Figure 1.
The ER stress-initiated pathway does not contribute to phenylalanine-induced apoptosis.
(A) The eIF4 blocker salubrinal (SAL) did not block apoptosis induced by phenylalanine. Cells were pretreated with 100 ng/ml SAL prior to phenylalanine treatment (0.9 mM for 18 h). Apoptosis was measured by TUNEL assay. (B) Quantification of apoptosis induced by phenylalanine from (A). (C) SAL pretreatment did not affect activated caspase-3 or caspase-12 expression following phenylalanine treatment as measured by Western blotting. (D) Quantification of band intensity in (C). Values are mean ± SD of three independent experiments. * P<0.05.
Figure 2.
The death receptor-initiated pathway is involved in phenylalanine-induced apoptosis.
(A) Phenylalanine-induced apoptosis is dependent on caspase activity. Cells were pretreated with 20 µM Z-VAD-FMK before phenylalanine (0.9 mM for 18 h) and apoptosis measured by TUNEL assay. (B) Quantification of apoptosis induced by phenylalanine from (A). (C) Z-VAD-FMK blocked caspase-3 and caspase-8 activation induced by phenylalanine. Cells were pretreated with 20 µM Z-VAD-FMK before phenylalanine treatment (0.9 mM for 18 h) and activated caspase-3 and caspase-8 activation measured by Western blotting. (D) Quantification of band intensity in (C). (E) Z-IETD-FMK blocked phenylalanine-induced apoptosis. Cells were pretreated with 20 µM Z-VAD-FMK before phenylalanine exposure as above. Apoptosis was measured by TUNEL assay. (F) Z-IETD-FMK blocked the phenylalanine-induced increase in activated caspase-3 and caspase-8 expression. (G) Quantification of band intensity in (F). Values are mean ± SD of three independent experiments. * P<0.05.
Figure 3.
Fas/FasL signaling contributes to phenylalanine-induced apoptosis.
(A) Cell surface Fas expression was upregulated by phenylalanine and reduced by Z-VAD-FMK pretreatment. Cells were pretreated with 20 µM Z-VAD-FMK before phenylalanine treatment (0.9 mM for 18 h) and cell surface of Fas expression was assessed by flow cytometry. (B) Dissociated Fas and FasL was decreased by phenylalanine and increased by Z-VAD-FMK pretreatment as assessed by immunoprecipitation. (C) Z-VAD-FMK blocked caspase-8 activation by phenylalanine. Caspase-3 and caspase-8 activation were determined by Western blot. (D) Quantification of band intensity in (C). (E) Z-VAD-FMK blocked Bidcleavage by phenylalanine. Expression of Bid was determined by Western blot. (F) Quantification of band intensity in (E). Values are mean ± SD of three independent experiments. * P<0.05.
Figure 4.
Blocking Fas/FasL signaling pathway prevents apoptosis induced by phenylalanine.
Anti-Fas blocked phenylalanine-induced apoptosis. Cells were pretreated with 1 µM anti-Fas antibody 2 h before phenylalanine treatment (0.9 mM for 18 h) and apoptosis measured by TUNEL assay. (B) Anti-Fas antibody prevents caspase-8 and caspase-3 activation by phenylalanine. Caspase-3 and caspase-8 activation was determined by the Western blot. (C) Quantification of band intensity in (B). Values are mean ± SD of three independent experiments. * P<0.05.