Figure 8

Subtypes of VSMCs determine expression levels of PDI and proliferation or apoptosis of VSMCs induced by SS, AGEs or both. The quiescent cultured VSMCs were subjected to SS and/or AGEs for 1 h, and continually cultured for additional 24 h. These cells were then stained with primary PDI and Ki-67 antibodies, FITC or Cy3-conjugated secondary antibodies, and a TUNEL kit, and then counterstained with DAPI. (a–d) The triple-labeling immunofluorescent shows Ki-67-positive (red), and PDI-labeling cells (green). (e–h) TUNEL-positive (green) and PDI-labeling cells (red). (i–l) SM-α-actin positive (green) and PDI-positive cells (red). (m–p) SM-α-actin positive (green) and Ki67-positive cells (red); (q–t) SM-α-actin positive (red) and TUNEL-positive cells (green). Using ImageJ program, the cells were sorted into two groups, cells with low and negative PDI or SM-α-actin expression and high and strong PDI or SM-α-actin expression. Then the Ki67-positive or TUNEL-positive cells were counted, respectively. DAPI stained cells were used for total cell numbers. (u and v) Statistical results of the ratio of Ki-67- or TUNEL-positive cells and PDI from (a to d) and (e to h,) respectively; (w and y) show statistical results of the ratio of Ki-67- or TUNEL-positive cells and SM-α-actin from (m to p) and (q to t), respectively; (x) shows statistical results of the ratio of PDI and SM-α-actin from (i to l). These statistical results were obtained from three independent experiments. a, P<0.05 versus negative control (NC); and b, P<0.05 versus SS or AGEs. Scale bar, 100 μm. Data are shown as the means±SEM