Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN reduces survival in GBM cell lines but does not affect the survival of the normal human brain cells or normal human MSCs. Established U87MG, SF767, U118, U373 cell lines (A), and MHBT32, MHBT161, GBM10, and GBM43 early-passage GBM primary cultures (B) were treated with 5 to 50 μM SFN for 48 hours. Cell survival was determined by the methylene blue cytotoxicity assay (*p < 0.0001; **p < 0.0001; ***p < 0.0001; ****p < 0.0001for SFN vs vehicle [n = 6]). C: Normal brain cells were treated with 1 to 50 μM SFN for 48 hours, and cell survival was measured by the methylene blue cytotoxicity assay (*p < 0.0001 vs vehicle [n = 6]; bars and whiskers indicate median survival ± SD, respectively). D: MSCs were treated with 10 and 30 μM SFN for 24 hours. Cell survival was determined by the methylene blue cytotoxicity assay (p > 0.05 for SFN vs vehicle).

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Cytotoxicity Assay

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: Molecular profile of the GBM cell lines

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Amplification, Expressing, Mutagenesis

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN triggered apoptosis as well as caspase 3, caspase 7, and caspase 9 activation in SFN-treated GBM cells. SFN induced apoptosis in U87MG (A) and M-HBT-32 cells (B) treated with 5 to 30 μM for 24 hours. Apoptotic cells with condensed and fragmented nuclei were identified by DAPI staining. A minimum of 300 cells were counted for each treatment, and the percentage of apoptotic cells was calculated (*p < 0.0001 vs vehicle [n = 6]). C and D: U87MG and MHBT32 cells were treated with SFN for 24 hours and processed for Western blotting using antibodies to caspase 3, caspase 7, and caspase 9 as described in the Methods.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Activation Assay, Staining, Western Blot

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN mediated ROS formation and the effect of NAC. A: U87-MG cells were plated at a density of 5 × 105 in 60-mm dishes, allowed to attach overnight, and exposed to either 5 to 30 μM SFN for 24 hours or pretreated with 10 mM NAC for 3 hours and 5 to 30 μM SFN for 24 hours, and ROS was determined as described in the Methods. B: U87 cells were treated with 10 mM NAC, and the survival fractions of the NAC-treated cells compared with the untreated control sample were determined as described in the Methods section (*p < 0.001; **p < 0.0001 for SFN + NAC vs SFN [n = 3]). C: U87 cells were pretreated with 1 mM CCCP, rotenone, or CCCP + rotenone, respectively, then treated with 10 μM SFN, and ROS was determined as described in the Methods (*p < 0.01 for SFN + CCCP vs SFN alone; **p < 0.0001 for SFN + rotenone vs SFN alone; **p < 0.0001 for SFN + rotenone + CCCP vs SFN alone [n = 3]). Bars and whiskers indicate median survival ± SD, respectively.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Control

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN induced DNA SSBs and triggered the induction of γ-H2AX and DSBs in U87 glioblastoma cells. A: Control vehicle-treated U87-MG cells (a) and cells treated with 10 and 30 μM SFN, respectively (b and c), were processed for the comet assay under alkaline conditions as described in the Methods. B: The comet assay was performed under neutral conditions. Control U87 cells (a) and cells treated with 10 and 30 μM SFN, respectively (b and c), were processed for the comet assay under neutral conditions. C: Under neutral conditions, SFN treatment increased the DNA tail length. At least 200 comet cells were counted and compared with vehicle-treated cells (*p < 0.001 for 10, 30, and 50 μM SFN vs vehicle alone [n = 3]). D: U87 cells (3 × 105 cells) were treated with the vehicle (0.05% DMSO) (Lane 1) or 10 μM SFN (Lane 2) for 24 hours. Cell lysates (50 μg protein) were analyzed via Western blot using the antiphospho-H2AX antibody (Ser139) and anti–β-actin.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Control, Single Cell Gel Electrophoresis, Western Blot

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN triggers apoptosis in CD133-positive GSCs. A: The frequency of CD133-positive cells in unsorted GBM cell populations. The GBM cells were stained with the CD133 antibody conjugated with phycoerythrin (anti–CD133-PE) and analyzed by flow cytometry. B: The CD133-positive cells were isolated from U87 cells by FACS and cultured in sphere-forming neural growth media, as described in the Methods (a). The spheroids formed after 7 days and were processed for fluorescence microscopy for CD133 detection (b) and CD133 and DAPI staining (c). C: SFN induced apoptosis in GBM CD133-positive cells. The CD133-positive stem cells were treated with 30 and 70 μM SFN for 16 hours; after harvesting the cells, apoptosis was determined by DAPI staining. At least 3 microscopic fields of 300 cells were scored per condition per experiment by counting the total number of CD133-positive cells, and the percentage of those that showed a fragmented or dense DAPI-stained nucleolus representing the apoptotic cells was calculated (*p < 0.0001 vs vehicle [n = 3]).

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Staining, Flow Cytometry, Isolation, Cell Culture, Fluorescence, Microscopy

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: Confocal microscopy of U87 spheroids and the effect of SFN or SFN + NAC on the survival of the spheroids. A: The spheroids were stained with the CD133 or SOX2 antibody, and the confocal images were acquired with a confocal/2-photon Olympus FLOVIEW FV1000 MPE system, as described in the Methods (a, DAPI staining; b, CD133 expression; c, SOX2 expression; d, overlapping CD133 and SOX2 expression; e, isotype control antibody [magnification ×20]; scale bar = 100 μm). B: SFN inhibits the survival of U87 spheroids. The U87 primary spheroids were isolated as described in the Methods and dispersed to single cells by treating them with Accutase for 15 to 20 minutes. The cells were spun down by centrifugation at 1000g for 4 minutes and seeded in fresh sphere-forming media in 96-well plates at a range of 20 to 50 cells per well. After 2 to 3 days, the spheroids containing 6 to 8 cells were formed, which were not treated (a, control), treated with 5 to 30 μM SFN (b–d,) pretreated with 1 mM NAC for 3 hours (e), or pretreated with 1 mM NAC and then treated 5 to 30 μM SFN (f–h) for 8 to 10 days. C: SFN inhibits growth and reduces the survival of the spheroids isolated from U87 cells, and NAC prevents SFN’s effect (*p < 0.001 SFN vs vehicle alone; **p < 0.001 SFN + NAC [n = 3]). Tumor primary neurospheres from M-HBT-161 (D) and GBM43 (E) early-passage primary cultures isolated and treated with SFN as described above. The untreated spheroids (a) or spheroids treated with 10, 30, and 50 μM SFN (b, c, and d, respectively) inhibits growth and reduces the survival of spheroids isolated from early-passage GBM primary cultures.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Confocal Microscopy, Staining, Expressing, Control, Isolation, Centrifugation

Journal: Oncology Reports

Article Title: Inhibition of ATR potentiates the cytotoxic effect of gemcitabine on pancreatic cancer cells through enhancement of DNA damage and abrogation of ribonucleotide reductase induction by gemcitabine

doi: 10.3892/or.2017.5580

Figure Lengend Snippet: AZ20 impairs cell viability and induces limited cell death in pancreatic cancer cell lines. (A) The chemical structure of AZ20, a novel ATR-selective inhibitor. (B and C) Pancreatic cancer cell lines were cultured in 96-well plates with variable concentrations of AZ20 for 72 h and viable cells were determined using MTT reagent. IC50 values for AZ20 were calculated as the drug concentration necessary to inhibit 50% OD590 compared to the vehicle control-treated cells. The data are presented as means ± standard errors from at least 3 independent experiments. (D) BxPC-3 and HPAC cells were treated with variable concentrations of AZ20 for 48 h. The cells were fixed with 80% ethanol, stained with PI and subjected to flow cytometric analysis. Dead cells are expressed as the percentage of PI+ cells with sub-G1 DNA content. The data are presented as means of triplicates ± standard errors from one representative experiment.

Article Snippet: Cell lines and treatments The AsPC-1, BxPC-3, CFPAC-1, HPAC and MIAPaCa-2 human pancreatic cancer cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were authenticated by the University of Arizona Genetics Core Facility (Tucson, AZ, USA).

