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Fig. 4. HRK contributes to NGF-deprivation-induced apoptosis in DRG neurons. (A) DRG neurons were isolated from control (wt and Hrk^+/–, n=4) or Hrk^–/– (n=5) littermates, cultured for 24 hours in NGF, then transferred to medium with or without NGF (supplemented with anti-NGF antibodies) for 72 hours. Cell survival was assessed by counting the number of viable neurons in an assigned field before NGF withdrawal (0 hours), then counting the same field again at 48 and 72 hours after NGF withdrawal. Percent survival was determined relative to the viable neuron number at 0 hours. Asterisks indicate statistically significant survival difference between control and Hrk^–/– neurons in the absence of NGF (P<0.05, Student's t-test). (B) SCG neurons were isolated from WT, Hrk^–/– or Bim^–/– mice and cultured with NGF for 5 to 6 days, then washed and cultured in medium with or without NGF (supplemented with anti-NGF antibodies) for up to 48 hours. Cell survival was assessed from the number of viable neurons remaining in each well at each time point relative to the number present in the same well before NGF withdrawal and were expressed in percent. Data represent the mean ± s.d. Asterisks indicate statistically significant difference between survival of wild-type (WT) and Bim^–/– neurons in the absence of NGF (P<0.05, Student's t-test). (C) Q-PCR analysis of Hrk mRNA expression in wt SCG neurons cultured with or without NGF for 16 hours. Expression of neurofilament 3 was used for normalisation. Data are the mean ± s.d. of nine cultures of neurons from three mice (triplicates).

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