Enhanced differentiation of human pluripotent stem cells into cardiomyocytes by bacteria-mediated transcription factors delivery
Fig 2
T3SS-based injection of transcription factors into hESC.
(A) hESCs were co-cultured with strain Δ8 containing indicated ExoS54-TF fusions at indicated MOI (0, 30 or 150) for 3 hours. Cells were lysed, nuclear protein extracted and examined for injected proteins by anti-Flag immunoblot. The T3SS deficient strain ΔexsA was infected at MOI of 150. (B) hESCs were co-cultured with indicated strains for 3 hours at MOI 50 and immunostained with anti-Flag to illuminate translocated ExoS54-Flag-TF proteins. The hESCs were infected with Δ8 (MOI = 150) or ΔexsA containing each of the ExoS54-TF fusion at MOI of 30 per strain (total MOI of 150, ΔexsA/GMTEM) as negative controls. Nuclei were stained with DAPI. Scale bar = 100 μm. (C) Translocation efficiency of ExoS54-Flag-TF fusions into hESCs was analyzed by FACS. (D) hESCs was co-cultured with Δ8 at MOI of 150 for 3 hours. Supernatants and adherent ES cells of each well were collected and serially diluted, then plated on LB-agar plates to enumerate the total bacterial cell number (CFU/well) and bacteria attached to the hES cells (residual), respectively. Co-culture was terminated by washing cells with PBS and continued to grow the hES cells on culture medium containing 20 μg/mL ciprofloxacin. After antibiotic treatment for 2 and 4 h, hES cell colonies were scraped and lysed with 0.2% Triton-X100 (bacterial cells do not get lysed), the lysates were serially diluted and plated on LB-agar plates to calculate the residual bacterial cell numbers (CFU/well). Error bars represent SD of triplicate assays.