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.
Figure 2.
Basal autophagy targets TBK1-binding proteins Ndp52 and Tax1bp1. a)
A549 GFP-LC3B cells were stained for Ndp52 and imaged. b,c) A549 cells were treated with PBS or 5 µM chloroquine (CQ) overnight and b) cell extracts blotted for indicated proteins (α-tub = alpha tubulin) or c) cells co-stained for Tax1bp1 and LC3B, and imaged by confocal microscopy. d) 293FT cells were transfected with myc-TAX1BP1 expression vector, or empty vector control, and lysates subjected to pull-down with indicated fusion protein, as described in Materials and Methods (- = GST only, B = GST-LC3B, C = GST-LC3C, G = GST-GABARAP). e) A549 FLAG-HA-TAX1BP1 cell lines stably expressing the indicated variants of Tax1bp1 protein (see text) were treated with 5 µM chloroquine overnight and stained for HA puncta. f) A549 GFP-TBK1 mCherry-LC3C cells were imaged (arrows indicate colocalisation). Scale bars = 25 µm in all panels.
Figure 3.
TBK1, autophagy and the Tax1bp1/Ndp52 cargo receptors maintain pro-survival/proliferation non-canonical NF-κB signalling in A549 NSCLC cells. a–c)
A549 cells were treated overnight with DMSO or 10 µM MRT68601 (TBKi) and a) stained for RelB (scale bar = 50 µm), nuclear localisation quantified in b) (n = 3, ± S.E.M., ** = p<0.01) or c) nuclear extracts prepared and blotted for indicated proteins. d–f) A549 cells were transfected with indicated siRNA (NTC = non-targeting control) for 72 h and e) cell extracts blotted for indicated proteins or d,f) cells were stained and quantified for nuclear RelB (n = 3, ± S.E.M., * = p<0.05). g) A549 cells were treated with PBS or 10 mM 3-methyladenine (3-MA) for 24 hours and then stained and quantified for nuclear RelB (n = 3, ± S.E.M., *** = p<0.005). h,i) A549 cells were infected with indicated lentivirus and, at 72 h post infection, h) cell extracts blotted for indicated proteins or i) total RNA quantified for BIRC3 mRNA (n = 3; ± S.D.). j) A549 cells were transfected with indicated siRNA for 72 h and total RNA extracts were subjected to qRT-PCR for BIRC3 mRNA (n = 3; ± S.D.). k) A549 cells were infected with indicated lentivirus and at 120 h post infection cell number counted as described in Materials and Methods (n = 3, ± S.E.M., * = p<0.05).
Figure 4.
Analysis of roles of Optineurin in RelB signalling and Tax1bp1 in RelA signalling in A549 NSCLC cells. a,b)
A549 cells were transfected with indicated siRNA (NTC = non-targeting control) for 72 h and a) cell extracts blotted for indicated proteins or b) cells stained and quantified for nuclear RelB (n = 3, ± S.E.M., * = p<0.05). c,d) A549 cells were transfected with indicated siRNA (NTC = non-targeting control) and stimulated with 5 ng/ml TNF-α for 3 h and c) stained and d) quantified for nuclear RelA (n = 3, ± S.E.M.). Scale bar = 25 µm.
Figure 5.
Basal autophagy drives nuclear RelB localisation in NCI-H23 NSCLC cells. a)
NCI-H23 cells were transfected with indicated siRNA (NTC = non-targeting control) for 72 h and a) cell extracts blotted for indicated proteins. b,c) NCI-H23 tandem-fluorescent LC3B cells were transfected with indicated siRNA and analysed as described in Materials and Methods. d,e) NCI-H23 cells were transfected with indicated siRNA for 72 h and cells were d) stained and e) quantified for nuclear RelB (n = 3, ± S.E.M., * = p<0.05, ** = p<0.01). Scale bar = 50 µm in all panels.
Figure 6.
Model of autophagy function in K-Ras addiction versus xenophagy.
See text for detailed discussion. Ub = ubiquitin.