Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: Effect of NS5A expression on IFN signal transduction. (A) Tetracycline (Tc)-regulated expression of full-length and mutant NS5A proteins. FL-NR denotes a full-length NS5A construct derived from an IFN-nonresponsive patient, while FL-CR denotes a full-length NS5A construct derived from an IFN-responsive patient. ΔN-110, ΔN-222, ΔN-334, ΔC-117, and ΔC-230 represent NS5A deletion mutants lacking 110, 222, 334, amino acids from the amino terminus and 117 and 230 amino acids from the carboxy terminus, respectively. Protein positions are shown by arrows. Western blots were probed with HCV-infected patient serum as described in Materials and Methods. (B) Effect of NS5A expression on ISGF-3 induction. Plasmids were transiently transfected into HeLa Tet-Off cells, grown in the absence and presence of tetracycline to induce and repress NS5A, respectively, and treated with IFN to induce ISGF-3. Protein extracts were hybridized to a 32P-labeled oligonucleotide corresponding to the consensus ISRE. “puc” represents a control transfection with the pTRE-puc parental plasmid with no insert. “E3L,” “PKR,” and “E1A” represent expression of the E3L, PKR, and E1A proteins, respectively. The p48 monoclonal antibody used to form supershifts (denoted by “ss” and an arrow) is denoted by “p48 Mab”. “n” and “c” represent nuclear and cytoplasmic extracts, respectively. ISGF-3 is induced only with IFN treatment and is indicated with arrows.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Expressing, Transduction, Mutagenesis, Construct, Derivative Assay, Western Blot, Infection, Transfection, Labeling, Plasmid Preparation

Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: NS5A expression is associated with increased IL-8 protein production and inhibition of the antiviral effects of IFN. (A) Correlation among levels of IL-8 in culture supernatants, amounts of NS5A protein expression, and levels of EMCV rescue in the trans rescue assay. HeLa cells expressing FL-NS5A-NR were grown for 48 h in medium containing 0, 0.001, 0.01, and 1.0 μg of tetracycline per ml for 48 h, treated with 20 U of IFN per ml for 24 h, and infected with EMCV at an MOI of 0.01 for 24 h. The amount of IL-8 protein in culture supernatants, determined by ELISA, is indicated in the bar graph. Changes in EMCV titers were determined 24 h postinfection by viral plaque assay. The levels of NS5A protein expression were determined by Western blot analysis of equal amounts of total cellular protein extracts and are presented as +++, ++, +, and −, which correspond to high, intermediate, low, and undetectable levels of NS5A protein, respectively, as determined by computerized scanning of the chemiluminescent signal. Fold EMCV rescue represents the difference in EMCV titers in the presence of IFN among cells treated with 0, 0.001, and 0.01 μg of tetracycline per ml versus the 1.0-μg/ml concentration. (B) IL-8 inhibits the antiviral actions of IFN in vitro. HeLa Tet-Off cells were pretreated with or without 33 ng of recombinant human IL-8 per ml for 6 h and then with 20 U of IFN per ml for 18 h. Cells were then infected with EMCV at an MOI of 0.1 for 24 h. Supernatants were harvested, and the amount of EMCV was determined by titration on L929 cells. Error bars represent standard deviations, and P values derived from Student's t tests are indicated.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Expressing, Inhibition, Rescue Assay, Infection, Enzyme-linked Immunosorbent Assay, Viral Plaque Assay, Western Blot, Concentration Assay, In Vitro, Recombinant, Titration, Derivative Assay

Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: NS5A transactivates the IL-8 promoter. (A) Effect of NS5A expression on luciferase expression under the control of the 546-luc IL-8 promoter. The 546-luc plasmid and pTRE-FL-NS5A-NR (10 μg each) were transfected into HeLa Tet-Off cells, and cells were split in triplicate and grown in media with or without tetracycline for 48 h. Cells were then treated or not treated with IFN for 24 h. The amount of luciferase activity in cell lysates was determined using a luminometer. (B) Effect of NS5A expression on luciferase activity under the control of truncated IL-8 promoters. Ten micrograms of the relevant reporter plasmid was transfected into HeLa FL-NS5A-NR cells, and cells were grown in media with or without tetracycline for 48 h. The amount of luciferase activity in cell lysates was determined using a luminometer. 546-luc, 133-luc, and 98-luc contain 546, 133, and 98 bases of the IL-8 promoter, respectively. Error bars represent standard deviations, and P values derived from Student's t tests are indicated.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Expressing, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Derivative Assay

Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: Characterization of domains on NS5A and the IL-8 promoter required for transactivation. (A) Effect of NS5A mutant proteins on luciferase expression under the control of the full-length (546-luc) IL-8 promoter. Ten-microgram quantities of the 546-luc plasmid and pTRE-FL-NS5A-NR, NS5A-ΔN110, NS5A-ΔN222, or NS5A-ΔC117 were transfected into HeLa Tet-Off cells, and cells were split in triplicate and grown in media with or without tetracycline for 48 h. The amount of luciferase activity in cell lysates was determined using a luminometer. The data shown are the fold changes in luciferase activity with the gene in the induced versus the uninduced state. (B) Im- munofluoresence analysis of HeLa cells expressing FL-NS5A-NR, NS5A-ΔN222, or NS5A-ΔC117. HeLa Tet-Off cells stably transfected with FL-NS5A-NR (top panels), NS5A-ΔN222 (middle panels), or NS5A-ΔC117 (lower panels) were grown in the absence or presence of tetracycline (Tc) to induce or repress NS5A expression for 48 h. Cells were fixed and stained with a monoclonal NS5A antibody and then with Cy3-linked anti-mouse secondary antibodies, as described in Materials and Methods. Images are at a ×100 amplification. (C) Effect of NS5A-ΔN222 expression on luciferase activity under the control of mutated IL-8 promoters. Ten-microgram quantities of NS5A-ΔN222 and the relevant reporter plasmid were cotransfected into HeLa Tet-Off cells; cells were split in triplicate and grown in media with or without tetracycline for 48 h. Cells were then treated with 4 ng of TNF-α for 6 h. The amount of luciferase activity in cell lysates was determined using a luminometer. 546-luc, 133-luc, and 98-luc contain 546, 133, and 98 bases of the IL-8 promoter, respectively. NF-κB–luc, AP-1–luc, and NF–IL-6–luc represent the 133-luc plasmid with point mutations in the NF-κB, AP-1, and NF–IL-6 binding sites.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Mutagenesis, Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay, Stable Transfection, Staining, Amplification, Binding Assay

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Steady-state mRNA levels of firefly luciferase variants. Shown is a northern blot, probing for the common 3’UTR within different firefly luciferase reporter mRNAs, which vary by optimal codon content, and U6 snRNA loading control. Quantification of Firefly luciferase mRNA (relative to 0% optimal construct) is shown at bottom (error bars denote standard deviation; n=3). (B) More optimal luciferase mRNA variants are more stable. Transcriptional shut-off experiments were performed in Tet-Off HEK293 cells, and firefly luciferase mRNA levels were determined by northern blots. Timepoints correspond to the time after the addition of doxycycline, which shut-offs transcription of the reporter. t½ corresponds to the half-life (min) ± standard deviation (n=3). See for loading control. (C) More optimal MECP2 mRNA variants are more stable. As in B, expect for MECP2 . See for loading control. (D) More optimal CFTR ΔF508 mRNA variants are more stable. As in B, except for CFTR ΔF508. See for loading control. See also and Tables S1 and S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Luciferase, Northern Blot, Construct, Standard Deviation

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Schematic of the ORFeome workflow. The ORFeome collection contains ~16,000 full-length coding sequences corresponding to 14,000 genes in a lentiviral background. Each ORF derived from the ORFeome is flanked by invariant UTRs, and also contains a C-terminal V5 tag. Lentiviral pools containing ~3000 ORFeome clones were used to infect HEK293T and generate stable cell lines. Metabolic labeling was used to determine stabilities of endogenous and ORFeome-derived mRNAs in these stable lines. (B) Changing coding regions changes mRNA stability. Plotted are boxplots of half-lives for endogenous HEK293T (End.) and ORFeome-derived mRNAs (in blue and gray, respectively). The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (C) ORFeome mRNAs show as much variation in stability as endogenous mRNAs. Plotted are density destributions of median-centered half-lives for endogenous HEK293T and ORFeome-derived mRNAs (in blue and gray, respectively). (D) Treatment with 4EGI-1 inhibits translation. Shown are A 254 traces from sucrose density gradients of cell lysates from HEK293T cells treated with the translation inhibitor 4EGI-1 (orange) or DMSO (grey). (E) Transaltion inhibition destabilizes endogenous mRNAs. Plotted are boxplots of half-lives for endogenous HEK293T mRNAs with DMSO or 4EGI-1 treatment (in grey and orange, respectively). The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (F) Translation inhibition has a minor effect on the variation in stability for endogenous mRNAs. Plotted are density destributions of median-centered half-lives for endogenous HEK293T in cells treated with DMSO or 4EGI-1 (in gray and orange, respectively). (G) Inhibition of translation destabilizes ORFeome-derived mRNAs and reduces the variation in stability. As in E, except for ORFeome mRNAs. DMSO, in blue; 4EGI-1, in orange. (H) Translation inhibition reduces the variation in stability for ORFeome-derived mRNAs. As in F, except for ORFeome mRNAs. DMSO, in blue; 4EGI-1, in orange. See also and Table S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay, Clone Assay, Stable Transfection, Labeling, Inhibition

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) ORFeome complexity was maintained through stable cell line generation. Shown is a western blot probing lysates from the pooled ORFeome stable lines with V5 (the common C-terminal tag in the ORFeome collection). WT, parental HEK293T line; S, supernatant; P, pellet. (B) ORFeome-derived mRNAs are expressed in the stable cell lines. Shown is a scatter plot comparing steady-state RNA-seq reads (with a +1 pseudocount) for each gene between the two pooled lines used in this study. In black, genes in neither pool; in blue, genes in pool 1; in orange, genes in pool 4; in green, genes in both pools. Red dashed lines represents y = 3X and y = X/3, which were used as cut-offs to classify genes as ORFeome-expressed. ORFeome genes that did not pass threshold were not used for subsequent analysis (see Methods for more details). Numbers refer to the total number of genes in each pool and the number passing the 3-fold threshold. (C) ORFeome mRNAs are expressed in a pool-dependent fashion. Shown are boxplots of normalized read counts (with a +1 pseudocount) for ORFeome-derived mRNAs (split into pool 1 and pool 4) in the two pooled stable cell lines. Abundance in cell line 1 is shown in blue; in cell line 4, in orange. Note that the ORFeome pools are expressed in the appropriate cell line. (D) 4EGI-1 substantially reduces translation. Cells were treated with DMSO, 4EGI-1, or cyclohexamide (as a positive control) for the indicated times and then briefly treated with puromycin, which is incorporated into nascent peptides. Lysates were separated by gel electrophoresiss and probed for puromycin (top) or tubulin (bottom). (E) DMSO treatment does not substantially affect mRNA stability. Shown are scatterplots comparing half-lives for endogenous genes (averaged from both pools) from the original experiment and DMSO-treated cells. Red dashed line represents x = y. (F) 4EGI-1 treatment affects mRNA stability. As in E, except comparing half-lives from the original experiment and 4EGI-1-treated cells.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Stable Transfection, Western Blot, Derivative Assay, RNA Sequencing Assay, Positive Control

