Several DNA coding for different proteins, CRISPR, or siRNAs against different genes were expressed in cultured normal human somatic cells (A–G; “-” = control: no expression of exogenous gene/siRNA). The cells were either untreated (-) or irradiated to induce DNA damage (+). After 24 hours, the cells were harvested for preparing cell-free extracts. Different proteins were then detected using SDS-PAGE followed by Western blotting. These include p53, the phosphorylated form of pRb as well as total pRb, the phosphorylated form of lamin B as well as total lamin B, and caspase 3 (note that the darkness of the bands roughly corresponds to the intensity of the bands in the Western blot). Fig.1. The cells were also analysed using flow cytometry. The x-axis is DNA contents (2N represents the position of DNA as in G1 phase; 4N represents the position of DNA as in G2/M phase); the y-axis is the accumulated cell number. Fig.2. (1) The first four samples are known (the rest, see below, are all mixed up): Samples 1-2: (-) Control (A) siRNA against ATM (siATM) (B) Human papillomavirus (HPV) E6 protein. Discuss how the Western blot and flow cytometry results confirms these treatments as well as any additional information you can deduced from these results.
Several DNA coding for different proteins, CRISPR, or siRNAs against different genes were expressed in cultured normal human somatic cells (A–G; “-” = control: no expression of exogenous gene/siRNA).
The cells were either untreated (-) or irradiated to induce DNA damage (+). After 24 hours, the cells were harvested for preparing cell-free extracts. Different proteins were then detected using SDS-PAGE followed by Western blotting. These include p53, the phosphorylated form of pRb as well as total pRb, the phosphorylated form of lamin B as well as total lamin B, and caspase 3 (note that the darkness of the bands roughly corresponds to the intensity of the bands in the Western blot).
Fig.1.
The cells were also analysed using flow cytometry. The x-axis is DNA contents (2N represents the position of DNA as in G1 phase; 4N represents the position of DNA as in G2/M phase); the y-axis is the accumulated cell number.
Fig.2.
(1) The first four samples are known (the rest, see below, are all mixed up):
Samples 1-2: (-) Control
(A) siRNA against ATM (siATM)
(B) Human papillomavirus (HPV) E6 protein.
Discuss how the Western blot and flow cytometry results confirms these treatments as well as any additional information you can deduced from these results.
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