TBK1 Kinase Addiction in Lung Cancer Cells Is Mediated via Autophagy of Tax1bp1/Ndp52 and Non-Canonical NF-κB Signalling (original) (raw)
Figure 1
TBK1 kinase activity engages basal autophagy in A549 NSCLC cells. a)
A549 GFP-LC3B or GFP-LC3BΔG->A cells were treated with PBS or 5 µM chloroquine (CQ) for 8 h and cells imaged for GFP. b, c) A549 GFP-LC3B cells were transfected with indicated siRNA for 72 h (NTC = non-targeting control) and b) cell extracts blotted for indicated proteins (α-tub = α-tubulin) or cells were c) imaged for GFP-LC3B puncta. d) A549 FLAG-HA-LC3B cells were transfected with indicated siRNA and HA-positive puncta immunostained, imaged and counted at 72 h (n = 3, ± S.E.M., * = p<0.05, ** = p<0.01). e,f) A549 tandem-fluorescent-LC3B (tfLC3B) cells were treated with DMSO or 1 µM MRT68601 (TBKi) for 24 h and e) imaged and f) quantified for total number of autophagic puncta (green+red) and the subset of these puncta with undetectable GFP signal (red), as described in detail in Materials and Methods. g) A549 cells were treated with DMSO or 10 µM MRT68601 (TBKi) for 24 h in the presence or absence of PBS or 5 µM (CQ) chloroquine and cell extracts blotted for indicated proteins. Scale bars = 50 µm in all panels.