PurA sensitizes cells to toxicity induced by oxidative stress

Cell culture

Mouse embryonal fibroblast (MEF) cells were isolated and cultured from the PURA WT and PURA K/O mouse models [2]. MEF cells were grown in DMEM medium with 10% FBS and 1% P/S and maintained in 37 °C humidified incubator with 7% CO₂.

Immunoblotting

Whole cell extracts were obtained from both treated or untreated PurA WT and PurA KO MEFs using RIPA buffer and protease inhibitor cocktail. Protein concentration was determined using Bradford assay. We used fifty micrograms of each sample which were denatured by boiling at 95 °C in SDS dye and water. Protein samples were loaded in and separated by 12% SDS-PAGE and then were transferred on either 0.2 or 0.45 μM nitrocellulose membranes. Membranes were blocked with 10% dry milk for 1 h then probed overnight with 0.5% dry milk plus the desired primary antibody, usually 1:1,000 dilution. Next day, after washing the membranes three times with 1X PBST and one time with 1X PBS, 5 min each, we probed membranes with specific secondary antibodies in dry milk, usually 1:10,000 dilution for 1 or 2 h depending on the primary antibody. Subsequently, membranes were scanned using the LICORE CLx instrument.

Immunocytochemistry and microscopy

PurA−/− and PurA +/+ MEFs in two-well chamber slides were washed with PBS and fixed in 4% paraformaldehyde for 15 min and blocked in 5% BSA in PBST. The MEFs were probed with the following antibodies anti-GPCR GPR7 (1:200) (1:1,000, Abcam, Cambridge, MA), and anti- β3-tubulin (1:500) overnight in 4 °C and washed with cold PBST. Alexa Flour Secondary antibodies donkey anti-mouse IgG 484 and donkey anti-rabbit IgG 568 have been applied for fluorescent labeling (Thermo Fisher Scientific, Eugene, OR). VECTASHIELD medium (Vector, Laboratories, Burlingame, CA) was used for DAPI labeling and mounting. Leica fluorescent microscope (Leica Microsystems, IL.) was used for imaging.

MTT assay

MEF cells from PurA ^+/+ and PurA ^−/− mice were seeded in 12 well plates (200,000 cells/well) to perform dose response and time course experiment of Paraquat treatment. After treatment with Paraquat, MTT reagent was prepared by dissolving 5 mg of MTT reagent/1 mL of 1X sterile PBS and kept in dark due to its light sensitivity. 1:10 dilution of MTT reagent was added to each well and incubated for 2 h at 37 °C. The MTT solvent was than prepared which contained: 50 mL of 100% isopropanol, 50 μL of NP40, 200 μL of 1 M HCL. After 2 h incubation with MTT reagent, media with MTT reagent were aspirated and 1 mL of MTT solvent was added to each well and was shaken for 30 min at RT covered with foil. Plates were checked every 5 min; the reaction was completed when cells became colorless and the supernatant was purple. We took 1 mL of each well and transferred it to Eppendorf tubes, spun down, and then transferred 200 μL to a 96 well plate to read on spectrophotometer or plate reader.

Reagents

Oxidative stress was induced by Paraquat dichloride hydrate, Sigma Aldrich, CAS # 75365-73-0. Heat shock response was induced by GGA, Geranylgeranylacetone, Sigma Aldrich, CAS# 6809-52-5.

Statistical analysis

Results are expressed as Mean ± SEM. T-test was used for the comparison of two unpaired groups. One-way ANOVA was used to compare three non-paired groups followed by a post-hoc Tukey HSD test. p value <0.05 was considered significant.

Effects of heat-shock and oxidative stress on MEF cells derived from PURA-WT and K/O

PurA mouse model was generated by [2] research group through targeted disruption of PURA gene by homologous recombination in embryonic stem cells (ES). The transgenic animals were lacking either one allele of PurA (PURA^+/−) or both alleles (PURA^−/−). In our study, we used the primary mouse embryo fibroblasts (MEFs) which were derived from the embryos of mouse models PurA WT (PURA^+/+) and PurA KO (PURA^−/−). The PURA phenotype of both cells was confirmed by PurA Western blotting (Figure 1A). As expected, PURA k/o MEF cells were negative for PurA expression. To gain insight into the effect of stress induced by Geranylgeranylacetone (GGA) which mimics heat-shock conditions, cells were treated with GGC and immunocytochemistry was performed for PurA in both WT and k/o cells. Results suggested that GGA treatment had no visible effects on PurA expression in WT cells (Figure 1B) and their morphology was indistinguishable in both WT and k/o cells (Figure 1C). In addition, PURA WT and k/o cells were also treated with Paraquat which induces oxidative stress, and phase contrast images were taken to monitor cellular responses to the treatment. As shown in Figure 1D, while paraquat treatment caused a significant alteration in cellular morphology with obvious cytotoxicity in PurA-WT cells, there were limited cytotoxicity observed in k/o cells.

Figure 1:

Effects of heat-shock and oxidative stress on PURA WT and PURA K/O MEF cells. (A) PurA expression in MEF cells. Western blot analysis of PurA expression in PURA WT and PURA K/O cells. (B) and (C) Immunocytochemical analysis of PurA expression in control and GGA treated PURA WT (B) and PURA K/O (C) MEF cells. (D) Phase contrast images of PURA WT and PURA K/O MEF cells treated with Paraquat. All images are representative of three independent experiments.

