Journal: Stem cell research

Article Title: Establishment and characterization of three human pluripotent stem cell lines from Charcot-Marie-Tooth disease Type 4B3 patients bearing mutations in MTMR5/Sbf1 gene

doi: 10.1016/j.scr.2024.103599

Figure Lengend Snippet: Reagents details

Article Snippet: , Rabbit anti-HB9 , 1:800 , Thermo Fisher Cat# PA5–23407 , AB_2540929.

Techniques: Immunocytochemistry, Reverse Transcription Polymerase Chain Reaction, Touchdown PCR

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 is upregulated in LUAD. GEPIA database exhibited (A) the MNX1 expression level in tumor tissues of LUAD and (B) the overall survival of LUAD patients with high or low expression of MNX1. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines (PC-9, A549, H1975 and H3255) were used to examine the (C) mRNA level and (D) protein expression of MNX1 using reverse transcription-quantitative PCR and western blotting, respectively. *P<0.05, **P<0.01, ***P<0.001 vs. BEAS-2B. MNX1, Motor neuron and pancreas homeobox 1; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: After being blocked with 5% skimmed milk at room temperature for 2 h, the membranes were probed overnight with primary antibodies against MNX1 (dilution 1:1,000; cat. no. 41983; Cell Signaling Technology, Inc.), Ki67 (dilution 1:1,000; cat. no. ab16667; Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:3,000; cat. no. ab29; Abcam), LaminB1 (dilution 1:10,000; cat. no. ab16048; Abcam), MMP2 (dilution 1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (dilution 1:1,000; cat. no. 10375-1-AP; Proteintech Group, Inc.), Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), p-Histone H2A.X (dilution 1:1,000; cat. no. 9718; Cell Signaling Technology, Inc.), Bcl-2 (dilution 1:1,000; cat. no. ab196495; Abcam), Bax (dilution 1:1,000; cat. no. ab32503; Abcam), cleaved-caspase3 (dilution 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), cleaved-caspase9 (dilution 1:1,000; cat. no. 7237; Cell Signaling Technology, Inc.), CCDC34 (dilution 1:1,000; cat. no. ab122396; Abcam) and GAPDH (dilution 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit (dilution 1:3,000; cat. no. ab6721; Abcam) or goat anti-mouse (dilution 1:2,000; cat. no. Ab6789; Abcam) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Gene Expression

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown represses proliferation, migration and invasion of A549 cells. Following transfection with sh-NC or sh-MNX1-1/sh-MNX1-2 in A549 cells, (A) the mRNA level and (B) protein expression of MNX1 were examined using reverse transcription-quantitative PCR and western blotting, respectively. (C) CCK-8 assay was used to examine cell viability. (D) Cell colony formation assay was conducted to assay formed colonies. (E-G) Wound-healing and Transwell assays were performed to examine cell migration and invasion, respectively. Magnification, ×100. (H) Protein expression of Ki67, PCNA, MMP2 and MMP9 was examined using western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; NC, negative control (empty vector); CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen.

Article Snippet: After being blocked with 5% skimmed milk at room temperature for 2 h, the membranes were probed overnight with primary antibodies against MNX1 (dilution 1:1,000; cat. no. 41983; Cell Signaling Technology, Inc.), Ki67 (dilution 1:1,000; cat. no. ab16667; Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:3,000; cat. no. ab29; Abcam), LaminB1 (dilution 1:10,000; cat. no. ab16048; Abcam), MMP2 (dilution 1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (dilution 1:1,000; cat. no. 10375-1-AP; Proteintech Group, Inc.), Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), p-Histone H2A.X (dilution 1:1,000; cat. no. 9718; Cell Signaling Technology, Inc.), Bcl-2 (dilution 1:1,000; cat. no. ab196495; Abcam), Bax (dilution 1:1,000; cat. no. ab32503; Abcam), cleaved-caspase3 (dilution 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), cleaved-caspase9 (dilution 1:1,000; cat. no. 7237; Cell Signaling Technology, Inc.), CCDC34 (dilution 1:1,000; cat. no. ab122396; Abcam) and GAPDH (dilution 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit (dilution 1:3,000; cat. no. ab6721; Abcam) or goat anti-mouse (dilution 1:2,000; cat. no. Ab6789; Abcam) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature.

