Figure 7. hGBP1 and hGBP2 played a role in the regulation of the host autophagic machinery. (A–C) Anti-LC3 and SQSTM1 immunoblot analysis of total lysates from uninfected control (empty), hGBP1- and hGBP2-stable knockdown macrophages or from cultures infected for the indicated time points in the presence or absence of 100 U/ml IFNG for 24 h. Other monolayers were pretreated with IFNG for 24 h prior to infection and then infected in the presence of IFNG. Host ACTB was used as loading control. IFNG induced autophagy in control THP1-derived macrophages; similarly treated cells infected with C. trachomatis induced autophagy as indicated by the higher amount of LC3II and reduced amount of SQSTM1. (B and C) hGBP1 or hGBP2 knockdown impaired autophagy induction. Only very low amounts of cellular LC3II, but increasing amounts of SQSTM1 can be detected in hGBP2-knockdown cells, compared with those in control THP1-derived macrophages, while hGBP1 knockdown had a minimal stimulatory effect on autophagy. (D) Anti-ATG5 and ATG12 immunoblots of total lysates from control (empty), hGBP1- and hGBP2-stable knockdown macrophages. Monolayers were pretreated with IFNG for 24 h prior to infection and then infected with C. trachomatis (MOI 10) in the presence of IFNG or left without treatment. Host ACTB was used as loading control. IFNG induced ATG12–ATG5 conjugate formation in all cells. hGBP2, and to a lesser extent hGBP1, knockdown impaired autophagy induction. Monomeric ATG5 and ATG12 were detected in cells lacking hGBP2, and to lesser extents in cells lacking hGBP1, but were absent in control THP1-derived macrophages. Blot shown in (A) is representative of three independent experiments and blots in (B–D) are representatives from two independent experiments.