Fig. 3

C. difficile toxins A (TcdA) and B (TcdB) increase TRPV4 gene and protein expression in enteric glial cells (ECGs) in vitro. A) Administration of TcdA (50ng/mL) and TcdB (1ng/mL) led to a significant elevation in TRPV4 gene expression in EGCs. The gene expression was evaluated by qPCR after incubating EGCs with the toxins for 2, 12 and 18 h. The data are presented as mean ± SD of the relative expression of TRPV4. Statistical significance was determined using the two-way ANOVA and Tukey tests; **p < 0.006 and ***p < 0.0001. B) TcdA and TcdB significantly increase TRPV4 protein expression in EGCs, assessed by Western Blotting. Data are presented as mean ± SEM, normalized to α-tubulin for comparison. Statistical significance was determined using the one-way ANOVA and Tukey tests. *p < 0.001 and ***p < 0.0001. C) Exposure to TcdA and TcdB increases the cytoplasmic immunostaining of TRPV4 in EGCs. Photomicrographs showcase the cytoplasmic immunostaining of TRPV4 (green), the nuclear staining with DAPI (blue), and the merged images (MERGE). D) illustrate the quantification of the mean fluorescence intensity of TRPV4 immunostaining in EGCs, measured using Zeiss software. The results are expressed as mean ± SEM of TRPV4 immunostaining fluorescence intensity. Statistical significance was determined using the one-way ANOVA and Tukey tests. *p < 0.001 and ***p < 0.0001. The entire blotting membranes are available in the supplementary material (Figures S2 and S3)