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. 2020 Nov 16;11:5799. doi: 10.1038/s41467-020-19584-1

Fig. 3. Molecular analyses of clonal populations reveal CL31-specific genetic alterations.

Fig. 3

a Heatmap representation of copy number alterations in the parental OCI-C5x cell line and clonal populations. b, c Representative images from one of three independent experiments of FISH staining for ERBB2 (red) and CEP17 (green) of b CL31 and c parental OCI-C5x cells in vitro. Scale bar 20 μm. d Quantification of cells with ERBB2:CEP17 copy number ratio equal or greater than two (FISH data) in the OCI-C5x cell line in vitro from experiment in (b) and in solid metastases derived from the OCI-C5x cell line. Data shown as mean ± SEM from three independent experiments, p Value was computed using the Mann–Whitney test, two-tailed. e Representative Western blot of ERBB2 protein in the OCI-C5x cell line and each of the clonal populations. β-actin was used as a loading control, representative blots from one of two independent experiments. f Maximum parsimony tree generated from all filter-passing substitutions detected by whole-exome sequencing of all single-cell derived clonal populations, and an autologous DNA sample from the patient’s blood. Branches colored by mean VAF of mutations in parental clone. g Western blot showing ERBB2 knockdown in CL31 using four distinct shRNAs, representative blots from one of three independent experiments. h Representative images and i quantification of colony formation in soft agar by CL31 expressing a control shRNA (GFP) or one of two distinct ERBB2 shRNAs (Sh2 and Sh4). Scale bar, 500 μm. The number of colonies were normalized to that of shGFP control. Three independent experiments were summarized by mean ± SEM. p Values were computed using the one-way ANOVA test and corrected for multiple comparisons using the Dunnett’s method. j OCI-C5x parental cells were sorted into ERBB2-low and ERBB2-high expressing populations by FACS. k Representative images of colony formation in soft agar by the unsorted OCI-C5x parental line and the ERBB2-low and ERBB2-high populations. Scale bar, 250 μm. l Quantification of colony formation in soft agar in (k) by the indicated OCI-C5x populations. Data were normalized to that of the unsorted parental line. Three independent experiments were shown as mean ± SEM. p Values were computed using the one-way ANOVA test and corrected for multiple comparisons using the Dunnett’s method.