Journal: Annals of Medicine and Surgery

Article Title: Targeting aerobic glycolysis by quercetin inhibits acute monocytic leukemia SHI-1 cells

doi: 10.1097/MS9.0000000000004180

Figure Lengend Snippet: Influence of Que on the glycolysis and energy metabolism-related protein expression level by western blotting and RT-qPCR in SHI-1 cells (mean ± SD, n = 3) . (A) Que down-regulated the expression of c-Myc, GLUT1, HK2, PKM2, LDHA, and PDK2 proteins in SHI-1 cells. (B) The expression of AMPK, mTOR, and p-mTOR protein in SHI-1 cells was significantly reduced after the treatment of Que. (C) Que decreased mRNA expression of c-Myc, GLUT1, HK2, PKM2, LDHA, and PDK2 in SHI-1 cells (mean ± SD, n=3). * P < 0.5, ** P < 0.01 vs untreated control group.

Article Snippet: The membranes were incubated with primary antibodies, including c-Myc (1:1000), GLUT1 (1:2000), HK2 (1:1000), PKM2 (1:2000), LDHA (Hangzhou HuaAn Biotechnology Co., Ltd; 1:800 M1007-7), pyruvate dehydrogenase kinase 2 (PDK2) (Hangzhou Jingjie Biotechnology Co., LTD; 1:1000; PTM-6321), AMPK (Hangzhou HuaAn Biotechnology Co.,Ltd; 1:1000; HA600078), mTOR (Hangzhou Jingjie Biotechnology Co., LTD; 1:1000; PTM-6594), p-mTOR (Hangzhou HuaAn Biotechnology Co.,Ltd; 1:2000; HA600094), and β-actin (Proteintech Group, Inc; 1:10 000; 66009-1-lg) overnight at 4°C.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: CytoJournal

Article Title: Nobiletin alleviates brain injury in uremic mice and inhibits indoxyl sulfate-induced neurotoxicity in HT22 cells through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

doi: 10.25259/Cytojournal_233_2024

Figure Lengend Snippet: NOB activates the PI3K/Akt signaling pathway. (a-d) WB analysis and quantification of p-PI3K, p-PDK1, and p-Akt protein expression in mouse kidney tissue. n = 3; ✶ ✶ ✶ P < 0.001, ✶ ✶ P < 0.01 versus Control, ### P < 0.001, ## P < 0.01, # P < 0.05 versus DDP. The bar represents mean ± S.D. NOB: Nobiletin, PI3K: Phosphatidylinositol 3-kinase, PDK1: Pyruvate dehydrogenase kinase 1, Akt: Protein kinase B, DDP: Cisplatin, WB: Western blot.

Article Snippet: The membrane was then blocked using 5% nonfat milk at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies targeting α-tubulin (2144), Bcl2 (15071), poly ADP-ribose polymerase (PARP) (9532), PI3K (4292), pyruvate dehydrogenase kinase 1 (PDK1) (3062), Akt (4691), p-PI3K (17366), p-PDK1 (3438), p-Akt (4060), and Bax (5023) (1:1000 each, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Control, Western Blot

Journal: CytoJournal

Article Title: Nobiletin alleviates brain injury in uremic mice and inhibits indoxyl sulfate-induced neurotoxicity in HT22 cells through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

doi: 10.25259/Cytojournal_233_2024

Figure Lengend Snippet: NOB inhibits apoptosis through the PI3K/Akt signaling pathway. (a-h) WB detection and quantitative analysis of the protein expression of p-PI3K, p-PDK1, and p-Akt in different groups of HT22 cells. (i-l) WB detection and quantitative analysis of the protein expression of Bcl2, Bax, and PARP in different groups of HT22 cells. (m and n) DCFH-DA probe measurement of intracellular ROS levels in different groups. ×100 Scale bar = 100 μm (o) CCK-8 assay assessing the effect of various LY concentrations on HT22 cell viability. n = 3; ✶ ✶ ✶ P < 0.001, ✶ ✶ P < 0.01, ✶ P < 0.05 versus DDP; ## P < 0.01, # P < 0.05 versus DDP + NOB. The bar represents mean ± S.D. NOB: Nobiletin, DDP: Cisplatin, PI3K: Phosphatidylinositol 3-kinase, PDK1: Pyruvate dehydrogenase kinase 1, Akt: Protein kinase B, Bcl2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, PARP: Poly ADP-ribose polymerase, WB: Western blot, DCFH-DA: Dichlorodihydrofluorescein diacetate, ROS: Reactive oxygen species, LY: LY294002, CCK-8: Cell counting kit-8.

