The human MDS cell line SKM-1, was kindly provided by Professor Zhou at the Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). The erythroleukemia K562 and the histiocytic lymphoma U937 cell lines were kept frozen in our laboratory. Cell line SKM-1, U937 and K562 were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and no antibiotic in 5% CO₂, 95% air incubator at 37°C. For the infection protocol, SKM-1 cells at the exponential stage were plated in 6-well plates (5×10⁴ cells/well), and were infected with the SPAG6-shRNA lentivirus or NC-shRNA lentivirus at a multiplicity of infection (MOI) of 20 in the concentration of 5 µg/ml polybrene, respectively. After 10 h, the cells were washed and then resuspended in complete medium. After 5 days, the transfection efficiencies were evaluated by flow cytometry.

SKM-1, K562 and U937 were seeded in 6-well plates (1×10⁵), and were treated with recombinant human sTRAIL (310–04; Peprotech, Rocky Hill, NJ, USA), respectively. The treated concentrations were as follows: 0, 20, 40, 60, 80, 100 and 50 ng/ml. After 24 h, the cells were collected, and the apoptosis rates were measured by flow cytometry.

The apoptosis rates were detected using Annexin V and 7-ADD double-staining by flow cytometry. Following the cell treatments, the cells were collected and washed twice with phosphate-buffered saline (PBS). The early apoptotic cells contained Annexin V⁺/PI⁻, while the late apoptotic cells contained Annexin V⁺/PI⁺. Both of the two stages of apoptotic cells were counted, and the results were illustrated as a percentage of the total cell count. The techniques were supported by the Life Science Department of Chongqing Medical University (Chongqing, China).

The total cellular RNA from each group was extracted using the TRIzol reagent, and the reverse transcription reaction was performed using the Prime Script™ RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The cDNA was amplified in a 10-µl PCR mix with 5 µl of SYBR-Green super mixture. The PCR reactions were performed in a CFX-Connect Real-Time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The cycling parameters were: 95°C for 30 sec, then 40 cycles at 95°C for 5 sec and 60°C for 30 sec. PCR primers were as follows: SPAG6 forward, 5′-CCTTTCAGCTCTCAGTCAGGTTTC-3′ and reverse, 5′-TCTTCACGTTTCATCCTTGTCCTT-3′; death receptor 4 (DR4) forward, 5′-TCGCTGTCCACTTTCGTCT-3′ and reverse, 5′-GGCGTTCCGTCCAGTTTTG-3′; DR5 forward, 5′-AAGACCCTTGTGCTCGTTGT-3′ and reverse, 5′-GCTGCAACTGTGACTCCTAT-3′; and GAPDH forward, 5′-CTTTGGTATCGTGGAAGGACTC-3′ and reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′. The PCR reactions were performed in a CFX-Connect Real-Time PCR system for 40 cycles (Bio-Rad Laboratories, Inc.). The relative gene expression levels were calculated by the 2^−ΔΔCt method.

The cells were collected and lysed in RIPA lysis buffer (Beyotime, Beijing, China) containing 1 µM PMSF, and the concentration was measured using the BCA Protein Assay kit (Beyotime). The total cell lysate was denatured via boiling and a total of 50 µg of protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin for 90 min at room temperature and then incubated overnight at 4°C with specific antibodies. The primary antibodies were as follows: GAPDH (AG019, 1:1,000; Beyotime) cleaved caspase-8 (9496, 1:500; Cell Signaling Technology, Inc., Beverly, MA, USA); SPAG6, PARP, cleaved PARP, caspase-8, DR4, DR5 and FADD (ab155653, ab32138, ab32561, ab32397, ab8414, ab181846, ab108601, 1:1,000; all from Abcam, Cambridge, UK); caspase-3 and BID (YT0656, YT0488, 1:500; both from Immunoway, Newark, DE, USA); and BAX and BAK (RLT0456, RLT0449, 1:500; both from Ruiying Biological, Suzhou, China). After five washes with TBS-Tween-20, the membranes were incubated with a goat anti-rabbit of goat anti-mouse peroxidase-conjugated second antibody (A0216, A0208, 1:1,000; Beyotime) for 1 h at 37°C. Then, the excess antibody was removed from the blots with TBS-Tween-20 three times before incubation in ECL. The protein expression levels were analysed using Vilber Fusion software (Fusion FX5 Spectra; Vilber Lourmat, Marne-La-Vallée, France).

