Extended Data Fig. 4: Both PVCs and payloads are necessary for efficient killing of insect cells.
From: Programmable protein delivery with a bacterial contractile injection system
a, Unloaded PVCs, as well as separately purified payloads (toxins Pdp1 and Pnf), are insufficient to produce efficient cytotoxicity in Sf9 cells. Pdp1 and Pnf were purified independently of PVC complexes by affinity purification and were administered to Sf9 cells at doses roughly equivalent to the amount loaded into PVCs (determination of biologically-relevant payload doses in (b)). Efficient cytotoxicity was observed only when both the PVC complex and the payload proteins were co-purified. Values are mean ± s.d. with n = 3 biological replicates. Statistical significance was computed using one-way ANOVA with Bonferroni post hoc test; ****P < 0.0001. b, Dose determination for cytotoxicity assay from (a). PVCs loaded with HiBiT-tagged payload proteins were run alongside a titration of a control protein (HiBiT control protein; Promega N3010); band intensities (quantified with ImageJ) were then compared to determine the approximate quantity of payload protein loaded into PVCs. From this analysis, we observed that Pdp1 is loaded in higher quantities than Pnf (~1% and ~0.1% of total protein, respectively); we thus dosed Sf9 cells in (a) with purified Pdp1 and Pnf protein at 1% and 0.1% of the dose of loaded PVCs. c, Representative flow cytometry analysis of PVC-mediated protein delivery in cultured cells. After gating the cells on FFC/SSC to remove debris, we set a GFP-/+ threshold using a positive control condition (cells transfected with both loxP-GFP and Cre). We then applied this threshold to experimental data to determine the proportion of cells expressing GFP.
