Journal: Journal of Virology

Article Title: Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA

doi: 10.1128/JVI.02230-18

Figure Lengend Snippet: Effects of topoisomerase poisons on cccDNA synthesis

Article Snippet: Topotecan (HY-13768A) and idarubicin (HY-17381) were purchased from MedChemExpress (MCE).

Techniques: Control

Journal: Journal of Virology

Article Title: Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA

doi: 10.1128/JVI.02230-18

Figure Lengend Snippet: TOP1 and TOP2 inhibitors block cccDNA synthesis. (A) Schematic presentation of the experimental schedule. HepAD38 cells were cultured in the absence of Tet, and 2 mM PFA was added to the culture medium 2 days after Tet removal to arrest viral DNA synthesis. Four days later, while PFA was withdrawn, Tet was added back to the culture medium to stop viral pgRNA transcription from the transgene. Cells were left untreated or treated with topotecan (TPT; 1 μM) or doxorubicin (Doxo; 1 μM) at 16 h after PFA removal and harvested at the indicated time points. (B) Hirt DNA after heat denaturalization at 88°C for 8 min and EcoRI digestion was resolved by agarose gel electrophoresis and HBV DNA species were detected by Southern blot hybridization with a riboprobe specifically hybridizing to negative-strand DNA. mtDNA served as a loading control. (C) The amounts of cccDNA were quantified by phosphorimager, normalized to the amount of mtDNA, and plotted as the percentage of that in the mock-treated (UT) cells harvested at 16 h post-PFA removal. Means and standard deviations (n = 4) are presented. *DP-rc, denatured deproteinized rc DNA; ccc*, EcoRI-linearized cccDNA.

Article Snippet: Topotecan (HY-13768A) and idarubicin (HY-17381) were purchased from MedChemExpress (MCE).

Techniques: Blocking Assay, Cell Culture, DNA Synthesis, Agarose Gel Electrophoresis, Southern Blot, Hybridization, Control

Journal: Journal of Virology

Article Title: Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA

doi: 10.1128/JVI.02230-18

Figure Lengend Snippet: TOP1 and TOP2 inhibitors inhibited HBV cccDNA synthesis in de novo infection. (A) Schematic representation of the experimental schedule. C3AhNTCP cells were infected with HBV at an MOI of 250 genome equivalents for 24 h. The cells were mock treated or treated with 200 nM doxorubicin (Doxo), 200 nM topotecan (TPT), 200 nM doxorubicin and 200 nM topotecan (Doxo + TPT), 200 nM aclarubicin (Acla), or 1 μg/ml myrcludex B (Myr-B) starting from HBV infection for a total of 36 h. (B) Hirt DNA was resolved by agarose gel electrophoresis after heat denaturalization at 88°C for 8 min and EcoRI digestion. HBV DNA species were detected by Southern blot hybridization with a riboprobe specifically hybridizing to negative-strand DNA. mtDNA served as a loading control of Hirt DNA analysis. (C) cccDNAs were quantified by a phosphorimager. The data from three independent experiments are presented. P values calculated by Student's t test are presented.

Article Snippet: Topotecan (HY-13768A) and idarubicin (HY-17381) were purchased from MedChemExpress (MCE).

Techniques: Infection, Agarose Gel Electrophoresis, Southern Blot, Hybridization, Control

Journal: Molecular Psychiatry

Article Title: Frontotemporal dementia patient-derived iPSC neurons show cell pathological hallmarks and evidence for synaptic dysfunction and DNA damage

doi: 10.1038/s41380-025-03272-x

Figure Lengend Snippet: NUPR2 , potentially related to the DNA damage pathway, is the only differentially expressed gene when comparing the C9-HRE and sporadic FTD neurons ( A ). Both sporadic (C9-) and C9-HRE-carrying FTD (C9+) neurons display nuclei, which are significantly rounder in shape as well as significantly smaller compared to healthy neuron nuclei, indicated by altered nuclear eccentricity ( B ), and a higher number of micronuclei ( C ). A representative image of a micronucleus next to the nucleus is shown (arrow). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used. n [Control] = 74; n [C9−] = 17; n [C9+] = 21. In addition, the distribution of larger and smaller sized nuclei is different in FTD neurons than that in control neurons ( D – F ). Representative images of control, C9-, and C9+ neurons treated with vehicle (upper panel) or 10 µM topotecan (lower panel). The neurons were stained with antibodies against MAP2 (red) and γH2A.X (green). Nuclei were stained with DAPI (blue) ( G ). Topotecan treatment significantly increases the number of γH2A.X-positive foci in the nuclei in all neurons, indicating increased DNA damage. At baseline, C9+ neurons display a slightly higher number of γH2A.X foci compared to control neurons, although this difference is not statistically significant ( H – J ). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used. n [Control] = 26–28; n [C9−] = 15–18; n [C9+] = 44–64. Statistically significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: To study the DNA damage response, iPSC-derived neurons were treated with 10 μM topotecan (Topotecan hydrochloride, Tocris, 4562) or DMSO vehicle for 1 h. The cells were fixed with 4% PFA and stained with conjugated MAP2 and γH2A.X antibody as described above.

Techniques: Control, Staining

Journal: Cancer science

Article Title: Histone deacetylase inhibitors enhance the chemosensitivity of tumor cells with cross-resistance to a wide range of DNA-damaging drugs.

doi: 10.1111/j.1349-7006.2007.00669.x

Figure Lengend Snippet: Fig. 5. HC-toxin selectively enhances the induction of cell death by a variety of DNA-damaging agents. (a) OVCAR-8 or SKOV-3 cells were incubated for 48 h with the indicated concentrations of bleomycin (BLM), mitomycin-C (MMC), topotecan (TPT), etoposide (VP-16), doxorubicin (DXR), 5-fluorouracil (5-FU), vincristine (VCR), or paclitaxel (PTX), in the absence (–HC) or presence (+HC) of 0.1 µM HC-toxin. The proportion of cells in sub-G1 phase was then determined using flow cytometry. Data are means ± SD from three separate experiments, each performed in duplicate. (b) OVCAR-8 cells were incubated for 9 h with 50 µM BLM, 2 µM MMC, 0.5 µM TPT, 20 µM VP-16, or 0.5 µM DXR, in the absence or presence of 0.1 µM HC-toxin. They were then fixed and subjected to immunofluorescence analysis with antibodies to phosphorylated H2AX. The cells were counterstained with antibodies to β-actin. Bar = 100 µm. Data are representative of three separate experiments. (c) OVCAR-8 cells were incubated for 24 h (upper panel) or 48 h (lower panel) as in (b) but in the additional absence or presence of 10 mM NAC, as indicated. The proportion of cells manifesting ROS accumulation (upper panel) or a fractional DNA content (lower panel) was then determined using flow cytometry. Data are means ± SD from three separate experiments, each performed in duplicate. *P < 0.01, **P < 0.001.

Article Snippet: Mitomycin-C was from ICN Biochemicals (Irvine, CA, USA), topotecan was from LKT Laboratories (St Paul, MN, USA), MS-275 was from Calbiochem (La Jolla, CA, USA), and FK228 was kindly provided by Fujisawa Pharmaceutical Co. (Osaka, Japan).

Techniques: Incubation, Flow Cytometry, Immunofluorescence

Journal: Molecular Medicine Reports

Article Title: XIAP underlies apoptosis resistance of renal cell carcinoma cells

doi: 10.3892/mmr.2017.7925

Figure Lengend Snippet: Cell viability percentage of ClearCa-2 and ClearCa-6 cell lines following CH11, Topotecan, CAPE and Etoposide treatment. Data are expressed as the mean ± standard deviation. *P<0.05 ClearCa-2 vs. ClearCa-6. CAPE, caffeic acid phenethyl ester; CH11, CD95-specific antibody.

Article Snippet: CH11 (CD95-specific CH11 antibody) was purchased from Immunotech; Beckman Coulter, Inc. (Brea, CA, USA); Etoposide was from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany), CAPE and Topotecan hydrochloride was from Merck KGaA.

Techniques: Standard Deviation

Journal: Molecular Medicine Reports

Article Title: XIAP underlies apoptosis resistance of renal cell carcinoma cells

doi: 10.3892/mmr.2017.7925

Figure Lengend Snippet: XIAP protein expression following Topotecan and CH11 treatment, as assessed by western blot analysis. Representative western blot images of Clear-Ca-2 and −6 cells following (A) CH11 and (B) Topotecan treatment. XIAP, X-linked inhibitor of apoptosis; CH11, CD95-specific antibody.

Article Snippet: CH11 (CD95-specific CH11 antibody) was purchased from Immunotech; Beckman Coulter, Inc. (Brea, CA, USA); Etoposide was from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany), CAPE and Topotecan hydrochloride was from Merck KGaA.

Techniques: Expressing, Western Blot

Journal: Molecular Vision

Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

doi:

Figure Lengend Snippet: HIF-1α inhibition does not affect VEGF expression. A : western blot analysis of HIF-1α expression in HLE-B3 cells treated with topotecan. Cell lysates were collected from cells treated with 500 nM topotecan in 0.01% DMSO after 8 h of hypoxic incubation. Control cells were mock treated with 0.01% DMSO and maintained in hypoxia as the topotecan-treated cells. Twenty μg protein/lane of cell lysates were analyzed with western blot analysis, and lane loading was normalized using a 1:1,000 dilution of rabbit anti-pan-actin antibody. Topotecan inhibited the expression of HIF-1α (1:1,000 dilution of rabbit anti- HIF-1α antibody) while a compensatory increase in HIF-2α was noted (1:1,000 dilution of rabbit anti- HIF-2α antibody). B, C : Densitometry analysis of HIF-1α and HIF-2α, respectively. D : Effect of HIF-1α inhibition on VEGF synthesis in hypoxia. HLE-B3 cells were cultured in 25 cm 2 flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media containing 500 nM topotecan or 0.01% DMSO for 8 h of hypoxic exposure. Cell-free supernatants were collected in triplicate at the end of hypoxic incubation and analyzed for VEGF levels with ELISA. There was no significant difference in the VEGF levels between the topotecan-treated cells and control cells. (A Student t test was performed to compare the VEGF levels between control and treated sample, and the p value was greater than 0.05.) E : HIF-2α protein from HLE-B3 control cells compared with a standard lysate prepared from human embryonic kidney cells (HEK) 293 (Novus Biologicals, Litton, CO). The HIF-2α found in the standard lysate migrated at about 97 kDa. The HIF-2 α from HLE-B3 cells was about 80 kDa.

Article Snippet: Topotecan hydrochloride, a recognized topoisomerase I inhibitor, was purchased from AvaChem Scientific LLC (San Antonio, TX).

Techniques: Inhibition, Expressing, Western Blot, Incubation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

doi:

Figure Lengend Snippet: Loss of HIF-1α does not influence mitochondrial membrane potential. JC-1 analysis of HLE-B3 cells treated with topotecan. Cells were incubated in serum-free media containing 500 nM of topotecan in 0.01% DMSO for 3 h in hypoxia. Control cells were treated with 0.01% DMSO in serum-free media and likewise exposed for 3 h in hypoxia. After 3 h of hypoxic exposure, fresh, oxygenated media without the inhibitor but with the addition of 5 µg/ml of JC-1 dye were added and incubated at 37 °C for 30 min. JC-1 is a potentiometric dye that exhibits a membrane potential dependent loss as J-aggregates (polarized mitochondria) when transitioned to JC-1 monomers (depolarized mitochondria), as indicated by a fluorescence emission shift from red to green. Therefore, mitochondrial depolarization can be indicated by an increase in the green/red fluorescence intensity ratio. The media were removed, and fresh serum-free media without inhibitor or potentiometric dye were again added to the cells. A : Confocal imaging of mitochondrial membrane depolarization after inhibition of HIF-1α. Note the proportionally equivalent red and green fluorescence between topotecan-treated and mock-treated cells, indicating that the membrane potential was not altered by inhibiting HIF-1α expression. These images were taken from a randomly chosen field. (The bar represents 20 µm.) B : There was no significant difference in the green/red fluorescence ratio between the control and topotecan-treated cells. Student t test, p>0.05.

Article Snippet: Topotecan hydrochloride, a recognized topoisomerase I inhibitor, was purchased from AvaChem Scientific LLC (San Antonio, TX).

Techniques: Membrane, Incubation, Control, Fluorescence, Imaging, Inhibition, Expressing

Journal: Molecular Vision

Article Title: Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

doi:

Figure Lengend Snippet: Inhibition of HIF-1α does not influence BAX or Bcl-2 levels. Cell lysates collected from cells treated with 500 nM topotecan were analyzed with western blot analysis for BAX and Bcl-2 levels. There was no change in the protein levels of BAX and Bcl-2 (A). (B) represents the corresponding densitometry analysis.

Article Snippet: Topotecan hydrochloride, a recognized topoisomerase I inhibitor, was purchased from AvaChem Scientific LLC (San Antonio, TX).

Techniques: Inhibition, Western Blot