Fig. 4: Molecular target of ERX-41 is LIPA.
a, Visualization of CRISPR–Cas9 screen in MDA-MB-231 cells showing genes associated with resistance to ERX-41; the top five genes are highlighted. b, KO clones of LIPA, SLC5A3, TMEM208, SOAT1 and ARID1A in MDA-MB-231 cells were evaluated for dose response to ERX-41 using CellTiter-Glo assays. Data presented as mean ± s.e.m., n = 3 biologically independent samples. Each gene was knocked out by two different sgRNAs. c,d, Effect of knockout of LIPA in SUM-159 (c) and MDA-MB-436 (d) on dose response to ERX-41 using CellTiter-Glo assays in vitro. Data presented as mean ± s.e.m., n = 3 biologically independent samples. e,f, Dose responses of parental SUM-159 and clones with LIPA KO to paclitaxel (e) and thapsigargin (f). Data presented as mean ± s.e.m., n = 3 biologically independent samples. g,h, Live-cell imaging studies with SYTOX Green showing ability of 1 μM ERX-41 to induce cell death in parental SUM-159 and SUM-159 clones with LIPA KO, with quantitation (g) and time-lapsed photomicrographs at 0 h and 30 h (h). Data presented as mean ± s.d., n = 4 fields per group. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Adjusted P values of last time points are shown. i–l, SCID mice were implanted with either parental SUM-159 or SUM-159 clones with LIPA KO and tumors allowed to establish (150 mm3). Following daily i.p. administration of vehicle or 10 mg kg–1 ERX-41, tumor size was measured and graphed (i), with mice body weights (j). Data presented as mean ± SEM, n = 5 mice per group. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Adjusted P values of last time points are shown. Xenograft tumors were harvested and processed for IHC with Ki67. Representative IHC staining is shown (k) and proliferative indices for each tumor are quantitated and graphed (l). Data presented as mean ± s.d., n = 5 per group. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Adjusted P values are shown. Experiment shown in h was done twice independently, with similar results, while that shown in i,j was done once. Numerical source data for a–g,i,j,I are provided.
