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

Journal: The Journal of Biological Chemistry

Article Title: Muscle-specific Knock-out of NUAK Family SNF1-like Kinase 1 (NUAK1) Prevents High Fat Diet-induced Glucose Intolerance *

doi: 10.1074/jbc.M111.302687

Figure Lengend Snippet: NUAK1 is preferentially expressed in highly oxidative tissues. A , quantitative RT-PCR analysis of Nuak1 and Nuak2. RNA was isolated from 10-week-old C57BL/6 mice. The data are the means ± S.E. ( n = 3). B , immunoblotting analysis of NUAK1 and LKB1 in heart, Sol, TA, and EDL muscles. Actin was used as a loading control. Protein extracts were from three individual 10-week-old C57BL/6 mice. C , schematic representation of floxed and knock-out alleles of Nuak1. The black arrowheads represent loxP sites. Gray arrows indicate PCR primers P1 and P2 used for the genotyping. Amplification with the P1 and P2 primers yields the 1166-bp product from the floxed allele and the 378-bp product from the knock-out allele. D , genotyping of Nuak1 floxed mice without (control) or with (MNUAK1KO) the Mck-Cre transgene. M , molecular marker. E , real time RT-PCR for Nuak1 mRNA in muscles. RNA was isolated from 10-week-old control and MNUAK1KO mice. The mRNA levels are expressed relative to that in soleus muscle of control mice. The data are the means ± S.E. ( n = 3). *, p < 0.05; **, p < 0.01 (Student's t test). F , immunoblot analysis of NUAK1 protein in muscles. Protein extracts of each type of muscle were prepared from three individual mice. Sol , soleus; Quad , quadriceps; Cont , control; MNKO , MNUAK1KO.

Article Snippet: The TaqMan gene expression assays used in this study were Nuak1 (Mm01250701_m1), Nuak2 (Mm00546961_m1), PGC-1α (Mm_01208835_m1), TNNC1 (Mm00437111_m1), TNNC2 (Mm00437116_m1), and 18 S rRNA (4319413E).

Techniques: Quantitative RT-PCR, Isolation, Western Blot, Muscles, Control, Knock-Out, Amplification, Marker

Journal: The Journal of Biological Chemistry

Article Title: Muscle-specific Knock-out of NUAK Family SNF1-like Kinase 1 (NUAK1) Prevents High Fat Diet-induced Glucose Intolerance *

doi: 10.1074/jbc.M111.302687

Figure Lengend Snippet: MNUAK1KO mice show improved glucose homeostasis under HFD conditions. A and B , fasting blood glucose levels of control and MNUAK1KO mice at 13–15 weeks ( A ) and 18–19 weeks of age ( B ). The data are the means ± S.E. ( n = 8). C , plasma insulin levels of control and MNUAK1KO mice. The data are the means ± S.E. ( n = 12). D , oral glucose tolerance of control and MNUAK1KO mice under HFD conditions. The data are the means ± S.E. ( n = 12). E , insulin tolerance of control and NUAK1 KO mice under HFD conditions. The data are the means ± S.E. ( n = 8). Mice at the age of 13–15 weeks were used for C–E . *, p < 0.05; **, p < 0.01 (Student's t test).

Article Snippet: The TaqMan gene expression assays used in this study were Nuak1 (Mm01250701_m1), Nuak2 (Mm00546961_m1), PGC-1α (Mm_01208835_m1), TNNC1 (Mm00437111_m1), TNNC2 (Mm00437116_m1), and 18 S rRNA (4319413E).

Techniques: Control, Clinical Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Muscle-specific Knock-out of NUAK Family SNF1-like Kinase 1 (NUAK1) Prevents High Fat Diet-induced Glucose Intolerance *

doi: 10.1074/jbc.M111.302687

Figure Lengend Snippet: Phosphorylation of TBC1D4 is up-regulated in NUAK1-deficient muscle after glucose administration. Immunoblot analysis of TBC1D4, GLUT4, and NUAK1 in soleus muscle of HFD-fed control and MNUAK1KO mice. Mice at the age of 13–15 weeks were fasted overnight and sacrificed 40 min after oral administration of water (basal control) or glucose. The graphs show the intensities of phospho-TBC1D4, GLUT4, and NUAK1 bands. Phospho-TBC1D4 protein levels were normalized to total TBC1D4. GLUT4 and NUAK1 protein levels were normalized to actin. The protein levels are expressed relative to those in the water-administered control mice. The data are the means ± S.E. ( n = 4 for NUAK1 and 5 for TBC1D4 and GLUT4). **, p < 0.01 (Student's t test). Cont , control; MNKO , MNUAK1KO.

