Journal: Bioactive Materials

Article Title: Remodeling of senescent macrophages in synovium alleviates trauma- and aging-induced osteoarthritis

doi: 10.1016/j.bioactmat.2025.09.016

Figure Lengend Snippet: Schematic illustration of the mechanisms of senotherapeutic nanoparticle in the treatment of OA. Based on the evidence that synovial macrophages in OA showed an increased expression of senescent biomarkers and functional decline, senotherapeutic nanoparticles containing pCQ and SOD termed as pCQ/SOD were prepared. Functionally, pCQ/SOD led to M1-to-M2 repolarization, reduced ROS-mediated mtDNA release and SASPs secretion to block cellular senescence, and rescued the impaired efferocytosis against apoptotic fibroblast-like synoviocytes (FLS) via STAT3/ADAM17/MerTK signaling to remodel local aging microenvironment. As a result, pCQ/SOD nanoparticles efficiently inhibited macrophage senescence and relieved aging-induced and trauma-induced OA in vivo .

Article Snippet: Raw264.7 cells were treated in different conditions and labeled with the following reagents for identification of M1 or M2 macrophages: PE/Cyanine5 anti-mouse CD86 Antibody (Biolegend, 105015), FITC anti-mouse CD206 (MMR) Antibody (Biolegend, 141703).

Techniques: Expressing, Functional Assay, Blocking Assay, In Vivo

Journal: Bioactive Materials

Article Title: Remodeling of senescent macrophages in synovium alleviates trauma- and aging-induced osteoarthritis

doi: 10.1016/j.bioactmat.2025.09.016

Figure Lengend Snippet: The effects of pCQ/SOD nanoparticles on macrophage senescence and polarization in vitro . (a) Representative images of Raw264.7 cells treated with pCQ, SOD or pCQ/SOD, respectively after 10 g/L D-gal induction for 24 h (Scale bar, 250 μm) (b) Relative quantifications of β-gal staining. (c) Transcriptive levels of p53 of Raw264.7 cells measured via qPCR. (d) Raw264.7 cells were treated with pCQ, SOD or pCQ/SOD, respectively after 10 g/L D-gal induction for 24 h, and then induced to M1-polarized macrophages via 100 ng/mL LPS. Representative images of iNOS (red) were shown. (Scale bar, 100 μm). (e) Relative quantifications of IF intensity of iNOS. (f) Transcriptive levels of iNOS of Raw264.7 cells measured via qPCR. (g) Raw264.7 cells were treated with pCQ, SOD or pCQ/SOD, respectively after 10 g/L D-gal induction for 24 h, and then induced to M2-polarized macrophages via 1000 U/mL IL4. Representative images of CD206 (green) were shown. (Scale bar, 100 μm). (h) Relative quantifications of IF intensity of CD206. (i) Transcriptive levels of CD206 of Raw264.7 cells measured via qPCR. (j) Flow cytometry and related quantification of CD86 and CD206 of macrophages. (k) KEGG enrichment of differentially expressed genes. (l) Heatmap of genes correlated with p53 signaling pathway and cellular senescence, and BAX-related genes in RNA-seq. (m) Schematic illustration of the regulatory effect of pCQ/SOD on polarization of senescent macrophages. Data are shown as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. p values were determined using ANOVA tests. The significant differences are determined by comparison with the senescence induction group.

Article Snippet: Raw264.7 cells were treated in different conditions and labeled with the following reagents for identification of M1 or M2 macrophages: PE/Cyanine5 anti-mouse CD86 Antibody (Biolegend, 105015), FITC anti-mouse CD206 (MMR) Antibody (Biolegend, 141703).

Techniques: In Vitro, Staining, Flow Cytometry, RNA Sequencing, Comparison

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: The polarization of M2 macrophages was higher in TNBC, which might be related to TCEB2 upregulation . TNBC tumor tissues and primary breast cancer tissues (non-TNBC), as well as paired adjacent normal tissues, were collected. (A) The mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tissues were determined by qRT-PCR( N = 40). (B) Western blot was employed to detect iNOS, CD80, Arg1, and CD163 protein levels in tissues ( N = 40). (C-D) The mRNA and protein levels of TCEB2 were determined by qRT-PCR and western blot, respectively ( N = 40). (E) The correlation between TCEB2 expression and the expressions of M2 macrophage cytokines (Arg1, VEGF, and IL-10), and the correlation between TCEB2 expression and the expressions of M1 macrophage cytokines (iNOS, IL-23, and IL-1β) were analyzed by Pearson correlation analysis ( N = 40). (F-G) The mRNA and protein levels of TCEB2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively ( N = 3). The measurement data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: As expected, the inhibitory effect of TCEB2 silencing on the ability of TNBC cells to induce M2 macrophage polarization was reversed by Slit2 knockdown.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 enhanced TNBC cell proliferation and invasion and promoted TNBC-induced M2 macrophage polarization . (A) qRT-PCR was performed to examine TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-TCEB2#1, sh-TCEB2#2, or sh-TCEB2#3 transfection. MDA-MB-231 and BT-549 cells were transfected with sh-NC or sh-TCEB2#3. (B-C) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (D-E) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. THP-1 cells were incubated with 100 ng/mL PMA for 24 h to obtain macrophages, and macrophages were then co-cultured with TNBC cells. (F-G) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (H) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: As expected, the inhibitory effect of TCEB2 silencing on the ability of TNBC cells to induce M2 macrophage polarization was reversed by Slit2 knockdown.

Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: 5. TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively. Both TCEB2 knockdown and Slit2 knockdown were induced in MDA-MB-231 and BT-549 cells. (C-D) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (E-F) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. (G) The level of Slit2 in conditioned media was detected using ELISA. (H-I) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (J) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: As expected, the inhibitory effect of TCEB2 silencing on the ability of TNBC cells to induce M2 macrophage polarization was reversed by Slit2 knockdown.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 knockdown inhibited TNBC-induced M2 macrophage polarization in vivo . The tumor implantation experiment was performed. (A-C) The size, volume, and weight of tumors were measured. (D) TCEB2 and Slit2 protein levels in tumor tissues were examined by western blot. (E) Ki67, CD80, and CD163 protein levels in tumor tissues were measured by IHC. (F) qRT-PCR was employed to determine the mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tumor tissues. The measurement data were presented as mean ± SD. N = 5. ** p < 0.01, *** p < 0.001.

Article Snippet: As expected, the inhibitory effect of TCEB2 silencing on the ability of TNBC cells to induce M2 macrophage polarization was reversed by Slit2 knockdown.

Techniques: Knockdown, In Vivo, Tumor Implantation, Western Blot, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: The polarization of M2 macrophages was higher in TNBC, which might be related to TCEB2 upregulation . TNBC tumor tissues and primary breast cancer tissues (non-TNBC), as well as paired adjacent normal tissues, were collected. (A) The mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tissues were determined by qRT-PCR( N = 40). (B) Western blot was employed to detect iNOS, CD80, Arg1, and CD163 protein levels in tissues ( N = 40). (C-D) The mRNA and protein levels of TCEB2 were determined by qRT-PCR and western blot, respectively ( N = 40). (E) The correlation between TCEB2 expression and the expressions of M2 macrophage cytokines (Arg1, VEGF, and IL-10), and the correlation between TCEB2 expression and the expressions of M1 macrophage cytokines (iNOS, IL-23, and IL-1β) were analyzed by Pearson correlation analysis ( N = 40). (F-G) The mRNA and protein levels of TCEB2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively ( N = 3). The measurement data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: • TCEB2 facilitated TNBC-induced M2 macrophage polarization by regulating Slit2.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 enhanced TNBC cell proliferation and invasion and promoted TNBC-induced M2 macrophage polarization . (A) qRT-PCR was performed to examine TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-TCEB2#1, sh-TCEB2#2, or sh-TCEB2#3 transfection. MDA-MB-231 and BT-549 cells were transfected with sh-NC or sh-TCEB2#3. (B-C) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (D-E) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. THP-1 cells were incubated with 100 ng/mL PMA for 24 h to obtain macrophages, and macrophages were then co-cultured with TNBC cells. (F-G) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (H) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: • TCEB2 facilitated TNBC-induced M2 macrophage polarization by regulating Slit2.

Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 ubiquitination degradation in TNBC . (A-B) The mRNA and protein levels of Slit2 in tissues were measured by qRT-PCR and western blot, respectively. (C-D) The mRNA and protein levels of Slit2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively. (E-F) The mRNA and protein levels of Slit2 in MDA-MB-231 and BT-549 cells after sh-NC or sh-TCEB2 transfection were measured by qRT-PCR and western blot, respectively. (G) Slit2 protein degradation in TNBC cells after sh-NC or sh-TCEB2 transfection was detected by CHX chase assay. (H) TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection was examined by qRT-PCR. (I) Western blot was performed to detect Slit2 protein in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection combined with MG132 treatment. (J) Slit2 ubiquitination level in tissues was measured by Slit2 ubiquitination analysis. (K) Slit2 ubiquitination level in MDA-MB-231 and BT-549 cells following sh-NC or sh-TCEB2 transfection combined with MG132 treatment was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: • TCEB2 facilitated TNBC-induced M2 macrophage polarization by regulating Slit2.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Western Blot, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 K63-ubiquitination degradation in TNBC by recruiting NEDD4 . (A) The interaction between TCEB2 and Slit2 in MDA-MB-231 or BT-549 cells was analyzed by Co-IP assay. (B) The E3 ubiquitination ligases acting on Slit2 were predicted using bioinformatics. (C-D) Co-IP assay was employed to analyze the interaction between NEDD4 and Slit2 and the interaction between NEDD4 and TCEB2. (E) qRT-PCR was performed to examine NEDD4 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-NEDD4#1, sh-NEDD4#2, or sh-NEDD4#3 transfection. (F) MDA-MB-231 and BT-549 cells were co-transfected with sh-NEDD4 and OE-TCEB2, and Slit2 ubiquitination level in cells was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. ** p < 0.01, *** p < 0.001.

Article Snippet: • TCEB2 facilitated TNBC-induced M2 macrophage polarization by regulating Slit2.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: 5. TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively. Both TCEB2 knockdown and Slit2 knockdown were induced in MDA-MB-231 and BT-549 cells. (C-D) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (E-F) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. (G) The level of Slit2 in conditioned media was detected using ELISA. (H-I) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (J) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: • TCEB2 facilitated TNBC-induced M2 macrophage polarization by regulating Slit2.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 knockdown inhibited TNBC-induced M2 macrophage polarization in vivo . The tumor implantation experiment was performed. (A-C) The size, volume, and weight of tumors were measured. (D) TCEB2 and Slit2 protein levels in tumor tissues were examined by western blot. (E) Ki67, CD80, and CD163 protein levels in tumor tissues were measured by IHC. (F) qRT-PCR was employed to determine the mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tumor tissues. The measurement data were presented as mean ± SD. N = 5. ** p < 0.01, *** p < 0.001.

Article Snippet: • TCEB2 facilitated TNBC-induced M2 macrophage polarization by regulating Slit2.

Techniques: Knockdown, In Vivo, Tumor Implantation, Western Blot, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: The polarization of M2 macrophages was higher in TNBC, which might be related to TCEB2 upregulation . TNBC tumor tissues and primary breast cancer tissues (non-TNBC), as well as paired adjacent normal tissues, were collected. (A) The mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tissues were determined by qRT-PCR( N = 40). (B) Western blot was employed to detect iNOS, CD80, Arg1, and CD163 protein levels in tissues ( N = 40). (C-D) The mRNA and protein levels of TCEB2 were determined by qRT-PCR and western blot, respectively ( N = 40). (E) The correlation between TCEB2 expression and the expressions of M2 macrophage cytokines (Arg1, VEGF, and IL-10), and the correlation between TCEB2 expression and the expressions of M1 macrophage cytokines (iNOS, IL-23, and IL-1β) were analyzed by Pearson correlation analysis ( N = 40). (F-G) The mRNA and protein levels of TCEB2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively ( N = 3). The measurement data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Collectively, TCEB2 upregulation promoted TNBC-induced M2 macrophage polarization by mediating Slit2 ubiquitination degradation.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 enhanced TNBC cell proliferation and invasion and promoted TNBC-induced M2 macrophage polarization . (A) qRT-PCR was performed to examine TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-TCEB2#1, sh-TCEB2#2, or sh-TCEB2#3 transfection. MDA-MB-231 and BT-549 cells were transfected with sh-NC or sh-TCEB2#3. (B-C) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (D-E) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. THP-1 cells were incubated with 100 ng/mL PMA for 24 h to obtain macrophages, and macrophages were then co-cultured with TNBC cells. (F-G) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (H) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Collectively, TCEB2 upregulation promoted TNBC-induced M2 macrophage polarization by mediating Slit2 ubiquitination degradation.

Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 ubiquitination degradation in TNBC . (A-B) The mRNA and protein levels of Slit2 in tissues were measured by qRT-PCR and western blot, respectively. (C-D) The mRNA and protein levels of Slit2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively. (E-F) The mRNA and protein levels of Slit2 in MDA-MB-231 and BT-549 cells after sh-NC or sh-TCEB2 transfection were measured by qRT-PCR and western blot, respectively. (G) Slit2 protein degradation in TNBC cells after sh-NC or sh-TCEB2 transfection was detected by CHX chase assay. (H) TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection was examined by qRT-PCR. (I) Western blot was performed to detect Slit2 protein in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection combined with MG132 treatment. (J) Slit2 ubiquitination level in tissues was measured by Slit2 ubiquitination analysis. (K) Slit2 ubiquitination level in MDA-MB-231 and BT-549 cells following sh-NC or sh-TCEB2 transfection combined with MG132 treatment was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Collectively, TCEB2 upregulation promoted TNBC-induced M2 macrophage polarization by mediating Slit2 ubiquitination degradation.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Western Blot, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 K63-ubiquitination degradation in TNBC by recruiting NEDD4 . (A) The interaction between TCEB2 and Slit2 in MDA-MB-231 or BT-549 cells was analyzed by Co-IP assay. (B) The E3 ubiquitination ligases acting on Slit2 were predicted using bioinformatics. (C-D) Co-IP assay was employed to analyze the interaction between NEDD4 and Slit2 and the interaction between NEDD4 and TCEB2. (E) qRT-PCR was performed to examine NEDD4 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-NEDD4#1, sh-NEDD4#2, or sh-NEDD4#3 transfection. (F) MDA-MB-231 and BT-549 cells were co-transfected with sh-NEDD4 and OE-TCEB2, and Slit2 ubiquitination level in cells was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. ** p < 0.01, *** p < 0.001.

Article Snippet: Collectively, TCEB2 upregulation promoted TNBC-induced M2 macrophage polarization by mediating Slit2 ubiquitination degradation.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: 5. TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively. Both TCEB2 knockdown and Slit2 knockdown were induced in MDA-MB-231 and BT-549 cells. (C-D) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (E-F) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. (G) The level of Slit2 in conditioned media was detected using ELISA. (H-I) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (J) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Collectively, TCEB2 upregulation promoted TNBC-induced M2 macrophage polarization by mediating Slit2 ubiquitination degradation.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 knockdown inhibited TNBC-induced M2 macrophage polarization in vivo . The tumor implantation experiment was performed. (A-C) The size, volume, and weight of tumors were measured. (D) TCEB2 and Slit2 protein levels in tumor tissues were examined by western blot. (E) Ki67, CD80, and CD163 protein levels in tumor tissues were measured by IHC. (F) qRT-PCR was employed to determine the mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tumor tissues. The measurement data were presented as mean ± SD. N = 5. ** p < 0.01, *** p < 0.001.

Article Snippet: Collectively, TCEB2 upregulation promoted TNBC-induced M2 macrophage polarization by mediating Slit2 ubiquitination degradation.

Techniques: Knockdown, In Vivo, Tumor Implantation, Western Blot, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: The polarization of M2 macrophages was higher in TNBC, which might be related to TCEB2 upregulation . TNBC tumor tissues and primary breast cancer tissues (non-TNBC), as well as paired adjacent normal tissues, were collected. (A) The mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tissues were determined by qRT-PCR( N = 40). (B) Western blot was employed to detect iNOS, CD80, Arg1, and CD163 protein levels in tissues ( N = 40). (C-D) The mRNA and protein levels of TCEB2 were determined by qRT-PCR and western blot, respectively ( N = 40). (E) The correlation between TCEB2 expression and the expressions of M2 macrophage cytokines (Arg1, VEGF, and IL-10), and the correlation between TCEB2 expression and the expressions of M1 macrophage cytokines (iNOS, IL-23, and IL-1β) were analyzed by Pearson correlation analysis ( N = 40). (F-G) The mRNA and protein levels of TCEB2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively ( N = 3). The measurement data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 enhanced TNBC cell proliferation and invasion and promoted TNBC-induced M2 macrophage polarization . (A) qRT-PCR was performed to examine TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-TCEB2#1, sh-TCEB2#2, or sh-TCEB2#3 transfection. MDA-MB-231 and BT-549 cells were transfected with sh-NC or sh-TCEB2#3. (B-C) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (D-E) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. THP-1 cells were incubated with 100 ng/mL PMA for 24 h to obtain macrophages, and macrophages were then co-cultured with TNBC cells. (F-G) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (H) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively.

Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 ubiquitination degradation in TNBC . (A-B) The mRNA and protein levels of Slit2 in tissues were measured by qRT-PCR and western blot, respectively. (C-D) The mRNA and protein levels of Slit2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively. (E-F) The mRNA and protein levels of Slit2 in MDA-MB-231 and BT-549 cells after sh-NC or sh-TCEB2 transfection were measured by qRT-PCR and western blot, respectively. (G) Slit2 protein degradation in TNBC cells after sh-NC or sh-TCEB2 transfection was detected by CHX chase assay. (H) TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection was examined by qRT-PCR. (I) Western blot was performed to detect Slit2 protein in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection combined with MG132 treatment. (J) Slit2 ubiquitination level in tissues was measured by Slit2 ubiquitination analysis. (K) Slit2 ubiquitination level in MDA-MB-231 and BT-549 cells following sh-NC or sh-TCEB2 transfection combined with MG132 treatment was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Western Blot, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 K63-ubiquitination degradation in TNBC by recruiting NEDD4 . (A) The interaction between TCEB2 and Slit2 in MDA-MB-231 or BT-549 cells was analyzed by Co-IP assay. (B) The E3 ubiquitination ligases acting on Slit2 were predicted using bioinformatics. (C-D) Co-IP assay was employed to analyze the interaction between NEDD4 and Slit2 and the interaction between NEDD4 and TCEB2. (E) qRT-PCR was performed to examine NEDD4 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-NEDD4#1, sh-NEDD4#2, or sh-NEDD4#3 transfection. (F) MDA-MB-231 and BT-549 cells were co-transfected with sh-NEDD4 and OE-TCEB2, and Slit2 ubiquitination level in cells was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. ** p < 0.01, *** p < 0.001.

Article Snippet: TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: 5. TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively. Both TCEB2 knockdown and Slit2 knockdown were induced in MDA-MB-231 and BT-549 cells. (C-D) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (E-F) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. (G) The level of Slit2 in conditioned media was detected using ELISA. (H-I) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (J) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 knockdown inhibited TNBC-induced M2 macrophage polarization in vivo . The tumor implantation experiment was performed. (A-C) The size, volume, and weight of tumors were measured. (D) TCEB2 and Slit2 protein levels in tumor tissues were examined by western blot. (E) Ki67, CD80, and CD163 protein levels in tumor tissues were measured by IHC. (F) qRT-PCR was employed to determine the mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tumor tissues. The measurement data were presented as mean ± SD. N = 5. ** p < 0.01, *** p < 0.001.

