Figure 5. CXCL12γ contributes activation of CXCR4-mediated PKCα/NFκB signaling in CSC and NE inductions and resistance in PCa.

A, Levels of total PKC activity in CXCL12γ overexpressing PCa cells relative to control cells. B, Effect of CXCL12γ on NFκB-reporter luciferase activity. Firefly luciferase was normalized against dsDNA. C, FACS analyses of the CSC (CD133+/CD44+) phenotype in CXCL12γ overexpressing or control PCa cells following NFκB inhibitor (IKK-2 inhibitor VI (IKK2 inh)) treatments. Data in Fig. 5A-5C are representative of mean ± SD (Student’s t-test). D, Immunoblot analysis showing PKCα and its phosphorylation in CXCL12γ overexpressing or control PCa cells as quantified by Western blots. E, The formation of prostatospheres in CXCL12γ overexpressing or control PCa cells following the pan-PKC inhibitor (Go6983) treatments as shown by bright-field microscopy and F, quantification of Fig. 5E. G, Immunoblot analysis showing NFκBp65 and its phosphorylation in CXCL12γ overexpressing or control PCa cells as quantified by Western blots. H, The formation of prostatospheres in CXCL12γ overexpressing or control PCa cells following treatment with an NFκB inhibitor (IKK2 inhibitor VI (IKK2 inh)) as shown by bright-field microscopy and I, quantification of Fig. 5H. Data in Fig. 5F and 5I are representative of mean ± SD (Student’s t-test). J, Immunoblot analysis showing NFκBp65 and its phosphorylation in PCa cells following the CXCL12 and/or AMD3100 treatments as quantified by Western blots and K, in CXCL12γ overexpressing or control PCa cells following AMD3100 treatments. L, Immunoblot analysis showing phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) at Ser159/163 (phospho-MARCKS) in CXCL12γ overexpressing PCa cells or control PCa cells. M, Immunoblot analysis showing reduction of phosphorylation of p-MARCKS, p-PKCα, and p-NFkB-p65 in CXCR4 knock-downed CXCL12γ overexpressing PCa cells.