Techniques: Cell Culture, Concentration Assay, Control, Staining

Journal: Oncology Reports

Article Title: Inhibition of ATR potentiates the cytotoxic effect of gemcitabine on pancreatic cancer cells through enhancement of DNA damage and abrogation of ribonucleotide reductase induction by gemcitabine

doi: 10.3892/or.2017.5580

Figure Lengend Snippet: AZ20 induces cell cycle arrest, inhibits the activity of CHK1, and increases DNA damage in pancreatic cancer cells. (A and B) BxPC-3 and HPAC cells were treated with variable concentrations of AZ20 for 48 h. The cells were fixed with 80% ethanol, stained with PI, and subjected to flow cytometric analysis to determine cell cycle distribution. Representative histograms are shown. (C and D) BxPC-3 and HPAC cells were treated with variable concentrations of AZ20 for 48 h. Whole cell lysates were subjected to western blotting and probed with anti-PARP-1, γH2AX, -p-CHK1, -CHK1, -p-CDC25C, -p-CDK1, -CDK1, -p-CDK2, -CDK2, -RRM1, -RRM2 or -GAPDH antibody.

Article Snippet: Cell lines and treatments The AsPC-1, BxPC-3, CFPAC-1, HPAC and MIAPaCa-2 human pancreatic cancer cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were authenticated by the University of Arizona Genetics Core Facility (Tucson, AZ, USA).

Techniques: Activity Assay, Staining, Western Blot

Journal: Oncology Reports

Article Title: Inhibition of ATR potentiates the cytotoxic effect of gemcitabine on pancreatic cancer cells through enhancement of DNA damage and abrogation of ribonucleotide reductase induction by gemcitabine

doi: 10.3892/or.2017.5580

Figure Lengend Snippet: AZ20 enhances GEM-induced cell death in pancreatic cancer cells. (A) BxPC-3 and HPAC cells were treated with 1 µM AZ20 and 80 nM GEM alone or simultaneously for 48 h. The cells were fixed with 80% ethanol, stained with PI and subjected to flow cytometric analysis to determine cell death (sub-G1) and cell cycle distribution. The data are presented as means of triplicates ± standard errors from 1 representative experiment. (B-E) Cell cycle results particularly the percentage of cells in the S phase in BxPC-3 and HPAC cell lines and G2/M phase in the BxPC-3 cells are graphed as means of triplicates ± standard errors from 1 representative experiment. (F and G) BxPC-3 and HPAC cells were treated with 1 µM AZ20 and 80 nM GEM alone or simultaneously for 48 h. Whole cell lysates were subjected to western blotting and probed with anti-PARP-1, -γH2AX, -p-CHK1, -CHK1, -p-CDC25C, -p-CDK1, -CDK1, -p-CDK2, -CDK2, -RRM1, -RRM2 or -GAPDH antibody; *p<0.05 (GEM vs. control), **p<0.005 (GEM vs. control), ***p<0.001 (GEM vs. control), ##p<0.005 (combo vs. GEM), ###p<0.001 (combo vs. GEM). (H) Relative RRM2 protein levels are expressed as means ± standard errors from 3 independent experiments; *p<0.05 (GEM vs. control), **p<0.005 (GEM vs. control), ***p<0.001 (GEM vs. control), ##p<0.005 (combo vs. GEM), ###p<0.001 (combo vs. GEM).

Article Snippet: Cell lines and treatments The AsPC-1, BxPC-3, CFPAC-1, HPAC and MIAPaCa-2 human pancreatic cancer cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were authenticated by the University of Arizona Genetics Core Facility (Tucson, AZ, USA).

Techniques: Staining, Western Blot, Control

Journal: Oncology Reports

Article Title: Inhibition of ATR potentiates the cytotoxic effect of gemcitabine on pancreatic cancer cells through enhancement of DNA damage and abrogation of ribonucleotide reductase induction by gemcitabine

doi: 10.3892/or.2017.5580

Figure Lengend Snippet: AZ20 enhances GEM-induced DNA damage in pancreatic cancer cells. (A and B) BxPC-3 and HPAC cells were treated with 1 µM AZ20 and 80 nM GEM alone or simultaneously for 8 h. Whole cell lysates were subjected to western blotting and probed with anti-PARP-1, -γH2AX, -p-CHK1, -CHK1, -p-CDC25C, -p-CDK1, -CDK1, -p-CDK2, -CDK2, -RRM1, -RRM2 or -GAPDH antibody. (C) Relative RRM2 protein levels are graphed as means ± standard errors from 3 independent experiments. (D and E) BxPC-3 and HPAC cells were treated with 1 µM AZ20 and 80 nM GEM alone or simultaneously for 8 h, and then the cells were subjected to alkaline comet assay. Representative comets are shown. (F and G) Comet assay results are graphed as the median percentage of DNA in the tail from 3 replicate gels ± SEM; ***p<0.001 (GEM vs. control), ##p<0.005 (combo vs. GEM), ###p<0.001 (combo vs. GEM).

