Figure 7. Effects of LGG and Butyrate (But) on TGFb1 Production by BM T Cells, on NFAT1/2 and SMAD2/3 Activation, and on PI3K and Akt Signaling in BM CD8⁺ T Cells.

(A–C) TGFb1 mRNA expression by FACS-sorted BM conventional eGFPCD4⁺ T cells, eGFPCD8⁺ T cells, and eGFP⁺ Treg cells. DEREG (eGFP.Foxp3) reporter mice were treated with vehicle, LGG, or butyrate for 4 weeks. BM cells were sorted at the end of the treatment period.

(D) Immunoblot analysis for the detection of NFAT1, NFAT2, pSMAD2, and pSMAD3 in purified BM CD8⁺ T cells.

(E) Immunoblot analysis for the detection of c-Jun and c-Fos in purified BM CD8⁺ T cells.

(D and E) Fresh BM CD8⁺ T cells were pooled together and then used for obtaining nuclear and cytoplasmic fractions. Laminin B1 was used as nuclear loading control. Tubulin was used as cytoplasmic loading control.

(F) Immunoblot analysis for the detection of Phospho-PI3K p85 in the whole lysate from BM CD8⁺ T cells.

(G and H) pAKT levels in BM CD8⁺ T cells and percent of pAKT⁺ BM CD8⁺ T cells, as determined by flow cytometry.

(D–F) Conventionally raised WT mice were treated with vehicle, LGG, or butyrate for 4 weeks. BM CD8⁺ T cells were purified at the end of the treatment period using the EasySep Mouse CD8⁺ T Cell Isolation Kit. One representative experiment of 3 experiments.

Data were expressed as mean ± SEM, n = 5–10 mice per group in (A)–(C), (G), and (H). Data were analyzed by two-way ANOVA and post hoc tests applying the Bonferroni correction for multiple comparisons. **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to vehicle or the indicated group. ns = not significant.