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. 2020 Aug 5;12(8):2189. doi: 10.3390/cancers12082189

Figure 4.

Figure 4

Zfra peptide suppresses WWOX phosphorylation at Y33 and Y61 that leads to Z cell expansion in the spleen. (A,B) Spleen cells were isolated from eight naïve BALB/c mice, followed by treating with or without 20 μM Zfra4-10 for 24 h in cell culture and then processing purification of TMR-Zfra+ cells using a cell sorter. The resulting naïve TMR-Zfra- (NZ-), naïve TMR-Zfra+ (NZ+), Zfra-activated TMR-Zfra- (AZ-), and Zfra-activated TMR-Zfra+ (AZ+) were isolated and injected to each indicated recipient naïve nude mouse via its tail vein. One week later, breast 4T1 cancer cells were inoculated into both flanks for measuring tumor growth with time. (C–E) Nude mice received Zfra peptide (2 mM in PBS) or PBS only via tail vein injections, followed by inoculating B16F10 cells in both flanks one week later and mice sacrificed a month later. Zfra suppressed WWOX phosphorylation at Y33 and Y61 (> 95%), and inhibited the level of CD27+ B cells by 78% in the spleen (whole mount scans). Statistical analysis for E: *** p < 0.001, n = 5, Student’s t test (all groups versus pS14 group). (F–H) Z cell levels were low in the spleen of mice post treatment with Zfra for two months, as the spleen cells had reduced expression of Hyal-2 and Zfra. Statistical analysis for H: * p < 0.05, *** p < 0.001, n = 5, Student’s t test (all groups versus CD44 group).