Thank you for your comments:

To clarify your query please see Fig. 7 - the answer is clearly stated ; here we have clearly interpreted our data with regard to autophagy.

As you know the hallmark of autophagy is the formation of autophagosomes. This formation is universally monitored using blot against LC3 (importantly the amount of LC3 II correlates to the amount of autophagosomes formation). Also, we have done another experiment to prove that by autophagosome quantification.

In the manuscript page 4 under the headline 'Irga6 is important in the induction of autophagy independent of C. trachomatis infection':

‘To answer these questions, we used immunoblots to monitor the formation of early autophagosomal precursors and newly formed autophagosomes by following changes in LC3 expression. LC3 exists in two forms: the cytosolic LC3-I form, which has a molecular weight of approximately 18 kDa and the membrane-bound LC3-II form, with a molecular weight of 16 kDa (Kabeya, et al., 2000). LC3-II is bound to the membrane of nascent autophagosomes and correlates with the amount of LC3-positive autophagosomes. In WT cells, LC3- II levels increased in response to IFNc stimulation (Figure 7A, lane 3). Notably, levels of IFNγ-induced LC3-II were comparable to those induced by rapamycin (Rapa), a conventional inducer of autophagy (Figure 7A, lane 2). Infection with C. trachomatis for 4 h or 8 h did not influence LC3-II expression (Figure 7A, lanes 4 and 6, respectively)….

To confirm the importance of Irga6 in autophagy, we quantified the number of autophagosomes in the cytoplasm. In contrast to control untreated WT cells (Figure 7D), numbers of GFP-LC3- decorated structures per cell in infected and uninfected WT cells exposed to either IFNγ or Rapa were increased. In agreement with our immunoblot analysis, IFNγ or Rapa induction did not increase the amount of autophagosomes and we observed only low numbers of decorated vesicles in Irga62/2 cells.’

We hope this clarifies your query

Best wishes

Thomas F. Meyer