Article Snippet: Cell lines and treatments The AsPC-1, BxPC-3, CFPAC-1, HPAC and MIAPaCa-2 human pancreatic cancer cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were authenticated by the University of Arizona Genetics Core Facility (Tucson, AZ, USA).

Techniques: Western Blot, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Control

Journal: Oncology Reports

Article Title: Inhibition of ATR potentiates the cytotoxic effect of gemcitabine on pancreatic cancer cells through enhancement of DNA damage and abrogation of ribonucleotide reductase induction by gemcitabine

doi: 10.3892/or.2017.5580

Figure Lengend Snippet: AZ20 enhances GEM sensitivity in a synergistic manner in pancreatic cancer cell lines.

Article Snippet: Cell lines and treatments The AsPC-1, BxPC-3, CFPAC-1, HPAC and MIAPaCa-2 human pancreatic cancer cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were authenticated by the University of Arizona Genetics Core Facility (Tucson, AZ, USA).

Techniques:

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2-induced different cell viability in M17, PC12 and MN9D cells as measured by MTT assay (A & B) and trypan blue dye exclusion assay (C). Each bar from Figs. represents data obtained from 5 separate experiments (N = 5 of independent cell culture preparations, which is the same in the following legends). *p < 0.05, **p < 0.01, ***p < 0.001, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: MTT Assay, Exclusion Assay, Cell Culture, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on DDRs in M17 and MN9D cells as demonstrated by measurements of γH2AX (A) and p53 (B). Cells were exposed to 100–400 μM H2O2 for 3 h. Top panels: autoradiograph obtained by western blotting. Button panel: the quantitative analysis of band densities in western blotting. Each bar from Figs. represents data obtained from 5–7 separate experiments (N = 5–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Autoradiography, Western Blot, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2 treatment induces single-strand DNA breaks as determined by the Comet assay in M17 and MN9D cells. Cells were exposed to different concentrations of H2O2 for 3 h. The cells were processed for comet assays run under alkaline conditions to identify DNA SSBs. Tail moment (B), tail length (C) and % DNA in the tail (D) were analyzed to evaluate DNA damage. Each bar from Figs. represents data obtained from 5 separate experiments (N = 5). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Single Cell Gel Electrophoresis, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on ROS production in M17 and MN9D cells. Cells were exposed to different concentrations of for 3 h. ROS was measured by the DCFH2-DA assay. Each bar from Figs. represents data obtained from 6 separate experiments (N = 6). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Ctr1 (A) and OGG1 (B) protein levels in M17 and MN9D cells. Each bar represents data obtained from 5 separate experiments (N = 5) in (A), and 4 separate experiments (N = 4) in (B). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Cav1.2 (A) and Cav1.3 (B) protein levels in M17 and MN9D cells as determined by western blotting. Cells were exposed to different concentrations of H2O2 for 3 h. Each bar in Figs. represents data obtained from 6 to 7 separate experiments (N = 6–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Western Blot, Control

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: A , B HUVEC ( n = 3) were infected with T. gondii tachyzoites (MOI 0.5:1) of Tg Δhce1, Tg Δmyr1 mutants and wt parasites ( Tg Δku80) and fixed at 15 min, 1 h and 3 h p. i. Cells were stained in parallel for geminin (G2-phase marker, magenta), PCNA (S-phase marker, green) and DAPI (DNA maker, blue) ( B ). The total number of cells in G2-phase (geminin-positive/PCNA negative), S-phase (PCNA-positive/geminin negative) and G1-phase (geminin- and PCNA-negative) were determined. The data show that both, T. gondii wt and HCE1-deficient strain infection arrest the host cells in S-phase whilst the MYR1-deficient strain fails to do so. The scale bar represents 5 µm. Graph bars represent the median ± SD of three biological donors. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Staining, Marker, Comparison, Control

