α sma

For single antibody staining, formalin-fixed, paraffin-embedded 5-μm skin tissue sections were de-paraffinized and rehydrated through a graded series of ethanol. Antigens were retrieved by incubation with a proteinase K solution (EMD Millipore, Billerica, MA, USA) for 5 minutes. Blocking was done by 2.5% normal horse serum for 1 h. Tissue sections were then incubated with primary antibody to αSMA (1:100, Novus Biologicals, Littleton, CO, USA), PDGFRβ (1:50, Cell Signaling Technology, Danvers, MA, USA), rat anti-mouse CD45 Ab (1:100, BD Pharmingen, San Diego, CA, USA), phospho-Smad2 (Ser465/467) Ab (1:100, Cell Signaling), or vWF (1:1000, DAKO, Santa Clara, CA, USA) at 4 °C for 16 h. Next, sections were incubated with ImmPress horseradish peroxidase (HRP) anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for 30 minutes. The color was developed using 3,3-diaminobenzidine (DAB) substrate (DAKO). Immunohistochemical images were collected using a microscope (BH-2; Olympus, Center Valley, PA, USA).

HSCs were grown on chamber slides, and the transfection experiments were carried out as described before. The cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS three times. The cells were permeated with X-Triton100 for 20 min and washed with PBS three times. Then the cells were blocked with 5% bovine serum albumin in PBS for 1 h followed by incubation with primary antibodies against desmin (ProteinTech Group, Chicago, USA) and α-SMA (Novus Biologicals, Littleton, USA) for 16 h at 4 °C overnight. After washing with PBS three times, secondary antibody was applied and incubated for 1 h. After additional washing, the cells were analyzed by fluorescence microscopy.

The following antibodies were used in this study for either western blotting, ChIP-sequencing, immunohistochemistry and chromatin immunoprecipitation. SOD2 (The Binding site, PC096), Gpx1 and Gpx4 (gift from L. Chavatte), ChREBP (Novus, NB400), SCD1 (Cell Signaling, C12H5), Phf2 (Cell Signaling, D45A2), H3K9me1 (Abcam, ab8896), H3K9me2 (Abcam, ab1220), H3K9me3 (Abcam, ab8898), histone H3 (Diagenode, C15200011), p(S473)-AKT (Cell signaling, D9E), Akt (Cell Signaling, 9272), P-p70S6K (Cell Signaling, 108D2), p70S6K (Cell Signaling, 49D7), p-GSK3β (Cell Signaling, D17D2), GSK3β (Cell Signaling, 27C10), LPK (Abcam, ab6191), ACC (Cell Signaling, 3662), FAS (Cell signaling, C20G5), p62 (gift from A. F. Burnol), Nrf2 (Santa Cruz Biotechnology, 13032), G6PDH (Cell Signaling, 12263), TKT (Cell Signaling, 8616), α-SMA (Novus, NB-600-531), collagen I (Novus, NB600-408), GAPDH (Santa Cruz Biotechnology, FL-335), malic enzyme (Novus, 68578), MTTP (Novus, 62489), NQO1 (Novus, A180), RNA PolII (Santa Cruz, sc899), HSP90 (Cell Signaling, 4874), Cidec (Abcam, ab77115), Plin2 (Progen, ap125), G0S2 (Santa Cruz Biotechnology, N13), and FLAG (Sigma, F1804).

In the in vitro experiment with cultured VPCs divergent from mouse iPSCs, CD-31 (1:1,000; Fitzgerald), PDGFRα, αSMA and p-VEGFR2 (1:1,000, Novus Biologicals) were stained for differentiated αSMA-positive vasculogenic cells. The area of positive staining was measured using the fraction area measurement function of the Image J software (NIH).
In immunohistochemical experiments, formalin (10 %) fixed brain slides with a thickness of 10 μm were stained for NeuN (1:400; Millipore, Billerica, MA) to label neurons, GFP (1:200; Novus Biologicals, Littleton, CO) for αSMA-positive arteries, or BrdU (1:400; AbD Serotec, Oxford, UK) to label newborn cells. Based on the NeuN staining, the peri-infarct region was morphologically determined in the regions adjacent to the stroke core. Staining was visualized by a fluorescent microscopy (Olympus, Japan). Z-stack imaging was used to confirm co-localization using the Olympus Stream. For systematic random sampling in design-based stereological cell counting, six coronal brain sections per mouse were selected, spaced 90 µm apart across the region of interest in each animal. For multistage random sampling, six fields per brain section were randomly chosen in the peri-infarct/penumbra regions of the brain. All counting assays were performed under blind condition.

Cells were lysed using RIPA buffer and the protein content was quantified by using Bradford’s reagent (Bio‐Rad, Hercules, CA) as per manufacturer’s instructions. Equal quantity of protein per lane was separated by SDS-PAGE. The separated proteins were then transferred onto the PVDF membrane. The membranes were blocked for 2 h with 5% blocking buffer (5% non-fat dry milk powder in 1X PBST). After washing in 1X PBST, membranes were incubated in respective primary antibodies, Fibronectin, α-SMA, NOX-4, AGXT2 (Novus Biologicals, Littleton, CO), p-eNOS, p-ERK, ERK, (Cell Signaling Technology (Beverly, MA, USA)), DDAH1 and GAPDH, (Santa Cruz Biotechnology, Santa Cruz, CA, USA), eNOS (Abcam, Cambridge, UK ),overnight at 4 °C and following the secondary antibody for 3 h. Protein bands were visualized using enhanced chemiluminescent reagent (Bio‐Rad, Hercules, CA) using Bio-Rad gel doc instrument. Images of the band intensity were quantified using the ImageJ software (NIH, Bethesda, MD).

Cells were washed in PBS solution, after which proteins were extracted using an established protocol. The proteins were then mixed with Laemmli sample buffer, heated at 65°C for 10 min, loaded (20 μg for each sample), separated by sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis under denaturing conditions, and electroblotted onto nitrocellulose membranes. The membranes were blocked by incubation in blocking buffer (1% BSA in Tris-buffered saline-0.1% Tween 20), then incubated with primary antibodies, washed, and incubated with anti-rabbit peroxidase-conjugated secondary antibody (1:10,000; Sigma). Immunoblots were developed using a BeyoECL (Beyotime) and Tanon 5200 system. The primary antibodies were as followed: α-SMA (Novus, 1:500), Col1a1 (Abcam, 1:1000), TGF-β (Abcam, 1:500), Cyclin D1 (Abcam, 1:200) p-IκB (Santa crus, 1:200), MyD88 (Abcam, 1:500), TLR4 (Abcam, 1:300).