Fig 2: L. johnsonii L531 reverses the disruption of iron metabolism caused by S. Typhimurium in IPEC-J2 cells. (A) S. Typhimurium (MOI = 40) burden was tested by plating. Data were obtained from three independent replicates of each sample at 2, 4, 6, 8, 10, and 12 hpi. hpi: hours post infection. (B,D) The cellular free iron content of IPEC-J2 cells. CN, Control. ST, S. Typhimurium. L, L. johnsonii L531. LS, L. johnsonii L531 + S. Typhimurium. (C,G) Western blot analysis of DMT1, TfR1, and FPN. (E) Free ferrous iron in IPEC-J2 cells was stained by FerroOrange. Scale bar, 20 µm. (F) The bacterial load was detected by plating. Values are expressed as the means ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (ns, not significant).

Fig 3: Iron homeostasis disorder triggered by S. Typhimurium promotes its growth and provokes oxidative stress in IPEC-J2 cells. (A) Cell viability assay of IPEC-J2 cells. CN, Control. (B) The intracellular bacterial load in IPEC-J2 Cells. (C) The cellular free iron content of IPEC-J2 cells. (D) Free ferrous iron fluorescence staining. Scale bars, 20 µm. (E) Analysis of S. Typhimurium intracellular localization. Blue, DAPI. Green, S. Typhimurium. Scale bars, 10 µm. Western blot analysis of DMT1, TfR1, FPN (F), Nrf2, and HO-1 (H). (G) ROS levels were determined by DCFH-DA kit. Scale bars, 100 µm. (I) The NQO1, SOD1, and GPX1 mRNA expression levels in IPEC-J2 cells. Values are expressed as the means ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: The effect on the colony formation of ovarian cancer cell lines. (a) Detection of overexpression efficiency: Wild type (WT) vs Overexpressed (OE). (b) Quantification of colony formation (OE vs WT). Statistical methods: independent samples t-test. (c) Images of colony formation. (Overexpression). (d) Verification of knock-down efficiency by western blot, SLC11A2, and ß-actin. Blots were cropped prior to incubation with primary antibody hybridization. Original blots are presented in Supplementary Fig. S1. (e) Quantification of colony formation, Wild type (WT) vs knockdown (KD). Statistical methods: independent samples t-test. (f) Images of colony formation (knockdown).

Fig 5: L. johnsonii L531 relieves dysregulation of iron homeostasis and oxidative stress caused by S. Typhimurium via IRP2 in Hela cells. (A) Western blot analysis of IRP2. (B) The intracellular bacterial count was tested by plating. (C) The cellular free iron content. (D) Free ferrous iron fluorescence staining. Scale bar, 20 µm. (E) Western blot analysis of IRP2, DMT1, TfR1, and FPN. (F) DHE staining to assess ROS level. Scale bar, 100 µm. (G) Western blot analysis of Nrf2 and HO-1. (H) The NQO1, SOD1, and GPX1 mRNA expression levels. Values are expressed as the means ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (ns, not significant).