Journal: Journal of Biological Chemistry

Article Title: c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation

doi: 10.1074/jbc.m115.679563

Figure Lengend Snippet: FIGURE 1. Mouse INSC is transiently expressed during ES cell differentiation into mesoderm and endoderm. A, culture condition for differentiation of ES cells into mesoderm and endoderm through a bipotent mesendoderm state. B, FACS analysis for detecting Gsc-gfp expression after 4 days of differentiation. C, immunofluorescence images of Hoechst (blue), VEGFR-2 (green), and Sox17 (red) of mES cells at differentiation days 0 (top) and 4 (bottom). Arrowheads, Vegfr2-positive cells. Scale bars, 75 m. D–F, immunofluorescence images of Hoechst (blue) and the indicated proteins (Gsc-gfp (green) (D), Nanog (red) (E), and Oct4 (red) (F)) of mES cells at differentiation days 0 (top) and 4 (bottom). Scale bars, 75 m. G, qPCR analysis for mRNA expression of the indicated genes at differentiation days 0, 2, 3, and 4. H, Western blotting analysis for Eomes, Foxa2, T-bra, and control -tubulin proteins in ES cells at differentiation days 0, 2, 3, and 4. I, qPCR analysis for expression of Par-3, LGN, and mouse Insc at differentiation days 0, 2, 3, and 4. J, Western blotting analysis for Par-3, LGN, mouse INSC, and control -tubulin proteins in ES cells at differentiation days 0, 2, 3, and 4. Arrowheads show each indicated protein. In all qPCR analysis, values are normalized to expression of G3pdh (mean S.D. (error bars) from three experiments). *, p 0.05; **, p 0.01; ***, p 0.001 analyzed by Dunnett’s multiple-comparison test.

Article Snippet: Quantitative RT-PCR was performed with KAPATM SYBR Fast qPCR Master Mix (KAPA Biosystems) on an ABI7500 cycler (Applied Biosystems).

Techniques: Cell Differentiation, Expressing, Immunofluorescence, Western Blot, Control, Comparison

Journal: Journal of Biological Chemistry

Article Title: c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation

doi: 10.1074/jbc.m115.679563

Figure Lengend Snippet: FIGURE 4. Mouse INSC regulates mesoderm differentiation of ES cells. A, Western blotting analysis of FLAG-mouse INSC and control -tubulin proteins in HEK293TcellstransfectedwithpENTR/pEF1a/FLAG-mouseINSCvectorandmouseINSCsiRNAs(mInscsi_1andmInscsi_2)orcontrolLucisiRNA(Lucisi).B,qPCR analysis for expression of mouse Insc in ES cells transfected with mouse INSC siRNAs or Luci siRNA at differentiation days 0 and 2. C, Western blotting analysis of mouse INSC and control -tubulin proteins in ES cells transfected with mouse INSC siRNAs or Luci siRNA at differentiation days 0, 2, 3, and 4. Arrowhead, mouse INSC protein. D, qPCR analysis for expression of Gsc, Eomes, Foxa2, T-bra, Sox17, and VEGFR-2 at differentiation days 0 and 6. In all qPCR analysis in B and D, values are normalized to the expression of G3pdh (mean S.D. (error bars) from three experiments; **, p 0.01; ***, p 0.001, analyzed by Dunnett’s multiple-comparison test). E, FACS analysis for detecting Gsc-gfp (left, top) and VEGFR-2 (left, bottom) expression in ES cells transfected with mouse INSC siRNAs or Luci siRNA at differentiation day 6. The quantitative results are shown on the right (mean S.D. from three experiments; **, p 0.01, analyzed by Dunnett’s multiple-comparison test).

Article Snippet: Quantitative RT-PCR was performed with KAPATM SYBR Fast qPCR Master Mix (KAPA Biosystems) on an ABI7500 cycler (Applied Biosystems).

