Fig 2: CEACAM1 is upregulated by NCS in an ATM-dependent manner. (a) Parental MCF-10A cells (MCF-10A) or MCF-10A cells stably expressing an ATM shRNA vector (ATM-KD) or a scrambled shRNA vector (CTRL) as a control were incubated for 4 h in the presence or the absence of 20 nM NCS. At the end of the incubation, total RNA was extracted and analyzed for the levels of CEACAM1 or ATM expression by quantitative real-time PCR. The values in the graph represent the mean±s.e.m. from three independent experiments. §P<0.005; #P<0.001. (b) MCF-10A cells stably expressing an ATM shRNA vector (ATM-KD) or a scrambled shRNA (CTRL) were incubated for 16 h in the presence or the absence of 5.46 nM NCS. At the end of the incubation, total proteins were extracted and analyzed for the levels of CEACAM1 or Nijmegen breakage syndrome 1 (NBS1) expression by western blotting. (c) Parental MCF-10A cells were incubated for 1 h in the presence of 10 µM KU-55933 or the same dilution of dimethyl sulfoxide (DMSO (solvent)) as a control, followed by 16 h incubation in the presence or the absence of NCS 5.46 nM. At the end of the incubation, total proteins were extracted and analyzed for the levels of CEACAM1 or NBS1 expression by western blotting. In b and c, numbers on the left indicate kDa. (d) MCF-10A cells were incubated in the presence of 5.46 nM NCS for the times indicated. At the end of the incubation, the cells were split into two parts and either processed for protein extraction or for total RNA purification. RNAs were analyzed for the levels of CEACAM1, CEACAM1 primary transcript (CEACAM1 int-ex), p21/Waf1 or BTG2 by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. Proteins were analyzed for the levels of CEACAM1, p53 Ser 15-p, p53 or ß-actin (lower panel). Numbers on the left indicate kDa. (e) MCF-10A cells were incubated for 1 h in the presence of 10 µM KU-55933 or the same volume of DMSO (solvent) as a control, followed by 4 h in the presence of the indicated concentrations of NCS. At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates.

Fig 3: ATM/ATR checkpoint independently suppresses gene amplification proficiency. (A) Experimental design. Cells with wild-type Mre11 were transiently transfected with either I-SceI endonuclease expression vector (inducing a site-specific chromosomal DSB) or a control, GFP expressing vector. 105 cells were plates and selected with MTX for 12 days. (B) MTX-resistant colonies arise more frequently cells with checkpoint abrogated by the treatment with caffeine. Left: representative plates from each experimental setting. Right: the average number of MTX-resistant colonies from three independent experiments. (C) Palindromic gene amplification in cells resistant to MTX. Top: double digest of DNA with KpnI and NheI confirmed that all the resistant cells underwent palindromic DHFR duplication. Briefly, the KpnI site was located distal to the I-SceI site and, if a DSB occurred at the I-SceI site and induces palindromic duplication, the KpnI site would be lost and the NheI fragment would be detected by a probe (DHFR). Consistent with palindromic duplication, all the resistant cells exhibited fragments of roughly 5 kb in size. Bottom: double digest of DNA with PstI and NheI confirmed DHFR amplification. This double digest yielded a 2.5 kb fragment from both parental and resistant cells, and the intensity of the fragment represented the copy number of DHFR. In all resistant cells, the intensities were much higher than that of parental cells (designated as P).

Fig 4: Proposed model for the regulation and role of the IFI16 protein in glucose restriction-induced activation of the ATM/AMPK/p53 pathway and autophagy.

Fig 5: BCA2 knockdown up-regulated several DDR proteins. (A) Transcriptional levels of BCA2, ATM and Rad51 genes were analyzed with quantitative real-time PCR at 72 h post-transfection with scrambled or BCA2-targeted siRNAs; (B) Replicate cultures of MCF-7 cells were transfected with siRNA against BCA2 or non-targeting control siRNA prior to irradiation and post-irradiation incubation. At 2 h post-irradiation, cell extracts were isolated and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. * denotes significant difference between control siRNA- and BCA2 siRNA-treated groups with p < 0.05.