Fig. 6

Dram1 Modulates an Autophagic Defense Mechanism that Requires Bacterial RD1 Virulence, Host p62/Sqstm1, and Sting

(A) GFP-Lc3 embryos were injected with 100 pg dram1 RNA. Representative confocal micrographs of epithelial cells at 3 dpf are shown.

(B) The number of GFP-Lc3 punctae was determined for n = 50 cells per group and quantified based on confocal micrographs of control- and dram1 RNA-injected embryos (n e 5, blinded).

(C) Bacterial pixel counts were determined at 3 dpi for infected control- or dram1 RNA-injected embryos. Data (mean ± SEM) is pooled from two individual experiments (n e 94 embryos per group).

(D) Representative stereo micrographs of the tail of infected control- or dram1 RNA-injected embryos at 3 dpi.

(E) Representative confocal micrographs of GFP-Lc3 embryos infected with Mm M ΔRD1 (400 cfu) and wild-type Mm M bacteria (200 cfu).

(F) Bacterial pixel counts following these infections were determined with or without dram1 knockdown. Data (mean ± SEM) are pooled from two individual experiments (n > 62 embryos per group).

(G) Representative confocal micrographs of GFP-Lc3 embryos injected with control morpholino, myd88 morpholino, dram1 morpholino 1, dram1 RNA (100 pg; inset in the image is a micrograph from a different RNA-injected embryo), sqstm1 morpholino, dram1 RNA + sqstm1 morpholino, sting morpholino, and dram1 RNA + sting morpholino. All groups were injected with Mm.

(H–J) Confocal micrographs were used to quantify GFP-Lc3 punctae per infected cell (n e 5 embryos per group, blinded) to evaluate the effect of dram1 RNA (H), dram1 RNA combined with sqstm1 Mo (I), or dram1 RNA combined with sting Mo (J). See also Figure S6.