Journal: Toxics

Article Title: Integrated ceRNA Network Analysis in Silica-Induced Pulmonary Fibrosis and Discovery of miRNA Biomarkers

doi: 10.3390/toxics14010063

Figure Lengend Snippet: Identification of core ncRNAs and genes in key metabolic pathways. ( A ) Core ncRNAs and genes associated with the ECM–receptor interaction pathway, NF-κB signaling pathway, TNF signaling pathway, and PI3K-Akt signaling pathway. ( B ) Validation of expression levels for the core ncRNAs and genes by quantitative real-time PCR ( n = 3, *** p < 0.001 vs. the saline group).

Article Snippet: Amplification and detection were carried out on an Applied Biosystems QuantStudio 3 Real-Time PCR System using the Tiangen SuperReal PreMix Plus (SYBR Green) Kit (Beijing, China) (Cat. Nos.

Techniques: Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Saline

Journal: Molecular oncology

Article Title: APOBEC3C-mediated NF-κB activation enhances clear cell renal cell carcinoma progression.

doi: 10.1002/1878-0261.13721

Figure Lengend Snippet: Fig. 3. A3C associates with NF-jB signaling pathway regulator mRNAs. (A) 786-O A3C recovery (Rec) cells were used for immunoprecipitation (IP) with an anti-GFP antibody and subjected to western blot analyses (n = 3). Ribosomal protein L7 (RPL7) and vinculin (VCL) served as positive and negative controls, respectively. Beads incubated with a non-targeting antibody (IgG) were used as specificity control. (B) Inputs and normalized RNA co-immunoprecipitation (RIP)-seq reads of A3C-IPs (n = 3) were investigated at four Y RNA loci to assess the reported association of A3C with Y RNAs. (C) The venn diagram shows the overlap of RIP-seq enriched transcripts (FC (fold change) ≥2; P < 0.05; RPKM (reads per kilobase of transcript per million mapped reads) in input > 0.1) with reported NF-jB signaling regulators (gene list obtained from www.gsea-msigdb.org/gsea/msigdb/). (D) Scatter plot shows results of the RIP-seq in 786-O A3C Rec cells. High-confidence binding partners of A3C are marked in blue (A3C-IP/input > 1). Within this group, NF-jB signaling regulators are col- ored in yellow. Transcripts with a ratio of A3C-IP/input ≤1 are considered as background (gray). Transcripts with low expression (average RPKM in input < 0.1) are depicted in light gray. (E) Bar plot presents examples of enriched transcripts detected in RIP-seq of the A3C-IPs (IDS, GNG5 and ZFP36 within top 30). Reported NF-jB signaling pathway regulators are depicted in yellow. (F) Bar plot indicates the same transcripts as in (E) obtained in separate A3C-IPs (n = 3) and analyzed with real time quantitative PCR (RT-qPCR). Note that the majority of enriched NF-jB signaling regulator transcripts identified in the RIP-seq also shows enrichment in A3C-IPs analyzed by RT-qPCR (A3C-IP/ input > 1). Data are representative of three independent experiments (mean SEM in E and F).

Article Snippet: Quantitative PCR was performed based on SYBRgreen I technology using the ORA qPCR Green ROX L Mix (HighQu, Kraichtal, Germany) on a LightCycler 480 II 384 format system (Roche, Basel, Switzerland).

Techniques: Immunoprecipitation, Western Blot, Incubation, Control, Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Molecular oncology

Article Title: APOBEC3C-mediated NF-κB activation enhances clear cell renal cell carcinoma progression.

doi: 10.1002/1878-0261.13721

Figure Lengend Snippet: Fig. 4. A3C depletion results in reduced expression of NF-jB signaling pathway regulators and impaired nuclear translocation of NF-jB subunits. (A) Transcript levels of NF-jB signaling pathway regulators (marked in yellow) were analyzed upon stable A3C knockdown (shA3C) in 786-O by real time quantitative PCR (RT-qPCR, n = 3). Beta actin (ACTB) and eukaryotic elongation factor 2 (EEF2, light gray) served as negative controls. IDS and GNG5 (dark gray) are putative binding partners of A3C, but not considered NF-jB signaling pathway regulators. (B) Western blot (WB) analyses confirmed decreased expression of CDK6 and IKBKA in 786-O shA3C cells (n = 5). (C) WB indicates protein levels of the unprocessed (p100) and processed (p52) forms of NF-jB2 in 786-O shA3C cells (n = 6). (D) Phosphorylation status at Ser536 and total protein levels of RelA in 786-O shA3C cells were characterized by WB analyses (n = 3). The phosphorylation signal was normalized to total RelA protein level. (E) Subcellular fractionation was performed using 786-O shC and shA3C cells (n = 3). The distribution of the NF- jB subunits NF-jB2 and RelA among the cytoplasmic and nuclear fractions is depicted in the WB. To verify the subcellular fractionation process, EEF2 and PTB were used as positive controls for the cytoplasmic and nuclear fraction, respectively. Note that due to the usage of different buffers in the cytoplasmic and nuclear protein pool, we observed slight differences in the running behavior of the proteins. (F) The schematic depicts a putative regulatory mechanism of the NF-jB signaling pathway by A3C. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired, two-tailed Student’s t test compared to 786-O shC (A). Data are representative of three independent experiments (5–95 percentile in A). Protein levels were normalized to vinculin (VCL) and 786-O control cells (shC) in five (B), six (C) or three (D, E) biological replicates; mean SD is indicated below the representative WB (B–E).

Article Snippet: Quantitative PCR was performed based on SYBRgreen I technology using the ORA qPCR Green ROX L Mix (HighQu, Kraichtal, Germany) on a LightCycler 480 II 384 format system (Roche, Basel, Switzerland).

Techniques: Expressing, Translocation Assay, Knockdown, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Western Blot, Phospho-proteomics, Fractionation, Two Tailed Test, Control