Fig 1: 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.
Fig 2: 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.
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