Fig 2: Thiol antioxidant NAC blocks PDTC-induced suppression of ACE2 in H322M cells. (A) H322M cells were treated without (black line) or with (red line) 5 mM NAC or 200 µM PDTC alone or in combination for 24 h. Then, cells were stained using the Cellular ROS assay kit (deep red). Intracellular ROS levels were analyzed with a Becton-Dickinson FACSVerse Flow Cytometer. Cells treated with H2O2 for 30 min as a positive control. (B) H322M cells, with or without NAC pretreatment for 30 min, were treated with PDTC for 24 h. Total RNA was collected for ACE2 measurement by real-time qRT-PCR. The data were normalized to GAPDH. Error bars represent the mean ± SD (n = 3). (*P < 0.05, ** P < 0.005, ***P < 0.0005, and n.s. indicates non-significance.) (C) The treated cells were lysed for Western blot analysis of ACE2 and PARP; ß-actin was used as a loading control. The bands indicating full-length PARP (f-PARP) and the cleaved fragment (c-PARP) are indicated. (D) H322M cells were electroporated with control or p50 siRNA for 24 h, and then, cells were treated with H2O2 for an additional 24 h. The treated cells were lysed for Western blot analysis of ACE2, p50 and PARP; ß-actin was used as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig 3: Clinically approved drugs, triclabendazole and emetine, suppress endogenous ACE2 expression in H322M cells. (A) H322M cells were seeded in 96-well plates and treated with various concentrations of triclabendazole (0, 12.5, 25, 50, 100 or 200 µM) or emetine (0, 31.2, 62.5, 125, 250, or 500 nM) 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 designated as 100%, and the viabilities of other groups are expressed as percent of the control. (n.s. indicates non-significance, *P < 0.05, and ****P < 0.0001.) (B) H322M cells were treated with various concentrations of triclabendazole (0, 50, or 100 µM) or emetine (0, 50, or 100 nM) for 24 h, and total RNA was collected for ACE2 measurement by real-time qRT-PCR. The data were normalized to GAPDH. Error bars represent the mean ± SD (n = 3). (*P < 0.05, **P < 0.005, ***P < 0.0005, and n.s. indicates non-significance.) (C) H322M cells were treated with 100 µM triclabendazole or 100 nM emetine. Total protein was harvested at different time points for Western blot analysis of ACE2 and PARP; ß-actin was used as a loading control. (D) H322M cells were pretreated with NAC or vehicle for 30 min, and subsequently treated with triclabendazole (50 µM) or emetine (100 nM) for 48 h. The treated cells were lysed for Western blot analysis of ACE2 and PARP; ß-actin was used as a loading control. The bands for full-length PARP (f-PARP) and the cleaved fragment (c-PARP) are indicated.

Fig 4: Effects of TGF-ß1 treatment on cell proliferation, the expression of CD44, GSN, and EMT markers (i.e. N-cadherin, vimentin, and E-cadherin) in MDA-MB231 breast cancer cells. a MTT assay of cell viability after incubation of MDA-MB231 breast cancer cells with TGF-ß1 from 1 to 5 ng/ml for 0, 24, 48, 72, and 96 h. b Western blotting (top) with quantitative analyses (bottom) showed a dose dependent increase in protein expressions for CD44 and GSN in MDA-MB231 cells treated with 0, 1, 2, 5, 10, 20 ng/ml of TGF-ß1 for 72 h. c Western blotting (top) with quantitative analyses (bottom) of vimentin, N-cadherin, CD44, and E-cadherin levels in MDA-MB231cells treated with 0, 1, 2, 5 ng/ml of TGF-ß1 for 72 h. GAPDH used as an internal control. d Real-time quantitative PCR (qPCR) analysis showed the mRNA level for GSN, E-cadherin, N-cadherin, and vimentin in MDA-MB231 cells treated with or without 2 ng/ml TGF-ß1 for 3 days. In a, b, c, and d, the values are the mean ± SEM (n = 6), with * indicating a significant difference compared to the untreated cells

Fig 5: The loss of DNMT3b and the acquired p53 mutation cause prominent DNA damage in HCT116 cells.a NGS targeting sequencing of 54 genes in HCT116 and BKO cells. HCT116 and BKO shared common mutations in four genes. Additional mutations at p53 (TP53) were found in BKO cells. b BKO cells were infected with Tet-on Flag-p53-IRES-GFP virus followed by treatment with or without 2 µM doxycycline for 3 days for western blot (left) and viability assay (right, means ± SEM, n = 3, *** indicated P < 0.001 by two-tailed unpaired Student’s t-test. ns. P > 0.05). c BKO cells stably expressing Flag-DNMT3b were selected and infected with Flag-p53-IRES-GFP virus. Afterward, cells were treated with or without 1 µM doxycycline for 48 h and fixed for ?H2AX IF staining (scale bar, 10 µm, left). Relative intensity of ?H2AX in cells (n > 80), *** P < 0.001 by Mann–Whitney test (upper right). Western blots of Flag-DNMT3b, p53, p21, and Actin (bottom right). d ICF LCLs were transfection with 2 µg control siRNA or p53 siRNA using Amaxa® Cell Line Nucleofector® Kit V. After post-transfection at 48 h, cells were fixed for ?H2AX IF staining (scale bar, 10 µm, left). Fluorescent intensity of ?H2AX in cells (n > 100) was quantitated by Image J and relative intensity is expressed, ***P < 0.001 by the Mann–Whitney test (middle). Western blots of p53 and GAPDH (right).