Journal: The Science of the Total Environment

Article Title: Sampling methods and assays applied in SARS-CoV-2 exposure assessment

doi: 10.1016/j.scitotenv.2021.145903

Figure Lengend Snippet: Data obtained from the chosen articles.

Article Snippet: , 34. SARS-CoV-2 RNA contamination on surfaces of a COVID-19 ward in a hospital of Northern Italy: what risk of transmission? , Italy , No , Surface samples from ward in University Hospital of Ferrara , Sampling performed with sterile rayon swabs pre-moistened in sterile phosphate-buffered solution , Viral RNA extraction with Patho Gene-spin Extraction kit (Generon) RT-qPCR targeted the RNA-dependent RNA polymerase ( RdRp ) gene (Generon), and the orf1ab , spike ( S ), and nucleocapsid ( N ) genes (ThermoFisher) , • SARS-CoV-2 was only detected in 3 samples of two floors and one-bathroom sink. • Reported to persist for a longer duration on surfaces under controlled laboratory conditions. , ( ) .

Techniques: Sampling, Lysis, RNA Extraction, Environmental Monitoring, Virus, Multiplex Assay, Northern Blot, Marker, RNA Detection, Isolation, Membrane, Control, Environmental Sampling, Amplification, Transmission Assay, Aerosol, Diagnostic Assay, Infection, Sterility, Real-time Polymerase Chain Reaction, Nested PCR, Reverse Transcription, Extraction, Purification, Digital PCR, Preserving, Quantitative RT-PCR, cDNA Synthesis, Magnetic Beads, Incubation, Modification, One Step RT-PCR, Cell Culture, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

doi: 10.1084/jem.20181762

Figure Lengend Snippet: Regnase-3 –deficient mice develop hypertrophic lymph nodes. (A) Frequency of mice showing lymphadenopathy in a cohort of 24 Regnase-3 −/− mice and 24 Regnase-3 +/+ littermate controls at 3–6.5 mo of age. (B) Photography of skin-draining lymph nodes of four Regnase-3 −/− mice and their Regnase-3 +/+ littermate controls at 5 mo of age. (C) Representative photography of inguinal lymph nodes of a Regnase-3 −/− mouse at 5 mo of age. Arrow indicates hypertrophic lymph node. (D) H&E staining and immunohistochemical analysis of B cells (B220), T cells (CD3), and macrophages (F4/80) in skin-draining lymph nodes of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images from n = 3/3). Magnification of images is indicated in brackets. Bars, 1,000 µm. (E) Immunohistochemical analysis of macrophages (CD68) in skin-draining lymph nodes of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images from n = 6/6). Images of enlarged and small lymph nodes are taken from the identical Regnase-3 −/− mouse. Top right: Frequency of strong positive (pos.) pixels in CD68 immunohistochemical sections of the lymph nodes was determined by Definiens software ( n = 6/6). Bars, 500 µm. (F) Top: Frequencies of B cells (CD19 + ) and T cells (CD90 + ) in enlarged and normal-sized lymph nodes of the same Regnase-3 −/− mouse and its Regnase-3 +/+ littermate control at 6 mo of age, assessed by flow cytometry (representative blots of n = 6/6). Number of total cells in lymph nodes of Regnase-3 +/+ mice and Regnase-3 −/− littermates ( n = 6/6). Bottom: Frequencies of B cells (CD19 + ), T cells (CD90 + ), CD4 + and CD8 + T cells, and CD11b + cells in enlarged lymph nodes of Regnase-3 −/− mice and their Regnase-3 +/+ littermate controls at 6 mo of age, assessed by flow cytometry ( n = 6/6). Data are represented as mean ± SEM and were compared by Mann–Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant).

