Journal: International Journal of Biological Sciences

Article Title: USP33 Promotes Lung Adenocarcinoma Brain Metastasis by Inhibiting the K48-Linked Ubiquitination and Degradation of S100A9 and Facilitating Vimentin Secretion

doi: 10.7150/ijbs.127941

Figure Lengend Snippet: Expression of S100A9 in LUAD samples and its association with prognosis and validation of the stably transfected S100A9 LUAD cell lines. (A) Multiplex immunohistochemistry showing S100A9-positive and EPCAM-positive cells in brain metastases of LUAD patients. Blue: DAPI; red: S100A9; green: EPCAM. (B) Western blot showing S100A9 protein levels in human bronchial epithelial (BEAS-2B) cells and NSCLC cells (A549, H1975, PC-9, H1299, and H2009). (C, F, I) Western blot showing S100A9 protein levels in H1975 cells (C) from the Vector and S100A9-OE groups, and in A549 cells (F) and PC-9 cells (I) from the sg-NC and sg-S100A9 groups. n = 3 independent biological replicates. Data are presented as mean ± SD. (D, E, G, H, J) RT‒qPCR and ELISA results showing S100A9 RNA levels (D, G) and secreted S100A9 protein levels (E, H) in H1975 cells (D, E) and A549 cells (G, H) from the Vector and S100A9-OE groups, and S100A9 RNA levels in PC-9 cells (J) from the sg-NC and sg-S100A9 groups. **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 3 independent biological replicates. Data are presented as mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey's post hoc test. Full-length blots/gels are presented in the raw image.

Article Snippet: The human LUAD cell line A549 (CL-0016, Procell) was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin (P/S) to maintain optimal proliferation.

Techniques: Expressing, Biomarker Discovery, Stable Transfection, Transfection, Multiplex Assay, Immunohistochemistry, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: USP33 Promotes Lung Adenocarcinoma Brain Metastasis by Inhibiting the K48-Linked Ubiquitination and Degradation of S100A9 and Facilitating Vimentin Secretion

doi: 10.7150/ijbs.127941

Figure Lengend Snippet: Effects of S100A9-OE on tumorigenesis in vivo and on the brain metastatic potential of H1975 and A549 cells. (A-H) A549 cells and H1975 cells were divided into a Vector group and an S100A9-OE group and used in the following experiments. (A) A549 and H1975 cells were injected into BALB/c nude mice to establish xenograft models. On day 32, the mice were euthanized, and the tumors were dissected and photographed (n = 8 per group). (B, C) Weights of tumors formed 32 days after subcutaneous injection of 5×10⁶ A549 cells (B) and H1975 cells (C) into the right axilla. *P < 0.05; ***P < 0.001; n = 8 independent biological replicates. Data are presented as mean ± SD. (D, E) Tumor volumes measured with a Vernier caliper at the indicated time points after subcutaneous injection of 5×10⁶ A549 cells (D) and H1975 cells (E) into the right axilla. ****P < 0.0001; n = 8 independent biological replicates. Data are presented as mean ± SD. (F, G) A549 cells (F) and H1975 cells (G) stably transfected with Vector-Luc or S100A9-Luc (expressing luciferase) (n = 6 per group) were injected into NOD-SCID mice via left cardiac ventricle injection, followed by small animal in vivo imaging. n = 6 independent biological replicates. Data are presented as mean ± SD. (H) The brain region with the strongest luminescence signal was imaged, and blood-brain barrier permeability was evaluated by Evans blue extravasation (n = 4 per group). n = 4 independent biological replicates. Data are presented as mean ± SD. Statistical significance was determined by Student's t test and two-way ANOVA followed by Bonferroni post hoc correction.

Article Snippet: The human LUAD cell line A549 (CL-0016, Procell) was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin (P/S) to maintain optimal proliferation.

Techniques: In Vivo, Plasmid Preparation, Injection, Stable Transfection, Transfection, Expressing, Luciferase, In Vivo Imaging, Permeability

Journal: International Journal of Biological Sciences

Article Title: USP33 Promotes Lung Adenocarcinoma Brain Metastasis by Inhibiting the K48-Linked Ubiquitination and Degradation of S100A9 and Facilitating Vimentin Secretion

doi: 10.7150/ijbs.127941

Figure Lengend Snippet: S100A9 mediates BBB dysfunction by regulating the malignant behavior of LUAD cells. A549 cells from the Vector group and S100A9-OE group and PC-9 cells from the sg-NC group and sg-S100A9 group were used in the following experiments. HBMECs and LUAD cells were cocultured in a Transwell system (0.4 μm) for 48 hours. (A, B) CCK-8 assay results showing OD values at 450 nm of HBMECs after 0, 24, 48, and 72 h of co-culture with A549 (A) or PC-9 (B) cells. ****P < 0.0001; n = 3 independent biological replicates. Data are presented as mean ± SD. (C, D) Na-F permeability assay results showing the permeability of HBMECs co-cultured with A549 (C) or PC-9 (D) cells. **P < 0.01; ***P < 0.001; n = 3 independent biological replicates. Data are presented as mean ± SD. (E-L) Western blot showing the expression levels of the tight junction proteins ZO-1, Occludin, and Claudin-5 in HBMECs co-cultured with A549 (E-H) or PC-9 cells (I-L). **P < 0.01; n = 3 independent biological replicates. Data are presented as mean ± SD. (M-R) Tube formation assay results showing the number of branches and total tube lengths formed by HBMECs co-cultured with A549 (M, O, P) or PC-9 cells (N, Q, R). *P < 0.05; **P < 0.01; n = 3 independent biological replicates. Data are presented as mean ± SD. Data were analyzed by Student's t test and two-way ANOVA followed by Bonferroni post hoc correction. Full-length blots/gels are presented in the raw image.

Article Snippet: The human LUAD cell line A549 (CL-0016, Procell) was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin (P/S) to maintain optimal proliferation.

Techniques: Plasmid Preparation, CCK-8 Assay, Co-Culture Assay, Permeability, Cell Culture, Western Blot, Expressing, Tube Formation Assay

Journal: International Journal of Biological Sciences

Article Title: USP33 Promotes Lung Adenocarcinoma Brain Metastasis by Inhibiting the K48-Linked Ubiquitination and Degradation of S100A9 and Facilitating Vimentin Secretion

doi: 10.7150/ijbs.127941

Figure Lengend Snippet: Effects of S100A9 overexpression on the transcriptome, differentially expressed genes, and signaling pathways in A549 cells. (A) Volcano plot showing genes differentially expressed between the Vector and S100A9-Luc groups of A549 cells. Each data point represents a single gene from three independent biological replicates. (B) KEGG analysis of enriched signaling pathways between the S100A9-Luc and Vector groups. Notably, the focal adhesion and ECM signaling pathways were regulated by S100A9. (C) GO analysis of differences in biological processes, cellular components, and molecular functions between the Vector and S100A9-Luc groups. (D-G) RT‒qPCR results showing RNA levels of S100A9 and Vimentin in A549, H1975, and PC-9 cells. ***P < 0.001; ****P < 0.0001; n = 3 independent biological replicates. Data are presented as mean ± SD. (H-O) Western blot showing protein expression levels of ITGB1, FAK, and Vimentin in A549 or H1975 (H-K) and PC-9 (L-O) cells. **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 3 independent biological replicates. Data are presented as mean ± SD. (P-W) Western blot showing protein expression levels of ITGB1, FAK, and Vimentin in A549 or H1975 cells between Vector group, S100A9-OE group and S100A9-OE+ITGB1 inhibitor group. **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 3 independent biological replicates. Data are presented as mean ± SD. Statistical significance was determined by Student's t test and one-way ANOVA followed by Tukey's post hoc test. Full-length blots/gels are presented in the raw image.

Article Snippet: The human LUAD cell line A549 (CL-0016, Procell) was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin (P/S) to maintain optimal proliferation.

Techniques: Over Expression, Protein-Protein interactions, Plasmid Preparation, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: USP33 Promotes Lung Adenocarcinoma Brain Metastasis by Inhibiting the K48-Linked Ubiquitination and Degradation of S100A9 and Facilitating Vimentin Secretion

doi: 10.7150/ijbs.127941

Figure Lengend Snippet: S100A9 exacerbates BBB damage and promotes End-MT in HBMECs by upregulating Vimentin expression. Groups (A-Q): The following groups of cells were used in the experiments: Vector group A549 cells cocultured with HBMECs; S100A9-OE group A549 cells cocultured with HBMECs; S100A9-OE+si-NC group A549 cells cocultured with HBMECs; and S100A9-OE+si-Vimentin group A549 cells cocultured with HBMECs. (A-C) RT‒qPCR (A) and Western blotting (B, C) results showing Vimentin RNA and protein levels in A549 cells transfected with si-1, si-2, and si-3. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant; n = 3 independent biological replicates. Data are presented as mean ± SD. (D) Na-F permeability assay results showing the permeability of HBMECs co-cultured with A549 cells. **P < 0.01; ***P < 0.001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. (E) Immunofluorescence staining analysis of ZO-1, Claudin-5, and Occludin fluorescence intensities in HBMECs co-cultured with A549 cells. (F-I) Western blot showing protein expression levels of ZO-1, Claudin-5, and Occludin in HBMECs co-cultured with A549 cells. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. (J-L) Tube formation experiments showing the number of branch points and tube lengths of HBMECs cocultured with A549 cells. *P < 0.05; ***P<0.001; ****P < 0.0001; N=3 independent biological replicates; ns, not significant; Data are the mean ± SD. (M) ROS levels in HBMECs cocultured with A549 cells. (N-Q) Western blot showing protein expression levels of VE-Cadherin, N-Cadherin, and α-SMA in HBMECs co-cultured with A549 cells. ****P < 0.0001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. Full-length blots/gels are presented in the raw image.

Article Snippet: The human LUAD cell line A549 (CL-0016, Procell) was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin (P/S) to maintain optimal proliferation.

Techniques: Expressing, Plasmid Preparation, Western Blot, Transfection, Permeability, Cell Culture, Immunofluorescence, Staining, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: USP33 Promotes Lung Adenocarcinoma Brain Metastasis by Inhibiting the K48-Linked Ubiquitination and Degradation of S100A9 and Facilitating Vimentin Secretion

doi: 10.7150/ijbs.127941

Figure Lengend Snippet: S100A9 positively regulates Vimentin expression to promote LUAD metastasis and increase HBMEC dysfunction. Groups (A-N): The following groups of cells were used in the experiments: Vector group A549 cells cocultured with HBMECs; S100A9-OE group A549 cells cocultured with HBMECs; S100A9-OE+si-NC group A549 cells cocultured with HBMECs; and S100A9-OE+si-Vimentin group A549 cells cocultured with HBMECs. (A) CCK-8 assay results showing OD values at 450 nm of HBMECs after 0, 24, 48, and 72 h of co-culture with A549 cells. ***P < 0.001; ****P < 0.0001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. (B-C) EdU assay results showing proliferation rates of HBMECs co-cultured with A549 cells. ***P < 0.001; ****P < 0.0001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. (D-E) Scratch assay results showing migration distances of HBMECs co-cultured with A549 cells. ****P < 0.0001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. (F-H) JC-1 assay results showing mitochondrial membrane potentials in HBMECs co-cultured with A549 cells. *P < 0.05; ****P < 0.0001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. (I-J) Confocal immunofluorescence results showing lysosomal pH in HBMECs co-cultured with A549 cells. *P < 0.05; ***P < 0.001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. (K-N) Western blot showing protein expression of P62, p-Beclin 1/Beclin 1, and LC3-II/I ratios in HBMECs co-cultured with A549 cells. *P < 0.05; ***P < 0.001; ****P < 0.0001; ns; n = 3 independent biological replicates. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test or two-way ANOVA followed by Bonferroni post hoc correction, as appropriate. Full-length blots/gels are presented in the raw image.

Article Snippet: The human LUAD cell line A549 (CL-0016, Procell) was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin (P/S) to maintain optimal proliferation.

Techniques: Expressing, Plasmid Preparation, CCK-8 Assay, Co-Culture Assay, EdU Assay, Cell Culture, Wound Healing Assay, Migration, Membrane, Immunofluorescence, Western Blot

Journal: International Journal of Biological Sciences

Article Title: USP33 Promotes Lung Adenocarcinoma Brain Metastasis by Inhibiting the K48-Linked Ubiquitination and Degradation of S100A9 and Facilitating Vimentin Secretion

doi: 10.7150/ijbs.127941

Figure Lengend Snippet: USP33 removes K48-linked polyubiquitin chains on S100A9 to stabilize its expression and promote Vimentin secretion. (A-C) ELISA results showing extracellular Vimentin secretion levels in A549 and H1975 cells from the Vector and S100A9-OE groups, and in PC-9 cells from the sg-NC and sg-S100A9 groups. **P < 0.01; n = 3 independent biological replicates. Data are presented as mean ± SD. (D, E) Co-IP assay results showing the interaction between USP33 and S100A9 when V5-tagged USP33 and Flag-tagged S100A9 were co-transfected into HEK293T cells. n = 3 independent biological replicates. Data are presented as mean ± SD. (F, G) Co-IP assay results showing the endogenous interaction between USP33 and S100A9 in A549 (F) and PC-9 (G) cells. n = 3 independent biological replicates. Data are presented as mean ± SD. (H-K) Western blot analysis showing S100A9 protein half-life in A549 cells from the Vector and USP33-OE groups after treatment with MG132 (H, I) or CHX (J, K). ****P < 0.0001; n = 3 independent biological replicates. Data are presented as mean ± SD. (L-M) IP results showing K48-linked polyubiquitination levels of S100A9 regulated by USP33 in Vector and USP33-OE A549 cells. n = 3 independent biological replicates. Data are presented as mean ± SD. Statistical significance was determined by Student's t test and two-way ANOVA followed by Bonferroni post hoc correction. Full-length blots/gels are presented in the raw image.

Article Snippet: The human LUAD cell line A549 (CL-0016, Procell) was cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin (P/S) to maintain optimal proliferation.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Co-Immunoprecipitation Assay, Transfection, Western Blot