*4.11. Endothelial Permeability Assay*

An endothelial permeability assay was performed as previously described with minor modifications [58]. Briefly, culture inserts (0.4 μm pore size, 6.5 mm diameter; Corning) were coated with 0.2% gelatin (Sigma-Aldrich) for 30 min at room temperature. HUVECs (50,000 cells per well) were plated on the inserts and cultured to form a tight monolayer with 100 μL M199 complete media in the upper chamber and 600 μL in the lower chamber at 37 ◦C, 5% CO2 for 72 h. Inserts were then transferred to a fresh plate and cell monolayers were treated in fresh media containing 100 ng/mL recombinant TNFα alone or with 1 μg/mL hydroxychloroquine for 16–22 h. Treatment groups were compared with untreated HUVECs. The conditioned media were collected and 100 μL fresh media containing fluorescein isothiocyanate (FITC)-conjugated dextran (MW 40000, final concentration 1 mg/mL, Sigma-Aldrich) was added to the upper chamber. The plate was incubated while protected from light for 60 min. The media from the lower chamber were diluted (1:20) in HBSS for measurement of fluorescence at 485/535 nm using a plate reader (SpectraMax i3, Molecular Devices). Results (fluorescence units) were expressed as percent changes relative to control.

Assessment of cell permeability when treated with 20% sera from healthy or preeclamptic women was performed using in vitro permeability assay kit from Millipore (Merck Millipore) in the absence or presence of 1 μg/mL hydroxychloroquine for 16–22 h. The treatment groups were compared with HUVECs treated with serum from women who had normal pregnancies (NP). Briefly, the transwells, which were coated with collagen, were rehydrated with 250 μL endothelial growth media (EGM, Lonza) and left at room temperature for 15 min. Subsequently, 200 μL of the media was removed and replaced with an equal volume of cell stock (1 x 105). Then, 500 μL of media was added to the receiver plate and incubated for 72 h to form a tight monolayer. Following this, fresh media was replaced in the receiver plate. The cells were treated accordingly and further incubated for 16–22 h. Media in the upper chamber was replaced with fresh media (150 μL) containing fluorescein isothiocyanate (FITC)-conjugated dextran, and the plate was incubated for 30 min and protected from light. The media from the lower chamber was diluted (1:20) with HBSS for measurement of fluorescence at 485/535 nm using a plate reader (SpectraMax i3, Molecular Devices). Results (fluorescence units) were expressed as percent changes relative to control.

#### *4.12. Zonula Occludens (ZO-1) Immunohistochemistry for the Assessment of Endothelial Integrity*

HUVECs were grown on 14 mm glass coverslips (4 × 10<sup>4</sup> cells/well) placed in 24 well plates. Cells were treated with 100 ng/mL recombinant TNFα or 20% sera from preeclamptic women in the presence or absence of 1 μg/mL hydroxychloroquine for 16–22 h prior to fixing with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. The treatment groups were compared with untreated HUVECs or cells treated with 20% normal pregnancy sera. Cells were blocked with 0.5% bovine serum albumin (BSA, Sigma-Aldrich) for 30 min, incubated first with rabbit anti-ZO-1 (1:50, Zymed) overnight at 4◦C, then with donkey anti-rabbit Alexa Fluor 568 (1:100, Invitrogen) for 1 h in the dark. Cell nuclei were stained with 2 μm <sup>4</sup>,6-diamidino-2-phenyindole dilactate (DAPI, Sigma Aldrich) for 10 min and mounted with fluorescent mounting media (DakoCytomation). Staining was examined with an Olympus BX60 fluorescent microscope and images were taken using an Olympus DP70 camera and Olympus CellSens software (Olympus). The primary antibody was replaced with an isotype-matched control antibody in the negative controls. The mean intensity of the staining was assessed using ImageJ software (version 2.0.0-rc-43/1.50i, http://imagej.net/Fiji/Downloads, Bethesda, MD).
