Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 4 CPT1A promotes microtubule formation and angiogenesis in BC and HUVECs. (A) HUVECs were cultured with the supernatants of BC cells, and the microtubule formations were detected by matrigel tube formation assay. (B) The vascular mimicry ability of the BC cells was detected by the vasculogenic mimicry assay. (C) The effect of CPT1A on ex vivo angiogenesis was assessed by CAM assay. The white arrows indicate the morphological changes in CAM. (D) Protein expression of angiogenesis markers (MMP2, MMP9, VEGF) was detected by Western blot in CPT1A differentially expressed BC cells. (E-G) HUVECs were cultured with medium and the supernatants of BC cells respectively. The migration ability of HUVEC cells was assessed by wound healing (E) and transwell assays (F), and the proliferation ability of HUVECs was detected by CCK8 assay (G). *P < 0.05, **P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Cell Culture, Tube Formation Assay, VM Assay, Ex Vivo, Chick Chorioallantoic Membrane Assay, Expressing, Western Blot, Migration, CCK-8 Assay

Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 8 High levels of CPT1A/VEGF expression are correlated with poor prognosis of patients with BC. (A) Chipbase database was used to search the cor relation of CPT1A with VEGF, MMP2 and MMP9 in blood vessels. (B) Representative IHC staining images showing CPT1A and VEGF expression in normal tissues, DCIS, and BC (n = 31). Scale bar: 200 μm (Up), 50 μm (Down). (C) Correlation between CPT1A expression and VEGF expression based on the IHC scores. (D) JMP software was used for statistics and cartogram. (E) Expression level of VEGF in BC and adjacent tissues. (F) The expression level of VEGF in BC tissues was found by Ualcan database. (G) Distribution of existing and absent LN metastasis in the VEGF-low and VEGF-high groups. Chi-square test was used to test VEGF and clinicopathological parameters of BC. (H) Ualcan database was used to search the relationship between VEGF and LN metasta sis in BC tissues. (I) Overall survival of BC patients with low or high VEGF expression in the Kaplan-Meier database. *P < 0.05 and **P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Expressing, Immunohistochemistry, Software

Journal: Experimental and Therapeutic Medicine

Article Title: Protective effects of gastrodin pretreatment on mouse hepatic ischemia-reperfusion occurring through antioxidant and anti-apoptotic mechanisms

doi: 10.3892/etm.2021.9902

Figure Lengend Snippet: Gas pretreatment upregulates Nrf2 and HO-1 expression levels in the mouse liver. (A) Western blotting was used to determine Nrf2 and HO-1 protein expression levels at 2 h following hepatic IR modeling (n=3 mice). (B) Reverse transcription-quantitative PCR was used to analyze the expression levels of Nrf2, HO-1 and SOD at 2 h post hepatic IR modeling (n=6). **P<0.01, ***P<0.001 vs. the Sham + vehicle group; #P<0.05, ##P<0.01 vs. the IR + vehicle group. Gas, gastrodin; IR, ischemia-reperfusion; Nrf2, nuclear erythroid 2-related factor 2; HO-1, heme oxygenase-1; SOD, superoxide dismutase.

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated with the following primary antibodies at 4˚C overnight: Anti-Nrf2 (cat. no. 16396-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-HO-1 (cat. no. 10701-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-Bax (cat. no. ab32503; 1:1,000; Abcam), anti-Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), anti-CASP-3 (cat. no. ab32351; 1:1,000; Abcam) and anti-β-actin (cat. no. MA1-140; 1:5,000; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Protective effects of gastrodin pretreatment on mouse hepatic ischemia-reperfusion occurring through antioxidant and anti-apoptotic mechanisms

doi: 10.3892/etm.2021.9902

Figure Lengend Snippet: Gas pretreatment upregulates Nrf2 and HO-1 expression levels in the mice liver. (A) Western blotting was used to determine Nrf2 and HO-1 protein expression levels at 24 h post hepatic IR modeling (n=3 mice). (B) Reverse transcription-quantitative PCR was used to analyze the expression levels of Nrf2 and HO-1 at 24 h post hepatic IR modeling (n=6 mice). *P<0.05, **P<0.01, ***P<0.001 vs. the Sham + vehicle group; #P<0.05, ##P<0.01, ###P<0.001 vs. the IR + vehicle group. Gas, gastrodin; HO-1, heme oxygenase-1; IR, ischemia-reperfusion; Nrf2, nuclear erythroid 2-related factor 2; SOD, superoxide dismutase.

