Fig. 5.

Effects of acute hepatic heme depletion and repletion on hepatic content of GCN2 and PERK and their autophosphorylated species in cultured MGB wild-type [WT; HRI (+/+)] and HRI knockout [KO; HRI (−/−)] mouse hepatocyes. A, untreated rat or mouse (C57BL/6J, BALB/c, MGB WT, or KO) hepatocytes were cultured as detailed under Materials and Methods. A, a representative example of GCN2 and GCN2-P Western immunoblotting analyses of these hepatocyte lysates (50 μg of protein) are shown at the top, with corresponding smaller aliquots used for actin immunoblotting analyses as loading controls. Densitometric quantification of total hepatic GCN2 (GCN2 + GCN2-P) and autophosphorylated GCN2 (GCN2-P) content (mean ± S.D.) of three individual experiments is shown at the bottom. Statistically significant differences in hepatic GCN2 content were found between WT and KO lysates at p < 0.001, between WT and BALB/c at p < 0.001, or KO and BALB/c at p < 0.001, KO and rat at p < 0.001, and KO or WT and C57BL/6J at p < 0.001. Statistically significant differences in hepatic GCN2-P content were found between WT and KO lysates at p < 0.05, between WT and BALB/c at p < 0.001, between KO and BALB/c at p < 0.001, between KO and rat at p < 0.001, and between KO or WT and C57BL/6J at p < 0.001. B, WT or KO mouse hepatocyte cultures were untreated, heme (H)-depleted or heme-repleted after heme depletion, as detailed under Materials and Methods. A representative example of total GCN2 and GCN2-P Western immunoblotting analyses of these hepatocyte lysates (50 μg of protein) is shown at the top, with corresponding smaller aliquots used for actin immunoblotting analyses as loading controls. Densitometric quantification of total hepatic GCN2 (GCN2 + GCN2-P) and phosphorylated GCN2 (GCN2-P) content and corresponding statistically significant differences between mean ± S.D. of three individual experiments are shown at the bottom. Statistically significant differences were observed in the total hepatic GCN2 and GCN2-P content of untreated and H-depleted WT hepatocytes between the two mean ± S.D. values each marked with the same symbol as follows: §, p < 0.001; *, p < 0.001, respectively. Total hepatic GCN2 content of H-depleted WT cells was significantly different from that of H-repleted WT cells (#) at p < 0.001. Total hepatic GCN2 content of WT hepatocytes was significantly different from that of KO hepatocytes (**) at p < 0.05. No significant differences were found between any other values. C, WT or KO mouse hepatocyte cultures were untreated, treated with heme (20 μM; H-control), heme (H)-depleted, or heme-repleted after heme depletion, as detailed under Materials and Methods. PERK and PERK-P Western immunoblotting analyses of these hepatocyte lysates (100 μg of protein) are shown at the left, with corresponding smaller aliquots (10 μg of protein) of these same SDS-PAGE sample buffer-solubilized cell lysates used for actin immunoblotting analyses as loading controls. The densitometric quantification of the relative PERK-P content (solid bars) to the total PERK immunochemically detectable content (open bars) is shown at the right. Values represent mean ± S.D. of three separate experiments. Statistically significant differences in either PERK or PERK-P content between the two mean ± S.D. values each marked with the same symbol were as follows: *, p < 0.001; §, p < 0.001; ‡, p < 0.001; ¶, p < 0.001; and **, p < 0.001.