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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: Increasing intratumor C/EBP-β LIP and nitric oxide levels overcome resistance to doxorubicin in triple negative breast cancer

Fig. 6

Lysosome and proteasome inhibitors and C/EBP-β LIP overexpression cooperate to induce doxorubicin-triggered immunogenic cell death. MDA-MB-231 cells (−, ctrl) were stably transfected with a doxycycline-inducible vector encoding C/EBP-β LIP. Cells were cultured in the absence (−) or presence (+) of doxycycline (doxy; 1 μg/ml) for 24 h, to induce C/EBP-β LIP. When indicated, cells were co-incubated with the lysosome inhibitor chloroquine (CQ; 1 μM) or with the proteasome inhibitor bortezomib (B; 1 μM), alone or in combination, followed by 5 μM doxorubicin (dox) for further 24 h. a. ChIP was performed to evaluate the binding of 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. b. The relative expression of CRT gene was measured in triplicates by qRT-PCR. Data are presented as means±SD (n = 3). *p < 0.01: treatments vs un-induced, untreated (“- doxy,-”) cells; °p < 0.05: “+ doxy” cells vs corresponding “- doxy” cells. c. 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. d. Surface CRT was detected by flow cytometry. The histograms represent the results obtained from 1 out of 3 experiments. Anti-ISO: anti-isotype antibody. 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 < 0.002: treatments vs un-induced, untreated (“- doxy,-”) cells; °p < 0.001: “+ doxy” cells vs corresponding “- doxy” cells. 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: treatments vs un-induced, untreated (“- doxy,-”) cells; °p < 0.001: “+ doxy” cells vs corresponding “- doxy” cells

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