injection of 1×106 CT26 cells in 100 mL of Hank’s balanced salt solution (HBSS) into the right hind flank. When the tumor reached a minimal volume of 200 mm3, the animals were subcutaneously treated three times with control bovine serum albumin (BSA) or rHSPB1 (2.5 mg/leg) on days 0, 3, and 6. After the first treatment, local radiation (8 Gy) was delivered to the region of the tumors. The tumor length (L) and width (l) were measured with calipers, and tumor volumes were determined according to the formula: (L × l2)/2. Tumors were measured every 3 days and allowed to grow for 15 days. For assessment of hypoxia, pimonidazole-1 was delivered intravenously at a dose of 60 mg/kg. After 90 min, the mice were killed and the tumors harvested, fixed, and immunostained with Hypoxyprobe™ (Hypoxyprobe Inc.) in accordance with the manufacturer’s instructions.

Supplementary Fig. 1

a In vitro binding assays: 125I-VEGF165 was incubated with recombinant HSPB1 or HSP70 protein and immunoprecipitated with the indicated antibodies.

b Neutralizing activity of the HSPB1 antibodies (Stressgene) was assessed in C.M. from HUVECs. HSPB1 antibodies (1ug/ml) were incubated with 3 ml of C.M. for 6 hr and the immune complexes precipitated with protein A-Sepharose (80uL; Sigma) and analyzed by SDS-PAGE.

c Effects of recombinant HSPB1 on Angiopoietin1-induced Tie2 phosphorylation in HUVECs are shown.

d Effects of recombinant HSPB1 on EGF-induced EGFR phosphorylation in HUVECs are shown.

Supplementary Fig. 2

a In capillary tube formation assays, tube formation was imaged.

b The intradermal regions of the backs of treated mice were imaged.

Supplementary Fig. 3

a HSPB1 secretion after VEGF165 treatment in HUVECs transfected with Flag-tagged HSPB1. The graph shows ± SEM of band intensities (soluble HSPB1 signal normalized to no VEGF treatment) for 3 independent experiments.

b Membrane integrity of HUVECs treated with VEGF for 24 hr was measured using an LDH (Lactate Dehydrogenase Assay) kit (Biovision) in accordance with the manufacturer’s instructions.

Supplementary Fig. 4

a HSPB1 Immunofluorescence of HSPB1(green) and CD31 (red) in KrasLSL-G12D/WT; p53Flox/Flox mice; Cre-lung adenocarcinoma.

b FACs analysis for CD31 following isolation from KrasLSL-G12D/WT; p53Flox/Flox mice; Cre-sarcoma-derived cells.

c Immunofluorescence of HSPB1 (green) and CD31 (red) in isolated tumor cells (STC) and endothelial cells (SEC). Scale bar = 5uM.

d p-HSPB1(Ser15, Ser82) immunohistochemistry in KrasLSL-G12D/WT; p53Flox/Flox mice; Cre-lung adenocarcinoma.

Supplementary Fig. 5

The mean lung weights in normal and CT26 colon carcinoma lung metastasis with or without the injection of GFP adenovirus.

Supplementary Fig. 6

a Growth curves of CT26 tumor xenografts in Balb/C nude mice. BSA and recombinant HSPB1 protein (each 2.5mg/leg, three times) were applied to tumors with or without 8 Gy radiation treatment (IR) (n=6 per group).

b Tumor vasculature was imaged and the hypoxic region of the tumor was stained with pimindazol-1 hypoxic probe (FITC). Scale bars = 20 uM.

Supplementary Fig. 7

Neutralizing activity of mouse HSPB1 antibodies were assessed in mouse blood serum. Control IgG and HSPB1 antibodies (1 ug/ml) were incubated with 20 ul of mouse blood in a final volume of 200 ul at 4°C overnight and the immune complexes precipitated with protein A-Sepharose (80 mL; Sigma) and analyzed by SDS-PAGE. Blood serum for Western blots or immunoprecipitation was used after albumin and IgG removal (using Biorad kit).

Supplementary References

Lee YJ, Koch M, Karl D, Torres-Collado AX, Fernando NT, Rothrock C, Kuruppu D, Ryeom S, Iruela-Arispe ML, Yoon SS (2010) Variable inhibition of thrombospondin 1 against liver and lung metastases through differential activation of metalloproteinase ADAMTS1. Cancer Res 70: 948-956

Lee YJ, Lee DH, Cho CK, Bae S, Jhon GJ, Lee SJ, Soh JW, Lee YS (2005)HSP25 inhibits protein kinase C delta-mediated cell death through direct interaction. J Biol Chem 280: 18108-18119

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