Journal: PLoS ONE

Article Title: Identification of Small Molecules with Type I Interferon Inducing Properties by High-Throughput Screening

doi: 10.1371/journal.pone.0049049

Figure Lengend Snippet: A. IFN-β (black bars) and Mx (white bars) mRNA induction in primary human tracheobronchial epithelial (hTBE) cells after 24 hrs incubation with C3. B. Percentage of ATP (representative of cell viability) in hTBE cells after 24 hrs incubation with C3 at increasing concentrations. The amount of ATP in mock-treated cells was set to 100%. Error bars represent standard deviation of three replicates.

Article Snippet: Primary hTBE cells (Lonza, http://www.lonza.com ) were cultured in Bronchial Epithelial Cell Growth Medium supplemented with the BEGM SingleQuot kit (Lonza).

Techniques: Incubation, Standard Deviation

Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: Description of datasets used in this study.

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Infection

Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: Unique network modules in ( A ) human MDM cells, ( B ) HTBE cells, ( C and D ) mouse lung, and ( E ) ferret lower lung are shown. Red nodes denote up-regulation, and blue nodes denote down-regulation. Diamond-shaped nodes are key regulators. Species unique MEGENA modules were identified by enrichment for SRG (JTG) intersection signatures from all eight systems. Modules with significant enrichment in one species but not in any other were deemed unique. (A) The human (MDM) M104 unique module with 30 nodes and 78 edges is ranked number 75 and is responsible for the negative regulation of leukocyte activation. CR1L , HLA-G , MNDA , and RBM34 are key regulators in this module. Most of the genes in this pathway are up-regulated. (B) The number 67 ranked regulation of endocannabinoid signaling/endosomal vacuolar pathway with 114 nodes, 326 edges, and key regulators HDGFRP2 , PDGFC , SMPDT , TIPARP , TJAP1 , and U2AF2 is shown. (C) The number 136 ranked and predominantly down-regulated mouse module M473 relevant for sphingolipid signaling has 27 nodes and 67 edges. The sole key regulator is ANXA11 . (D) M631 is a number 49 ranked and up-regulated mouse module responsible for positive regulation of T cell cytokine production with CTSS as a single key regulator. M631 has 23 nodes and 57 edges. (E) The number 170 ranked ferret lower lung module M14 with 202 nodes and 565 edges shows minichromosome maintenance (MCM) and cell cycle functionality. Key regulators are ARHGAP21 , ARPC1B , ATP6V0C , CD3D , CD3E , CD3G , CDC42BPG , CFL1 , DPP3 , ECT2 , ENO1 , ENSMPUG00000006004 (putative MELK ), ENSMPUG00000008504 (putative TRBC2 ), IFT81 , MCM10 , MCM4 , MKI67 , TPPP3 , TRAC , TRADD , UBE2V1 , and UNC93B1 .

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Activation Assay

Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: A549 cells were transfected with four individual siRNAs targeting TDRD7, the negative control scrambled or the cytotoxic siRNA Allstars. At 48 hours after transfection, ( A ) RNA was isolated and analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using primers specific for TDRD7 and TBP. ( B ) Cell viability was assessed using CellTiter-Glow. ( C ) A549 cells were transfected with indicated siRNAs. At 48 hours after transfection, cells were infected with WSN virus at a multiplicity of infection of 0.01. Supernatants were collected at 48 hours postinfection (hpi) and analyzed by plaque assay using MDCK cells. ( D ) A549, 293T, and HTBE cells were seeded overnight and then treated with increasing doses of universal IFN-β (0, 10, 100, and 1000 IU/ml) for 24 hours. RNA was then isolated and analyzed by qRT-PCR using primers specific for TDRD7 and TBP. (A to D) Data represent means ± SD of at least two independent experiments run in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. Five-week-old female BALB/c mice were administered phosphate-buffered saline (PBS), nontargeting control (NTC), or TDRD7 PPMOs (100 μg in 40 μl of PBS; the equivalent of approximately 5 mg/kg) intranasally for two consecutive days before infection. On day 0, mice were infected with A/Puerto Rico/8/34 [40 plaque-forming units (PFU)] intranasally. On days 3 and 6 after infection, five mice per condition were euthanized to harvest the lungs and determine virus titer. The graph shows the mean lung virus titer ± SD on day 3 ( E ) and day 6 ( F ) after infection. Data shown are from two independent experiments with five mice per condition. ns, not significant.

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Transfection, Negative Control, Isolation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Virus, Plaque Assay, Saline, Control

Journal: Science Advances

Article Title: Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans

doi: 10.1126/sciadv.abm5859

Figure Lengend Snippet: The transcriptional response and induced biological processes during influenza infection in WT and TDRD7 KD HTBE cells are shown. ( A ) The multiset intersections between the TDRD7 network neighborhood (up to layer 2) and significantly responding TDRD7 enhanced (T + ) or restricted genes (T − ) by SuperExact Test are depicted. The networks of all eight systems have been used to construct the consensus network. The layer 2 network neighborhood includes all next and second next neighboring genes of TDRD7 . The heights of the bars indicate set size, and colors refer to FET P values (see scale). Green-filled circles below the bars denote corresponding intersected sets (left). The size of the network layer 2 ∩ T + intersection is 13 genes (red rectangle), whereas the network layer 2 ∩ T − intersection includes 50 members (blue rectangle). Both intersections are significant after FET (layer 2 ∩ T + : 2.28-fold enrichment (FE), P = 3.63 × 10 −3 ; layer 2 ∩ T − : 1.93-FE, P = 4.70 × 10 −6 ). The biological functions of the intersections have been assessed by functional enrichment for BioPlanet (2019) pathways. ( B ) The functional enrichment of TDRD7 -enhanced genes in the network neighborhood (layer 2 ∩ T + ) is shown involving IFN signaling. ( C ) The layer 2 ∩ T − intersection has TNF/NF-κB and IL signaling functionality. Two examples, one each of a T + and T − gene in the layer 2 neighborhood, are depicted. ( D ) Antiviral GBP6 is an IFN-inducible guanosine triphosphatase (GTPase) that requires the IFN modulation by TDRD7 for significant up-regulation at 24 hpi. ( E ) PIM1 is responsible for cell survival and may benefit the viral life cycle. ( F ) The expression of TDRD7 in WT and TDRD7 KD HTBE cells is shown. Significance between WT and TDRD7 KD expression at 24 hpi is indicated as follows: *** P < 0.001 and **** P < 0.0001, which was assessed using edgeR’s generalized linear models together with a quasi-likelihood F test.

Article Snippet: HTBE cells were purchased from Lonza and passaged twice to generate a stock of cells stored in liquid nitrogen.

Techniques: Infection, Construct, Functional Assay, Expressing