Figure 2 | Scientific Reports

Figure 2

From: The proto-oncogene survivin splice variant 2B is induced by PDGF and leads to cell proliferation in rheumatoid arthritis fibroblast-like synoviocytes

Figure 2

The expression of survivin splice variants in RA and OA synovial tissues.(a) The survivin pre-mRNA generates some mature mRNA transcripts (splice variants) which result from alternative splice. Arrows indicate the stop codon. (b) RT-PCR of whole synovial tissues showed the expression of survivin splice variants (n = 9 different RA patients, n = 6 different OA patients and the HL-60 cell line). The forward primer was located in Exon 2 and the reverse primer was located in Exon 4. Forty cycles of PCR were performed. The data shown are representative of the samples and cropped image was used. The gels have been run under the same experimental conditions. (c,d) The relative expression levels determined by real-time PCR with the TaqMan method. The relative expression levels were corrected based on the expression level of HPRT. (c) Whole synovial tissues (n = 9 from different RA patients, n = 6 from different OA patients) (RA, 68.4 ± 7.2 years old; OA, 74.1 ± 7.9 years old; t-test, p = 0.183). The Dunnett method, ∗p < 0.05 (WT; p < 0.01, 2B; p = 0.018 and ΔEx3; p > 0.05, respectively). (d) Separated cells from the synovium from different RA patients (n = 3). The CD55+ cells indicate RA-FLS (CD3–CD14–CD16–CD19–CD20–CD56–CD45–CD55+), the CD19+ cells indicate B cells (CD45+CD19+) and the CD4+ cells indicate T cells (CD45+CD4+). The results are the Means ± SEM. (e) The results of the Western blot analysis to confirm whether anti-survivin splice variant antibodies react with the three types of GST-fusion survivin proteins. The anti-survivin-2B (chicken and rabbit polyclonal antibodies) and anti-survivin-ΔEx3 (chicken polyclonal antibodies) antibodies recognized the GST fusion human survivin variants corresponding to each antibody. The anti-survivin antibodies (rabbit monoclonal and polyclonal antibodies) reacted against all three GST-fusion splice variants. The cropped blots were used in the figure. (f–g) Immunohistochemistrical (IHC) staining of survivin-2B and -ΔEx3 in RA and OA synovial tissues (red, x40, x20). The primary antibodies against survivin-2B and -ΔEx3 were used. (h–l) The dual IHC staining of lineage markers (red) and survivin-2B (green) in RA synovial tissues (x40). Arrows indicate lineage marker-positive cells. (h) CD55 and survivin-2B. (i) CD4 and survivin-2B. (j) CD8 and survivin-2B. (k) MPO and survivin-2B. (l) CD19 and survivin-2B.

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