Fig 1.
Expression of selected HSPs genes at basal level in normal Tasmanian devil skin, primary DFTD tumours and DFTD cell lines.
Expression of the HSPS genes HSPA1L, HSPA2, HSPA4L, HSP90B1, HSPAA1, HSP90AB1, HSPD1, and HSPB1 was analysed using qRT-PCR. Expression was measured relative to the control gene RPS18. Results are the mean and standard error of three biological replicates. Significance is defined as * p<0.05, ** p<0.01 and *** p<0.001. No-cDNA controls and no-RT controls were also included and did not show any amplification of product. Expression of HSPB1 was not detected at basal level therefore, the figure is not shown. Results are representative of two experiments.
Table 1.
Selected study heat shock protein (HSP) genes annotated in the Tasmanian devil reference genome.
Fig 2.
Expression of selected HSP proteins at basal level in DFTD cell lines and DFTD primary tumours.
HSPs expression was detected by western blot using total protein isolated from three DFTD cell lines (C5065, 1426 and 4946) and three primary DFTD tumour biopsies (TD1, TD2 and TD3). The anti-HSP70 antibody detects different members of the HSP70 family; the anti GRP94 antibody detects the HSP90B1, the anti-HSP90 antibody detects various members of the HSP90 family and the anti-HSP60 detects the HSPD1. Bands represent two technical replicates for each biological sample. Bands from the same blot in the HSP90 panel were reorganised (vertical black lines) to have a consistent order in the figure. Original images of the blots are presented in S4 Fig. Results are representative of two experiments.
Fig 3.
Studies of DFTD cell viability after heat shock treatment.
a. DFTD cells growing in culture were exposed to heat shock (42 oC and 45 oC) or control temperature (35 oC) in a water bath for 30, 60 or 120 min. Cell viability was analysed immediately after the treatment by flow cytometry. Each marker represents the mean and standard error of three biological replicates. b. DFTD cells in culture were treated as before but returned to normal incubation conditions (35 oC) to allow recovery for 24 hours. Viability was then measured with flow cytometry. Bars represent the mean and standard error of three biological replicates. Experiments were repeated twice with reproducible results.
Fig 4.
Expression of HSPs after heat shock treatment.
Cultured DFTD cells were heat shocked at 42 oC for 30 min in a water bath and then recovered in an incubator under normal cell culture conditions (35 oC) for 4, 8, or 24 hours. a. HSP gene expression was analysed using qRT-PCR. Expression was measured relative to the control gene RPS18. Results are the mean and standard error of three biological replicates. Significance is defined as * p<0.05, ** p<0.01 and *** p<0.001. b. Protein expression of HSPs was analysed using western blot. Expression was measured relative to the untreated control. Results are the mean and standard error of two biological replicates. Statistical significance is defined as * p<0.05, ** p<0.01 and *** p<0.001. Results are representative of two experiments.
Fig 5.
HSP expression after radiation.
Cultured DFTD cells were exposed to gamma radiation (20 Gy) and then recovered for 24 hours in an incubator under normal cell culture conditions. a. HSP gene expression was analysed using qRT-PCR. Expression was measured relative to the control gene RPS18. Results are the mean and standard error of three biological replicates. Significance is defined as * p<0.05, ** p<0.01 and *** p<0.001. b. Protein expression of HSPs was analysed using western blot. Expression was measured relative to the untreated control. Results are the mean and standard error of two biological replicates. The results from one single experiment are shown. Statistical significance is defined as * p<0.05, ** p<0.01 and ***p<0.001.
Fig 6.
Screening of antibodies against DFTD tumour antigens using serum from an immunised Tasmanian devil.
Total cell proteins from cultured DFTD cells were separated by two-dimensional gel-electrophoresis, transferred to nitrocellulose membrane and probed with pre-immune serum or post-immunisation serum. Negative control (no serum) is also shown. Arrows indicate spots detected by the immune serum that were selected for further identification by mass spectrometry (see Table 2). Experiments were repeated twice with reproducible results.
Table 2.
DFTD tumour antigens recognised by serum of an immunised Tasmanian devil and identified by mass spectrometry.