The novel protein C9orf116 promotes rat liver cell line BRL-3A proliferation - Fig 7

<p>Effect of C9orf116 on mitotic entry (A) EdU incorporation assays. The Click-it reaction revealed EdU staining (red). Cell nuclei were stained with Hoechst 33342 (blue). (B) The percentage of EdU-positive cells was quantified. (C) The expression of p-H3 (Ser10) was evaluated by Western blot analysis in C9orf116 knockdown and overexpression BRL-3A cells compared to their control, respectively. (D) The expression of p-H3 (Ser10) was quantified. The experiments were performed three times and three replicates in each one. The data were shown as the means ± SD. Representative images were shown *<i>P</i><0.05, **<i>P</i><0.01.</p

The effect of C9orf116 siRNAs on the mRNA expression level of C9orf116.

<p>NC. Negative siRNA; siR1, siR2 and siR3 represented cells treated with C9orf116 siRNA 1, siRNA 2 and siRNA 3, respectively. The experiments were performed three times and three replicates in each one, the data were shown as the means ± SD, *<i>P</i><0.05, **<i>P</i><0.01.</p

The sequence of C9orf116 siRNAs.

<p>The sequence of C9orf116 siRNAs.</p

Effect of C9orf116 on BRL-3A cell proliferation.

<p>(A) The cell viability was assessed at 24, 48 and 72 h after transfected with siRNA and control by MTT assay. (B) The cell viability was assessed at 24, 48 and 72 h in C9orf116 over-expression cells and control by MTT assay. (C) Proliferation measurement by counting live cells in haemocytometer chamber after trypan blue staining, the number of live cells was counted at 24, 48 and 72 h after transfected with siRNA and control. (D) The number of live cells was counted at 24, 48 and 72 h in C9orf116 over-expression cells and control. NC, C9-siR2 represents the cells treated with negative siRNA, siR2-C9orf116, respectively; pCDH, pCDH-C9 represents the cells treated with Empty vector (pCDH), pCDH-C9orf116, respectively. The above results were presented as means ± SD. The experiments were performed three times and three replications per group. * Indicates <i>p</i> <0.05.</p

The primer sequences used in the RT-PCR.

<p>The primer sequences used in the RT-PCR.</p

Effect of C9orf116 on apoptosis of BRL-3A cells.

<p>(A) Representative graphs obtained by flow cytometric analysis after double-staining with Annexin V-PE/7-AAD. (B) The percentage of late and early apoptotic cells were summed to give the total number of apoptotic cells. The experiments were performed three times and three replicates in each one. The data were shown as the means ± SD.</p

Effect C9orf116 on the expression of cell proliferation related genes.

<p>(A) The expression of cell proliferation related genes in C9-siR2 group at the mRNA level by qRT-PCR analysis. (B) The expression of cell proliferation related genes in C9-siR2 group at the protein level by western blot analysis. (C) The expression of cell proliferation related genes in pCDH-C9 group at the mRNA level by qRT-PCR analysis. (D) The expression of cell proliferation related genes in pCDH-C9 group at the protein level by western blot analysis. The experiments were performed three times and three replicates in each one, the data were shown as the means ± SD. Representative images were shown *<i>P</i><0.05, **<i>P</i><0.01.</p