Characterization of undifferentiated rESC and riPSC under feeder-dependent and feeder-free culture conditions.

<p>(A) Rat PSCs form floating or loosely attached colonies on mitotically inactivated MEFs (MEF-2iLIF). Both cell types are positive for alkaline phosphatase activity and show SSEA-1 and Oct4 expression. Scale bars: 200 μm. (B) Flow cytometry analyses of both rPSC types for Oct4 expression. (C) Representative diploid karyograms of rPSCs originating from MEF-2iLIF conditions. P indicates the passage number under MEF-2iLIF conditions. rESCs in passage 14 showed a normal female rat karyotype (42, XX). riPSCs in passage 27 presented with an aberrant male karyotype characterized by a translocation between one homolog of chromosome 3 and the X chromosome (arrows) and two marker chromosomes (mar), presumably composed of material from chromosomes 17 and 20 which are missing. (D) No significant difference in population doubling time was detected between rESC and riPSC. Mean ± SEM, n = 20–21, unpaired t-test, P = 0.897. (E) Also in feeder-free monolayer culture (Geltrex-2iLIF), undifferentiated rPSCs express pluripotency markers. Phase contrast images reveal the typical high nucleus-to-cytoplasm ratio of pluripotent stem cells. In addition, cells show alkaline phosphatase activity, and are immuno-positive for SSEA-1 and Oct4. Scale bars: 200 μm. (F) Flow cytometry analysis revealed Oct4^pos expression levels comparable to feeder-based cultures for both rPSC types. (G) Representative karyograms of rPSCs after several passages in Geltrex-2iLIF conditions. P indicates the passage number of cells originating from MEF-2iLIF conditions plus additional passages in Geltrex-2iLIF. After 16 passages, rESC still show a normal rat karyotype in the majority of metaphase plates. After 5 passages, riPSCs also showed no differences to the karyotype found under MEF-2iLIF conditions. However, after 18 passages in Geltrex-2IiLIF, the majority of cells showed a tetraploid karyotype (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192652#pone.0192652.s001" target="_blank">S1 Fig</a>). (H) Proliferation rates show a significant difference of rESCs versus riPSCs in Geltrex-2iLIF culture. Mean ± SEM, n = 21, unpaired t-test, *P < 0.001.</p

Expression pattern of rPSCs during differentiation.

<p>Semiquantitative RT-PCR analyses showing the expression of pluripotency (Rex-1, Nanog, Oct4), mesoderm (T-Bra), CM-specific (GATA4, Nkx2.5, α-MHC, β-MHC, Mlc2v, Mlc2a, ANP) and gap junction (Cx40, Cx43, Cx45) genes in differentiating rESCs and riPSCs derived from AMW-based EB-formation and AA-2P enhanced differentiation in dynamic suspension culture. Expression of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal control; water and reverse transcriptase minus (RT-) indicate negative controls.</p

Detection of sarcomeric structures and gap junctions in rPSC-derived cardiomyocytes.

<p>(A) Immunofluorescence stainings of cryosections (thickness: 10 μm) of 14 days old beating EBs derived from rESCs and riPSCs differentiated in scalable suspension culture. Sarcomeric α-Actinin and gap junction protein Cx43. Nuclei are counterstained with DAPI. Scale bars: 100 μm. (B) Immunofluorescence staining of sarcomeric α-Actinin and Cx43 on cells after dissociation of EBs (day 14) and re-seeding on fibronectin coated glass-bottom dishes. Scale bars: 100 μm. (C) Transmission electron microscopy images of ultra-thin sections (approx. 70 nm) of EBs on day 14 of differentiation showing distinct Z-bands (z) of myofibrils, abundant mitochondria (m), and dark granules of glycogen (gly) depositions. Arrowheads indicate cisternae of sarcoplasmic reticulum in close proximity to Z-bands. Scale bars: 500 nm.</p

Cardiac differentiation of rPSCs is enhanced by ascorbic acid-2-phosphate and is scalable using agarose microwells.

<p>(A) The differentiation protocol started (D0, day 0) with preparation of hanging drops using 3x10³ undifferentiated rPSCs (expanded in MEF-2iLIF conditions) per droplet in serum-based differentiation medium (DM). After 2 days (D2), resulting EBs were transferred individually to agarose coated 96-well plates with or without addition of 100 μM AA-2P. Numbers of beating EBs were quantified on day 8, 10, 12 and 14. (B) Cell culture plate with approximately 160 hanging drops. (C,D) Efficiency of cardiac differentiation of rPSC-EBs determined by the emergence of beating EBs over time with and without AA-2P supplementation. Mean ± SEM, (n = 4–10 independent experiments with 48 EBs per biological repetition). (E) Fold increase of beating EB number after of AA-2P supplementation analyzed on day 14. Mean ± SEM, (n = 5–10 independent experiments). Unpaired t-test, *P < 0.05. (F) Protocol for rPSC differentiation expanded in Geltrex-2iLIF conditions using forced aggregation in agarose microwells for two days. Subsequent differentiation was conducted in dynamic suspension culture. (G) rPSC aggregates with different starting cell numbers after 48 h on agarose microwells. Scale bar: 200 μm. (H) Efficiency of cardiac differentiation determined by the quantification of beating EBs on day 14 (left Y-axis). Mean ± SEM, n = 9–26 independent experiments, *P < 0.02. Cardiac differentiation efficiency determined by flow cytometry analysis of cardiac troponin T (cTnT)-stained EB-derived cells on day 14 (right Y-axis). n = 3–7. (I) Exemplary histograms of cTnT-flow cytometry analyses on differentiation day 14. Isotype controls are shown in gray.</p

3D structural modeling of 4OI-XPO1 interactions based on the co-crystal structure of XPO1 (CRM1) with leptomycin B (PDB ID: 6TVO).

