Extended Data Fig. 7. Analysis of c-Myc and L-Myc in Kdm6a-Mutant vs. Kdm6a-WT Tumor Derived Cell Lines.

(a) Immunoblot analysis for c-Myc with a c-Myc antibody (Y69) in Kdm6a-WT and Kdm6a-Mutant SCLC cell lines cultured in ultra-low attachment flasks at late times (>2 months) after cell line generation. NCI-H82 and CORL279 human SCLC cell lines are included as benchmark controls for SCLCs with high c-Myc expression. (b, c) RT-qPCR for Myc mRNA expression in the early vs. late passage Kdm6a-WT and Kdm6a-Mutant tumor-derived cell lines shown as individual lines (b) or grouped by genotype (c). (d) Immunoblot analysis for L-Myc with a specific L-Myc antibody (Cell Signaling E3M5P) in Kdm6a-WT and Kdm6a-Mutant SCLC cell lines cultured in ultra-low attachment flasks at late times (>2 months) after cell line generation. CORL47 and NCI-H1092 human SCLC cell lines are included as benchmark controls for SCLC cell lines with high L-Myc expression. CORL47 and NCI-H1092 express the RLF-MYCL fusion and hence MYCL migrates at higher molecular weights compared to WT L-Myc. (e-h) RT-qPCR for Mycl (e,f) or Mycn (g,h) mRNA expression in the early vs. late passage Kdm6a-WT and Kdm6a-Mutant lines shown as individual lines (e,g) or grouped by genotype (f,h). For all RT-qPCR experiments, n=6 biological independent experiments for each genotype (2 biological independent experiments for each cell line). Data are presented as mean values ± SEM. Statistical significance was calculated using unpaired, two-tailed students t-test and p-values are indicated. For all t-tests comparing Kdm6a-Mutant vs. Kdm6a-WT cell lines, early passage Kdm6a-Mutant vs. Kdm6a-WT were compared. (I,j) Tracks of H3K4me1, and KDM6A ChIP-seq for Mycl (i), and Myc (j) from ChIP-seq data from Fig. 5. Each track is the sum of 2 Kdm6a-WT cell lines (1014, 159-1) and 2 Kdm6a-Mutant cell lines (236L,236R) peaks with their respective input. (k) Immunoblot analysis for c-Myc and L-Myc in a Kdm6a-Mutant SCLC cell line (672-2) using highly specific antibodies with benchmark controls above. (l) Immunoblot analysis of 672-2 cells transduced with 4 independent Myc sgRNAs or a non-targeting control (sgControl) acutely after transduction and puromycin selection.