rig i reporter cell line  (InvivoGen)

Journal: bioRxiv

Article Title: Template-Directed RIG-I Agonist Assembly for Targeted Cancer Immunotherapy

doi: 10.1101/2022.12.08.519592

Figure Lengend Snippet: a) RIG-I protein expression in HEK-Lucia™ RIG-I cells. The whole cell lysates were collected and assayed (50 μg) for the RIG-I proteins by Western analysis using specific antibody. HEK-Lucia™ RIG-I cells were found to express high levels of RIG-I protein. b) RIG-I activation by ds-ppp-RNA (+ive control) in HEK-Lucia™ RIG-I and HEK-Lucia™ Null control cells. Cells were transfected with Lyovec control or stated concentrations of ds-ppp-RNA with Lyovec. Luciferase activity was measured in supernatants after 48 h as per manufacturer’s recommendations. The commercially available ds-ppp-RNA was shown to robustly induce RIG-I activation (n = 3; *, p < 0.05; **, p < 0.01). c) Comparison of ss-ppp-miRNA-21 and ss-miRNA-21-mediated dose-dependent RIG-I activation in Null vs RIG-I expressing reporter cell lines. HEK293 RIG-I cells or Null cells were treated with the indicated concentrations of agonists. Luciferase activity was measured in supernatants after 48 h. At all tested concentrations, luciferase activity was significantly higher in the HEK-Lucia™ RIG-I cells than in the HEK-Lucia™ Null cells, indicating RIG-I specific activation. Both the ss-ppp-miRNA-21 and ss-miRNA-21 designs led to RIG-I activation (n = 3; *, p < 0.05; **, p < 0.01). d) Relative expression of miRNA-21 in B16-F10 cells. B16-F10 cells express moderate levels of miR-21, as compared to a reference cell line (murine breast 4t1), known to express abundant miR-21 (n = 3; *, p < 0.05; **, p < 0.01). e) Caspase-3/7 activation (dose dependent) by ppp-ss-miRNA-21 in B16-F10 cells. Cells were transfected with varying concentrations of ppp-ss-miRNA-21 or ss-miRNA-21 with Lyovec. After 48 h cell death was measured by a CaspaseGlo assay. ss-ppp-miRNA-21 induced a dose-dependent increase in caspase activation that was not observed when using ss-miRNA-21 (n = 3, p ≤ 0.05). f) Cell viability reduction (dose dependent) by ss-ppp-miR21 in B16-F10 cells. Cells were transfected with varying concentrations of ss-ppp-miRNA-21 or ss-miRNA-21. Cell viability was determined using CellTiter-Glo® 2.0 Assay after 48 h of incubation. There was a dose-dependent decrease in tumor cell viability in the presence of ss-ppp-miRNA-21 but not ss-miRNA (n = 3; p ≤ 0.05).

Article Snippet: The RIG-I reporter cell line HEK-Lucia™ RIG-I cells (Catalog Code, hkl-hrigi, InvivoGen) and the control cell line HEK-Lucia™ Null cells (Catalog No. hkl-null, InvivoGen), and Mouse skin melanoma cells (B16-F10) were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Gibco).

Techniques: Expressing, Western Blot, Activation Assay, Transfection, Luciferase, Activity Assay, Comparison, Caspase-Glo Assay, Incubation

Journal: bioRxiv

Article Title: Template-Directed RIG-I Agonist Assembly for Targeted Cancer Immunotherapy

doi: 10.1101/2022.12.08.519592

Figure Lengend Snippet: a-b) RIG-I activation by ss-ppp-miRNA-21 in HEK-Lucia™ RIG-I and HEK-Lucia™ Null control cells. Cells were transfected with ss-ppp-miRNA-21 or ss-miRNA-21 (4 μg/mL) along with varying concentrations of miRNA-21 mimic. Luciferase activity was measured in the supernatants after 48 h as per manufacturer’s recommendations. a) There was a dose-dependent RIG-I activation with increasing concentrations of the miR-21 mimic in the in HEK-Lucia™ RIG-I but not the HEK-Lucia™ Null cells, indicating RIG-I-specific activation that is miRNA-21 template dependent (n = 3; *, p < 0.05; **, p < 0.01). b) The response curves to the ss-ppp-miRNA-21 and ss-miRNA-21 resulted in the determination of an EC50 of 83.4 ng/mL for the miRNA-21 mimic when using ss-ppp-miRNA-21. By contrast, the calculated EC50 when using the 5’-ppp-deficient ss-miRNA-21 was 357.9 ng/mL, indicating that the presence of the 5’-ppp rendered the response more potent in this cell line (n = 3; **, p < 0.01; ***, p < 0.001). c) IFN-β expression by miRNA-21 transfected B16-F10 cells. B16-F10 cells (with or without transfection with a miRNA-21 mimic) were treated with indicated concentrations of ss-ppp-miRNA-21 or ss-miRNA-21 for 48 h. IFN-β concentrations in the culture supernatant were measured using ELISA. Transfection with a miRNA-21 mimic led to an increase in IFN-β secretion in the ss-ppp-miRNA-21 treated cells, relative to cells devoid of the mimic (n = 3; **, p < 0.01); d) IP-10/CXCL-10 concentration in the supernatants was quantified by ELISA in cells treated as in c. There was a highly but significant increase in IP-10/CXCL-10 secretion in cells transfected with the miRNA-21 mimic, when the cells were treated with ss-ppp-miRNA-21 relative to cells treated with ss-miRNA-21 (n = 3; ***, p < 0.001; ****, p < 0.0001). IP-10/CXCL-10 secretion in cells transfected with the miRNA-21 mimic, when the cells and treated with ss-ppp-miRNA-21 was higher than in cells treated with the ds-ppp-miRNA positive control (n = 4; p = 0.0001). e) Caspase-3/7 activation (dose dependent) in miRNA-21 mimic transfected B16-F10 cells. Cells were treated with ss-ppp-miRNA-21 or ss-miRNA-21 (4ug/mL) along with increasing concentrations of miRNA-21 mimic. After 48 h, cell death was measured by a CaspaseGlo assay. There was a dose-dependent increase in caspase 3/7 activation by ss-ppp-miRNA-21 in cells transfected with increasing concentrations of the miRNA-21 mimic (n = 4; *, p < 0.05; **, p < 0.01; ***, p < 0.001). f and g) western blot analysis of RIG-I; and phosphorylation of NF-kB (p65) protein. B16-F10 cells (with or without transfection with miRNA-21 mimic) were treated with ss-ppp-miRNA-21 (8 μg/mL) and respective controls. Whole cell lysates (50 μg) were analyzed over 8-20% SDS-PAGE and assayed using specific antibodies for indicated proteins. f) There was a moderate increase in RIG-I protein in cells treated with ds-ppp-miRNA-21. Protein induction was most notable in cells transfected with miRNA-21 mimic and treated with ss-ppp-miRNA-21. g) The abundance of phospho-P65 was increased in cells treated with ss-ppp-miRNA-21 relative to control treatments. This effect was seen both in cells transfected with miRNA-21 mimic prior to treatment with ss-ppp-miRNA-21 and cells that had not been transfected with the mimic. No differences in P65 protein were observed, indicating that the effect reflected protein phosphorylation.

Article Snippet: The RIG-I reporter cell line HEK-Lucia™ RIG-I cells (Catalog Code, hkl-hrigi, InvivoGen) and the control cell line HEK-Lucia™ Null cells (Catalog No. hkl-null, InvivoGen), and Mouse skin melanoma cells (B16-F10) were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Gibco).

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Positive Control, Caspase-Glo Assay, Western Blot, SDS Page