Supplementary methods
1. Primary culture of BMSCs
3-4 week–old female and male C57BL/6J mice were sacrificed by cervical dislocation under anaesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. Anaesthesia of mice was considered sufficient when the paw pinch test was negative. BMSCs were obtained from the bone marrow of female and male by flushing the femur and tibia diaphisis with Dulbecco’s modified Eagle’s medium (DMEM) and were cultured in DMEM supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin.
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2. In vivo experimental protocol
Female and male C57BL/6J mice, 20-25 g, 8-10-week old, received monocrotaline (MCT, 60 mg/kg) via tail vein to produce pulmonary arterial hypertension (PAH) model. 3 days after PAH mouse model produced, the mice were either untreated or treated with female or male BMSCs (2

10
6
cells in 100
 l PBS via tail vein).
2,3 Four experimental groups studied in either gender mice were as follows: 1)
PBS-injected mice treated with PBS (blank control group); 2) MCT-injected mice treated with PBS (MCT-treatment group); 3) MCT-injected mice treated with female
BMSCs (MCT + Female-BMSCs group); 4) MCT-treated mice injected with male
BMSCs (MCT + Male-BMSCs group).
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3. Assessment of pulmonary hypertension
Mice were fully anaesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection and sacrificed as mentioned above. After isolation of the heart and dissection of the attached vessels and both atria, the right ventricular wall (RV) was cut out and weighed, and then the left ventricular wall and septum
(LV+S) were treated in the same way.
4,5 The RV/ (LV+S) weight ratio was calculated as an index of RV hypertrophy.
After the lungs were fixed in formalin fixation for 24 h, a midsagittal slice was processed for paraffin embedding. Then 5 μm-thick sections were cut and stained with hematoxylin-eosin for pulmonary morphometry. In each mouse, 30 intraacinar vessels were analyzed by categorizing them as muscular and nonmuscular by a pathologist blinded to the treatment.
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4. Identification of BMSCs by morphology and flow cytometry
BMSCs obtained from the bone marrow were cultured, passed and observed by light microscope. BMSCs at passage 3 were homogenous and appeared in long rhombus shape.
1 Flow cytometry (Becton Dickenson, USA) was used for further characterization of BMSCs. Briefly, the cultured BMSCs at passage 3 were trypsinized
(0.25% trypsin) and adjusted to the density of 2

l0 6 /ml, and then incubated with fluorescence-conjugated antibodies for CD29, CD34, CD45 and their isotype controls in a black chamber at 4 °C for 30 min, respectively; after washing with PBS, cells analysis was performed with a flow cytometry.
2

5. Measurement of [Ca 2+ ] i
concentration in response to histamine and H
2
O
2
BMSCs monolayers on glass coverslips were incubated with culture medium containing 5 μmol/L fura-2 (acetoxymethyl ester form; Invitrogen) in a room temperature and 95% air/5% CO
2
atmosphere for 30 minutes. The coverslips were then gently washed for 30 min with indicator-free 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid-buffered saline (HBS) composed of (in mmol/L) NaCl
137, KCl 4.9, CaCl
2
1.5, MgSO
4
1.2, NaH
2
PO
4
1.2, D-glucose 15, and HEPES 20, pH
7.40, at room temperature to allow deesterification of the indicator. Monolayers were exposed to 10

M histamine or 100

M H
2
O
2
in HBS (made from a 3% stock solution;
Sigma) and the [Ca 2+ ] i
was continuously monitored before and after the stimulations.
6. Cell proliferation and migration assay
BMSCs at passage 3 were seeded in a 96-well plate (Corning) with 5

10 3 cells per well in triplicate. The cells were collected from each well 1–8 days after seeding and counted microscopically to produce cell growth curves. According to the growth curves, we chose day 2 (the time point on which there was no distinct difference between male and female BMSCs) to investigate BMSCs proliferation with or without histamine or H
2
O
2
treatment. Cell proliferation was measured by a MTS colorimetric assay according to manufacturer’s instructions (Cell Titer, Promega Corporation,
Madison, WI). After 1h incubation at 37 °C with MST labeling reagent, the number of living cells was determined by measuring the ratio absorbance at 470 nm.
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3

BMSCs migration assays were performed using a 48-well microchemotaxis chamber. Briefly, after 1 h treatment of histamine or H
2
O
2
, cells were trypsinized and dispersed into homogeneous single cell suspension, then washed extensively and resuspended in DMEM at a concentration of 10
6 cells/ml. 10
5 cells (100 µl cell suspension) were dispersed onto 0.5% gelatin/PBS-coated chemotaxis filters (Costar,
Amherst, MA; 8-mm pore size) within Boyden chamber inserts and cultured for 1 h at at 37 °C for adhesion. Then 600 μl chemoattractant solution was added to the lower compartments. A following 3h-incubation was allowed at 37 °C in 5% CO
2
and 95% air for cell migration. After incubation, the filter was removed and the upper surface of the filter containing the non-migrating cells was carefully scraped with a cotton swab. Cells migrated to the lower surface of the filters were fixed with formaldehyde, 0.1% crystal violet and 20% methanol, and counted manually in at least four random high-power fields (

