Figure 1.
Gene modulation in HT-29 cells upon exposure to different stimuli.
Heat map representation of gene expression determined by RT-qPCR. HT-29 cells were treated with Shh (Sonic Hedgehog), Purm (purmorphamine), But (butyrate), LPS, IFN-γ, EGF, with or without the addition of cyclopamine (Cyc), GANT 61, or DMSO (vehicle) for 24 hours (A). Histograms of individual gene expression fold changes in HT-29 cells: IHH and SHH (B); GLI1, GLI2 and GLI3 (C); SMO and PTCH1 (D); HHIP and WNT1 (E); and BMP4 and BMP7 (F). The graphs use the same samples as in (A). Values represent the means ± SEM of three independent experiments and are normalized to GAPDH, beta-actin, and RPL32 RNA genes. Significant changes in relation to the control group are highlighted.
Figure 2.
Distribution and levels of Gli-1 and β-catenin in HT-29 cells.
Confocal microscopy of cytospin preparations showing the relative nuclear and cytoplasmic distribution and levels of Gli-1 (A) and β-catenin (B) in HT-29 cells exposed to different stimuli for 24 hours. Analysis of protein staining densities confirm that Gli-1 significantly increases after treatment with Shh (Sonic Hedgehog), Purm (purmorphamine), or But (butyrate), compared to cells treated with DMSO (vehicle) (*P<0.04), and compared to cells treated with Cyc (cyclopamine) or GANT 61 (**P<0.02). Density of β-catenin significantly decreases upon treatment with Shh (# P<0.04) compared to DMSO, Cyc, or GANT 61 (C). Nuclei are stained with DAPI (blue). Micrograph panel is representative of 3–4 experiments for each condition (Original magnification ×1000).
Figure 3.
Production of cytokines by HT-29 cell cultures upon exposure to different stimuli.
Concentration of cytokines in supernatants of 24 hour cultures of HT-29 cells measured by ELISA. Histograms show the levels of IL-8 (A) and MCP-1 (B), under various conditions: treatment with Shh (Sonic Hedgehog), Purm (purmorphamine), Butyrate, LPS, IFN-γ, EGF, with or without the addition of cyclopamine, GANT 61, or DMSO (vehicle control) for 24 hours. IL-8 levels are significantly lower in cells treated with either Shh, purmorphamine, or butyrate compared to those treated with cyclopamine (*P<0.02), GANT 61 (#P<0.04), or LPS (**P<0.03), respectively (A). MCP-1 levels are significantly lower in cells treated with Shh, purmorphamine, or butyrate compared to the ones treated with GANT 61 (#P<0.02), LPS (*P<0.03) IFN-γ (**P<0.03), or EGF (***P<0.04), respectively (B). Data are expressed as the mean ± SEM of 6 independent experiments.
Figure 4.
Monocyte chemotaxis induced by cell culture supernatants of HT-29 cells upon exposure to different stimuli.
Monocytes were incubated in the upper chambers of a 5 µm-pore-size polycarbonate filter in a transwell system, with the 24 h-culture supernatants of HT-29 cells treated with rShh, cyclopamine, LPS, or IFN-γ in the lower chambers. Supernatants of HT-29 untreated cells were used as controls and values were arbitrarily normalized to 1. Additional controls were obtained by the incubation with the chemoattractant MCP-1, and anti-MCP-1 antibody, as positive and negative controls, respectively. *Cell migration is significantly lower in cells treated with Shh compared to controls (P = 0.01), and those treated with GANT61 (P = 0.005), cyclopamine (P = 0.003), LPS (P = 0.042), IFN-γ (P = 0.017), and MCP-1 (P = 0.001), respectively. **Anti-MCP-1 significantly decreased chemokinesis compared with GANT61, cyclopamine, LPS, IFN-γ, or MCP-1 (P<0.02),. Data are expressed as the mean ± SEM of 3 independent experiments.
Figure 5.
Survival and proliferative activity of HT-29 cells upon exposure to different stimuli. HT-29 cell viability was analyzed at different time points with different treatments, using the MTT assay.
When HT-29 cells were exposed to cyclopamine, cell viability decreased significantly comparing 48 with 24 hours (*P<0.05) (A). For the purpose of analyzing changes in the proliferative activity of HT-29 cells upon different treatments, the cellular incorporation of BrdU was measured at 48 and 72 hours. After 72 hours, BrdU incorporation was maintained in vehicle-treated cells (DMSO), while it significantly decreased in all other treatment groups (*P<0.04). Within the group of cyclopamine exposed HT-29 cells, BrdU incorporation decreased significantly from 48 to 72 hours (**P<0.05) (B). Data are expressed as the mean ± SEM of 3 independent experiments.
Figure 6.
Relationship between apoptosis and the activity of Hedgehog pathway in HT-29 cells.
Flow cytometric demonstration of HT-29 apoptosis after 24 hours of exposure to different stimuli, as assessed by annexin-V/7-AAD. Left lower quadrant indicates double negative cells while right lower quadrant indicates annexin-V-positive cells (A). Treatment with Shh, purmorphamine or butyrate significantly abrogates the anti-CD95 induced apoptosis (*P<0.02), which is partially restored by the addition of cyclopamine (B). Data are expressed as the mean ± SEM of 3 independent experiments.