Figures & data
Table 1. HCT-116, HT-29, and KM12C cells were grown and either transfected with Par-4 expression plasmid or control plasmid for 48 hrs. Par-4 was immunoprecipitated from whole cell lysates using anti-Par-4 antibody and protein G magnetic beads. An in-solution trypsin digestion was performed on the eluted proteins, and the digests were sent for mass spectrometry analysis. A representative list of the most abundant proteins identified in the digest reveals TRIM21 as a potential novel interacting partner of Par-4.
Figure 1. TRIM21 is a novel interacting partner of Par-4. HCT-116, HT-29, and KM12C cells were grown and transfected with either Par-4 expression plasmid or control plasmid for 48 hrs. Co-immunoprecipitations were performed in HT-29, HCT-116, and KM12C cells (a, b, c, respectively) in order to validate the TRIM21/Par-4 interaction.
Figure 2. TRIM21 interacts with Par-4 via its PRY-SPRY domain. HCT-116 cells were co-transfected with Par-4 plasmid and different TRIM21 constructs for 48 hrs. Par-4 was immunoprecipitated from whole cell lysates using anti-Par-4 antibody and protein G magnetic beads. The proteins were eluted from the beads and a western blot analysis is performed using anti-TRIM21 antibody. (a) A diagram representing the various TRIM21 constructs that were used (b) Western blots demonstrating that when co-transfecting with the TRIM21 construct that does not contain the PRY-SPRY domain, TRIM21 does not interact with Par-4; in contrast, when co-transfecting with the TRIM21 constructs that contain the PRY-SPRY domain, TRIM21 does interact with Par-4.
Figure 3. TRIM21 is not sufficient to downregulate Par-4 protein levels. (a) Western blots of endogenous expression levels of Par-4 and TRIM21 in colon cancer cell lines show an inverse correlation in expression. (b) SW480 cells were transfected with either a control plasmid or various TRIM21 constructs for 48 hours. Whole cell lysates were collected and Par-4 protein levels are analyzed by Western blot with actin as a loading control.
Figure 4. Ectopic expression of TRIM21 downregulates Par-4 in the presence of cisplatin in colon cancer cells. Colon cancer cell lines, transfected with or without TRIM21 expression plasmid for 48 hrs, were treated with increasing doses of cisplatin for 24 hrs. The cisplatin concentrations are in units of μg/ml. Western blots of Par-4 and TRIM21 levels are shown with actin shown as a loading control (a) HT-29 (b) HCT-116.
Figure 5. Cisplatin downregulates Par-4 in a dose- and proteasome-dependent manner. (a) Western blots showing Par-4 expression levels in TRIM21-transfected HCT-116 cells over time at different doses of cisplatin. Cisplatin doses are in units of μg/ml. Time is in units of hours. Actin is shown as a loading control. (b) HCT-116 cells were transfected with or without TRIM21 expression plasmid for 48 hrs, then treated with the indicated doses of cisplatin in μg/ml for 24 hrs, and with or without 10 μM MG132. Blot shows Par-4 expression levels, and actin is shown as a loading control.
Figure 6. Cisplatin downregulates Par-4 in both the cytoplasmic and nuclear compartments. HCT-116 cells were either transfected with plasmid encoding TRIM21 or control plasmid for 48 hrs and treated with or without 3.75 μg/ml of cisplatin. Then, nuclear-cytoplasmic fractionation was performed and Par-4 and TRIM21 levels were examined by Western blotting, with Lamin A/B and β-tubulin serving as markers validating the integrity of the fractionation.
Figure 7. Cisplatin downregulates Par-4 in pancreatic cancer cells. Pancreatic cancer cell lines were either transfected with TRIM21 plasmid or control plasmid for 48 hrs, and then treated with increasing concentrations of cisplatin for 24 hrs. Cisplatin doses are in units of μg/ml. Western blots showing Par-4 expression are shown with actin as a loading control. Western blots validating TRIM21 overexpression are also included. (a) BxPc-3 (b) MiaPaca-2 (c) AsPc-1.
Figure 8. TRIM21 is a potential therapeutic target in colon and pancreatic cancer. (a) HCT-116 cells were either transfected with a plasmid encoding TRIM21 or control plasmid for 48 hrs, and then treated with increasing doses of cisplatin for 24 hrs, and viability was assessed by MTT assay. Viability is plotted as a percentage of control samples. Asterisks indicate statistically significant differences in viability between TRIM21 and control transfected cells. (b) Panc-1 cells were either transfected with a plasmid encoding Par-4 or control plasmid for 48 hrs, and then treated with increasing doses of cisplatin. Cisplatin doses are shown in units of μg/ml. Levels of PARP and Par-4 levels were examined by Western blotting. Percentage of cleaved PARP, as determined by densitometric analysis, is shown below the blots. Kaplan-Meier survival curves showing overall survival, , and progression-free survival, , of a cohort of pancreatic cancer patients stratified by TRIM21 mRNA expression levels. Data was obtained.