PurA expressing MEFs are more sensitive to oxidative stress compared to PurA knockout MEFs

Oxidative stress was induced on both PurA expressing MEFs and PurA knockout MEFs using Paraquat drug in a dose (0.5, 1.0, and 1.5 mM) and time (4, 8, 18, 24, and 24 h) dependent manner. PurA WT and KO MEFs were treated with paraquat when the cells were about 85% confluent. MTT assays were performed to determine relative cell’s viability under all examined conditions in both PurA WT and PurA K/O MEFs. MTT results showed that the relative cell viability in the presence of PurA was significantly reduced in PurA WT MEFs in a time and dose dependent manner when compared to the control or non-treated samples. By the 24 h treatments, cell viability was reduced to 20% viable cells with 1.5 mM Paraquat treatment. On the other hand, although paraquat treatments resulted in a significant reduction in cell viability in a dose and time dependent manner, cell viabilities at 24 h with 1.5 mM paraquat treatment was about 50% in PurA K/O cells compared to the WT cells (Figure 2). These results suggest that cytotoxicity induced by oxidative stress is clearly more pronounced in PurA expressing MEF cells than cells with no PurA expression.

Figure 2:

Time and dose dependent effect of Paraquat treatment on cellular viability of PURA WT and PURA K/O MEF cells. PURA WT (A) and PURA K/O (B) MEF cells were treated with increasing doses of Paraquat (0, 0.5, 1, 1.5 mM). At 0, 4, 8, 18, and 24 h post treatments, MTT assays were performed to determine cellular viabilities and presented as bar graph. Data are shown as mean ± SEM (n=3).

Apoptosis and autophagy activation in PurA-WT and K/O cells in response to oxidative stress

The effect of oxidative stress on apoptosis was determined by biochemically analyzing the expression of apoptotic marker, cleaved caspase 3, in the presence and absence of PurA in MEFs. PurA expressing MEFs and PurA knockout MEFs were treated with increasing doses of paraquat (0, 0.5, 1, 1.5 mM) and whole cell protein lysates were extracted at 4,8,18, and 24 h post treatments. Western blot analyses were performed to determine the activation of apoptosis under all treatment conditions using an antibody against the Cleaved caspase 3. In both WT and KO MEFs, Cleaved caspase 3 was not detected at 4 and 8 h of paraquat treatments (Figures 3 and 4). Cleaved caspase 3 expressions were mainly induced at 18 and 24 h post treatments. Western blot analysis revealed a notable increase in the expression of cleaved caspase 3, and correlated with the increased concentrations of Paraquat treatments in MEF cells (Figures 3 and 4, panels B and C). In agreement with the viability assays (Figure 2), cleaved caspase 3 activation in response to paraquat treatments was significantly higher in PurA expressing MEFs than PurA k/o MEFs (compare Figure 3B with Figure 4B).

Figure 3:

Effect of oxidative stress on induction of apoptosis and autophagy in PURA WT MEF cells. (A) PURA WT MEF cells were treated with increasing doses of Paraquat (0, 0.5, 1, 1.5 mM). At 0, 4, 8, 18, and 24 h post treatments, protein lysates were prepared and western blot analyses were performed for the detection of PurA, cleaved caspase 3, total caspase 3, and LC3-I/II. Beta-actin was probed in the same membranes as loading control. The band intensities of cleaved caspase 3 (B), total caspase 3 (C), LC3-I (D), and LC3-II (E) were determined, normalized to B-actin, and represented as bar graph. (F). The LC3-II/LC3-I ratios were calculated and shown as bar graph. Data are shown as mean ± SEM (n=3).

Figure 4:

Effect of oxidative stress on apoptosis and autophagy induction in PURA K/O MEF cells. (A) PURA K/O MEF cells were treated with increasing doses of Paraquat (0, 0.5, 1, 1.5 mM). At 0, 4, 8, 18, and 24 h post treatments, protein lysates were prepared and western blot analyses were performed for the detection of PurA, cleaved caspase 3, total caspase 3, and LC3-I/II. Beta-actin was probed in the same membranes as loading control. The band intensities of cleaved caspase 3 (B), total caspase 3 (C), LC3-I (D), and LC3-II (E) were determined, normalized to B-actin, and represented as bar graph. (F). The LC3-II/LC3-I ratios were calculated and shown as bar graph. Data are shown as mean ± SEM (n=3).

In the same set of experiments, expression of autophagy marker LC3-I and LC3-II was also analyzed in parallel with cleaved caspase 3. As shown in Figure 3D and E, while there was no significant alteration in LC3-I expression, LC3-II expressions were induced in PurA expressing MEF in response to increasing doses of paraquat treatment at 24 h post treatments. Analysis of LC3-II/LC3-I ratios further verified the autophagy induction in those cells (Figure 3F). Interestingly, unlike PurA expressing MEFs, PurA k/o MEFs showed no significant alteration in either LC3-I or LC3-II expression under the same paraquat treatment conditions (Figure 4, lanes D–F). These results suggest that autophagy induction in PurA expressing cells under oxidative stress conditions is also more pronounced than those with PurA k/o cells.