Techniques: Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Colony Assay, shRNA, Negative Control, Plasmid Preparation, Cell Counting

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown hinders cell cycle progression and promotes apoptosis of A549 cells. Following transfection with sh-NC or sh-MNX1 in A549 cells, (A) Cell cycle distribution was assessed using flow cytometry analysis. (B) Protein expression of cyclin B1 and p-Histone H2A.X was detected using western blotting. (C) Cell apoptosis rate was detected using flow cytometry analysis. (D) Protein expression of Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9 was detected using western blotting. **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; NC, negative control (empty vector); p-, phosphorylated.

Article Snippet: After being blocked with 5% skimmed milk at room temperature for 2 h, the membranes were probed overnight with primary antibodies against MNX1 (dilution 1:1,000; cat. no. 41983; Cell Signaling Technology, Inc.), Ki67 (dilution 1:1,000; cat. no. ab16667; Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:3,000; cat. no. ab29; Abcam), LaminB1 (dilution 1:10,000; cat. no. ab16048; Abcam), MMP2 (dilution 1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (dilution 1:1,000; cat. no. 10375-1-AP; Proteintech Group, Inc.), Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), p-Histone H2A.X (dilution 1:1,000; cat. no. 9718; Cell Signaling Technology, Inc.), Bcl-2 (dilution 1:1,000; cat. no. ab196495; Abcam), Bax (dilution 1:1,000; cat. no. ab32503; Abcam), cleaved-caspase3 (dilution 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), cleaved-caspase9 (dilution 1:1,000; cat. no. 7237; Cell Signaling Technology, Inc.), CCDC34 (dilution 1:1,000; cat. no. ab122396; Abcam) and GAPDH (dilution 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit (dilution 1:3,000; cat. no. ab6721; Abcam) or goat anti-mouse (dilution 1:2,000; cat. no. Ab6789; Abcam) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature.

Techniques: Knockdown, Transfection, Flow Cytometry, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown inhibits tumor growth in vivo. An in vivo LUAD animal model was established and the (A) body weight and (B) tumor volume were monitored every 3 days. (C) After sacrifice, the tumors were taken out and images captured. (D) Tumor weight was recorded. (E) Protein expression of Ki67 and PCNA in tumor tissues was detected using western blotting. **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; LUAD, Lung adenocarcinoma; sh, short hairpin RNA; NC, negative control (empty vector); PCNA, proliferating cell nuclear antigen.

Article Snippet: After being blocked with 5% skimmed milk at room temperature for 2 h, the membranes were probed overnight with primary antibodies against MNX1 (dilution 1:1,000; cat. no. 41983; Cell Signaling Technology, Inc.), Ki67 (dilution 1:1,000; cat. no. ab16667; Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:3,000; cat. no. ab29; Abcam), LaminB1 (dilution 1:10,000; cat. no. ab16048; Abcam), MMP2 (dilution 1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (dilution 1:1,000; cat. no. 10375-1-AP; Proteintech Group, Inc.), Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), p-Histone H2A.X (dilution 1:1,000; cat. no. 9718; Cell Signaling Technology, Inc.), Bcl-2 (dilution 1:1,000; cat. no. ab196495; Abcam), Bax (dilution 1:1,000; cat. no. ab32503; Abcam), cleaved-caspase3 (dilution 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), cleaved-caspase9 (dilution 1:1,000; cat. no. 7237; Cell Signaling Technology, Inc.), CCDC34 (dilution 1:1,000; cat. no. ab122396; Abcam) and GAPDH (dilution 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit (dilution 1:3,000; cat. no. ab6721; Abcam) or goat anti-mouse (dilution 1:2,000; cat. no. Ab6789; Abcam) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature.