400) per filter by microscopic examination. All groups were studied in triplicate.
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7. Western blot assay
Whole cell protein samples were prepared by lysing the desired female and male
BMSCs with or without 1h treatment of 10 μM histamine or 100 μM H
2
O
2
stimulation or GAPDH plasmids transfection. Equal amounts of protein extracted from all samples were western-blotted for specific antibodies against GAPDH. β-actin and α-tubulin together served as control for equal protein loading.
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8. Plasmid and stable transfection
Female and male BMSCs were separately seeded in 6-well plate in DMEM supplemented with 10% FBS. Transfection was performed using the Lipofectamine
2000 reagent (Invitrogen, CA, USA) according to the manufacturer’s recommendations. At 48 hours after transfection, the cells were trypsinized and plated for clonal selection of stable transfectants in 400 µg/ml G-418. Several clones with good expression of the genes were selected out of 48 individual clones in each group.
9. Statistical analysis
Data are reported as mean ± SD (standard deviation) of at least 3 independent experiments. SNK-q test and one-way analysis of variance analysis were used for two or multiple group comparisons. P < 0.05
was accepted as indicating statistical significance.
References
1.
Leng S, He J, Fan W, Cheng S, Long D, He H. Bone mesenchymal stem cells for gene transfer of NGF to the adult rat brain: Rescue the NGFR p75 positive neurons from fimbria–fornix lesion-induced degeneration. Neurosci Lett
2008; 448: 282-287.
2.
Nagaya N, Okumura H, Uematsu M, Shimizu W, Ono F, Shirai M, et al. Repeated inhalation of adrenomedullin ameliorates pulmonary hypertension and survival in monocrotaline rats. Am J Physiol Heart Circ Physiol 2003; 285: H2125-2131.
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3.
Raoul W, Wagner-Ballon O, Saber G, Hulin A, Maecos E, Giraudier S, et al.
Effects of bone marrow-derived cells on monocrotaline- and hypoxia-induced pulmonary hypertension in mice.
Respir Res 2007; 8: 8.
4.
Young KC, Torres E, Hatzistergos KE, Hehre D, Suguihara C, Hare JM. Inhibition of the SDF-1/CXCR4 axis attenuates neonatal hypoxia-induced pulmonary hypertension. Circ Res 2009; 104: 1293-1301.
5.
Dahal BK, Kosanovic D, Pamarthi PK, Sydykov A, Lai YJ, Kast R, et al.
Therapeutic efficacy of azaindole-1 in experimental pulmonary hypertension. Eur
Respir J 2010; 36: 808-818.
6.
Hong L, Sultana H, Paulius K, Zhang G. Steroid regulation of proliferation and osteogenic differentiation of bone marrow stromal cells: a gender difference.
J
Steroid Biochem Mol Biol 2009; 114: 180-185.
7.
Buttke TM, McCubrey JA, Owen TC. Use of an aqueous soluble tetrazolium/formazan assay to measure viability and proliferation of lymphokine-dependent cell lines. J Immunol Methods 1993; 157: 233-240.
8.
Annabi B, Naud E, Lee YT, Eliopoulos N, Galipeau J. Vascular progenitors derived from murine bone marrow stromal cells are regulated by fibroblast growth factor and are avidly recruited by vascularizing tumors.
J Cell Biochem
2004; 91: 1146-1158.
9.
Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Sliver M, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res
1999; 85: 221-228.
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Supplementary figure, table and legends
Figure S1
Figure S1.
Identification of BMSCs cultured from the bone marrow mice.
( A ) BMSCs (third passages) exhibited long rhombus shape by light microscopy. Scale bar = 50 µm. (
B F ) Representative FACS analyses showed that BMSCs were positive for diagnostic surface marker CD29 (99.96% ± 0.03%), and were negative for specific hematopoietic markers CD45 (1.97% ± 0.38%) and CD34 (0.34% ± 0.13%). n = 7 for each experiment.
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Figure S2
Figure S2. Regulation of [Ca 2+ ] i
signal kinetics in female and male BMSCs by
GAPDH. ( A ) Representative [Ca
2+
] i
oscillations from a siRNA vector-transfected female BMSCs (F-BMSCs) monolayer exposed to 100 µM H
2
O
2
in HBS (with 1.5 mmol/L Ca
2+
). ( B ) Representative [Ca
2+
] i elevation from a siRNA-GAPDH-transfected-F-BMSCs monolayer exposed to 100 µM H
2
O
2
in HBS.
( C ) Representative [Ca 2+ ] i
elevation from a GAPDH overexpression vector (OE vector)
-transfected male BMSCs (M-BMSCs) monolayer exposed to 100 µM H
2
O
2
in HBS.
( D ) Representative [Ca
2+
] i
oscillations from a OE-GAPDH-transfected-M-BMSCs monolayer exposed to 100 µM H
2
O
2
in HBS. ( E , F ) The detailed summarization of
[Ca
2+
] i
kinetics of female and male BMSCs with or without GAPDH manipulations in response to 10 μM histamine ( E ) or 100 μM H
2
O
2
( F ).
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Figure S3.
Figure S3. Western blot assay of GAPDH expression in BMSCs.
( A ) After treated with or without 10 µM histamine or 100 µM H
2
O
2 for 1 h, the cells were lysed. The lysates (50
 g for each) were subjected to electrophoresis, then simultaneously probed with monoclonal antibodies against GAPDH (1:2000 dilution), β-actin (1:250 dilution) and
α-tubulin (1:2000 dilution) and finally visualized by ECL. (
B ) After transfected with or without GAPDH-overexpressing (OE-GAPDH) or siRNA (siRNA-GAPDH) plasmids, the cells were lysed, and the expressions of GAPDH were analyzed following the same procedures.
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