Extended Data Fig. 6: Assessment of apoptotic pathways activated by pyrimidine synthesis inhibitors.
a. Immunoblot analysis of PARP, cleaved caspases-8/−9 and BID in Jurkat cells treated brequinar, PALA or RTX ± thymidine (dT, 10 μM) or uridine (50 μg/ml). b, Immunoblot analysis of replication stress and apoptosis in Jurkat cells upon FAS activation (anti-Fas antibody 50 ng/ml) ± Z-VAD (50 μM) in high and low glucose. c, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with staurosporine (STS) ± Z-VAD (50 μM) for 12 hr in high and low glucose. d. Immunoblot analysis of replication stress and apoptosis in Jurkat cells cultured in conditioned media collected from cells treated with the indicated inhibitors or fresh media containing brequinar (B), RTX (R) or anti-Fas antibody (F) for 24 hr. Uridine (50 μg/ml) or thymidine (dT, 10 μM) were added following media collection. e, Immunoblot analysis of cFLIP, caspase-8 and apoptosis in Jurkat-FLAG-cFLIP cells treated with anti-Fas antibody (50 ng/ml). f, Immunoblot analysis of cFLIP, caspase-8, replication stress and apoptosis in Jurkat-FLAG-cFLIP cells treated with brequinar (B, 400 nM) or RTX (R, 10 nM) for 48 hr in high and low glucose. g, Immunoprecipitation of FLAG-cFLIP::procaspase-8::FADD complexes in Jurkat-FLAG-cFLIP cells treated with RTX (25 nM) for 48 hr. Z-VAD (50 μM) was added to stabilize FLAG-cFLIP::procaspase-8::FADD complexes. h, Flow cytometric analysis of DNA fragmentation (DAPI, upper) and TOM20 (lower) in Jurkat cells treated with RTX (25 nM) for 48 hr in high and low glucose. i, Flow cytometric analysis of cytochrome c (CYCS) release (left) and immunoblot analysis of BAK, replication stress and apoptosis (right) in Jurkat BAK KO cells treated with brequinar (B, 400 nM), RTX (R, 25 nM for flow cytometry; 10 nM for immunoblot) or aphidicolin (A, 4 μg/ml for flow cytometry; 5 μg/ml for immunoblot) for 48 hr. j, Immunoblot analysis of tBID, replication stress and apoptosis in Jurkat cells with DOX-inducible expression of tBID in high and low glucose. Cells were treated with 500 ng/ml doxycycline for the indicated time. k, Immunoblot analysis of BAK1, tBID and apoptosis in Jurkat BAK1 KO cells treated with ABT-263 (5 μM for 24 hr), staurosporine (250 nM for 4 hr) or expressing DOX-inducible tBID (0.5 μg/ml DOX for 6 hr). l,m, Immunoblot analysis of replication stress and apoptosis in Jurkat cells treated with 200 nM brequinar ± AZD6738 (ATRi, l) or MK8776 (Chk1i, m) in high and low glucose for 48 hr.
