Fig 1: Targeting PBX3 inhibits GBM invasion in vivo. a Representative images of Hematoxylin and eosin (H&E) staining of tissues from mice with orthotopic tumors derived from LV-siRNA-NC-U87 or LV-siRNA-PBX3-U87 cells. Scale bar = 500 µm. b Images of orthotopic tumors derived from LV-siRNA-NC-U87 or LV-siRNA-PBX3-U87 cells. c The tumors derived from LV-siRNA-NC-U87 or LV-siRNA-PBX3-U87 cells were weighed after imaging. **p < 0.01. d H&E staining and immunohistochemistry for PBX3, N-cadherin, ZEB1, Slug and CD44 in tumors derived from LV-siRNA-NC-U87 or LV-siRNA-PBX3-U87 cells. Scale bar = 50 µm. e Western blot analysis of PBX3, p-MEK, MEK, p-ERK1/2, ERK1/2, c-myc, LIN28, IL-6, p-STAT3, and STAT3 in tumors derived from LV-siRNA-NC-U87 or LV-siRNA-PBX3-U87 cells. f qRT-PCR analysis of let-7b expression in tumors derived from LV-siRNA-NC-U87 or LV-siRNA-PBX3-U87 cells. **p < 0.01
Fig 2: PBX3 promotes GBM mesenchymal transition, migration and invasion via activating MEK/ERK1/2 signaling pathway. a U87 and U251 cells stably expressing LV-PBX3 or LV-NC were treated with U0126 for 24 h and immunoblotting analysis of p-MEK, MEK, p-ERK1/2, ERK1/2, N-cadherin, ZEB1, Slug and CD44 were performed. b and c Quantification of wound-healing (b) and transwell (c) assays. **indicates a statistical significant difference (p < 0.01) between LV-NC + U0126 (-) group and LV-PBX3 + U0126 (-) group. $ indicates a statistical significant difference (p < 0.01) between LV-NC + U0126 (-) group and LV-NC + U0126 (+) group. # indicates a statistical significant difference (p < 0.01) between LV-NC + U0126 (+) group and LV-PBX3 + U0126 (+) group. d U87 and U251 cells stably expressing LV-siRNA-PBX3 or LV-siRNA-NC were treated with PMA for 24 h and immunoblotting analysis of p-MEK, MEK, p-ERK1/2, ERK1/2, N-cadherin, ZEB1, Slug and CD44 were performed. e and f Quantification of wound-healing (e) and transwell (f) assays. **indicates a statistical significant difference (p < 0.01) between LV-siRNA-NC + PMA (-) group and LV-siRNA-PBX3 + PMA (-) group. $ indicates a statistical significant difference (p < 0.01) between LV-siRNA-NC + PMA (-) group and LV-siRNA-NC + PMA (+) group. # indicates a statistical significant difference (p < 0.01) between LV-siRNA-NC + PMA (+) group and LV-siRNA-PBX3 + PMA (+) group
Fig 3: miR-224 inhibits PBX3 expression. (a) Information maps of miR-224 and PBX3 binding sites. (b) Dual luciferase reporter gene assay verified the combination of miR-224 and PBX3. (c) miRNA pull-down assay. (d) Verification of miR-224 expression. (e) miR-224 overexpression mimics inhibited the expression of PBX3. The content of PBX3 increased with the addition of the miR-224 inhibitor. (f) Correlation analysis of PBX3 and miR-224 coexpression. (g) Correlation analysis of circNBPF10 and PBX3 coexpression. (h) PBX3 expression in tumors detected by qRT-PCR. **P < 0.01.
Fig 4: PBX3 is a direct target of miR-653-5p. (a) The binding site between miR-653-5p and the 3'-UTR of PBX3. (b) The PBX3 expression in PTC cell lines was determined by qRT-PCR assay. (c) Luciferase reporter assay was conducted to investigate the connection between miR-653-5p and PBX3 in HEK-293T. (d) RIP assay was carried out to determine the relationship among miR-653-5p, circNRIP1, and PBX3. (e) The success of PBX3 inhibition with sh-PBX3 in PTC cells was confirmed by qRT-PCR analysis. (f) EdU incorporation assay was used to detect the cell proliferation. (g) Tunel staining was used to evaluate the apoptosis of tumor cells. (h, i) The cell migration and invasion abilities were detected with transwell assay. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 5: Let-7b directly targets PBX3 3'-UTR and forms a positive feedback loop. a Predicted binding sites of wild-type (WT) and mutant sequences of let-7b in the 3'-UTR of PBX3 mRNA. b Luciferase reporter assays were performed in U87 and U251 cells with co-transfection of indicated WT or mutant 3'-UTR constructs and pre-let-7b mimic or miR-NC mimic. **p < 0.01. c Western blot analysis of PBX3, p-MEK, MEK, p-ERK1/2, ERK1/2, c-myc, and LIN28 protein levels in let-7b overexpressing- or depleting-cells. d Western blot analysis of HMGA2, p-STAT3, and STAT3 protein levels in let-7b overexpressing- or depleting-cells and ELISA analysis of IL-6 protein levels in the supernatants of let-7b overexpressing- or depleting-cells. **p < 0.01. e qRT-PCR analysis of HMGA2 and IL-6 mRNA levels and western blot analysis of p-STAT3 and STAT3 protein levels in U87 and U251 cells transfected with LV-siRNA-NC or LV-siRNA-PBX3. f Pearson’s correlation analyses indicated that let-7b expression was negatively associated with PBX3, HMGA2 and IL-6 mRNA levels in GBM tissues. g Schematic diagram of the positive feedback loop involves PBX3, MEK/ERK1/2, c-myc and LIN28/let-7b
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