Fig 1: Overexpression of CIAPIN1 in breast cancer cells promotes cell proliferation. MCF7 and MDA-MB-468 cells were transfected with pcDNA3.1-CIAPIN1 or vector, and were cultured for 12, 24 48 and 72 h. (A, B) The cell viability was determined by MTT assay in MCF7 and MDA-MB-468 cells, respectively. (C) 5-ethynyl-20-deoxyuridine (EdU) assay was used to assess cell proliferation of MCF7 and MDA-MB-468 cells at 24 h after transfection. (D) Quantification of EdU-positive cells relative to DAPI-positive cells. Data are presented as mean ± SD in triplicates and analyzed using the Student t-test. ***P < 0.01 vs. control group.
Fig 2: Inhibition of CIAPIN1 suppresses glycolysis in breast cancer cells through STAT3/PKM2 pathway. The energy metabolism by inhibition of CIAPIN1 was measured in the lysate of MDA-MB-231 and MDA-MB-453 cells on (A) pyruvate, (B) lactate and (C) ATP. (D) Representative gel blots depicting levels of PKM2 (normalized to GAPDH) and phosphorylated STAT3 (p-STAT3, normalized to total STAT3). Quantification analysis shows that Inhibition of CIAPIN1 reduced the PKM2 protein expression and phosphorylation of STAT3 (E, F). Data represent the average of three independent experiments (mean ± SD). ***P < 0.001 vs. si-NC group.
Fig 3: Inhibition of CIAPIN1 in breast cancer cells by siRNA suppresses cell proliferation. (A) Three siRNAs targeting CIAPIN1 mRNA (siRNA-87, siRNA-299 and siRNA-505) were introduced into MDA-MB-231 and MDA-MB-453 cells for transient knockdown of CIAPIN1. (A, B) The levels of CIAPIN1 mRNA were detected by RT-qPCR in MDA-MB-231 and MDA-MB-453 cells at 48 h after transfection. (C, D) Western blotting was performed to determine the levels of CIAPIN1 protein in MDA-MB-231 and MDA-MB-453 cells. The expression of CIAPIN1 was normalized against endogenous GAPDH levels. (E) Cell viability was suppressed by CIAPIN1 knockdown in MDA-MB-231 and MDA-MB-453 cells detected by MTT assay 72 h after transfection. Data represent the average of three independent experiments (mean ± SD), and analyzed using ANOVA, followed by the Bonferroni post hoc test for multiple-group comparison. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group or si-NC group.
Fig 4: Expression of CIAPIN1 in breast cancer cells. (A) The mRNA expression of CIAPIN1 in four breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-453, and MDA-MB-468) by RT‑qPCR. (B) The representative bands of CIAPIN1 protein in these four breast cancer cell lines. MCF7 and MDA-MB-468 cells were transfected with pcDNA3.1-CIAPIN1 or vector. (C) The levels of CIAPIN1 mRNA were detected by RT-qPCR in MCF7 and MDA-MB-468 at 48 h after transfection. (D) The levels of CIAPIN1 protein were detected by Western blot, and representative gel blots of CIAPIN1 are shown. Data are presented as mean ± SD and analyzed using the T-test. ***P < 0.01 vs. control group.
Fig 5: Inhibition of CIAPIN1 enhances cellular ROS production and oxidative stress in breast cancer cells. (A) Representative images of DHE staining of MDA-MB-231 and MDA-MB-453 cells. (B) Quantification of DHE-positive cells relative to DAPI-positive cells. Three oxidative stress indicators were measured in the lysate of MDA-MB-231 and MDA-MB-453 cells using the colourimetric method, including (C) MDA, (D) SOD and (E) CAT. Data represent the average of three independent experiments (mean ± SD), and analyzed using Student t test.. ***P < 0.001 vs. si-NC group.
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