s frugiperda sf9 cells  (ATCC)

Journal: Environmental Toxicology

Article Title: Mono(2‐Ethylhexyl) Phthalate Induces Inflammatory and Angiogenic Alterations Mediated by the PI3K / AKT Pathway in HTR ‐8/ SVneo Trophoblastic Cells

doi: 10.1002/tox.24570

Figure Lengend Snippet: MEHP induces AKT phosphorylation and p65 nuclear translocation in HTR‐8/Svneo cells. p‐AKT and total AKT were measured by western blot in cultured cells treated with DMSO (control), MEHP (5 and 200 μM), and LY294002 (20 μM). GAPDH was used as a loading control. A representative blot of phosphorylated AKT, total AKT, and GAPDH (A) and the p‐AKT/AKT ratio are shown (B). VEGF (1 μg/μl) was used as positive control of AKT phosphorylation. Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test. Results are expressed as mean ± SE. * p ≤ 0.05, *** p ≤ 0.001. Immunofluorescence labeling of the NF‐κB subunit, p65, was used to determine the localization of p65. DAPI (4′,6‐diamidino‐2‐phenylindole, 1 μg/mL) was used to stain nuclei. Images were collected by an optical microscope (100×). Representative photomicrographs of immuno‐stained HTR‐8/Svneo cells are shown (C). p65 is shown in green and nuclei in blue; cells with nuclear localization of p65 are highlighted with red arrows.

Article Snippet: HTR‐8/Svneo cells (#CRL‐3271, ATCC, Manassas, VA, USA) were cultured in RPMI‐1640 medium (#30–2001, ATCC, Manassas, VA, USA) supplemented with fetal bovine serum (FBS) 5% (#S1620, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (#15240062, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Cell Culture, Control, Positive Control, Immunofluorescence, Labeling, Staining, Microscopy

Journal: Environmental Toxicology

Article Title: Mono(2‐Ethylhexyl) Phthalate Induces Inflammatory and Angiogenic Alterations Mediated by the PI3K / AKT Pathway in HTR ‐8/ SVneo Trophoblastic Cells

doi: 10.1002/tox.24570

Figure Lengend Snippet: Low and high doses of MEHP induce gene expression and secretion of different proinflammatory cytokines in HTR‐8/Svneo cells. Relative expression levels of IL6 (A), IL1B (B), CXCL8 (C), and TNF (D) are shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP at 5 and 200 μM. Data were obtained using the ∆∆Ct method, normalizing mRNA levels with ACTB expression. The concentration of IL‐6 (E) and IL‐8 (F) in culture medium is shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM). Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test. Results are expressed as mean ± SE. * p ≤ 0.05, *** p ≤ 0.001.

Article Snippet: HTR‐8/Svneo cells (#CRL‐3271, ATCC, Manassas, VA, USA) were cultured in RPMI‐1640 medium (#30–2001, ATCC, Manassas, VA, USA) supplemented with fetal bovine serum (FBS) 5% (#S1620, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (#15240062, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Gene Expression, Expressing, Control, Concentration Assay

Journal: Environmental Toxicology

Article Title: Mono(2‐Ethylhexyl) Phthalate Induces Inflammatory and Angiogenic Alterations Mediated by the PI3K / AKT Pathway in HTR ‐8/ SVneo Trophoblastic Cells

doi: 10.1002/tox.24570

Figure Lengend Snippet: High doses of MEHP increase gene expression and secretion of proinflammatory cytokines in HTR‐8/Svneo cells via PI3K/AKT/p65. Relative expression levels of IL6 (A), IL1B (B), CXCL8 (C), and TNF (D) are shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM) in the presence and absence of LY294002 (20 μM). Data were obtained using the ∆∆Ct method, normalizing mRNA levels with ACTB expression. The concentration of IL‐6 (E), IL‐8 (F), IL‐1β (G), and TNF‐α (H) in culture medium is shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM) in the presence and absence of LY294002 (20 μM). Concentration of IL‐1β (G) and TNF‐α (H) is only presented in the presence of LY294002, as values in its absence were below the detection limits of the assay. Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test. Results are expressed as mean ± SE. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: HTR‐8/Svneo cells (#CRL‐3271, ATCC, Manassas, VA, USA) were cultured in RPMI‐1640 medium (#30–2001, ATCC, Manassas, VA, USA) supplemented with fetal bovine serum (FBS) 5% (#S1620, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (#15240062, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Gene Expression, Expressing, Control, Concentration Assay

Journal: Environmental Toxicology

Article Title: Mono(2‐Ethylhexyl) Phthalate Induces Inflammatory and Angiogenic Alterations Mediated by the PI3K / AKT Pathway in HTR ‐8/ SVneo Trophoblastic Cells

doi: 10.1002/tox.24570

Figure Lengend Snippet: MEHP upregulates proangiogenic markers in HTR‐8/Svneo cells. Relative expression levels of ANGPTL4 (A), PGF (B), and VEGFA (C) are shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM). Data were obtained using the ∆∆Ct method, normalizing mRNA levels with ACTB expression. Protein levels of VEGF, determined by western blot (D, E), are shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP at 5 and 200 μM. GAPDH was used as a loading control. Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test. Results are expressed as mean ± SE. *** p ≤ 0.001.

