To begin, seed DAKIKI cells in a six-well plate at a density of six million cells per well. Set up the different experimental groups, and after the corresponding treatment, based on the grouping method, incubate the plates for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Extract total RNA from the DAKIKI cells, following the instructions in the total RNA extraction kit.
Take one microliter of the extracted RNA from each group of samples to measure its concentration. Reverse transcribe one microgram of total RNA from each sample into complementary DNA, following the kit instructions. Perform reverse transcription polymerase chain reaction, or RT-PCR amplification to detect the expression of each gene.
For western blotting after the initial 24-hour incubation, collect the cells from each group. Add an appropriate volume of cell lysis solution to the collected cells before incubating them on ice for 30 minutes. Centrifuge the samples at 13, 500g for 10 minutes at four degrees Celsius, and collect the supernatant for further analysis.
Vortex the protein samples with 5X SDS-PAGE Loading Buffer at a ratio of four to one and heat the mixed samples at 100 degrees Celsius for five minutes to denature the protein before electrophoresis. Once the run is complete, transfer the gel onto the PVDF membranes. To block the membrane, incubate it in 5%nonfat milk for two hours at room temperature.
Then, incubate the membrane with the corresponding primary antibodies for 24 hours while using beta-actin antibody as an internal control. Subsequently, incubate the membranes with the corresponding secondary goat anti-rabbit IgG antibody at room temperature for two hours. Finally, treat the membranes with an appropriate amount of enhanced chemoluminescence working solution for protein band detection.
The quantitative RT-PCR results showed that the relative mRNA expression of C1GalT1 and Cosmc was downregulated in DAKIKI cells in the model group, compared with the control group. Dioscin upregulated the relative mRNA of both two different degrees compared with the model group. Western blotting results also showed that compared with the control group, the protein expression of C1GalT1 and Cosmc in DAKIKI cells in the model group decreased.
The protein expressions were upregulated after dioscin intervention.