Techniques: Knockdown, In Vivo, Animal Model, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 binds to CCDC34 promoter and transcriptionally regulates CCDC34 expression. (A) GEPIA database exhibited the CCDC34 expression level in tumor tissues of LUAD. *P<0.05. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines A549 were applied to examine the (B) mRNA level and (C) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. BEAS-2B. A549 cells were transfected with oe-NC or oe-MNX1 and the (D) mRNA level and (E) protein expression of MNX1 using reverse RT-qPCR and western blotting, respectively. A549 cells were transfected with oe-MNX1 or sh-MNX1 or their negative controls and the (F) mRNA level and (G) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. oe-NC; ## P<0.01, ### P<0.001 vs. sh-NC. (H) As predicted from JASPAR website, MNX1 might bind to CCDC34 promoter. (I) Luciferase reporter assay was performed to verify the interaction between MNX1 and CCDC34 promoter and the luciferase activity was examined using dual-luciferase reporter system. ***P<0.001 vs. oe-NC. (J) ChIP assay was used to verify the interaction between MNX1 and CCDC34 promoter. ***P<0.001 vs. anti-IgG. MNX1, Motor neuron and pancreas homeobox 1; CCDC34, coiled-coil domain-containing 34; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector); RT-qPCR, Reverse transcription-quantitative PCR; ChIP, chromatin immunoprecipitation.

Article Snippet: After being blocked with 5% skimmed milk at room temperature for 2 h, the membranes were probed overnight with primary antibodies against MNX1 (dilution 1:1,000; cat. no. 41983; Cell Signaling Technology, Inc.), Ki67 (dilution 1:1,000; cat. no. ab16667; Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:3,000; cat. no. ab29; Abcam), LaminB1 (dilution 1:10,000; cat. no. ab16048; Abcam), MMP2 (dilution 1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (dilution 1:1,000; cat. no. 10375-1-AP; Proteintech Group, Inc.), Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), p-Histone H2A.X (dilution 1:1,000; cat. no. 9718; Cell Signaling Technology, Inc.), Bcl-2 (dilution 1:1,000; cat. no. ab196495; Abcam), Bax (dilution 1:1,000; cat. no. ab32503; Abcam), cleaved-caspase3 (dilution 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), cleaved-caspase9 (dilution 1:1,000; cat. no. 7237; Cell Signaling Technology, Inc.), CCDC34 (dilution 1:1,000; cat. no. ab122396; Abcam) and GAPDH (dilution 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit (dilution 1:3,000; cat. no. ab6721; Abcam) or goat anti-mouse (dilution 1:2,000; cat. no. Ab6789; Abcam) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Reporter Assay, Activity Assay, Gene Expression, shRNA, Over Expression, Negative Control, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: CCDC34 overexpression abolishes the inhibitory effect of MNX1 knockdown on proliferation, migration and invasion in A549 cells. Following transfection with oe-NC or oe-CCDC34 in A549 cells, (A) the mRNA level and (B) protein expression of CCDC34 were examined using reverse transcription-quantitative PCR and western blotting, respectively. ***P<0.001 vs. oe-NC. (C) CCK-8 assay was used to examine cell viability. (D) Cell colony formation assay was conducted to assay formed colonies. (E-G) Wound-healing and Transwell assays were performed to examine cell migration and invasion, respectively. Magnification, ×100. (H) Protein expression of Ki67, PCNA, MMP2 and MMP9 was examined using western blot. *P<0.05, ***P<0.001 vs. sh-NC. ## P<0.01, ### P<0.001 vs. sh-MNX1+oe-NC. CCDC34, coiled-coil domain-containing 34; MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector); PCNA, proliferating cell nuclear antigen.

