Quantity one quantitation software version 4

For analysis of different proteins involved in CeO₂ NPs induced toxicity, 2 × 10⁵ cells/mL/well grown in 6-well culture plate were exposed with NPs concentrations (1 μg/mL–100 μg/mL) for 24 h. After completion of exposure, cells were washed three times with PBS and protein was extracted from cells for electrophoresis. The amount of protein was estimated using Bradford method [33 (link)] and resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. For the detection of specific proteins, PVDF membrane was incubated with anti-BAX, anti-BCl-2, anti-caspase-3, anti-caspase-9, anti-cytochrome C, anti-PARP, anti-p53, and anti-β-actin after blocking in 3% BSA solution. Detection of protein bands was carried out using chemiluminiscence and densitometric analysis was done using Quantity One Quantitation Software version 4.3.1 (Bio-Rad, USA).

Table 1 represents primer sequences that were used for amplification. Normalization was carried out using housekeeping gene, beta actin. The PCR cycling conditions (for CCKAR) were of initial denaturation of 5 min at 94°C, followed by 35 cycles at 94°C for 1 min and 60°C for 1 min and 72°C for 1 min and 30 s, and final extension of 72°C for 10 min. For beta actin, the PCR cycle conditions were of initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s, and final extension of 72°C for 10 minutes. Gene Amp PCR system 9700 (Applied Biosystems, USA) was used for amplification and products were analysed in 2% agarose gel stained with ethidium bromide in VERSA DOC Imaging system, Model 1000 (Biorad, USA). Densitometric analysis of the PCR products was done using Quantity One Quantitation Software version 4.3.1 (Biorad, USA).