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. Author manuscript; available in PMC: 2022 Feb 7.
Published in final edited form as: Nat Chem. 2021 Aug 9;13(10):992–1000. doi: 10.1038/s41557-021-00766-3

Fig. 4. Mach miniproteins are highly active in vitro and in vivo and deliver other biomacromolecules into the cytosol.

Fig. 4

(a-c) Shown are dose-response curves corresponding to activity in the EGFP assay and toxicity in the LDH assay for PMO-Mach3, 4, and 7. Activity is shown as fluorescence intensity relative to unconjugated PMO at 5 μM, and toxicity is shown as LDH release relative to a lysis control. (d) The relative fluorescence of Mach conjugated to PNA 654 are compared to PNA alone, as determined by EGFP assay. (e) Comparison of the toxicity of wild-type and inactive mutant DTA and DTA(E148S) alone or conjugated to Mach3 or Mach7. Delivery of the active toxin to the cytosol results in toxicity as measured by luminescence. (f) Confocal micrographs displaying green fluorescence produced by EGFP, Mach3-EGFP, or Mach7-EGFP in HeLa cells after 3 h incubation at 10 μM. (g-i) EGFP synthesis in EGFP transgene mice after treatment with PMO-Mach. Dose-response EGFP protein level in (g) quadriceps (h) diaphragm and (i) heart. (a-c) Points show mean ± SD, for EGFP assay n = 3, and mean for LDH assay, n = 2 distinct samples. EGFP assay experiments were repeated at different concentration ranges with similar results, reported in Supplementary Figure 21 (d) Each bar represents group mean ± SD, n = 3 distinct samples, p-values calculated from student’s two-tailed t-test (PNA-Mach3 p = 0.0005, PNA-Mach7 p = 0.0002. (e) Each bar represents group mean ± SD, n = 3, except for Mach3-DTA(E148S) and Mach7-DTA(E148S) which show mean, n = 2 distinct samples. Full concentration curves are reported in Supplementary Figure 25. (f) Scale bar shows 10 μm. This experiment was conducted twice independently with similar results. (g-i) Saline (n = 6), Mach3 and Mach4 at 5 mg/kg (n = 4), all other n = 8 mice. Each bar represents group mean ± SD, p-values calculated from two-tailed Mann-Whitney U test. (Quadriceps: PMO-Mach3 10 mg/kg p = 0.0003, PMO-Mach3 30 mg/kg p < 0.0001, PMO-Mach4 30 mg/kg p < 0.0001; Diaphragm: PMO-Mach3 10 mg/kg p < 0.0001, PMO-Mach3 30 mg/kg p <0.0001, PMO-Mach4 30 mg/kg p < 0.0001; Heart: PMO-Mach3 5 mg/kg p = 0.0001, PMO-Mach3 10 mg/kg p < 0.0001, PMO-Mach3 30 mg/kg p < 0.0001, PMO-Mach4 10 mg/kg p < 0.0001, PMO-Mach4 30 mg/kg p < 0.0001.)