Phosphate buffered saline (pbs)

All examined embryos were cultured in sequential media G1 and G2 (Vitrolife, Västra Frölunda, Sweden) to blastocyst stage under 6% CO₂ and 5% O₂ at 37 °C. On day 5 after fertilization, blastocysts were graded according to the Istanbul Consensus Workshop on Embryo Assessment [37 (link)]. Trophectoderm blastocyst biopsies were performed only for blastocysts with grade over 211. Slower-developing blastocysts were cultured until day 6 or 7 and, when they reached good morphology (over 211), subjected to trophectoderm biopsy. Approximately 5–7 TE cells located opposite to the inner cell mass were aspirated with a biopsy pipette and dissected with a laser (Hamilton Thorne Inc., Beverly, MA, USA). Biopsied TE cells were washed in phosphate-buffered saline (PBS) (Cell Signaling Technology, Danvers, MA, USA)/polyvinylpyrrolidone (PVP) (Sigma-Aldrich, Saint Louis, MO, USA) and stored at −196 °C for subsequent PGT-A analysis. Blastocysts were vitrified immediately after the biopsy using the Kitazato vitrification kit (Kitazato, Shizuoka, Japan) according to the manufacturer’s protocols.

Genomic DNA was extracted from 40 cell lines purchased from Coriell Cell Repositories (CCR, New Jersey, USA) and from 92 unrelated and anonymized cord bloods of Chinese babies born at the National University Hospital. The Caucasian Human Variation DNA panel (HD100CAU) was purchased from CCR. Single cells were isolated from GM03620, GM06581, and six other CCR cell lines derived from two SMA parent–child trios. The first trio of cell lines comprise GM03813 (affected child) and GM03814 and GM03815 (his carrier parents), while the second trio comprise GM23686 (affected child) and GM23687 and GM23688 (her carrier parents). Single-cell isolation, processing, and WGA have been described elsewhere (Chen et al., 2015 (link)). Briefly, isolated single cells in 2 µl of 1× phosphate-buffered saline (PBS, Cell Signaling Technology, Massachusetts, USA) were lysed by adding 1.5 µl of 0.6 M potassium hydroxide (KOH, Sigma-Aldrich, Missouri, USA), heated at 65°C for 10 min, rapidly cooled to 4°C, and neutralized by the addition of 1.5 µl of 0.6 M Tricine (Sigma-Aldrich). The lysed cells were subjected to WGA using illustra GenomiPhi^™ V2 DNA Amplification Kit (GE Healthcare, Illinois, USA) according to manufacturer’s instructions, except that the incubation time was 4 h. This study was approved by the Institutional Review Board of the National University of Singapore (07-123E and 13-309E).

HCC cell lines were cultured for 24 h in 1% FBS medium and seeded in 96 multi-well plates. After attachment of the cells to the dishes, in addition to the described treatments, IGF1 75 ng/mL and VEGF 20 ng/mL recombinant molecules were also used as single treatment. The proliferating cells were evaluated by the staining with anti-human Ki-67 (BioLegend Inc., San Diego, CA, USA) diluted 1:200 in PBS (Cell Signaling, Beverly, MA, USA) for 2h in a humidified dark chamber. The staining was performed as previously described [32 (link)]. The ImageJ software (http://rsb.info.nih.gov/ij/) was used for the analysis of fluorescent signals and the values relative to three independent experiments were plotted in graphs using GraphPad Prism 5.0 software (La Jolla, CA, USA).

The experiment was conducted according to the method reported by Price et al. (30 (link)). Specifically, the total protein of peritoneal macrophage of BALB/c mice was extracted using radio immunoprecipitation assay lysis buffer according to the instruction of manufacture. After incubation of macrophages in 6-well plates (1 × 10⁵ cells/mL) for 36 h, the macrophages were used for the protein extraction. All the primary antibodies were diluted with PBS for 1000 times (Cell Signaling Technology, Danvers, MA, USA).
In brief, cell lysates were subjected to 10% SDS-PAGE and transferred to nitrocellulose NC membranes, and then incubated overnight at 4°C with anti-TLR4, anti-TRIF, anti-TRAF6, anti-P-NF-kB p65, and anti-GAPHD monoclonal antibodies after a 1 h blocking on (5% (w/v) non-fat milk. The membranes were subsequently washed with Tris Buffered Saline Tween (TBST) and incubated for 1 h at room temperature with corresponding secondary anti-bodies. Immunoreactive bands were detected using enhanced chemiluminescence (ECL) kit (Millipore Co., Billerica, MA, USA), GAPHD was used as internal control.

After 9 days in culture, primary neurons were treated with 50 μM chloroquine (Cell Signaling; 14774s) or PBS (Veh) for 18 h. After treatment, cells were washed with PBS and lysed in radioimmunoprecipitation buffer with protease/phosphatase inhibitor for 15 min on ice. Lysed cells were centrifuged at full speed for 15 min. Cell lysates were quantified and analyzed by Western blot as described earlier for brain lysates.