Cell Culture

The JEG-3 human choriocarcinoma cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) growth medium complete with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, USA) and 1% antibiotic-antimycotic (Thermofisher Scientific). The cells were cultured in 21% oxygen conditions (21% O2, 5% CO2 at 37 °C) and medium was changed every 2 to 3 days. The cells were passaged using trypsin-ethylenediaminetetraacetic acid (Wisent, Saint-Jean-Baptiste, Canada) then seeded at 300,000 cells per well in 6-well plates and grown for 42 h in the same 21% oxygen culture conditions.

Cell Treatment and 1.5% Oxygen/Reoxygenation Conditions

To induce oxidative stress similar to that experienced in the PE placenta, JEG-3 cells were cultured according to a low oxygen/reoxygenation protocol adapted from methods previously published by our lab [5]. Briefly, JEG-3 cells were plated and incubated in 21% oxygen conditions with either 10 or 50 µM of rosiglitazone (Selleck Chemicals, Houston, USA), or with dimethyl sulfoxide (MilliporeSigma Canada, Oakville, Canada) vehicle control for 18 h. After replacing the media, cells were supplemented with rosiglitazone or vehicle control and placed in 1.5% oxygen conditions (1.5% O2, 5% CO2 at 37 °C) for 6 h. The media was replenished with rosiglitazone and returned to 21% oxygen conditions for 18 h of reoxygenation.

RNA Extraction & RT-qPCR

Cells were lysed in QIAzol lysis reagent (Qiagen, Germantown, USA), and total RNA was extracted using the manufacturer’s protocol. The extracted RNA was quantified and assessed using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific). Using 1 µg of RNA, cDNA was generated with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Rt-qPCR was performed at 14 µl total reaction volume containing 25 ng of template cDNA, 7 µl SYBR-green Master Mix (Applied Biosystems, Waltham, USA), and 286 nM of forward and reverse primers (See Table 1). Relative changes in gene expression were analyzed using the DDCt method as described in [22].

Table 1 Quantitative real-time PCR primer sequencesProtein Extraction & Immunoblotting

Protein was extracted from the JEG-3 cells using RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitor cocktails (MilliporeSigma Canada). Protein concentration was then quantified with a BCA protein assay kit (Thermo Fisher Scientific). Western blot analyses were performed for each sample set using 25 µg of protein that were denatured (5 min, 95 °C) in Laemmli sample buffer (Bio-Rad Laboratories, Hercules, USA) and separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by a wet transfer onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20, then incubated overnight at 4 °C with anti-GCM1 (1:1000; Aviva Biosystems, San Diego, USA), anti-HO1 (1:500; Cell Signaling Technology, Danvers, USA), or anti-PPARγ (1:1000; Cell Signaling Technology) primary antibodies. Horseradish peroxidase-conjugated secondary antibodies were added to the membranes for 2 h at room temperature (RT) and developed with Pierce™ ECL western blotting substrate (Thermo Fisher Scientific). Subsequently, signals were visualized on a ChemiDoc™ imaging system (Bio-Rad Laboratories) and Image Lab Version 5.1 software (Bio-Rad Laboratories). Protein levels were normalized to a housekeeping protein, β‐actin (1:5000; Cell Signaling Technology).

Immunological Assay

To measure PlGF secretion, enzyme-linked immunosorbent assay (ELISA) was performed on JEG-3 cell culture media. The media was centrifuged at 1500 rpm for 10 min at 4 °C and the supernatant collected. Levels of PlGF were assessed according to the manufacturer’s instructions (R&D Systems, Minneapolis, USA) and optical density of the assay product was measured at 450 and 540 nm (for correction) using a Synergy H1 microplate reader (BioTek, Winooski, USA). A standard curve was generated to calculate the concentration of PlGF, and values were normalized to total number of cells plated or total protein concentration.

siRNA-mediated Suppression of PPARγ and GCM1

To demonstrate the knockdown of PPARγ and GCM1, Silencer™ Select pre-designed siRNA assays (Thermo Fisher Scientific) were utilized and Silencer™ Select negative control No. 1 siRNA (Thermo Fisher Scientific) was used as negative control. Silencing experiments were performed according to the manufacturer’s instruction. Briefly, JEG-3 cells were seeded in a 6-well plate and grown to 50–60% confluency in antibiotic-free DMEM media. The next day, culture media was removed, washed with PBS, and cells were subsequently incubated with fresh antibiotic-free DMEM media. A solution of 50–75 pmol siRNAs were prepared using Opti-MEM™ I reduced serum medium (Thermo Fisher Scientific) and Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), incubated at RT for 20 min and added dropwise to the cells. Transfection media was diluted after 5 h and replaced with complete DMEM media containing antibiotics after 24 h. Cells were cultured for another 24 h before protein extraction.

Immunocytochemistry

To show PPARγ nuclear translocation upon rosiglitazone treatment, cells were plated on an 8-well chamber slide (SPL Life Science, Pocheon-si, Korea) and grown to 50% confluency before treatment. Following treatment, cells were washed 3 times with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min at RT. After fixation, PFA was removed, and cells were washed 3 times with PBS, then permeabilized with 0.1% Tween-20 for 10 minutes. Cells were then blocked with 5% bovine serum albumin in PBS for 1 h at RT and incubated with anti-PPARγ primary antibody (1:250, Thermo Fisher Scientific) for 2 h. Cells were washed 3 times with PBS and incubated with Alexa-fluor 488 anti-rabbit secondary antibody (Thermo Fisher Scientific, 4 ug/ml concentration) for 45 min. Excess secondary antibody was removed by washing 3 times with PBS washes. Excess PBS was wiped carefully from the slides with Kimwipes. ProLong™ Diamond antifade mountant with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) was added and coverslips were mounted. Slides were left to dry for 24 h before imaging by confocal microscopy (Nikon A1 laser scanning confocal microscope).

Statistical Analysis

All statistical analysis was performed with GraphPad Prism 10.4.1 software. mRNA and protein expression values were normalized to respective housekeeping genes or protein. Relative mRNA and protein expression values from cells treated with either vehicle, rosiglitazone, T0070709, or PPARγ-siRNA and scramble siRNA were first normalized to respective housekeeping genes or protein. Subsequently, the relative expression values for each biological replicate set were normalized to respective vehicle control or scramble siRNA control. ELISA data was adjusted based on cell number or total protein concentration. Groups were analyzed by student’s t-test or one-way ANOVA. P < 0.05 is considered significant and indicated with (*) on each graph. Bar plot data were represented as mean ± SEM.