Fig 1.
Expression of MYO1C in human embryonic kidney HEK-293, cervical cancer HeLa, and normal breast epithelium MCF10A cell lines.
Immunoblot of MYO1C protein was performed in embryonic non-cancerous HEK-293, adult non-cancerous MCF10A and adult cancerous (HeLa) cell lines. HeLa and HEK-293 cells transfected with MYO1C expression construct were included as positive control and also to examine the specificity of the MYO1C antibody. De novo expression of MYO1C was found to be low in the embryonic HEK-293 and the cancerous HeLa cells, and high in the normal breast epithelial MCF10A cells. Three independent experiments were performed and the figure shows a representative of these repeats.
Table 1.
List of primary antibodies and the optimized dilution.
Fig 2.
Protein levels of MYO1C in endometrial hyperplasia and endometrial adenocarcinomas of stage I-III.
a) Representative images for the IHC staining of MYO1C in tissue samples of endometrial carcinoma with low, medium and high MYUO1C protein levels. b) Significant association was found between MYO1C protein level and tumor grade (P = 0.035, linear-by-linear association in the SPSS chi-square test). The proportion of samples with high MYO1C protein level was greatest in hyperplasias (Hyp) and smallest in stage III tumors, whereas the proportion of tumors with low abundance of MYOC1 was smallest in hyperplasias and highest in stage III tumors. Pairwise differences between sample groups were not significant. c) When the samples were regrouped as tumors and hyperplasia, the Fisher’s exact test showed significant difference in MYO1C expression between these two groups of samples (P = 0.0303).
Fig 3.
Over-expression of MYO1C significantly decreased viability of HEK-293 cells.
(a) Immunoblot of MYO1C proteins in HEK-293 cells shows increased expression in the cells transfected with different amounts of MYO1C-construct at 48 hours post-transfection in comparison with the cells transfected with corresponding empty plasmid, serving as control. The image is a representative from at least three independent experiments. (b) Dose-response effect of over-expression of MYO1C protein on cell proliferation was observed at 48 hours post-transfection in comparison with cells transfected with empty vector. Each bar represents mean value ± SEM of all repeats, a Student’s t-test was used to compare the differences in mean values, *P-value < 0.05 versus the corresponding empty plasmid. (c) Immunoblot of MYO1C proteins in HEK-293 cells shows increased expression in MYO1C-transfected cells at 24, 48, 72 and 96 hours post-transfection. The image is a representative from at least three independent experiments. (d) Relative number of living cells was reduced in MYO1C-transfected cells. Significant decline was observed at 48, 72 and 96 hours post-transfection compared to cells transfected with the empty vector. Each bar represents mean value ± SEM of all repeats (at least three independent experiments, each in quadruplicates). A Student’s t-test was used to compare the differences in mean values, ****P-value < 0.001 versus the corresponding empty plasmid.
Fig 4.
siRNA-knockdown of MYO1C enhanced viability.
(a) Immunoblot of MYO1C protein expression in MCF10A cells transfected with MYO1C-siRNA and negative control (scrambled siRNA) to the final concentration of 10nM at 24, 48, 72 and 96 hours post-transfection. The image is a representative from at least three independent experiments. (b) Histogram showing percentages of relative cell viability of MCF10A cells transfected with MYO1C-siRNA and scrambled siRNA to the final concentration of 10nM. Significant increase in the number of live cells was observed at 48, 72 and 96 hours post-transfection compared to cells transfected with the scrambled siRNA. Each bar represents mean value ± SEM of all repeats (at least three independent experiments, each in quadruplicates). A Student’s t-test was used to compare the differences in mean value, ****P-value < 0.001 versus cells treated with scrambled siRNA as well as untreated cells (time point zero).
Fig 5.
Decrease in cell migration and adhesion of MCF10A cells after knockdown of MYO1C.
(a) The expression levels of MYO1C in the MCF10A cells subjected to 20 nM siRNA transfection were verified with RT-PCR. (b) Geometry on gap immediately after ripping of the stopper (I) and geometry on gap of the control (scrambled siRNA) (II), and MYO1C-siRNA transfected (III) after 24-hour incubation were shown. The white circles show the area of the geometry on gap at each measurement. (c) The analyses of the geometry on gap closure (migration assay) of MCF10A cells transfected with MYO1C-specific siRNA was evaluated after ripping off cell seeding stopper followed by 24-hour incubation in comparison to the geometry of control cells. (d) Immunoblot of MYO1C protein levels in MCF10A cells transiently transfected with different concentrations of MYO1C-siRNA (1, 3, 5 and 10 nM) and control (mock transfection) at 48 hours post-transfection. (e) Histogram showing percentages of relative cell index of MCF10A cells transfected with MYO1C-siRNA and control cells (received mock transfection). A dose-dependent decrease in relative cell index response to MYO1C depletion was observed reaching to a significant value with concentrations of 5 and 10 nM of MYO1C-siRNA at 48 hours post-transfection compared to control cells (mock transfection). Images shown in (b) and (d) are representatives from three and six independent experiments, respectively. Each bar in (a), (c) and (e) represents the mean value ± SEM from three and six independent experiments, respectively. A Student’s t-test was used to compare the differences in mean value, *P-value < 0.05, ***P-value < 0.005 and ****P-value < 0.001 versus control cells.
Fig 6.
Increased expression of MYO1C protein affects expression of PI3K/AKT components.
(a) The immunoblot of proteins from cell lysates of the HeLa cells transfected with serial dilution of MYO1C-gene expression constructs and (b) derived relative protein expressions in response to serial transfection of MYO1C that showed a decreasing in AKT, pAKTS473, pAKTT308 and PTEN at the maximal dose but increasing in p110α protein expression in comparison with cells transfected with empty construct (control); each bar represents mean value (± SEM), a Student’s t-test was used to compare the differences in mean value, *P-value < 0.05, ***P-value < 0.005 and ****P-value < 0.001 versus control cells. The images shown are representative of Western blotting from at least three independent experiments.
Fig 7.
Fast activation of AKT in response to serum activation after knockdown of MYO1C in HeLa cells.
(a) The immunoblot of proteins from cell lysates of the HeLa cells transfected with MYO1C-siRNA in comparison with non-transfected HeLa cells and subjected to serum activation for 2, 5 and 20 minutes. Histogram showing percentages of relative protein expression of pAKTS473 (b) and pERK (c) from HeLa cells after serum supplement for 2, 5 and 20 minutes. The expression of pAKTS473 was significantly higher after serum supplement at 2 minutes and significantly lower after 20 minutes in comparison to control cells (scrambled siRNA); there was no significant difference in activation of ERK. Each bar in (b) and (b) represents the mean value ± SEM for at least three independent repeats. A Student’s t-test was used to compare the differences in mean value, *P-value < 0.05 versus control cells. The images shown are representative of Western blotting and serum activation assay from at least three independent experiments.