Fig. 5

C/EBP-β LIP induces calreticulin expression and immunogenic cell death. MDA-MB-231 cells were left untreated (−, ctrl) or transfected with an empty pcDNA4/TO vector (em/empty), with a pcDNA4/TO expression vector encoding C/EBP-β LAP or C/EBP-β LIP, respectively. a. Whole cell lysates were probed with an antibody recognizing both C/EBP-β LAP and LIP isoforms. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. b. ChIP was performed to evaluate the binding of LAP or LIP to the CRT promoter (sites: 831–843; 1302–1313). no Ab: no anti-C/EBP-β antibody; bl: blank; DNA input: genomic DNA. The figure is representative of 1 out of 3 experiments. c. The relative expression of CRT gene was measured in triplicates by qRT-PCR. Data are presented as means±SD (n = 3). *p < 0.001: LIP-expressing cells vs all the other experimental conditions. d. Whole cell lysates were probed with an anti-CRT antibody. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. e. Surface CRT was detected by flow cytometry. The histograms represent the results obtained from 1 out of 3 experiments. f. Tumor cells were stained with PKH2-FITC, DC were stained with an anti-HLA-DR-PE antibody. Tumor cells were co-incubated with DC for 24 h. Double-stained cells were counted by flow cytometry. Data are presented as means±SD (n = 3). *p < 0.001: LIP-expressing cells vs all the other experimental conditions. g. T-lymphocytes were co-cultured with DC after phagocytosis, then incubated with MDA-MB-231 cells. The percentage of CD8⁺CD107⁺T-cells was measured by flow cytometry. Data are presented as means±SD (n = 3). *p < 0.001: LIP-expressing cells vs all the other experimental conditions