human lung adenocarcinoma epithelial cells  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: NITAC-mediated ISGylation of eIF4E2 attenuates GSK3β proline-directed kinase activity, conferring cytoprotection

doi: 10.1016/j.jbc.2025.110777

Figure Lengend Snippet: NITAC-enabled activation of eIF4E2 ISGylation. A , schematic diagram of the Nb.30C7-NITAC structure. B and E , Nb.30C7-NITAC enhanced the ISGylation of Flag-eIF4E2. B , A549 or ( E ) HeLa cells were transfected with plasmids expressing FLAG-eIF4E2, HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT and HA-Nb.30C7-NITAC as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-ISG15 antibody, followed by IB analysis. C and F , Nb.30C7-NITAC enhances the ISGylation of endogenous eIF4E2. C , A549 or ( F ) HeLa cells were transfected with plasmids expressing HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-ISG15 antibody, followed by IB analysis. D and G , Nb.30C7-NITAC enhances the ISGylation of exogenous eIF4E2 in a dose-dependent manner. D , A549 or ( G ) HeLa cells were transfected with plasmids (0, 0.2, 0.4, 0.6, or 0.8 μg) expressing HA-Nb.30C7-NITAC for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-ISG15 antibody, followed by IB analysis. Co-IP, coimmunoprecipitation; eIF4E2, eukaryotic translation initiation factor 4E2; IB, immunoblot; IFNβ, interferon β; NITAC,Nanobody-based ISGylation Targeting Chimera.

Article Snippet: HEK293T human embryonic kidney cell line, A549 human lung adenocarcinoma epithelial cell line, HeLa human cervical cancer cell line, HCT116 colon cancer cell line, HT22 mouse hippocampal neuronal cell line, and BV2 mouse microglial cell lines were obtained from American Type Culture Collection and China Center for Type Culture Collection.

Techniques: Activation Assay, Transfection, Expressing, Co-Immunoprecipitation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: NITAC-mediated ISGylation of eIF4E2 attenuates GSK3β proline-directed kinase activity, conferring cytoprotection

doi: 10.1016/j.jbc.2025.110777

Figure Lengend Snippet: ISGylation of eIF4E2 enhances its interaction with GSK3β. A and B , GSK3β interacts with ISGylated eIF4E2. A , A549 cells were transfected with plasmids expressing HA-eIF4E2(WT), HA-eIF4E2(4KR), FLAG-GSK3β, and HERC5 as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to Co-IP with anti-FLAG antibody, followed by IB analysis. ( B ) HEK293T cells cotransfected components of the His 6 -ISGylation system (UBE1L, UBCH8, and His 6 -ISG15), plasmids expressing Myc-eIF4E2(WT/4KR), FLAG-GSK3β, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 48 h, and then cell extracts were subjected to Co-IP with anti-FLAG antibody and Ni-NTA pull down to precipitate His 6 -ISG15 and its complexes, followed by IB analysis. C , depletion of ISG15 inhibits GSK3β from interacting with ISGylated eIF4E2. A549 cells were first transfected with siRNA to knock down the ISG15. After 24 h, plasmids expressing FLAG-eIF4E2, HA-GSK3β, and HERC5 were transfected into the cells, and simultaneously, the cells treated with IFNβ (1000 U/ml) for 36 h. Cell extracts were subjected to co-IP with anti-FLAG antibody, followed by IB analysis. D and E , ISGylation of eIF4E2 enhances its interaction with GSK3β. D , HEK293T cells cotransfected components of the His 6 -ISGylation system (UBE1L, UBCH8, and His 6 -ISG15), plasmids expressing FLAG-eIF4E2(WT/4KR) and HERC5 as indicated for 48 h, and then cell extracts were subjected to co-IP with anti-FLAG antibody and Ni-NTA pull down to precipitate His 6 -ISG15 and its complexes, followed by IB analysis. E , A549 cells were transfected with plasmids expressing FLAG-eIF4E2(WT/4KR), HERC5, and HA-Nb.30C7-NITAC as indicated for 12 h and then treated with IFNβ (1000 U/ml) for another 36 h. Then, cell extracts were subjected to co-IP with anti-FLAG antibody, followed by IB analysis. Co-IP, coimmunoprecipitation; eIF4E2, eukaryotic translation initiation factor 4E2; GSK3β, glycogen synthase kinase 3β; IB, immunoblot; IFNβ, interferon β; NITAC, Nanobody-based ISGylation Targeting Chimera.

Article Snippet: HEK293T human embryonic kidney cell line, A549 human lung adenocarcinoma epithelial cell line, HeLa human cervical cancer cell line, HCT116 colon cancer cell line, HT22 mouse hippocampal neuronal cell line, and BV2 mouse microglial cell lines were obtained from American Type Culture Collection and China Center for Type Culture Collection.

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Knockdown, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: NITAC-mediated ISGylation of eIF4E2 attenuates GSK3β proline-directed kinase activity, conferring cytoprotection

doi: 10.1016/j.jbc.2025.110777

Figure Lengend Snippet: ISGylation of eIF4E2 inhibits GSK3β kinase activity. A , ISGylation inhibited the S/T-P phosphorylation of downstream substrates of the eIF4E2–GSK3β pathway. HEK293T cells cotransfected components of the ISGylation system (UBE1L, UBCH8, and ISG15), plasmids expressing FLAG-eIF4E2and HHARI as indicated for 48 h, and then cell extracts were subjected to co-IP with anti-FLAG antibody, followed by IB analysis. B and C , ISGylated eIF4E2 inhibited the S/T-P phosphorylation of downstream substrates of the eIF4E2–GSK3β pathway. B , HCT116 cell transfected plasmids expressing HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 24 h and treated with CPT (0.5 μM) for another 24 h. Co-IP with anti-ISG15 and IB analysis of whole-cell extracts. C , A549 cell transfected plasmids expressing HA-Nb.30C7, HA-HECT, HA-Nb.30C7/HA-HECT, and HA-Nb.30C7-NITAC as indicated for 12 h and treated with IFNβ (1000 U/ml) for another 36 h. Co-IP with anti-ISG15 and IB analysis of whole-cell extracts. D , ISGylated eIF4E2 inhibits GSK3β′s proline-directed kinase activity. E3 ligase HERC5, E2 conjugating enzyme UBE1L, and UBCH8 protein were synthesized by in vitro transcription and translation. In vitro ISGylation assays were conducted in the presence of ISG15, E1, E2, HERC5, and FLAG-eIF4E2. FLAG-GSK3β, FLAG-eIF4E2, and FLAG-eIF4E2(4KR) proteins were overexpressed and purified from HEK293T cells. His 6 -tagged p53 and ISG15 were expressed and purified from Escherichia coli . eIF4E2, HERC5, UBCH8, UBE1L, and ISG15 were incubated in an ATP-containing buffer at 37 °C for 1 h to induce ISGylation of eIF4E2. After the reaction, the resulting components were combined with GSK3β and p53 in an ATP-containing buffer and incubated at 30 °C for 1 h to perform the in vitro kinase assay, followed by IB analysis. Co-IP, coimmunoprecipitation; CPT, camptothecin; IB, immunoblot; eIF4E2, eukaryotic translation initiation factor 4E2; GSK3β, glycogen synthase kinase 3β; NITAC, Nanobody-based ISGylation Targeting Chimera; S/T-P, proline-directed serine/threonine.

Article Snippet: HEK293T human embryonic kidney cell line, A549 human lung adenocarcinoma epithelial cell line, HeLa human cervical cancer cell line, HCT116 colon cancer cell line, HT22 mouse hippocampal neuronal cell line, and BV2 mouse microglial cell lines were obtained from American Type Culture Collection and China Center for Type Culture Collection.

Techniques: Activity Assay, Phospho-proteomics, Expressing, Co-Immunoprecipitation Assay, Transfection, Synthesized, In Vitro, Purification, Incubation, Kinase Assay, Western Blot

Journal: iScience

Article Title: Transcriptomic analysis of phage-mammalian cell interaction reveals diverse phage immunobarcodes

doi: 10.1016/j.isci.2025.113513

Figure Lengend Snippet: Internalization, survival, and antibacterial activity of phages in A549 and RAW264.7 cell lines (A and B) Bar graphs showing the number of internalized phage particles after 12 h, and the number of viable-surviving intracellular phage particles after 24-h in A549 (A) and RAW264.7 cells (B). The initial concentration of phage added to the cells was 10 9 PFU/well. Statistical significance for each treatment is presented relative to the CK01-treated group. (C) Fluorescence microscopy images show intracellular A . baumannii strain WHG40042 , WHG40090 , WHG40149 LB8R, and WHG40004 in A549 and RAW264.7 cells. The cell membranes are stained with Dil (red), the nuclei are stained with DAPI (blue), and the bacteria are stained with SYBR green dye (green). Scale bars, 5 μm. (D and E) Intracellular bacterial loads in A549 (D) and RAW264.7 cells (E) infected with A. baumannii host strains and treated with respective phage for 8 h. Phage and bacterial quantification results are presented as mean ± SD of three independent experiments. ns: not significant; ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; ∗∗∗: p ≤ 0.001 by one-way ANOVA. Dashed line represents the limit of detection.

Article Snippet: The human lung adenocarcinoma epithelial cell line A549 (CCL-185) and murine macrophage cell line RAW264.7 (TIB-71) cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium F12 (DMEM-F12; Fisher Scientific, 11320-033, GIBCO), and Dulbecco’s Modified Eagle Medium (DMEM; Fisher Scientific, 11965-092, GIBCO) respectively, supplemented with 10% fetal bovine serum (FBS, Sigma, F4135) and antibiotic-antimycotic solution (100 U/mL penicillin G, 100 mg/mL streptomycin and 0.25 μg/mL amphotericin B) (Fisher Scientific, 15240062) at 37°C and 5% CO 2 .

Techniques: Activity Assay, Concentration Assay, Fluorescence, Microscopy, Staining, Bacteria, SYBR Green Assay, Infection

Journal: iScience

Article Title: Transcriptomic analysis of phage-mammalian cell interaction reveals diverse phage immunobarcodes

doi: 10.1016/j.isci.2025.113513

Figure Lengend Snippet: Differential gene expression profiles in A549 and RAW264.7 cells after phage challenge Bar graphs representing the number of differentially expressed genes per sample after stimulation with phages, compared with PBS-treated A549 cells for 12 h (A) and RAW264.7 cells for 6 and 24 h (B). Criteria for determining expression: log 2-fold change ≥1, significance p-value of ≤0.05. Each bar represents the number of genes up-regulated or down-regulated following the stimulation of cells with phage.

Article Snippet: The human lung adenocarcinoma epithelial cell line A549 (CCL-185) and murine macrophage cell line RAW264.7 (TIB-71) cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium F12 (DMEM-F12; Fisher Scientific, 11320-033, GIBCO), and Dulbecco’s Modified Eagle Medium (DMEM; Fisher Scientific, 11965-092, GIBCO) respectively, supplemented with 10% fetal bovine serum (FBS, Sigma, F4135) and antibiotic-antimycotic solution (100 U/mL penicillin G, 100 mg/mL streptomycin and 0.25 μg/mL amphotericin B) (Fisher Scientific, 15240062) at 37°C and 5% CO 2 .

Techniques: Gene Expression, Expressing

Journal: iScience

Article Title: Transcriptomic analysis of phage-mammalian cell interaction reveals diverse phage immunobarcodes

doi: 10.1016/j.isci.2025.113513

Figure Lengend Snippet: Unique and common gene expression profiles across phage-treated samples The Venn diagrams show the number of genes uniquely expressed and the gene-overlap relationships among varied phage treatments in A549 cells (A) and RAW264.7 cells (B). (C and D) PCA analysis of DEGs of different phage and control treatments in A549 cells (C) and RAW264.7 cells (D).

Article Snippet: The human lung adenocarcinoma epithelial cell line A549 (CCL-185) and murine macrophage cell line RAW264.7 (TIB-71) cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium F12 (DMEM-F12; Fisher Scientific, 11320-033, GIBCO), and Dulbecco’s Modified Eagle Medium (DMEM; Fisher Scientific, 11965-092, GIBCO) respectively, supplemented with 10% fetal bovine serum (FBS, Sigma, F4135) and antibiotic-antimycotic solution (100 U/mL penicillin G, 100 mg/mL streptomycin and 0.25 μg/mL amphotericin B) (Fisher Scientific, 15240062) at 37°C and 5% CO 2 .

Techniques: Gene Expression, Control

Journal: iScience

Article Title: Transcriptomic analysis of phage-mammalian cell interaction reveals diverse phage immunobarcodes

doi: 10.1016/j.isci.2025.113513

Figure Lengend Snippet: Anti-inflammatory transcripts in A549 cells challenged with phage (A) Heat maps representing repressed expression (blue) of inflammatory genes and upregulation (black) of anti-inflammatory genes after phage treatment. Gene expression is shown as log 2 (fold change). (B) GO enrichment analysis of downregulated genes shows the enrichment of inflammatory terms. Each term is represented as -log 10 ( p -value). (C) KEGG enrichment analysis of downregulated genes, indicating the enrichment of pro-inflammatory signaling pathways. Enrichment of each pathway is represented as -log 10 ( p -value). (D) GO enrichment analysis of upregulated genes indicating the negative regulation of inflammatory pathways in A549 cells after treatment with phage for 12 h. Enrichment of each pathway is shown as -log 10 ( p -value).

Article Snippet: The human lung adenocarcinoma epithelial cell line A549 (CCL-185) and murine macrophage cell line RAW264.7 (TIB-71) cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium F12 (DMEM-F12; Fisher Scientific, 11320-033, GIBCO), and Dulbecco’s Modified Eagle Medium (DMEM; Fisher Scientific, 11965-092, GIBCO) respectively, supplemented with 10% fetal bovine serum (FBS, Sigma, F4135) and antibiotic-antimycotic solution (100 U/mL penicillin G, 100 mg/mL streptomycin and 0.25 μg/mL amphotericin B) (Fisher Scientific, 15240062) at 37°C and 5% CO 2 .

Techniques: Expressing, Gene Expression, Protein-Protein interactions

Journal: iScience

Article Title: Transcriptomic analysis of phage-mammalian cell interaction reveals diverse phage immunobarcodes

doi: 10.1016/j.isci.2025.113513

Figure Lengend Snippet: Effects of phage on cellular physiological processes Heat maps representing GO (A) and KEGG (B) enrichment analysis of downregulated genes in A549 cells and GO (C) and KEGG (D) enrichment analysis of downregulated genes in RAW264.7 cells. The figures show pathways involved in cellular growth, proliferation, and development, and the enrichment of each pathway is represented as -log 10 ( p -value).

Article Snippet: The human lung adenocarcinoma epithelial cell line A549 (CCL-185) and murine macrophage cell line RAW264.7 (TIB-71) cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium F12 (DMEM-F12; Fisher Scientific, 11320-033, GIBCO), and Dulbecco’s Modified Eagle Medium (DMEM; Fisher Scientific, 11965-092, GIBCO) respectively, supplemented with 10% fetal bovine serum (FBS, Sigma, F4135) and antibiotic-antimycotic solution (100 U/mL penicillin G, 100 mg/mL streptomycin and 0.25 μg/mL amphotericin B) (Fisher Scientific, 15240062) at 37°C and 5% CO 2 .

Techniques: