Fig. 6: A positive charge at the N-termini of LRRC8A or LRRC8D reduces Pt drug uptake.

a, b Western blot rescue control of the cell lines, where either wild-type (wt) or N-terminally mutated LRRC8A (p.I2R) or LRRC8D (p.F2R) constructs were reintroduced into LRRC8A or LRRC8D-deficient cells. c Immunofluorescence imaging of the clonal rescue cell lines, expressing the C-terminally MYC tagged LRRC8 subunits, the scale bar represents 20 µm. Clonogenic survival assays of the different LRRC8A or LRRC8D rescue cell lines treated with cisplatin (d–f), carboplatin (g–i), and blasticidin S (j–l) for 24 h with the indicated drug concentrations. Representative images of selected lines and concentrations are shown. The data represent mean ± SD of three independent replicates and were fitted to a non-linear regression dose-response curve (log(inhibitor) vs. normalized response -Variable slope). p values are calculated by one-way ANOVA followed by Tukey’s multiple comparisons test for the log(IC50) values of the survival curves. m, n Representative yH2AX immunofluorescence images of the LRRC8A or LRRC8D-deficient cells expressing either the wt or mutated Lrrc8a or Lrrc8d rescue constructs treated with 2 µM cisplatin for 24 h. The scale bar represents 10 µm. o, p Quantification of yH2AX foci in the nucleus of LRRC8A or LRRC8D-deficient cells expressing either the wt or mutated Lrrc8a or Lrrc8d rescue constructs treated with 2 µM cisplatin for 24 h. Per cell line and condition, approximately 100 nuclei were quantified. Lines at median and quartiles of three independent replicates are shown (ordinary one-way ANOVA followed by Tukey’s multiple comparisons test).