Article Snippet: Staining antibodies were anti-B220 (RA3-6B2, rat-IgG2a; BD), anti-CD3 (SP7, rabbit IgG; Zytomed), anti-F4/80 (BM8, rat-IgG2a; Linaris), anti-MHC-II (M5/114.15.2, rat IgG; Novus Biologicals), anti-CD68 (Ab125212, rabbit IgG; Abcam) anti-ki67 (SP6, rabbit IgG; Thermo Fisher Scientific), and anti-Tyr701-phospho-STAT1 (58D6, rabbit IgG; Cell Signaling).

Techniques: Staining, Immunohistochemical staining, Software, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

doi: 10.1084/jem.20181762

Figure Lengend Snippet: Systemic IFN signaling in Regnase-3 −/− mice. (A) CD19 + B cells were isolated from enlarged lymph nodes of Regnase-3 −/− mice and their Regnase-3 +/+ littermates ( n = 3/3), and RNA was isolated and subjected to RNA sequencing. Heatmap of RNA sequencing data for all significantly up-regulated (≥2 log2 fold) genes in Regnase-3 −/− B cells is shown. GO term association to “response to IFNβ” and “response to IFNγ” is indicated for each gene. (B) Serum cytokines in Regnase-3 −/− mice and Regnase-3 +/+ littermates at 6 mo of age ( n = 8/8), assessed by multiplex assay. (C) Stat1 mRNA expression in tissues from Regnase-3 +/+ and Regnase-3 −/− mice at 8 mo of age, assessed by quantitative RT-PCR, normalized to Hprt relative (rel.) to their expression in Regnase-3 +/+ mice ( n = 5/5). (D) Left: Immunohistochemical analysis of MHC-II in liver sections of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ controls (representative images). Magnification of images is indicated in brackets. Bars, 250 µm. Right: Frequency of strong positive pixels in MHC-II immunohistochemical sections of lung, kidney, and liver were determined by Definiens software ( n = 6 Regnase-3 −/− mice and 6 Regnase-3 +/+ littermate controls). (E) Frequency of MHC-II–positive macrophages (CD68 + ) of all CD68 + macrophages in the lung of Regnase-3 −/− mice with lymphadenopathy and their Regnase-3 +/+ littermate controls, assessed in immunohistochemical, consecutive sections ( n = 5/6). (F) Left: Immunohistochemical analysis of pSTAT1 in skin-draining lymph nodes from Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images). Right: Frequency of strong positive pixels in pSTAT1 immunohistochemical sections of lymph nodes, determined by Definiens software ( n = 6 Regnase-3 −/− mice and 6 Regnase-3 +/+ littermate controls). Bars, 500 µm. Data are represented as mean ± SEM and were compared by Mann–Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant).

Article Snippet: Staining antibodies were anti-B220 (RA3-6B2, rat-IgG2a; BD), anti-CD3 (SP7, rabbit IgG; Zytomed), anti-F4/80 (BM8, rat-IgG2a; Linaris), anti-MHC-II (M5/114.15.2, rat IgG; Novus Biologicals), anti-CD68 (Ab125212, rabbit IgG; Abcam) anti-ki67 (SP6, rabbit IgG; Thermo Fisher Scientific), and anti-Tyr701-phospho-STAT1 (58D6, rabbit IgG; Cell Signaling).

Techniques: Isolation, RNA Sequencing Assay, Multiplex Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Software, MANN-WHITNEY

