Extended Data Fig. 2: Immunogenic cell death in human cancer cells and pathways of interferon stimulation in response to treatments with TT3 or TT3-Rep.
a–c, Human A549 lung carcinoma (a), SK-MEL-5 melanoma (b), or Hela cervical cancer cells (c) were treated with 10 µg/mL empty TT3 LNPs (TT3), TT3 LNPs carrying replicons encoding GFP (TT3-Rep), or left untreated as indicated. Three days post treatment, the percentage of viable cells was enumerated (left column). One day post treatment, the ICD markers extracellular ATP (second column), CRT+ cells (third column), extracellular HMGB1 (fourth column) were measured. Shown are mean (box) of different treatments from 3 technical cell culture replicates from a single experiment. d, Twelve hours post treatment of 10 µg/mL TT3 LNP (LNP) or TT3 LNP carrying replicons encoding mCherry (LNP-Rep), levels of MDA-5 and TLR3 mRNA in B16F10 cells were assessed by qPCR. Shown are fold changes of MDA-5 and TLR3 expression in different treatments (n = 6 from 3 technical cell culture replicates and 2 technical qPCR replicates) in comparison to untreated cells (n = 6 from 3 technical cell culture replicates and 2 technical qPCR replicates) normalized by actin B expression from a single experiment. e, One day post treatment of A549 type I interferon reporter cells or MDA-5 knockout A549 reporter cells with 10 µg/mL TT3 LNP (LNP) or TT3 LNP carrying replicons encoding mCherry (LNP-mCherry), the supernatants were collected and incubated to detect stimulation the expression of the luciferase reporter. Shown are relative luminescence units from n = 4 technical cell culture replicates from a single experiment.
