![Fig. 5. PNU156804 selectively inhibits Jak3-γc-cytokine–mediated cell proliferation compared with Jak2 growth factors. / (A) Proliferation of quiescent PHA-activated human T cells (5.0 × 104 cells/well) were cultured in the absence or presence of 1 nM human IL-2 (●), IL-4 (○), IL-7 (▾), or IL-15 (▿) with increasing concentrations of PNU156804 (ordinate) for 16 hours at 37°C. Cells were then pulsed with [3H]-thymidine (0.5 μCi [0.0185 MBq]/200 μL) for 4 hours, and the incorporated radiolabeled probe plotted on the abscissa was expressed as percentage inhibition of total cpm from DMSO-treated sample sets (n = 6). (B) Quiescent rat T cells (5.0 × 104 cells/well) were cultured in the presence of the Jak2 activator (1 nM PRL [○]) or Jak3 activator (1 nM IL-2 [●]), with increasing concentrations of PNU156804 (ordinate, 0-100 μM) for 16 hours at 37°C. Cells were pulsed with [3H]-thymidine (0.5 μCi [0.0185 MBq]/200 μL) during the final 4 hours of the assay, and the DNA-incorporated radiolabeled probe was plotted on the abscissa and expressed as percentage inhibition of total cpm from DMSO-treated sample sets (n = 6). (inset) Antiphosphotyrosine immunoblot of Jak2-Jak3–activated signaling pathway. Nb2-11c cells treated with PNU156804 or DMSO (as described in Figure 1A) were stimulated with 100 nM IL-2 (lanes a-f) or 100 nM PRL (lanes g-l). Jak3 or Jak2 (upper panel), Stat5a (middle panel), and Stat5b (lower panel) were immunoprecipitated from lysates, separated by SDS-PAGE, transferred to PVDF membrane, and blotted with specific antiphosphotyrosine mAb.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/2/10.1182_blood.v99.2.680/6/h80222002005.jpeg?Expires=1726826947&Signature=h4SOO1Uhm9B8CNwwvLRGf0mm~GiPjnv2cuGCN~x98rbOQfBMwaSQVCzCOQZsUNimwQ~9nYA0gwKn0S8E48C0dkNsRzUsZDX~SijiC8Fi4ExIsDKJQUFNuaapMTYhyWE940mMmr9QisAfo5aGaHETjaUf9zkrD-OWSwbFz-rgaCqq6z52-ra~0SM~cus85tmd-x62EeSXMU7QyPiSJVqjqU2nlWWXz2S7K-OWBGzzBlEvnuuJeLYuBoRdLnJ0KiUYj-XC7QKqGcHQq~t5ImbNlYSqlxh7EGCWgFNRcKSCOiCKWbsnHMc~wNoOBqerr4OlrLfO5Umyvd5OWWEUiI-0DA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PNU156804 selectively inhibits Jak3-γc-cytokine–mediated cell proliferation compared with Jak2 growth factors.
(A) Proliferation of quiescent PHA-activated human T cells (5.0 × 104 cells/well) were cultured in the absence or presence of 1 nM human IL-2 (●), IL-4 (○), IL-7 (▾), or IL-15 (▿) with increasing concentrations of PNU156804 (ordinate) for 16 hours at 37°C. Cells were then pulsed with [3H]-thymidine (0.5 μCi [0.0185 MBq]/200 μL) for 4 hours, and the incorporated radiolabeled probe plotted on the abscissa was expressed as percentage inhibition of total cpm from DMSO-treated sample sets (n = 6). (B) Quiescent rat T cells (5.0 × 104 cells/well) were cultured in the presence of the Jak2 activator (1 nM PRL [○]) or Jak3 activator (1 nM IL-2 [●]), with increasing concentrations of PNU156804 (ordinate, 0-100 μM) for 16 hours at 37°C. Cells were pulsed with [3H]-thymidine (0.5 μCi [0.0185 MBq]/200 μL) during the final 4 hours of the assay, and the DNA-incorporated radiolabeled probe was plotted on the abscissa and expressed as percentage inhibition of total cpm from DMSO-treated sample sets (n = 6). (inset) Antiphosphotyrosine immunoblot of Jak2-Jak3–activated signaling pathway. Nb2-11c cells treated with PNU156804 or DMSO (as described in Figure 1A) were stimulated with 100 nM IL-2 (lanes a-f) or 100 nM PRL (lanes g-l). Jak3 or Jak2 (upper panel), Stat5a (middle panel), and Stat5b (lower panel) were immunoprecipitated from lysates, separated by SDS-PAGE, transferred to PVDF membrane, and blotted with specific antiphosphotyrosine mAb.
