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. 2016 Jul 6;7(30):48533–48546. doi: 10.18632/oncotarget.10406

Figure 3. Puma and Noxa induction and cell death following ΔNp63 inhibition are p53 independent but require p73.

Figure 3

A. Puma and Noxa protein expression correlates with ΔNp63 inhibition. Cell extracts were harvested from cell lines 72 hr following infection with a si-ΔNp63 or the control siRNA and analyzed by western blot with antibodies as shown. B. Puma and Noxa induction following ΔNp63 inhibition are not inhibited by p53DD expression. After Abrams cells were infected with the control vector or p53DD expressing lentiviral vectors, infected cells were selected with hygromycin. Abrams expressing either p53DD or the control lentiviral vector were treated with a si-ΔNp63 or the control siRNA for 72 hr. Subsequently, cell extracts were analyzed by western blot with antibodies as shown. C. Cell death following si-ΔNp63 is p53 independent. Quantitation of annexin V- and/or PI-positive cells treated and analyzed as in B. Error bars show standard deviation for two independent experiments. D. Puma and Noxa induction following inhibition of ΔNp63 require endogenous p73. Abrams cells were infected with the control shRNA or p73-directed shRNA expressing lentiviral vectors. Stable pools of Abrams infected with expressing a p73-directed shRNA or control shRNA were transfected with si-ΔNp63 or the control siRNA for 72 hr. Subsequently, cell extracts were analyzed by western blot with antibodies as shown. Note that endogenous p73 levels are unchanged following ΔNp63 inhibition. E. ΔNp63 mediates apoptosis by p73 dependent manner. Quantitation of annexin V- and/or PI-positive Abrams cells treated as in D, Error bars show standard deviation for two independent experiments. F. ΔNp63 binds p73. Abrams and OSA8 cells were transfected with HA- tagged p73 plasmid. After 48 hr of transfection, immunoprecipitation was performed by using HA- tag or IgG control antibodies. Following immunoprecipitation, extracts were analyzed by western blot with p63 antibody as shown. G. Both induction of Noxa and Puma are prerequisite for inducing apoptosis following inhibition of ΔNp63. To silence ΔNp63 expression, OSA16 cells were treated with 200 μg/ml Doxycycline in presence of indicated siRNAs for 72h and cell extracts were analyzed by western blot with antibodies as shown.