Journal: Advanced Science

Article Title: Excessive MYC Orchestrates Macrophages induced Chromatin Remodeling to Sustain Micropapillary‐Patterned Malignancy in Lung Adenocarcinoma

doi: 10.1002/advs.202403851

Figure Lengend Snippet: MYC in collaboration with M2‐like macrophages gains unique binding sites that exhibit alternative transcriptional regulatory activity. A) The DNase I hypersensitivity site identified via DNase I treated‐PCR in promoters of MP‐pattern genes in E‐V cells, M‐V cells, E‐M2 cells and M‐M2 cells (A549 cell based). B) HOMER de novo motif analysis on altered H3K27ac occupied sites in AC‐subtype tissue versus MP‐subtype tissue, and M‐V cells versus M‐M2 cells. C) Western Blots of FOSL2 and JUN in cells with different treatment. Three biological replicates were performed for each cell. D) ChIP‐PCR of IgG, MYC and FOSL2 for binding promoters of MP‐pattern genes in A549 cells with different treatment. E) The DNase I hypersensitivity site identified via DNase I treated‐PCR in promoters of MP‐pattern genes in A549 cells with different treatment. The dependence experiment revealed that the high DNase I sensitivity of MP‐pattern gene promoters induced by M2‐like macrophages depends on TGFβ‐FOSL2 axis. F) ChIP‐PCR of IgG, MYC and FOSL2 for binding promoters of MP‐pattern genes in A549 cells with different treatment. The dependence experiment revealed that MYC binding MP‐pattern gene promoters in M‐M2 A549 cells depends on TGFβ‐FOSL2 axis. G) The DNase I hypersensitivity site identified via DNase I treated‐PCR in promoters of MP‐pattern genes in A549 cells with different treatment. The rescue experiment revealed that the high DNase I sensitivity of MP‐pattern gene promoters in solely MYC‐overexpression A549 cells were rescued by FOSL2 overexpression or exogenous addition of TGFβ. H) ChIP‐PCR of IgG, MYC and FOSL2 for binding promoters of MP‐pattern genes in A549 cells with different treatment. The rescue experiment revealed that MYC binding MP‐pattern gene promoters in M‐V A549 cells were rescued by FOSL2 overexpression or exogenous addition of TGFβ. I) Western Blots of FOSL2 in cancer cells with different time‐point of coculture with M2‐like macrophages. Three biological replicates were performed for each cell. J) Western Blots of FOSL2 in cancer cells coculture with M2‐like macrophages and several antibodies were used to neutralize cytokines. Three biological replicates were performed for each cell. K,L) Western Blots of FOSL2 in cancer cells exogenous addition of TGFβ with different concentration (K) and treatment time (L). M) Representative images of immunohistochemistry of FOSL2 in tissues micro‐array from MAPes cohort. Right: quantitative statistics. Scale bars: 100 µm. N) Relative expression of FOSL2 in SCISSOR+ cells (MP‐subtype proneness, mean expression: 0.4277) and SCISSOR‐ cells (AC‐subtype proneness, mean expression: 0.3369) from single‐cell RNA‐seq data in Figure . O) The correlation analysis in expression of MYC and expression of FOSL2 from TCGA‐LUAD mRNA dataset. P) Representative images of immunofluorescence of FOSL2 (green), MYC (red) and DAPI (blue) in M‐M2 cells, Scale bars: 1µm (Upper). The detected fluorescence intensity at the white line (Bottom). Q) Virtual docking was performed based on the protein structure of MYC and the tertiary structure of FOSL2 protein predicted by Alphafold2. R) Co‐immunoprecipitation of c‐Myc antibody and IgG antibody indicated that MYC binding FOSL2 after redundant expression when cancer cells were co‐cultured with M2‐like macrophages. For each group, n = 5. The p values were determined by Student's t ‐test (unpaired two‐tailed), n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM.

Article Snippet: For certain experiments, cells were transfected with a full‐length c‐Myc expression vector (pcDNA3‐cmyc, Addgene, #16011, RR_ID: Addgene_16011). pcDNA3 empty vector (Corues Biotechnology Co.), targeting FOSL2 short hairpin RNA and control scribble (Sangon Biotech), FOSL2‐expression vector (Addgene, #187907, RR_ID: Addgene_187907), pcDNA3.1 empty vector (Corues Biotechnology Co.), FOSL2 tet‐off system vector (pLV3rsv‐hPGK‐FOSL2‐Tetoff‐IRES‐Puro, Corues Biotechnology Co.), dual‐luciferase reporter system plasmid (Corues Biotechnology Co.), CASP3‐promoter‐dsRed tracing system plasmid (Corues Biotechnology Co.) and MYC‐truncated plasmid (Corues Biotechnology Co.).

Techniques: Binding Assay, Activity Assay, Western Blot, Over Expression, Concentration Assay, Immunohistochemistry, Microarray, Expressing, RNA Sequencing, Immunofluorescence, Fluorescence, Immunoprecipitation, Cell Culture, Two Tailed Test

Journal: Advanced Science

Article Title: Excessive MYC Orchestrates Macrophages induced Chromatin Remodeling to Sustain Micropapillary‐Patterned Malignancy in Lung Adenocarcinoma

doi: 10.1002/advs.202403851

Figure Lengend Snippet: Inhibition of the TGFβ‐FOSL2 axis effectively diminishes the malignancy associated with the MP‐pattern. A–D) The dependence experiment revealed that detachment‐induced cell death resistance (A), non‐anchored clonality ability (B), anti‐shearing force (C) and MP‐pattern genes expression in protein level (D) induced by M2‐like macrophages depends on TGFβ‐FOSL2 axis in cancer cell lines in A549 cells. For detachment‐induced cell death assay, Lung cancer cell lines were cultured attached or detached on TC‐treated plates or covalently bound hydrogel layer‐treaded plates. For non‐anchored clonality ability, indicated cells were allowed to grow in soft agar for 2 weeks and colonies were counted. E) An experimental illustration showing the rescue experiment in BALB/c Nude athymic mice with A549 cells xenograft, which transfected with FOSL2 tet‐off system. F,G) Tumor images (F) and growth kinetics (G) of subcutaneously implanted tumors in 2‐week‐after‐implanted FOSL2 tet‐off system transfected A549 cells and treated/not treated with doxycycline. n = 6. Scale bars: 1 cm. H) CTCs detected from venous blood in BALB/c Nude athymic mice 2‐week‐after‐implanted FOSL2 tet‐off system transfected A549 cells, which treated/not treated with doxycycline. Left: Representative images, right: quantitative statistics. n = 6. I) Dissemination index detected from venous blood in BALB/c Nude athymic mice bearing 2‐week‐after‐implanted FOSL2 tet‐off system transfected A549 cells, which treated/not treated with doxycycline. n = 6. J) An experimental illustration showing efficacy of Galunisertib against the malignancy of MP‐pattern with redundant expression of MYC via orthotopic lung injection. n = 5. K) Lung tumor growth was detected using in vivo imaging. Left: Representative images, right: quantitative statistics. n = 5. L,M) CTCs (L) and Dissemination index (M) detected from venous blood in BALB/c Nude athymic mice bearing MYC basal/redundant expression tumor, treated or not treated with Galunisertib. n = 5. N) Representative images of immunofluorescence staining of CTOS derived from indicated tumor tissue. Red, villin; blue, DAPI. Scale bars: 20 µm. O) Working model for MP/AC‐pattern malignancy orchestration. The p values were determined by Student's t ‐test (unpaired two‐tailed), n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM.

Article Snippet: For certain experiments, cells were transfected with a full‐length c‐Myc expression vector (pcDNA3‐cmyc, Addgene, #16011, RR_ID: Addgene_16011). pcDNA3 empty vector (Corues Biotechnology Co.), targeting FOSL2 short hairpin RNA and control scribble (Sangon Biotech), FOSL2‐expression vector (Addgene, #187907, RR_ID: Addgene_187907), pcDNA3.1 empty vector (Corues Biotechnology Co.), FOSL2 tet‐off system vector (pLV3rsv‐hPGK‐FOSL2‐Tetoff‐IRES‐Puro, Corues Biotechnology Co.), dual‐luciferase reporter system plasmid (Corues Biotechnology Co.), CASP3‐promoter‐dsRed tracing system plasmid (Corues Biotechnology Co.) and MYC‐truncated plasmid (Corues Biotechnology Co.).

Techniques: Inhibition, Expressing, Cell Culture, Transfection, Injection, In Vivo Imaging, Immunofluorescence, Staining, Derivative Assay, Two Tailed Test

Journal: Heliyon

Article Title: Possible toxicity of chronic carbon dioxide exposure associated with face mask use, particularly in pregnant women, children and adolescents – A scoping review

doi: 10.1016/j.heliyon.2023.e14117

Figure Lengend Snippet: Significant increase in CO 2 levels in mask wearers under various conditions in scientific intervention studies.

Article Snippet: Bharatendu 2020 [ ] , PETCO 2 , N95 masks: 37.3 vs 40.4 , +3.1 , <0.001.

Techniques: