Ionizing radiation induces degradation of p21^Cip1. A, HEK293 cells were irradiated with 10 Gy IR and collected at the indicated times. A Western blot was performed on equal amounts of whole cell lysate. NS is a nonspecific band of the p21^Cip1 antibody (C-19) used as a loading control. B, Western blot of HEK293 cells pretreated with 25 μg/ml CHX prior to treatment with no IR or 10 Gy IR. C, Western blot of HEK293 cells pretreated with 25 μg/ml CHX for 1 h prior to irradiation with 0, 0.5, 2, or 10 Gy IR. D, Western blot of HEK293 cells pretreated with 50 μm Z-VAD-fmk, 25 μg/ml CHX, and 100 μg/ml phleomycin (or vehicle) for 1 h.

IR-induced degradation of p21^Cip1 is independent of ATM. A, Western blot of whole cell lysates from HEK293 cells pretreated for 1 h with DMSO or 10 μm KU55933 prior to 10 Gy IR. Phospho-specific antibodies to ATM or the ATM target Chk2 were used to verify the efficacy of the inhibitor. B, Western blot of whole cell lysates from immortalized A-T fibroblasts or reconstituted A-T fibroblasts pretreated with 25 μg/ml cycloheximide for 1 h prior to 10 Gy IR. C, Western blot of whole cell lysate from HEK293 cells pretreated with 25 μg/ml CHX and DMSO or 100 μm wortmannin for 1 h prior to 10 Gy IR. Phospho-specific antibodies to ATM or ATR target sites were used to verify the efficacy of the inhibitor. D, A-T cells were transfected with siRNAs to ATR or DNA-PK, either individually or in combination. 60 h post-transfection, cells were pretreated with 25 μg/ml CHX for 1 h prior to irradiation with 10 Gy IR. Cells were collected 1 h after IR, and equal amounts of whole cell lysate were analyzed by Western blot.

DDB1, but not Skp2, is required for the IR-induced degradation of p21^Cip1. A, HEK293 cells were infected with a nontarget (NT) lentiviral shRNA or an shRNA against Skp2. Infected cells were selected with 2.0 μg/ml puromycin. Knockdown of Skp2 was assessed by Western blot using dilutions of the nontarget sample. B, nontarget or Skp2 knockdown cells were pretreated with 25 μg/ml CHX for 1 h prior to irradiation with 10 Gy IR. Cells were collected at the indicated time points and analyzed by Western blot. C, HEK293 cells were transfected with a nontarget siRNA or two separate siRNAs against Skp2. 60 h post-transfection, cells were irradiated with 10 Gy. Cells were collected 1 h after IR, and whole cell lysates were analyzed by Western blot. D, HEK293 cells were infected with a nontarget lentiviral shRNA or an shRNA against DDB1 and assessed for DDB1 knockdown and p21^Cip1 up-regulation as in A. E, nontarget or DDB1 knockdown cells were pretreated with 25 μg/ml CHX for 1 h prior to irradiation with 10 Gy IR. Cells were collected at the indicated time points and analyzed by Western blot. F, HEK293 cells were transfected with a nontarget siRNA or an siRNAs against DDB1. 60 h post-transfection, cells were pretreated with 25 μg/ml CHX for 1 h prior to irradiation with 10 Gy. Cells were collected 1 h after IR and whole cell lysates were analyzed by Western blot. Vertical lines indicate gel lanes that were spliced together. G, HEK293 cells were transfected with a nontarget siRNA or an siRNAs against Cdt2. 60 h post-transfection, cells were irradiated with 10 Gy. Cells were collected 1 h after IR, and whole cell lysates were analyzed by Western blot.

IR-induced degradation of p21^Cip1 occurs in transformed cells. A and B, indicated cells were pretreated with 25 μg/ml CHX and left untreated or irradiated with 10 Gy IR. All immunoblots were run using equal amounts of whole cell lysate and are representative of multiple experiments. C, same as in A and B, except LICOR secondary antibodies were used, and LICOR imaging software was used to quantify p21 levels. p21 levels were normalized to tubulin. Graphs are from three independent experiments with representative experiments shown below. D, Western blot of HCT116 or HCT116 p53^-/- cells pretreated with 25 μg/ml CHX for 1 h prior to 10 Gy IR. E, Western blot of BJ human foreskin fibroblasts infected with a control retrovirus or a retrovirus expressing SV40 T-antigen. Infected cells were selected with 2.0 μg/ml puromycin until control cells were dead and detached from the dish. Selected cells were cultured to confluence and pretreated with 25 μg/ml CHX for 1 h prior to 10 Gy IR. Vertical lines indicate gel lanes that were spliced together.

p21^Cip1 is degraded by the ubiquitin-proteasome system following IR. A, Western blot of whole cell lysates from HEK293 cells pretreated with 25 μg/ml CHX and DMSO or 25 μm MG132 for 1 h prior to 10 Gy IR. B, Western blot of whole cell lysates from HEK293 cells pretreated with 25 μg/ml CHX and DMSO or 5 μm epoxomicin for 1 h prior to 10 Gy IR. C, HEK293 cells were pretreated with DMSO or 25 μm MG132 for 30 min prior to IR. Cells were collected at the indicated time points as described under “Experimental Procedures.” p21^Cip1 was immunoprecipitated from 1.0 mg of whole cell lysate using a polyclonal antibody to p21^Cip1, and a Western blot was performed using a p21^Cip1 monoclonal antibody. D, schematic of the p21-HA and p21(K6R)-HA constructs. E, HEK293 cells were transfected with 5.0 μg of total DNA, including 50 ng of GFP and 100 ng of p21-HA or p21(K6R)-HA. Cells were pretreated with 25 μg/ml CHX for 1 h prior to irradiation with 10 Gy IR. Cells were collected 1 h after IR, and equal amounts of whole cell lysate were analyzed by Western blot.

p21^Cip1 lacking the ability to bind PCNA is not degraded following IR. A, HEK293 cells were transfected with 5.0 μg of DNA, including 500 ng of p21-HA or p21^ΔPCNA-HA and 100 ng of GFP. 60 h post-transfection, p21^Cip1 was immunoprecipitated from 1.0 mg of whole cell lysate, and immunoblots were performed with antibodies to p21^Cip1 or PCNA. B, HEK293 cells were transfected with 5.0 μg of DNA, including 100 ng of p21-HA or p21^ΔPCNA-HA and 50 ng of GFP. 60 h after transfection, cells were treated with 25 μg/ml CHX for 1 h prior to irradiation with 10 Gy of IR. Samples were collected 1 h after IR. Equal amounts of whole cell lysate were analyzed by Western blot for expression of the indicated proteins. C, upper panel, HEK293 cells were transfected with 5.0 μg of DNA, including 100 ng of p21-HA or p21(K6R)-HA and 50 ng of GFP. 60 h later cells were irradiated with the indicated doses of IR. 6 h post-IR, cells were trypsinized and GFP⁺ cells were sorted (in triplicate) directly into 6-well dishes containing conditioned DMEM. 10–14 days later, surviving colonies were counted as those with >50 cells. Shown are the results of an experiment representative of multiple independent repeats. Lower panel, Western blot of 10 Gy samples taken at 1 h post-IR. In this experiment, 30% of cells were GFP⁺, so exogenous p21 levels are about 3-fold higher than shown. D, model depicting degradation of p21^Cip1. Both Skp2 and DDB1^Cdt2 can regulate the normal turnover of p21^Cip1, but only DDB1^Cdt2 is responsible for the IR-inducible turnover.