*2.3. The Loss of DG Induces the Expression of Senescence-Associated Features*

Lamin B1 downregulation occurs at the onset of cell transition to senescence [23,30]. Thus, we tested whether the reduction in lamin B1 as a consequence of DG loss would lead to a senescent phenotype. To approach this idea, we searched for senescence characteristics in DG-null cells. Decreased proliferative potential and a higher percentage of cells at the G0/G1 phase of the cell cycle were found in DG-KO1 and DG-KO2 cell cultures, compared with WT cells, as shown by MTT and flow cytometry analyses, respectively (Figure 4A,B). G0/G1 arrest might be indicative of premature senescence; thus, the identification of senescent cells in DG-KO cultures was carried out using senescence-associated β-galactosidase activity [SA-β-gal]. Interestingly, increased numbers of senescent cells were found in DG-KO1 and DG-KO2 cultures (20–25%), compared with WT cells (5%) (Figure 4C). In addition to increased β-galactosidase activity, senescent cells display large-flat cell morphology and undergo both nucleolar stress and the loss of perinuclear chromatin [28], among other characteristics. To corroborate the increase in senescent cells in DG-KO cultures, we searched for the presence of these features in DG-null cell cultures, using WT cells induced to senescence by treatment with sodium butyrate (NaBu) for ten days as a positive control for the senescent phenotype. NaBu is a histone deacetylase inhibitor that elicits senescence via irreversible induction of cell cycle arrest [31]. WT cells treated with NaBu showed a flattened and expanded morphology, which markedly contrasts with the typical polygonal morphology of untreated WT cells (Figure 5A). DG-KO2 but not DG-KO1 cells showed a subtle but statistically significant increase in cell surface, compared with WT cells, as determined by phalloidin staining of F-actin and estimation of cell surface area (Figure 5A and right graph). On the other hand, a marked decrease in H3K9me3 foci immunostaining (heterochromatin marker) was observed in DG-null cells in a similar fashion to that observed in NaBu-induced senescent cells (Figure 5B), as determined by confocal laser scanning microscopy (CLSM) and fluorescence intensity quantification (Figure 5B and right graph). Finally, disaggregated nucleoli with a smaller area were found in DG-KO1 and DG-KO2 cell cultures, compared with WT cells, as revealed by immunostaining for the nucleolar protein B23 and nucleolar area quantification (Figure 5C and right graph). However, nucleolar disaggregation was much greater in NaBu-induced senescent cells.

Collectively, the aforementioned data imply that incipient senescence is present in DG-KO cell cultures in the absence of any detectable senescence-inducing stimuli. Thus, we assessed whether the lack of DG sensitizes C2C12 cells to senescence induction. In line with this, the percentage of senescent cells was significantly higher in DG-KO1 cultures (60–70%) than that in WT cells (30%) upon five days of NaBu treatment (Figure 6A). Long-term treatment (10 days) rendered a similar percentage of senescent cells (~75%) between WT and DG-KO cell cultures (Figure 6A).
