Fig. 2: Lysosomal killing of bacteria in ADSCs and effect of inflammatory cytokines. | Nature Communications

Fig. 2: Lysosomal killing of bacteria in ADSCs and effect of inflammatory cytokines.

From: Mesenchymal stem cells offer a drug-tolerant and immune-privileged niche to Mycobacterium tuberculosis

Fig. 2

a, b H37Rv CFU from infected ADSCs after bafilomycin A1 (BafA1, 100 nM), 3-methyladenine (3MA, 5 mM), chloroquine (100 µM), or rapamycin (100 nM) treatment for the final 6 h in INH treated (24 h) (a) or untreated (b) groups, n = 4 (c, d) 3rd day CFU from H37Rv-infected dTHP-1 treated with IFNγ (5 ng/ml) or TNFα (20 ng/ml) for 24 h in the presence (c) or absence (d) of INH, n = 4. Dose-dependent effect of IFNγ (5, 12.5, 25 ng/ml) and TNFα (10, 20, 50 ng/ml) treatment (24 h) on H37Rv survival in the infected ADSCs in the presence (e) or absence (f) of INH, n = 4 independent experiments with 2–3 biologically independent samples. CFU assay of infected ADSCs (g) and infected THP-1 macrophages (h) upon IFNγ and TNFα treatment along with doses of IFNγR1 and anti-TNFα purified proteins, respectively, n = 5. i Representative confocal images of PKH67-labeled H37Rv- and BCG-infected ADSCs (3rd dpi), stained with LysoTracker Red and LAMP1. Scale bars, 10 µm. Images are representative of >50 fields across three independent experiments. Percent colocalization of H37Rv with LysoTracker Red, LAMP1, LysoTracker + LAMP1 (j, n = 5, 5–8 independent fields each) and CatD +  LAMP1+lysoTracker triple positive (l, n = 3, 3–4 independent fields each) in ADSCs which were either untreated control or treated with IFNγ (5 ng/ml), TNFα (20 ng/ml) or MK571 (50 µM) for 24 h prior to samples processing at 3rd dpi, k Percent colocalization of BCG with LysoTracker Red, LAMP1, and LAMP1 + LysoTracker compartments at 3rd dpi in ADSCs, n = 3, three independent fields each. m Percent colocalization of H37Rv with ABCC1 in ADSCs control or IFNγ, TNFα, MK571 treated 3rd day samples, n = 3, 3–5 independent fields each. a–h: mean ± SD, j–m: mean ± SEM. Data were analyzed using two-tailed unpaired Student’s t test (b, d, f, j–m) and one-way ANOVA (a, c, e, g, h). *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001, NS ‘not significant’ Source data are included in Source data File.

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