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: A) Schematic of lung epithelial development. As the airways extend, bud tip progenitors are maintained as progenitors in the tips of branching buds, and leave cells behind that give rise to the intrapulmonary airways. Late in development, remaining bud tip progenitors differentiate into alveolar cells. No bud tip progenitors are present in the adult lung. B) Schematic of creation of epithelium-only bud tip progenitor organoids from 12 week fetal lungs. C) Feature plots for bud tip progenitor marker genes SFTPC, ID2 and HMGA1 from scRNA-seq of day 0 bud tip progenitor organoids maintained for 3 weeks in culture. Additional feature plots from this same data set are shown in Figure 4A. D) mRNA expression by QRT-PCR of basal cell marker TP63 in bud tip progenitor organoids treated for 3 days with serum-free basal medium supplemented with DMSO (control) or with signaling factors known to be important for lung development and cellular differentiation. DMSO (1:1,000 dilution), FGF7 (10 ng/mL), ‘3F’ (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), Dexamethasone (25 ng/mL), IL6 (10 ng/mL), IL2 (50 U/mL), Smoothened Agonist (SAG; 500 nM), EGF (100 ng/mL), FGF10 (500 ng/mL), CHIR99021 (2 μM), IFNγ (10 ng/mL), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), DAPT (10 μM), Hydrocortisone (100 ng/mL). Gene expression is reported as arbitrary units. Treatment with TGFβ1 led to a significant increase in the expression of TP63 (one-way Analysis of Variance (ANOVA) (alpha=0.05, p<0.0001, F=14.7. Dunnett’s test of multiple comparison’s compared the mean of each group to the mean of the DMSO control group.) Estimated p values are shown on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. E) Bud tip progenitor organoids were treated with FGF7 (10 ng/mL), a permissive environment for TP63 expression compared to maintenance in bud tip progenitor medium (‘3F’), or with FGF7 (10 ng/mL) plus factors to inhibit SMAD signaling (A8301 [1 μM] and NOGGIN [100 ng/mL]) and TP63 gene expression was evaluated by QRT-PCR after 10 days in culture. A one-way Analysis of Variance was used followed by Tukey’s multiple comparison test to compare the means of each group to the mean of every other group. Estimated p values are reported on the graph. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment. F) Bud tip progenitor organoids were treated for 3 days with SMAD activation or inhibition conditions and expression of TP63 was evaluated by QRT-PCR for all treatment groups. All media contained 3F components (FGF7 10ng/mL, CHIR99021 3μM, ATRA 50 nM), with individual groups containing combinations of: DMSO (1:1000 dilution), TGFβ1 (100 ng/mL), BMP4 (100 ng/mL), SB431542 (10 μM), LDN212854 (200 nM). One-way ANOVA alpha=0.05, F=21.19, p<0.0001; Tukey’s multiple comparisons of the mean of each group versus the mean in all other groups, estimated p values are reported on the graph. 3 days TGFβ1 and BMP4 is referred to as ‘dual SMAD activation’, or ‘DSA’. Data is plotted as arbitrary units. Error bars are plotted to show mean +/− the standard error of the mean. N=3 independent biological specimens. Data is from a single experiment and is representative of n=3 experiments. G) QRT-PCR for markers of canonical differentiated lung epithelial cell types showing DMSO (gray bars) and DSA treated (blue bars) organoids after 3 total days of treatment. Data is plotted as fold change over DMSO controls. Two-sided Mann-Whitney Tests were performed to compare the mean of the DMSO group to the Dual Smad Activation group (alpha=0.05). Error bars represent the mean +/− the standard error of the mean. n=3 independent biological specimens, and data is from a single experiment and is representative of n=3 experiments. H) Protein staining of DMSO treated (control) fetal bud tip progenitor organoids (top row) and 3 days of DSA treatment (bottom row) for TP63+ protein (green), KRT5 (pink) and DAPI (blue). Scale bar represents 50 μm. I) Quantification of (g). Total number of TP63+ cells were counted for 3–9 individual organoids across 3 biological replicates. n=3 independent biological specimens. A two-sided Mann Whitney test (alpha=0.05) was used to compare the means of each sample. For all graphs, p values are reported as follows: * p<0.05; ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Marker, Expressing, Quantitative RT-PCR, Control, Cell Differentiation, Gene Expression, Comparison, Activation Assay, Inhibition, MANN-WHITNEY, Staining