Article Snippet: The membranes were blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated with the following primary antibodies at 4˚C overnight: Anti-Nrf2 (cat. no. 16396-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-HO-1 (cat. no. 10701-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-Bax (cat. no. ab32503; 1:1,000; Abcam), anti-Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), anti-CASP-3 (cat. no. ab32351; 1:1,000; Abcam) and anti-β-actin (cat. no. MA1-140; 1:5,000; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: CPT1A-driven FAO pathway promotes BC progression. ( A ) The production levels of FAO, ATP and NADPH were detected by kit assays in four groups of BC cells. Data were presented as the mean ± SD. ( B ) Protein expression of FAO markers (LCAD, MCAD) was detected by Western blot in CPT1A differentially expressed BC cells. ( C-D ) Colony formation ( C ) and wound healing ( D ) assays were used to test the proliferation and migration abilities of CPT1A overexpression cell after adding ETX. ( E-F ) The vascular mimicry ability of the CPT1A overexpression cell after adding ETX was detected by the microtubule formations assay ( E ). The effect of CPT1A on ex vivo angiogenesis was assessed by CAM assay. The white arrows indicate the morphological changes in CAM ( F ). ( G ) Metabolic kits were detected the FAO, ATP, and NADPH levels in CPT1A overexpression cell after adding ETX. ( H ) Protein expression of FAO, angiogenesis and EMT markers was detected by Western blot in CPT1A overexpression cell after adding ETX. * P < 0.05, ** P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210-1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1-AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Expressing, Western Blot, Migration, Over Expression, Ex Vivo, Chick Chorioallantoic Membrane Assay

Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 2 CPT1A enhances BC proliferation both in vivo and in vitro. (A) CPT1A knockdown and overexpression BC cell lines were constructed, and the trans fection efficiency was verified by Western blot. (B-D) BC cells proliferation ability was tested by CCK8 (B), colony formation (C) and EdU assays (D). Scale bar: 50 μm. (E) The ratio of G2/M%+S% in different groups was quantified by flow cytometry. Quantification of the flow cytometry result (n = 3). (F) Repre sentative fluorescence imaging and morphology of the nude mice xenograft. The down panel represents the analysis of tumor volume and weight. Size of each grid: 1 mm × 1 mm. (n = 5). (G) IHC staining showed Ki67 expression in tumor specimens from xenografts. Scale bar: 50 μm. *P < 0.05 and **P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: In Vivo, In Vitro, Knockdown, Over Expression, Construct, Western Blot, Flow Cytometry, Fluorescence, Imaging, Immunohistochemistry, Expressing

Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 4 CPT1A promotes microtubule formation and angiogenesis in BC and HUVECs. (A) HUVECs were cultured with the supernatants of BC cells, and the microtubule formations were detected by matrigel tube formation assay. (B) The vascular mimicry ability of the BC cells was detected by the vasculogenic mimicry assay. (C) The effect of CPT1A on ex vivo angiogenesis was assessed by CAM assay. The white arrows indicate the morphological changes in CAM. (D) Protein expression of angiogenesis markers (MMP2, MMP9, VEGF) was detected by Western blot in CPT1A differentially expressed BC cells. (E-G) HUVECs were cultured with medium and the supernatants of BC cells respectively. The migration ability of HUVEC cells was assessed by wound healing (E) and transwell assays (F), and the proliferation ability of HUVECs was detected by CCK8 assay (G). *P < 0.05, **P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Cell Culture, Tube Formation Assay, VM Assay, Ex Vivo, Chick Chorioallantoic Membrane Assay, Expressing, Western Blot, Migration, CCK-8 Assay

Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 5 CPT1A-driven FAO pathway promotes BC progression. (A) The production levels of FAO, ATP and NADPH were detected by kit assays in four groups of BC cells. Data were presented as the mean ± SD. (B) Protein expression of FAO markers (LCAD, MCAD) was detected by Western blot in CPT1A differ entially expressed BC cells. (C-D) Colony formation (C) and wound healing (D) assays were used to test the proliferation and migration abilities of CPT1A overexpression cell after adding ETX. (E-F) The vascular mimicry ability of the CPT1A overexpression cell after adding ETX was detected by the microtu bule formations assay (E). The effect of CPT1A on ex vivo angiogenesis was assessed by CAM assay. The white arrows indicate the morphological changes in CAM (F). (G) Metabolic kits were detected the FAO, ATP, and NADPH levels in CPT1A overexpression cell after adding ETX. (H) Protein expression of FAO, angiogenesis and EMT markers was detected by Western blot in CPT1A overexpression cell after adding ETX. *P < 0.05, **P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Expressing, Western Blot, Migration, Over Expression, Ex Vivo, Chick Chorioallantoic Membrane Assay

Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 6 The CPT1A/HIF-1α positive feedback loop promotes the proliferation and metastasis of BC. (A) KEGG pathway analyzed the signaling pathways of extracellular differential expression in CPT1A knockdown MDA-MB-231 cells. (B) The interaction between endogenous CPT1A and HIF-1α protein was analyzed by Co-IP assay. (C) MDA-MB-231 cells of vector and CPT1A overexpression groups were treated with 10 µM CHX for 0, 20, 40, and 60 min, then cells were collected and lysed respectively. The half-life of HIF-1α protein was detected by Western blotting and quantified by Prism Software. MDA- MB-231 cells of Control and sh-CPT1A groups were treated with 10 µM MG132 for 0, 2, 4, and 6 h, and the expression level of HIF-1α by Western blot and quantitatively analyzed. (D) MDA-MB-231 cells of control and sh-CPT1A groups were pretreated with 10 µM MG132 for 8 h. The ubiquitination levels of HIF-1α were measured by IP experiments. Meanwhile, the expression of CPT1A in the input samples was detected. (E) The binding site of HIF-1α and CPT1A promoter was predicted by the JASPAR website (UP). WT and MT1, MT2 luciferase reporter gene vectors in CPT1A 3’-UTR were co-transfected with HIF-1α control or overexpressed plasmid for 48 h in BC cells, and the luciferase activities were measured by dual-luciferase assay. Relative luciferase activ ity was computed by the ratio of Firefly and Renilla luciferase values. (F) The expression level of HIF-1α was detected by Western blot for confirming the transfected effects of HIF-1α overexpression in BC cells. (G-H) The proliferation ability of BC cells in the Con group, sh-CPT1A group, and sh-CPT1A + HIF-1α overexpression group was evaluated using the CCK8 assay (G) and the transwell migration assay (H). (I) The level of FAO in the supernatant of the three groups of cells was detected by FAO kit assay. (J) The levels of EMT and angiogenesis markers were detected in the three groups using Western blot. *P < 0.05 and **P < 0.01, N.S was represented for no statistical significance

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Protein-Protein interactions, Quantitative Proteomics, Knockdown, Co-Immunoprecipitation Assay, Plasmid Preparation, Over Expression, Western Blot, Software, Control, Expressing, Ubiquitin Proteomics, Binding Assay, Luciferase, Transfection, CCK-8 Assay, Transwell Migration Assay

Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 8 High levels of CPT1A/VEGF expression are correlated with poor prognosis of patients with BC. (A) Chipbase database was used to search the cor relation of CPT1A with VEGF, MMP2 and MMP9 in blood vessels. (B) Representative IHC staining images showing CPT1A and VEGF expression in normal tissues, DCIS, and BC (n = 31). Scale bar: 200 μm (Up), 50 μm (Down). (C) Correlation between CPT1A expression and VEGF expression based on the IHC scores. (D) JMP software was used for statistics and cartogram. (E) Expression level of VEGF in BC and adjacent tissues. (F) The expression level of VEGF in BC tissues was found by Ualcan database. (G) Distribution of existing and absent LN metastasis in the VEGF-low and VEGF-high groups. Chi-square test was used to test VEGF and clinicopathological parameters of BC. (H) Ualcan database was used to search the relationship between VEGF and LN metasta sis in BC tissues. (I) Overall survival of BC patients with low or high VEGF expression in the Kaplan-Meier database. *P < 0.05 and **P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Expressing, Immunohistochemistry, Software

Journal: Breast cancer research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer.

