Figure 6
siRNA vs.beclin-1 mediated inhibition of autophagy in mesenchymal stem cells enhances survival of intracellular M. tuberculosis. Untreated or treated BM-MSCs were infected with Mtb (MOI = 1). Lysates were plated for viability of intracellular Mtb by plating lysates on 7H11 agar plates at the indicated time intervals, and stem cell viability was evaluated using alamar blue. (a) Mtb infected MSCs retained approximately 90% viability over 7 days of culture. (b) Mtb counts of MSCs did not increase over the course of 7 days of culture. (c) BM-MSCs were treated with siRNA vs. belcin-1 or scrambled siRNA control, then infected with Mtb and CFU counts done over the course 7 days. Average CFU counts per 104 MSCs of triplicate wells per group of 3 identical experiments are shown in panels b and c (**p < 0.005,*p < 0.05, ANOVA). siRNA vs.beclin-1 decreased the initial uptake of Mtb, although intracellular Mtb increased in numbers after knockdown of beclin-1. (d) MSC lysates were tested for beclin-1 protein levels using western blotting to confirm siRNA knockdown.
