Fig. 2: MaTAR25 knockout affects 4T1 cell viability, migration, and invasion in vitro; all of which can be rescued by ectopic expression of MaTAR25 in knockout cells.

a CRISPR/Cas9 was used to generate MaTAR25 KO clones in 4T1 cells. Pairs of sgRNAs were introduced targeting upstream and downstream of the transcription start site of MaTAR25, resulting in 390–620 bp genomic deletions over this region, and a Renilla Luciferase sgRNA was used as a negative control. Knockout clones were selected by genomic PCR and Sanger sequencing for homozygous genomic deletion. Control clones were selected by qRT-PCR for expressing similar levels of MaTAR25 as parental 4T1 cells. qRT-PCR (n = 2), and representative images of single smRNA-FISH (red: MaTAR25) are shown to confirm MaTAR25 KO. Scale bars are 5 μm. b 4T1 cells were seeded at the same cell density in 12-well tissue culture plates at day 0, and cell counting was performed at different time points. The mean cell numbers of three independent replicates of 4T1 control groups and MaTAR25 KO groups are shown ± SD (n = 3). *P < 0.05 (paired Student’s t test; two-tailed). c Live-cell tracking was performed over time to examine cell migration. Images were collected every 5 min for a total of 8 h and analyzed by CellTracker image-processing software. The mean relative migration distance (μm) of 4T1 control groups and MaTAR25 KO groups is shown ± SD (n = 3 independent replicates). *P < 0.05 (paired Student’s t test; two-tailed). d Twenty-four-well Boyden chamber invasion assay (24 h). The mean relative cell invasion of three independent replicates of 4T1 control groups and MaTAR25 KO groups is shown ± SD (n = 3). *P < 0.05 (paired Student’s t test; two-tailed). e Selected clones with ectopic expression of MaTAR25 or GFP were used as positive and negative controls to assess rescue in a cell viability assay. Data are presented as mean values ± SD (n = 3). *P < 0.05 (paired Student’s t test; two-tailed). f Cell invasion assay. The mean cell numbers and mean relative cell invasion of three independent replicates of 4T1 control1, MaTAR25 KO1, MaTAR25 KO1 with GFP expression, and MaTAR25 KO1 with MaTAR25 ectopic expression is shown ± SD (n = 3). *P < 0.05 (paired Student’s t test; two-tailed).