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. Author manuscript; available in PMC: 2015 Jul 14.
Published in final edited form as: Cancer Cell. 2014 Jul 4;26(1):121–135. doi: 10.1016/j.ccr.2014.05.004

Figure 5. c-Myc-mediated metabolism in p62-deficient stroma.

Figure 5

(A-C) Immunoblot analysis of c-Myc levels in WT and p62 KO fibroblasts (A); in WT fibroblasts lentivirally infected with shRNA non-targeted control (shNT) or shRNA specific for p62 (shp62) (B); and in p62 KO fibroblasts retrovirally infected with control vector (control) or with c-Myc expression vector (c-Myc) (C). Results are representative of three experiments. (D) IL-6 ELISA in control and c-Myc cells (n = 4) as in (C). (E) RT-PCR of SLC7A5, SLC1A5, and GLS1 mRNA in control and c-Myc cells (n = 3). (F) Intracellular GSH levels in control and c-Myc cells (n = 3). (G) Immunoblot analysis of c-Myc and p-STAT3 in WT fibroblasts infected with non-targeting shRNA (shNT) or shRNA for c-Myc (shMyc). (H and I) RT-PCR of IL-6 mRNA (H) and IL-6 ELISA (I) in the same cells (n = 3) as in (G). (J) Quantification of intracellular GSH levels in WT shNT and shMyc cells (n = 3). (K) H&E-stained organotypic gels of TRAMP-C2Re3 cells with shNT or shMyc fibroblasts. Scale bar, 100 μm. (L and M) Quantification of PCa cell invasion (L) and proliferation index (M) of experiment shown in (K). (N) Invasion index determined by modified Boyden chamber assay of Myc-CaP cells co-cultured with shNT and shMyc fibroblasts. (O) RT-PCR of c-Myc and IL-6 mRNA levels in FACS-isolated prostate stromal cells from WT and p62 KO mice (n = 3). (P) Immunoblot analysis with the indicated antibodies of cell lysates of WT and p62 KO fibroblasts. (Q) Immunoblot analysis for the specified proteins of cell lysates from p62 KO fibroblasts retrovirally infected with control vector (control) or FLAG-RagBGTP expression vector (RagBGTP). Results are representative of three experiments. (R) IL-6 ELISA (n = 3) in cells shown in (Q). (S and T) Immunoblot analysis of c-Myc and p-S6K in fibroblasts treated with rapamycin (S) or Torin1 (T) for 12 hr. (U) Intracellular GSH levels in fibroblasts treated with Torin1. (V) IL-6 ELISA in fibroblasts treated with Torin1 (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Results are presented as mean ± SEM.