Apoptosis was measured by a CCK-8 assay. Cells in exponential growth were seeded in 96-well plates at a density of 8×10³ cells/well, and then, they were treated with recombinant human sTRAIL (rTRAIL) containing different concentrations for 24 h, respectively. Finally, 10 µl of CCK-8 (Dojindo Laboratories, Kumamoto, Japan) solution was added to each well, and the plates were incubated at 37°C for 100 min. Cell apoptosis was estimated by measuring the absorbance at 450 nm using the plate reader. All conditions were tested in five replicates.

The cells infected with the lentivirus were collected after infection and lysed in RIPA lysis buffer. The solution was centrifuged at 12,000 rpm for 10 min at 4°C and the supernatants were kept. Protein A agarose and antibody (ab108601, 1:100, ab14738, ab181846, 1:2,000; Abcam) diluted with RIPA buffer were mixed and incubated overnight at 4°C. Then the total protein was mixed and incubated overnight at 4°C. After incubation, the mixture was washed six times with different types of washing buffer. Finally, a western blot analysis was performed for immunoblot analysis.

All the results were technically repeated three times, and were expressed as the mean ± standard deviation (SD). The significant differences between groups were assessed by one-way analysis of variance (ANOVA) and Students t-test (SPSS version 20.0; SPSS, Inc., Chicago, IL, USA) and a value of P<0.05 was considered statistically significant.

The lentivirus expressing shRNA was used against SPAG6 for gene silencing in SKM-1 cells, and the SKM-1 cells infected with NC-shRNA lentivirus were used as a control group. Six days after infection, abundance of cells with green fluorescence could be observed under a fluorescence microscope (Fig. 1A). The transfection efficiencies were detected by FACS, and the results were 97.19±3.21% and 88.31±12.81% (Fig. 1B). This demonstrated that the SKM-1 cells were successfully infected by the lentivirus. The inhibitory degree of SPAG6 was measured by qRT-PCR and western blot analysis. The results showed that both the mRNA and protein levels were reduced in cells infected with SPAG6 shRNA-lentivirus as compared to the control group (Fig. 1C and D).

Apoptosis was analysed by Annexin V and 7-AAD assays, as illustrated in Fig. 2A and B, and the apoptosis rates in SKM-1 infected with SPAG6-shRNA were significantly higher than those in the control group (NC-shRNA, 8.65±2.07% vs. SPAG6-shRNA, 20.20±2.11%; P<0.05). To explore the activation of the TRAIL signal pathway, the expression of related proteins was examined. Western blot analysis revealed that the expression levels of cleaved PARP, caspase-8 and cleaved caspase-8 were obviously higher in SKM-1 infected with SPAG6-shRNA lentivirus than in the control group, whereas the expression of PARP showed no significant difference between the two groups (Fig. 2C and D). Furthermore, the expression of apoptotic factors, including BAK, BAX, BID and caspase-3, also showed statistical differences in the two groups (Fig. 3E and F). These data indicate that the TRAIL signal pathway was activated when the expression of SPAG6 was reduced.

Following rTRAIL treatment, the percentage of apoptosis cells increased, and the flow cytometry data indicated that the higher concentration of TRAIL led to higher apoptosis rates (Fig. 3A and B). To investigate the growth of SKM-1 cells treated with different concentrations of TRAIL, CCK-8 assays were performed to analyse cell proliferation. Cell proliferation was suppressed in the cells treated with TRAIL as compared to the normal control group (Fig. 3C). In addition, western blot analysis showed that the expression of related proteins was higher after treatment (Fig. 3D and E). However, as shown in Fig. 3F and G, in the SKM-1, U937 and K562 cells after treatment with the same concentration of TRAIL, the apoptosis rates were not significantly different except for that of SKM-1 (the expression of SPAG6, SKM-1<U937<K562), and similar results were also observed in the CCK-8 assays (Fig. 3H). These data illustrate that a high expression of SPAG6 may resist apoptosis induced by TRAIL.

TRAIL induces apoptosis through DR4 (TNFRSF10A) and DR5 (TNFRSF10B). Therefore, the degree of apoptosis induced by TRAIL is closely related to the levels of DR4 and DR5. As illustrated in the figure, both the mRNA and protein levels of the two receptors were not changed (Fig. 4A-C). However, the expression of FADD was statistically different (Fig. 4B and C), indicating that the regulation of apoptosis may not be through DR4 and DR5 expression.

As shown in the Fig. 4, the expressions of DR4 and DR5 showed no difference. Therefore, immunoprecipitation was used to explore the relationship between the FADD and TRAIL death receptors. FADD was used as the bait protein to immunoprecipitate DR4 and DR5. Then, DR4 and DR5 were blotted. As shown in the figure, the interaction between FADD and both death receptors increased in SKM-1 infected with SPAG6 shRNA-lentivirus as compared to the control group (Fig. 4D).