Techniques: Western Blot, Control, Expressing, Transfection, Comparison

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

Journal: Microbial Biotechnology

Article Title: Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

doi: 10.1111/1751-7915.14031

Figure Lengend Snippet: Schematic illustrating SARS‐CoV‐2 genome and regions targeted by RT‐qPCR primers and probes. A. Schematic overview portrays the SARS‐CoV‐2 genome with RdRP and E gene regions magnified to show the locations of primers and probes of the original Charité protocol, vDetect (v1 and v2), and rTEST RT‐qPCR assays. F, forward primer; P, probe; R, reverse primer. The inset boxes (from left to right) illustrate a SARS‐CoV‐2 particle with labelled structural proteins and RNA, legend describing panel A and the primers and probes used in each test to detect RNase P subunit p30 (RPP30). B. Diagram compares the sequences of RdRP and E gene primers and probes for the original Charité protocol, vDetect (v.1) and vDetect v.2 and rTEST RT‐qPCR assays to the Wuhan reference sequence. The numbers written above the Wuhan reference sequence correspond to the start and end base positions of the sequence Reverse primer sequences are written in the reverse complement (rc). Magenta lines and letters represent mixed bases found in the primers and probes in the Charité protocol that were replaced with the correct bases in vDetect v1 (blue lines and letters). Red lines and letters signify LNA‐modified bases.

Article Snippet: RT‐qPCR reactions were optimized on a CFX96 (Bio‐Rad), QuantStudio 5 (Agilent Technologies, CA, USA) and Mx3005P (Agilent Technologies) real time PCR detection systems using the 1Step RT qPCR Probe ROX L Kit (Cat. No. QOP0201, highQu, Germany).

Techniques: Quantitative RT-PCR, Sequencing, Modification

Journal: Microbial Biotechnology

Article Title: Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

doi: 10.1111/1751-7915.14031

Figure Lengend Snippet: Optimization, analytical sensitivity and clinical performance of a rapid, RNA extraction‐free, multiplexed RT‐qPCR assay. A. Analytical sensitivity of the triplexed E, RdRP and RNase P assay in the rTEST COVID‐19 qPCR Allplex kit. B. Clinical performance of the rTEST COVID‐19 qPCR Allplex kit. C. Optimization of gargle sample input for a rapid, RNA extraction‐free, triplexed rTEST. D. Comparison of four different thermal profiles using 8 μl of gargle input volume in rapid, direct RT‐qPCR. E. Analytical sensitivity of the triplexed E, RdRP and RNase P assay in the RNA extraction‐free rTEST COVID‐19 qPCR Rapid kit. F. Clinical performance of the rTEST COVID‐19 qPCR Rapid kit. The dotted line at C t = 40 (panels A and E) serves as a threshold after which amplification is considered invalid. The dotted lines and shaded areas (panels B and F) indicate samples that were not detected by either the evaluation test, index test or both tests. C t , cycle threshold; E, envelope gene; ND, not detected within 45 cycles; NTC, no template control; RdRP, RNA‐dependent RNA polymerase.

Article Snippet: RT‐qPCR reactions were optimized on a CFX96 (Bio‐Rad), QuantStudio 5 (Agilent Technologies, CA, USA) and Mx3005P (Agilent Technologies) real time PCR detection systems using the 1Step RT qPCR Probe ROX L Kit (Cat. No. QOP0201, highQu, Germany).

Techniques: RNA Extraction, Quantitative RT-PCR, Comparison, Amplification, Control

Journal: Microbial Biotechnology

Article Title: Sequential development of several RT‐qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS‐CoV‐2 from influenza A and B

doi: 10.1111/1751-7915.14031

Figure Lengend Snippet: Schematic illustrating influenza A and B genome and regions targeted by RT‐qPCR primers and probes. (A) Schematic overview portrays the influenza A and B genome with PB1 and PA gene regions magnified to show the locations of primers and probes. Nucleotides labelled in red text indicate mixed bases in the consensus sequences for influenza A and B. BHQ2, black hole quencher 2; F, forward primer; HA, haemagglutinin; M, matrix protein; NA, neuraminidase; NP, nucleoprotein; NS, non‐structural protein; P, probe; PA, polymerase acidic protein; PB1, polymerase basic 1 protein; PB2, polymerase basic 2 protein; R, reverse primer; seg., segment; YY, Yakima Yellow ® .

Article Snippet: RT‐qPCR reactions were optimized on a CFX96 (Bio‐Rad), QuantStudio 5 (Agilent Technologies, CA, USA) and Mx3005P (Agilent Technologies) real time PCR detection systems using the 1Step RT qPCR Probe ROX L Kit (Cat. No. QOP0201, highQu, Germany).

Techniques: Quantitative RT-PCR