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Endogenous mRNA stability negatively correlates with length. Shown are boxplots for half-lives of endogenous HEK293T mRNAs binned into quartiles by ORF length. (B) ORFeome mRNA stability does not correlate with length. As in A, except for ORFeome mRNAs. (C) Endogenous mRNA stability weakly correlates with local secondary structure. For each ORF, the folding energy in 100 bp sliding windows was calculated, and the minimum value taken. Shown are boxplots for half-lives of endogenous HEK293T binned into quartiles by folding energy (with increased secondary structure on the right). (D) ORFeome mRNA stability does not correlate with local secondary structure. As in B, except for ORFeome mRNAs. (E) microRNA-mediated regulation cannot explain the variation in ORFeome stability. ORFs were classified as containing or lacking seed-matched sites for the top five expressed mRNAs (site ORFs [orange] and no site ORFs [blue], respectively). Shown are boxplots for their half-lives. Significance was calculated by the Kolmogorov-Smirnov test. (F) AU-rich elements cannot explain the variation in ORFeome stability. As in E, except for AU-rich elements. (G) AU-rich elements in ORFs destabilize mRNAs upon translational repression. As in F, except for half-lives determined in the presence of 4EGI-1.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Codons are differentially associated with stability. Shown are spearman correlations, for each codon, of their frequency with mRNA stability (codon stability coefficient; CSC) for endogenous HeLa mRNAs, endogenous HEK293T mRNAs, and ORFeome mRNAs. The 15 codons most associated with stability (as defined by the ORFeome collection) were designated as “optimal” (blue), while 15 codons most associated with instability were designated as “non-optimal” (orange). (B) HeLa and HEK293T cells have similar codon stability coefficients (CSCs). Plotted are the CSC values for endogenous HEK293T mRNAs compared to endogenous HeLa mRNAs (C) As in B, except comparing endogenous HEK293T and ORFeome-derived CSC values. (D) ORFeome mRNAs with more optimal codons are more stable. Shown are boxplots of mRNA half-lives for ORFeome mRNAs, binned into quartiles by the frequency of optimal codons. The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (E) As in D, except for endogenous HEK293T mRNAs. (F) Endogenous HEK293T CSCs weakly correspond with pause scores. Using HeLa ribosome profiling, pause scores were calculated for each codon in the A site, and then codons were divided into three groups (slow in orange; neutral in green; fast in blue). Shown are boxplots for the corresponding CSC values as determined by endogenous HEK293T mRNAs. (G) As in F, except for ORFeome-derived CSCs. See also .

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Codon stability coefficients (CSCs) have little relationship to tRNA abundance. Plotted are the normalized tRNA abundance in comparison to CSC values for endogenous HEK293T and and ORFeome mRNAs (left and right panels, respectively). (B) tRNA abundance has little impact on elongation speed. Codons were divided into thirds by their A-site pause scores (slow in orange; neutral, green; fast, blue). Shown are boxplots for the abundance of corresponding tRNAs. The line represents the median half-life, and the box, 1 st and 3 rd quartiles. (C) Amino acids are differentially associated with stability. Shown are spearman correlations, for each amino acid, of their frequency with mRNA stability (amino acid stability coefficient or AASC) for endogenous HeLa mRNAs, endogenous HEK293T mRNAs, and ORFeome mRNAs. Polar amino acids (in pink) have charged or highly electronegative side chains; nonpolar amino acids (dark gray) have aliphatic and weakly electronegative side chains. (D) HeLa and HEK293T have similar AASCs. Plotted are the AASC values for endogenous HEK293T mRNAs compared to endogenous HeLa mRNAs (E) As in B, except comparing endogenous HEK293T and ORFeome-derived AASC values. (F) Hydrophobic amino acids are associated with stability. Plotted are the hydrophobicity scores for each amino acid compared to their stability coefficient for endogenous HEK293T mRNAs. (G) As in F, except for ORFeome-derived AASC values. (H) Schematic diagram of LSM8 reporter constructs. In the middle of the LSM8 coding region, five repeats of instability-associated amino acids (S, H and E) or stability-associated amino acids (V, I, and L) were inserted. (I) Insertion of instability-associated amino acids destabilizes the LSM8 reporter mRNA. Transcriptional shut-off experiments were performed in Tet-Off HEK293 cells, and LSM8 mRNA levels were determined by northern blots. Timepoints correspond to the time after the addition of doxycycline. t½ corresponds to the half-life (min) ± standard deviation (n=4). See for loading control. (J) The destabilized LSM8 reporter mRNA has shorter poly(A) tails. High resolution northern blotting was performed to measure poly(A)-tail lengths on the SHE and VIL LSM8 mRNAs. Arrow indicates deadenylated mRNA species; dT, oligo(dT)/RNase H treated mRNA control. (K) LSM8 reporter mRNAs are deadenylated. Transcription of the SHE and VIL LSM8 reporters was shut-off, as in I, and poly(A)-tail lengths were measured by high-resolution northern blotting. Timepoints represent time elapsed after transcription shutoff with 2 μg/mL doxycycline. Arrow indicates deadenylated mRNA species; dT, oligo(dT)/RNase H treated mRNA control. See also and Tables S1 and S2.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Derivative Assay, Construct, Northern Blot, Standard Deviation

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Valine frequency correlates with mRNA stability. Shown are boxplots of mRNA stabilities for HeLa, endogenous HEK293T, and ORFeome mRNAs binned into quartiles by valine frequency. (B) Serine frequency negatively correlates with mRNA stability. As in A, except for serine. (C) U6 snRNA northern analysis for transcription shut-off experiments for the LSM8 variants shown in . (D) U6 snRNA Northern analysis for LSM8 variable amino acid content transcription shutoff/Northern mRNA decay analyses shown in .

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques: Northern Blot

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: (A) Amino acids, when in the A-site, are translated at different rates. Plotted are the pause scores for each amino acid when in the predicted A-site (see methods for details). Nonpolar amino acids, grey; polar amino acids, pink. (B) Amino acid stability coefficients correlate with A-site pause scores. Shown are plots comparing A-site pause scores for each amino acid with its stability coefficient, as defined by endogenous HeLa, endogenous HEK293T, and ORFeome mRNAs (left, middle, and right, respectively). (C) As in A, except for the P site. (D) As in A, except for the E site. (E) A-site pause scores correlate poorly with P- and E-site pause scores. Plotted are the pairwise comparisons for A-, P-, and E-site pause scores. (F) ORFeome amino acid stability coefficients poorly correlate with P-site pause scores. Shown are plots comparing P-site pause scores for each amino acid with its stability coefficient, as defined by ORFeome mRNAs. (G) As in F, except for E-site pause scores.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: bioRxiv

Article Title: Codon usage and amino acid identity are major determinants of mRNA stability in humans

doi: 10.1101/488676

Figure Lengend Snippet: For each pair of codons, the absolute difference in the corresponding CSC values was calculated and then normalized to the maximal difference (to correct for differences in overall variance between organisms). Pairs of codons were binned into these encoding the same or different amino acid (n = 87, in grey, and n = 1742, in green, respectively). Shown are boxplots of those differences for S. pombe , trypanosomes, zebrafish, and endogenous HEK293T mRNAs. Significance determined by Wilcoxon rank sum test.

Article Snippet: HEK293 Tet-Off ® cells (Clontech 631152) were maintained in DMEM, high glucose, pyruvate (Thermo Fisher Scientific Cat #11995065) supplemented with 10% FBS (Gibco 1992275) and 1% Penicillin/Streptomycin.

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Manufacturing of recombinant adeno-associated viral vectors for clinical trials

doi: 10.1038/mtm.2016.2

Figure Lengend Snippet: Institutions and rAAV GMP manufacturing technologies

Article Snippet: Targeted Genetics Corporation, Seattle, WA , wtAd5 Infection , HEK293 Producer HeLa Cell line WAVE and stir Tank bioreactors , Depth filtration, Benzonase, Ion exchange, UF/TFF, Chrom step, Heat inactivation, Chrom step, NanofiltrationPolishingFormulation , 1, 2 , , .

Techniques: Purification, Transfection, Flocculation, Lysis, Filtration, Plasmid Preparation, Concentration Assay, Affinity Chromatography, Sonication, Clarification Assay, Chromatography, Diafiltration Assay, Infection, Ion Exchange Chromatography

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: Statistics for spatial transcriptomics outcome for Visium_FFPE_ HLTF -/- CDX in ID Hltf KO mice (A and B) and HLTF +/+ CDX in ID Hltf KO (C and D).

Article Snippet: Thus, System Biosciences (Palo Alto, CA) produced a custom stable cell line with shRNA sequence targeting of HLTF [ ].