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Growth factors and small molecules were used at the following concentrations: FGF10 (500 ng/mL, made in-house as previously described), A8301 (1 μM, Stem Cell Technologies cat. no. 72024), NOGGIN (100 ng/mL, R&D Systems, cat. no. 6057), Y27632 (APExBIO cat. no. A30008), LDN212854 (200 nM, R&D Systems cat. no. 6151/10), SB431542 (10 μM, Stemgent cat. no. 04–0010), TGFβ1(100 ng/mL, R&D systems cat. no. 240-B-002), BMP4 (100 ng/mL, R&D systems cat. no. 314-BP-050) Dexamethasone (25 ng/mL, Stem Cell Technologies cat. no. 72092), IL6 (10 ng/mL, R&D Systems, cat. no. 206-IL-010), IL2 (50 U/mL, R&D Systems, cat. no. 202-IL-010), Smoothened Agonist (SAG; 500 nM, R&D Systems, cat. no. 4366/1), EGF (100 ng/mL, R&D Systems cat. no. 236-EG-200), IFNγ (10 ng/mL, R&D Systems cat. no. {"type":"entrez-protein","attrs":{"text":"CAA31639","term_id":"32692","term_text":"CAA31639"}} CAA31639 ), DAPT (10 μM, R&D Systems cat. no. 2634/10), Hydrocortisone (100 ng/mL, Stem Cell Technologies cat. no. 74142).

Techniques: Transduction, Control, Virus, Plasmid Preparation, Recombinant, RNAscope, Multiplex Assay, In Situ Hybridization, Generated, Software

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 1 Proliferation and specific cytotoxic effects of CART-19 cells. A The design of the CAR-T cell construction experiments. B Morphological images of activated T cells clustered after 24 h and 72 h of incubation with TransAct CD3/28 beads. C Flow cytometric analysis of CAR expression on the surface of mock T, and CART-19 cells with biotin-conjugated anti-Fab antibody followed by PE-conjugated streptavidin. Gating was based on the same cells stained with isotype-matched antibody. The median fluorescence intensity (MFI) was calculated for CAR-T population in the PE fluorescence channel (right column). This result is the representative of three separate experiments using cells from healthy volunteer donors. D The phenotypic characterization of CART-19 cells by flow cytometry. The ratio of CD4+ / CD8+ T cells (left) and the proportion of TN/CM (right) are shown. E Growth curves of CAR-T cells. Data represent the mean ± s.d. of three separate experiments. F Cytolytic activities of CART-19 cells in cell assays. Nalm-6 cells were labeled with CFSE labeling reagent (Sigma-Aldrich, USA) and co-cultured with CART-19 cells at the E: T ratio of 1:1 for 30 h. The presence of CFSE-labeled cells was observed by mi croscopy. Bar, 100 μm. G Cytotoxic activity of mock NT and CART cells against Nalm-6 cells. The effector cells were co-cultured with target cells at E: T ratios of 1:5, 1:2, 1:1 and 5:1 with a total cell number of 1 × 106. H Dynamic changes of cytokine secretion profile of CART-19 cells during 24 h after co-culture with Nalm-6 cells at E: T ratios of 1:5 to 5:1. Data were visualized by heatmap. Concentrations (pg/ml) of cytokines and chemokines in the supernatant were detected by multiplex immunoassay and the values were log2 transformed

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Flow Cytometry, Labeling, Cell Culture, Activity Assay, Co-Culture Assay, Multiplex Assay, Transformation Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: RNA sequencing reveals that tomatidine modulates ATF4-dependent ER stress genes in pancreatic cancer cells Human and murine PDAC cell lines were treated for 40 h with 6.4 μg/mL tomatidine, RNA was isolated, and RNA sequencing was performed to analyze differences in gene regulation. N = 3 biological separate experiments. (A–D) (A) Heatmap of all genes and how they change in tomatidine-treated vs. untreated cells for Panc1 cells (FDR<0.05). Volcano plot highlighting ER stress-related genes in (B) Panc1 and (C) MT5 cells. (Upregulated genes on the right of the central axis and vice versa with higher fold change as we go away from the origin on y axis) (D) IPA upstream analysis of ATF4-related genes in Panc1 cells. (E) Heatmap elucidating targeting of ATF4-related genes in treated vs. untreated Panc1 cells. (Fold change: +2.5 to −1.5; FDR<0.03). (F) Top 10 pathways focusing on UPR, ATF4, and ER stress via Reactome analysis of the RNA-sequencing data of treated vs. untreated samples for Panc1 cells. (FDR< 0.03).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: RNA Sequencing, Isolation

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: ATF4 expression in PDAC (A) KM plotter was utilized to examine ATF4 expression in human PDAC specimens examining high vs. low expression compared to overall survival. (B) Data distribution elucidating significance of the overall survival curve prepared by KM Plotter. (C and D) (C) scRNA-seq datasets of pancreatic cancer tissue from metastatic patients were obtained from NIH dbGAP (accession phs002045.v1.p1) and (D) analyzed for ATF4 expression. (See also Table S1 ). (E) Pancreatic tumor tissue from KPC mice were stained by multiplex IF and imaged using Akoya Vectra Polaris and the Phenochart software to analyze ATF4 in multiple cellular compartments. Markers used to determine ATF4 (white), epithelial cells (CK19; red), stroma (αSMA; orange and PDGFRβ; Green), CD8 T+ cells (yellow), and macrophages (F4/80; turquoise). Scale bar = 100 μm.

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Staining, Multiplex Assay, Software

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine inhibits ATF4-dependent signaling in PDAC (A and B) (A) MiaPaca-2 tumor cells were treated with tomatidine for 72 h and cell lysates were immunoblotted for ATF4, 4EBP1, and phospho-4EBP1(p-4EBP1) protein expression and (B) p-4EBP1/4EBP1 levels quantified by densitometry. (C) Immunofluorescence (IF) was performed on vehicle (DMSO) or tomatidine-treated Panc1 cells to track ATF4 (FITC-Green) translocation from nucleus (DAPI-Blue) to cytoplasm. Scale bar = 50 μm. (D–G) (D) Nuclear to cytoplasmic translocation was quantified. Panc1 cells were treated with vehicle (DMSO) or tomatidine for 40 h and ATF4 transcriptional activity was analyzed by chromatin immunoprecipitation (ChIP) qPCR evaluating binding of ATF4 to the downstream promoter regions of (E) eIF4EBP1 (F) CHOP- B site and (G) ASNS. Data are reported as the means + SEMs. n = 3 or more independent biological replicates (4B, One-way ANOVA with Tukey’s test for pairwise comparisons was used to analyze the data; 4D-G, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: In vivo tomatidine treatment inhibits pancreatic tumor growth (A–C) (A) MT5 tumor-bearing C57BL/6 mice (5 mice/group) were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle control (40% HPBCD) and monitored for tumor growth. (n = 5/group). RNA isolated from the tumor tissues of MT5 tumor-bearing C57BL/6 mice treated with vehicle or 5 mg/kg daily i.p. injections of tomatidine were assessed for (B) ATF4 and (C) eIF4EBP1 expression via qPCR. Data are reported as the means + SEMs. n = 5 mice per group. (5A, mixed between-within subjects ANOVA shows a significant interaction between days of treatment and group; 5B-C, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: In Vivo, Control, Isolation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine mediated inhibition of ATF4 signaling can increase sensitivity to ferroptotic cell death in PDAC (A) Panc-1 tumor cells were treated with tomatidine (6.4 μg/mL) and analyzed by RNA sequencing. Ingenuity pathway analysis of the regulated genes suggested ferroptosis as a top hit for tomatidine-treated cells. (B–D) (B) Pancreatic cancer cells were treated with vehicle (DMSO), erastin (to induce ferroptosis), ferrostatin-1 (to inhibit ferroptosis), tomatidine, or in different combinations and lipid peroxidation of (C) Panc-1 and (D) MiaPaca-2 was analyzed by flow cytometry using Bodipy-11. (E) Panc-1 and MiaPaca-2 cells treated with vehicle (DMSO) or tomatidine and lysates collected after 24 h were immunoblot for GPX4 expression. (F) Panc1 cells were plated overnight and treated with tomatidine for 6 h and then assayed by Seahorse assay to analyze mitochondrial fitness. (G) Schematic showing how tomatidine can regulate ATF4-dependent signaling to induce ferroptosis in pancreatic cancer. Data are reported as the means + SEMs. n = 3 or more independent biological replicates. (7C-D, Two-tailed independent student’s test was used to analyze the data; 7F, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05, ∗∗p < 0.007).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Inhibition, RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet:

