Cell culture and transfection
Chondrogenic cell line ATDC5 from mouse was obtained from Sigma Aldrich (St. Louis, MO, USA). These cells were cultured with the medium which was the mixture (1:1) of Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F12. And the mixture medium was supplemented with 5% fetal bovine serum (Gibco, Thermo Fisher Scientific, USA) and 2 mM Glutamine (Sigma-Aldrich). These cells were placed in the humid atmosphere under 37°C with 5% CO2. Trypsin/EDTA solution was used to culture these cells for the production of subcultures. The overexpression BNIP3 lentivirus and corresponding negative control were purchased from Genechem (Shanghai, China). The polybrene (Genechem) was used to promote the transfection efficiency. All the operations of the transfection were followed the instruction. 3-MA (5mM/mL) (Sigma-Aldrich) was used to treat overexpression BNIP3 cells.
Cell Counting Kit-8 (CCK-8) assay
ATDC5 cells were planted into the 96 well plates. After the adhesion of these cells, the culture medium supplemented with the LPS (0, 1, 2.5, 5, 10µg/mL) was used for the culture of these cells for 6 h. Next, the CCK-8 solution (Dojindo, Kumamoto, Japan) was diluted with the culture medium (1:10) which then added into the 96 well plates. Then these cells were incubated in the incubator for 1.5h. At last, the absorbance was measured with the spectrophotometer (Thermo Fisher Scientific).
Detection of glycosaminoglycan (GAG)
The levels of total GAG were measured with the commercial kit from Jianglai Biotechnology Co., Ltd (Shanghai, China). Supernatant of ATDC5 cells was collected by centrifugal tube. 50µL supernatant and 50µL 1,9-dimethylmethylene blue (DMMB) were added to 1.5 mL centrifuge tube, which was treated with vortex agitation for 15 s and incubated at room temperature for 30 min in dark. Then, centrifuge tube was centrifuged at 16000g for 10 min and the supernatant was removed cleanly. 50µL propyl alcohol was added to the centrifuge tube for vortex agitation for 15 s and incubated at room temperature for 5 min in dark. The solution in centrifuge tube was transferred to a new cuvette, which immediately detected by spectrophotometer (Thermo Fisher Scientific). The relative levels of total GAG were determined according to standard curve.
Supernatant of ATDC5 cells was collected by centrifugal tube. The supernatant was then centrifuged to remove impurities and transferred to sterilized tubes. Next, Human TNF-α ELISA kit (ab181421, Abcam, UK), human IL-1β ELISA kit (RAB0273, Sigma Aldrich) and human IL-6 ELISA kit (ab178013, Abcam) were used to detect the secretion of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) of ATDC5 cells. All experimental operations were performed in accordance with the instructions.
Apoptosis kit (Beyotime, Nanjing, China) was used to determine the proportion of apoptosis cells. These cells were prepared into the cell suspension. Next, the cell suspension was washed with the cold phosphate buffer saline (PBS) for three times to eliminate the interference of fetal bovine serum. After that, these cells were incubated with the Annexin V and polyimide (PI) in the dark room for 40 minutes. At last, the flow cytometry was performed to detect the apoptosis rates of these cells.
Total protein was collected with the RIPA buffer (Beyotime). And the concentration of these proteins was measured with the BCA (Beyotime) method. Next, these proteins were separated with the 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime). After that, these proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). Then these membranes were blocked with the 5% skim milk powder and incubated with the primary anti-bodies at 4 °C overnight. The primary anti-bodies used in this research were BNIP3 (ab109362, Abcam), Bcl-2 (ab32124, Abcam), Bax (ab32503, Abcam), Cleaved caspase3 (ab49822, Abcam), Cleaved caspase9 (ab2324, Abcam), Little Computer 3 (LC3) I/II (ab62721, Abcam), Autophagy-related protein 7 (ATG7) (ab133528, Abcam), Beclin1 (#3495S, Cell Signaling Technology, USA), P62 (#5114S, Cell Signaling Technology) and GAPDH (ab8245, Abcam). In the second day, these membranes were washed with the phosphate buffered solution (PBST) for three times and then incubated with the second anti-body for 2h at room temperature. At last, these membranes were washed with the PBST again and the immunoreactive signal was detected with the LAS-3000 Image Analyzer (Fujifilm, Tokyo, Japan).
Total mRNA was extracted with the trizol (Thermo Fisher Scientific) method. Next, the appropriate amount of mRNA was reversely transcribed into cDNA by the reverse transcription kit (Roche, Basle, Switzerland). Next, the SYBE Green (Applied Biosystems, New York, USA) was mixed with cDNA in a certain proportion. At last those cDNA was amplified with the 7500 RT-PCR system (Applied Biosystems). The expression of targeted genes was calculated with the 2-∆∆Ct method. The primers used in this research was BNIP3 forward: 5′-CAGAATTCATGGAGCAGAAACTCATCTCTGAAGAGGATCTGATGTCGCAGAACGGAGCG-3′ Reverse: 5′-TAGGATCCTCAAAAGGTGCTGGTGGAGG-3′ and GAPDH forward: 5′-ACAACTTTGGTATCGTGGAAGG-3′ Reverse: 5′-GCCATCACGCCACAGTTTC-3′.
These cells were plated on the sterilized coverslips before the experiment. After the adhesion of these cells, 4% paraformaldehyde was used to fix these cells. Next, the 0.2% Trixton-X 100 (Beyotime) was used to increase the penetrability of cell membranes. Then these cells were incubated with the primary antibody LC3B (#2775S, Cell Signaling Technology). Next, these cells were washed with the PBST for 30 minutes and incubated with the second antibody Alexa Fluor 555 Conjugate (#3969S, Cell Signaling Technology). After that, these cells were washed with the PBST again and the DAPI (Invitrogen, California, USA) was dropped on those cells. At last, these cells were observed under the laser scanning confocal microscope (Olympus, Hachioji, Japan).
All the experiments in this research were repeated for three times. And the data in this paper was displayed as mean ± SD. All the data in this research was analyzed with the Graphpad Prism 7.0 (Graphpad Software Inc, California, USA). The difference between diverse groups was analyzed with the student’s t test. The difference was significant when the values of P was less than 0.05.