Fig. 9.

Time course experiments of BNIP3 and CTSE western blot analyses with protein extracts from L. m.-infected BMDM, total autophagy scores, determination of the nucleus-kinetoplast distances, and infection rates. Methods: (a–e) BMDM from BALB/c mice were infected with L. m. promastigotes up to 48 h p.i.. Uninfected controls were incubated for the same amount of time in RPMI medium. a, b Proteins were harvested and subjected to western blot analysis. Protein expressions of L. m.-infected BMDM were analyzed densitometrically. ACTB served as the internal loading control. c L. m.-infected and uninfected BMDM were subjected to TEM analyses. 50 BMDM of each sample were analyzed semiquantitatively for the grade of vacuolization (0 – 3) and the presence of MLS (+1) resulting in the total autophagy score (maximum = 4). d, e L. m.-infected BMDM were stained with a Diff-Quik kit to determine (d) the distances between the parasite nuclei and the kinetoplasts and (e) the infection rates for each investigated time point. Results: (a) BNIP3 and CTSE overexpression in L. m.-infected BMDM increased over time up to 24 h p.i. compared to uninfected controls. b This overexpression was confirmed densitometrically for BNIP3 (dark gray columns) and CTSE (light gray columns). c The total autophagy score was significantly increased in L. m.-infected BMDM (red columns) in all investigated samples compared to uninfected controls (blue columns). In L. m.-infected BMDM at 2 to 10 h p.i., a significant decline in the total autophagy score was detectable compared to L. m.-infected BMDM at 1 h p.i.. This decline might has been caused by a partial autophagy inhibition. The total autophagy score significantly rose in L. m.-infected BMDM at 24 h p.i. compared to 10 h p.i.. The total autophagy score declined in uninfected controls from 0.5 h until 24 h. d The distance between nucleus and kinteoplast shortened over time, which confirmed the differentiation of promastigotes into amastigotes (black dots). e The infection rates increased over time up to 24 h p.i. (green columns). Between 1 and 24 h p.i., there were no significant changes in the infection rates, which indicated an inhibition of autophagic activity. Finally, the infection rates declined from 24 h p.i. until 48 h p.i., which suggested that parasites were autophagically digested. L. m.-inf. = L. m.-infected, n.s. = not significant, p.i. = post infection, ⁽*⁾ p ≤ 0.1, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001