Figure 1.
SUMOylation increased the expression level of mouse p53 protein.
Pre-GCs were seeded in 6-well culture plates and transfected with 2 µg of HA-p53a, HA-p53b, HA-sumo-1, HA-ubc9, or empty HA expression plasmids. 20 µg total extracts were prepared and resolved by SDS–PAGE and analyzed by Western blot using anti-HA (the top group) or anti-p53 (the middle group) specific antibody. Positions of molecular weight markers, free HA-p53 or putative p53/HA–SUMO-1 and p53/HA-UBC9 conjugates are indicated. β-actin is a loading control.
Figure 2.
Identification of SUMOylation of p53b in vivo at Lys 375.
(A) Immunoprecipitation analysis of SUMOylation of p53 in vivo. Pre-GCs were transfected with pCMV-Flag-sumo-1 plasmid or pCMV-Flag plasmid. Flag-tagged protein were pulled down by anti-Flag affinity beads and then analyzed by Western blot using anti-p53 antibody. A visible band of p53 was detected in the sample of pCMV-Flag-sumo-1 transfected granulosa cells, but not in the control sample. (B) SUMOylation consensus site aligned in sequences of p53 from human and mouse. The SUMOylation consensus site is ψKXE with a hydrophobic residue, such as Phenylalanine (F), Isoleucine (I), or Leucine (L). Sequences of human p53 and mouse p53b were aligned. Lys386 in human p53 is SUMO-1 modified and the mouse p53b has a highly homologous sequence to this SUMOylation site. (C) Co-immunoprecipitation of SUMO-1 with p53a or p53b. Pre-GCs were co-transfected with Flag-tagged sumo-1 plasmid and HA-tagged p53a or HA-tagged p53b plasmid. Flag-tagged proteins were pulled down by anti-Flag affinity beads and then analyzed by Western blot using anti-HA antibody. A band of p53 (p53b) was detected in the sample of Flag-sumo-1 plasmid and HA-p53b plasmid co-transfected granulosa cells, but not in the HA-p53a plasmid and Flag-sumo-1 plasmid co-transfection group or control sample. (D) p53b is SUMOylated at lys375. Pre-GCs were co-transfected with 2 µg of wild type HA-p53b or mutant p53b (K375R) plasmid and 2 µg of Flag-sumo-1 plasmid. 20 µg total extracts were prepared and resolved by SDS–PAGE and analyzed by Western blot using the anti-HA-specific antibody. Positions of molecular weight markers, free p53 or p53-SUMO-1 conjugates are indicated. β-actin is a loading control. After transfection, two bands could be detected in the group with co-transfection of wild type p53b and sumo-1, the lower molecular weight below was considered as free p53b and the higher molecular weight above was p53b-SUMO-1 as the SUMOylated p53b, but only one band (mutant p53b) could be observed in the group with co-transfection of mutant p53b and sumo-1 plasmids, while the p53b (K375R)-SUMO-1- band was not observed.
Figure 3.
SUMO-1 modification enhanced the stability of p53b in a dose-dependent manner.
(A) Pre-GCs were co-transfected with 2 µg HA-p53b plasmid and different amounts (2 µg, 3 µg, 4 µg, and 6 µg) of Flag-sumo-1 plasmids. 20 µg total proteins were prepared and resolved by SDS–PAGE and analyzed by Western blot using anti-HA-specific antibody. Positions of molecular weight markers, free p53b and p53b-SUMO-1 conjugates are indicated. β-actin is a loading control. (B) Relative expression quantity of p53b and SUMO-1-p53b were determined by densitometric scans. The total amount of β-actin present in the lower set of lanes was used to standardize the amount of p53b and SUMO-1-p53b present in the upper set of lanes. The value expressed by each bar represents the mean ± SD (n = 3). Different letters indicated statistical difference (P<0.05). (C) Pre-GCs were co-transfected with 2 µg HA-p53b (K375R) and different amounts (2 µg, 3 µg, 4 µg, and 6 µg) of Flag-sumo-1 plasmids. HA antibody were used to detect the expression of p53b (K375R) after transfection. The results showed that elevating the amount of transfected Flag-sumo-1 resulted in a simultaneous increase in the level of SUMOylated p53b and free p53b, but not mutant p53b (K375R).
Figure 4.
SUMO-1 modification of p53b at Lys 375 was required for its nuclear accumulation in granulosa cells.
(A) Pre-GCs were transfected with HA-tagged p53b or mutant p53b (K375R) plasmid, respectively. Then immunofluorescence cytochemistry was used to detect the subcellular localization of p53b or p53b (K375R) by HA antibody. The immunostaining signal of p53b were observed in nucleus of granulosa cells, but p53b (K375R) were seen in both nucleus and cytoplasm of granulosa cells (green). The nucleus was stained by PI (red). (B) Pre-GCs were co-transfected with HA-tagged p53b or mutant p53b (K375R) plasmid with Flag-tagged sumo-1 plasmid. Wild type p53b and mutant p53b (K375R) were detected by HA antibody with FITC-conjugated secondary antibody (green) and SUMO-1 was detected by SUMO-1 antibody with Cy3-conjugated secondary antibody (red), and the nucleus was stained by DAPI (blue). Co-localization of SUMO-1 and p53b in the nucleus were observed, but p53b (K375R) were still localized in both nucleus and cytoplasm of granulosa cells.
Figure 5.
SUMOylation of p53b induced apoptosis in granulosa cells.
(A) Representative flow cytometric analysis of apoptotic cells stained with Annexin V-FITC and PI after transfection with 2 µg wild type p53b, mutant p53b (K375R) plasmid, control plasmid or non-transfection control group for 24 h. In each panel, the lower right quadrant contains apoptotic cells (positive for Annexin V and negative for PI). (B) The apoptosis rate of granulosa cells. The value expressed by each bar represents the mean ± SD (n = 3). Different superscripts denote statistical difference at a P<0.05. (C) Expression level of Bax mRNA. 24 h after transfection with 2 µg wild type p53b, mutant p53b (K375R), control plasmid or non-transfection control group, expression level of Bax mRNA was detected by quantitative real-time PCR. Fold changes were calculated from β-actin normalized Ct values. The value expressed by each bar represents the mean ± SD (n = 3). Different superscripts denote statistical difference at a P<0.05.