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Control, Recombinant, Viability Assay, Luciferase, ChIP-qPCR, Gene Expression, Software

Journal: Scientific Reports

Article Title: Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes

doi: 10.1038/s41598-022-05670-5

Figure Lengend Snippet: The graph represents raw Ct values (threshold cycles) of potential housekeeping genes (n = 10) analyzed for the 40 samples from 10 donors (four time points per donor), arranged in ascending order of raw unamplified Ct values. For each gene, the Ct values measured via qRT-PCR with cDNA without pre-amplification as well as after 14× pre-amplification is shown. Symbols represent geometric mean values and error bars reflect the standard error of mean per gene. Vertical and horizontal grey dashed lines show the cut-off (mean Ct ≤ 30). Three of the genes ( ACTB , ATP6 and B2M , highlighted in grey area) showed un-amplified Ct-values < 30 indicating that no pre-amplification for adequate detection will be required.

Article Snippet: In the present work, 10 different TaqMan® Gene Expression Assays ( ACTB , Hs01060665_g1; B2M , Hs00187842_m1; GUSB , Hs00939627_m1; MT-ATP6 , Hs02596862_g1; PGK1 , Hs00943178_g1; PP1A , Hs99999904_m1; RPL13A , Hs04194366_g1; RPLP0 , Hs02992885_s1; TBP , Hs00427620_m1; YWHAZ , Hs01122445_g1) were utilized and pooled to enable the multiplex amplification of specific cDNA targets.

Techniques: Quantitative RT-PCR, Amplification

Journal: Scientific Reports

Article Title: Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes

doi: 10.1038/s41598-022-05670-5

Figure Lengend Snippet: Raw Ct values (threshold cycles) of three candidate house-keeping genes ( ACTB , ATP6 and B2M ) as well as a combination of them (arithmetic mean) are depicted over time (four time point each: 9 am—0 h; 3 pm—6 h; 9 pm—12 h; 9 am next day—24 h) for each donor. They were fulfilling the criteria for being an appropriate housekeeping gene in this context.

Article Snippet: In the present work, 10 different TaqMan® Gene Expression Assays ( ACTB , Hs01060665_g1; B2M , Hs00187842_m1; GUSB , Hs00939627_m1; MT-ATP6 , Hs02596862_g1; PGK1 , Hs00943178_g1; PP1A , Hs99999904_m1; RPL13A , Hs04194366_g1; RPLP0 , Hs02992885_s1; TBP , Hs00427620_m1; YWHAZ , Hs01122445_g1) were utilized and pooled to enable the multiplex amplification of specific cDNA targets.

Techniques:

Journal: Scientific Reports

Article Title: Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes

doi: 10.1038/s41598-022-05670-5

Figure Lengend Snippet: Overview of the housekeeping gene expression results (raw Ct values either without pre-amplification or after 14× pre-amplification) and the significant correlations with sociodemographic and epidemiological characteristics.

Article Snippet: In the present work, 10 different TaqMan® Gene Expression Assays ( ACTB , Hs01060665_g1; B2M , Hs00187842_m1; GUSB , Hs00939627_m1; MT-ATP6 , Hs02596862_g1; PGK1 , Hs00943178_g1; PP1A , Hs99999904_m1; RPL13A , Hs04194366_g1; RPLP0 , Hs02992885_s1; TBP , Hs00427620_m1; YWHAZ , Hs01122445_g1) were utilized and pooled to enable the multiplex amplification of specific cDNA targets.

