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. 2019 Nov 20;20(23):5826. doi: 10.3390/ijms20235826

Figure 3.

Figure 3

Genetic inhibition of CALCRL counteracts the CGRP-induced increase in chemotherapy resistance. CALCRL was knocked down in HNT-34 cells using lentivirus-borne, doxycycline inducible shRNAs. Unless indicated otherwise, experiments were performed after pre-incubation with and in the presence of doxycycline. (a) Expression of CALCRL in HNT-34_shRen, HNT-34_shCALCRL-1, and HNT-34_shCALCRL-2 was determined by capillary-based protein quantification analysis (Wes). Left panel, Wes plot; right panel, quantification. Protein aggregates are frequently observed with G-protein coupled receptors. (b) cAMP levels after stimulation of serum starved HNT-34_shRen, HNT-34_shCALCRL-1, and HNT-34_shCALCRL-2 with 0 or 1 nM CGRP for 2 min. Means + SEM from 3 biological replicates. * p < 0.05; paired Student’s two-tailed t-test. (c) Proliferation of HNT-34_shRen, HNT-34_shCALCRL-1, and HNT-34_shCALCRL-2 in the absence and presence of doxycycline. Means +/− SEM from 3 biological replicates (error bars do not exceed data point symbols in most cases). (d,e) CALCRL knock-down diminishes CGRP mediated protection from araC- and daunorubicin (DNR)-induced apoptosis. Cells were pre-incubated with or without 100 nM CGRP for 1 h prior to addition of the indicated concentrations of cytostatic drugs. Means +/− SEM from 3–4 biological replicates. ns, not significant, * p < 0.05, paired Student’s two-tailed t-test. (d) AnnexinV/DAPI assay. Significance indicators refer to the proportion of viable cells. (e) Caspase 3/7 assay.