Figure 6.
Epigenetic control of cell adhesion molecule expression by CREBBP contributes to transformation of SCLC.
A, Knockdown of Crebbp decreases global H3K27ac levels in preSC cells. Histone H3 was used as loading control.
B, Knockout of CREBBP decreases global H3K27ac level in human SCLC cell line DMS53. Histone H3 was used as loading control.
C, Metaplots of the H3K27ac distribution across all transcripts and across the core 57 genes consistently downregulated upon Crebbp suppression in preSC cells and in the 3 neuroendocrine tumor types. Data from two controls (non-silencing shRNA and empty vector) and two Crebbp shRNAs (shCrebbp-1 and shCrebbp-2) along with input are shown. TSS, transcriptional start site. −5000 and 5000 represent base pairs upstream and downstream of the TSS.
D, ChIP-seq read density plots shows decreased H3K27ac levels in introns 1 and 2 of Cdh1 in two Crebbp knockdown preSC cells (shCrebbp-1 and shCrebbp-2; blue) compared to two control preSC cells (shNS and shEmpty; red).
E, Immunoblotting of E-CADHERIN protein in 2 control preSC cells and 2 sgRNA-mediated Cdh1 knockout preSC cells. Beta-ACTIN was used as loading control.
F, Anchorage-independent assay of sgRNA-mediated Cdh1 knockout on the growth ability of preSC cells in soft agar. Representative images of colonies in soft agar are shown. Cells were seeded at 2.5×105 cells/well (6 well plate). The number of colonies from 15 fields was counted. ***, p<0.001. n= 3 independent experiments. Scale bar = 100μm.
G, Colony formation assay of Cdh1 knockout on the growth ability of preSC cells. Cells were seeded at 6×103 cells/well (6 well plate). Representative images are shown. The number of colonies from 4 fields representing the entire well was counted. ****, p<0.0001. n= 3 biological replicates.