rt pcr  (Thermo Fisher)

Journal: OncoTargets and therapy

Article Title: Nuclear factor-κB p65 regulates glutaminase 1 expression in human hepatocellular carcinoma

doi: 10.2147/OTT.S167408

Figure Lengend Snippet: Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

Article Snippet: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Upon relative treatment, cells were lysed, and total RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Thoracic Cancer

Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

doi: 10.1111/1759-7714.12895

Figure Lengend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

Techniques: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

Journal: Thoracic Cancer

Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

doi: 10.1111/1759-7714.12895

Figure Lengend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

Techniques: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

Journal: Communications Biology

Article Title: APOL1 risk allele RNA contributes to renal toxicity by activating protein kinase R

doi: 10.1038/s42003-018-0188-2

Figure Lengend Snippet: APOL1 risk variants activated PKR and downstream signals. a HEK293FT cells over-expressing APOL1 were harvested for detection of APOL1 and eIF2α protein by Western blotting. Renal risk variants G1 and G2 increased phospho-eIF2α compared to the G0 allele. b HEK293FT cells over-expressing APOL1 were harvested to identify which of the four eIF2 kinases (HRI, PKR, PERK, GCN2, respectively) were activated. The G0 variant showed minimally increased phospho-PKR, indicating activation, while renal risk variants G1 and G2 manifested substantially increased phospho-PKR. c HEK293FT cells over-expressing APOL1 were treated with a PKR inhibitor or vehicle control, then harvested for detection of PKR and eIF2α. The APOL1 renal risk variants increased phospho-PKR and phospho-eIF2α compared to the G0 allele, effects that were abolished or greatly diminished by PKR inhibition. d Transfected HEK293FT cells expressing APOL1 RNA encoded by the G1 and G2 variants increased interferon α and interferon β RNA expression compared to the G0 variant, as measured by qRT-PCR. e Total protein synthesis was analyzed in transfected HEK293FT cells expressing APOL1 RNA with the G0, G1, and G2 variants. Translation initiation was measured using the L-azidohomoalanine protein synthesis assay. Quantification of protein synthesis, with the APOL1 risk variants measured against the G0 transfected cells (set to 100%) and puromycin exposure (set to 0%). Both risk variants G1 and G2 reduced protein synthesis by HEK293FT cells. f Quantification of cell viability of stable HEK293 cell line expressing APOL1. The cells were lysed and incubated with CellTiter-Glo to measure the amount of ATP in the cells. HEK293 cells with the G1 and G2 variants manifested decreased cell viability compared with the G0 variants. Each dot is calculated from one well. Each horizontal line represents mean. All results are presented as ratio of controls, normalized to 100% and P values were calculated using a Student one-tailed t -test. Welch correction was used for panel ( d )

Article Snippet: Samples were analyzed by PCR (RT) or quantitative RT-PCR (qRT-PCR) using Power SYBR Green PCR master mix (ThermoFisher).

Techniques: Expressing, Western Blot, Variant Assay, Activation Assay, Inhibition, Transfection, RNA Expression, Quantitative RT-PCR, Incubation, One-tailed Test

Journal: Cancer Management and Research

Article Title: ITLNI identified by comprehensive bioinformatic analysis as a hub candidate biological target in human epithelial ovarian cancer

doi: 10.2147/CMAR.S189784

Figure Lengend Snippet: Validation of hub genes. Notes: ( A ) Survival analysis indicated that ITLN1 was a positive prognosis factor in epithelial ovarian cancer, while patients with a higher expression of ITLN1 had significantly longer overall survival compared to those with higher expression ( P =2.593e–02). ( B ) ITLN1 validation using qRT-PCR analysis. ( C, D ) Validation of ITLN1 expression from the GEO databases. Two datasets showed lower expression of ITLN1 in epithelial ovarian cancer tissues compared with normal ovarian tissues ( P

Article Snippet: Quantitative real-time RT-PCR (qRT-PCR) analysis We extracted total RNA from tissue samples using TRizol reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR