Fig. 2

LCD knockout strategy and verification in HEK293T and PANC-1 cells. a Schematic representation of LCD knockout strategy using CRSPR-Cas9 technology. LCD1 (located between exon 8 and 11) was deleted with sgRNA primers: two upstream primers (sgRNA-A1/A2) and two downstream primers (sgRNA-B1/B2). This was then verified by genotyping with KMT2D-1-F/R primers. For LCD2 (located between exon 31 and 36), two upstream primers (sgRNA-C1/C2) and two downstream primers (sgRNA-D1/D2) were used to delete LCD2 and then the rest was tested with KMT2D-2-F/R primers. b, c Verification with genotyping assays. In HEK293T and PANC-1 cells, genotyping PCR was performed in transfected cells. The results are consistent with the predictions compared with the control group (vehicle). d, e RT-PCR analyses to measure LCD knockout. lcd1 mRNA expression only significantly decreased in LCD1-deleted cells, while that of lcd2 only in LCD2-deleted cells. The set gene located at the C-terminal of the KMT2D gene was selected as a control (error bars denote standard deviation). f, g Representative images of immunofluorescences and western blotting in HEK293T and PANC-1 cells. After LCD knockout, the KMT2D protein level did not significantly decrease, and KMT2D LCD1 or KMT2D LCD2 was successfully deleted. KMT2D, 593 KDa; LCD1 deletion-KMT2D, 483 KDa; LCD2 deletion-KMT2D, 447 KDa. All the values are represented as means ± SD. Data are representative of three independent experiments