Fig 2: Selinexor inhibits nuclear export and forces nuclear accumulation of ACE-2 in vitro. Vero E6 cells were incubated with 500 nM selinexor or DMSO for 24 h. a) Cells were fixed with 3% paraformaldehyde, incubated with anti-ACE-2 antibody (Invitrogen, #MA5-32307) followed by a rabbit secondary antibody, Alexa Fluor 488, green-fluorescent dye (Thermo Fisher, #A11008), then visualized with the Echo Revolve fluorescent microscope (ECHO) at 60× magnification. Membrane-bound ACE-2 receptors are present in DMSO-treated cells only (white arrows). b) Sub-cellular fractionation was performed post-treatment then analyzed by immunoblots and c) quantified by densitometric analysis. d) Vero E6 and HEK 293 cells were treated with selinexor (550 nM, 24 h) and whole protein lysates were analyzed on immunoblots.

Fig 3: Antibiotic resistance and mCherry expression in R. parkeri RPATATE_0245::pLoxHimar (3A2). Vero cells were infected with R. parkeri WT and 3A2 at the same multiplicity of infection (MOI) of 2. Infected cells were cultured with either plain medium (A,B), or with medium containing 100 µg/mL spectinomycin (C). At the indicated time points (A,C) or day 5 post infection (p.i.) (B), cells were collected for quantifying the kinetic intracellular growth of these rickettsiae by quantitative real-time PCR amplifying citrate synthase (CS) gene. (D) Vero cells were infected with R. parkeri WT and 3A2. Cells were collected when the infection rate reached greater than 80%, as shown by the light microscopy after Diff-Quik staining (I). A drop of cell suspension under a cover slip was imaged under mCherry fluorescence (II) using an Echo Revolve Microscope (Echo Inc., San Diego, CA, USA). In addition, R. parkeri WT and 3A2 were also visualized under grey scale (III). Uninfected Vero cells served as negative controls. Scale bars were shown as labeled. ns, not statistically significant. *, p < 0.01.

Fig 4: Sw71 spheroids’ propensity for invasion. (a–c) Representative triplets of invading Sw71 spheroids. Optimal results were gained in Opti-MEM followed by lsDMEM/F12 and hsDMEM/F12 (86%, 83% and 75% respectively). (d) Representative spheroid on D10 in hsDMEM/F12, showing a classical “path-generating” radial invasion pattern, seen in 97% of the cases. (e) Representative spheroid on D6 in hsDMEM/F12, showing a non-typical “path-finding” amoeboid invasion (zone marked in red: cells with rounded, deformable morphology squeeze through narrow spaces and smaller pores of the Matrigel). Of note, invading Sw71 spheroid trophoblasts form invadosomes (orange arrows) for the proteolysis-dependent ECM degradation which are numerous and well-developed in radial invasion (d) and only a few in the amoeboid invasion (e). Imaging: Echo Revolve microscope, Echo, San Diego, CA, USA, magnification 4× ± digital zoom. Seven independent experiments were performed.

Fig 5: Generation of Sw71 spheroids as a model of early human placenta. (a) Differentiation of spheroid from Sw71 cells visualized at different time-points: 2–8 h—aggregation; 8–24 h—compaction, 24–48 h—stable spheroid. Left column—formation of a single differentiated spheroid with intact border, right column—additional digital zoom to show the formation of intact periphery. (b) Reproducibility (exp. 1–3) and some obstacles (exp. 4–6) during differentiation of the models exemplified by representative Sw71 spheroids captured at D0, D1 and D2 (6 independent experiments): exp. 1—fine spheroid, brightfield view; exp. 2—fine spheroid, phase contrast view; exp. 3—fine Sw71-GFP spheroid, trans, fluorescent and overlaid view; exp. 4—mechanical non-organic contamination; exp. 5—nonoptimal humidity; exp. 6—plate defects. (c) Sw71 spheroids’ morphological characteristics: 1. Measurements; 2. Mean diameter values of Sw71 spheroids (n = 126) at D0, D1 and D2, and before and after their transfer; 3. Surface measured for the same spheroids. (d) Sw71 spheroids’ disintegration during long term culture without additional feeding presented by two representative spheroids. Left—Sw71-GFP spheroid on D7 in overlayed view. Right—Sw71 spheroid, pictured on D8 and D14. Imaging: (a) time-lapse live cells imaging system OMNI (CytoSmart Technology, The Netherlands), magnification 10× (+ manual digital zoom), pictures taken every 2 h, but only selected images were included. (b, c, d) ECHO Revolve microscope (RVL-100-M, Echo, San Diego, CA, USA), magnification 4×. Bars (b, c) = 530 µm. Statistics was processed with GraphPad Prism v.5.0 software.