Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 5. NaF exposure induces elevated phos phorylation of PRKAA1 expression in vivo and in vitro. (A) The hierarchical cluster heatmap of phosphorylated proteins in rat hippocampus. (B) Statistics of different phosphorylation sites and proteins in rat hippocampus. (C) Volcano plot of differentially phosphorylated proteins in rat hippocampus. The criteria for significance were set at logarithmic fold change (log2 FC) of > 1.2 or < 0.8 (P < 0.05). (D) Phosphorylation of PRKAA1 protein in rat hippocampus. (E-F) Representative western blot and relative quan tifications of PRKAA1 and p-PRKAA1 in rat hippocampus. (G-H) Representative western blot and relative quantifications of PRKAA1 and p-PRKAA1 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Expressing, In Vivo, In Vitro, Phospho-proteomics, Western Blot, Control

Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 6. DM inhibits aberrant mitophagy by pre venting the phosphorylation of PRKAA1 in vitro. (A-B) Representative western blot and relative quantifications of PRKAA1and p-PRKAA1 in SH- SY5Y cells. (C-D) Representative western blot and relative quantifications of PINK1 and Parkin in SH-SY5Y cells. (E-F) Representative images and relative quantifications of immunofluores cence staining of PINK1 and Mito-tracker, after DM intervention. The scale bar represents 50 µm. (G-H) Representative western blot and relative quantifications of TOMM-20 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group, @P < 0.05 compare with the NaF group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Phospho-proteomics, In Vitro, Western Blot, Staining, Control

Journal: Journal of Cancer

Article Title: LncRNA HOTAIR Enhances Epithelial-to-mesenchymal Transition to Promote the Migration and Invasion of Liver Cancer by Regulating NUAK1 via Epigenetic Inhibition miR-145-5p Expression

doi: 10.7150/jca.85335

Figure Lengend Snippet: Primers

Article Snippet: Then seal with 5% non-fat dry milk powder for 90min at room temperature and incubated using primary antibodies overnight at 4°C, which were NUAK1 (1:500, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:2000, Proteintech, USA), Vimentin (1:2000, Proteintech, USA), EZH2 (1:1000, Proteintech, USA), MMP2 (1:500, Wanleibio, China), MMP9 (1:1000, Wanleibio, China).

Techniques:

Journal: Journal of Cancer

Article Title: LncRNA HOTAIR Enhances Epithelial-to-mesenchymal Transition to Promote the Migration and Invasion of Liver Cancer by Regulating NUAK1 via Epigenetic Inhibition miR-145-5p Expression

doi: 10.7150/jca.85335

Figure Lengend Snippet: HOTAIR and NUAK1 are positively correlated. (A) Detected the NUAK1 expression levels in six cell lines. (B) Analyzed NUAK1 interacting genes through STRING website. (C) qPCR experiment to detect the mRNA expression of NUAK1 in HCC tissue samples. (D) Relative analysis of HOTAIR and NUAK1 expression in HCC tissues. (E-F) Detected the relationship between HOTAIR and NUAK1 by Western blot and qPCR. The data shown were representative of three independent experiments. Bars, SD (n=3), *p<0.05, **p<0.01, and ***p<0.001 vs NC group.

Article Snippet: Then seal with 5% non-fat dry milk powder for 90min at room temperature and incubated using primary antibodies overnight at 4°C, which were NUAK1 (1:500, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:2000, Proteintech, USA), Vimentin (1:2000, Proteintech, USA), EZH2 (1:1000, Proteintech, USA), MMP2 (1:500, Wanleibio, China), MMP9 (1:1000, Wanleibio, China).

Techniques: Expressing, Western Blot

Journal: Journal of Cancer

Article Title: LncRNA HOTAIR Enhances Epithelial-to-mesenchymal Transition to Promote the Migration and Invasion of Liver Cancer by Regulating NUAK1 via Epigenetic Inhibition miR-145-5p Expression

doi: 10.7150/jca.85335

Figure Lengend Snippet: HOTAIR regulates EMT through NUAK1 (A-B) Scratch test detected cell migration ability. (C) Added HTH-01-015 to SNU-387 and HepG2 cells, and Transwell test to detect cell invasion ability. (D) Added HTH-01-015 to SNU-387 and HepG2 cells, and detected the protein expression of E-cadherin, N-cadherin, Vimentin, MMP2, MMP9 by Western blot. (E) Added HTH-01-015 to SNU-387 and HepG2 cells, and detected the mRNA expression of E-cadherin, N-cadherin, Vimentin, MMP2, MMP9 by qPCR. (F) Immunofluorescence test to detect the protein expression level of N-cadherin. Added arrows to indicate cells of interest. (G) Transfected LZRS-HOTAIR plasmid in SNU-387 and HepG2 cells, and co-transfected LZRS-HOTAIR plasmid and HTH-01-015, used Western blot to detect the protein expression of E-cadherin, N-cadherin, Vimentin. The data shown were representative of three independent experiments. Bars, SD (n=3), *p<0.05, **p<0.01, and ***p<0.001 vs NC group.

Article Snippet: Then seal with 5% non-fat dry milk powder for 90min at room temperature and incubated using primary antibodies overnight at 4°C, which were NUAK1 (1:500, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:2000, Proteintech, USA), Vimentin (1:2000, Proteintech, USA), EZH2 (1:1000, Proteintech, USA), MMP2 (1:500, Wanleibio, China), MMP9 (1:1000, Wanleibio, China).

Techniques: Migration, Expressing, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation

Journal: Journal of Cancer

Article Title: LncRNA HOTAIR Enhances Epithelial-to-mesenchymal Transition to Promote the Migration and Invasion of Liver Cancer by Regulating NUAK1 via Epigenetic Inhibition miR-145-5p Expression

doi: 10.7150/jca.85335

Figure Lengend Snippet: miR-145-5p is low expressed in liver cancer (A) The target of NUAK1 predicted by TargetScan Human 7.2. (B) Firefly luciferase activity normalized to that of Renilla luciferase 48 h after transfection of SNU-387 and HepG2 cells with reporter vectors expressing wild-type or mutant NUAK1 3'UTR. (C) Detected miR-145-5p expression in five liver cancer cells by qPCR. (D) Analyzed the expression of miR-145-5p in HCC tissues by TCGA database. The data shown were representative of three independent experiments. Bars, SD (n=3), *p<0.05, **p<0.01, and ***p<0.001 vs NC group.

Article Snippet: Then seal with 5% non-fat dry milk powder for 90min at room temperature and incubated using primary antibodies overnight at 4°C, which were NUAK1 (1:500, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:2000, Proteintech, USA), Vimentin (1:2000, Proteintech, USA), EZH2 (1:1000, Proteintech, USA), MMP2 (1:500, Wanleibio, China), MMP9 (1:1000, Wanleibio, China).

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Mutagenesis

Journal: Journal of Cancer

Article Title: LncRNA HOTAIR Enhances Epithelial-to-mesenchymal Transition to Promote the Migration and Invasion of Liver Cancer by Regulating NUAK1 via Epigenetic Inhibition miR-145-5p Expression

doi: 10.7150/jca.85335

Figure Lengend Snippet: miR-145-5p is the upstream target gene of NUAK1. (A) Scratch test detected cell migration ability. (B) Transfected miR-145 mimic and inhibitor in SNU-387 and HepG2, Transwell test to detect cell invasion ability. (C) Transfected with miR-145 mimic and inhibitor, Western blot to detect the protein expression of E-cadherin, N-cadherin, and Vimentin. (D) qPCR experiment to detect mRNA expression of E-cadherin, N-cadherin, Vimentin, MMP2, MMP9. (E) Immunofluorescence test to detect the protein expression level of N-cadherin. Added arrows to indicate cells of interest. (F) In cells transfected with miR-145 mimic and inhibitor, Western blot was used to detect the protein expression of NUAK1. The data shown were representative of three independent experiments. Bars, SD (n=3), *p<0.05, **p<0.01, and ***p<0.001 vs NC group.

Article Snippet: Then seal with 5% non-fat dry milk powder for 90min at room temperature and incubated using primary antibodies overnight at 4°C, which were NUAK1 (1:500, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:2000, Proteintech, USA), Vimentin (1:2000, Proteintech, USA), EZH2 (1:1000, Proteintech, USA), MMP2 (1:500, Wanleibio, China), MMP9 (1:1000, Wanleibio, China).

Techniques: Migration, Transfection, Western Blot, Expressing, Immunofluorescence

Journal: Journal of Cancer

Article Title: LncRNA HOTAIR Enhances Epithelial-to-mesenchymal Transition to Promote the Migration and Invasion of Liver Cancer by Regulating NUAK1 via Epigenetic Inhibition miR-145-5p Expression

doi: 10.7150/jca.85335

Figure Lengend Snippet: HOTAIR regulates the expression of NUAK1 through miR-145-5p. (A-B) Transfected Sh-HOTAIR and LZRS-HOTAIR plasmids in SNU-387 and HepG2, and detected the expression level of miR-145-5p by qPCR. (C) Transfected si-EZH2 in SNU-387 and HepG2, and detected the expression level of miR-145-5p by qPCR. (D) Transfected LZRS-HOTAIR plasmid in SNU-387 and HepG2, and transfected LZRS-HOTAIR plasmid while adding si-EZH2, and detected the expression level of miR-145-5p by qPCR. (E-H) ChIP assays in SNU-387 and HepG2 transfected with sh-HOTAIR and si-EZH2 cells were performed on the miR-145-5p promoter regions using anti-H3K27me3 and EZH2 antibodies. Enrichment was determined relative to the input controls. (I) Transfected Sh-HOTAIR plasmid in SNU-387 and HepG2, and transfected Sh-HOTAIR plasmid while adding miR-145 inhibitor, Western blot experiment to detect the protein expression of NUAK1. The data shown were representative of three independent experiments. Bars, SD (n=3), *p<0.05, **p<0.01, and ***p<0.001 vs NC group.

Article Snippet: Then seal with 5% non-fat dry milk powder for 90min at room temperature and incubated using primary antibodies overnight at 4°C, which were NUAK1 (1:500, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:2000, Proteintech, USA), Vimentin (1:2000, Proteintech, USA), EZH2 (1:1000, Proteintech, USA), MMP2 (1:500, Wanleibio, China), MMP9 (1:1000, Wanleibio, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot