Fig. 4

Enhanced Autophagy in Nudt21-Ablated Macrophages Following Inflammatory Activation. A Volcano plot showing DEGs in Nudt21-cKO BMDMs compared with WT BMDMs after treatment with IFNγ (50 ng/ml) overnight, followed by LPS (50 ng/ml) stimulation for 4 hours. Genes with a fold change greater than 1.5 and P < 0.05 are highlighted; upregulated genes are shown in red, and downregulated genes are shown in blue. B Gene Ontology (GO) enrichment analysis of transcripts upregulated in Nudt21-cKO BMDMs relative to WT controls after treatment with IFNγ (50 ng/ml) overnight and LPS (50 ng/ml) for 4 hours. C Heatmap displaying the expression profiles of genes associated with autophagy pathways in WT and Nudt21-cKO BMDMs following treatment with IFNγ (50 ng/ml) overnight and LPS (50 ng/ml) for 4 hours. D RT‒qPCR analysis of autophagy-related gene expression in bone marrow monocytes from WT and Nudt21-cKO mice in the HLH model (6 h, n = 6 per group). E RT‒qPCR analysis of the mRNA levels of autophagy-related genes in BMDMs from WT and Nudt21-cKO mice following treatment with IFNγ (50 ng/mL) overnight and LPS (50 ng/mL) for 4 hours, with or without 5 μM actinomycin D (ActD) or 5 μg/mL α-amanitin. Representative transmission electron microscopy (TEM) images (F) and quantification of autolysosomes (G) in BMDMs primed with 50 ng/ml IFNγ overnight and 50 ng/ml LPS for 4 hours. Scale bar: 500 nm. Images represent macrophages pooled from 2–3 mice per genotype and condition, with at least 6 images analyzed per genotype. H Immunoblot analysis showing the LC3 protein levels in WT and Nudt21-cKO BMDMs following overnight stimulation with IFNγ (50 ng/mL) and LPS (50 ng/mL) for the indicated durations. I Fluorescence microscopy images of LC3B and LAMP1 staining, along with DAPI counterstaining, in WT and Nudt21-cKO BMDMs primed with 50 ng/ml IFNγ overnight and 50 ng/ml LPS for 2 hours. Scale bar in the zoomed image: 2 µm. Captions represent at least two independent experiments. J Quantification of colocalized puncta of LC3 and LAMP1 across 50 cells per group. The error bars represent the standard errors of the means (SEMs). P values were determined by unpaired Student’s t-test