Fig 2: MSI2a binds to TP53INP1 mRNA and increases TP53INP1 mRNA stability and protein expression. a Schematic map of construction of the 3′-UTR reporter constructs for TP53INP1 that were used for a dual-luciferase assay. TP53INP1 mRNA stability curves plotted using qPCR expression versus time. b-d Luciferase reporter assay. (b) MDA-MB-231 cells transfected with MSI2a plasmids and (c) Hs-578 T cells transfected with MSI2a siRNA were treated with actinomycin D (5 mg/mL) for the indicated periods of time. (d) MDA-MB-231 cells were cotransfected with MSI2-overexpressing plasmids, Renilla luciferase 3′-UTR constructs, and a control firefly luciferase control vector. e Hs-578 T cells were cotransfected with MSI2a-specific siRNAs, Renilla luciferase 3′-UTR constructs, and a control firefly luciferase control vector. f Luciferase reporter assay. MDA-MB-231 and Hs-578 T cells were cotransfected with MSI2a-overexpressing plasmids, Renilla luciferase, 3′-UTR constructs, and a control firefly luciferase control vector. Renilla luciferase activity following MSI2a overexpression was determined. g Luciferase reporter assay. Schematic map of construction of the MSI2a-Mut plasmids. MDA-MB-231 cells were cotransfected with MSI2a or MSI2a-Mut plasmids, Renilla luciferase, 3′-UTR constructs, and a control firefly luciferase control vector. Renilla luciferase activity was determined. The data were normalized to firefly luciferase activity. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3: Western blot analysis of OXTR, APAF1, and TP53INP1 in cells incubated with endosulfan for 24 and 48 h. The numbers under bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present data from one of three independent experiments (which yielded similar results).

Fig 4: (A) Relative levels of TP53INP1 mRNA and of miRNAs targeting it (miR-193b and miR-342) in MCF-7 cells exposed to o,p'-DDT. The Y-axis shows the ratio of the mRNA or miRNA amount in the cells treated with a DDT isomer to the mRNA or miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05). (B) Western blot analysis of TP53INP1 in cells incubated with o,p'-DDT. The numbers under TP53INP1 bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present findings from one of three independent experiments (which yielded similar results).

Fig 5: MSI2a inhibited TNBC progression in a TP53INP1-mediated manner. a Wound-healing assays. Representative images and quantification of the assay results show the wound-healing capacity of MSI2a-overexpressing MDA-MB-231 cells transfected with siTP53INP1 or MSI2a-knockdown Hs-578T cells transfected with TP53INP1 plasmids, respectively. Scale bar, 200 µm. b Representative images and quantification of the number of migrated cells of MDA-MB-231 cells coexpressing empty vector or MSI2a and control siRNA or siTP53INP1 as well as Hs-578T cells coexpressing control siRNA or siMSI2a and empty vector or TP53INP1. Scale bar, 100 µm. c MSI2a-overexpressing MDA-MB-231 cells and MSI2a-knockdown BT20 cells were analyzed by Western blot with anti-MSI2, anti-TP53INP1, anti-P73, anti-DUSP10, anti-ERK, and anti-p-ERK antibodies. Vinculin was used as a control. d MDA-MB-231 cells coexpressing empty vector or MSI2a and control siRNA or siTP53INP1 were analyzed by Western blot with anti-MSI2a, anti-TP53INP1, anti-ERK, and anti-p-ERK antibodies. Vinculin was used as a control. *p < 0.05