bhk21  (ATCC)

Journal: mBio

Article Title: Involvement of L polymerase and heat shock proteins in the biogenesis of viral circular RNAs derived from respiratory syncytial virus

doi: 10.1128/mbio.03980-25

Figure Lengend Snippet: Validation of rsv_circ_482/969-protein interactions. ( A–C ) RIP assay was conducted to determine the rsv_circ_482/969-protein interaction. BHK21 cells were transfected with an L-EGFP ( A ) or HSP70-EGFP ( B ) or HSP90-EGFP ( C ) overexpressing plasmid, followed by inoculation with RSV viruses. Cell lysates were subjected to IP with anti-EGFP antibodies, and IgG was used as a negative control. The protein expressions of L/HSP70/HSP90-EGFP were analyzed by WB. The expression of rsv_RNA and rsv_circ_482/969 in different groups was detected by qRT-PCR. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ( D ) HEK293T cells were transfected with rsv_circ_482 and MCP overexpression plasmids described in and then infected with RSV. Pull-down assays coupled with WB were used to validate the interaction between rsv_circ_482 and HSP70/90 proteins.

Article Snippet: HEK293T cells (ATCC), HEp-2 cells (ATCC, CCL-23), and BHK21 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, C11965500BT) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) penicillin-streptomycin and maintained at 37°C in a humidified 5% CO2 atmosphere.

Techniques: Biomarker Discovery, Transfection, Plasmid Preparation, Negative Control, Expressing, Quantitative RT-PCR, Over Expression, Infection

Journal: mBio

Article Title: Involvement of L polymerase and heat shock proteins in the biogenesis of viral circular RNAs derived from respiratory syncytial virus

doi: 10.1128/mbio.03980-25

Figure Lengend Snippet: Cellular HSP70/90 proteins are involved in viral circRNA production. ( A and B ) HEp-2 cells were transfected with three pooled siRNA for each HSP70/90 isoform and then subjected to RSV infection (MOI = 1). The efficiency of siRNAs and the effect of siRNAs on rsv_RNA and rsv_circ_482/969 expression levels were analyzed through qRT-PCR. ( C ) HEp-2 cells were transfected with HSP70-EGFP or HSP90-EGFP plasmid and then infected with RSV (MOI = 1). The protein levels of HSP70/90 were analyzed by WB, and the effect of HSP70/90 overexpression on rsv_RNA and rsv_circ_482/969 expression levels was analyzed through qRT-PCR. OE, overexpression. ***, P < 0.001. ( D ) CO-IP assay was conducted to determine the interaction of HSP70/90 with RSV L protein in BHK21 cells transfected with pL-EGFP plasmids. Cell lysates were subjected to IP with an anti-EGFP antibody and then immunoblotted with anti-HSP70/90 antibody. For all panels, NC means negative control.

Article Snippet: HEK293T cells (ATCC), HEp-2 cells (ATCC, CCL-23), and BHK21 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, C11965500BT) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) penicillin-streptomycin and maintained at 37°C in a humidified 5% CO2 atmosphere.

Techniques: Transfection, Infection, Expressing, Quantitative RT-PCR, Plasmid Preparation, Over Expression, Co-Immunoprecipitation Assay, Negative Control

Journal: mBio

Article Title: Involvement of L polymerase and heat shock proteins in the biogenesis of viral circular RNAs derived from respiratory syncytial virus

doi: 10.1128/mbio.03980-25

Figure Lengend Snippet: The flanking AT/TA sequence of RSV circRNAs functions as a cis -acting element. ( A and B ) Validation of T7 RNP protein expression in BHK21- and HEK293T-derived stable cell lines by WB. ( C ) The expression of support plasmids, pN, pP, pL, and pM2-1, which are required for reverse genetics system, was analyzed by WB. ( D and E ) RSV RNA levels were determined by qRT-PCR at 48 h after co-transfecting BHK21-T7 TNP cells or HEK293T-T7 RNP cells with minigenome replicon and support plasmids. ( F, G ) Effect of flanking sequence mutation on the RSV RNA levels was determined by qRT-PCR in the reverse genetics system described in panels D and E . For panels B to E , P values are as follows: **, P < 0.01; ***, P < 0.001; NS, no significance.

Article Snippet: HEK293T cells (ATCC), HEp-2 cells (ATCC, CCL-23), and BHK21 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, C11965500BT) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) penicillin-streptomycin and maintained at 37°C in a humidified 5% CO2 atmosphere.

Techniques: Sequencing, Biomarker Discovery, Expressing, Derivative Assay, Stable Transfection, Quantitative RT-PCR, Mutagenesis