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: Fig. 5 CPT1A-driven FAO pathway promotes BC progression. (A) The production levels of FAO, ATP and NADPH were detected by kit assays in four groups of BC cells. Data were presented as the mean ± SD. (B) Protein expression of FAO markers (LCAD, MCAD) was detected by Western blot in CPT1A differ entially expressed BC cells. (C-D) Colony formation (C) and wound healing (D) assays were used to test the proliferation and migration abilities of CPT1A overexpression cell after adding ETX. (E-F) The vascular mimicry ability of the CPT1A overexpression cell after adding ETX was detected by the microtu bule formations assay (E). The effect of CPT1A on ex vivo angiogenesis was assessed by CAM assay. The white arrows indicate the morphological changes in CAM (F). (G) Metabolic kits were detected the FAO, ATP, and NADPH levels in CPT1A overexpression cell after adding ETX. (H) Protein expression of FAO, angiogenesis and EMT markers was detected by Western blot in CPT1A overexpression cell after adding ETX. *P < 0.05, **P < 0.01

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210- 1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1- AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Expressing, Western Blot, Migration, Over Expression, Ex Vivo, Chick Chorioallantoic Membrane Assay

Journal: Breast Cancer Research : BCR

Article Title: CPT1A/HIF-1α positive feedback loop induced fatty acid oxidation metabolic pathway contributes to the L-ascorbic acid-driven angiogenesis in breast cancer

doi: 10.1186/s13058-025-02039-0

Figure Lengend Snippet: LAA uptake reduced by CPT1A/HIF-1α only promotes the angiogenesis via Nrf2/NQO1 pathway. ( A ) LC-MS/MS analysis of differential metabolites in the extracellular supernatant of MDA-MB-231 cells following CPT1A knockdown. ( B-C ) CCK8 ( B ) and colony formation ( C ) assays were used to evaluate the proliferation of CPT1A knockdown cells after the addition of LAA (5 µM) in the sh-CPT1A group. ( D ) Matrigel tube formation assay detected the HUVECs tube formation ability after adding LAA (5 µM) in sh-CPT1A group. ( E ) The vascular mimicry ability of the BC cells after adding LAA (5 µM) in sh-CPT1A group was detected by the vasculogenic mimicry assay. ( F ) The effect of CPT1A knockdown cells after adding LAA (5 µM) on ex vivo angiogenesis was assessed by CAM assay. The white arrows indicate the morphological changes in CAM. ( G-H ) HUVECs were cultured with medium and the supernatants of BC cells and LAA (5 µM) respectively. The migration ability of HUVECs was assessed by wound healing ( G ) and transwell assays ( H ). ( I ) Western blot was measured the expression levels of EMT and angiogenesis markers. ( J ) The level of GSH/GSSG in the supernatant of the three groups of cells was detected by GSH/GSSG kit assay. ( K ) Representative IF staining images showing ROS expression in sh-CPT1A after adding LAA (5 µM). Scale bar: 15 μm. ( L ) Protein expression of HIF-1α, Nrf2 and NQO1 were detected by Western blot in sh-CPT1A after adding LAA (5 µM). * P < 0.05 and ** P < 0.01. ns was represented for no statistical significance

Article Snippet: Antibodies against Ki67 (No. 27309, 1:200), ZEB2 (14026-1-AP, 1:1000), Snail (13099-1-AP, 1:1000), E-cadherin (20874-1-AP, 1:20000), MMP9 (10375-2-AP, 1:1000), MMP2 (10373-2-AP, 1:20000), MCAD (55210-1-AP, 1:2000), LCAD (17526-1-AP, 1:2000), TWIST (25465-1-AP, 1:1000), Vimentin (110366-1-AP, 1:5000), CPT1A (15184-1-AP, 1:5000), HIF-1α (20960-1-AP, 1:3000), β-Tubulin (10068-1-AP, 1:1000), Nrf2 (16396-1-AP, 1:2000), and NQO1 (11451-1-AP, 1:2000) were purchased from Proteintech (Humanzyme), China.

