human embryonic lung fibroblast cell line mrc 5 cells  (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