Both 4OI and leptomycin B are covalently bound to the reactive Cys528 (marked with an asterisk *) and interact extensively with the hydrophobic NES-binding groove. A. 4OI binds the site through hydrophobic interactions between the octyl chain and Ile521, Leu525, Met545, Val565 and Leu569 in the hydrophobic pockets Φ2 and Φ3 of the NES-binding site. The C1-carboxyl group further stabilizes binding through two hydrogen bonds with Lys537 and Lys568. These hydrophobic and electrostatic interactions optimally direct the methylene group of 4OI towards Cys528 and could be the driving force for the covalent Michael 1,4-addition. B. Overlay of 4OI (cyan) and leptomycin B (magenta) in the NES-binding groove showing about 70% occupancy by leptomycin B and 40% by 4OI. Lipophilicity protein surface at the NES-binding cleft: lipophilic (green), hydrophilic (violet), neutral (white), α-helices (gold). * = Cys528. (EPS)</p

Effects of XPO1 knock-down on IAV infection, cellular responses, and antiviral activity of the compounds.

A549 cells were transfected for 24 h with specific siRNA targeting XPO1 mRNA or nonspecific siRNA. Cells were then pretreated with the compounds (SEL, 1 μM; 4OI, 100 μM; BARD, 0.1 μM; SFN, 10 μM) for 12 h, infected with IAV PR8M (MOI = 1) for 2 h, and then incubated in fresh buffer containing the compounds for 22 h. A-C. Efficiency of XPO1 knock-down. A. XPO1 mRNA (RT-qPCR). B. XPO1 protein (immunoblot). C. Densitometry of B. D. Viral HA mRNA expression with reference to HPRT1 mRNA as internal control (RT-qPCR). E. Viral NP (immunoblot). F. IAV titers in cell culture supernatants (foci-forming assay, foci-forming units [FFU]/ml). G, H. IFIT1 and CXCL10 mRNA (RT-qPCR). I. Mitochondrial ROS (flow cytometry). J. Expression of NFE2L2, HMOX1, SLC7A11, AKR1B10, GCLM, and KEAP1 mRNAs (RT-qPCR, internal control HPRT1 mRNA). The heat map is based on log2 fold change (scale as indicated in the color legend) with respect to expression in wild-type uninfected cells. Bar graphs for each target gene are shown in S1 Fig for additional clarity. n = 3, means ±SEM. One-way ANOVA with Tukey’s post-hoc test, using infected untreated wild-type or knock-down cells as reference. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.</p

List of RT-qPCR primers.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand–target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a “noncanonical” mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.</div

Percentage of infected cells throughout an 8 h time course of IAV infection.

A549 cells were pretreated with the compounds (SEL, 1 μM; 4OI, 100 μM; BARD, 0.1 μM; SFN, 10 μM) for 12 h, were then infected with IAV PR8M (MOI = 1) for 1 h and subsequently incubated in fresh medium containing the compounds. Analysis based on the same images as used for Fig 2C–2F. Total number of cells was determined by counting DAPI-positive nuclei, and IAV infected cells by counting cells staining positive for NP in nucleus, cytoplasm or both. Data correspond to averages from 7 microscopic fields. A. Percentage of infected cells at 4, 6, and 8 h p.i. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. (EPS)</p

Biochemical and predicted ligand-target interactions with XPO1.

A,B. “Click-chemistry” pull-down assay demonstrating covalent binding of an alkynated 4OI probe (4-OI-alk) to XPO1 (A) and KEAP1 (B) in Calu-3 cells. At the indicated time points after addition of the probe to the cells, proteins complexed with the probe were detected by immunoblot for XPO1 or KEAP1. C-J. Ligand-target modeling studies of the compounds with the active site of XPO1 containing the functionally critical Cys528 (marked with a white asterisk *). Predicted binding energies are shown in Table 1. 3D models and the corresponding 2D interaction diagrams are shown in A,B (SEL), C,D (4OI), E,F (SFN), and G,H (BARD C1). A more detailed binding pose of 4OI to this site, as well as superimposed binding poses of 4OI and leptomycin B, are shown in S4 Fig. * = Cys528.</p

Chemical structures of the four compounds used.

The reactive electrophilic carbon atoms that can potentially undergo Michael addition from nucleophilic targets are highlighted in red or blue. A. Bardoxolone methyl (BARD) is unique in that it has two reactive carbon atoms at positions 1 (red) and 9 (blue). B. Sulforaphane (SFN). C. 4-Octyl itaconate (4OI). D. Selinexor (SEL). This bona fide XPO1 inhibitor is not known to be an NRF2 agonist, but also possesses one electrophilic double bond.</p