*4.1. Patient Samples*

The study was approved by the Local Ethics Committee at Nara Medical University (Kashihara, Japan; approval number 873, 24 July 2014), and all participants provided written informed consent. We included PE/HDP patients with a pregnancy and disease-free pregnan<sup>t</sup> women with pregnancy were the control (Table 1). The participants' BMI values before pregnancy were less than 25 kg/m<sup>2</sup> with gestational age-matched normal pregnan<sup>t</sup> women at 27 weeks' gestation or later. All subjects were Eastern Asian origin, and none of the subjects were taking any medication or showed evidence of any metabolic diseases or other complications besides PE/HDP. PE/HDP was defined as new onset and diagnosed based on two consecutive measurements of diastolic and systolic blood pressure, diastolic blood pressure greater than or equal to 90 mmHg, or systolic blood pressure was greater than or equal to 140 mmHg, with urine protein over 300 mg/day, occurring diagnosed after 20 weeks of gestation [67]. All subjects (4 patients and 4 controls) provided serum samples for analysis and did not have gestational diabetes mellitus, thyroid malfunction, or other complications. All venous blood samples were obtained after an overnight fast at routine medical examination. The sera were separated immediately and stored at −80 ◦C for 3 years at the longest and 6 months at the shortest. HMGB1 concentrations of the sera were measured using Human HMGB1 ELISA kit (Arigo Biolaboratories Corp., Hsinchu, Taiwan).


**Table 1.** Clinical characteristics of patients/controls involved in the study.

### *4.2. Cell Culture and Treatment*

Human primary visceral preadipocytes were purchased from ZenBio, Inc. (Research Triangle Park, NC, USA). The cells were differentiated to adipocytes according to the supplier's protocol, and their differentiation to mature adipocyte was confirmed by Oil Red O staining. The primary adipocytes were incubated with 10% individual PE/HDP patient serum (#1~#4) and control serum (#1~#4) for 24 h. Human SW872 adipocytes were purchased from American Type Culture Collection (ATCC, Manassas, VA), and cultured at 37 ◦C with 5% CO2 in DMEM medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin G (Wako) and 100 μg/mL streptomycin (Wako) as described [68]. Mouse 3T3-L1 preadipocytes were purchased from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Ibaraki, Japan), and cell culture and differentiation of 3T3-Ll preadipocytes were performed as described by Ntambi et al. [69,70]. Briefly, confluent 3T3-Ll pre-adipocytes monolayers were incubated for 72 h in DMEM medium containing 10% FCS, 0.5 mM methylisobutylxanthine (IBMX; Wako), 1 μM dexamethasone (Wako), and 10 μg/mL insulin (Wako). After 72 h the cells were washed free of IBMX and dexamethasone and maintained in DMEM medium containing 10% FCS and 10 μg/mL insulin for 72 h. Adipocyte morphology was monitored by the appearance of cytoplasmic triacylglycerol droplets, which is closely correlated with the acquisition of the adipocyte phenotype. For the stimulation experiments, SW872 and 3T3-L1 cells (undifferentiated preadipocytes and differentiated adipocytes) were treated with 150 μg/mL (for SW872) or 300 μg/mL (for 3T3-L1) AGE-bovine serum albumin (BSA) (Calbiochem®, Merck KGaA, Darmstadt, Germany), 1 μg/mL HMGB1 (Bio-Techne, Minneapolis, MN) or 100 ng/mL S100B (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan). SW872 cells were also treated with 10 ng/mL *E. coli* LPS (Wako) for 24 h as described [33].

#### *4.3. Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)*

Total RNA was isolated using a RNeasy Protect Cell Mini Kit (Qiagen, Hilden, Germany) from primary cultured human visceral adipocytes, SW872, and 3T3-L1 adipocytes/preadipocytes, and cDNA was synthesized from total RNA as template using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) as described [68,70–82]. Real-time polymerase chain reaction (PCR) was performed using SYBR® Fast qPCR kit (KAPA Biosystems, Boston, MA) and a Thermal Cycler Dice Real Time System (Takara Bio Inc., Kusatsu, Japan). All the PCR primers were synthesized by Nihon Gene Research Laboratories, Inc. (NGRL; Sendai, Japan), and the primer sequences for each primer set are described in Table 2. PCR was performed with an initial step of 3 min at 95 ◦C followed by 40 cycles of 3 s at 95 ◦C and 20 s at 60 ◦C for human β*-actin*, mouse *rat insulinoma gene*

*(Rig)*/*ribosomal protein S15* (*RpS15*), mouse *IL-6*, human and mouse *CCL2*, human *LEP*, human *ADIP*, human *RETN*, human *S100B,* human *HMGB1,* and human *RAGE*, and with an initial step of 3 min at 95 ◦C followed by 40 cycles of 3 s at 95 ◦C and 20 s at 62 ◦C for human *IL-6* and human *TNF*<sup>α</sup>. The mRNA expression levels were normalized to the mRNA level of *Rig*/*RpS15* in mouse samples or β*-actin* in human samples [68,70–85].


**Table 2.** Primers used for real-time reverse transcriptase-polymerase chain reaction (RT-PCR).

#### *4.4. Measurement of IL-6 and CCL2 Concentrations in Culture Medium via ELISA*

Cells were stimulated with HMGB1 (1 μg/mL), AGE (150 and 300 μg/mL), S100B (100 ng/mL), and LPS (10 ng/mL) for 24 h, culture medium was collected, and the concentrations of IL-6 and CCL2 were measured by using a Human IL-6 ELISA kit (RayBiotech, Norcross, GA, USA) for IL-6 and a Quantikine® ELISA Human CCL2/MCP-1 Immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA) for CCL2, according to the instructions of suppliers.