Article Snippet: The TaqMan gene expression assays used in this study were Nuak1 (Mm01250701_m1), Nuak2 (Mm00546961_m1), PGC-1α (Mm_01208835_m1), TNNC1 (Mm00437111_m1), TNNC2 (Mm00437116_m1), and 18 S rRNA (4319413E).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Muscle-specific Knock-out of NUAK Family SNF1-like Kinase 1 (NUAK1) Prevents High Fat Diet-induced Glucose Intolerance *

doi: 10.1074/jbc.M111.302687

Figure Lengend Snippet: Phosphorylation of insulin signaling proteins are up-regulated in NUAK1-deficient muscle. Immunoblot analysis of phosphorylation of IRS1 at Tyr-608 ( black arrowhead ) and AKT at Thr-308 in soleus muscle of HFD-fed control and MNUAK1KO mice. A white arrowhead denotes a nonspecific band. Graphs show the intensities of phospho-protein bands normalized to total protein levels in each case. The data are expressed relative to those from control mice. *, p < 0.05; **, p < 0.01 (Student's t test). A , mice under fed conditions. The data are the means ± S.E. ( n = 3). B , mice were fasted overnight and administered water or glucose (1 g/kg of body weight). Soleus was excised 40 min after the administration. The data are the means ± S.E. ( n = 6). C , mice were fasted overnight and intraperitoneally injected with PBS or insulin (1.5 units/kg of body weight). Soleus was excised 20 min after the injection. The data are the means ± S.E. ( n = 3). Cont , control; MNKO , MNUAK1KO.

Article Snippet: The TaqMan gene expression assays used in this study were Nuak1 (Mm01250701_m1), Nuak2 (Mm00546961_m1), PGC-1α (Mm_01208835_m1), TNNC1 (Mm00437111_m1), TNNC2 (Mm00437116_m1), and 18 S rRNA (4319413E).

Techniques: Phospho-proteomics, Western Blot, Control, Injection

Journal: Oncotarget

Article Title: microRNA-203 suppresses invasion and epithelial-mesenchymal transition induction via targeting NUAK1 in head and neck cancer.

doi: 10.18632/oncotarget.6972

Figure Lengend Snippet: Figure 3: miR-203 suppresses cancerous invasion of HNSCC cells. A. Expression of miR-203 was examined by real-time PCR in cells with epithelial phenotype (HaCaT, HSC2, and MSCC-1) and EMT-induced cells (MSCC-inv1, HOC313, KOSCC25B, KOSCC33A, and SpSCC). The graph shows miR-203/U6. The results are presented as means ± SD. *P < 0.05. B. Expression vector of pre-miR-203 or scramble negative control vector was transfected into MSCC-inv1 cells. miR-203/U6 was examined by real-time PCR and data were normalized to negative control-transfected samples. The results are presented as means ± SD. *P < 0.05. C. The figure shows the cell shape of control- and pre-miR-203-infected MSCC-inv1 cells. D. Expression of E-cadherin and NUAK1 mRNA was examined by real-time PCR in control- and pre-miR-203-infected MSCC-inv1 cells. The graph shows the expression of these mRNAs (mRNA/GAPDH). All results are presented as means ± SD. *P < 0.05. E. The graph shows invasion capability of negative control- or pre-miR-203-infected MSCC-inv1 cells. The invasiveness of the cells was determined by in vitro invasion assay for 9 h. **P < 0.01. F. HSC2 cells with miR-203 expression were transfected with miR-203 inhibitor (5, 25, 50, and 75 nM). The invasiveness of the cells was determined by in vitro invasion assay for 22 h. *P < 0.05.

Article Snippet: Cells were transfected with 5 nM hsa-miRNAs and with 5-100 nM anti-miR-203 using Lipofectamine RNAiMax (Invitrogen). siRNAs targeting NUAK1 and negative control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Negative Control, Transfection, Control, Infection, In Vitro, Invasion Assay

Journal: Oncotarget

Article Title: microRNA-203 suppresses invasion and epithelial-mesenchymal transition induction via targeting NUAK1 in head and neck cancer.

doi: 10.18632/oncotarget.6972

Figure Lengend Snippet: Figure 5: miR-203 suppresses NUAK1 expression. A. Luciferase assays were performed with pmirGLO vector containing the 3’-UTR of NUAK1 and the graph shows the relative luciferase activity in mature miR-203- or control miRNA-transfected cells. The data were normalized to control samples and results are presented as means ± SD. **P < 0.01. B. NUAK1 expression was examined in control- or mature miR-203-transfected HNSCC cells (KOSCC25B, SpSCC, and HOC313) by western blotting analysis. The graph shows the NUAK1/β-actin ratio by densitometric analysis. C. NUAK1 expression was examined in control- or miR-203 inhibitor-transfected HSC2 cells by western blotting analysis. The graph shows the NUAK1/β-actin ratio by densitometric analysis.