Article Snippet: TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively.

Techniques: Knockdown, In Vivo, Tumor Implantation, Western Blot, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: The polarization of M2 macrophages was higher in TNBC, which might be related to TCEB2 upregulation . TNBC tumor tissues and primary breast cancer tissues (non-TNBC), as well as paired adjacent normal tissues, were collected. (A) The mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tissues were determined by qRT-PCR( N = 40). (B) Western blot was employed to detect iNOS, CD80, Arg1, and CD163 protein levels in tissues ( N = 40). (C-D) The mRNA and protein levels of TCEB2 were determined by qRT-PCR and western blot, respectively ( N = 40). (E) The correlation between TCEB2 expression and the expressions of M2 macrophage cytokines (Arg1, VEGF, and IL-10), and the correlation between TCEB2 expression and the expressions of M1 macrophage cytokines (iNOS, IL-23, and IL-1β) were analyzed by Pearson correlation analysis ( N = 40). (F-G) The mRNA and protein levels of TCEB2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively ( N = 3). The measurement data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The current study found that TCEB2 promoted M2 macrophage polarization in TNBC by facilitating ubiquitination degradation of Slit2 through recruiting NEDD4.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 enhanced TNBC cell proliferation and invasion and promoted TNBC-induced M2 macrophage polarization . (A) qRT-PCR was performed to examine TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-TCEB2#1, sh-TCEB2#2, or sh-TCEB2#3 transfection. MDA-MB-231 and BT-549 cells were transfected with sh-NC or sh-TCEB2#3. (B-C) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (D-E) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. THP-1 cells were incubated with 100 ng/mL PMA for 24 h to obtain macrophages, and macrophages were then co-cultured with TNBC cells. (F-G) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (H) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The current study found that TCEB2 promoted M2 macrophage polarization in TNBC by facilitating ubiquitination degradation of Slit2 through recruiting NEDD4.

Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 ubiquitination degradation in TNBC . (A-B) The mRNA and protein levels of Slit2 in tissues were measured by qRT-PCR and western blot, respectively. (C-D) The mRNA and protein levels of Slit2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively. (E-F) The mRNA and protein levels of Slit2 in MDA-MB-231 and BT-549 cells after sh-NC or sh-TCEB2 transfection were measured by qRT-PCR and western blot, respectively. (G) Slit2 protein degradation in TNBC cells after sh-NC or sh-TCEB2 transfection was detected by CHX chase assay. (H) TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection was examined by qRT-PCR. (I) Western blot was performed to detect Slit2 protein in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection combined with MG132 treatment. (J) Slit2 ubiquitination level in tissues was measured by Slit2 ubiquitination analysis. (K) Slit2 ubiquitination level in MDA-MB-231 and BT-549 cells following sh-NC or sh-TCEB2 transfection combined with MG132 treatment was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The current study found that TCEB2 promoted M2 macrophage polarization in TNBC by facilitating ubiquitination degradation of Slit2 through recruiting NEDD4.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Western Blot, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 K63-ubiquitination degradation in TNBC by recruiting NEDD4 . (A) The interaction between TCEB2 and Slit2 in MDA-MB-231 or BT-549 cells was analyzed by Co-IP assay. (B) The E3 ubiquitination ligases acting on Slit2 were predicted using bioinformatics. (C-D) Co-IP assay was employed to analyze the interaction between NEDD4 and Slit2 and the interaction between NEDD4 and TCEB2. (E) qRT-PCR was performed to examine NEDD4 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-NEDD4#1, sh-NEDD4#2, or sh-NEDD4#3 transfection. (F) MDA-MB-231 and BT-549 cells were co-transfected with sh-NEDD4 and OE-TCEB2, and Slit2 ubiquitination level in cells was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. ** p < 0.01, *** p < 0.001.

Article Snippet: The current study found that TCEB2 promoted M2 macrophage polarization in TNBC by facilitating ubiquitination degradation of Slit2 through recruiting NEDD4.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: 5. TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively. Both TCEB2 knockdown and Slit2 knockdown were induced in MDA-MB-231 and BT-549 cells. (C-D) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (E-F) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. (G) The level of Slit2 in conditioned media was detected using ELISA. (H-I) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (J) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The current study found that TCEB2 promoted M2 macrophage polarization in TNBC by facilitating ubiquitination degradation of Slit2 through recruiting NEDD4.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 knockdown inhibited TNBC-induced M2 macrophage polarization in vivo . The tumor implantation experiment was performed. (A-C) The size, volume, and weight of tumors were measured. (D) TCEB2 and Slit2 protein levels in tumor tissues were examined by western blot. (E) Ki67, CD80, and CD163 protein levels in tumor tissues were measured by IHC. (F) qRT-PCR was employed to determine the mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tumor tissues. The measurement data were presented as mean ± SD. N = 5. ** p < 0.01, *** p < 0.001.

Article Snippet: The current study found that TCEB2 promoted M2 macrophage polarization in TNBC by facilitating ubiquitination degradation of Slit2 through recruiting NEDD4.

Techniques: Knockdown, In Vivo, Tumor Implantation, Western Blot, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: The polarization of M2 macrophages was higher in TNBC, which might be related to TCEB2 upregulation . TNBC tumor tissues and primary breast cancer tissues (non-TNBC), as well as paired adjacent normal tissues, were collected. (A) The mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tissues were determined by qRT-PCR( N = 40). (B) Western blot was employed to detect iNOS, CD80, Arg1, and CD163 protein levels in tissues ( N = 40). (C-D) The mRNA and protein levels of TCEB2 were determined by qRT-PCR and western blot, respectively ( N = 40). (E) The correlation between TCEB2 expression and the expressions of M2 macrophage cytokines (Arg1, VEGF, and IL-10), and the correlation between TCEB2 expression and the expressions of M1 macrophage cytokines (iNOS, IL-23, and IL-1β) were analyzed by Pearson correlation analysis ( N = 40). (F-G) The mRNA and protein levels of TCEB2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively ( N = 3). The measurement data were presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In addition, the treatment of exogenous recombinant Slit2 protein significantly promoted M2 macrophage polarization ( Fig. S2B-C ).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 enhanced TNBC cell proliferation and invasion and promoted TNBC-induced M2 macrophage polarization . (A) qRT-PCR was performed to examine TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-TCEB2#1, sh-TCEB2#2, or sh-TCEB2#3 transfection. MDA-MB-231 and BT-549 cells were transfected with sh-NC or sh-TCEB2#3. (B-C) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (D-E) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. THP-1 cells were incubated with 100 ng/mL PMA for 24 h to obtain macrophages, and macrophages were then co-cultured with TNBC cells. (F-G) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (H) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In addition, the treatment of exogenous recombinant Slit2 protein significantly promoted M2 macrophage polarization ( Fig. S2B-C ).

Techniques: Quantitative RT-PCR, Transfection, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 ubiquitination degradation in TNBC . (A-B) The mRNA and protein levels of Slit2 in tissues were measured by qRT-PCR and western blot, respectively. (C-D) The mRNA and protein levels of Slit2 in normal breast epithelial cells (MCF-10A cells) and human breast cancer cell lines (MCF-7, SK-BR-3, BT-549, MDA-MB-231, and MDA-MB-468 cells) were measured by qRT-PCR and western blot, respectively. (E-F) The mRNA and protein levels of Slit2 in MDA-MB-231 and BT-549 cells after sh-NC or sh-TCEB2 transfection were measured by qRT-PCR and western blot, respectively. (G) Slit2 protein degradation in TNBC cells after sh-NC or sh-TCEB2 transfection was detected by CHX chase assay. (H) TCEB2 mRNA level in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection was examined by qRT-PCR. (I) Western blot was performed to detect Slit2 protein in MDA-MB-231 and BT-549 cells following OE-NC or OE-TCEB2 transfection combined with MG132 treatment. (J) Slit2 ubiquitination level in tissues was measured by Slit2 ubiquitination analysis. (K) Slit2 ubiquitination level in MDA-MB-231 and BT-549 cells following sh-NC or sh-TCEB2 transfection combined with MG132 treatment was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In addition, the treatment of exogenous recombinant Slit2 protein significantly promoted M2 macrophage polarization ( Fig. S2B-C ).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Western Blot, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 mediated Slit2 K63-ubiquitination degradation in TNBC by recruiting NEDD4 . (A) The interaction between TCEB2 and Slit2 in MDA-MB-231 or BT-549 cells was analyzed by Co-IP assay. (B) The E3 ubiquitination ligases acting on Slit2 were predicted using bioinformatics. (C-D) Co-IP assay was employed to analyze the interaction between NEDD4 and Slit2 and the interaction between NEDD4 and TCEB2. (E) qRT-PCR was performed to examine NEDD4 mRNA level in MDA-MB-231 and BT-549 cells following sh-NC, sh-NEDD4#1, sh-NEDD4#2, or sh-NEDD4#3 transfection. (F) MDA-MB-231 and BT-549 cells were co-transfected with sh-NEDD4 and OE-TCEB2, and Slit2 ubiquitination level in cells was detected using Slit2 ubiquitination analysis. The measurement data were presented as mean ± SD. N = 3. ** p < 0.01, *** p < 0.001.

Article Snippet: In addition, the treatment of exogenous recombinant Slit2 protein significantly promoted M2 macrophage polarization ( Fig. S2B-C ).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Transfection

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: 5. TCEB2 promoted TNBC-induced M2 macrophage polarization by acting on Slit2 . (A-B) The mRNA and protein levels of Slit2 in MDA-MB-231 or BT-549 cells after sh-NC, sh-Slit2#1, sh-Slit2#2, or sh-Slit2#3 transfection were measured by qRT-PCR and western blot, respectively. Both TCEB2 knockdown and Slit2 knockdown were induced in MDA-MB-231 and BT-549 cells. (C-D) Cell viability and proliferation were analyzed by CCK8 assay and colony formation assay, respectively. (E-F) Cell migration and invasion were determined by wound healing assay and Transwell assay, respectively. (G) The level of Slit2 in conditioned media was detected using ELISA. (H-I) qRT-PCR and ELISA were adopted to measure the mRNA and secretion levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in macrophages. (J) CD163 and CD80 levels in macrophages were analyzed by flow cytometry. The measurement data were presented as mean ± SD. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In addition, the treatment of exogenous recombinant Slit2 protein significantly promoted M2 macrophage polarization ( Fig. S2B-C ).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Knockdown, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Transwell Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Translational Oncology

Article Title: TCEB2 promotes M2 polarization of macrophages in triple negative breast cancer by mediating ubiquitination degradation of Slit2 through recruiting NEDD4

doi: 10.1016/j.tranon.2025.102536

Figure Lengend Snippet: TCEB2 knockdown inhibited TNBC-induced M2 macrophage polarization in vivo . The tumor implantation experiment was performed. (A-C) The size, volume, and weight of tumors were measured. (D) TCEB2 and Slit2 protein levels in tumor tissues were examined by western blot. (E) Ki67, CD80, and CD163 protein levels in tumor tissues were measured by IHC. (F) qRT-PCR was employed to determine the mRNA levels of iNOS, IL-23, IL-1β, Arg1, VEGF, and IL-10 in tumor tissues. The measurement data were presented as mean ± SD. N = 5. ** p < 0.01, *** p < 0.001.

Article Snippet: In addition, the treatment of exogenous recombinant Slit2 protein significantly promoted M2 macrophage polarization ( Fig. S2B-C ).

Techniques: Knockdown, In Vivo, Tumor Implantation, Western Blot, Quantitative RT-PCR