Techniques: Liquid Chromatography with Mass Spectroscopy, Knockdown, Tube Formation Assay, VM Assay, Ex Vivo, Chick Chorioallantoic Membrane Assay, Cell Culture, Migration, Western Blot, Expressing, Staining

Journal: Hearing research

Article Title: Sustained Wnt signaling in the mouse inner ear after morphogenesis: In hair cells, supporting cells, and spiral ganglion neurons.

doi: 10.1016/j.heares.2025.109282

Figure Lengend Snippet: Fig. 1. Comparison of two Wnt reporter mice in E11.5: Coronal section of the otocyst. A: X-gal staining. Staining with the β-galactosidase substrate X-gal (5‑bromo-4‑chloro-3-indolyl-β-d-galactopyranoside) was observed in the dorsomedial part of the otocyst. The position of the embryo was noted. B: Fluorescence immunostaining of WntVIS mice. Green; WntVIS-GFP, Red; Sox2 (sensory epithelial progenitor cells), White; TuJ1 (βIII-tubulin, neuron), and DAPI (nuclear staining). B’: The grayscale image of the Sox2 channel. B”: The grayscale image of the GFP channel. Wnt signaling activity at the cellular level and with gradients can be observed. The yellow arrowheads indicate the immature spiral ganglion neurons, whereas the cyan arrowhead indicates the immature vestibular neurons. The yellow dotted line indicates the cochlear prosensory domain, whereas the cyan dotted line indicates the vestibular prosensory domain.

Article Snippet: The WntVIS signal was enhanced using a chick anti-GFP antibody (1:250, GFP-1010; Aves Labs, Davis, CA, USA).

Techniques: Comparison, Staining, Fluorescence, Immunostaining, Activity Assay

Journal: Hearing research

Article Title: Sustained Wnt signaling in the mouse inner ear after morphogenesis: In hair cells, supporting cells, and spiral ganglion neurons.

doi: 10.1016/j.heares.2025.109282

Figure Lengend Snippet: Fig. 2. Wnt signaling activity in supporting cells of the developing and developed Organ of Corti. Sox2 was co-stained with GFP for marking the prosensory epithelia in the embryonic stage (A–C) and the supporting cells (C, D). A; E15 basal turn, B; E17 apical turn, C; E17 basal turn, D; P60. Green; WntVIS-GFP, Red; Sox2 (prosensory epithelia), White; TuJ1 (βIII-tubulin; neuron), Blue: DAPI (nucleus). The 2nd column indicates the grayscale image of the GFP channel. The 3rd column indicates the grayscale image of the Sox2 channel. PSE; prosensory epithelia, GER; greater epithelial ridge, HCs; hair cells, SCs; supporting cells, IHC; inner hair cell, OHCs; outer hair cells, IPh; inner phalangeal cell, IP; inner pillar cell, OP; outer pillar cell, D (1–3); Deiters’ cell (1st – 3rd), H; Hensen’s cell, Cl; Claudius cell. Scale bars, 100 µm.

Article Snippet: The WntVIS signal was enhanced using a chick anti-GFP antibody (1:250, GFP-1010; Aves Labs, Davis, CA, USA).

Techniques: Activity Assay, Staining

Journal: Hearing research

Article Title: Sustained Wnt signaling in the mouse inner ear after morphogenesis: In hair cells, supporting cells, and spiral ganglion neurons.

doi: 10.1016/j.heares.2025.109282

Figure Lengend Snippet: Fig. 3. Wnt signaling activity in hair cells of the developing and developed Organ of Corti. A; E17 apical turn, B; E17 basal turn, C; P10, D; P30. Green; WntVIS-GFP, Red; Myo7A (hair cells), Blue; DAPI (nucleus). The 2nd column indicates the grayscale image of the GFP channel. The 3rd column indicates the grayscale image of the Myo7A channel. The yellow circle with a dotted line indicates the nucleus of the inner hair cell, while the blue circle with a dotted line indicates the nucleus of the outer hair cells. The yellow arrowhead indicates GFP fluorescence in outer hair cells. PSE; prosensory epithelia, GER; greater epithelial ridge, HCs; hair cells, SCs; supporting cells, IHC; inner hair cell, OHCs; outer hair cells, IPC; inner pillar cell, OPC; outer pillar cell, D; Deiters’ cell, H; Hensen’s cell, Cl; Claudius cell, TBC; tympanic border cell. Scale bars, 100 µm.

Article Snippet: The WntVIS signal was enhanced using a chick anti-GFP antibody (1:250, GFP-1010; Aves Labs, Davis, CA, USA).

