Fig. 4

Validation of GC-essential isoforms. a Crystal violet proliferation assay following CRISPRi-mediated suppression with sgRNAs targeting CIT, CCNE1 and MTA3. Data is shown as mean ± SD, n = 2; each dot is a different sgRNA. b Images from crystal violet proliferation assay of HFE145 (normal gastric cell line) or YCC3 (GC cell line) 7 days post-infection with sgRNAs targeting CIT, CCNE1 or MTA3. c BrDU proliferation assay. 7 DPI with the indicated sgRNAs. Data is shown as mean ± SD, n = 2; each dot is a biological replicate of the indicated sgRNA. pValue is calculated using two-tailed unpaired t test. (*p ≤ 0.05). d Anchorage-independent growth of YCC3 cells following CRISPRi-mediated suppression of the indicated genes. Following sgRNA infection, 50,000 cells were plated on a semisolid surface. A number of colonies were quantified by counting 5 different images for each sgRNA. Data is shown as mean ± SD; each dot is a different image. pValue is calculated using two-tailed unpaired t test. (*p ≤ 0.05). e Images of YCC3 colonies in control cells or following suppression of CIT expression. f WB showing the CIT expression in YCC3 cells following CRISPRi-mediated suppression. g H3K4me3 and H3K27Ac at the CIT promoter in GC tumours and adjacent normal tissue. Bottom panel shows the CIT PacBio sequencing in GC cell lines. h The indicated GC cell lines were infected with control or CIT-targeting sgRNAs (two sgRNAs). Seven days post-infection, cell proliferation was assessed using a crystal violet staining assay. The results are plotted as an average ± SD of two sgRNAs. Each dot represents a different sgRNA. pValue is calculated using two-tailed unpaired t test. (*p ≤ 0.05). i CIT RNA-Seq reads in GC cell lines. j CIT protein levels in GC cell lines