*4.7. HUVEC Viability Assay*

We first determined the effect of different concentrations of hydroxychloroquine on HUVEC viability. Cells were plated at 2 × 10<sup>4</sup> cells per well in 96-well plates (*n* = 8, Corning) and grown to confluence in 100 μL culture media with hydroxychloroquine added at different concentrations (0.1, 1, 10, 100 μg/mL) and further incubated for 24 h. Viability was assessed by adding 20 μL MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagen<sup>t</sup> (Promega, Madison, WI, USA) to each well. After 1 h at 37 ◦C, the absorbance at 490 nm was read using a plate reader (SpectraMax i3, Molecular Devices, San Jose, CA 95134 USA).

#### *4.8. Oxidative Stress as Assessed by 8-Isoprostane*

Cells were grown to confluence in 96-well plates (2 × 10<sup>4</sup> cells per well) for 24 h in M199 complete media. Cells were treated with media (control), 100 ng/mL TNFα (Life Technologies /Thermo Fisher Scientific, Waltham, MA, USA), 20% normal pregnancy sera, or 20% preeclampsia sera, in the presence or absence of hydroxychloroquine (0, 0.1, 1, and 10 μg/mL) for a further 24 h. Conditioned media were then stored at -80◦C in the presence of 0.005% butylated hydroxytoluene (BHT) as described above. Total 8-isoprostane was measured using a commercial enzyme immunoassay (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. Samples were assayed in duplicate after diluting 1:5 with assay diluent. On the basis of the results from this experiment, in all subsequent experiments 1 μg/mL hydroxychloroquine was used. The cells were treated with either 100 ng/mL of recombinant TNFα or 20% preeclampsia sera in combination with either 1 μg/mL hydroxychloroquine or 100 μM apocynin (NADPH oxidase inhibitor) (Sigma-Aldrich) for 24 h.

#### *4.9. Measurement of NADPH Oxidase (NOX2) mRNA Expression*

Cells were grown to confluence in 6-well plates (1 × 10<sup>5</sup> cells per well) for 48–72 h in M199 complete media. Cells were treated with 100 ng/mL recombinant TNFα or 20% preeclampsia serum combined with either 100 μM apocynin or 1 μg/mL hydroxychloroquine for 6 and 12 h, respectively. The treatment groups were compared with untreated HUVECs or cells treated with 20% sera from normotensive pregnan<sup>t</sup> women. Total cellular RNA was isolated with Ambion (Life Technologies /Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. The cDNA was prepared with 1 μg of cellular mRNA and reverse-transcribed using SuperScript III first strand synthesis system (Life Technologies). Quantitative PCR was performed on Rotorgene (Qiagen, Hilden, Germany) in a reaction mixture (20 μL) containing Sensimix SYBR Green PCR master mix (Bioline Meridian Biosciences, Heidelberg, Germany). The reactions were performed with the following conditions: 95 ◦C for 10 min then 40 cycles of 95◦C for 20 s, 60 ◦C for 30 s, and 72 ◦C for 30 s. NOX2 was amplified using primers 5-TGG CAC CCT TTT ACA CTG-3 and 5-CCA CTA ACA TCA CCA CCT CA-3. The housekeeping gene 18S was amplified using primers 5-GTC TGT GAT GCC CTT AGA TGT C-3 and 5-AAG CTT ATG ACC CGC ACT TAC-3. Relative gene expression was determined using the delta delta – cycle threshold (CT) method.

### *4.10. Measurement of NOX2 Protein Expression*

HUVECs were grown to confluence and treated with either 100 ng/mL recombinant TNFα or 20% preeclampsia serum combined with 100 μM apocynin or 1 μg/mL hydroxychloroquine for 6 and 12 h, respectively. HUVECs were assessed for total NOX2 protein. Protein extracts of nucleic and cytoplasmic fractions were obtained using the nuclear and cytoplasmic reagents (Life Technologies /Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. Protein quantification was performed using the Pierce Bicinchoninic acid (BCA) kit (Life Technologies /Thermo Fisher Scientific, Waltham, MA, USA)). For Western blots, 40μg protein was loaded for each sample. Membranes were then blocked with 5% ( *w*/*v*) skim milk in phosphate-bu ffered saline with 0.1% (*v*/*v*) Tween-20 for 1 h prior to probing with antibodies. Membranes were stripped in a mild stripping bu ffer (1.5% *w*/*v* glycine, 0.1% *w*/*v* sodium dodecyl sulfate, 1% *v*/*v* Tween-20 in distilled water) for 5 min between antibodies. The primary antibodies and concentrations used were Nox2 at 0.025 ng/mL (anti-NOX2/gp91phox antibody (ab80508, Abcam, Cambridge, United Kingdom) and the control β-actin at 0.01 ng/mL (IMG-5142A, Imgenex). Antibodies were diluted in blocking bu ffer and incubated overnight at 4 ◦C. Chemilumiscence detection was performed using Clarity Western Electrochemiluminescence (ECL) blotting Substrates (Bio-Rad, Hercules, CA, USA).