Techniques: Activity Assay, Fluorescence

Journal: Hearing research

Article Title: Sustained Wnt signaling in the mouse inner ear after morphogenesis: In hair cells, supporting cells, and spiral ganglion neurons.

doi: 10.1016/j.heares.2025.109282

Figure Lengend Snippet: Fig. 4. Wnt signaling activities in the developing and developed saccule. A–E: Fluorescent immunostaining. A; E15, B; E17, C; P0, D; P30, E; P60. Green; WntVIS-GFP, Red; Myo7A (hair cells). Yellow arrowheads indicate the Wnt-activated hair cells. F: Percentage of GFP-positive cells in hair cells (HCs) and supporting cells (SCs). HCs were counted based on Myo7A fluorescence, while SCs were counted based on DAPI without Myo7A fluorescence. The error bar indicates a standard error.

Article Snippet: The WntVIS signal was enhanced using a chick anti-GFP antibody (1:250, GFP-1010; Aves Labs, Davis, CA, USA).

Techniques: Immunostaining, Fluorescence

Journal: Hearing research

Article Title: Sustained Wnt signaling in the mouse inner ear after morphogenesis: In hair cells, supporting cells, and spiral ganglion neurons.

doi: 10.1016/j.heares.2025.109282

Figure Lengend Snippet: Fig. 5. Wnt signaling activities in the developing and developed spiral ganglion. Midmodiolar section of WntVIS mouse. Green; WntVIS-GFP, Red; Sox2 (glial cells), White; βIII-tubulin (TuJ1, neurons). A; E15, B; P0, C; P7, D; P15, E; P30. During E15 to P7, Wnt signaling activities were observed in SGNs.

Article Snippet: The WntVIS signal was enhanced using a chick anti-GFP antibody (1:250, GFP-1010; Aves Labs, Davis, CA, USA).

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats

doi: 10.3892/ijmm.2018.3866

Figure Lengend Snippet: Myocardial injury is accompanied by an increase in O-GlcNAc modification and inhibited autophagic flux in rats at 8 weeks post-STZ induction. (A) M-mode echocardiography shows the left ventricular diastolic function as the ratio of E/A in rats at 8 weeks post-STZ induction. Morphological changes in the myocardium were assessed by (B) Masson staining and (C) hematoxylin and eosin staining (scale bar=100 and 25 µ m). (D) Protein was extracted from heart tissue, and the expression levels of O-GlcNAc (RL2), OGT and OGA were detected by western blot analysis; (E) Expression levels of LC3II/I, LAMP2, Beclin-1 and P62 in each group were detected by western blot analysis in the vehicle group and the 8-week STZ-induced group. (F) Transmission electron microscopy shows autophagosomes, characterized by a double-layer membrane structure, as indicated with the red arrows (scale bar=1 µ m). Data are expressed as the mean ± standard error of the mean (n≥6). * P<0.05 and ** P<0.01, vs. Vehicle group. Tubulin was the loading control. O-GlcNAc, O-linked β-N-acetylglucosamine; STZ, streptozotocin; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; LAMP2, lysosome-associated membrane protein 2; LC3, microtubule-associated protein 1 light chain 3α; E/A, left ventricular filling peak velocity/atrial contraction flow peak velocity.

Article Snippet: The anti-P62 (cat. no. 18420-1-AP; 1:1,000) and anti-Tubulin (cat. no. 66240-1-Ig; 1:1,000) antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA).

Techniques: Modification, Staining, Expressing, Western Blot, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Medicine

Article Title: O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats

doi: 10.3892/ijmm.2018.3866

Figure Lengend Snippet: Increased O-GlcNAc modification in vivo inhibits autophagic flux and aggravates myocardial function in type I DM rats. Rats were randomly divided into five groups (n≥6 in each group) for treatment: Vehicle, STZ, STZ+NaCl, STZ+Don, and STZ+TG. (A) M-mode echocardiography showed the left ventricular diastolic function as the ratio of E/A. Morphological changes in the myocardium were assessed by (B) hematoxylin and eosin staining and (C) Masson staining (scale bar=100 and 25 µ m); (D) Expression levels of O-GlcNAc (RL2), OGA and OGT in each group were detected by western blot analysis. (E) Western blot analysis was used to detect the expression of autophagy markers LC3II/I, Beclin1, P62, and LAMP2. Data are expressed as the mean ± standard error of the mean (n≥6). * P<0.05 and *** P<0.001, vs. Vehicle group; # P<0.05 and ### P<0.001, vs. STZ+NaCl group. Tubulin was the loading control. (F) Transmission electron microscopy showed the autophagosomes, which are characterized by a double-layer membrane structure, as indicated by red arrows (scale bar=1 µ m). O-GlcNAc, O-linked β-N-acetylglucosamine; STZ, streptozotocin; Don, 6-diazo-5-oxo-L-norleucine; TG, thiamet G; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; LAMP2, lysosome-associated membrane protein 2; LC3, microtubule-associated protein 1 light chain 3α; E/A, left ventricular filling peak velocity/atrial contraction flow peak velocity.

