*2.5. LINC01133 Regulates Cell Cycle and the Levels of Expression of Cell Cycle Regulatory Proteins p21 and Cyclin A*

We further examined the viability of 12Z cells following *LINC01133* knockdown using an AnnexinV/Propidium Iodide FACS assay. We found that *LINC01133* knockdown did not influence the survival of the cells (Figure 4A). The mean percent of early apoptotic AnnexinV positive cells was about 25% for both cells transfected with a control or *LINC01133a* oligo. Analysis of the DNA profiles of the *LINC01133* siRNA transfected cells showed a slight but significant enrichment of the number of cells in the G1 phase (7%, adjp < 0.005), and a concomitant down-regulation of the number of cells entering S-phase of the cell cycle (5%, adjp < 0.05), compared to control siRNA transfected cells (Figure 4B). This effect on cell cycle in knockdown cells was associated with significant up-regulation of the levels of expression of the cell cycle checkpoint regulatory protein p21 (~2.5-fold, adjp < 0.005) and down-regulation of Cyclin A (~2-fold, adjp < 0.05) (Figure 4C), compared to control oligo transfected cells. These findings were consistent with the results of our RNA-seq (see Table S4), further indicating that changes in expression of these genes may be responsible for the cell cycle phenotype.

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 8 of 19

**Figure 4.** *LINC01133* knockdown leads to an increase in cells in the G1 phase and a decrease in cells in the S phase associated with an increase in p21 and a decrease in Cyclin A levels. (**A**) No significant changes in the number of apoptotic cells are seen 72 h after *LINC01133* knockdown. Top: Representative flow cytometry scatter plots for AnnexinV+ versus 7-AAD+ cells. Bottom: No significant change in the number of AnnexinV+ or 7AAD+ cells was observed between *LINC01133a* knockdown and control siRNA transfected cells. Mean values + SD of three biological replicates are shown. (**B**) *LINC01133* knockdown leads to an increase in cells in the G1 phase and a decrease in cells in the S phase. Top: Representative DNA profiles obtained from flow cytometry analysis of PI stained 12Z cells 72 h after transfection with *LINC01133b* siRNA or control siRNA (brown G1 peak, blue S phase, purple G2 + M). Bottom: The percentage of cells in each cell cycle phase are plotted as mean + SD of three independent experiments. Statistically significant differences between the groups in A and B are indicated with a star on the top of each panel. \*—adjp < 0.05 (two-way ANOVA test with Sidak's for multiple comparison), ns-not significant. (**C**) p21 protein is significantly upregulated and Cyclin A protein significantly down-regulated following *LINC01133* knockdown. Top: Representative examples of Western blot analysis following *LINC01133* knockdown of p21 (left) and Cyclin A (right) together with the α-tubulin loading control. Bottom: Densitometric analysis of p21 (left) and cyclin A (right) levels from Western blots normalized to the α-tubulin loading control. Data are displayed as bar graphs with the level from the control siRNA set to 1, and mean and + SD of biological triplicates shown. Statistically **Figure 4.** *LINC01133* knockdown leads to an increase in cells in the G1 phase and a decrease in cells in the S phase associated with an increase in p21 and a decrease in Cyclin A levels. (**A**) No significant changes in the number of apoptotic cells are seen 72 h after *LINC01133* knockdown. Top: Representative flow cytometry scatter plots for AnnexinV+ versus 7-AAD+ cells. Bottom: No significant change in the number of AnnexinV+ or 7AAD+ cells was observed between *LINC01133a* knockdown and control siRNA transfected cells. Mean values + SD of three biological replicates are shown. (**B**) *LINC01133* knockdown leads to an increase in cells in the G1 phase and a decrease in cells in the S phase. Top: Representative DNA profiles obtained from flow cytometry analysis of PI stained 12Z cells 72 h after transfection with *LINC01133b* siRNA or control siRNA (brown G1 peak, blue S phase, purple G2 + M). Bottom: The percentage of cells in each cell cycle phase are plotted as mean + SD of three independent experiments. Statistically significant differences between the groups in A and B are indicated with a star on the top of each panel. \*—adjp < 0.05 (two-way ANOVA test with Sidak's for multiple comparison), ns-not significant. (**C**) p21 protein is significantly upregulated and Cyclin A protein significantly down-regulated following *LINC01133* knockdown. Top: Representative examples of Western blot analysis following *LINC01133* knockdown of p21 (left) and Cyclin A (right) together with the α-tubulin loading control. Bottom: Densitometric analysis of p21 (left) and cyclin A (right) levels from Western blots normalized to the α-tubulin loading control. Data are displayed as bar graphs with the level from the control siRNA set to 1, and mean and + SD of biological triplicates shown. Statistically significant differences between the groups are indicated with adj *p*-values on the top of each graph (ANOVA, with Dunnett's multiple comparison test).
