Fig 1: Marked impairment of cisplatin-mediated apoptosis in S47 cells and mice. (A) Primary MEFs from the wild-type (Wt) or S47 Hupki mouse were treated with 10 µM cisplatin (CDDP) for the time points indicated, and protein lysates were analyzed by Western blot analysis for the proteins indicated. The data depicted are representative of three independent experiments in a minimum of three independent batches of MEFs. (B) Primary MEFs from the wild-type and S47 mice as well as the p53 knockout mouse (p53-/-) were treated with 10 µM CDDP for 24 h, and protein lysates were analyzed by Western blot analysis for the proteins indicated. (C) Flow cytometric analysis of Annexin V-positive cells from primary wild-type and S47 MEFs treated with 10 µM CDDP for 24 h. The totals represent an average of three independent experiments normalized to untreated controls. Error bars represent standard deviations. (D) IC50 analysis for cisplatin (CDDP) in primary wild-type (WT) and S47 MEFs treated with the indicated concentrations of cisplatin for 72 h and analyzed for viability by the Alamar blue assay. The depicted data represent an average of four independent experiments on independent batches of MEFs. Error bars represent standard deviation. (E) Human LCLs homozygous for wild-type p53 and the S47 variant were treated with 10 µM CDDP for 24 h, and protein lysates were analyzed by Western blot for the proteins indicated. (F) Flow cytometric analysis of Annexin V-positive cells from wild-type and S47 human LCLs treated with 10 µM CDDP for 24 h. The totals represent an average of three independent experiments normalized to untreated controls. Error bars represent standard deviations. (G) IC50 analysis for cisplatin (CDDP) in wild-type and S47 LCLs treated with the indicated concentrations of cisplatin for 48 h and analyzed for viability by Alamar blue staining. The depicted data represent an average of three independent experiments. Error bars represent standard deviation. (H) IC50 analysis for adriamycin in wild-type and S47 LCLs treated with the indicated concentrations of adriamycin for 48 h and analyzed for viability by Alamar blue staining. The depicted data represent an average of three independent experiments. Error bars represent standard deviation. (I) Cisplatin-mediated apoptosis, as assessed by cells positive for cleaved lamin A, in the kidneys of wild-type or S47 mice following injection with 20 mg/kg CDDP and analyzed after 48 h. Data are representative of n = 3 per mice group. Bar, 100 µm. (J) Clonogenic survival of shARF immortalized wild-type and S47 MEFs treated with the indicated concentrations of cisplatin (CDDP), plated at equal cell numbers 48 h later, and stained with crystal violet after 7 d. (K) Quantification of clonogenic survival of immortalized wild-type and S47 MEFs following cisplatin treatment. All values were normalized to the untreated control averaged from three independent experiments. Error bars represent standard deviation. (*) P-value < 0.05.
Fig 2: The S47 variant is impaired for transactivation of a subset of p53 target genes, including Gls2, Noxa (Pmaip1), and Sco2. (A) qRT–PCR analysis of p53 target genes in primary wild-type (Wt) and S47 MEFs treated with 10 µM cisplatin (CDDP) for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. (B) qRT–PCR analysis of the p53 target genes indicated in independent batches of primary MEFs from wild-type and S47 mice treated with 10 µM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. (C) qRT–PCR analysis of the p53 target genes indicated in human LCLs that are homozygous for wild-type p53 or S47 treated with 10 µM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P =0.05. SCO2 was not expressed in LCL cells, so these data are not depicted. (D) Western analysis for the proteins indicated in wild-type and S47 MEFs pretreated with 10 µM p38MAPK inhibitor SB203580 for 2 h followed by 10 µM cisplatin (CDDP) for 24 h. GAPDH served as the loading control. (E) qRT–PCR analysis of the cells in D for the p53 target genes indicated, normalized to control (cyclophilin A). The depicted data represent the average of three independent experiments. Error bars represent standard deviation. (*) P <0.05.
Fig 3: Impaired DNA-binding ability of the S47 variant. (A) ChIP of primary wild-type (Wt) and S47 MEFs treated with 10 µM CDDP for 24 h analyzed using antisera to p53 (CM5) or IgG. The percentage binding normalized to input from qPCR analysis is shown. The data depicted are averaged from three independent experiments normalized to input. Error bars represent standard deviation. (*) P-value < 0.05. (B) ChIP analysis of p53 binding to the consensus elements from the genes indicated in human H1299 (p53-null) cells containing doxycycline-inducible wild-type or S47 forms of p53 in the absence and presence of 100 ng/mL doxycycline plus 10 µM cisplatin for 24 h. ChIP was performed using antisera to p53 (fl393G) or normal rabbit IgG. The data depicted are averaged from three independent experiments normalized to input. Error bars represent standard deviation. (*) P-value < 0.05. The IgG results are depicted for the CDKN1A/p21 p53-binding site but were comparable for all other sites analyzed. NOXA was not expressed in this cell line, so this gene was not analyzed.
Fig 4: Impaired ferroptosis in S47 cells. (A) Representative phase-contrast images of primary MEFs containing wild-type p53 (WT), heterozygous S47/wild type, or S47 treated with 4 µM ferroptosis inducer erastin or vehicle (DMSO) for 8 h (magnification 10×). Data represent the average of three independent studies. Bar, 20 µm. (B) Cell viability (Alamar blue) analysis of wild-type, S47/wild-type, or S47 primary MEFs treated with erastin for 72 h. The data represent the average of four independent experiments. Error bars represent standard error of the mean. (C) Western blot analysis for GLS2 in wild-type MEFs, wild-type MEFs infected with a lentiviral short hairpin for Gls2 (shGls2), and S47 MEFs untreated or treated with 4 µM erastin for 24 h. GAPDH served as the loading control. In the bottom panel, the percent viability using the Trypan blue exclusion assay is shown. Error bars represent standard deviation. (D) qRT–PCR analysis of slc7a11 normalized to cyclophilin A. The data are averaged from three independent biological replicates. Error bars represent standard deviation. (E) qRT–PCR analysis of Ptgs2 normalized to cyclophilin A. The data are averaged from three independent biological replicates. Error bars represent standard deviation. (F) Immunoblot analysis for GPX4 in wild-type and S47 MEFs following treatment with 10 µM CDDP for 24 h. (G) Cell viability analysis of wild-type and S47 human LCLs treated with RSL3 for 48 h. The data represent the average of three independent experiments. Error bars represent standard deviation. (H) Trypan blue exclusion analysis of the percent viability in wild-type MEFs or wild-type LCLs exposed to 10 µM CDDP, CDDP plus 2 µM ferrostatin-1 (Fer-1), or CDDP plus 20 µM zVAD-fmk. The data represent the average of three independent experiments. Error bars represent standard deviation. (I) Proposed model depicting the relative abilities of wild-type p53 and S47 to induce senescence, apoptosis, and ferroptosis and suppress spontaneous tumor initiation.
Fig 5: Experimental results of two implementations of sequential chemistry to perform the complete IHC protocol on cell blocks.a and b involve the static head, while c and d involve the scanning head. a Photographs of a single HFC captured at three different times with different colored liquids. The liquids within the HFC can be switched within ~2 s. b IHC staining for P53 on a cell pellet section with varying incubation times of the primary Ab, secondary Ab, and streptavidin peroxidase at different locations. c Scanning of the reagents with a multi-HFC head allows fast sequential chemical treatment of a surface. This strategy was also applied for IHC on a cell pellet section (d), and good contrast was achieved with residence times as short as 30 s
Supplier Page from Abcam for Anti-p53 (phospho S46) antibody [36]