Fig. 7

Post-infection of BCG failed to stop autophagy induced by M. smegmatis. A Raw 264.7 WT cells or ATG16L knockdown cells were infected with M. smegmatis MOI 5 for 3 h and then infected with heat-killed or live BCG (MOI 10) for 16 h. Western blots were performed for phosphorylated ribosomal S6 (P-S6), total ribosomal S6 (S6), LC3B-I/II, and actin. B Shown is the LC3B-II/β-actin ratio (± SD). C Raw 264.7 expressing LC3B-GFP were infected with M. smegmatis at MOI 5 for 3 h and then incubated in the presence or absence of HK-BCG MOI 1, 5 10, or 20. Flow cytometry was used to detect GFP. D Densitometric summary analysis was calculated by LC3B-II normalized to β-actin as in (C). E M. smegmatis survival recovered from infected macrophages was determined as in (C). F C57BL/6 mice were immunized with BCG (10⁷) ± HK BCG (3 × 10⁷) or HK 155 (3 × 10⁷) via the subcutaneous route. Spleens were harvested 14 days later, and lymphocytes were re-stimulated for 36 h with BCG lysates (10 ug/ml). IFN production by CD4⁺ cells was evaluated by intracellular cytokine staining. G BCG survival in splenocytes was determined by CFU. The mean ± SD of a representative experiment of 3 independent assays is shown. Significance was calculated by one-way or two-way ANOVA corrected by Dunnett's Test for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001