The knockdown of CD137L attenuates cell proliferation, survival, and CXCR4 expression. (A-E) Hut78 cells were transfected with either CD137L-targeting sgRNA (sgCD137L) or control scrambled sgRNA (sgSCR). (A) CD137L expression levels were assayed by flow cytometry and quantitative RT-PCR. MFIs are shown for a representative experiment. (B) Hut78-sgCD137L and Hut78-sgSCR cells (1.0 × 10⁵ per well) were cultured with or without plate-coated recombinant CD137L (10 μg/mL) for 48 hours. Viable cells were counted at indicated time points. (C) Apoptosis (Annexin V⁺ and 7-AAD⁻) was evaluated with Annexin V and 7-AAD staining for flow cytometric analysis in Hut78-sgCD137L and Hut78-sgSCR cells. (D) Western blotting analysis was conducted on the lysates of Hut78-sgCD137L and Hut78-sgSCR cells. Phosphorylation of AKT, ERK1/2, p38 MAPK, and JNK was measured. The representative blot from 3 independent experiments. Band intensities were calculated by Image J software. Relative optical density of p-AKT/AKT, p-ERK1/2/ERK1/2, p-p38 MAPK/p38 MAPK, and p-JNK/JNK was calculated and normalized to the value of Hut78-sgSCR cells. Data are presented as mean plus or minus SD. *P < .05, **P < .01. (E) Hut78-sgCD137L or Hut78-sgSCR cells (5.0 × 10⁶) were injected into NSG mice. The tumor volume was calculated using the equation: V = π (L1 × L22)/6, where V = volume (mm³), L1 = longest diameter (mm), and L2 = shortest diameter (mm). Values are means plus or minus SEM (n = 12). **P < .01 by Mann-Whitney U test compared with control group. (F) CXCR4 expression was evaluated by flow cytometry, and quantitative RT-PCR. Representative results are shown. MFI expression was normalized to the MFI expression of isotype-treated cells. Data are presented as mean plus or minus SD. *P < .05, **P < .01.