htbe rnaseq  (ATCC)

Journal: bioRxiv

Article Title: Small molecule inhibition of the mitochondrial lipid transfer protein STARD7 attenuates influenza viral replication

doi: 10.64898/2026.01.20.700505

Figure Lengend Snippet: The M4 compound is a broadly-acting influenza virus inhibitor: ( A ) Chemical structure of M4. (B) After pretreatment with M4 for 16h at the indicated concentrations, A549 cells were infected with influenza A/WSN/33 virus at MOI of 0.1, (C) MEF cells were infected with at MOI of 0.5 and (D) HTBE cells were infected at MOI of 0.25. 48h post infection, cells were fixed, stained for NP, and analyzed with a high content immunofluorescence imager. Percent infection was calculated as the ratio of anti-NP-stained cells to DAPI stained cells. Data are normalized by the mean for DMSO-treated wells and are shown as means ± SEM from three independent experiments. Dose-response curves for infectivity (black) and cell number (red) are shown. (E) MDCK cells were pre-treated for 16hs with different concentrations of M4 followed by infection with the indicated influenza A subtypes at MOI of 0.1 or influenza B/Yamagata at MOI of 0.2. Supernatants were analyzed at 24h post infection by plaque assay and data are represented as means ± s.d. of three independent experiments. The IC 50 values are indicated in .

Article Snippet: HTBE cells (ATCC PCS-300-010) were cultured in commercially available airway epithelial cell basal media following the manufacturer’s protocol (ATCC).

Techniques: Virus, Infection, Staining, Immunofluorescence, Plaque 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: 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

Journal: Nature microbiology

Article Title: Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.

doi: 10.1038/s41564-021-00964-2

Figure Lengend Snippet: Fig. 3 | TBC1D5 restricts IAV replication in vitro, ex vivo and in vivo. a, A549 cells were transfected with indicated siRNAs for 48 h before infection with A/WSN/33 (MOI 0.01). Supernatants were analysed at 48 h postinfection (h p.i.) by plaque assay. Data represent mean ± s.d. of three independent experiments (n = 3). b, A549 cells were transfected with indicated plasmids (10 ng) for 36 h and infected with A/WSN/33 (MOI 0.01). At 48 h p.i., supernatants were analysed by plaque assay. Data show mean ± s.d. from one representative experiment in triplicate (n = 3). c,d, HTBE cells were transfected for 36 h with indicated siRNAs before A/WSN/33 infection (MOI 1). At 24 h p.i., cells were immunolabelled with NP antibody. Representative images are shown in c. Scale bar, 10 μm. Quantification (d) shows mean ± s.d. of three independent experiments (n = 3). e, Protein analysis of A549 parental and TBC1D5 KO clones. Blot is representative of three independent experiments. f, A549 parental and TBC1D5 KO cells were infected with A/WSN/33 (MOI 0.01) for 48 h and supernatants analysed by plaque assay. Data show mean ± s.d. from one representative experiment in triplicate (n = 3). g, Parental, TBC1D5 KO and TBC1D5 KO + 10 ng of TBC1D5 cells were infected with A/WSN/33 (MOI 0.1) for 24 h before immunolabelling with NP antibody. Data show mean ± s.d. normalized infection from one representative experiment in triplicate (n = 3). h, MEFs were treated with indicated PPMOs or PBS for 72 h and subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting. Blot is a representative from two independent experiments. i, Schematic representation of mouse experiments. j, Mice survival was monitored for 14 d p.i. Shown is percentage survival ± s.d. from two independent experiments, each with five mice per condition (n = 10). k,l, On days 3 and 6 p.i., mice were euthanized to harvest the lungs and determine TBC1D5 expression (k) and virus titre (l). Blot is a representative from two independent experiments (k). Graph shows mean lung virus titre ± s.d. from two independent experiments each with five mice per condition (n = 10) (l). Statistical significance was calculated using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test (a,b,d,f,g) or log rank Mantel–Cox test (j). l, Two-way ANOVA test and Dunnett post hoc test were conducted by adding experiment batch as a covariate along with PPMO treatment effect. NS, not significant.

Article Snippet: Eukaryotic cell lines Policy information about cell lines Cell line source(s) Cell lines were purchased from American Type Culture Collection (ATCC): A549 (ATCC CCL-185), MDCK (ATCC CCL-34), HEK293T (ATCC CRL-3216), MEF (ATCC CRL-2991), THP-1 (ATCC TIB-202), and Human tracheobronchial epithelial (HTBE) cells (ATCC PCS-300-010).

Techniques: In Vitro, Ex Vivo, In Vivo, Transfection, Infection, Plaque Assay, Clone Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Expressing, Virus

Journal: Molecular Cell

Article Title: Functional landscape of SARS-CoV-2 cellular restriction

doi: 10.1016/j.molcel.2021.04.008

Figure Lengend Snippet: IFN-mediated restriction of SARS-CoV-2 relies on a limited subset of ISGs (A) Schematic representation of the gain-of-function screen to identify ISGs that inhibit SARS-CoV-2 replication. (B) Ranked log2FC of the percentage of infected cells (SARS-CoV-2 N + cells, blue shading) and normalized cell number (pink shading) after individual overexpression of 399 human ISGs and controls. Values are relative to the negative control CAT . Dashed lines illustrate cut offs for antiviral ISG hit calling strategy; the dotted blue line indicates log2FC infection = 4 × standard deviations (SDs) log2FC of CAT log2FC, and the dotted pink line indicates cell number = 70% of CAT . Controls are shown ( CAT , negative; LY6E , positive). (C) Correlation plots between screens. r, Pearson correlation coefficient. (D) 293T-ACE2 cells transduced with lentiviruses carrying each of the identified ISGs were infected with SARS-CoV-2 (MOI = 0.25) for 40 h prior to immunostaining for viral SARS-CoV-2 nucleoprotein (N). Data represent mean log2FC values (percentage of N + cells relative to parental control wells) from three independent experiments (n = 3). (E) Representative images are shown. Scale bars, 10 μm. (F) Calu-3 cells transduced with lentiviruses encoding the indicated ISGs were infected with SARS-CoV-2 (MOI =1.5) for 48 h prior to immunostaining for viral N protein. Data show mean ± SEM normalized infection (percentage of infected cells relative to parental control wells) from one representative experiment in quadruplicate (n = 4). (G) Differentiated HTBE cells stably expressing the indicating ISGs or negative control GFP were infected with SARS-CoV-2 (MOI = 1) on the apical side. At 18 h post-infection, supernatants were collected and the amount of SARS-CoV-2 focus-forming units per milliliter (FFU/mL) analyzed using Vero E6 cells. Data show mean ± SD and are representative from two sets of HTBE cells per ISG (n = 2). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparison post hoc test (D) or one-way ANOVA with Dunnett’s post hoc test (F and G).

Article Snippet: For the ISG overexpression experiment, HTBE cells from a single donor (Lonza CC2540S) were expanded in PneumaCult Ex Plus medium and differentiated in PneumaCult ALI medium (Stemcell 05040, 05001).

Techniques: Infection, Over Expression, Negative Control, Transduction, Immunostaining, Control, Stable Transfection, Expressing, Comparison

Journal: Molecular Cell

Article Title: Functional landscape of SARS-CoV-2 cellular restriction

doi: 10.1016/j.molcel.2021.04.008

Figure Lengend Snippet: Comparative antiviral activities of SARS-CoV-2 restriction factors Heatmap showing normalized infection upon overexpression of indicated ISGs across 21 viruses. Data for SARS-CoV-2 were generated within this study (see ). Data for the remaining 20 viruses were obtained from previously published work ( Schoggins et al., 2011 , ). Data for SARS-CoV-1 were generated by infecting 293T-ACE2 stably expressing each of the indicated ISGs with SARS-CoV-1 (MOI = 0.01). At 48 h post-infection, supernatants were collected and used to calculate the median tissue culture infectious dose (TCID50). Data show TCID50/mL relative to parental control wells. Data show mean ± SD from one representative experiment in triplicate (n = 3) of two independent experiments. Virus families are indicated. Virus full names and abbreviations are described in .

Article Snippet: For the ISG overexpression experiment, HTBE cells from a single donor (Lonza CC2540S) were expanded in PneumaCult Ex Plus medium and differentiated in PneumaCult ALI medium (Stemcell 05040, 05001).

Techniques: Infection, Over Expression, Generated, Stable Transfection, Expressing, Control, Virus

Journal: Molecular Cell

Article Title: Functional landscape of SARS-CoV-2 cellular restriction

doi: 10.1016/j.molcel.2021.04.008

Figure Lengend Snippet: IFN-mediated restriction of SARS-CoV-2 relies on a limited subset of ISGs (A) Schematic representation of the gain-of-function screen to identify ISGs that inhibit SARS-CoV-2 replication. (B) Ranked log2FC of the percentage of infected cells (SARS-CoV-2 N + cells, blue shading) and normalized cell number (pink shading) after individual overexpression of 399 human ISGs and controls. Values are relative to the negative control CAT . Dashed lines illustrate cut offs for antiviral ISG hit calling strategy; the dotted blue line indicates log2FC infection = 4 × standard deviations (SDs) log2FC of CAT log2FC, and the dotted pink line indicates cell number = 70% of CAT . Controls are shown ( CAT , negative; LY6E , positive). (C) Correlation plots between screens. r, Pearson correlation coefficient. (D) 293T-ACE2 cells transduced with lentiviruses carrying each of the identified ISGs were infected with SARS-CoV-2 (MOI = 0.25) for 40 h prior to immunostaining for viral SARS-CoV-2 nucleoprotein (N). Data represent mean log2FC values (percentage of N + cells relative to parental control wells) from three independent experiments (n = 3). (E) Representative images are shown. Scale bars, 10 μm. (F) Calu-3 cells transduced with lentiviruses encoding the indicated ISGs were infected with SARS-CoV-2 (MOI =1.5) for 48 h prior to immunostaining for viral N protein. Data show mean ± SEM normalized infection (percentage of infected cells relative to parental control wells) from one representative experiment in quadruplicate (n = 4). (G) Differentiated HTBE cells stably expressing the indicating ISGs or negative control GFP were infected with SARS-CoV-2 (MOI = 1) on the apical side. At 18 h post-infection, supernatants were collected and the amount of SARS-CoV-2 focus-forming units per milliliter (FFU/mL) analyzed using Vero E6 cells. Data show mean ± SD and are representative from two sets of HTBE cells per ISG (n = 2). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparison post hoc test (D) or one-way ANOVA with Dunnett’s post hoc test (F and G).

Article Snippet: HTBE (RNAseq) , ATCC , Cat# PCS-300-010.

Techniques: Infection, Over Expression, Negative Control, Transduction, Immunostaining, Control, Stable Transfection, Expressing, Comparison

Journal: Molecular Cell

Article Title: Functional landscape of SARS-CoV-2 cellular restriction

doi: 10.1016/j.molcel.2021.04.008

Figure Lengend Snippet:

Article Snippet: HTBE (RNAseq) , ATCC , Cat# PCS-300-010.

Techniques: Virus, Recombinant, Transfection, Reverse Transcription, SYBR Green Assay, Infection, Negative Control, Expressing, Software, Imaging