Journal: Oncology Letters

Article Title: Quantitative proteomic analysis of the miR-148a-associated mechanisms of metastasis in non-small cell lung cancer

doi: 10.3892/ol.2018.8581

Figure Lengend Snippet: Primer sequences used in experiments.

Article Snippet: Subsequent to blocking (20°C for 1 h) in 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with rabbit primary antibodies against the following: DNA methyltransferase 1 (DNMT1, dilution 1:500, D160261), matrix metallopeptidase 15 (MMP15, 1:500 dilution, D120991), Rho-associated protein kinase 1 (ROCK1, 1:1,000 dilution, D221198), sphingosine-1-phosphate receptor 1 (S1PR1, 1:500 dilution, D161195), cholecystokinin B receptor (CCKBR, 1:500 dilution, D160389), WNT1 (1:200 dilution, D261302), (MMP7, 1:500 dilution, D120096), actinin α4 (ACTN4, 1:500 dilution, D221929), fumarate hydratase (FH, 1:500 dilution, D222390), heat shock protein β1 (HSPB1, 1:500 dilution, D163024), lactate dehydrogenase (LDHB, 1:1,000 dilution, D151002), fatty acid synthase (FASN, 1:200 dilution, D190620) and catalase (CAT, 1:500 dilution, D122036) (all from BBI Life Sciences Corporation, Shanghai, China), vimentin (VIM, 1:200 dilution, sc-5565) and laminin β3 (LAMB3, 1:200 dilution, sc-20775) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphoglycerate dehydrogenase (PHGDH; 1:200 dilution, AP2936c; Abgent, San Diego, CA, USA) and isocitrate dehydrogenase (NADP + ) 2 (IDH2, 1:200 dilution, ab131263; Epitomics, Burlingame, CA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Quantitative proteomic analysis of the miR-148a-associated mechanisms of metastasis in non-small cell lung cancer

doi: 10.3892/ol.2018.8581

Figure Lengend Snippet: Differentially expressed proteins in microRNA-148a inhibitor-transfected cells compared with control cells.

Article Snippet: Subsequent to blocking (20°C for 1 h) in 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with rabbit primary antibodies against the following: DNA methyltransferase 1 (DNMT1, dilution 1:500, D160261), matrix metallopeptidase 15 (MMP15, 1:500 dilution, D120991), Rho-associated protein kinase 1 (ROCK1, 1:1,000 dilution, D221198), sphingosine-1-phosphate receptor 1 (S1PR1, 1:500 dilution, D161195), cholecystokinin B receptor (CCKBR, 1:500 dilution, D160389), WNT1 (1:200 dilution, D261302), (MMP7, 1:500 dilution, D120096), actinin α4 (ACTN4, 1:500 dilution, D221929), fumarate hydratase (FH, 1:500 dilution, D222390), heat shock protein β1 (HSPB1, 1:500 dilution, D163024), lactate dehydrogenase (LDHB, 1:1,000 dilution, D151002), fatty acid synthase (FASN, 1:200 dilution, D190620) and catalase (CAT, 1:500 dilution, D122036) (all from BBI Life Sciences Corporation, Shanghai, China), vimentin (VIM, 1:200 dilution, sc-5565) and laminin β3 (LAMB3, 1:200 dilution, sc-20775) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphoglycerate dehydrogenase (PHGDH; 1:200 dilution, AP2936c; Abgent, San Diego, CA, USA) and isocitrate dehydrogenase (NADP + ) 2 (IDH2, 1:200 dilution, ab131263; Epitomics, Burlingame, CA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Quantitative proteomic analysis of the miR-148a-associated mechanisms of metastasis in non-small cell lung cancer

doi: 10.3892/ol.2018.8581

Figure Lengend Snippet: Identified differentially expressed proteins verification by RT-qPCR and western blot analysis. (A) MicroRNA levels of 8 upregulated proteins (MYH9, CD44, ITGB1, LAMB3, PHGDH, ACTN4, LMNA and VIM) and 8 downregulated proteins (PSMA7, MTHFD1, GSTM3, FH, HSPB1, LRPPRC, CPT2 and IDH2) were determined by RT-qPCR following the transfection of SPC-A-1 cells with an miR-148a inhibitor or control oligonucleotide. β-actin served as an internal control. (B) Protein levels of LAMB3, VIM, PHGDH, ACTN4, FH, HSPB1, IDH2, LDHB, FASN and CAT were determined by western blot analysis following the transfection of SPC-A-1 cells with a miR-148a inhibitor or control oligonucleotide. β-actin served as an internal control. The data were representative of three independent experiments. *P<0.05. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MYH9, myosin heavy chain 9; CD44, cluster of differentiation 44; ITGB1, integrin β1; LAMB3, laminin subunit β3; PHGDH, phosphoglycerate dehydrogenase; ACTN4, α-actinin-4; LMNA, lamin A/C; VIM, vimentin; PSMA7, proteasome subunit α7; MTHFD1, methylenetetrahydrofolate dehydrogenase; GSTM3, glutathione S-transferase M3; FH, fumarate hydratase; HSPB1, heat shock protein β1; LRPPRC, leucine rich pentatricopeptide repeat; CPT2, carnitine palmitoyltransferase 2; IDH2, isocitrate dehydrogenase 2; LDHB, lactate dehydrogenase B; FASN, fatty acid synthase; CAT, catalase.

Article Snippet: Subsequent to blocking (20°C for 1 h) in 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with rabbit primary antibodies against the following: DNA methyltransferase 1 (DNMT1, dilution 1:500, D160261), matrix metallopeptidase 15 (MMP15, 1:500 dilution, D120991), Rho-associated protein kinase 1 (ROCK1, 1:1,000 dilution, D221198), sphingosine-1-phosphate receptor 1 (S1PR1, 1:500 dilution, D161195), cholecystokinin B receptor (CCKBR, 1:500 dilution, D160389), WNT1 (1:200 dilution, D261302), (MMP7, 1:500 dilution, D120096), actinin α4 (ACTN4, 1:500 dilution, D221929), fumarate hydratase (FH, 1:500 dilution, D222390), heat shock protein β1 (HSPB1, 1:500 dilution, D163024), lactate dehydrogenase (LDHB, 1:1,000 dilution, D151002), fatty acid synthase (FASN, 1:200 dilution, D190620) and catalase (CAT, 1:500 dilution, D122036) (all from BBI Life Sciences Corporation, Shanghai, China), vimentin (VIM, 1:200 dilution, sc-5565) and laminin β3 (LAMB3, 1:200 dilution, sc-20775) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphoglycerate dehydrogenase (PHGDH; 1:200 dilution, AP2936c; Abgent, San Diego, CA, USA) and isocitrate dehydrogenase (NADP + ) 2 (IDH2, 1:200 dilution, ab131263; Epitomics, Burlingame, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Quantitative proteomic analysis of the miR-148a-associated mechanisms of metastasis in non-small cell lung cancer

doi: 10.3892/ol.2018.8581

Figure Lengend Snippet: Significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways associated with the identified differentially expressed proteins.

Article Snippet: Subsequent to blocking (20°C for 1 h) in 5% non-fat milk in PBS, the membranes were incubated overnight at 4°C with rabbit primary antibodies against the following: DNA methyltransferase 1 (DNMT1, dilution 1:500, D160261), matrix metallopeptidase 15 (MMP15, 1:500 dilution, D120991), Rho-associated protein kinase 1 (ROCK1, 1:1,000 dilution, D221198), sphingosine-1-phosphate receptor 1 (S1PR1, 1:500 dilution, D161195), cholecystokinin B receptor (CCKBR, 1:500 dilution, D160389), WNT1 (1:200 dilution, D261302), (MMP7, 1:500 dilution, D120096), actinin α4 (ACTN4, 1:500 dilution, D221929), fumarate hydratase (FH, 1:500 dilution, D222390), heat shock protein β1 (HSPB1, 1:500 dilution, D163024), lactate dehydrogenase (LDHB, 1:1,000 dilution, D151002), fatty acid synthase (FASN, 1:200 dilution, D190620) and catalase (CAT, 1:500 dilution, D122036) (all from BBI Life Sciences Corporation, Shanghai, China), vimentin (VIM, 1:200 dilution, sc-5565) and laminin β3 (LAMB3, 1:200 dilution, sc-20775) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphoglycerate dehydrogenase (PHGDH; 1:200 dilution, AP2936c; Abgent, San Diego, CA, USA) and isocitrate dehydrogenase (NADP + ) 2 (IDH2, 1:200 dilution, ab131263; Epitomics, Burlingame, CA, USA).

Techniques: