Figure 3.

Acid sphingomyelinase determines expression of cathepsin D, which is essential for BCG degradation. (A) Bone-marrow-derived macrophages (BMDMs) from wild-type (wt) and Asm-deficient (Asm^−/−) mice were infected with BCG for the indicated times or left uninfected and then subjected to Western blot analysis using antibodies against mature cathepsin D (CTSD) and β-actin. Western blot is representative of 3 independent experiments. (B) Expression of CTSD was normalized to actin levels and displayed as arbitrary units (a.u.) by using ImageJ. Shown are means ± SD of 3 experiments; p-values were calculated by ANOVA followed by Bonferroni’s multiple comparisons test, ** p < 0.01, * p < 0.05. (C) Wt or Asm-deficient macrophages were left uninfected or infected with GFP-BCG for 60 min, fixed, and stained with Cy3-labeled antibodies against CTSD. Samples were analyzed by confocal microscopy. Picture represents 3 independent studies. Scale bar = 25 µm. (D) Co-localization of GFP-BCG and CTSD was measured from at least 50 bacteria/sample and shown as mean ± SD of 3 experiments. *** p < 0.001 (Student’s t-test). (E) BMDMs were left untreated or pretreated with 10 μM pepstatin for 1 h and infected with BCG for 24 h. Number of BCG CFUs was determined after 2 weeks of culture. Shown are means ± SD of the CFU from 3 experiments; two-way ANOVA followed by Bonferroni’s multiple comparisons test, * p < 0.05. (F) BMDMs were transfected with either CTSD gRNA/CRISPR-cas9 plasmid or CRISPR-cas9 control plasmid, then infected with BCG for 24 h for CFU assay. Shown are means ± SD of CFU of 3 independent experiments. Quantitative analysis was performed with GraphPad and analyzed with two-way ANOVA followed by Bonferroni’s multiple comparisons test, * p < 0.05. ns = no significant.