Article Snippet: After being blocked with 5% skimmed milk at room temperature for 2 h, the membranes were probed overnight with primary antibodies against MNX1 (dilution 1:1,000; cat. no. 41983; Cell Signaling Technology, Inc.), Ki67 (dilution 1:1,000; cat. no. ab16667; Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:3,000; cat. no. ab29; Abcam), LaminB1 (dilution 1:10,000; cat. no. ab16048; Abcam), MMP2 (dilution 1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (dilution 1:1,000; cat. no. 10375-1-AP; Proteintech Group, Inc.), Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), p-Histone H2A.X (dilution 1:1,000; cat. no. 9718; Cell Signaling Technology, Inc.), Bcl-2 (dilution 1:1,000; cat. no. ab196495; Abcam), Bax (dilution 1:1,000; cat. no. ab32503; Abcam), cleaved-caspase3 (dilution 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), cleaved-caspase9 (dilution 1:1,000; cat. no. 7237; Cell Signaling Technology, Inc.), CCDC34 (dilution 1:1,000; cat. no. ab122396; Abcam) and GAPDH (dilution 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit (dilution 1:3,000; cat. no. ab6721; Abcam) or goat anti-mouse (dilution 1:2,000; cat. no. Ab6789; Abcam) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature.

Techniques: Over Expression, Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Colony Assay, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: CCDC34 overexpression abolishes the function of MNX1 knockdown on cell cycle and apoptosis in A549 cells. (A) Cell cycle distribution was assessed using flow cytometry analysis. (B) Protein expression of cyclin B1 and p-Histone H2A.X was detected using western blotting. (C) Cell apoptosis rate was detected using flow cytometry analysis. (D) Protein expression of Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9 was detected using western blotting. ***P<0.001 vs. sh-NC; # P<0.05, ### P<0.001 vs. sh-MNX1+oe-NC. CCDC34, coiled-coil domain-containing 34; MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector).

Article Snippet: After being blocked with 5% skimmed milk at room temperature for 2 h, the membranes were probed overnight with primary antibodies against MNX1 (dilution 1:1,000; cat. no. 41983; Cell Signaling Technology, Inc.), Ki67 (dilution 1:1,000; cat. no. ab16667; Abcam), proliferating cell nuclear antigen (PCNA; dilution 1:3,000; cat. no. ab29; Abcam), LaminB1 (dilution 1:10,000; cat. no. ab16048; Abcam), MMP2 (dilution 1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (dilution 1:1,000; cat. no. 10375-1-AP; Proteintech Group, Inc.), Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), p-Histone H2A.X (dilution 1:1,000; cat. no. 9718; Cell Signaling Technology, Inc.), Bcl-2 (dilution 1:1,000; cat. no. ab196495; Abcam), Bax (dilution 1:1,000; cat. no. ab32503; Abcam), cleaved-caspase3 (dilution 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.), cleaved-caspase9 (dilution 1:1,000; cat. no. 7237; Cell Signaling Technology, Inc.), CCDC34 (dilution 1:1,000; cat. no. ab122396; Abcam) and GAPDH (dilution 1:2,500; cat. no. ab9485; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit (dilution 1:3,000; cat. no. ab6721; Abcam) or goat anti-mouse (dilution 1:2,000; cat. no. Ab6789; Abcam) secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature.

Techniques: Over Expression, Knockdown, Flow Cytometry, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 is upregulated in LUAD. GEPIA database exhibited (A) the MNX1 expression level in tumor tissues of LUAD and (B) the overall survival of LUAD patients with high or low expression of MNX1. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines (PC-9, A549, H1975 and H3255) were used to examine the (C) mRNA level and (D) protein expression of MNX1 using reverse transcription-quantitative PCR and western blotting, respectively. *P<0.05, **P<0.01, ***P<0.001 vs. BEAS-2B. MNX1, Motor neuron and pancreas homeobox 1; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis.

Article Snippet: Subsequently, 2% of the supernatant served as the input, and the remaining 100 μl of the supernatants were cultured overnight with 60 μl A/G agarose beads (Cell Signaling Technology, Inc.) at 4°C, followed by incubation with anti-MNX1 antibody (dilution 1:50; cat. no. 41983; Cell Signaling Technology, Inc.) or the negative control IgG (dilution 1:50; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Gene Expression

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown represses proliferation, migration and invasion of A549 cells. Following transfection with sh-NC or sh-MNX1-1/sh-MNX1-2 in A549 cells, (A) the mRNA level and (B) protein expression of MNX1 were examined using reverse transcription-quantitative PCR and western blotting, respectively. (C) CCK-8 assay was used to examine cell viability. (D) Cell colony formation assay was conducted to assay formed colonies. (E-G) Wound-healing and Transwell assays were performed to examine cell migration and invasion, respectively. Magnification, ×100. (H) Protein expression of Ki67, PCNA, MMP2 and MMP9 was examined using western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; NC, negative control (empty vector); CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen.

Article Snippet: Subsequently, 2% of the supernatant served as the input, and the remaining 100 μl of the supernatants were cultured overnight with 60 μl A/G agarose beads (Cell Signaling Technology, Inc.) at 4°C, followed by incubation with anti-MNX1 antibody (dilution 1:50; cat. no. 41983; Cell Signaling Technology, Inc.) or the negative control IgG (dilution 1:50; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Colony Assay, shRNA, Negative Control, Plasmid Preparation, Cell Counting

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown hinders cell cycle progression and promotes apoptosis of A549 cells. Following transfection with sh-NC or sh-MNX1 in A549 cells, (A) Cell cycle distribution was assessed using flow cytometry analysis. (B) Protein expression of cyclin B1 and p-Histone H2A.X was detected using western blotting. (C) Cell apoptosis rate was detected using flow cytometry analysis. (D) Protein expression of Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9 was detected using western blotting. **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; NC, negative control (empty vector); p-, phosphorylated.

Article Snippet: Subsequently, 2% of the supernatant served as the input, and the remaining 100 μl of the supernatants were cultured overnight with 60 μl A/G agarose beads (Cell Signaling Technology, Inc.) at 4°C, followed by incubation with anti-MNX1 antibody (dilution 1:50; cat. no. 41983; Cell Signaling Technology, Inc.) or the negative control IgG (dilution 1:50; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Knockdown, Transfection, Flow Cytometry, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 knockdown inhibits tumor growth in vivo. An in vivo LUAD animal model was established and the (A) body weight and (B) tumor volume were monitored every 3 days. (C) After sacrifice, the tumors were taken out and images captured. (D) Tumor weight was recorded. (E) Protein expression of Ki67 and PCNA in tumor tissues was detected using western blotting. **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; LUAD, Lung adenocarcinoma; sh, short hairpin RNA; NC, negative control (empty vector); PCNA, proliferating cell nuclear antigen.

Article Snippet: Subsequently, 2% of the supernatant served as the input, and the remaining 100 μl of the supernatants were cultured overnight with 60 μl A/G agarose beads (Cell Signaling Technology, Inc.) at 4°C, followed by incubation with anti-MNX1 antibody (dilution 1:50; cat. no. 41983; Cell Signaling Technology, Inc.) or the negative control IgG (dilution 1:50; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Knockdown, In Vivo, Animal Model, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: MNX1 binds to CCDC34 promoter and transcriptionally regulates CCDC34 expression. (A) GEPIA database exhibited the CCDC34 expression level in tumor tissues of LUAD. *P<0.05. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines A549 were applied to examine the (B) mRNA level and (C) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. BEAS-2B. A549 cells were transfected with oe-NC or oe-MNX1 and the (D) mRNA level and (E) protein expression of MNX1 using reverse RT-qPCR and western blotting, respectively. A549 cells were transfected with oe-MNX1 or sh-MNX1 or their negative controls and the (F) mRNA level and (G) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. oe-NC; ## P<0.01, ### P<0.001 vs. sh-NC. (H) As predicted from JASPAR website, MNX1 might bind to CCDC34 promoter. (I) Luciferase reporter assay was performed to verify the interaction between MNX1 and CCDC34 promoter and the luciferase activity was examined using dual-luciferase reporter system. ***P<0.001 vs. oe-NC. (J) ChIP assay was used to verify the interaction between MNX1 and CCDC34 promoter. ***P<0.001 vs. anti-IgG. MNX1, Motor neuron and pancreas homeobox 1; CCDC34, coiled-coil domain-containing 34; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector); RT-qPCR, Reverse transcription-quantitative PCR; ChIP, chromatin immunoprecipitation.

Article Snippet: Subsequently, 2% of the supernatant served as the input, and the remaining 100 μl of the supernatants were cultured overnight with 60 μl A/G agarose beads (Cell Signaling Technology, Inc.) at 4°C, followed by incubation with anti-MNX1 antibody (dilution 1:50; cat. no. 41983; Cell Signaling Technology, Inc.) or the negative control IgG (dilution 1:50; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Reporter Assay, Activity Assay, Gene Expression, shRNA, Over Expression, Negative Control, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: CCDC34 overexpression abolishes the inhibitory effect of MNX1 knockdown on proliferation, migration and invasion in A549 cells. Following transfection with oe-NC or oe-CCDC34 in A549 cells, (A) the mRNA level and (B) protein expression of CCDC34 were examined using reverse transcription-quantitative PCR and western blotting, respectively. ***P<0.001 vs. oe-NC. (C) CCK-8 assay was used to examine cell viability. (D) Cell colony formation assay was conducted to assay formed colonies. (E-G) Wound-healing and Transwell assays were performed to examine cell migration and invasion, respectively. Magnification, ×100. (H) Protein expression of Ki67, PCNA, MMP2 and MMP9 was examined using western blot. *P<0.05, ***P<0.001 vs. sh-NC. ## P<0.01, ### P<0.001 vs. sh-MNX1+oe-NC. CCDC34, coiled-coil domain-containing 34; MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector); PCNA, proliferating cell nuclear antigen.

Article Snippet: Subsequently, 2% of the supernatant served as the input, and the remaining 100 μl of the supernatants were cultured overnight with 60 μl A/G agarose beads (Cell Signaling Technology, Inc.) at 4°C, followed by incubation with anti-MNX1 antibody (dilution 1:50; cat. no. 41983; Cell Signaling Technology, Inc.) or the negative control IgG (dilution 1:50; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Over Expression, Knockdown, Migration, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Colony Assay, shRNA, Negative Control, Plasmid Preparation

Journal: Oncology Letters

Article Title: MNX1 facilitates the malignant progress of lung adenocarcinoma through transcriptionally upregulating CCDC34

doi: 10.3892/ol.2023.13911

Figure Lengend Snippet: CCDC34 overexpression abolishes the function of MNX1 knockdown on cell cycle and apoptosis in A549 cells. (A) Cell cycle distribution was assessed using flow cytometry analysis. (B) Protein expression of cyclin B1 and p-Histone H2A.X was detected using western blotting. (C) Cell apoptosis rate was detected using flow cytometry analysis. (D) Protein expression of Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9 was detected using western blotting. ***P<0.001 vs. sh-NC; # P<0.05, ### P<0.001 vs. sh-MNX1+oe-NC. CCDC34, coiled-coil domain-containing 34; MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector).

Article Snippet: Subsequently, 2% of the supernatant served as the input, and the remaining 100 μl of the supernatants were cultured overnight with 60 μl A/G agarose beads (Cell Signaling Technology, Inc.) at 4°C, followed by incubation with anti-MNX1 antibody (dilution 1:50; cat. no. 41983; Cell Signaling Technology, Inc.) or the negative control IgG (dilution 1:50; cat. no. 2729; Cell Signaling Technology, Inc.) at 4°C overnight.

Techniques: Over Expression, Knockdown, Flow Cytometry, Expressing, Western Blot, shRNA, Negative Control, Plasmid Preparation