Fig. 3

Chloroquine and bortezomib induce C/EBP-β LIP/CHOP/TRB3/caspase 3 axis by increasing NO levels. MDA-MB-231 and JC cells were cultured for 24 h in the absence (ctrl, −) or presence (+) of the lysosome inhibitor chloroquine (CQ; 1 μM) or of the proteasome inhibitor bortezomib (B; 1 μM), alone or in combination. When indicated, the lysosome activator torin-1 (To; 1 μM) or the proteasome activator betulinic acid (BA; 10 μM) were added. a. Whole cell lysates were probed for NOS I, NOS II, NOS III. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. b. The activity of NOS enzyme in cell lysate and the levels of nitrite in the supernatants were measured in triplicates by spectrophotometric assays. Data are presented as means±SD (n = 3). *p < 0.05: treated cells vs ctrl cells; °p < 0.02: CQ + B-treated cells vs CQ/B-treated cells. c. MDA-MB-231 cells were cultured for 24 h in the absence (−) or presence (+) of the lysosome inhibitor chloroquine (CQ; 1 μM), the proteasome inhibitor bortezomib (B; 1 μM), the lysosome activator torin-1 (To; 1 μM), the proteasome activator betulinic acid (BA; 10 μM), the NO donor sodium nitroprusside (SNP; 10 μM), the NO scavenger carboxy-PTIO (PTIO; 100 μM), alone or co-incubated in different combinations. Whole cell lysates were immunoprecipitated (IP) with the anti-C/EBP-β antibody, which recognizes both C/EBP-β-LAP and C/EBP-β-LIP, then immunoblotted (IB) with the anti-mono/poly-ubiquitin (UQ) antibody; alternatively, lysates were directly immunoblotted with the indicated antibodies. No Ab: lysate from untreated cells immunoprecipitated in the absence of the anti-C/EBP-β antibody, as control of specificity. The expression of β-tubulin was used as control of equal protein loading before immunoprecipitation. The figure is representative of 1 out of 3 experiments