Techniques: Sequencing

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: A. The relative abundance of each microbial species was estimated based on the depth and coverage of sequencing across every available reference genome. The top 10 species are provided in bar graph format for individuals subdivided into two cohorts, i.e. ID male mice that were either Hltf +/+ (control) or Hltf KO. B. The Shannon Diversity Index was used to measure the diversity of taxa within the samples and between the cohorts. Box-and-whisker plots summarize numerical taxonomic (family) data based on quartiles. The inner quartile range < 2 indicates the values are not significantly different despite the differences in median values of 2.99 and 2.63, respectively, for Hltf +/+ and Hltf KO.

Article Snippet: Thus, System Biosciences (Palo Alto, CA) produced a custom stable cell line with shRNA sequence targeting of HLTF [ ].

Techniques: Sequencing, Control, Whisker Assay

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: Shown is A , the total number of BM cells, and the percentages of B , B cells and C , Gr1 + Mac1 + myeloid cells, where each point on the graph represents one mouse. Values in panels B and C are expressed as percentages of total cells. D , Representative H&E stains from one individual Mll3 +/+ and Mll3 Δ/Δ mouse of sternum sections taken at 400X magnification. Images depict the entire field of view (top panels) and a zoomed-in view (bottom panels). Scale bars are 50μm. E-I , Similar to panels B and C , except that the percentages of LSK cells, CLP, CMP, MEP, and GMP are shown, respectively. B, C, I , *p<0.05, **p<0.01, ***p<0.005 as determined by the Student’s t -test. G , *p<0.05 as determined by the Mann-Whitney test.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: Absolute gene expression profiling adapted from Gene Expression Commons evaluating Mll3 expression across 39 different hematopoietic cell populations in the mouse BM, spleen, and thymus. Shown are the normalized values of Mll3 expression activity for several cell populations from 2–4 biological replicates. Positive values indicate high Mll3 expression compared to the common reference, and negative values denote relatively low Mll3 expression in comparison to the common reference. Error bars represent mean + SD. HSC, hematopoietic stem cell; Gra, granulocyte; Fr, fraction; Mz, marginal zone; Fo, follicular.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: Gene Expression, Expressing, Activity Assay, Comparison

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: The percentage of total cells (top) and absolute number (bottom) of A , B cells, B , T cells, and C , Gr1 + Mac1 + myeloid cells is shown, where each point represents one mouse. D , Representative H&E stains of spleen sections (50X magnification, top panels; 400X magnification, middle panels) from one individual Mll3 +/+ and Mll3 Δ/Δ mouse. Images in the bottom panels are zoomed-in views of the middle panels. Scale bars are 500μm (top panels) and 50μm (middle panels). E , Quantification of splenic follicle size. Each point on the graph represents the average follicle size (black, ≥10 follicles measured; red, <10 follicles measured) for one mouse. *p<0.05, **p<0.01 as determined by the Student’s t -test.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: Shown is the percentage of total cells (top) and absolute number (bottom) of A , B cells, B , T cells, and C , Gr1 + Mac1 + myeloid cells. D , Similar to panels A-C , except the total numbers of CD4 + (left) and CD8 + (right) T cells are shown. Each point represents one mouse. B-C , *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by the Student’s t -test. D , *p<0.05, **p<0.005 as determined by the Mann-Whitney test. E , Representative H&E stains (top panels) and IHC for CD3 (bottom panels) from two individual Mll3 +/+ and Mll3 Δ/Δ mice of LN sections taken at 50X magnification. Scale bars are 500μm.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: A , Relative MLL3 expression in human KBM7 cells as determined by qPCR. TaqMan probes (indicated in red) recognized exon junctions before (exons 18–19) and after (exons 39–40 and 55–56) the gene-trap (exon 38), indicated in green. B , Relative adhesion of KBM7 cells to fibronectin as measured in fluorescence units. Shown is the average of technical replicates from four individual experiments + SEM. C , The number of KBM7 cells that migrated across a trans-well membrane in response to FBS after 24h. Data are the combination of technical replicates from four individual experiments + SD. Ref, control gene-trap; MLL3, MLL3 -targeted gene-trap. *p<0.05, **p<0.01 as determined by the Mann-Whitney test.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: Expressing, Fluorescence, Membrane, Control, MANN-WHITNEY