Article Snippet: The anti-P62 (cat. no. 18420-1-AP; 1:1,000) and anti-Tubulin (cat. no. 66240-1-Ig; 1:1,000) antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA).

Techniques: Modification, In Vivo, Staining, Expressing, Western Blot, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Medicine

Article Title: O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats

doi: 10.3892/ijmm.2018.3866

Figure Lengend Snippet: Increased O-GlcNAc modification inhibits autophagic flux in NRCMs under high-glucose conditions. NRCMs were exposed to high glucose (25 mM) and were treated with TG (5 µ M) or Don (40 µ M) for 24 h. (A) Supernatants were extracted, and the expression of O-GlcNAc (RL2) was analyzed by western blot analysis. (B) Expression levels of LAMP2, Beclin-1, P62, and LC3II/I in each group were detected by western blot analysis. NRCMs were transfected with sh-OGT or ad-OGA for 48 h and were exposed to high glucose (25 mM, Glu group) for 24 h. (C) Supernatants were extracted, and the expression levels of O-GlcNAc (RL2), OGT, and OGA were analyzed by western blot analysis. (D) Expression levels of LAMP2, Beclin-1, P62, and LC3II/I in each group were detected by western blot analysis. Data are expressed as the mean ± standard error of the mean (n≥6). * P<0.05, ** P<0.01 and *** P<0.001, vs. Vehicle group; # P<0.05 and ## P<0.01, vs. Glu group. Tubulin was the loading control. NRCMs, neonatal rat cardiomyocytes; O-GlcNAc, O-linked β-N-acetylglucosamine; STZ, streptozotocin; sh-OGT, OGT-knockdown adenovirus; ad-OGA, OGA-overexpression adenovirus; Ctrl, control; Glu, glucose; Don, 6-diazo-5-oxo-L-norleucine; TG, thiamet G; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; LAMP2, lysosome-associated membrane protein 2; LC3, microtubule-associated protein 1 light chain 3α.

Article Snippet: The anti-P62 (cat. no. 18420-1-AP; 1:1,000) and anti-Tubulin (cat. no. 66240-1-Ig; 1:1,000) antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA).

Techniques: Modification, Expressing, Western Blot, Transfection, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats

doi: 10.3892/ijmm.2018.3866

Figure Lengend Snippet: High glucose inhibits the degradation stage of autophagy. (A) NRCMs were transfected with LAMP2 siRNA for 48 h and were exposed to high glucose for another 24 h. The expression of LAMP2, Beclin-1, P62, and LC3II/I in each group was detected by western blot analysis. (B) NRCMs were pretreated with 3-MA (5 mM) for 24 h and with high glucose for another 24 h; the expression levels of P62 and LC3II/I in each group were detected by western blot analysis. Data are expressed as the mean ± standard error of the mean (n≥6). * P<0.05 and ** P<0.01, vs. Vehicle group; # P<0.05 and ## P<0.01, vs. Glu group. Tubulin was the loading control. NRCMs, neonatal rat cardiomyocytes; O-GlcNAc, O-linked β-N-acetylglucosamine; Glu, glucose; siRNA, small interfering RNA; LAMP2, lysosome-associated membrane protein 2; LC3, microtubule-associated protein 1 light chain 3α; 3-MA, 3-methyladenine.

Article Snippet: The anti-P62 (cat. no. 18420-1-AP; 1:1,000) and anti-Tubulin (cat. no. 66240-1-Ig; 1:1,000) antibodies were obtained from ProteinTech Group, Inc. (Chicago, IL, USA).

Techniques: Transfection, Expressing, Western Blot, Small Interfering RNA