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. Author manuscript; available in PMC: 2024 Apr 22.
Published in final edited form as: Mucosal Immunol. 2024 Jan 6;17(2):155–168. doi: 10.1016/j.mucimm.2024.01.001

Fig. 4.

Fig. 4

E-ALF-exposed M.tb has decreased endolysosomal trafficking within ATs. ATs were infected with either A-ALF or E-ALF-exposed GFP-M. tb for 2 hours at MOI of 100:1 followed by 1 hour of gentamicin to kill extracellular M.tb. Monolayers were stained with different intracellular markers at the indicated time points (6 hpi, 12 hpi, 24 hpi, and 72 hpi). (A) Co-localization events indicative of compartment fusion of M.tb with LAMP-1+ (represents M.tb in lysosomes) or LC3+ (represents M.tb in autophagosomes), n = 4 (mean ± SEM) in duplicate, using pooling of four different A-ALFs or E-ALFs. (B) Percent reduction in co-localization of E-ALF-exposed M.tb relative to A-ALF-exposed M.tb at the indicated time points. (C) Representative confocal images of ATs infected with A-ALF and E-ALF-M.tb stained with intracellular markers LAMP-1 and LC3 at the indicated time points. Events were enumerated by counting at least 200 independent events (200–300 bacteria). The region indicated by the gray dashed line is shown expanded on the right; and co-localization events are indicated by white arrowheads. Open arrowheads indicate double co-localization events (LAMP-1+LC3+). <scale bars represent 10 μm>. Student’s unpaired t test analysis of Adult versus Elderly, *p < 0.05, **p < 0.01. The “n” values represent the number of biological replicas using pooling of ALF samples from different adult or elderly human donors. A = adult ALF-exposed M.tb; ALF = alveolar lining fluid; ATs = alveolar epithelial type cells; DAPI = 4’,6-diamidino-2-phenylindole (ATs nuclear DNA); DIC = differential interference contrast; DNA = deoxyribonucleic acid; E = elderly ALF-exposed M.tb; GFP = green fluorescent protein; hpi = hours post-infection; LC3 = microtuble-associated protein light chain 3; LAMP = Lysosomal-associated membrane protein; MOI = multiplicity of infection; M.tb = Mycobacterium tuberculosis; ns = no significant differences; SEM = standard error of the mean.