Fig 2: GBP5 knockout downregulated the expression of proinflammatory mediator genes in THP-1 cells. The transcriptomes of wild-type and GBP5–/– THP-1 cells, with or without stimulation [IFNγ and lipopolysaccharide (LPS)], respectively, were analyzed by RNAseq technique. N = 3 for each treatment group. (A) Generation of GBP5 knockout (GBP5–/–). THP-1 cell line by CRISPR/Cas9-mediated genome editing. The two CRISPR target sites are highlighted in red. Loss of GBP5 was confirmed by Western blot. WT, wild-type control. (B) Messenger RNA expression of GBP5 and related genes in wild-type and GBP5–/– THP-1 cells (clone B2), with or without stimulation (IFNγ and LPS), respectively. FPKM, fragments per kilobase million. ***P < 0.001; ****P < 0.0001; unpaired Student’s t-test. (C) Differential gene expression between wild-type and GBP5–/– THP-1 cells: volcano plot. Vertical and horizontal dashed lines indicate the cutoff values for differentially expressed genes:| Log2 (Fold Change)| > 1 and p.adjust < 0.05. (D) GO (Gene Ontology) analysis of downregulated genes in GBP5 knockout cells. The cutoff values for selecting differentially expressed genes for the input gene list: Log2(Fold Change) < -2, and p. adjust < 0.05. BP, biological process; CC, cellular compartment; MF, molecular function. (E) Gene set enrichment analysis (GSEA) of GBP5–/– THP-1 cells (clone B2) transcriptome compared to wild-type THP-1 cells. Upper panel: the enrichment score curves of the top KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways exhibiting decreased expression in GBP5–/– THP-1 cells: immune related pathways. Middle panel: distribution of the genes related to the pathways indicated in the upper panel. The genes were ranked according to their differential expression between wild-type and GBP5–/– THP-1 cells. Genes of higher rank (left) exhibit relatively higher expression in wild-type THP-1 cells. Lower panel: Graphical representation of the correlations of the gene expression levels with the phenotypes: wild-type or GBP5 knockout. Genes on the right are more negatively correlated with GBP5 deficiency.

Fig 3: Flow chart of this study (A) Data sources of this work. (B) The correlation between GBPs (guanylate-binding proteins) and the effect of immunotherapy (C) Correlation between GBP5 and TME (tumor microenvironment) in pan-cancer (D) Single-cell analysis of GBP5 in CRC (colorectal cancer). (E) Construction of a GBP5-related prognostic model in CRC.

Fig 4: Memory of Prior IFN? Induction Is Reversible but Persists for up to 14 Days in Continuously Cycling Cells(A) Scheme outlining the experiment to determine the duration of IFN?-mediated transcriptional memory.(B) Cells subjected to the IFN? treatment regimen outlined in (A) were processed for western blotting and probed for GBP5 protein expression. a-TUB, loading control.(C) Cells as in (B) but processed for RT-qPCR of GBP5 and HLA-DRA mRNA. Expression levels were normalized to ACTB and internally to cells after priming. Error bars, SD; n = 3 biological replicates; numbers represent the p values.

Fig 5: GBP2 and 5 do not localize to Toxoplasma vacuoles. (A) Immunofluorescence images of THP-1?GBP1+Tet-mCH-GBP1, ?GBP2+Tet-mCH-GBP2 or ?GBP5+Tet-mCH-GBP5 cells treated with IFN? and Doxycycline (Dox) and stained for Golgi marker GM-130 to illustrate Golgi localization of GBP5 in uninfected cells. Magenta: mCherry (mCH)-GBP1/2/5; green: GM-130 (Golgi) and blue: nuclei. Scale bar, 10 µm. (B) Immunofluorescence images of THP-1?GBP1+Tet-mCH-GBP1, ?GBP2+Tet-mCH-GBP2 or ?GBP5+Tet-mCH-GBP5 cells expressing the indicated GBPx mutant treated with IFN?+Dox to illustrate localization of the respective protein in uninfected cells. Magenta: mCherry (mCH)-GBP1/2/5 and blue: nuclei. Scale bar, 10 µm. (C) Immunofluorescence images (top) and HRMAn-based quantification of GBP recruitment to Tg (bottom) in THP-1?GBP1+Tet-mCH-GBP1, ?GBP2+Tet-mCH-GBP2 or ?GBP5+Tet-mCH-GBP5 cells treated with IFN?+Dox and infected with type I (RH) or type II (PRU) T. gondii (Tg) for 6 h. Magenta: mCherry (mCH)-GBP1/2/5; grey: Tg and blue: nuclei. Scale bar, 10 µm. (D) Images (left) and HRMAn-based quantification of GBP1 recruitment to Tg-vacuoles (right) in THP-1?GBP1+Tet-mCH-GBP1 cells expressing the indicated GBP1 mutant treated with IFN?+Dox and infected for 6 h. Magenta: mCherry (mCH)-GBP1; grey: pathogen and blue: nuclei. Scale bar, 10 µm. Data information: images in (A + C-D) representative of n = 3 and in (B) representative of n = 2 independent experiments. Graph in (C + D) show mean ± SEM from n = 3 independent experiments. ****P = 0.0001 for indicated comparisons in (C) from one-way ANOVA comparing to Dox-only treated cells following adjustment for multiple comparisons; n.d. not detected.