Determine the effects of Wdr4 on global translation efficiency via ribosome profiling (Ribo-seq)

Tumor-associated macrophages (TAMs) play a pivotal role in shaping the tumor microenvironment in hepatocellular carcinoma (HCC), significantly influencing disease progression and patient outcomes. WDR4, a tRNA-binding scaffold essential for N7-methylguanosine (m7G) methylation, has an undefined role in HCC-associated TAMs. Here, we found that WDR4 expression is markedly upregulated in HCC-infiltrating TAMs and correlates with poor prognosis in patients. Using HCC mouse models, we demonstrated that WDR4 depletion reprograms TAMs toward an anti-tumor phenotype and suppresses HCC progression. To investigate the impact of WDR4 on translational efficiency, we performed ribosome profiling (Ribo-seq) on Wdr4-deficient TAMs. To ensure reproducibility and minimize potential off-target effects, we included TAMs sorted from orthotopic HCC-bearing Wdr4 conditional knockout mice, as well as immortalized bone marrow-derived macrophages (iBMDMs) with Wdr4 knocked out using two independent sgRNAs. An orthotopic HCC model was established in Wdr4^ff and myeloid-specific Wdr4 knockout (Wdr4^cKO) mice by inoculating Hepa1-6 cells. After tumor development, tumor tissues were harvested, and F4/80+ TAMs were isolated via flow cytometric sorting. For in vitro experiments, monoclonal immortalized bone marrow-derived macrophage (iBMDM) lines were infected with lentiviruses expressing Wdr4-targeting sgRNAs. Following puromycin selection for at least one week, cells were collected at 70–80% confluence. This included two independent Wdr4-targeting sgRNAs and one wild-type (WT) control group. Ribo-seq was performed on both F4/80+ TAMs and iBMDM samples. Both ribosome-protected fragments (footprint RNA) and input RNA were subjected to sequencing.