immunofluorescence  (Novus Biologicals)

Journal: International Journal of Oncology

Article Title: KMO downregulation promotes hepatocellular carcinoma growth via 3-HAA-mediated mitochondrial mass and function imbalances

doi: 10.3892/ijo.2026.5846

Figure Lengend Snippet: Downregulation of KMO promotes the decrease of NR4A1 in HCC cells. (A) Venn diagram among KMO related DEGs, transcription factors and mitochondrial related genes. The overlapped four genes are the screened mitochondrial related transcription factors from KMO related DEGs. (B) Correlation analysis between KMO and four mitochondrial related transcription factors screened using TCGA database. (C) mRNA expression of four mitochondrial related transcription factors in KMO-knockdown, overexpression and 3-HAA treated KMO-knockdown HCC cells and the corresponding controls. (D) Survival analysis of NR4A1 in HCC patients in TCGA database. (E) The correlation analysis between NR4A1 and mitochondrial DNA-encoded genes in HCC. Final working concentration of 3-HAA: 100 µ M. * P<0.05, ** P<0.01, ns: P>0.05. KMO, kynurenine 3-monooxygenase; NR4A1, nuclear receptor subfamily 4 group A member 1; HCC, hepatocellular carcinoma; DEGs, differentially expressed genes; TCGA, the Cancer Genome Atlas; 3-HAA, 3-hydroxyanthranilic acid.

Article Snippet: Cells were incubated with 1:100 diluted KMO or NR4A1 primary antibody (cat no. 10698-1-AP and 12235-1-AP, Proteintech Group, Inc.) overnight at 4°C, followed by fluorescent secondary antibody (1:200; cat no. SA00013-2; Proteintech Group, Inc.) at room temperature for 1 h. After DAPI staining (1:200) at room temperature for 15 min, cells were imaged at a magnification of ×100.

Techniques: Expressing, Knockdown, Over Expression, Concentration Assay

Journal: International Journal of Oncology

Article Title: KMO downregulation promotes hepatocellular carcinoma growth via 3-HAA-mediated mitochondrial mass and function imbalances

doi: 10.3892/ijo.2026.5846

Figure Lengend Snippet: Downregulation of KMO promotes translocation of NR4A1 to mitochondria by 3-HAA. Effect of KMO and 3-HAA on subcellular distribution of NR4A1 in (A) MHCC-97H and (B) LM3 cells. NR4A1 is shown in green and mitochondria are stained red. The yellow color indicates the co-localization of NR4A1 with mitochondria. Scale bar, 20 µ m. (C) The overlap rate of NR4A1 and mitochondria (orange color rate) was quantified and compared between the indicated HCC cells. Final working concentration of 3-HAA: 100 µ M. Confocal microscope magnification, ×100. * P<0.05, *** P<0.001. KMO, kynurenine 3-monooxygenase; NR4A1, nuclear receptor subfamily 4 group A member 1; 3-HAA, 3-hydroxyanthranilic acid; HCC, hepatocellular carcinoma.

Article Snippet: Cells were incubated with 1:100 diluted KMO or NR4A1 primary antibody (cat no. 10698-1-AP and 12235-1-AP, Proteintech Group, Inc.) overnight at 4°C, followed by fluorescent secondary antibody (1:200; cat no. SA00013-2; Proteintech Group, Inc.) at room temperature for 1 h. After DAPI staining (1:200) at room temperature for 15 min, cells were imaged at a magnification of ×100.

Techniques: Translocation Assay, Staining, Concentration Assay, Microscopy

Journal: International Journal of Oncology

Article Title: KMO downregulation promotes hepatocellular carcinoma growth via 3-HAA-mediated mitochondrial mass and function imbalances

doi: 10.3892/ijo.2026.5846

Figure Lengend Snippet: KMO affects cell growth, mitochondrial quality and ATP production through NR4A1. (A) Effect of NR4A1 downregulation on cell viability in KMO overexpression HCC cells was observed by using CCK8 test on 0, 24, 48, 72 and 96 h. The cell viability rates between KMO overexpression cells transfected with siKMO and transfected with siControl were compared. (B) Effect of NR4A1 downregulation on cell proliferation in KMO overexpression HCC cells was observed by using EdU test. EdU labeled newly proliferated cells (red) and Hoechst33342 labeled cell nucleus (blue). Scale bar, 100 µ m. Positive cells rate (red/blue) were counted and compared between KMO overexpression cells and KMO overexpression cells with NR4A1 knockdown. (C) Effect of NR4A1 downregulation on mitochondrial mass in KMO overexpression HCC cells was observed by using confocal microscopy after MitoTracker staining. The parts in white box were magnified. Scale bar, 20 µ m. Mitochondrial mass was analyzed by using the value of average fluorescence intensity per cell. (D) Effect of NR4A1 downregulation on ATP production in KMO overexpression HCC cells. (E) The mechanism of KMO downregulation promoting tumor growth in HCC: KMO downregulation reduced 3-HAA levels, which suppressed expression of the transcription factor NR4A1 and drove its mitochondrial translocation, thereby disrupting mitochondrial homeostasis and promoting the proliferation of HCC cells. * P<0.05, ** P<0.01. KMO, kynurenine 3-monooxygenase; NR4A1, nuclear receptor subfamily 4 group A member 1; HCC, hepatocellular carcinoma; 3-HAA, 3-hydroxyanthranilic acid; ROS, reactive oxygen species; 3-HK, 3-hydroxykynurenine QUIN, quinolinic acid; Try, tryptophan; Kyn, kynurenine.

Article Snippet: Cells were incubated with 1:100 diluted KMO or NR4A1 primary antibody (cat no. 10698-1-AP and 12235-1-AP, Proteintech Group, Inc.) overnight at 4°C, followed by fluorescent secondary antibody (1:200; cat no. SA00013-2; Proteintech Group, Inc.) at room temperature for 1 h. After DAPI staining (1:200) at room temperature for 15 min, cells were imaged at a magnification of ×100.

Techniques: Over Expression, Transfection, Labeling, Knockdown, Confocal Microscopy, Staining, Fluorescence, Expressing, Translocation Assay

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Nr4a1 is a key gene in maintaining microglial homeostasis during cognitive impairment in TN rats. ( a ) Volcano plot of transcriptomic differentially expressed genes (DEGs). ( b ) Protein–protein interaction network of cognition-related DEGs. ( c ) Heatmap of cognition-related DEGs. ( d ) mRNA expression of Nr4a1 in the hippocampus measured using RT-qPCR ( n = 3). ( e ) Protein expression of Nr4a1 was analyzed using western blotting ( n = 6). ( f - g ) Representative immunofluorescence images and quantitative analysis of Nr4a1 expression in the hippocampus ( n = 6). Scale bar = 20 μm. ( h ) Flowchart of the experimental timeline for in vivo Part II. ( i ) Changes in the facial mechanical pain threshold ( n = 8). ( j ) Schematic diagram of the empty vector control and Nr4a1 -overexpressing AAV constructs. ITR, inverted terminal repeat; CX3CR, ZsGreen and Nr4a1 promoter. (k) Image showing AAV- Nr4a1 virus infusion into the hippocampus. Scale bar = 200 μm. Data are expressed as mean ± SD. # P < 0.05, ^ P < 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, In Vivo, Plasmid Preparation, Control, Construct, Virus

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Nr4a1 overexpression alleviates microglial overactivation and its mediated neuroinflammation in the hippocampus of TN rats. ( a - d ) Representative immunofluorescence images and quantitative analysis of Iba1 expression, microglial number, and morphological changes in the hippocampal CA1 region ( n = 6). Scale bar = 20 μm. ( e - k ) Protein expression levels of Iba1, TNF-α, IL-6, IL-1β, IL-4, and IL-10 were analyzed using western blotting ( n = 6). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Over Expression, Immunofluorescence, Expressing, Western Blot

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Nr4a1 overexpression improved neuronal autophagy and synaptic plasticity in the hippocampus of TN rats. ( a ) Representative TEM images of hippocampal neuronal ultrastructure (orange arrows indicate autophagosomes) ( n = 3). Scale bar = 2 μm. ( b - d ) Protein expression levels of LC3-II and p62 were analyzed using western blotting ( n = 6). ( e - j ) Protein expression levels of BDNF, TrkB, SYN, and PSD95 were analyzed using western blotting ( n = 6). ( k ) Representative Golgi staining images of hippocampal dendritic spines (different arrow types indicate spine morphologies). Scale bar = 10 μm. ( l - t ) Quantitative analysis of total dendritic spine density, mature spine density, and immature spine density in the hippocampus ( n = 6). Data are expressed as mean ± SD. ** P < 0.01, *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Over Expression, Expressing, Western Blot, Staining

Journal: The Journal of Headache and Pain

Article Title: Nr4a1 regulates microglia overactivation-mediated neuroinflammation to ameliorate cognitive impairment in trigeminal neuralgia rats

doi: 10.1186/s10194-026-02293-3

Figure Lengend Snippet: Co-IP/MS identified Akt2 binding to Nr4a1 . Restoring Nr4a1 expression rescued downregulated PI3K/Akt2 signaling in the hippocampus of TN rats and in an LPS-stimulated BV2 cell model. ( a ) Flowchart of the Co-IP/MS experiment. ( b ) Potential protein interactors of Nr4a1 predicted using the STRING database. ( c ) Silver staining of Co-IP products (target protein: Nr4a1 ). ( d ) Protein identification results from mass spectrometry analysis of Co-IP products. ( e ) Molecular docking models of Nr4a1 with different Akt isoforms. ( f ) Representative immunofluorescence images of Nr4a1 and p-Akt2 in each group of cells. Scale bar = 20 μm. ( g - j ) Protein expression levels of p-PI3K, PI3K, p-Akt2, Akt2, p-mTOR, and mTOR in BV2 cells from each group were analyzed using western blotting ( n = 6). ( k - n ) Protein expression levels of p-PI3K, PI3K, p-Akt2, Akt2, p-mTOR, and mTOR in the hippocampus of rats from each group were analyzed using western blotting ( n = 6). Data are expressed as mean ± SD. *** P < 0.001

Article Snippet: Sections were blocked using a solution containing 10% donkey serum and 0.3% Triton X-100 in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: Iba1 (1:300; ab178846, Abcam), Iba1 (1:300; ab283319, Abcam), GFAP (1:300; ab7260, Abcam), NeuN (1:300; ab177487, Abcam), and Nr4a1 (1:100; sc-365113, Santa Cruz).

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Expressing, Silver Staining, Mass Spectrometry, Immunofluorescence, Western Blot