Sm22α

Lungs were inflated with 2% paraformaldehyde under constant pressure of 30 cm water and allowed to fix overnight. Tissue was embedded in paraffin and sectioned. Hematoxylin and eosin staining was performed to examine tissue morphology. Immunohistochemistry was used to detect protein expression using the following antibodies on paraffin sections: GFP (chicken, Aves, 1:500), GFP (goat, Abcam, 1:100), RFP (rabbit, Rockland, 1:250), Nkx2.1 (rabbit, Santa Cruz, 1:50), Sox2 (rabbit, Seven Hills, 1:500), Sox9 (rabbit, Santa Cruz, 1:100), Pecam (rat, HistoBioTec, 1:20), SM22α (goat, Abcam, 1:100), Pdgfrα (rabbit, Cell Signaling, 1:50), Pdgfrα (goat, R&D Systems, 1:50), Pdgfrβ (rabbit, Cell Signaling, 1:100), Pdgfrβ (goat, R&D Systems, 1:400), Scgb1a1 (goat, Santa Cruz, 1:20), Tubb4 (mouse, BioGenex, 1:20), Sftpc (rabbit, Millipore, 1:250), Sftpc (goat, Santa Cruz, 1:50), Pdpn (mouse, Hybridoma Bank, 1:50), Aqp5 (rabbit, Abcam, 1:100), and Ki67 (rabbit, Abcam, 1:50).

Western blot (WB) analysis for proteins isolated from wound tissue of animals with or without CD34⁺ therapy was performed by following the standard procedures. Primary antibodies used were for MMP1, α-SMA (from Santa Cruz, CA), MMP3, MMP9, MMP13, SM22α (all from Abcam, USA), β actin, and GAPDH (both from Cell Signaling, Beverly, MA). Mouse, rabbit IgG-HRP conjugated (Cell Signaling, Beverly, MA) and goat IgG-HRP conjugated (Santa Cruz, CA) secondary Abs were used and specific bands were detected using enzyme-linked chemiluminescences (Pierce, IL). Densitometric analysis of developed bands was performed by using UN-SCAN-IT (gel 6.1 version) software. Relative density was calculated using respective GAPDH/ β-actin bands.
In a separate experiment, fibroblasts were cultured under serum deprived (1% FBS) conditions. Total protein was extracted in lysis buffer containing protease and phosphatase inhibitors from 5 different conditions of fibroblast cultures, such as added MG132 (10 µM), CD34⁺ cells, CD34⁺ cells plus MG132, or MG132 plus SP 600125 (JNK Inhibitor II, 20 µM) (from Calbiochem, Darmstadt, Germany), with medium alone serving as a control at both 6 h and 12 h time points. Twenty micrograms of total proteins were tested by WB analysis for levels of c-Jun and GAPDH (all from Cell Signaling, Beverly, MA) following the above-mentioned techniques.

Mice were sacrificed at various time points (days 3, 5, and 7) of the experiments, and skin tissues were harvested so that part of the tissue was fixed in formalin-PBS buffer, paraffin-embedded, and sectioned to generate wound-edge specimens of 4 µm diameter. After de-paraffinized, sections were stained with Masson’s trichrome by standard procedures and examined under light microscopy. For immunofluorescence staining, antigen retrieval was performed using citrate buffer, pH to 6.0, and microwaving for 5 min then cooling for 3 min. After non-specific blocking, specific staining was performed by using α-SMA (Sigma-Aldrich, St. Louis, MO, USA), Pro-collagen 1A1 (Santa Cruz, CA) Ab, or SM22α (Abcam, USA) Ab followed by incubation with secondary antibody alexa fluor 594-conjugated IgG or Texas red (Invitrogen, Molecular Probe, Carlsbad, CA). Counterstaining was performed with DAPI (Invitrogen) and imaged by using a fluorescent microscope (Nikon E800 with MetaMorph version 4.5 software, Universal Imaging Corp.). The GFP staining procedure was carried out according to the protocol of VECTASTAIN Elite ABC kit (Vector laboratories Inc, Burlingame, CA) after using anti- GFP primary antibody (Zymed, Invitrogen). Immunohistochemical images were analyzed using an image analysis software program (ImageJ, NIH).

Embryos were dissected, fixed in 4% paraformaldehyde overnight at 4°C, dehydrated through increasing gradient of ethanol washes, and embedded in the paraffin wax for tissue sectioning. Hematoxylin and Eosin (H&E) staining was performed using standard procedures. Immunohistochemistry was performed using the following antibodies: anti-HDAC3 (Santa Cruz, 1:10), anti-Aqp5 (abcam,1:100), T1-alpha(HybridomaBank,1:50), anti-Scgb1a1 (Santa Cruz, 1:20), anti-Sftpc (Millipore, 1:50), β-tublinIV (BioGenex1:20), SM22α (Abcam,1:100), anti-Sox2 (Seven Hills Bioreagents, 1:500), anti-phosphohistone 3 (Cell Signaling Technology, 1:200), anti-Nkx2-1 (Santa Cruz,1:50), anti-BrdU (Abcam,1:100), and anti-Ki67 (Abcam,1:50). Slides were mounted with Vectashield mounting medium containing DAPI (VectorLaboratories, Burlingame, CA, USA). For BrdU staining, pregnant mice were administrated intraperitoneally at 0.1mg/g body weight, 2 hours prior to harvest. Embryos were processed and sectioned as above.

Cells were lysed in SDS buffer (60mM TrisHCl pH 6.8, 5% glycerol, 2% SDS). Twenty-five to 50 μg of cell lysates were run on SDS-polyacrylamide gels, transferred to PVDF membranes (Millipore, Billerica, MA), and incubated 1 hour in a blocking buffer (5% Bovine Serum Albumin (BSA), 10 mM Tris–HCl, pH 7.6, 150 mM NaCl, and 0.1%Tween 20). Each membrane was incubated overnight against a specific antibody: SMAD2 (Cell Signaling), pSMAD2 (Cell Signaling), SMAD1 (Cell Signaling), SMAD5 (Cell Signaling), pSMAD1/5 (Cell Signaling), PAI-1(Santa Cruz Inc., Dallas, TX), VE-cadherin (Cell Signaling), Calponin (Millipore), PECAM1 (Abcam), SM22α (Abcam). After incubation with peroxidase conjugated secondary antibodies, detection was performed with enhanced chemiluminescence reagents (GE Healthcare, Sweden). Protein levels of GAPDH (R&D Systems, Minneapolis, MN) were used to normalize the results.