Figure 2: STAT activation in haemocytes impairs Atg2 expression to reduce mycobactericidal activity.
From: Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection
(a) M. marinum burden in control (w1118; tub-Gal80ts/+; crq-Gal4/+) and inducible haemocyte-specific Stat92E-knockdown (w1118, UAS-Stat92E-IR/+; UAS-Stat92E-IR/tub-Gal80ts; crq-Gal4/+) flies, assayed by qRT–PCR. Flies were initially infected with 500 CFU M. marinum and cultured at 29 °C. P-value for difference in bacterial numbers by two-way analysis of variance (ANOVA), n=5 per timepoint. (b) Survival of control (w1118; tub-Gal80ts/+; crq-Gal4/+) and haemocyte-specific Stat92E-knockdown (w1118, UAS-Stat92E -IR/+; UAS-Stat92E -IR/tub-Gal80ts; crq-Gal4/+) flies, injected with PBS or infected with 500 CFU M. marinum, as indicated. Infectious dose and temperature as in a. P-value for difference between infected survival curves by log-rank test. (c) Fold change in expression of several autophagy genes in Stat92E-IR (w1118, UAS-Stat92E-IR; UAS-Stat92E-IR/tub-Gal80ts; crq-Gal4/+) flies compared with controls (w1118; tub-Gal80ts/+; crq-Gal4/+and w1118, UAS-Stat92E-IR; UAS-Stat92-IR/+) 5 days after injection with 500 CFU M. marinum. The value shown is the geometric mean of two ratios (knockdown/driver-only control and knockdown/STAT-IR only control). Atg2 fold change is statistically different from other autophagy genes (***P<0.001 by Mann–Whitney test, n=14). (d) Atg2 expression by qRT–PCR in S2R+ cells overexpressing HA (control) or upd3. Normalized to Rpl1. Values are statistically different (*P<0.05 by Mann–Whitney test, n=6). (e) Expression of Atg2 by qRT–PCR in S2R+ cells with Luciferase or Stat92E knocked down, with or without M. marinum infection. Normalized to Rpl1 and then to the uninfected Luciferase control. Values are statistically different as indicated (**P<0.01 by Mann–Whitney test, n=7). (f) Intracellular dsRed-expressing M. marinum in S2R+ cells overexpressing HA (control) or Atg2. Picture is a representative; graph shows normalized M. marinum fluorescence from 37 (Atg2 O/E) or 45 (control) cells. Values are statistically different (*P<0.05 by Mann–Whitney test). Scale bar, 5 μm. (g) Intracellular dsRed-expressing M. marinum in S2R+ cells with Luciferase or Stat92E knocked down by RNAi. The graph shows normalized M. marinum fluorescence from 44 (Stat dsRNA) or 76 (Luc dsRNA) cells. Values are statistically different (**P<0.01 by Mann–Whitney test).
