human fetal lung fibroblasts mrc5  (ATCC)

Journal: bioRxiv

Article Title: A novel role of circCPSF6 regulating antiviral innate immunity via miR-665 and PCBP2-IPS-1 axis

doi: 10.1101/2025.11.03.686289

Figure Lengend Snippet: (A) Heatmap showing clustering of log2 transformed reads per million (RPM) value of circRNAs from RNA seq data (GSE172315) in IAV H9N2 infected A549 cells with relative expression values indicated by blue to red scale (blue, high; red, low). (B) CircCPSF6 expression in control and infected sample from same data. (C) RT-PCR validation of circCPSF6 expression level in A549 cells, infected with PR8 at indicated multiplicity of infection (MOI) and post infection time points. (D) PCR amplification of circCPSF6 and linear CPSF6 using divergent (circRNA specific) and convergent (linear RNA) primers in cDNA or genomic DNA (gDNA) from A549 cells; GAPDH used as control. (E) Schematic representation of circCPSF6 genomic location generated by the back splicing of CPSF6 exons and Sanger sequencing of divergent primer amplicon showing back splice junction (BSJ). (F) Expression of circCPSF6, linear CPSF6 and GAPDH measured with or without RNase R (2U/μg RNA) treatment at 37℃ for 20 mins in A549 cells. (G) Expression of circCPSF6 and linear CPSF6 measured in actinomycin D (2μg/mL) treated A549 cells for indicated time. (H) CircCPSF6 amplification from cDNA prepared using random hexamer and oligo-dT primer. (I) CircCPSF6 copy number quantified by digital PCR (dPCR) in PR8 infected (MOI 1; 24h) A549 cells. (J – L) CircCPSF6 expression in human cell lines (J) MRC5 and THP1, (K) human primary PBMCs, (L) murine primary lung fibroblasts (mpLF) and BMDCs, post PR8 infection (MOI 1; 24h). (M) Schematic representation of in vivo virus challenge and expression of circCpsf6 in mice lung tissue after PR8 infection (PFU 100; 3 days). (N) RNA-FISH using Cy3 labelled antisense-probe designed specific to circCPSF6 BSJ (red) for the cellular localization. Nuclei were stained with DAPI (blue). Scale bar, 10μm. Data are presented as the mean ± SEM from triplicate samples of a single experiment and representative of three independent experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01 by two-tailed unpaired Student’s t -test or two-way ANOVA test. (Schematics are created with BioRender.com)

Article Snippet: A549 human alveolar epithelial cells (ATCC CCL-185), HEK293T human embryonic kidney cells (ATCC CRL-3216), MRC5 human embryonic lung fibroblast cells (ATCC CCL-171) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Transformation Assay, RNA Sequencing, Infection, Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Amplification, Generated, Sequencing, Random Hexamer, Digital PCR, In Vivo, Virus, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: A novel role of circCPSF6 regulating antiviral innate immunity via miR-665 and PCBP2-IPS-1 axis

doi: 10.1101/2025.11.03.686289

Figure Lengend Snippet: (A) Sequence alignment of hsa_circCPSF6 and mmu_circCpsf6 was performed using basic local alignment search tool (BLAST). (B - C) CircCPSF6 expression level determined by RT-PCR in NDV infected (MOI 1; 24h) (B) A549 cells and (C) MRC5 cells.

Article Snippet: A549 human alveolar epithelial cells (ATCC CCL-185), HEK293T human embryonic kidney cells (ATCC CRL-3216), MRC5 human embryonic lung fibroblast cells (ATCC CCL-171) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: bioRxiv

Article Title: A novel role of circCPSF6 regulating antiviral innate immunity via miR-665 and PCBP2-IPS-1 axis

doi: 10.1101/2025.11.03.686289

Figure Lengend Snippet: (A) CircCPSF6 knockdown and viral load was measured by RT-PCR in PR8 infected (MOI 1; 24h) MRC5 cells. (B) Knockdown of circCPSF6 measured by RT-PCR in PR8 infected (MOI 1; 24h) human PBMCs, mice mpLFs and BMDCs. (C – D) Viral load measured by RT-PCR in circCPSF6 knockdown A549 cells followed by (C) NDV infection (MOI 1; 24h) and (D) SeV infection (MOI 2; 24h). (E) Overexpression level of circCPSF6 and viral load measured by RT-PCR in PR8 infected (MOI 1; 24h) MRC5 cells. (F) Viral load measured by RT-PCR in circCPSF6 overexpressing, NDV infected (MOI 1; 24h) A549 cells. (G) IL-6 transcript expression measured by RT-PCR in circCPSF6 knockdown, PR8 infected A549 cells (MOI 1; 24h).

Article Snippet: A549 human alveolar epithelial cells (ATCC CCL-185), HEK293T human embryonic kidney cells (ATCC CRL-3216), MRC5 human embryonic lung fibroblast cells (ATCC CCL-171) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Infection, Over Expression, Expressing

Journal: bioRxiv

Article Title: Nanoscopy Reveals Heparan Sulfate Clusters as Docking Sites for SARS-CoV-2 Attachment and Entry

doi: 10.1101/2025.09.08.674976

Figure Lengend Snippet: hACE2-independent internalization of pseudo-typed SARS-CoV-2. A, Upper: Schematic drawing of a pseudo-typed SARS-CoV-2 virion, VSV EGFP -S-A647, in which the EGFP -coding sequence was inserted into the genomic RNA of VSV, the G protein of VSV was replaced with S of SARS-CoV-2, and A647 was conjugated with primary amines of superficial envelop proteins. Lower: Confocal images of A647 of purified VSV EGFP -S-A647 (V-A647, red) on coverslips and immunofluorescent staining with antibody against S2 subunit of S-protein (green). B, Bright-field and confocal images of VSV EGFP -S-A647’s A647 (V-A647) and EGFP expression ( V-EGFP ) in BHK21 cells stably expressing hACE2 (BHK hACE2 ), BHK21 cells without ACE2 expression (BHK), and BHK cells transiently overexpressed with hACE2- BFP plasmid (BHK+hACE2). Top: Incubation protocol – cells were incubated with VSV EGFP -S-A647 for 1 h at 37°C, followed by washout for 24 h (applies to panels B-G). C, The white dotted box in panel B enlarged to show V-A647 spots in EGFP-labelled cytosol. D, Similar to panel B, except in 293T hACE2 and 293T cells. E, Similar to panel B, except in Vero (containing ACE2), Hela (no ACE2) and MRC5 (no ACE2) cells. F, Similar to panel B, except VSV EGFP -S-A647 was replaced with Lenti EGFP -S Omi -A647. G, Fluorescence intensity of VSV EGFP -S-A647’s V-A647 (F 647 ) and V-EGFP (F EGFP ) in BHK hACE2 (471 cells, 3 experiments), BHK (370 cells, 3 experiments), BHK+hACE2 (515 cells, 3 experiments). 293T hACE2 (404 cells, 3 experiments), 293T (234 cells, 3 experiments), Vero (295 cells, 3 experiments), Hela (305 cells, 3 experiments), and MRC5 (237 cells, 3 experiments) cells. Lenti EGFP -S Omi -A647’s F 647 and F EGFP in BHK hACE2 (364 cells, 3 experiments) and BHK (335 cells, 3 experiments) cells are also plotted. Virus incubation protocol is shown in panel B. F 647 : mean ± s.e.m., reflecting viral uptake; F EGFP : mean ± s.e.m., reflecting viral infection. F 647 and F EGFP were measured per cell identified in the bright field and were normalized to cells expressing ACE2 in each subgroup (see gaps between subgroups). ***: p < 0.001, t-test.

Article Snippet: We used baby hamster kidney fibroblast BHK21 (BHK, ATCC, #CCL-10), human embryonic kidney 293T (293T, ThermoFisher Scientific, R70007), African green monkey kidney Vero E6 (Vero, ATCC, #C1008 [clone E6]), Hela (ATCC, #CCL-2), and human lung fibroblast MRC5 (ATCC, #CCL-171) cells.

Techniques: Sequencing, Purification, Staining, Expressing, Stable Transfection, Plasmid Preparation, Incubation, Fluorescence, Virus, Infection

Journal: Scientific Reports

Article Title: Immune factors produced by PBMCs upon stimulation with lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1-derived exopolysaccharides inhibit HCoV-229E and SARS-CoV-2 replication

doi: 10.1038/s41598-025-17308-3

Figure Lengend Snippet: R-1 EPS-stimulated PBMC supernatant suppresses HCoV-229E replication. ( A ) Schematic diagram of PBMC supernatant treatment. ( B ) The antiviral effect of R-1 sup on HCoV-229E was evaluated using MRC5 as host cells. MRC5 cells were pre-, post-, or both pre- and post- (whole-time) treated by Control sup or R-1 sup (derived from donor #1) upon HCoV-229E infection. Viral genome RNA copy number in culture supernatant were titrated by RT-qPCR at 48 h post infection (hpi). Data are shown as mean ± SD ( n = 8). *: P < 0.05, **: P < 0.01 by Kruskal-Wallis test. ( C ) The time course of HCoV-229E viral genome RNA replication in PBMC sup-pretreated cells. ( D ) Live-cell imaging in HCoV-229E infected MRC5 cells. Untreated, or Control sup or R-1 sup (derived from donor #1)-pretreated MRC5 cells were infected with HCoV-229E, and Incucyte ® Cytotox Reagents were applied to the cell culture medium and cell viability was monitored using Incucyte after infection. Merged images of phase contrast and Cytotox staining of dead cells (green) are shown. Scale bars indicate 200 μm. ( E ) Time course of the dead cell count in Live-cell imaging. Number of green-fluorescence positive dead cells were measured using the Incucyte software. Data are represented as mean ± SD ( n = 8). ( F ) Immunostaining of HCoV-229E spike protein in virus infected cells (48 hpi), which were pretreated with Control or R-1 sup (derived from donor #4). Fluorescence of spike protein is shown in green and DAPI staining of cellular nuclear is shown in blue. Representative 20 × confocal images are shown. Scale bars indicate 100 μm. ( G ) Green-fluorescence positive spike protein area (µm 2 /cell) of HCoV-229E infected cells were measured. Bars represent average area of eight fields of view in each experimental condition. ( H ) Inhibitory effect of R-1 sup pretreatment on HCoV-229E replication were confirmed using PBMCs derived from five individual donors. Data are shown as mean ± SD ( n = 8). ***: P < 0.001 by Mann–Whitney U test.

Article Snippet: MRC5 fatal lung normal diploid fibroblasts (CCL-171, ATCC; Manassas, VA, USA) and human peripheral blood mononuclear cells (PBMC, purchased from Precision for medicine, MD, USA) were maintained in Eagle’s minimal essential medium (EMEM) with L-glutamine, phenol red, sodium pyruvate, non-essential amino acids and 1,500 mg/L sodium bicarbonate (Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA).

Techniques: Control, Derivative Assay, Infection, Quantitative RT-PCR, Live Cell Imaging, Cell Culture, Staining, Cell Counting, Fluorescence, Software, Immunostaining, Virus, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Immune factors produced by PBMCs upon stimulation with lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1-derived exopolysaccharides inhibit HCoV-229E and SARS-CoV-2 replication

doi: 10.1038/s41598-025-17308-3

Figure Lengend Snippet: R-1 EPS-stimulated PBMC supernatant suppresses SARS-CoV-2 replication. The antiviral effect of R-1 sup on SARS-CoV-2 were evaluated using ACE2-expressing MRC5 (MRC5/hACE2) as host cells. ( A ) MRC5/hACE2 cells were pre-, post-, or both pre- and post- (whole-time) treated by Control sup or R-1 sup (derived from donor #1), upon SARS-CoV-2 infection. Viral genome RNA copy number in culture supernatant were titrated by RT-qPCR at 48 h post infection (hpi). Data are shown as mean ± SD ( n = 8). ***: P < 0.001 by Kruskal-Wallis test. ( B ) Time course of SARS-CoV-2 viral genome RNA replication in PBMC sup-pretreated cells. ( C ) Immunostaining of SARS-CoV-2 spike protein in virus infected cells (24 hpi), which were pretreated with Control or R-1 sup (derived from donor #4). Fluorescence of spike protein is shown in green and DAPI staining of cellular nuclear is shown in blue. Representative 20 ×confocal images are shown. Scale bars indicate 100 μm. ( D ) Green-fluorescence positive spike protein area (µm 2 /cell) of SARS-CoV-2 infected cells were measured. Bars represent average area of eight fields of view in each experimental condition. ( E ) Inhibitory effect of R-1 sup pretreatment on SARS-CoV-2 replication were confirmed using PBMCs derived from five individual donors. Data are shown as mean ± SD ( n = 8). *: P < 0.05, **: P < 0.01, ***: P < 0.001 by Mann–Whitney U test.

Article Snippet: MRC5 fatal lung normal diploid fibroblasts (CCL-171, ATCC; Manassas, VA, USA) and human peripheral blood mononuclear cells (PBMC, purchased from Precision for medicine, MD, USA) were maintained in Eagle’s minimal essential medium (EMEM) with L-glutamine, phenol red, sodium pyruvate, non-essential amino acids and 1,500 mg/L sodium bicarbonate (Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA).

Techniques: Expressing, Control, Derivative Assay, Infection, Quantitative RT-PCR, Immunostaining, Virus, Fluorescence, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Immune factors produced by PBMCs upon stimulation with lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1-derived exopolysaccharides inhibit HCoV-229E and SARS-CoV-2 replication

doi: 10.1038/s41598-025-17308-3

Figure Lengend Snippet: R-1 sup induces type I IFN and inflammatory responses in MRC5 cells. Cellular RNA was extracted from untreated, or Control sup or R-1 sup-treated MRC5 cells to perform RNA-Seq analysis. ( A ) Principal component analysis (PCA) was performed using normalized gene counts. Genes with low expression levels were excluded from the analyses. Each point represents an individual sample. Ellipses denote 95% confidence intervals assuming a multivariate normal distribution for each group. ( B ) The number of differentially expressed genes (DEGs, |log2 fold change| > 1, adjusted P value < 0.05) are indicated. ( C ) Top 10 enriched pathways of upregulated and downregulated DEGs by P value are shown (R-1 sup vs. Control sup). Pathway analysis was performed by MetaCore. Pathways are ranked by Log P values. Count indicates the number of DEGs in each pathway. Percent DE indicates the percentage of DEGs in the total object in each pathway. ( D ) Pathway map of “Interferon alpha/beta signaling via JAK/STAT pathway”. The meters next to the object indicate the DEGs in the dataset (R-1 sup vs. Control sup). Red and blue meters represent upregulated genes and downregulated genes, respectively. The level of the meter represents the intensity of the log2 fold change. Image generated using MetaCore. Detailed figure legends of pathway map are available in the MetaCore reference guide ( https://portal.genego.com/legends/MetaCoreQuickReferenceGuide.pdf ).

Article Snippet: MRC5 fatal lung normal diploid fibroblasts (CCL-171, ATCC; Manassas, VA, USA) and human peripheral blood mononuclear cells (PBMC, purchased from Precision for medicine, MD, USA) were maintained in Eagle’s minimal essential medium (EMEM) with L-glutamine, phenol red, sodium pyruvate, non-essential amino acids and 1,500 mg/L sodium bicarbonate (Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA).

Techniques: Control, RNA Sequencing, Expressing, Generated

Journal: Scientific Reports

Article Title: Immune factors produced by PBMCs upon stimulation with lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1-derived exopolysaccharides inhibit HCoV-229E and SARS-CoV-2 replication

doi: 10.1038/s41598-025-17308-3

Figure Lengend Snippet: R-1 EPS-stimulated PBMC supernatant activates type I IFN signaling via JAK/STAT pathway in MRC5 cells. MRC5 cells were treated with Control sup or R-1 sup (derived from donor #1), and the concentrations of type I IFN in culture supernatant ( A ), gene expression levels of interferon stimulated genes (ISGs) ( B ), and signal transduction factors downstream of the IFN-alpha/beta receptor ( C ) were monitored every 6 h. The gene expression levels are shown relative to GAPDH expression. The red line and blue line indicate R-1 sup and Control sup-treated cells, respectively. ( D ) Inhibitory experiment for HCoV-229E replication using recombinant IFN-β. MRC5 cells were pretreated with 0.5, 1, and 5 pg/mL of recombinant human IFN-β (rIFN-β) for 24 h. Viral genome RNA copy number of HCoV-229E were quantified by qRT-PCR at 48 hpi. (E, F) Inhibitory experiment with IFN signaling using ruxolitinib (Rux), a JAK inhibitor, was performed. Rux was added to MRC5 or MRC5/hACE2 cells during the pre-treatment by R-1 sup. Viral genome RNA copy number of HCoV-229E ( E ) or SARS-CoV-2 ( F ) were quantified by qRT-PCR at 48 hpi. Data are shown as mean ± SD ( n = 8). *: P < 0.05, ****: P < 0.0001 by Kruskal-Wallis test.

Article Snippet: MRC5 fatal lung normal diploid fibroblasts (CCL-171, ATCC; Manassas, VA, USA) and human peripheral blood mononuclear cells (PBMC, purchased from Precision for medicine, MD, USA) were maintained in Eagle’s minimal essential medium (EMEM) with L-glutamine, phenol red, sodium pyruvate, non-essential amino acids and 1,500 mg/L sodium bicarbonate (Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest, Nuaillé, France), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA).

Techniques: Control, Derivative Assay, Gene Expression, Transduction, Expressing, Recombinant, Quantitative RT-PCR