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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Cancer Discov. 2019 Apr 30;9(7):944–961. doi: 10.1158/2159-8290.CD-18-1393

Figure 5: Cyclophosphamide Induces ER Stress in DHL cells that Drives Cytokine Secretion.

Figure 5:

(A) Gene-set enrichment analysis (GSEA) of ER-stress signatures from RNA sequencing of DFBL-20954 cells collected from spleens or BM. The comparison reflects genes overexpressed after cyclophosphamide (CTX) treatment as compared to PBS treatment. P-values have a false discovery rate (FDR) adjustment. Abbreviations: ES, enrichment score; NES, normalized enrichment score.

(B) Immunoblotting for the indicated targets using lysates from purified, viable BM DFBL-20954 cells collected 16 hours after in vivo treatment with PBS (vehicle), doxorubicin (Dox) or CTX. Each lane represents an individual mouse.

(C) Levels of human VEGF-A in cells (left) or media (right) of DFBL-20954 cells isolated from BM and treated ex vivo for 24 hours with DMSO, thapsigargin (TG) or tunicamycin (TM). Two-sided Welch t-test * p<0.05, ** p<0.01.

(D) Levels of human VEGF-A from whole BM collected 16 hours after in vivo treatment of DFBL-20954-engrafted mice as indicated. Unpaired two-sided-t-test.

(E) Immunoblotting, as in (B)

(F) ChIP-qPCR of ATF4 localization to exon 1 of human VEGF-A or exon 7 of human ASNS in purified, viable BM DFBL-20954 and DFBL-69487 cells collected 16 and 48 hours, respectively, after treatment with Veh, Dox or CTX (n=3 replicates per condition with multiple mice pooled for each replicate). Unpaired two-sided-t-test.