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[Preprint]. 2024 Aug 30:rs.3.rs-4844371. [Version 1] doi: 10.21203/rs.3.rs-4844371/v1

Figure 5. Amiloride enhances the sensitivity of HSPC and CRPC cell lines to low concentrations of lapatinib.

Figure 5

(A) 22Rv1 cells were treated with varying concentrations of lapatinib, 10μM amiloride or a combination of the two for 72h before being collected and lysed into cytoplasmic and nuclear fractions as described in earlier figure legends. Co-administration of lapatinib and amiloride does not increase the accumulation of ErbB3 in the cytoplasmic fraction. (B) 22Rv1 cells were treated with 1–10μM lapatinib and assayed for viability. Lapatinib was dissolved in 100% DMSO. Experiments were done in triplicate. Error bars represent standard deviation. (C) In a head-to-head comparison of lapatinib and amiloride, the combination was additive when cell viability was assayed with the MTT reagent. Experiments were done in triplicate. Error bars represent standard deviation. Coloured dotted line estimates 50% viability. Table shows p-values with respect to FBS DMSO. (D) 22Rv1 (CRPC) cells were treated for 72h with lapatinib, amiloride or the combination or 100% sterile DMSO and stimulated with PBS, EGF or HRG for 15 minutes prior to collection to observe activation of ErbB family members and their downstream targets. Cells were lysed in denaturing lysis buffer before being analysed by immunoblotting. 25μg of protein were loaded per lane. Tubulin was used as a loading control. Tables show p-values with respect to PBS DMSO in each cell line tested. (E) PC-346C cells were treated with varying concentrations of amiloride and assayed for viability. Amiloride was dissolved in 100% DMSO. Experiments were done in triplicate. Error bars represent standard deviation. (F) PC-346C cells were treated with 1–10μM lapatinib and assayed for viability. Lapatinib was dissolved in 100% DMSO. Experiments were done in triplicate. Error bars represent standard deviation. (G) PC-346C cells were treated with lapatinib, amiloride or the combination, which is shown to be additive when cell viability was assayed with the MTT reagent. Experiments were done in triplicate. Error bars represent standard deviation. Table shows p-values with respect to FBS DMSO. (H) PC-346C cells were treated with varying concentrations of lapatinib, 10μM amiloride or a combination of the two for 72h before being collected and lysed into cytoplasmic and nuclear fractions as described in earlier figure legends.