Journal: bioRxiv

Article Title: FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies

doi: 10.1101/2023.03.02.530819

Figure Lengend Snippet: A . Overall percentage of CG:TA transitions in sequencing reads after library amplification using Q5 (left), Q5U® (center) and Taq DNA polymerase (right), with (DNA repair (orange), UDG (brown)) and without (blue) DNA repair treatment of the lung tumor FFPE sample. Experiments were done in duplicate and error bars are reflecting the variability between replicates. B . Profiles of C to T transitions on read 1 (R1, left) and read 2 (R2, right) as a function of read position (in bp). Libraries were amplified using Q5 (top), Q5U® (middle) and Taq DNA polymerase (bottom). Experiments were done in duplicate. Fraction of C to T is a global count of the of C (on the reference genome) to T (on the mapped reads) normalized to the total number of C to C matches. C . Schema representing the fate of the deaminated strand (gray) and the un-damaged strand (purple) after end repair (red) and amplification with Q5 (left) and Taq or Q5U (right).

Article Snippet: The DNA repair step commonly used in this manuscript corresponds to the NEBNext FFPE DNA Repair v2 Module and contains enzymes and buffers that are optimized to repair FFPE DNA in next generation sequencing workflow including UDG, Endonuclease IV, Fpg, Endonuclease VIII, T4 PDG, Taq DNA ligase and Bst DNA polymerase.

Techniques: Sequencing, Amplification

Journal: bioRxiv

Article Title: FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies

doi: 10.1101/2023.03.02.530819

Figure Lengend Snippet: A . Overall percentage of CG:TA transitions in sequencing reads for lung (left) and pancreatic (right) tumor FFPE samples. Samples were either left untreated (orange) or treated with DNA repair mix before (red) and after (blue) end repair. B . Breakdown of the fraction of C:T transitions according to Read types, Read 1 (R1, top) and Read 2 (R2, bottom) and position on the read starting from the 5’ end. Two FFPE samples have been used for these experiments : Lung tumor FFPE sample (Blue) and pancreatic tumor FFPE sample (red) treated with DNA repair after end repair (left), before end repair (right) and no treatment (middle). Fraction of C to T is a global count of the of C (on the reference genome) to T (on the mapped reads) normalized to the total number of C to C matches. Red arrows indicate positions of increased C to T transition rate.

Article Snippet: The DNA repair step commonly used in this manuscript corresponds to the NEBNext FFPE DNA Repair v2 Module and contains enzymes and buffers that are optimized to repair FFPE DNA in next generation sequencing workflow including UDG, Endonuclease IV, Fpg, Endonuclease VIII, T4 PDG, Taq DNA ligase and Bst DNA polymerase.

Techniques: Sequencing

Journal: bioRxiv

Article Title: FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies

doi: 10.1101/2023.03.02.530819

Figure Lengend Snippet: Substitution profiles for low (less than 1% A ) and high (more than 10% B ) allelic frequency substitution. Barplot representing the number of genomic positions per megabases (mb). The barplots are color coded according to the substitution types and organized by contexts (XpA, XpT, XpC and XpG with X= A, C, T or G) and reads (read1, top and read2, bottom). Sequencing depth has been downsample to 300-fold coverage for comparison, thus the variant read number for high (>10%) allelic frequency is above 30 reads supporting the presence of a variant and low (<1%) allelic frequency is below 3 reads supporting the presence of a variant. The equivalent result for medium (1 to 10%) allelic frequency substitution is plotted on and . All reads substitution have a Phred Quality Scores Q>30. CpA context has no high allelic frequency substitution in Read 2 in the DNA repair sample. The excess of G to T can be attributed to oxidative damage.

Article Snippet: The DNA repair step commonly used in this manuscript corresponds to the NEBNext FFPE DNA Repair v2 Module and contains enzymes and buffers that are optimized to repair FFPE DNA in next generation sequencing workflow including UDG, Endonuclease IV, Fpg, Endonuclease VIII, T4 PDG, Taq DNA ligase and Bst DNA polymerase.

