We have taken this into account in our experiments by using a control cell line (pLKO vector cells) that was cultured in the same puromycin-containing media as our shRNA cells.
The sensogram data were corrected by subtracting data from a control cell injected with Tris buffer containing 100 µM calcium chloride.
Relative telomere length was determined by comparing isolated PBMC with a control cell line (GM03671C; Coriell Institute of Medical Research, Camden, NJ, USA), a tetraploid cell line, which served as an internal control and was assigned a telomere length of 100%.
Fig. 6 Apoptosis ratio detection by Annexin V/PI assay on the Hep-G2 cells treated by conjugate 4c: a control cells; b, c cells treated by 4c for 24 h.
After 24 h of compound dosing, the cells were analysed by indirect immunofluorescence using an anti-Flag tag antibody to identify cells overexpressing KDM4A, and an antibody for endogenous H3K9me3 to quantify the level of this histone modification, known to be regulated by KDM4A.[ 7] As a control, cells overexpressing the H188A catalytically deficient KDM4A variant were also analysed.
Fig. 7 Collapse of mitochondrial membrane potential in the Hep-G2 cells treated by conjugate 4c. a Control cells; b, c, d cells treated by 4c for 24 h; cells were stained with Rhodamine 123 for 30 min.
As can be seen in Figure 3B-C and Figure 7K, when we overexpress a cherry control followed by a venus control, cells always end up at the same AP position (with very minimal variations) which shows that this assay is particularly robust.
Finally, within 12 hours of incubation on Matrigel, late-EPCs formed characteristic tube-like structures assembled in a branching reticular network, indistinguishable from those generated by a control endothelial cell line (Fig. 3).
The ability of the cells to invade Matrigel-coated filters is presented as the invasion index, the ratio of invasion through Matrigel divided by a control for cell motility.
The levels of VEGF-A excretion reached by control cells was 5174 pg±1323 per 10 cells; the average value of control cells was set at 100%.
