Figure 5.

Deletion of tsRNA target site leads to increased gene expression. (A) Diagram representing the CRISPR/Cas9 strategy for generating a mutant HEK293T-based cell line in which the predicted target site and its flanking sequence for SPINT1 in intron 2 is removed. (B) Bar chart showing the fold change of expression of steady state SPINT1 and GK transcripts, measured by qRT-PCR, in wild type, heterozygous SPINT1^+/– and homozygous SPINT1^–/– cells. (C) Western blot images showing signals of SPINT1 in wild type, and homozygous SPINT1^mut cells with ß-tubulin as loading control. Numbers represent quantification of the SPINT1 signal normalized to ß-tubulin. (D) Bar chart showing the fold change of nascent expression of SPINT1 upon mock transfection and transfection of tsRNA against SPINT1 in wt and SPINT1^mut cells. (E) Western blot images showing signals of SPINT1 in wild type, and homozygous SPINT1^mut cells mock or tsRNA transfected of with H3 as loading control. Numbers represent quantification of the SPINT1 signal normalized to H3. (F) Diagram representing the CRISPR/Cas9 strategy for generating a mutant HEK293T-based cell line in which the predicted target site and its flanking sequence for BCL2 in intron 2 is removed. (G) Bar chart showing the fold change of nascent and steady state expression of BCL2 upon mock transfection and transfection of tsRNA against BCL2 in wt and BCL2^mut cells. (H) Western blot images showing signals of BCL2 in wild type, and homozygous BCL2^mut cells mock or tsRNA transfected of with ß-tubulin as loading control. Numbers represent quantification of the SPINT1 signal normalized to ß-tubulin. (I) As in F for EGFR in intron 21. (J) As in G for EGFR. (K) As in H for EGFR.