toluene  (Chem Impex International)

Journal: Acta pharmaceutica Sinica. B

Article Title: The suppression of cervical cancer ferroptosis by macrophages: The attenuation of ALOX15 in cancer cells by macrophages-derived exosomes.

doi: 10.1016/j.apsb.2023.03.025

Figure Lengend Snippet: Figure 3 Attenuation of cervical cancer ferroptosis by TAM is involved in downregulation of ALOX15. (AeC) HeLa and SiHa cells were treated with CM from M0 macrophage and TAM for 48 h. Ferroptosis-associated gene was detected by RT-PCR. The ratio of genes expressions in cells treated with TAMs-CM relative to M0-CM were shown in the heatmap (A). Volcano map indicated the relationship between the observed fold change in gene expression and the P value significance of such changes in cells treated with CM. The horizontal dotted lines represented the P Z 0.05, and vertical dotted line represented the 2-fold change cut-offs, the red dots represented the selected ALOX15 gene (B, C). (D, E) HeLa cells were treated with CM or exosome (EXO) from PBMC or THP-1-derived M0, M2 macrophage and TAM for 48 h. ALOX15 mRNA levels was detected by RT-PCR. (F) HeLa cells were treated with CM or EXO from PBMC or THP-1-derived M0, M2 macrophage and TAM for 48 h. ALOX15 protein levels was detected by Western blot. The relative quantification of protein was analyzed by scanning densitometry using Image J software and b-actin was used as the loading control. (G) TAM (THP-1) and HeLa in combination or HeLa alone were inoculated subcutaneously in nude mice. And then, the ALOX15 and macrophage marker CD68 expression was detected by immunofluorescence. (H, I) HeLa and SiHa cells were transfected with ALOX15 siRNAs for 48 h. Cell death induced by ERA was detected (H). ALOX15 protein levels were detected by Western blot. The relative quantification of protein was analyzed by scanning densitometry using Image J software and b-actin was used as the loading control (I). (J, K) HeLa and SiHa cells were transfected with ALOX15 siRNAs and treated by TAM-CM for 48 h. Cell death induced by ERA was detected. Data represented the mean SEM of at least three times biological replicates. NS, not significant; ***P < 0.001, compared to control.

Article Snippet: For primary antibody, the anti-ALOX15 mouse monoclonal antibody (1:200, Novus Biologicals, NBP2-01740) was used.

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Derivative Assay, Western Blot, Software, Control, Marker, Expressing, Transfection

Journal: Acta pharmaceutica Sinica. B

Article Title: The suppression of cervical cancer ferroptosis by macrophages: The attenuation of ALOX15 in cancer cells by macrophages-derived exosomes.

doi: 10.1016/j.apsb.2023.03.025

Figure Lengend Snippet: Figure 4 TAMs-derived exosomal miR-660-5p attenuates ALOX15 expression to inhibit ferroptosis in cervical cancer cells. (A, B) Flowchart for the selection of ten miRNAs. (C) HeLa cells were transfected with the ten miRNAs mimics, and then ALOX15 mRNA levels were detected by RT-PCR. (D) The miR-660-5p levels were detected by RT-PCR in PBMC-derived monocytes (Mo), M0, TAM, M2, HeLa, SiHa cells and exosomes (EXO) from PBMC and THP-1-derived Mo, M0, TAM, M2. (E, F) HeLa cells were transfected with miR-660-5p inhibitor, and then treated with CM and EXO from M0 and TAM. ALOX15 mRNA levels were detected by RT-PCR (E). ALOX15 protein levels were detected by Western blot. The relative quantification of protein was analyzed by scanning densitometry using Image J software and b-actin was used as the loading control (F). (G) The predicted miR-660-5p target sites in the coding sequence (CDS) of ALOX15 mRNA and the mutated version. (H) HeLa cells were co-transfected with pGL3 vector containing the wild type (WT) or mutated (MT) CDS of ALOX15, or pGL3-control vector, along with miR-660-5p mimics and inhibitor. After 48 h, luciferase activity was detected. Data were normalized to luciferase activity in the corre- sponding cells transfected with NC. (I) HeLa and SiHa cells were transfected with miR-660-5p mimics and inhibitor. ALOX15 mRNA levels were detected by RT-PCR. (J) HeLa cells were co-transfected with pGL3 vector containing the WT or MT CDS of ALOX15, and then cells were treated with CM from THP-1 derived M0, M2 macrophages and TAM. After 48 h, luciferase activity was detected. (K, L) HeLa and SiHa cells were transfected with miR-660-5p inhibitor, and then treated with TAM-CM or TAM-EXO and indicated dose of ERA or RSL3 for 48 h. Cell death was

Article Snippet: For primary antibody, the anti-ALOX15 mouse monoclonal antibody (1:200, Novus Biologicals, NBP2-01740) was used.

Techniques: Derivative Assay, Expressing, Selection, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Software, Control, Sequencing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Acta pharmaceutica Sinica. B

Article Title: The suppression of cervical cancer ferroptosis by macrophages: The attenuation of ALOX15 in cancer cells by macrophages-derived exosomes.

doi: 10.1016/j.apsb.2023.03.025

Figure Lengend Snippet: Figure 6 ALOX15 is associated with macrophages infiltration and good prognosis. (A) Infiltration of immune cells including TAMs in cervical cancer tissues with high or low expression of ALOX15 by using ssGSEA and CIBERSORT. The relative numerical values corresponding to the height of the histogram indicated the different levels of abundance and the proportions. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001, high expression of ALOX15 compared to low expression. (B) Single-sample GSEA analysis identifying the relative infiltration of immune cell populations for 311 cervical cancer samples with available RNA-sequencing data in TCGA. The relative infiltration of each cell type is normalized into a z-score. (C, D) Association of ALOX15 mRNA expression with the disease-free survival (DFS) and overall survival (OS) in cervical cancer patients, respectively. These data of cervical cancer samples were acquired from TCGA. (E) Representative immunohistochemical staining for high and low expression of ALOX15 in human cervical cancer sample. (F) Association of ALOX15 protein expression with the OS in 147 cervical cancer patients.

Article Snippet: For primary antibody, the anti-ALOX15 mouse monoclonal antibody (1:200, Novus Biologicals, NBP2-01740) was used.

Techniques: Expressing, RNA Sequencing, Immunohistochemical staining, Staining

Journal: Acta pharmaceutica Sinica. B

Article Title: The suppression of cervical cancer ferroptosis by macrophages: The attenuation of ALOX15 in cancer cells by macrophages-derived exosomes.

doi: 10.1016/j.apsb.2023.03.025

Figure Lengend Snippet: Figure 7 Schematic diagram that TAM enhanced cervical cancer resistance to ferroptosis. A proposed model illustrating the role of TAM- derived exosomal miR-660-5p in regulating ALOX15-induced ferroptosis in cervical cancer cells.

Article Snippet: For primary antibody, the anti-ALOX15 mouse monoclonal antibody (1:200, Novus Biologicals, NBP2-01740) was used.

Techniques: Derivative Assay