Fig 1: Cyclin D1, E1, and A2 are involved in MGB1‐regulated aggressiveness in resistant cells. (A) Representative western blots (left) and quantification (right) of cyclin D1, cyclin E1, cyclin A2, and α‐tubulin (loading control) in resistant cells after siCyclin D1 (siD1), siCyclin E1 (siE1), and siCyclin A2 (siA2) transfection relative to the negative control (siNC). N = 3 independent experiments, once per experiment. (B) Cell viability. Bar represents mean with SEM, N = 3 independent experiments, thrice for each experiment, *P value <0.05 unpaired Welch's t‐test. (C) Representative figure of migration and invasion assay with 20× magnification. Scale bar of 100 μm. (D) Quantification of migration. N = 3 independent experiments, thrice for each experiment, and (E) invasion of resistant cells after transfection with siD1, siE1, and siA2 relative to the negative control (siNC). The bar represents the mean with SEM, N = 3 independent experiments, thrice for each experiment. *Statistical significance of figure A, D, and E was determined with 95% confidence interval.
Fig 2: MGB1 is upregulated in resistant cells and is critical for their aggressiveness. (A) The top 10 upregulated genes (mRNA level) of resistant cells relative to wild‐type cells in the microarray assay. N = 1, experiment. (B) Comparison of the MGB1 mRNA level in SK‐BR‐3 (wild‐type cells, HER2 positive/ER negative), SK‐BR‐3 R (resistant cells), MCF7 (HER2 negative/ER positive), and MDA‐MB‐231 (HER2 negative/ER negative) cells. The bar represents mean with SEM, N = 3 independent experiments, thrice for each experiment, *statistical significance with CI of 95%. (C) Representative western blots (left) and quantification (right) of MGB1 and α‐tubulin (loading control) in resistant cells relative to wild‐type cells. The bar represents mean with SEM, N = 3, once per experiment, *statistical significance with CI of 95%. (D) Cell viability, the bar represents mean with SEM, N = 3 independent experiments, thrice for each experiment, *P < 0.05, unpaired Welch's t‐test. (E) Migration, and (F) invasion of resistant cells after transfection with siMGB 1 relative to the negative control (siNC). The bar represents mean with SEM, N = 3 independent experiments, thrice for each experiment, *statistical significance with CI of 95%. Scale bar of 100 μm. (G) Viability of resistant cells after transfection with siMGB1 or siNC followed by trastuzumab treatment (50 μg·mL −1 for 4 days). The bar represents mean with SEM, N = 2 independent experiments, thrice per experiment with *P value < 0.05 unpaired Student's t‐test.
Fig 3: Schematic depicting how MGB1 regulates the progression of resistant cells through cyclin and NF‐κB regulation. The surviving cells after long‐term treatment with trastuzumab showed resistance. MGB1 is upregulated in resistant cells and regulates progression (cell proliferation, migration, and invasion ability; proliferation is critical for cell viability) through the induction of NF‐κB‐dependent cyclin D1 expression and cyclin E1 expression. Cyclin D1 and E1 expression affected cyclin A2 expression.
Fig 4: MGB1 does not regulate apoptosis and senescence cells but p‐p65 expression. (A) Representative images (left) of senescence cells (red arrow), and their quantification (number of senescence cells per 100 cells) 4 and 5 days posttransfection (right). Scale bar of 100 μm. (B) Representative western blots (left), and quantification (right) of caspase 3, cleaved caspase 3, and α‐tubulin (loading control) expression in resistant cells after siMGB1 transfection, relative to that of the negative control (siNC). (C) Graph bars showing the percentage of living cells and dead cells in wild‐type and the resistant group treated with siMGB1. (D) Representative western blots (left) and quantification (right) of p‐p65 and GAPDH (loading control) expression in resistant cells relative to that of wild‐type cells. (E) p‐p65 and GAPDH (loading control) expression in resistant cells after siMGB1 transfection, relative to that of the negative control (siNC). All statistical analysis in this figure is shown as the bar represents the mean with SEM, N = 3 independent experiments, once for each experiment. *Statistical significance was determined with 95% confidence interval.
Fig 5: MGB1 regulates cyclin expression in resistant cells but not in wild‐type cells overexpressing MGB1. (A) Representative western blots (left) and quantification (right) of cyclin D1, cyclin E1, cyclin A2, and α‐tubulin (loading control) in resistant cells relative to wild‐type cells. (B) Representative western blots (left) and quantification (right) of cyclin D1, cyclin E1, cyclin A2, cyclin B1, p‐histone, MGB1, and α‐tubulin (loading control) in resistant cells after siMGB1 transfection relative to negative control (siNC). (C) Representative images of western blots (left) and quantification (right) of cyclins and GAPDH (loading control) in untreated wild‐type cells (WT UT), wild‐type cells with empty vector (WT‐EV), wild‐type cells with MGB1 overexpression (WT‐MGB1ox), and resistant cells (R). All statistical analysis in this figure is shown as the bar represents the mean with SEM, N = 3 independent experiments, once for each experiment. *Statistical significance was determined with 95% confidence interval.
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