Article Snippet: Cells were transfected with 5 nM hsa-miRNAs and with 5-100 nM anti-miR-203 using Lipofectamine RNAiMax (Invitrogen). siRNAs targeting NUAK1 and negative control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Luciferase, Plasmid Preparation, Activity Assay, Control, Transfection, Western Blot

Journal: Oncotarget

Article Title: microRNA-203 suppresses invasion and epithelial-mesenchymal transition induction via targeting NUAK1 in head and neck cancer.

doi: 10.18632/oncotarget.6972

Figure Lengend Snippet: Figure 6: Role of NUAK1 in invasion and EMT induction. A. A549 cells were treated with 10 ng/mL of TGF-β. The upper panel shows the cell shape at 0 and 48 h after TGF-β treatment. The lower panel shows expression of NUAK1, E-cadherin, N-cadherin, SNAI1, SNAI2, ZEB1, and vimentin. Expression of indicated proteins was examined by western blotting at 0, 1, 2, 4, 8, 12, 24, and 48 h after TGF-β treatment. β-actin was used as a control. B. A stable clone of pre-miR-203-transfected A549 cells was obtained. Control- or pre-miR- 203-transfected A549 cells were treated with 10 ng/mL of TGF-β. Expression of the indicated proteins was examined by western blotting at 0, 2, 12, 24, and 48 h after TGF-β treatment. β-actin expression was used as a loading control. C. NUAK1 or empty vector was transfected into HSC2 cells. The expression of NUAK1 was examined by western blotting. β-actin was used as a loading control. D. The invasiveness of NUAK1-overexpressing HSC2 cells was examined by in vitro invasion assay for 10 h. The upper panel shows that the cells penetrated to the lower side of the membrane. The lower graph shows the results of the in vitro invasion assay presented as means ± SD. *P < 0.05. E. SpSCC cells were transfected by NUAK1 siRNA or control siRNA. A scrambled sequence that did not show significant homology to rat, mouse or human gene sequences was used as a control. The effect of knockdown was evaluated by western blotting and real-time PCR, and β-actin and GAPDH were used as loading controls, respectively. **P < 0.01. F. The invasiveness of NUAK1-knocked-down SpSCC cells was examined by in vitro invasion assay for 9 h. The upper panel shows that the cells penetrated to the lower side of the membrane. The lower graph shows the results of in vitro invasion assay presented as means ± SD. *P < 0.05.

Article Snippet: Cells were transfected with 5 nM hsa-miRNAs and with 5-100 nM anti-miR-203 using Lipofectamine RNAiMax (Invitrogen). siRNAs targeting NUAK1 and negative control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation, In Vitro, Invasion Assay, Membrane, Sequencing, Knockdown, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: microRNA-203 suppresses invasion and epithelial-mesenchymal transition induction via targeting NUAK1 in head and neck cancer.

doi: 10.18632/oncotarget.6972

Figure Lengend Snippet: Figure 8: NUAK1 expression and its correlation with invasion pattern in HNSCC cases. A. NUAK1 expression was examined by immunohistochemistry in 54 HNSCC cases. Representative pictures of NUAK1 positive or negative HNSCC case are shown. B. The graph shows the number of cases with or without NUAK1 expression in each grade of the YK classification (YK-2, -3, -4C, and -4D). C. Schematic model of the role of miR-203 in HNSCC. In HNSCC cells with epithelial phenotype, miR-203 suppresses the expression of NUAK1 and SNAI2. In HNSCC cells with EMT phenotype, miR-203 is downregulated by hypermethylation. Therefore, miR-203 cannot suppress the expression of NUAK1 and SNAI2. Elevated expression of NUAK1 may induce the invasion of HNSCC cells. In addition, the miR-200 family (miR-200a, -200b, -200c, and -141) is also downregulated and target genes including ZEB1, ZEB2, SNAI1, etc. are upregulated. Downregulation of miR-203 may be caused by downregulation of the miR-200 family and/or upregulation of SNAI1/SNAI2 and ZEB1/ZEB2. Elevated expression of NUAK1, ZEB1, ZEB2, SNAI1, and SNAI2 may induce EMT in HNSCC cells.

Article Snippet: Cells were transfected with 5 nM hsa-miRNAs and with 5-100 nM anti-miR-203 using Lipofectamine RNAiMax (Invitrogen). siRNAs targeting NUAK1 and negative control siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities

doi: 10.1038/s41419-020-02885-0

Figure Lengend Snippet: a Expression levels of NUAK1 were screened in the Broad Institute Cancer Cell Line Encyclopedia (CCLE). b NUAK1 expression levels were analyzed by western blot in 12 cancer cell lines and 2 normal cell lines. c A549, NCI-H460, MNK45, and U251 cells were treated with 20 μM NUAK1 inhibitor WZ4003 for 0, 4, 8, and 12 h and analyzed by western blot. WZ4003 treatment induces phosphorylation of ULK1 and puncta of LC3 together with downregulation of p62 and upregulation of phosphorylated ATG13. Phosphorylation of NUAK1 downstream target MYPT1 is downregulated by WZ4003. d Phosphorylated ULK1 protein levels (normalized to total ULK1), phosphorylated ATG13 protein levels (normalized to total ATG13), phosphorylated MYPT1 protein levels (normalized to total MYPT1), p62 protein levels (normalized to GAPDH), and LC3B protein levels (normalized to GAPDH), n = 3. e Representative images of LC3B fluorescence in U251 cells and A549 cells treated with 20 μM WZ4003 for 0–12 h.

Article Snippet: Fig. 1 NUAK1 is overexpressed in different types of cancer cells, and NUAK1 inhibition induces ULK1-dependent autophagy formation. a Expression levels of NUAK1 were screened in the Broad Institute Cancer Cell Line Encyclopedia (CCLE). b NUAK1 expression levels were analyzed by western blot in 12 cancer cell lines and 2 normal cell lines. c A549, NCI-H460, MNK45, and U251 cells were treated with 20 μM NUAK1 inhibitor WZ4003 for 0, 4, 8, and 12 h and analyzed by western blot.

Techniques: Expressing, Western Blot, Phospho-proteomics, Fluorescence

Journal: Cell Death & Disease

Article Title: Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities

doi: 10.1038/s41419-020-02885-0

Figure Lengend Snippet: a The average cell death and CI value quantification for the 7 × 6 matrices of WZ4003 and SBI-0206965 in MNK45, U251, A549, and NCI-H460 cells. b The results of average cell death induced by WZ4003 + MRT68921 combinations evaluated in the 5 × 6 matrices and corresponding quantification of synergy CI values. c A schematic of the combination treatment of WZ4003 plus reported ULK1 inhibitors. The CI value was calculated for cell death induced by each combination group. CI combination index.

Article Snippet: Fig. 1 NUAK1 is overexpressed in different types of cancer cells, and NUAK1 inhibition induces ULK1-dependent autophagy formation. a Expression levels of NUAK1 were screened in the Broad Institute Cancer Cell Line Encyclopedia (CCLE). b NUAK1 expression levels were analyzed by western blot in 12 cancer cell lines and 2 normal cell lines. c A549, NCI-H460, MNK45, and U251 cells were treated with 20 μM NUAK1 inhibitor WZ4003 for 0, 4, 8, and 12 h and analyzed by western blot.

Techniques:

Journal: Cell Death & Disease

Article Title: Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities

doi: 10.1038/s41419-020-02885-0

Figure Lengend Snippet: The cancer cell lines MNK45, U251, A549, and NCI-H460 were treated with WZ4003 (20 μM), SBI-0206965 (20 μM) or a combination of WZ4003 plus SBI-0206965, chloroquine (CQ, 20 μM) was set as controls. a The induction of apoptosis was analyzed by Annexin V-FITC and propidium iodide staining after 12 h of treatment. b Western blot analysis for p-ULK1/ULK1, p-ATG13/ATG13, cleavage of apoptosis marker PARP1, LC3A/B, and p62 after 8 h of treatment. c Quantifications of P-ULK1/ULK1, p-ATG13/ATG13, p-MYPT1/MYPT1, p62/GAPDH, and LC3B/GAPDH ( n = 3). d Representative and merge images showing LC3B fluorescence (green), mitotracker fluorescence (red), and DAPI fluorescence (blue) in U251 cell after treatment of WZ4003 (20 μM), SBI-0206965 (20 μM), CQ (20 μM), or combinations for 8 h. Enlarged merge images representative the co-localizations of LC3B-labeled autophagosome and mitotracker-labeled mitochondria. e ROS levels were analyzed by DCFH-DA staining after 8 h of treatment. Combination treatment induced lethal enhancement of ROS levels. ROS reactive oxygen species.