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: HUVEC ( n = 3) were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80) ( A ) Comet assay was used to detect parasite-driven general DNA strand breaks induction (at 12 h p. i.) and showed that T. gondii infection induced DNA damage in a MYR1- and HCE1-independent fashion. Scale bar: 100 µm. B , C Host cell genome instability was evaluated by cell binucleation (indicating cytokinesis failure) and micronuclei formation. Current data show an increased percentage of binucleated cells and micronuclei in HUVEC infected with all, Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80). D Exemplary illustrations of a T. gondii -infected host cell experiencing binucleated phenotype, and cells with micronuclei and simultaneous staining of γH2AX-based DNA damage foci. The results show that most of the binucleated cells were positive for DNA damage in contrast to micronuclei. Micronuclei are indicated by white arrowheads. On average, 1400 cells were counted in each non-infected and T. gondii strain infection in ( A − C ). The scale bar represents 5 µm. Graph bars represent the median ± SD of three biological donors. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Single Cell Gel Electrophoresis, Staining, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: A , B HUVEC ( n = 3) were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80), fixed at 15 min, 1 and 3 h p. i. and stained for γH2AX (cyan) to quantify DNA damage foci. Tachyzoites were detected by specific antibodies (red) and the nuclei were stained by DAPI (blue). B The percentage of DNA damage-positive cells was quantified in comparison to the total number of cells in the field of view at 15 min, 1 h and 3 h p. i. Data show that T. gondii infection-driven double-strand DNA breaks occur already at 15 min p. i. in a MYR1-dependent manner. The p-values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Staining, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: A , B HUVEC ( n = 3) were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku 80). B T. gondii -infected cells (3 h p. i.) were stained in parallel for γH2AX (DNA damage foci marker), geminin and PCNA to study whether DNA damage foci formation was linked to a distinct cell cycle phase. γH2AX-positive cells were assigned to each cell cycle phase and plotted as the percentage of DNA damage-positive cells. The scale bar represents 5 µm. Graph bars represent the median ± SD of three biological donors. The p-values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Staining, Marker, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: HUVEC were infected with tachyzoites (MOI 0.5:1) of Tg Δ hce 1, Tg Δ myr 1 mutants and control parasites ( Tg Δ ku8 0) and analysed for key molecules of the ATM or ATR pathway by Western blotting-based protein quantification at 12 h p. i. Graph bars represent the median ± SD of four to six biological replicates. Vinculin quantification was used as a loading control of the assay. The results show that exclusively the ATM-dependent pathway was activated by T. gondii -infection in a MYR1- and HCE1-independent fashion. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Western Blot, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: HUVEC ( n = 6) were infected with control tachyzoites (MOI 0.5:1) and both intracellular and extracellular ROS production was evaluated after 3 h p. i. A Exemplary histogram using flow cytometry-based quantification of intracellular ROS via the probe DCFH-DA. NAC treatment was used to block ROS production. DCFH-DA-based quantification of intracellular ROS production in T. gondii -infected HUVEC in the presence and absence of the ROS inhibitor NAC. A control of cells without DCFH-DA staining was included (WOS: without staining). Data showed that T. gondii- infected cells produced lower levels of ROS than uninfected controls. MFI: mean of fluorescence. FACS gating strategy in Supplementary Fig. . B Quantification of extracellular ROS (Amplex red) in T. gondii -infected HUVEC and control cells showed no infection-driven changes. RFI: relative fluorescence intensity. C Quantification of γH2AX-based DNA damage foci in NAC-treated and untreated T. gondii -infected HUVEC. No changes in DNA damage induction were detected when cells were pre-treated with the ROS inhibitor (NAC), suggesting that T. gondii -driven DNA damage occurs ROS-independently. The p values were calculated using a one-way ANOVA followed by a Kruskal Wallis multiple comparison test of variance, using the non-infected condition (n. i.) as control. A p < 0.05 was considered as significant.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: Infection, Control, Flow Cytometry, Blocking Assay, Staining, Produced, Fluorescence, Comparison

Journal: Communications Biology

Article Title: Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism

doi: 10.1038/s42003-024-07374-0

Figure Lengend Snippet: Hypothetic illustration of T. gondii -driven effects on host cellular genome integrity.

Article Snippet: T. gondii tachyzoites (RH, Δ ku 80, Δ myr 1 and Δ hce 1 strain) were maintained by serial passages in primary HFF cells (ATCC, maximum passage: 10).

Techniques: