PMC

Determination of dynamic alteration of cell proliferation in activated HTM cells after exposure to Tm or STS by MTS assay. Data were presented as the percentage of viable cells compared with untreated (control) cells (mean±SD, n=6).

GRP78 and myocilin (MYOC) were expressed in both NTM and GTM cells, and exhibited a diffused distribution throughout the cytoplasm in NTM cells. The expression pattern of GRP78 and MYOC was most overlapping in GTM and treated NTM cells. Perinuclear aggregation of GRP78 expression was observed in GTM and treated NTM cells (white arrow). The Tm slightly upregulated GRP78 expression and STS down-regulated it, while the expression of MYOC did not show much difference. Scale bar=10 μm.

Both NTM and GTM cells were treated as indicated, and normalized GRP78 mRNA levels were measured using real time PCR and were shown relative to levels in untreated cells. All experiments were performed in triplicate, and the relative amount of mRNA in each sample was calculated using the 2-ΔC_t method in individual experiments. Values represent the means±SDs, ANOVA test, the double asterisk indicates a p<0.05; Student’s t-test, and the asterisk indicates a p<0.05.

Morphology of TM cells before and after Tm or STS treatment. NTM and GTM cells were obtained and cultured using identical culture protocols. GTM cells were slightly larger compared to NTM cells. The morphology of the cells became thin and slender after treatment with 1 μM Tm or 0.1 μM STS for 24 h as compared to untreated controls, especially in the STS-treated group. Gross morphological alterations (cell shrinkage, low density) were apparent at 24 h after STS treatment. Scale bar=30 μm.

Normalized GRP78 protein levels were measured in cells (treated with Tm or STS) using western blot analysis and were shown relative to levels in no treatment cells. Meanwhile, the expression peak of GRP78 protein appeared at 24 h after treatment with Tm. After exposure to Tm and following ER stress, increased GRP78 protein levels were detected in all cells, however, a lower fold change of the protein (2.564 versus 2.710 for 24 h exposure) was found in GTM cells compared to NTM cells (p<0.05). Error bars represent SDs from three individual experiments, ANOVA test, the double asterisk indicates a p<0.05; Student’s t-test, and the asterisk indicates a p<0.05.