Article Snippet: Fig. 1 NUAK1 is overexpressed in different types of cancer cells, and NUAK1 inhibition induces ULK1-dependent autophagy formation. a Expression levels of NUAK1 were screened in the Broad Institute Cancer Cell Line Encyclopedia (CCLE). b NUAK1 expression levels were analyzed by western blot in 12 cancer cell lines and 2 normal cell lines. c A549, NCI-H460, MNK45, and U251 cells were treated with 20 μM NUAK1 inhibitor WZ4003 for 0, 4, 8, and 12 h and analyzed by western blot.

Techniques: Staining, Western Blot, Marker, Fluorescence, Labeling

Journal: Cell Death & Disease

Article Title: Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities

doi: 10.1038/s41419-020-02885-0

Figure Lengend Snippet: a The homology model of NUAK1. b Ramachandran plot for NUAK1. Dark green dots represent the residues in favored regions; yellow dots represent the residues in allowed regions, and the red cross represents the residues in irrational regions. c The 2D-binding mode of MRT68921 with NUAK1. d The 3D-binding mode of MRT68921 with NUAK1. e The surface-binding mode of MRT68921 with NUAK1. MRT68921 is colored in cyan, and the surrounding residues in the binding pockets are colored in green. The backbone of the receptor is depicted as a light blue cartoon. f The docking scores of MRT68921, WZ4003, and HTH-01-015 binding with human protein NUAK1.

Article Snippet: Fig. 1 NUAK1 is overexpressed in different types of cancer cells, and NUAK1 inhibition induces ULK1-dependent autophagy formation. a Expression levels of NUAK1 were screened in the Broad Institute Cancer Cell Line Encyclopedia (CCLE). b NUAK1 expression levels were analyzed by western blot in 12 cancer cell lines and 2 normal cell lines. c A549, NCI-H460, MNK45, and U251 cells were treated with 20 μM NUAK1 inhibitor WZ4003 for 0, 4, 8, and 12 h and analyzed by western blot.

Techniques: Binding Assay

Journal: Cell Death & Disease

Article Title: Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities

doi: 10.1038/s41419-020-02885-0

Figure Lengend Snippet: a Twelve cancer cell lines were treated with MRT68921 at different concentrations (from 0 to 10 μM) for 24 h, followed by analysis for cytotoxic effects with CCK-8 assay ( n = 3). b 293 T and HUVECs were treated with MRT68921 (from 0 to 40 μM) for 24 h and evaluated by CCK-8 assay. MRT68921 has a significantly stronger cytotoxic effect on cancer cells than normal cells ( n = 3). c NCI-H460 and MNK45 cells were treated with MRT68921 (from 0 to 10 μM) for 24 h, followed by apoptosis analysis with Annexin V/PI staining. A significant increase in both early and late apoptotic populations was observed. d NCI-H460 cells were treated with three different concentrations of MRT68921 (0, 1, and 5 μM) for 8 h, followed by ROS detection with DCFH-DA staining. Treatment with 5 μM MRT68921 induces elevated ROS levels. e U251 and MNK45 cells were treated with different concentrations of MRT68921 (from 0 to 5 μM) for 8 h, followed by western blot. f , Quantifications of cleavage PARP1/PARP1, p-Gsk3β/Gsk3β, p-MYPT1/MYPT1, NUAK1/GAPDH, p62/GAPDH, and LC3B/GAPDH ( n = 3).

Article Snippet: Fig. 1 NUAK1 is overexpressed in different types of cancer cells, and NUAK1 inhibition induces ULK1-dependent autophagy formation. a Expression levels of NUAK1 were screened in the Broad Institute Cancer Cell Line Encyclopedia (CCLE). b NUAK1 expression levels were analyzed by western blot in 12 cancer cell lines and 2 normal cell lines. c A549, NCI-H460, MNK45, and U251 cells were treated with 20 μM NUAK1 inhibitor WZ4003 for 0, 4, 8, and 12 h and analyzed by western blot.

Techniques: CCK-8 Assay, Staining, Western Blot