Fig 6.

PikA disturbs autophagy by interacting with Beclin 1. (A) PikA induced the production of LC3B-II. Lysates of HEK293T cells transfected to express mCherry, mCherry-PikA, or mCherry-PikA_H171A for 14 h were probed for LC3B and p62 by immunoblotting, the expression of the PikA and its mutant was also probed, and actin was used as a loading control (left). Densitometry of LC3B using Fiji software from three independent experiments. Band intensity quantitation was performed to calculate the fold change of LC3B-II/LC3B-I ratio (right). (B) The effects of inhibitors on autophagy induced by PikA. HEK293T transfected to express mCherry, mCherry-PikA, or mCherry-PikA_H171A for 14 h were treated with 100 nM Bafilomycin A1 for 4 h, and LC3B processing was probed by immunoblotting. Actin was detected as a loading control. Data shown were one representative from three independent experiments with similar results. (C and D) PikA induced a notable increase in autophagosome formation. HeLa cells transfected to express GFP-LC3B and mCherry-PikA or mCherry-PikA_H171A for 14 h (C) were analyzed using a fluorescence microscope for image acquisition (D, left) (bars, 5 µm) and quantitation of the number of autophagosomes labeled by GFP-LC3B. Nuclei were labeled by Hoechst staining. Results (mean ± s.e.) shown were from three independent experiments done in triplicate, at least 100 cells were scored for each sample (D, right). ns, not significant; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. (E and F) Interaction between PikA and Beclin1. Lysates of HEK293T cells transfected to express Flag-PikA and HA-Beclin1 for 14 h were subjected to immunoprecipitation with antibody specific for Flag (E) or HA (F), and co-purification of the interacting protein was detected by immunoblotting with the respective antibodies. Images shown were one representative of three independent experiments with similar results.