Fig 6.

Fibroblast-derived conditioned media promotes Caco-2 cell rounding and is CSPG4 dependent. (A) Caco-2 cells were intoxicated with 1 pM TcdB2 in complete media (Dulbecco’s modified Eagle’s medium [DMEM]) or conditioned media from siRNA-treated 18Co cells targeting a non-targeting control (Control siRNA) or CSPG4. Rounding was quantified from images acquired over the course of 24 hours. Images to the right are representative images acquired at the end of the experiment. A representative western blot of CSPG4 in the 18Co conditioned media from the non-targeting control siRNA (Control) and CSPG4 siRNA (siCSPG4) conditions used in these experiments is shown below. A fluorescent image of the “stain-free” gel used in this western blot is shown to demonstrate equal loading of protein. Statistical analysis was performed using a two-tailed ANOVA with a Sidak post hoc test for multiple comparisons. N = 3 *P < 0.05, ****P < 0.0001. See also Fig. S4. (B) Conditioned media from 18Co cells were treated with antibodies targeting CSPG4 prior to the intoxication of Caco-2 cells with 1 pM TcdB2. Conditioned media lacking antibodies targeting CSPG4 rounded over a 24-hour period. Representative images from these assays are shown on the right that were acquired at the end of the experiment. Statistical analysis was performed using a two-tailed ANOVA with a Sidak post hoc test for multiple comparisons. Images to the right of the graph are representative images acquired at 24 hours. N = 3, *P < 0.05, ***P < 0.001.