PMC

YAMC (A, D), kiKSR (B), or HT29 (C) cells were treated with TNF (100 ng/ml), IL-1α (10 ng/ml), or γ-IFN (100 ng/ml) as indicated in the presence or absence of a 1-h pretreatment with viable LGG, hk-LGG, or LGG-cm. Cellular lysates were prepared for Western blot analysis with anti-Thr(P)-p38, anti-p38, anti-phospho-SAPK/JNK, and anti-SAPK/JNK antibodies.

YAMC (A, B, D, F), HT29 (C), and kiKSR-expressing cells (E) were treated with viable LGG, hk-LGG, LGG-cm, or LGG-s as indicated for 1 h followed by TNF (100 ng/ml, 15 min) treatment. PI 3-kinase inhibitors, wortmannin (100 nm) or LY294002 (10 μm), or MEK1 inhibitor PD98059 (10 μm) was used to pre-treat cells for 1 h before TNF or LGG treatment (D). Akt activation was detected by Western blot analysis of cellular lysates with anti-Ser(P)-473-Akt and anti-Akt antibodies. YAMC cells were treated with LGG for the indicated times, and IκBα degradation and ERK1/2 activation were detected by Western blot analysis of cellular lysates with anti-IκBα or anti-phospho (P) ERK1/2 antibodies (F).

YAMC cells were treated with LGG, L. casei (LC), or L. acidophilus (LA) for 1 h followed by 15 min of TNF (100 ng/ml) treatment as indicated in A. Akt and p38 activation was detected as before. LC, LA (B), or S. pullorum (C and D), which were prepared as indicated under “Experimental Procedures,” were incubated with kiKSR-expressing cells for 1 h prior to the addition of TNF (100 ng/ml) and then treated for 6 h. The cells were prepared for TUNEL staining as before (C). The percentage of cells undergoing apoptosis in a representative experiment is shown (B and D).

YAMC cells (A and B) or kiKSR-expressing YAMC cells (C–E) were treated with TNF for 6 h in the absence or presence of 1-h pretreatment with wortmannin (100 nm), LY294002 (10 μm), SB203580 (10 μm), or SB202190 (10 μm) as indicated. TUNEL staining was used to identify apoptotic nuclei by peroxidase. The cells were visualized by differential interference contrast microscopy (A and C). The percentage of cells undergoing apoptosis from representative experiments is shown (B and D). p38 phosphorylation was detected as described in .

kiKSR-expressing YAMC cells (A–C) or human colonic epithelial carcinoma cell line (HT29) cells (D) were treated with TNF (100 ng/ml) or the “cytokine mixture” combination of TNF (100 ng/ml), IL-1α (10 ng/ml), and γ-IFN (100 ng/ml), respectively, for 6 h in the presence or absence of a 1-h pretreatment with viable LGG, hk-LGG, or concentrated supernatant recovered from LGG culture MRS broth (LGG-s) at the concentrations indicated. Then, cells were fixed for terminal deoxynucleotidyltransferase TUNEL with apoptotic nuclei labeled with fluorescein isothiocyanate and DAPI staining (A and D). Fluorescein isothiocyanate- and DAPI-labeled images were taken from the same field. The percentage of cells undergoing apoptosis from a representative experiment is shown in B. Caspase activity in living cells was detected using a multi-caspase activity assay kit (C). Arrows indicate representative apoptotic nuclei. All experiments in this and subsequent figures were performed on at least three separate occasions.