Article Snippet: HTR‐8/Svneo cells (#CRL‐3271, ATCC, Manassas, VA, USA) were cultured in RPMI‐1640 medium (#30–2001, ATCC, Manassas, VA, USA) supplemented with fetal bovine serum (FBS) 5% (#S1620, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (#15240062, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Control, Western Blot

Journal: Environmental Toxicology

Article Title: Mono(2‐Ethylhexyl) Phthalate Induces Inflammatory and Angiogenic Alterations Mediated by the PI3K / AKT Pathway in HTR ‐8/ SVneo Trophoblastic Cells

doi: 10.1002/tox.24570

Figure Lengend Snippet: MEHP upregulates the gene expression of proangiogenic molecules in HTR‐8/Svneo cells via PI3K/AKT. Relative expression levels of ANGPTL4 (A), PGF (B), and VEGFA ( C) are shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM) in the presence or absence of LY294002 (20 μM). Data were obtained using the ∆∆Ct method, normalizing mRNA levels with ACTB expression. Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test. Results are expressed as mean ± SE. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Article Snippet: HTR‐8/Svneo cells (#CRL‐3271, ATCC, Manassas, VA, USA) were cultured in RPMI‐1640 medium (#30–2001, ATCC, Manassas, VA, USA) supplemented with fetal bovine serum (FBS) 5% (#S1620, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (#15240062, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Gene Expression, Expressing, Control

Journal: Environmental Toxicology

Article Title: Mono(2‐Ethylhexyl) Phthalate Induces Inflammatory and Angiogenic Alterations Mediated by the PI3K / AKT Pathway in HTR ‐8/ SVneo Trophoblastic Cells

doi: 10.1002/tox.24570

Figure Lengend Snippet: High concentrations of MEHP and LY294002 decrease endothelial‐like network formation in HTR‐8/Svneo cells. An endothelial tube‐like formation assay was performed in Matrigel. The endothelial‐like networks (highlighted in blue (A)) are shown for HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM) in the presence or absence of LY294002 20 μM. Semiquantitative analysis performed in WimTube ( https://www.wimasis.com/en/WimTube , Wimasis GmbH, Munich, Germany) for Mean Loop Area (B), Mean Tube Length (C), and Total Branching Points (D) are exhibited for HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM) in the presence or absence of LY294002 (20 μM). Data were analyzed by one‐way ANOVA followed by the Tukey post hoc test. Results are expressed as mean ± SE. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Article Snippet: HTR‐8/Svneo cells (#CRL‐3271, ATCC, Manassas, VA, USA) were cultured in RPMI‐1640 medium (#30–2001, ATCC, Manassas, VA, USA) supplemented with fetal bovine serum (FBS) 5% (#S1620, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (#15240062, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Tube Formation Assay, Control

Journal: Environmental Toxicology

Article Title: Mono(2‐Ethylhexyl) Phthalate Induces Inflammatory and Angiogenic Alterations Mediated by the PI3K / AKT Pathway in HTR ‐8/ SVneo Trophoblastic Cells

doi: 10.1002/tox.24570

Figure Lengend Snippet: MEHP promotes migration of HTR‐8/Svneo cells in a PI3K/AKT‐dependent manner. Cell migration was assessed through the wound‐healing assay (A) in HTR‐8/Svneo cells treated with DMSO (control) and MEHP (5 and 200 μM) in the presence or absence of LY294002 (20 μM). Images were captured at 0, 24, and 48 h. The percentage of wound closure (B) was calculated using the control group as a reference. Data were analyzed by two‐way ANOVA followed by Bonferroni's post hoc test. Results are expressed as mean ± SE. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05.

Article Snippet: HTR‐8/Svneo cells (#CRL‐3271, ATCC, Manassas, VA, USA) were cultured in RPMI‐1640 medium (#30–2001, ATCC, Manassas, VA, USA) supplemented with fetal bovine serum (FBS) 5% (#S1620, Biowest, Nuaillé, France) and 1% penicillin/streptomycin (#15240062, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Migration, Wound Healing Assay, Control