Techniques: Sequencing, Variant Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 2. TGFβ induces TGFβ2 expression in GBM and non-GBM cell lines. A, qRT-PCR of TGFB 1 , TGFB 2 , and TGFB 3 in LN229 cells treated with TGFβ1 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, qRT-PCR of TGFB 2 in LN229 cells treated with TGFβ1, TGFβ2, and TGFβ3 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in culture supernatant from LN229 cells treated with TGFβ for 72 hours. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGF b 2 in LN229 cells treated with TGFβ1 and/or the TβRI inhibitor (TβRI inh.) LY-2109761 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. E, qRT-PCR of TGFB 2 in GBM and non-GBM cell lines treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 3. CREB1 regulates the autocrine induction of TGFβ2 by TGFβ. A, nucleotide sequence of the proximal region of the TGFB 2 promoter. The SBEs and CREB1 site (CRE) are indicated relative to the transcription start site. ClustalW sequence alignment for 3 animal species [ Homo sapiens ( H.s .), Pan troglodytes ( P.t. ), and Mus musculus ( M.m .)] shows the conservation of the binding sites. B, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an shRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an siRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGFβ2 in LN229 cells expressing ICER treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. The molecular weights are shown.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 5. PI3K and RSK regulate the TGFβ- mediated induction of TGFβ2 through CREB1. A, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with TGFβ for 3 hours and the PI3K inhibitor (inh) LY-294002 for 24 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with increasing amounts of the RSK inhibitor BI-D1870 for 24 hours and TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with TGFβ for 48 hours and the PI3K inhibitor for 72 hours. *, P < 0.05, using the Student t test; data, mean ± SD. D, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with the RSK inhibitor BI-D1870 for 72 hours and TGFβ for 48 hours. *, P < 0.05, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 6. TGFβ2 correlates with CREB1 expression in GBM patient tumors. A and B, graphs showing the correlation between CREB1 and TGFB 1 (B) or TGFB 2 (A) mRNA levels in patient GBM tumor samples. Data obtained from the REMBRANDT database. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed P value are shown. C, graph showing the correlation between p-CREB1 and TGFβ2 protein levels in tissue microarrays (TMA) from patient GBM samples. Not all spots were evaluable in all stainings. A Spearman test was used, and the correlation coeffi cient (ρ) and the two- tailed signifi cance are shown. Representative images from the TMAs are shown; scale bar, 50 μm. D, Kaplan–Meier curves showing the OS of patients with TGFB2 mRNA levels upregulated ≥3-fold and CREB1 mRNA levels upregulated ≥2-fold. Statistical signifi cance was assessed by the log-rank test. Data obtained from the REMBRANDT database.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Two Tailed Test

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 7. CREB1 regulates TGFβ2 expression in PDX models. A, scheme showing the experimental procedure. B, IHC of p-CREB1 and TGFβ2 from mouse tumors 60 days after inoculation with neurospheres expressing shRNAs targeting CREB1 and control shRNAs; scale bar, 50 μm (C). Kaplan–Meier survival curves from mice in B. D, the TGFβ2 malignant autocrine loop. In GBM, TGFβ collaborates with the PI3K and RSK pathways through a CREB1– SMAD3 transcriptional complex to induce TGFβ2 expression. This leads to the generation of an autocrine loop and accumulation of TGFβ2 in the tumor, hyperactivation of TGFβ, and tumor progression.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Control

Journal: Discover. Oncology

Article Title: Is serum thymidine kinase 1 a prognostic biomarker in primary tumor location of colorectal carcinomas?

doi: 10.1007/s12672-023-00614-5

Figure Lengend Snippet: Characteristics of TK1-IgY-pAb. A . Example of a patient with RC (T1N2M0). a Western blot of STK1p. The serum samples, presurgery (Lines 1 and 2, duplicate), and 6 months after surgery (Line 3); Serum sample from a disease-free person (Lines 4 and 5, duplicate). b ) TK1 immunohistochemistry (IHC) staining of RC tissue postsurgery (T1N2M0). Brownish-yellow TK1 was mainly in the cytoplasm. Blue staining was used to counterstain nuclei with hematoxylin. Magnification 200 × . B . Receiver operation characteristic (ROC) analysis. The analysis was based on STK1p values of 488 CRC patients and 488 tumor-free persons

Article Snippet: Briefly, Sections. (4-μm-thick) were prepared from formalin-fixed paraffin-embedded surgical specimens from the RC patient by deparaffinization and rehydration and then incubated with TK1-IgY-pAb (1.0 μg/ml, in PBS) overnight at 4 °C followed by incubation with biotinylated donkey anti-chicken IgY antibody at room temperature for 60 min. SA-HRP (streptavidin-HRP conjugate, Invitrogen, SA10001) was added and incubated at room temperature for 90 min. Fresh diaminobenzidine (DAB) solution was used for color rendering, and the slides were lightly counterstained with hematoxylin.

Techniques: Western Blot, Immunohistochemistry, Staining