Doubling Graphs
Figure 1: Doubling rate of Immortalized, GFP-Modified, and Unmodified cells.
Doubling rate of immortalized cells took some passages to get up to speed, but remained approximately constant through the rest of the project. The GFP-modified cells also maintained a high doubling rate, while the cells with no genetic modifications (SI) began to fall off by the latest passages.
Figure 2: Total doublings of Immortalized, GFP-Modified, and Unmodified cells.
Total doublings for GFP-modified and TERT/CDK4 Immortalized have now passed the Hayflick Limit with no signs of slowing, while the Spontaneously Immortalized control cells appear to be tapering at ~40 doublings, as expected.
Immunostaining
Figure 3: Images from staining for CDK4 and TERT in proliferative and differentiated BSCs from same groups as above.
Flag staining appears to be non-specific for TERT expression, with broad appearance across all cell types, but HA staining shows a strong specific signal in a subset of Immortalized cells, potentially indicating plasmid integration. Some Flag staining appears to show nuclear localization in those cells as well. Interestingly, nuclear HA expression appears to fade in differentiated Immortalized cells. Images suggest successful integration of TERT and CDK4.
Figure 4: Images from staining for muscle cell markers Pax7 and MF20 in differentiated BSCs from same groups as above.
Pax7 stains on our immortalized cells show their successfully differentiated structure relative to the controls, with clear muscle-like formations as cells grow together. Immortalized cells also show MF20 staining of myotubes, not seen in GFP-modified or unmodified control cells. Both types of structures are also defined enough that they can be seen in brightfield imaging, confirming their presence. Images indicate strong myogenicity in immortalized cells compared to controls.