Supplementary Materials and Methods s1

Supplementary Materials and Methods

Cell culture and treatment

Human pulmonary arterial ECs (HPAECs), human pulmonary microvascular ECs (HPMECs), Human pulmonary artery smooth muscle cells (HPASMCs), human pulmonary fibroblasts (HPFs), and human small airway epithelial cells (HPSAEpCs) were obtained from PromoCell (Heidelberg,

Germany) and used within nine passages. All ECs were validated by testing positive expression of vWF and CD31. A Smad3 inhibitor, SIS3 was purchased from Santa Cruz Biotechnology (Santa

Cruz, CA, USA).

SiRNAs against HSPB1, VEGFR1, and VEGFR2 and a control siRNA were purchased from

Santa Cruz Biotechnology (Dallas, TX, USA) and were transfected into cells using Lipofectamine

2000 (Invitrogen, Carlsbad, CA, USA). Recombinant human IL-1β, stromal cell-derived factor

(SDF)-1α, and plasminogen activator inhibitor (PAI)-1 were purchased from Sigma-Aldrich (St.

Louis, MO, USA). Recombinant human TGF-β1 was purchased from PeproTech (Rocky Hill, NJ,

USA). Cells were irradiated with gamma rays from a [137Cs] source (Atomic Energy of Canada,

Mississauga, ON, Canada) at a dose rate of 3.81 Gy per min.

Lentiviral vectors and transduction

Lentiviral expression vectors were generated using the ViraPower Promoterless Lentiviral Gateway

Expression system (Invitrogen). Briefly, cDNA-encoding mouse HSPB1 was inserted into the

EcoRI/XhoI-digested pcDNA3-EGFP plasmid (Addgene, Cambridge, MA, USA) to obtain pcDNA3-

HSPB1-EGFP, which was subcloned into the pENTR/D-TOPO entry vector. The pENTR-EGFP and pENTR-HSPB1-EGFP vectors were recombined with pENTR5'/CMVp and pLenti6.4/R4R2/V5-

DEST according to the manufacturer’s instructions. Lentiviral particles were produced in 293FT cells

(Invitrogen) by transfection of the recombined plasmid using the ViraPower Packaging Mix. For transduction, cells were cultured in OPTI-MEM (Invitrogen) with EGFP or HSPB1-EGFP lentiviral particles.

Reverse transcription (RT)-PCR For RT-PCR, total RNA was isolated from ECs using TRI reagent (MRC, Cincinnati, OH, USA), and

1 μg RNA was used to synthesize cDNA with an Omniscript RT kit (Qiagen, Hilden, Germany), followed by amplification using TaKaRa Ex Taq polymerase (TaKaRa Bio Inc., Otsu, Japan).

Bleomycin-induced fibrosis model

Bleomycin sulfate (Sigma-Aldrich) was dissolved in sterile PBS, and a 50-µL volume of the chemical

(0.5 U/kg) or PBS was administered intratracheally to 9-week-old mice as previously described (1).

qRT-PCR analysis

RNA was isolated using TRI reagent (MRC, Cincinnati, OH, USA), and 1 μg RNA was used to synthesize cDNA with an Omniscript RT kit (Qiagen, Hilden, Germany), PCR was performed un triplicate using the CFX96 TM Real-Time system (Bio-Rad, Hercules, CA, USA) with qPCR SYBR

Green master mix (Invitrogen). For each sample, the expression level was normalized against the geometric mean of the housekeeping gene encoding GAPDH. Primer sequences are listed in

Supplementary Table S1.

Immunoblotting and immunocytochemistry

Immunoblotting and immunocytochemistry were performed as previously described (2) using antibodies against HSPB1, VEGFR1, VEGFR2, CD31, VE-cadherin, TIE2, and TGFβ1 (Santa Cruz

Biotechnology); α-SMA (Abcam, Cambridge, MA, USA); p-VEGFR2 (Tyr1175; Cell Signaling

Technology, Beverly, MA, USA) ); β-actin (Sigma-Aldrich); p-Smad2/3 and Smad2/3 (Cell Signaling

Technology). The band intensities of immunoblot were analysed using Image J program.

Transgenic mice

The cDNA encoding mouse HSPB1 was subcloned into the pTRE-Tight-BI-ZsGreen1 vector

(Clontech Laboratories, Inc., Mountain View, CA, USA), and the doxycycline-dependent induction of

HSPB1 and ZsGreen1 expression was tested in vitro using CT26 and B16F10 cell lines (kindly provided Dr. Sam S. Yoon, Massachusetts General Hospital, Boston, MA, USA). TRE-

HSPB1/ZsGreen1 transgenic mice were generated by microinjecting the transgene into fertilized eggs of FVB mice; the resulting offspring were crossed with VE-cadherin-tTA mice (Jackson Laboratory,

Bar Harbor, ME, USA; Stock# 013585) to obtain double-transgenic mice. Doxycycline (Sigma-

Aldrich) was added to the drinking water (2 mg/mL with 5% sucrose). Continuous doxycycline administration was initiated before mating of the parental mice, and the offspring were administered doxycycline until 1 week before treatment. Primer sequences and PCR conditions for genotyping are listed in Supplementary Table S1.

Histology and immunohistochemistry

Mice were euthanized, and lung tissue was harvested and fixed in 10% (v/v) neutral-buffered formalin prior to prepare paraffin sections. Paraffin-embedded sections were deparaffinized and stained with hematoxylin and eosin (Sigma-Aldrich) or a Masson’s trichrome staining kit (Sigma-Aldrich) to detect collagen.

