immunofluorescence staining raw 264 7  (ATCC)

Journal: International Journal of Molecular Medicine

Article Title: MOTS-c protects against placental injury via Nrf2 activation in hypoxia-induced intrauterine growth restriction mice

doi: 10.3892/ijmm.2025.5697

Figure Lengend Snippet: MOTS-c mitigates hypoxia-mediated oxidative stress in placenta. (A) MDA content in placenta. (B) SOD activity in placenta. (C) Nrf2 mRNA expression levels in placenta. (D) Representative western blotting images. (E) Total Nrf2 expression in placental tissues. (F) Nuclear Nrf2 expression in placental tissues. (G) Nrf2 expression in cytoplasm of placental tissues. (H) Representative immunofluorescence images and (I) quantification of Nrf2 in placenta. Scale bar, 50 μ m. (J) Relative mRNA expression levels of HO-1 and NQO-1. Data are expressed as the mean ± SD. Normal, n=6; IUGR, n=6; IUGR + MOTS-c, n=5. * P<0.05 vs. normal, ** P<0.01 vs. normal, *** P<0.001 vs. normal; # P<0.05 vs. IUGR; ## P<0.01 vs. IUGR; ### P<0.001 vs. IUGR. IUGR, intrauterine growth restriction; SOD, superoxide dismutase; MDA, malondialdehyde; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase 1; NQO-1, NAD(P)H quinone dehydrogenase 1.

Article Snippet: For Nrf2 immunofluorescence staining in HUVECs, cells were fixed with 100% methanol for 15 min at −20°C, followed by permeabilization with 0.1% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) (cat. no. HY-D0842; MedChemExpress) for 60 min at room temperature.

Techniques: Activity Assay, Expressing, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: MOTS-c protects against placental injury via Nrf2 activation in hypoxia-induced intrauterine growth restriction mice

doi: 10.3892/ijmm.2025.5697

Figure Lengend Snippet: MOTS-c exposure attenuates oxidative stress in HUVECs. (A) Relative intensity of DCFH-DA staining. (B) Cellular SOD activity. (C) Cellular MDA content. (D) Representative images of MitoSOX staining. Scale bar, 10 μ m. (E) Representative images of JC-1 staining. Scale bar, 50 μ m. (F) Representative immunofluorescence images of Nrf2 in HUVECs. Scale bar, 5 μ m. (G) Relative nuclear to cytoplasm fluorescence ratio. Results are representative of three independent experiments. Data are expressed as the mean ± SD, n=4. *** P<0.001, # P<0.05 vs. PBS under hypoxic conditions, ## P<0.01 vs. PBS under hypoxic conditions; ### P<0.001 vs. PBS under hypoxic conditions. SOD, superoxide dismutase; MDA, malondialdehyde; DCFH-DA,2'7'-dichlorodihydro fluorescein diacetate ; Nrf2, nuclear factor erythroid 2-related factor 2.

Article Snippet: For Nrf2 immunofluorescence staining in HUVECs, cells were fixed with 100% methanol for 15 min at −20°C, followed by permeabilization with 0.1% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) (cat. no. HY-D0842; MedChemExpress) for 60 min at room temperature.

Techniques: Staining, Activity Assay, Immunofluorescence, Fluorescence

Journal: International Journal of Molecular Medicine

Article Title: MOTS-c protects against placental injury via Nrf2 activation in hypoxia-induced intrauterine growth restriction mice

doi: 10.3892/ijmm.2025.5697

Figure Lengend Snippet: Nrf2 inhibitor ML385 offset the protective effect of MOTS-c against hypoxia-induced dysregulated angiogenesis and oxidative stress in HUVECs. (A) Representative images and (B) quantitative analysis of the in vitro tube formation. Scale bar, 10 μ m. (C) Representative images of DCFH-DA staining and (D) quantification of cellular ROS. Scale bar, 50 μ m. Results are representative of three independent experiments. Data are expressed as the mean ± SD. * P<0.05, ** P<0.01. NS, not significant; DCFH-DA,2',7'-dichlorodihydro fluorescein diacetate; Nrf2, nuclear factor erythroid 2-related factor 2.

Article Snippet: For Nrf2 immunofluorescence staining in HUVECs, cells were fixed with 100% methanol for 15 min at −20°C, followed by permeabilization with 0.1% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) (cat. no. HY-D0842; MedChemExpress) for 60 min at room temperature.

Techniques: In Vitro, Staining

Journal: International Journal of Molecular Medicine

Article Title: MOTS-c protects against placental injury via Nrf2 activation in hypoxia-induced intrauterine growth restriction mice

doi: 10.3892/ijmm.2025.5697

Figure Lengend Snippet: Nrf2 overexpression does not enhance the beneficial effects of MOTS-c on hypoxia-stimulated HUVECs. (A) Relative Nrf2 mRNA expression levels. (B) Cell viability. (C). Representative images of DCFH-DA staining and (D) quantification of cellular ROS. Scale bar, 10 μ m. Data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, # P<0.05, ## P<0.01. NS, not significant; Nrf2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; OE, overexpression; DCFH-DA, 2',7'-dichlorodihydro fluorescein diacetate.

Article Snippet: For Nrf2 immunofluorescence staining in HUVECs, cells were fixed with 100% methanol for 15 min at −20°C, followed by permeabilization with 0.1% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) (cat. no. HY-D0842; MedChemExpress) for 60 min at room temperature.

Techniques: Over Expression, Expressing, Staining

Journal: International Journal of Molecular Medicine

Article Title: MOTS-c protects against placental injury via Nrf2 activation in hypoxia-induced intrauterine growth restriction mice

doi: 10.3892/ijmm.2025.5697

Figure Lengend Snippet: MOTS-c protects against hypoxia-induced placental insufficiency in an Nrf2-dependent manner. (A) Morphology of fetal mice on GD17.5 in WT and Nrf2 KO mice. (B) Placental efficiency, which represents the ratio of fetal to placenta weight. (C) Representative images of H&E staining of placental tissues. Scale bar, 100 μ m. (D) Quantification of the placental blood sinus area. (E) Western blotting analysis of CD31, VEGFA and VEGFR2 protein expression levels in placenta. (F) Relative mRNA expression levels of Pgf , Igf2 , Glut1 , Fatp4 and Snat2 in placenta. Data are expressed as mean ± SD. n=4-6. * P<0.05 vs. normal, ** P<0.01 vs. normal, *** P<0.001 vs. normal, # P<0.05 vs. IUGR, ### P<0.001 vs. IUGR.. NS, not significant; IUGR, intrauterine growth restriction; GD, gestational day; Pgf, placental growth factor; Nrf2, nuclear factor erythroid 2-related factor 2; Igf2, insulin-like growth factor 2; Glut1, glucose transporter type 1; Fatp4, fatty acid transporter 4; Snat2, sodium-dependent neutral amino acid transporter-2; VEGFA, vascular endothelial growth factor A; VEGFR2, VEGF receptor 2.

Article Snippet: For Nrf2 immunofluorescence staining in HUVECs, cells were fixed with 100% methanol for 15 min at −20°C, followed by permeabilization with 0.1% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) (cat. no. HY-D0842; MedChemExpress) for 60 min at room temperature.

Techniques: Staining, Western Blot, Expressing