p53 dependence of senescence markers p21v1 and p21v2 in aging and acute injury

Ethics approval statement

The animal use and experiments including hemorrhagic shock injury (HI) described in the study were approved by the Institutional Animal Care and Use Committee (IACUC) at Augusta University and were performed in accordance with the relevant guidelines and regulations.

Animals

The young (3 months old) and old (22–24 months old) C57BL/6 male mice used were either bred in-house or purchased from the National Institute on Aging. All the mice were housed in the vivarium at the Augusta University with a 12 h light/ dark cycle.

Hemorrhagic shock injury procedure

The animals were subjected to sham or HI procedure as described earlier^33,34. Briefly, the animals were anesthetized with isoflurane (Henry Schein, Dublin, OH, USA). Both the femoral arteries were cannulated for bleeding, blood pressure monitoring, or fluid resuscitation. Sham animals did not undergo hemorrhage or fluid resuscitation. HI was induced by bleeding rapidly to a MAP of 35 ± 5 mmHg in 45 min by removing ~60% of the total blood volume. The animals were then maintained in a state of shock by maintaining the low MAP for another 45 min, after which fluid resuscitation was carried out with Ringer lactate (RL; twice the volume of shed blood in 1 h). The animals were euthanized at the end of 2 h post-fluid resuscitation, the heart was perfused with ice-cold PBS, and the tissues were collected.

RNA extraction and PCR

RNA was extracted from young, aged and HI tissues using Trizol-reagent (Invitrogen, CA, USA). The RNA quality was ascertained by A260/280 as well as by resolving on 2% agarose gel to assess the 28S and 18S bands. cDNA synthesis was carried out using ImProm-II reverse transcriptase (Promega, WI, USA). The transcripts were analyzed using Bio-rad iTaq Universal SYBR Green Master mix (Bio-rad, CA, USA) in an Agilent real-time PCR machine (Agilent Technologies, CA, USA). The PCR results were normalized to the geometric mean of β-actin, β-glucuronidase and ribosomal protein large P0 (Rplp0)^35,36,37. Primer sequences are listed in Supplementary Table 1. 2^ΔCt values were used for statistical analyses, and the graphs are represented in log₂ scale.

Cell culture and induction of senescence

p53^−/− and wild-type littermate MEF (WT-MEF) cells were treated with 250 nM–1μM concentrations of doxorubicin for 48 h and harvested for molecular studies. Additionally, an H₂O₂-induced stress model was used by treating p53^−/− and wild-type littermate MEF (WT-MEF) cells with 250 μM concentration of H₂O₂ for 24 h. To examine the expression of senescence markers in replicative senescence, MEF cells from passage three (P3, denoted as early passage) were serially passaged after 80% confluence until passage 13 (P13, denoted as late passage). Cells from early as well as late passage were then used to check the expression of senescence markers p16, p21 and p53.

Indirect immunofluorescence and confocal microscopy

Protein expression of p16 and p21 was assessed by indirect immunofluorescence. Briefly, the cryo-sections were blocked using 2% BSA for 1 h and a cocktail of p21 (1:150, Cat. No sc-6246, Santa Cruz Biotechnology, CA, USA) and p16 (1:100, Cat. No. PA5-20379, Invitrogen, CA, USA) unconjugated primary antibodies were used. The slides were then washed with PBS with and without 0.075% Brij-35 detergent (Thermo Fisher Scientific) and incubated with a cocktail of secondary anti-mouse 488 for p21 (1:1500, Abcam, MA, USA), anti-rabbit 594 for P16 (1:1500, Invitrogen) for 2 h. The slides were again washed and incubated with DAPI (1 µg/ml, Sigma, MO, USA) and mounted with Vectastain mountant (Vector Labs, CA, USA). The images were captured on Leica Stellaris Confocal Microscope (Leica, Hamburg, Germany). The hepatocytes were stained using ASGPR primary antibody tagged with Alexa fluor 594 (1:200, Cat. No. PA5-32030, Invitrogen, CA, USA).

Western blot

Immunoblotting was performed to assess the difference in p21 protein expression among young, aged and HI tissues. Briefly, the tissue samples were first homogenized in the RIPA buffer added with protease and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA USA), sonicated and clarified by centrifugation. The protein concentrations were determined by the DC (detergent compatible) protein assay (Bio-Rad Laboratories, Hercules, CA). Then, 40 µg protein was loaded onto a 12% SDS–PAGE, transferred onto polyvinylidene difluoride (PVDF) membrane and probed with p21 (1:200, Cat. No sc-6246, Santa Cruz Biotechnology, CA, USA) and actin (1:1000, Cat. No. ab 179467, Abcam) primary antibody and anti-mouse secondary antibody conjugated with horseradish peroxidase (1:10,000, Cat. No. 7076P2, Cell Signaling, Danvers, MA). The immune complexes were detected by chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA USA).

Statistical analysis

Experiment appropriate statistical methods were used to analyze the results. The 2^ΔCt values for amplified senescence markers in tissues and doxorubicin-treated MEF cells were assessed using the Kruskal–Wallis test. The results of the H₂O₂-induced stress model were analyzed using a 2-way ANOVA (Uncorrected Fisher’s LSD), while p53 expression for H₂O₂-induced stress was analyzed using Wilcoxon’s and that of replicative senescence were analyzed by Mann–Whitney test. A p-value of <0.05 was considered as significant. The mRNA expression was assessed in seven mice/group with at least two technical replicates. The in vitro experiments were carried out in triplicates. The statistical analysis was performed using Graph Pad Prism software.