Techniques: Gene Expression

Journal: Frontiers in Immunology

Article Title: Long-Lasting T Cell Responses in BNT162b2 COVID-19 mRNA Vaccinees and COVID-19 Convalescent Patients

doi: 10.3389/fimmu.2022.869990

Figure Lengend Snippet: Cytokine mRNA expression in PBMCs and secretion of cytokines after stimulation with SARS-CoV-2 spike-specific peptides. Cytokine analysis was performed for PBMCs collected from BNT162b2 vaccinated HCWs 6 weeks (n=15), 3 months (n=15), and 6 months (n=14) after the first vaccine dose and from convalescent COVID-19 patients (n=10) and negative controls (n=10). PBMCs were treated with DMSO or stimulated with tetanus toxoid (10 µg/ml), SARS-CoV-2 wt or Delta S protein peptide pools (0.5 µg/ml) for 48h. Delta variant S peptide stimulation was performed to 7/15 vaccinated, 6/10 COVID-19 patients, and 3/10 negative controls. Cells and supernatants were collected, and total cellular RNA was isolated from stimulated cells. (A) IFN-γ and IL-2 mRNA expression was quantitated from total cellular RNA with RT-qPCR and the data is presented as fold change in comparison to DMSO treated cells. (B) Secreted cytokines (IFN-γ and IL-2) were analyzed with multiplex immunoassay from PBMCs. IFN-γ measurement was unsuccessful from 1 6wk-sample, 1 3mo-sample, and 3 6mo-samples stimulated with DMSO or S_wt. IL-2 measurement was unsuccessful from 3 6wk-samples, 5 3mo and 6mo-samples, and 3 negative samples stimulated with DMSO or S_wt. Data is represented as median and interquartile range. Statistical analysis was performed by Wilcoxon signed rank test for comparison of wt S peptide pool stimulations with Delta S peptide pool stimulations (A, B) and tetanus and SARS-CoV-2 wt and Delta S peptide pool stimulations with DMSO control (B) . Samples with no data on both data points were excluded from analysis. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: For amplification and quantitation, 5 μl of purified RNA was used in One Step PrimeScript III RT-qPCR Kit (Takara Bio Inc) with predesigned TaqMan FAM-MGB IFN-γ (Hs00989291_m1), IL-2 (Hs00174114_m1), IL-4 (Hs00174122_m1), TNF-α (Hs00174128_m1) and β-actin (Hs01060665_g1) primer/probe sets (Thermo Fisher Scientific) in Rotor-Gene Q (Qiagen).

Techniques: Expressing, Variant Assay, Isolation, Quantitative RT-PCR, Comparison, Multiplex Assay, Control

Journal: Frontiers in Immunology

Article Title: Long-Lasting T Cell Responses in BNT162b2 COVID-19 mRNA Vaccinees and COVID-19 Convalescent Patients

doi: 10.3389/fimmu.2022.869990

Figure Lengend Snippet: Correlation of spike-specific T cell responses. Nonparametric Spearman correlation analysis of secreted IFN-γ and IL-2 concentrations with AIM + CD4 + and CD8 + T cells in wild type (wt) SARS-CoV-2 spike protein-specific peptide pool stimulated PBMCs. Results are shown from 15 BNT162b2 vaccinated health care workers at three timepoints after two doses of BNT162b2 vaccine (n=37 fro IFNg and 32 for IL-2), 10 COVID-19 patients, and 8 for IFNG and 3 for IL-2 healthy non-vaccinated controls. Dotted lines indicate 95% CI.

Article Snippet: For amplification and quantitation, 5 μl of purified RNA was used in One Step PrimeScript III RT-qPCR Kit (Takara Bio Inc) with predesigned TaqMan FAM-MGB IFN-γ (Hs00989291_m1), IL-2 (Hs00174114_m1), IL-4 (Hs00174122_m1), TNF-α (Hs00174128_m1) and β-actin (Hs01060665_g1) primer/probe sets (Thermo Fisher Scientific) in Rotor-Gene Q (Qiagen).

Techniques: