Figure 2.

Exogenous Hsp70 and Hsc70 promote motoneuron survival. Representative phase-contrast photomicrographs of motoneurons are shown after 3 d in culture plated in the absence of trophic support but with addition of rhHsp70 (75 μg/ml; A), MEx (B), or neither (C). Cultures contained healthy cells as indicated by round phase-bright cell bodies with extensive neurite outgrowth in A and B, whereas most of the cells in C are not healthy, as evidenced by their phase-dark appearance and very short neurites. D, Numbers of surviving cells were determined after 3 d in culture. Results are expressed as a percentage of control, in which control represents cultures supplied with MEx (n = 3 independent experiments with 3-4 wells per condition per experiment; error bars represent SEMs). ^*p < 0.05 compared with -MEx as determined by ANOVA. E, Uptake of biotinylated Hsc70 by cultured motoneurons. Confocal, fluorescent (E), differential interference contrast (F), and overlay (G) images are presented of a motoneuron in culture for 20 h in the presence of biotinylated Hsc70. Motoneurons were incubated with 10 μg/ml biotin-labeled Hsc70, and the protein was detected with Cy3-streptavidin. The arrows in E and G indicate a region of cytoplasmic uptake of Hsc70. Control cultures with no protein added showed no fluorescent staining (data not shown). H, Numbers of surviving cells were determined after 3 d in culture after addition of Hsp27, -40, -70, or -90 (1 μm), or Hsp70 (1 μm) plus MEx. Results are expressed as a percentage of control, in which control represents cultures supplied with MEx (average ± SD; n = 2 independent experiments with 3-4 wells per condition per experiment).