Prior to immunohistochemistry, deparaffinized sections were boiled in 0.1 M citrate buffer

(pH 6.0) for 30 min and then incubated with 0.3% (v/v) hydrogen peroxide in methanol for 15 min.

Sections were blocked in normal horse serum at room temperature for 30 min and labeled overnight at

4°C with primary antibodies against HSPB1, TGF-β, CD31 (1:100; Santa Cruz Biotechnology), α-

SMA, or fibroblast-specific protein (FSP)-1 (1:100; Abcam). Immunoreactivity was visualized using

ABC and DAB kits (Vector Laboratories, Burlingame, CA, USA), and sections were counterstained with hematoxylin. For immunocytochemistry, sections treated with primary antibodies were incubated with appropriate fluorescently labeled secondary antibodies (1:250; Molecular Probes) and counterstained with DAPI (3 µM). Images were acquired using a camera fitted to an epifluorescence microscope (Zeiss, Oberkochen, Germany).

For human cancer tissue analysis, a lung cancer tissue Accumax array (catalog no. A202) comprising 40 cases was purchased from ISU ABXIS Co., Ltd. (Seoul, Korea), and a non-small cell lung cancer tissue array (catalog no. LC10012) comprising 45 cases was purchased from US Biomax

(San Francisco, CA, USA). Microarray analysis

Three days after HSPB1 siRNA transfection, total RNA was isolated from HPMECs, with RNA quality assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

Gene expression profiling was performed using a Human GE 4×44K v2 Microarray kit (Agilent

Technologies). Fluorescent cRNA was generated and hybridized using an Agilent Low RNA Input

Linear Amplification kit PLUS and an Agilent Gene Expression Hybridization kit according to the manufacturer’s protocols. Images were acquired using an Agilent DNA microarray scanner and analyzed using Feature Extraction software (Agilent Technologies). Normalization and cluster analysis were performed using Agilent GeneSpring software. Normalized intensities of irradiated samples were divided by the corresponding normalized intensities of nonirradiated (control) samples to calculate fold changes in gene expression levels.

Tube-formation assays

One hundred microliters of Matrigel matrix (BD Biosciences, Bedford, MA, USA) was transferred to each well of a 96-well plate and incubated at 37°C in an atmosphere containing 5% CO 2 for 30 min.

Next, 1.5 × 104 HPMECs in complete medium (100 μL) were added to each well containing the

Matrigel matrix . The plate was incubated at 37°C in an atmosphere containing 5% CO2 for 4–16 h.

Aortic ring assay

Sprague-Dawley rats were sacrificed at 7 months after thoracic irradiation (12.5 Gy, Co60 irradiator).

Rat thoracic aortas were isolated, the connective tissue was removed, and aorta segments were embedded in growth factor-reduced Matrigel matrix (BD Biosciences) diluted in reduced serum media (Opti-MEM, Invitrogen) with or without VEGF-A (20 ng/mL) on 48-well plates. Microvessel outgrowth was observed for 3–7 days (3).

ELISA ELISA kits for VEGF, MMP-9, and FGF-7 were purchased from R&D Systems. Direct ELISAs for

IGFBP3 and proliferin were performed using anti-IGFBP3 and anti-proliferin antibodies from Santa

Cruz Biotechnology (Santa Cruz, CA, USA). All ELISAs were performed according to the manufacturers’ protocols.

Endothelial Cell Isolation

Lung tumor were obtained, minced, and digested in 625 U/ml collagenase II (GIBCO/Life

Technologies, Carlsbad, CA, USA) in HBSS for 30 min at 37°C. Cells were strained through a 100

μm strainer, and collagenase activity was stopped with equal volume fetal calf serum. The interface containing endothelial cell was seperated using Histopaque (Sigma). After centrifugation, cells were resuspended in 400 μl HBSS + 0.5% BSA and 2 μl CD31-FITC mAb (PharMingen, San diego, CA) and rotated for 1hour at 4°C. Endothelial cells were washed and isolated using anti-FITC mAb conjugated to magnetic beads (Miltenyi Corp., Auburn, California, U.S) as described in (4). We used non-selected cells with magnetic beads as tumor cells. Tumor cells were washed with PBS, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. All isolated cells were used within five passages.

Statistical analysis

Student’s t-tests and analysis of variance were used to evaluate the statistical significance of differences between experimental groups. Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). Differences with P values of less than

0.05 were considered significant.

Supplementary reference 1. Corbel M, Caulet-Maugendre S, Germain N, Molet S, Lagente V, Boichot E. Inhibition of

bleomycin-induced pulmonary fibrosis in mice by the matrix metalloproteinase inhibitor

batimastat. The Journal of pathology 2001;193(4):538-45.

2. Lee YJ, Lee HJ, Choi SH, Jin YB, An HJ, Kang JH, et al. Soluble HSPB1 regulates VEGF-

mediated angiogenesis through their direct interaction. Angiogenesis 2012;15(2):229-42.

3. Nicosia RF, Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A

quantitative assay of angiogenesis in vitro. Laboratory investigation; a journal of technical

methods and pathology 1990;63(1):115-22.

4. Ryeom S, Baek KH, Rioth MJ, Lynch RC, Zaslavsky A, Birsner A, et al. Targeted deletion of

the calcineurin inhibitor DSCR1 suppresses tumor growth. Cancer cell 2008;13(5):420-31.