Extended Data Fig. 5: circFAM53B encodes a unique peptide.
From: Tumour circular RNAs elicit anti-tumour immunity by encoding cryptic peptides
(a) The putative IRES activity of circFAM53B, determined by relative luciferase activity of Luc/ Rluc, in the vectors was tested. **P = 0.0016. (b) Immunoblotting for Flag expression in HEK293T cells transfected with P-circ vector carrying an expression cassette for circFAM53B with a 3×Flag-coding sequence. For gel source data, see Supplementary Fig. 4. (c) Immunoblotting for circFAM53B-219 in HEK293T cells transfected with empty vector (vec), linFAM53B or circFAM53B. For gel source data, see Supplementary Fig. 4. (d) MS analysis identified circFAM53B-219 unique sequences in HEK293T cells transfected with circFAM53B. (e) HLA-I immunoprecipitation followed by MS analysis identified circFAM53B(192-200) as the HLA binding peptide in HEK293T cells transfected with circFAM53B. (f) Immunoblotting for circFAM53B-219 in 34 cases of primary breast tumour tissues (T) and the paired normal breast tissues (N). For gel source data, see Supplementary Fig. 5. (g) Immunoblotting for circFAM53B-219 in normal breast epithelial cells and several breast cancer cell lines. For gel source data, see Supplementary Fig. 4. (h-m) DCs from HLA-A*02+ breast cancer patients (h-j) or healthy donors (k-m) were pulsed with linFAM53B(264-302), circFAM53B(181-219) and then co-cultured with autologous T cells. The in vitro primed T cells were rechallenged by autologous breast cancer cells (h-j) or circFAM53B(192-200)-pulsed T2 cells, MCF-7 and MDA-MB-231 cells, respectively (k-m). (h, k) Flow cytometric analysis for the markers of effector T cells (CD45RO+CCR7−) in the primed T cells and the percentages of the stained T cells are shown. (i, l) Quantification of the spot count per 5 × 104 T cells determined by IFNγ ELISpot is shown. (l) ***P = 0.0003. (j, m) Percentages of the PI+ dead target cells, determined by flow cytometry, are shown. (j) ***P = 0.0002 (day 6), 0.0002 (day 8), 0.0001 (day 21). (m) ***P = 0.0009 (MCF-7), 0.0006 (MDA-MB-231). Results are mean ± s.d. of n = 3 (a, h-m) independent experiments producing similar results. Representative image of n = 3 independent experiments (b-g). ****P < 0.0001. P values, compared with HEK293T cells transfected with empty vector (vec) (a), untreated T cells (UT) (h-m), were determined by two-tailed one-way ANOVA with Dunnett’s multiple-comparisons test.
