Journal: Frontiers in Physiology

Article Title: Transcriptomic analysis and experimental verification reveal the involvement of PI3K/AKT signaling pathway in high-altitude cognitive dysfunction

doi: 10.3389/fphys.2026.1781613

Figure Lengend Snippet: Primer sequences for the genes observed in qRT-PCR.

Article Snippet: Primary antibody against β -actin were purchased from Zhongshan Golden Bridge Biological Technology Co., Ltd. (Beijing, China).

Techniques:

Journal: Frontiers in Physiology

Article Title: Transcriptomic analysis and experimental verification reveal the involvement of PI3K/AKT signaling pathway in high-altitude cognitive dysfunction

doi: 10.3389/fphys.2026.1781613

Figure Lengend Snippet: Effects of HH on the BBB integrity in mouse hippocampus. (A) Representative images of EB extravasation in the hippocampus. (B) Quantitative analysis of EB leakage. (C) Representative western blots of ZO-1 and occludin. Quantitative analysis of the ZO-1/β-actin (D) and occludin/β-actin (E) ratios. Data are presented as mean ± SD. Statistical comparisons were performed by Student’s t test. For EB, n = 6 per group; for Western blot analysis, n = 3 per group, ** p < 0.01 vs. Con group.

Article Snippet: Primary antibody against β -actin were purchased from Zhongshan Golden Bridge Biological Technology Co., Ltd. (Beijing, China).

Techniques: Western Blot

Journal: Frontiers in Physiology

Article Title: Transcriptomic analysis and experimental verification reveal the involvement of PI3K/AKT signaling pathway in high-altitude cognitive dysfunction

doi: 10.3389/fphys.2026.1781613

Figure Lengend Snippet: Effects of HH on the expression of genes and proteins related to PI3K/AKT signaling pathway in mouse hippocampus. (A) The relative mRNA expression levels of Kdr , Spp1 , Vwf , and Vegfa screened via qRT-PCR. (B) Representative western blots of p-PI3K, PI3K, p-AKT, and AKT. Quantitative analysis of the p-PI3K/PI3K (C) and p-AKT/AKT (D) ratios. (E) Representative western blots of Nrf2, HO-1, p-NF-κB, and NF-κB. Quantitative analysis of the Nrf2/β-actin (F) , HO-1/β-actin (G) , and p-NF-κB/NF-κB (H) ratios. Data are presented as mean ± SD. Statistical comparisons were performed by Student’s t test. For qRT-PCR, n = 6 per group; for Western blot analysis, n = 3 per group, * p < 0.05, ** p < 0.01 vs. Con group.

Article Snippet: Primary antibody against β -actin were purchased from Zhongshan Golden Bridge Biological Technology Co., Ltd. (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Tramadol promoted hypoxia signaling in a dose-dependent manner by stabilizing HIF-1α, enhancing its nuclear translocation, and upregulating HIF-1α target gene expression in breast cancer cells. A Hypoxia levels were elevated in MDA-MB-231 and MCF-7 cells following short-term tramadol exposure. Cells were incubated with 0, 0.5, or 1 mg/mL tramadol for 4 h, stained with a hypoxia-sensitive fluorescent dye (Hypoxia Red), and analyzed via flow cytometry. Quantification of the hypoxic population (M2 gate) is shown as mean ± SEM from three independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons (** p < 0.01, **** p < 0.0001 vs. untreated control). B Tramadol prolonged the stability of HIF-1α protein. MDA-MB-231 and MCF-7 cells were treated with 1 mg/mL tramadol or vehicle for 4 h prior to cycloheximide (CHX) addition. Protein degradation kinetics of HIF-1α were monitored at various time points (0–40 min) by western blot. β-actin served as the internal loading control. C Immunofluorescence staining revealed tramadol-induced nuclear localization of HIF-1α in breast cancer cells. MDA-MB-231 and MCF-7 cells were exposed to 1 mg/mL tramadol or vehicle control for 4 h, followed by staining with an anti-HIF-1α antibody (red) and nuclear counterstaining with DAPI (blue). Representative fluorescence images were acquired using a fluorescence microscope. Scale bar: 10 μm. D Subcellular fractionation followed by western blot analysis further confirmed the nuclear enrichment of HIF-1α following tramadol treatment (1 mg/mL, 4 h). Nuclear (Nuc) and cytoplasmic (Cyto) protein fractions were isolated from MDA-MB-231 and MCF-7 cells. GAPDH and histone H3 served as markers for cytoplasmic and nuclear compartments, respectively. E Tramadol induced the transcription of HIF-1α and its downstream targets BHLHE40, HMOX1, and VEGFA in a dose-dependent manner. Cells were treated with 0, 0.5, or 1 mg/mL tramadol for 4 h, with or without the HIF-1α inhibitor PX-478 (20 μM). Gene expression was measured by qPCR, normalized to ACTB, and shown as mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. untreated control).

Article Snippet: Primary antibodies against β-actin (catalog no.: sc-47778), GAPDH (catalog no.: sc-47724), p62 (catalog no.: sc-28359), ALG2 (catalog no.: sc-376950), Alix (catalog no.: sc-53540), and α-actinin (ACTN, catalog no.: sc-17829) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and primary antibodies against HIF-1α (catalog no.: #14179), PERK (catalog no.: #3192), p-eIF2α (catalog no.: #9721), ATF4 (catalog no.: #11815), CHOP (catalog no.: #2895), ATF6 (catalog no.: #65880), XBP1 (catalog no.: #40435), IRE1 (catalog no.: #3294), H3 (catalog no.: #9715), LC3B (catalog no.: #2775), PARP (catalog no.: #9546), p-ERK (catalog no.: #4370), ERK (catalog no.: #4695), p-p38 (catalog no.: #9211), p-38 (catalog no.: #9212), p-JNK (catalog no.: #4671), and JNK (catalog no.: #67096) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Translocation Assay, Targeted Gene Expression, Incubation, Staining, Flow Cytometry, Control, Western Blot, Immunofluorescence, Fluorescence, Microscopy, Fractionation, Isolation, Gene Expression

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Effects of tramadol on the ER stress pathways and ROS levels in MDA-MB-231 and MCF-7 cells. A MDA-MB-231 and MCF-7 cells were treated with 0, 0.05, 0.1, 0.5, 1, or 1.5 mg/ml tramadol. β-Actin was used as a loading control. B MDA-MB-231 and MCF-7 cells were treated with 0 or 1 mg/ml tramadol. Western blot analysis was performed on the nuclear and cytosolic fractions, with Histone 3 and GAPDH serving as internal controls for the nuclear and cytosolic proteins, respectively. C MDA-MB-231 and MCF-7 cells were treated with 0 or 1 mg/ml tramadol. qPCR analysis was performed to assess the mRNA expression of downstream genes regulated by ATF4. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control). D MDA-MB-231 and MCF-7 cells were treated with 0, 0.5, 1, or 1.5 mg/ml tramadol. DCFH-DA staining was conducted to assess intracellular ROS levels, whereas MitoSox Red staining was used to evaluate mitochondrial ROS levels, both of which were analyzed via flow cytometry. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons (* p < 0.05, ** p < 0.01 vs. control).

Article Snippet: Primary antibodies against β-actin (catalog no.: sc-47778), GAPDH (catalog no.: sc-47724), p62 (catalog no.: sc-28359), ALG2 (catalog no.: sc-376950), Alix (catalog no.: sc-53540), and α-actinin (ACTN, catalog no.: sc-17829) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and primary antibodies against HIF-1α (catalog no.: #14179), PERK (catalog no.: #3192), p-eIF2α (catalog no.: #9721), ATF4 (catalog no.: #11815), CHOP (catalog no.: #2895), ATF6 (catalog no.: #65880), XBP1 (catalog no.: #40435), IRE1 (catalog no.: #3294), H3 (catalog no.: #9715), LC3B (catalog no.: #2775), PARP (catalog no.: #9546), p-ERK (catalog no.: #4370), ERK (catalog no.: #4695), p-p38 (catalog no.: #9211), p-38 (catalog no.: #9212), p-JNK (catalog no.: #4671), and JNK (catalog no.: #67096) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control, Western Blot, Expressing, Staining, Flow Cytometry

Journal: Redox Report : Communications in Free Radical Research

Article Title: Tramadol induced hypoxia signaling and paraptosis-like cell death in breast cancer cells via HIF-1α and ATF4 dependent pathways

doi: 10.1080/13510002.2025.2588866

Figure Lengend Snippet: Tramadol influenced cell death pathways and paraptosis-associated signaling in breast cancer cells. MDA-MB-231 and MCF-7 cells were treated with 0, 0.05, 0.1, 0.5, 1, or 1.5 mg/ml tramadol. β-Actin was used as a loading control.

Article Snippet: Primary antibodies against β-actin (catalog no.: sc-47778), GAPDH (catalog no.: sc-47724), p62 (catalog no.: sc-28359), ALG2 (catalog no.: sc-376950), Alix (catalog no.: sc-53540), and α-actinin (ACTN, catalog no.: sc-17829) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and primary antibodies against HIF-1α (catalog no.: #14179), PERK (catalog no.: #3192), p-eIF2α (catalog no.: #9721), ATF4 (catalog no.: #11815), CHOP (catalog no.: #2895), ATF6 (catalog no.: #65880), XBP1 (catalog no.: #40435), IRE1 (catalog no.: #3294), H3 (catalog no.: #9715), LC3B (catalog no.: #2775), PARP (catalog no.: #9546), p-ERK (catalog no.: #4370), ERK (catalog no.: #4695), p-p38 (catalog no.: #9211), p-38 (catalog no.: #9212), p-JNK (catalog no.: #4671), and JNK (catalog no.: #67096) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control