blotswere probedwith primary anti p-akt  (Cell Signaling Technology Inc)

Journal: OncoTargets and Therapy

Article Title:

LINC00958 Promotes The Malignancy Of Nasopharyngeal Carcinoma By Sponging microRNA-625 And Thus Upregulating NUAK1

doi: 10.2147/ott.s216342

Figure Lengend Snippet: Figure 6 NUAK1 restoration rescues CNE-1 and SUNE-1 cells from miR-625 overexpression–induced inhibition of growth and metastasis in vitro. (A) MiR-625– overexpressing CNE-1 and SUNE-1 cells that were next cotransfected with either pc-NUAK1 or pcDNA3.1 were subjected to Western blotting for the measurement of NUAK1 protein expression. *P < 0.05 vs group agomir-NC. #P < 0.05 vs the agomir-625+pcDNA3.1 group. (B–D) The proliferation, apoptosis, migration, and invasiveness of CNE-1 and SUNE-1 cells treated as described above were investigated by the CCK-8 assay, flow cytometry, and Transwell assays, respectively. *P < 0.05 vs group agomir- NC. #P < 0.05 vs the agomir-625+pcDNA3.1 group.

Article Snippet: Next, the membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% of Tween 20 at 37°C for 2 h. The membranes were probedwith primary antibodies against NUAK1 (cat. No. sc-271827; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (cat. No. sc-66163; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Over Expression, Inhibition, In Vitro, Western Blot, Expressing, Migration, CCK-8 Assay, Cytometry

Journal: OncoTargets and Therapy

Article Title:

LINC00958 Promotes The Malignancy Of Nasopharyngeal Carcinoma By Sponging microRNA-625 And Thus Upregulating NUAK1

doi: 10.2147/ott.s216342

Figure Lengend Snippet: Figure 5 NUAK1 is a direct target gene of miR-625 in NPC. (A) The binding sequences of miR-625 in the 3′-UTR of NUAK1 mRNA predicted by miRDB and TargetScan. The positions of mutated nucleotides (red) in the 3′-UTR of NUAK1 mRNA are also shown. (B) CNE-1 and SUNE-1 cells that were cotransfected with either agomir-625 or agomir-NC and either NUAK1-Wt or NUAK1-Mut were harvested at 48 h post-transfection and subjected to the detection of luciferase activity. *P < 0.05 vs group agomir- NC. (C, D) RT-qPCR and Western blotting were carried out to assess the expression of NUAK1 mRNA and protein in CNE-1 and SUNE-1 cells transfected with either agomir-625 or agomir-NC. *P < 0.05 vs the agomir-NC group. (E) Quantification of NUAK1 expression in 59 pairs of NPC tissue samples and matched adjacent non-tumor nasopharyngeal epithelial tissue samples. *P < 0.05 vs non-tumor nasopharyngeal epithelial tissue samples. (F) Spearman correlation analysis was conducted to determine the correlation of miR-625 with NUAK1 expression among the NPC tissue samples. R2 = 0.3739, P < 0.0001.

Article Snippet: Next, the membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% of Tween 20 at 37°C for 2 h. The membranes were probedwith primary antibodies against NUAK1 (cat. No. sc-271827; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (cat. No. sc-66163; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: OncoTargets and Therapy

Article Title:

LINC00958 Promotes The Malignancy Of Nasopharyngeal Carcinoma By Sponging microRNA-625 And Thus Upregulating NUAK1

doi: 10.2147/ott.s216342

Figure Lengend Snippet: Figure 7 The influence of LINC00958 downregulation on the malignant characteristics of CNE-1 and SUNE-1 cells was partially reversed by the miR-625 knockdown. (A) The efficiency of antagomir-625 in CNE-1 and SUNE-1 cells was evaluated by RT-qPCR. *P < 0.05 vs the antagomir-NC group. (B, C) CNE-1 and SUNE-1 cells were cotransfected with si-LINC00958 and either antagomir-625 or antagomir-NC. The miR-625 and NUAK1 protein levels were analyzed by RT-qPCR and Western blotting, respectively. *P < 0.05 vs group si-NC. #P < 0.05 vs the si-LINC00958+antagomir-NC group. (D–F) The proliferation, apoptosis, migration, and invasiveness of the aforementioned cells were assessed by the CCK-8 assay, flow cytometry, and Transwell assays, respectively. *P < 0.05 vs group si-NC. #P < 0.05 vs the si-LINC00958 +antagomir-NC group.

Article Snippet: Next, the membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% of Tween 20 at 37°C for 2 h. The membranes were probedwith primary antibodies against NUAK1 (cat. No. sc-271827; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (cat. No. sc-66163; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Migration, CCK-8 Assay, Cytometry

Journal: OncoTargets and Therapy

Article Title:

LINC00958 Promotes The Malignancy Of Nasopharyngeal Carcinoma By Sponging microRNA-625 And Thus Upregulating NUAK1

doi: 10.2147/ott.s216342

Figure Lengend Snippet: Figure 8 LINC00958 knockdown impairs the tumor growth of NPC cells in vivo. (A) A representative image of tumor xenografts derived from si-LINC00958–transfected or si-NC–transfected CNE-1 cells. (B) Either si-LINC00958–transfected or si-NC–transfected CNE-1 cells were subcutaneously injected into nude mice. Tumor volumes were monitored every 2 days starting at 2 weeks after the injection. *P < 0.05 vs group si-NC. (C) The weight of tumor xenografts was determined at 4 weeks after the cancer cell inoculation. *P < 0.05 vs group si-NC. (D, E) The expression levels of miR-625 and of the NUAK1 protein were measured in tumor xenografts by RT-qPCR and Western blotting, respectively. *P < 0.05 vs the si-NC group. (F) RT-qPCR analysis of LINC00958 expression in tumor xenografts formed by si-LINC00958–transfected or si- NC–transfected CNE-1 cells. *P < 0.05 vs the si-NC group.

Article Snippet: Next, the membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% of Tween 20 at 37°C for 2 h. The membranes were probedwith primary antibodies against NUAK1 (cat. No. sc-271827; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (cat. No. sc-66163; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Knockdown, In Vivo, Derivative Assay, Transfection, Injection, Expressing, Quantitative RT-PCR, Western Blot