Article Snippet: The membrane was then blocked using 5% nonfat milk at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies targeting α-tubulin (2144), Bcl2 (15071), poly ADP-ribose polymerase (PARP) (9532), PI3K (4292), pyruvate dehydrogenase kinase 1 (PDK1) (3062), Akt (4691), p-PI3K (17366), p-PDK1 (3438), p-Akt (4060), and Bax (5023) (1:1000 each, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, CCK-8 Assay, Western Blot, Cell Counting

Journal: CytoJournal

Article Title: Nobiletin alleviates brain injury in uremic mice and inhibits indoxyl sulfate-induced neurotoxicity in HT22 cells through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

doi: 10.25259/Cytojournal_233_2024

Figure Lengend Snippet: NOB alleviates DDP-induced brain damage in uremic mice. (a) Body weight of mice. (b) Kidney coefficient of mice. (c-f) Serum levels of BUN, SCr, Kim-1, and Nagl in mice. (g) Representative images of hematoxylin & eosin staining morphological analysis of kidney tissue, showing renal injury features including cellular debris, tubular necrosis, and inflammatory cells ×100. Scale bar = 100 μm (h-k) WB analysis and quantification of Bcl-2, Bax, and PARP protein expression in mouse brain tissue. n = 3; ✶ ✶ ✶ P < 0.001, ✶ ✶ P < 0.01 versus Control, ### P < 0.001, ## P < 0.01, # P < 0.05 versus DDP. The bar represents mean ± S.D. NOB: Nobiletin, BUN: Blood urea nitrogen, SCr: Serum creatinine, Bcl2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, PARP: Poly ADP-ribose polymerase, DDP: Cisplatin, WB: Western blot, S.D.: Standard deviation.

Article Snippet: The membrane was then blocked using 5% nonfat milk at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies targeting α-tubulin (2144), Bcl2 (15071), poly ADP-ribose polymerase (PARP) (9532), PI3K (4292), pyruvate dehydrogenase kinase 1 (PDK1) (3062), Akt (4691), p-PI3K (17366), p-PDK1 (3438), p-Akt (4060), and Bax (5023) (1:1000 each, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Expressing, Control, Western Blot, Standard Deviation

Journal: CytoJournal

Article Title: Nobiletin alleviates brain injury in uremic mice and inhibits indoxyl sulfate-induced neurotoxicity in HT22 cells through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

doi: 10.25259/Cytojournal_233_2024

Figure Lengend Snippet: NOB exhibits anti-apoptotic effects on DDP-induced HT22 cells. (a and b) CCK-8 assay evaluating the impact of DDP and NOB on HT22 cell viability. ✶ ✶ ✶ P < 0.0001, ✶ ✶ P < 0.001, ✶ P < 0.01 versus 0 (c) CCK-8 assay assessing the effect of various concentrations of NOB on DDP-induced HT22 cell viability. (d-g) WB analysis and quantification of Bcl-2, Bax, and PARP protein expression in HT22 cells across different groups. (h-i) DCFH-DA probe measurement of intracellular ROS levels in different groups. ×100 Scale bar = 100 μm. n = 3; ✶ ✶ ✶ P < 0.001, ✶ ✶ P < 0.01, ✶ P < 0.05 versus DDP; ### P < 0.001 versus Control. The bar represents mean ± S.D. NOB: Nobiletin, DDP: Cisplatin, CCK-8: Cell counting kit-8, Bcl2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, PARP: Poly ADP-ribose polymerase, WB: Western blot, DCFH-DA: Dichlorodihydrofluorescein diacetate, ROS: Reactive oxygen species.

Article Snippet: The membrane was then blocked using 5% nonfat milk at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies targeting α-tubulin (2144), Bcl2 (15071), poly ADP-ribose polymerase (PARP) (9532), PI3K (4292), pyruvate dehydrogenase kinase 1 (PDK1) (3062), Akt (4691), p-PI3K (17366), p-PDK1 (3438), p-Akt (4060), and Bax (5023) (1:1000 each, Cell Signaling Technology, Danvers, MA, USA).

Techniques: CCK-8 Assay, Expressing, Control, Cell Counting, Western Blot

Journal: CytoJournal

Article Title: Nobiletin alleviates brain injury in uremic mice and inhibits indoxyl sulfate-induced neurotoxicity in HT22 cells through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway

doi: 10.25259/Cytojournal_233_2024

Figure Lengend Snippet: NOB inhibits apoptosis through the PI3K/Akt signaling pathway. (a-h) WB detection and quantitative analysis of the protein expression of p-PI3K, p-PDK1, and p-Akt in different groups of HT22 cells. (i-l) WB detection and quantitative analysis of the protein expression of Bcl2, Bax, and PARP in different groups of HT22 cells. (m and n) DCFH-DA probe measurement of intracellular ROS levels in different groups. ×100 Scale bar = 100 μm (o) CCK-8 assay assessing the effect of various LY concentrations on HT22 cell viability. n = 3; ✶ ✶ ✶ P < 0.001, ✶ ✶ P < 0.01, ✶ P < 0.05 versus DDP; ## P < 0.01, # P < 0.05 versus DDP + NOB. The bar represents mean ± S.D. NOB: Nobiletin, DDP: Cisplatin, PI3K: Phosphatidylinositol 3-kinase, PDK1: Pyruvate dehydrogenase kinase 1, Akt: Protein kinase B, Bcl2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, PARP: Poly ADP-ribose polymerase, WB: Western blot, DCFH-DA: Dichlorodihydrofluorescein diacetate, ROS: Reactive oxygen species, LY: LY294002, CCK-8: Cell counting kit-8.

Article Snippet: The membrane was then blocked using 5% nonfat milk at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies targeting α-tubulin (2144), Bcl2 (15071), poly ADP-ribose polymerase (PARP) (9532), PI3K (4292), pyruvate dehydrogenase kinase 1 (PDK1) (3062), Akt (4691), p-PI3K (17366), p-PDK1 (3438), p-Akt (4060), and Bax (5023) (1:1000 each, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, CCK-8 Assay, Western Blot, Cell Counting

Journal: bioRxiv

Article Title: Regulation of fatty acid oxidation involved in high altitude hypoxia induced cardiomyocyte ferroptosis via the SIRT1-PPARα-GPX4 signaling pathway

doi: 10.1101/2025.01.20.633884

Figure Lengend Snippet: (A) Celluar study design. (B) After PPARα activation, western blotting was performed on H9c2 cells to determine PDK4, CPT1, GPX4, and SLC7A11 levels, and grayscale values were analyzed, with β-actin serving as a protein loading control. Intracellular levels of FFA (C) and A-CoA (D) after treatment of H9c2 cells with WY14643. (E) Intracellular ATP levels in H9c2 cells after PPARα activation. (F) Intracellular GSH/GSSG levels in H9c2 cells after PPARα activation. (G) After PPARα activation, representative images of TMRE staining and quantitative analysis of fluorescence (I) in H9c2 cells. Scale bar = 100 μm. (H) After PPARα activation, representative images of DCFH-DA staining and quantitative analysis of fluorescence (J) in H9c2 cells. Scale bar = 100 μm. (K) CCK-8 assays were employed to assess the viability of H9c2 cells after PPARα activation. Statistical significance was indicated as *P < 0.05, **P < 0.01 vs. Control, #P < 0.05, ##P < 0.01 vs. Hypoxia.

Article Snippet: Antibodies against PPARα, pyruvate dehydrogenase kinase 4 (PDK4), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α), SIRT1, GPX4, SLC7A11, and β-actin were obtained from Proteintech (Wuhan, China) and Abcam (Cambridge, UK).

Techniques: Activation Assay, Western Blot, Control, Staining, Fluorescence, CCK-8 Assay

Journal: bioRxiv

Article Title: Regulation of fatty acid oxidation involved in high altitude hypoxia induced cardiomyocyte ferroptosis via the SIRT1-PPARα-GPX4 signaling pathway

doi: 10.1101/2025.01.20.633884

Figure Lengend Snippet: (A) Celluar study design. (B) After SIRT1 overexpression, western blotting was performed on H9c2 cells to determine PGC1α, PPARα, PDK4, CPT1, GPX4, and SLC7A11 levels, and grayscale values were analyzed, with β-actin serving as a protein loading control. Intracellular levels of FFA (C) and A-CoA (D) after SIRT1 overexpression in H9c2 cells. (E) Intracellular ATP levels in H9c2 cells after SIRT1 overexpression. (F) Intracellular GSH/GSSG levels in H9c2 cells after SIRT1 overexpression. (G) Representative images of TMRE staining and quantitative analysis of fluorescence (I) in H9c2 cells after SIRT1 overexpression. Scale bar = 100 μm. (H) Representative images of DCFH-DA staining and quantitative analysis of fluorescence (J) in H9c2 cells after SIRT1 overexpression. Scale bar = 100 μm. (K) CCK-8 assays were employed to assess the viability of H9c2 cells after SIRT1 overexpression. Statistical significance was indicated as *P < 0.05, **P < 0.01 vs. Control, #P < 0.05, ##P < 0.01 vs. Hypoxia.

Article Snippet: Antibodies against PPARα, pyruvate dehydrogenase kinase 4 (PDK4), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α), SIRT1, GPX4, SLC7A11, and β-actin were obtained from Proteintech (Wuhan, China) and Abcam (Cambridge, UK).

Techniques: Over Expression, Western Blot, Control, Staining, Fluorescence, CCK-8 Assay

Journal: bioRxiv

Article Title: Regulation of fatty acid oxidation involved in high altitude hypoxia induced cardiomyocyte ferroptosis via the SIRT1-PPARα-GPX4 signaling pathway

doi: 10.1101/2025.01.20.633884

Figure Lengend Snippet: (A) Celluar study design. (B) After overexpression of SIRT1, PPARα was inhibited by GW6471 in H9c2 cells. Western blotting was conducted to measure the levels of PGC1α, PPARα, PDK4, CPT1, GPX4, and SLC7A11 in H9c2 cardiomyocytes, and grayscale values were analyzed. β-actin was used as a protein loading control. Intracellular levels of FFA (C), A-CoA (D), ATP (E), and GSH/GSSG (F) in H9c2 cells after SIRT1 overexpression and PPARα inhibition. (G) Representative images of TMRE staining and quantitative analysis of fluorescence (I) in H9c2 cells after SIRT1 overexpression and PPARα inhibition. Scale bar = 100 μm. (H) Representative images of DCFH-DA staining and quantitative analysis of fluorescence (J) in H9c2 cells after SIRT1 overexpression and PPARα inhibition. Scale bar = 100 μm. (K) CCK-8 assays were employed to assess the viability of H9c2 cells after SIRT1 overexpression and PPARα inhibition. Statistical significance was indicated as *P < 0.05, **P < 0.01 vs. Control, #P < 0.05, ##P < 0.01 vs. Hypoxia, &P < 0.05, &&P < 0.01 vs. Hypoxia+SIRT1 OE .

Article Snippet: Antibodies against PPARα, pyruvate dehydrogenase kinase 4 (PDK4), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α), SIRT1, GPX4, SLC7A11, and β-actin were obtained from Proteintech (Wuhan, China) and Abcam (Cambridge, UK).

Techniques: Over Expression, Western Blot, Control, Inhibition, Staining, Fluorescence, CCK-8 Assay

Journal: bioRxiv

Article Title: Regulation of fatty acid oxidation involved in high altitude hypoxia induced cardiomyocyte ferroptosis via the SIRT1-PPARα-GPX4 signaling pathway

doi: 10.1101/2025.01.20.633884

Figure Lengend Snippet: (A) Animal study design. (B) Echocardiography in rats. (C-E) RSV decreased LVPWd and LVPWs levels and increased LVEDV, LVESV, SV, and CO levels of the heart in the HH test. (F) RSV decreased the cardiac coefficient compared with the HH group. (G) Transmission electron microscopy was utilized to visualize the left ventricular myocardium ultrastructure in rats (8,000×). Scale bar = 1 μm. (H, I) A detailed analysis was conducted to determine the number of mitochondria per unit area and the average mitochondrial size. Myocardial FFA (J), A-CoA (K), and ATP (L) levels in rats after treatment with RSV. (M) DHE staining of rat myocardial tissue (40×) after RSV treatment and quantitative analysis of fluorescence (O). (N) Western blotting was performed on rat myocardial tissue extracts to determine SIRT1, PGC1α, PPARα, PDK4, CPT1, GPX4, and SLC7A11 levels, and grayscale values were analyzed (Q), with β-actin serving as a protein loading control. (P) Myocardial GSH/GSSG levels in rats after treatment with RSV. Statistical significance was indicated as *P < 0.05, **P < 0.01 vs. Control, #P < 0.05, ##P < 0.01 vs. HH.

Article Snippet: Antibodies against PPARα, pyruvate dehydrogenase kinase 4 (PDK4), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α), SIRT1, GPX4, SLC7A11, and β-actin were obtained from Proteintech (Wuhan, China) and Abcam (Cambridge, UK).

Techniques: Transmission Assay, Electron Microscopy, Staining, Fluorescence, Western Blot, Control

Journal: bioRxiv

Article Title: Regulation of fatty acid oxidation involved in high altitude hypoxia induced cardiomyocyte ferroptosis via the SIRT1-PPARα-GPX4 signaling pathway

doi: 10.1101/2025.01.20.633884

Figure Lengend Snippet: Under conditions of HH exposure, the downregulation of SIRT1 triggers FAO and ferroptosis through the PPARα-PDK4/CPT1-GPX4/SLC7A11 pathway. This results in mitochondrial dysfunction, ultimately contributing to the development of cardiac impairment, as depicted in the pathway map.

Article Snippet: Antibodies against PPARα, pyruvate dehydrogenase kinase 4 (PDK4), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α), SIRT1, GPX4, SLC7A11, and β-actin were obtained from Proteintech (Wuhan, China) and Abcam (Cambridge, UK).

Techniques: