Inhibition of growth factor-stimulated DNA synthesis in human erythroid progenitors in the presence of thrombin (throm) and Gö 6976 (Gö, an inhibitor of Ca²⁺-dependent PKC subtypes). (A) Inhibition of Epo (0.5 U/mL)-induced DNA synthesis by thrombin (2 U/mL), Gö 6976 (1 μmol/L), thrombin receptor peptide SFLLRN (TRP, 100 μmol/L) and by lowering the extracellular Ca²⁺ concentration from 1.2 mmol/L to about 10⁻⁷ mol/L by adding 4 mmol/L EGTA. Reduction of Epo-stimulated ³H-thymidine incorporation was significant for all agents (**P < .01; *P < .05 by ANOVA followed by Dunnett’s test). Data show mean values ±SEM of six to nine different experiments. The inhibition by EGTA was measured in six cultures from two batches of cells. The dotted line gives basal thymidine incorporation in the absence of added growth factors. (B) Experiment analogous to (A) but measuring the inhibition of SCF (50 ng/mL)-stimulated DNA synthesis. Columns show mean values ±SEM of three to nine different experiments, except for the effect of thrombin alone, which was measured in three separate cultures from one batch of cells. All agents significantly reduced SCF-stimulated DNA synthesis (*P < .05; **P < .01 by paired t-test). (C) Effects of thrombin (2 U/mL), Gö 6976 (1 μmol/L), Epo (0.5 U/mL), and PMA (10 nmol/L) on cells maintained for 24 hours in serum-free medium with SCF (50 ng/mL) as only growth factor (no starving). Groups normalized with respect to DNA-synthesis in the sole presence of SCF. Differences between groups were significant onP < .05 (*) and P < .01 ($$, &&, ##, ++) levels (ANOVA/Bonferroni). (D) Effects of thrombin and of Epo on phorbol ester (PMA)-stimulated DNA synthesis. PMA (10 to 100 nmol/L) was present during both the 16-hour starvation and the 24-hour experimental periods, while thrombin (2 U/mL) and Epo (0.5 U/mL) were first added at the end of the starvation period. The dotted line corresponds to basal thymidine incorporation in the absence of PMA. The values give means ±SEM of six to nine cultures from two to three different batches of cells. Thrombin and Epo caused significant changes of PMA-induced DNA synthesis (*P < .05; **P < .01 by ANOVA and Dunnett’s test). Addition of thrombin (2 U/mL) to the combination of PMA and Epo did not result in a significant change of thymidine incorporation.