Figure 4.

Extracellular Ca²⁺ influx and a Ca²⁺ channel are required for the collapse-inducing activity of Sema 3A. (A–E) E13 mouse embryo DRG explant cultures were incubated with or without various calcium channel blocking or depleting agents for 7 min prior to addition of control conditioned medium (A, C, and E) or Sema 3A-AP-conditioned medium (B, D, and E). (A and B) Control conditioned medium only; (C and D) with 2 mM CoCl₂. (Scale bar, 50 μm.) (E) The relative responsiveness of DRG growth cones to Sema 3A-AP in the presence of 2 mM CoCl₂, 200 μM CdCl₂, 10 μM hanatoxin, and 1 mM BAPTA or thrombin (50 units/ml) in the presence or absence of 2 mM CoCl₂. The percentage of collapsed growth cones with (black bars) or without (gray bars) Sema 3A-AP or with thrombin (white bars) is shown. (F) E13 mouse embryo DRG explant cultures were incubated with or without 2 μM BAPTA-AM and 0.08 μM calmidazolium chloride for 60 min prior to addition of control conditioned medium or Sema 3A-AP-conditioned media. The percentage of collapsed growth cones with (black bars) or without (gray bars) Sema 3A-AP in the presence of these reagents is presented. (G) Control PAE cells or PAE-NP-1 cells were treated with medium containing Sema 3A-AP with or without 2 mM CoCl₂ or 200 μM CdCl₂. Bars indicate SEM for triplicates. Where not obvious, the error bars are smaller than the symbols.