Fig 2: PDTC treatment inhibits endogenous ACE2 expressions in H322M and Calu-3 lung cancer cells. (A) The endogenous ACE2 protein levels in six different lung cancer cells were measured by Western blot with ß-actin as a loading control. The intensity of the bands was measured by VisionWorks software (UVP). Fold change relative to control was calculated using ACE2: ß-actin ratio. (B) H322M and Calu-3 cells were seeded in 96-well plates and treated with various concentrations of PDTC (0, 12.5, 25, 50, 100 or 200 µM) for 48 h. Then, the relative cell viabilities were determined by MTS assay. Error bars represent the standard deviation. The viability of control cells was taken as 100%, and the other groups are reported as percent of the control. (n.s. indicates non-significance, and **P < 0.005.) (C) H322M and Calu-3 cells were treated with various concentrations of PDTC (0, 50, 100 or 200 µM) for 24 h, and RNA was isolated for ACE2 measurement by real-time qRT-PCR. The data were normalized to GAPDH. Error bars represent the mean ± SD (n = 3). (***P < 0.0005; and n.s. indicates non-significance.) (D) H322M and Calu-3 cells were treated with 200 µM PDTC, and protein was harvested at different time points for Western blot analysis of ACE2; ß-actin was used as a loading control. (E) Calu-3 cells were treated with 100 µM PDTC (solid line) or DMSO (dashed line) for 48 h, and cells were subjected to a flow cytometry assay to measure ACE2 protein on the cell surface. (F) A549, H322M and Calu-3 cells were treated with or without 200 µM PDTC for 24 h, and cytoplasmic and nuclear fractions were prepared for Western blotting to detect changes in nuclear localization of NF-?B subunits, p65 and p50. (G) H322M cells were electroporated with control or p50 siRNA. After 48 h, the cells were lysed for Western blot analysis of ACE2 and p50; ß-actin was used as a loading control.

Fig 3: Expression of NF-?B1, NF-?B2 RelA, RelB, and c-Rel proteins in the thymus from non-pregnant and pregnant ewes (n = 6 for each group). Note: DN16 = day 16 of the estrous cycle; DP13 = day 13 of pregnancy; DP16 = day 16 of pregnancy; DP25 = day 25 of pregnancy. Significance is denoted by letters; if two groups have different letters, this indicates that the difference between these groups was significant with P < 0.05.

Fig 4: miR-329 regulates PDG-induced trophoblast apoptosis and inhibition of IL-6 expression by targeting NF-?B p65.(A) TLR6- trophoblast cells were transfected with an anti-miR-inhibitor scramble sequence labeled with Cy3. After 24h, uptake efficiency was evaluated by fluorescent microscopy and a 99% transfection efficiency was observed. (B - D) TLR6- trophoblast cells were transfected with either an anti-miR scramble sequence or a specific anti-miR-329 inhibitor. Post-transfection, cells were treated with either NT or PDG (80µg/ml) for 12h, after which (B) NF-?B p65 mRNA and (C) IL-6 mRNA levels were measured by qRT-PCR. (D) After 48h caspase-3 activity was measured. Treatment of scramble control cells with PDG significantly reduced NF-?B p65 and IL-6 mRNA expression levels and upregulated caspase-3 activity, and these responses were significantly reversed by the presence of the anti-miR-329 inhibitor. *p<0.05 relative to the NT control unless otherwise indicated (n=3-5).

Fig 5: 6-acetylmonodethiogliotoxin (1) reduces binding affinity of p65 to DNA in a dose dependent manner. Dose-dependent effects of 6-acetylmonodethiogliotoxin (Compound 1) on p65 binding affinity to DNA. Jurkat cells were pre-incubated with 6-acetylmonodethiogliotoxin at indicated concentrations for 4 h and treated with TNFa (20 ng/mL) for 30 min, and then subjected to TransAM assay. Negative control (Co-) corresponds to nuclear extracts of DMSO treated cells, without TNFa activation, positive control (Co+) corresponds to nuclear extracts of DMSO treated cells activated by TNFa, wild-type (WT) and mutated type (MT) corresponds to nuclear extracts of DMSO treated cells activated by TNFa loaded on plate comprising wild-type or mutated consensus oligonucleotide, respectively. Goniothalamin (GTN) at concentration 7 µM was used as a positive inhibitory control. Shown data are mean ± SD of three independent experiments. Asterisks indicate a significant difference compared to control positive as analyzed by t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).